Practical guidance of chlorine dosage for efficient … › bio › docs ›...

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Practical guidance of chlorine dosage for efficient antifouling strategies

Jaeeun Kim, Sung Min Mun, Seok Hun Hong, Jee Yeon Kim, Jeyong Yoon*

School of Chemical and Biological Eng., College of Eng., Seoul National University,

San 56-1, Sillim-dong, Gwanak-gu, Seoul, 151-742, KOREA * Corresponding author e-mail: [email protected] Presenting author e-mail: [email protected]

The industrial systems contain many sites and components susceptible to biofilms,

which play an important role, win microbial growth and industrial fouling. Thus, the

control of the biofims in industrial systems has been an important issue. However the

efficient controlling strategy was not provided yet, since the resistance of biofilms to

biocides makes many methods less effective.

Although the prevention of biofouling can be achieved by periodical application

of biocides at suitable condition, the perfect removal of biofilm from industrial system

is almost impossible. Thus a new strategy that is keeping biofilm formation below

certain threshold with proper monitoring and biocide usage has been introduced and

emphasized. Nevertheless, the development of alternative biocides, chlorine is still the

most used biocide in the industrial systems cause of its low cost and convenience,

although excessive dosage of chlorine might cause corrosion and toxic byproduct

problems.

Reportedly, the popular CT concept which is usually used for planktonic cell

disinfection dose not work for biofilms, therefore a new practical guidance of chlorine

dosage for controlling biofilm is needed.

In this study, we attempted to suggest a guide that helps to determine the proper

concentration of chlorine and reaction time effective for antifouling control. The

biocidal efficiency of chlorine was examined with P. aeruginosa biofilm as an

indicating microorganism in CDC biofilm reactor. This study suggests the CT contour

diagram that can provide the proper chlorine concentration and reaction time needed to

achieve a certain level of inactivation of P. aeruginosa biofilm. This guidance might

help practicians to control biofouling effectively.

Keywords: Biocides, Chlorine, Antifouling, Pseudomonas aeruginosa

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Complex biofilms degrading Polychlorinated Biphenyls

Alexandre J. Macedo, Kenneth N. Timmis, Wolf-Rainer Abraham

GBF - National Research Centre for Biotechnology, Environmental Microbiology, Mascheroder Weg 1, 38124 Braunschweig, GERMANY

Worldwide 1.5 million tons of polychlorinated biphenyls were produced, from which a

substantial amount have been released into the environment since the early 1930's. No

natural products are known which are similar to PCBs. Their chemical stability and

toxicity make PCBs a serious and persistent environmental pollutant. Constant efforts

on PCB metabolism and degradation has been made, but less is know about the ability

of microbial communities to degrade PCB of polluted and non-polluted sites and how

these communities are composed. In our presentation we will show that several

communities are able to build biofilms on PCB oil, we identified the major members of

the biofilms by Single-Strand Conformation Polymorphism technique. Distinct

communities were observed depending on the origin of the samples. Regarding the PCB

degradation, communities from PCB contaminated sites showed a better efficacy to

degrade PCBs. Nevertheless, the communities from non-polluted sites degraded up to

45% of monochlorinated biphenyls and up to 17% of a pentachlorinated congener.

Furthermore, by using Confocal Laser Scanning Microscopy, we will present structure

and dynamics of complex biofilms growing on PCB oil.

Keywords: Polychlorinated Biphenyls (PCB), Single-Strand Conformation Polymorphism (SSCP), Biofilms

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Biofilm of Pseudomonas fluorescens growing under various environmental conditions

Irina A. Ryzhikova*, Olga L. Chrushcheva

Department of Botany and Microbiology, Faculty of Biology and Ecology

Yaroslavl State Universrty, pr Matrosova, 9,150057, RUSSIA * Corresponding author e-mail: [email protected]

UNDER REVISION

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Simulation of the effects of biofouling growth in a virtual heat exchanger

E. Eguía López*, Milad M. Milad, M. A. Girón Portilla, B. Río-Calonge, Trueba

Ruiz

Department of Sciences & Techniques of Navigation and Ship Construction, University of Cantabria, Gamazo nº 1, 39004, Santander. SPAIN

*Corresponding author e-mail: [email protected]

The importance of the problem of the biological deposit in the tube walls of heat

exchanger is very known due to the economic impact, and the lost which caused in the

industry and in the all device and installation that use water of the refrigeration.

The objective of this investigation work is the developing a computer program to

optimise the design, simulation an analysis of the heat exchanger (cooler, evaporator

and condenser) of the Tube-shell type which service under the effect of fouling and

especially the biofouling that is characteristic in the marine installation.

In this paper it is proposed the design and the simulation of a heat exchanger

operation with computer through three different methods. For this objective a computer

program has been created for the calculation of the heat exchanger that defines all its

dimensions as well as the flow characteristics of the fluids, following the requirements

of the thermal process that will carry out the heat exchanger in question. After the

design it is simulated the operation of the heat exchanger under normal and critical

conditions to correct the design failure.

Keywords: Biofouling, Marine Installation, Heat Exchanger, Computer Program

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Attachment of Desulfovibrio desulfuricans to stainless steel and nickel surfaces – the influence of nickel on the bioadhesion

F.A. Lopes(1)*, P. Morin(1), R. Oliveira(1), L.F. Melo(2)

(1) Centro de Engenharia Biológica, Universidade do Minho, 4710-057 Braga, PORTUGAL

(2) Faculdade de Engenharia do Porto, Departamento de Engenharia Química, Universidade do Porto, Porto, PORTUGAL

* Corresponding author e-mail: [email protected]

The presence of micro-organisms such as sulphate-reducing bacteria (SRB) on metal

surfaces and their ability to carry out specific biochemical reactions can alter the

physical/chemical conditions at the surface and lead to microbiologically influenced

corrosion (MIC). To prevent the deterioration of material caused by biofilms it is

necessary to understand the processes governing biofilm development including

mechanisms of cell adhesion and to take into account the dissolution of metallic

elements that may further affect adhesion and biofilm development. The present work

investigated the influence of nickel as part of the substratum composition on the

adhesion ability of Desulfovibrio desulfuricans by studying the attachment of the

bacteria to stainless steel 304 (SS 304), metallic nickel (Ni) and polymethylmetacrylate

(PMMA), which is a non-metallic control material. The influence of nickel on SRB

growth and its relation to adhesion was also assessed. A statistically significant

difference across the colonization of the three tested materials was detected, with the

highest number of cells adhered on Ni, followed by SS 304 and by PMMA. The higher

number of SRB adhered to the stainless steel compared to PMMA may be explained by

differences in hydrophobicity, roughness, and by the larger (γ+) surface tension

component of stainless steel. Bacterial growth was found to be positively affected by

the presence of nickel which may explain the preferential adhesion to this metal. It is

suggested that the specific polymeric structures at the bacteria surface not only mediate

the microbial adhesion to nickel substratum, but are also part of a metal-uptake system

for this essential element.

Keywords: Desulfovibrio desulfuricans, Sulphate Reducing Bacteria (SRB), Stainless

Steel, Nickel, Microbiologically Influenced Corrosion (MIC)

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Bioleaching – an interfacial process mediated by acidophilic bacteria, iron(III)ions and exopolymers

Wolfgang Sand

Universität Duisburg-Essen, Biofilm Centre, Aquatische Biotechnologie Geibelstrasse 41, D - 47057 Duisburg, GERMANY

e-mail: [email protected]

Extracellular polymeric substances (EPS) seem to play a pivotal role in bioleaching. For

a better control of this process structure and function of EPS from leaching bacteria are

of crucial importance. Our research focussed on the extremophilic bacteria

Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans. For this purpose the

composition of the corresponding EPS and their function was analyzed. The EPS of

both species consist mainly of neutral sugars and lipids. The functions of the

exopolymers seem to be (I) to mediate attachment to a (metal) sulfide surface, (II) to

concentrate iron (III) ions by complexation through uronic acids or other residues at the

mineral surface and, thus, allowing for an oxidative attack on the sulfide. Consequently,

dissolution of the metal sulfide is enhanced, which may result in an acceleration of 20-

to 100-fold of the bioleaching process over the chemical leaching. Experiments were

performed to elucidate the importance of the iron (III) ions complexed by EPS for strain

specific differences in oxidative activity for pyrite. Strains of Acidithiobacillus

ferrooxidans with a high amount of iron (III) ions in their EPS, possess a higher

oxidation activity than those with less iron (III) ions. These data provide insight into the

function and, consequently, the advantage of extracellular polymeric substances for

bacteria. Furthermore, the physiological properties of the bacteria depend on genetic

characteristics. As shown by a molecular approach using sequence analysis of 16S

rRNA genes (DGGE) Acidithibacillus ferrooxidans and Leptospirillum ferrooxidans

contain at least five different subgroups each, which can be correlated with the pyrite

oxidation activity of the bacteria.

Keywords: Extracellular Polymeric Substances (EPS), Extremophilic Bacteria

Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, Bioleaching

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Influence of papermaking conditions in the formation of biofilms

A. Blanco, C.E. Torres, C. Negro, J. Tijero

Chemical Engineering Department. Chemistry Faculty. Complutense University. 28040 Madrid. SPAIN

e-mail: [email protected]

Microbiological problems in the paper industry have been enhanced nowadays due to

the actual trends in the papermaking processes. A higher consumption of secondary

fibres, the progressive closure of water circuits and the manufacture in neutral/alkaline

pH have converted paper mills in an optimum environment for the development of

microorganims. The attachment of these microorganisms to paper machine surfaces

originates the formation of biofilms which produce important problems during

papermaking. These problems may affect the runnability of the machine and the quality

of the final product and, therefore, the productivity of the mill.

On the other hand, the control of microbial activity in the paper industry is

becoming more complicated nowadays due to the limitations in the use of biocides.

Therefore, new environmental friendly strategies are being developed to control the

problems in the mills producing a minimum final impact on the environment. Some of

the new concepts are based on dispersants, enzymatic treatments, nutrients limitation or

combinations between different systems.

This paper will present the results obtained with new environmental friendly

control systems. Slime growth conditions have been optimised for getting biofilm

samples at lab scale, of adequate nature, in order to evaluate different control systems.

Several dispersants and enzymatic treatments have been studied and the synergic effect

of dual systems has been evaluated at alb and mill scale. Furthermore, the influence of

papermaking conditions on biofilm formation has been also studied.

Keywords: Paper Industry, Biocide, Biofilm, Biofouling, Slime

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Release of cells by the biofilm under glutaraldehyde treatment

S. Gómez de Saravia1, M. Disiervi2, M. Fernández Lorenzo de Mele1

1 INIFTA, Departamento de Química, Facultad de Ciencias Exactas, UNLP-CONICET, C.C. 16, Sucursal 4, 1900 La Plata, ARGENTINA

2 Instituto de Limnología (ILPLA), Facultad de Ciencias Naturales y Museo, UNLP, C.C. 712, 1900 La Plata ARGENTINA

Presenting author e-mail: [email protected] Corresponding author e-mail: [email protected]

Although frequently disregarded, the release of cells by the biofilms has practical

importance because it reveals a survival mechanism that allows the spread of the cells

and the initiation of a new biofilm in another place. The aim of this paper is to compare

the release of cells from a biofilm immersed in a glutaraldehyde-containing saline

medium and in a basal control. With this purpose biofilms were formed on stainless

steel sheets dipped in a Pseudomonas fluorescences culture medium and then removed

to submerge them in sterile saline medium. The survival sessile and released cells (rc)

were enumerated after different periods. Plate counting method and vital staining using

epifluorescence microscopy were employed. It could be observed that in the presence of

glutaraldehyde the number of surviving rc reaches a maximum and then decreased with

time while an increasing sigmoidal curve was shown by the control. The slope of the

curves is associated to the rate of bacterial release. In the presence of glutaraldehyde

this slope was lower than that of the control. The balances of living and dead, sessile

and released cells were analyzed. The decrease of the number of survival rc in the

presence of glutaraldehyde was interpreted as the result of both the reduction of the

release rate and the killing action of glutaraldehyde.

Keywords: Glutaraldehyde, Stainless Steel, Pseudomonas fluorescences,

Epifluorescence Microscopy, Biofilm

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Evaluation of antifouling performance of two copper saturated layer applied on carbon steel panels

P. S. Guiamet 1,2 , S. G. Gómez de Saravia 1,3

1 Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA) Departamento de Química, Facultad de Ciencias Exactas, UNLP-CONICET,

C.C. 16, Suc.4, (1900), La Plata, ARGENTINA 2 CONICET 3 CICBA

e-mail: [email protected]; [email protected]

Biofouling is a colonization occurring in aquatic systems and due to sessile organisms

fixed to man made substrata. The resultant biofouling can affect economic cost. The aim

of this paper was evaluated the performance of two antifouling copper saturated layers.

Bioassays were carried out in Argentina harbour (38º02’ S-57º32W). Six series of two

panels with two copper saturated layers and without they were vertically hung from a

floating dock, about 0.3 and 1.5 m below the water surface to provide the record of

fouling organisms. Every month (during six months) one series of panels was removed

so as to estimate the recruitment of macro and microsessile species (bacteria, fungi,

microalgae, protozoan, etc.) and was immediately replaced by clean one (monthly

panels or recruitment panels). The other six series were removed on a monthly basis,

one every month, following the development of the established community from the

beginning of the exposition (accumulative panels). Microscopic and scanning electron

microscopic observations of biological community were made. Studies of abiotic factors

were made too. Data obtained from control panels and copper saturated panels were

compared in order to estimate performance of the coating. The panels covered with one

of two a saturated copper layer as well as a control panel (without copper layer) have

been showed an important levels adherence on the surface by the different

microorganisms investigated (different groups of bacteria and fungi). Panel covered

with the other saturated copper layer shown a good antifouling performance

(macrofouling).

Keywords: Copper, Carbon Steel, Antifouling, Scanning Electron Microscopy (SEM)

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Succession of microbial communities in a biofilm on the protective coating

Zh.Kopteva, V. Zanina, I.Kozlova

Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine

154 Zabolotny str., 03143 Kyiv, UKRAINE e-mail: [email protected]

It was studied the succession of microbial communities in the system of coating-

microorganisms. Successional analysis of microbial communities in the biofilm formed

on the coating surface on the base of polyethylene was done in the conditions of a

laboratory experiment. Bacteria were isolated by us earlier from biodeteriorated

coatings used for gas pipelines protection from corrosion.

It was determined that the biofilm on the surface of coating Polyken 980-25

begins to formation during 1 hour and consists of iron reducing bacteria, the number of

which increases during the experiment on 1-3 order. On the first stages of succession it

has been revealed bacteria of Pseudomonas genus, iron reducing, oxidizing

hydrocarbon. After 2 hours the denitrifying bacteria begin to grow in the biofilm and

after 6 hours of the experiment – the bacteria of Bacillus genus.

It is necessary to note that iron reducing bacteria as dominants in the biofilm are

agents of biodeterioration not only of a protective material but a metal. Penetrating

through the coating pores or after its destruction on the metal surface they can cause

corrosion.

The reliability of metal underground structures primarily depends on the status

of the anti-corrosive protection that in addition to the cathodic protection takes place

due to insulation coatings. One of the reasons of degradation of materials protective

properties is vital functions of soil bacteria capable to initiate and promote corrosive

processes.

The use of successional analysis is perspective for estimation of different coating

types bioresistance.

Keywords: Polyethylene, Metal, Corrosion, Pseudomonas, Bacillus, Iron Reducing

Bacteria

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Microbial degradability of poly(ethylene terephthalate) and some pet nitrated copolymers

M. Soledad Marqués-Calvo, Marta Cerdà-Cuéllar

Optics & Optometrics Department, EUOOT,

Polytechnique University of Catalonia, E-08222 Terrassa (Barcelona), SPAIN Corresponding author e-mail: [email protected]

With the intention of combining biodegradability and good material properties, many

attempts are being made with aromatic polyesters introducing aliphatic components into

the aromatic polyester chains. In this sense, a series of PET copolymers containing 5-

nitroisophthalic units (PET70NI30) and nitroterephthalic units (PET70NT30) were

synthesized and characterized previously. Microbial degradability of these films was

investigated in laboratory cultures. Discs of the films with a diameter of 5 mm and a

thickness ranging from 225 to 275 µm were used in this study. The microorganisms and

the discs were cultured in minimum mineral medium. Fungal attack of the discs by

Aspergillus niger (CECT 2700) and Penicillium pinophilum (CECT 2912) was tested by

light microscopy, scanning electron microscopy, and gel permeation chromatography.

Bacterial biodegradability by Pseudomonas fluorescens and Bacillus spp. from

environmental isolates, were estimated by weight loss of films, and gel permeation

chromatography. Surface roughness of the three polymers was quantified by optical

imaging profiler. All the discs had one bright and slightly concave upper surface and

one dull and slightly concave lower surface after their synthesis. The roughness of both

surfaces of each polymer was different after their synthesis, mineral medium cultures,

and microorganisms cultures. In general, roughness was higher in lower surfaces of the

different polymers. However, biodegradability was only present in upper surface of

PET70NT30 discs with A. niger cultures. Anchorage to and growth on this surface by A.

niger were observable by scanning electron microscopy, and loss of surface molecular

weight was present.

Keywords: Polymers, Biodegradability, Aspergillus niger, Penicillium pinophilum,

Pseudomonas fluorescens

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Manganese-oxidizing bacteria of biofilm in power plant lamellar heat exchanger

M. Peltola1, J. Ekman1, T. Kuosmanen1, M. Pulliainen2, T. Laurila3, A. Kiviharju4,

J. Nikkonen4, J.-F. Selin4, H. Huopalainen4, M. S. Salkinoja-Salonen1

1 Department of Applied Chemistry and Microbiology, University of Helsinki, FINLAND

2 Savcor Process Oy, Mikkeli, FINLAND 3 Savcor ART Oy, Mikkeli, FINLAND 4 Fortum Oyj, Porvoo, FINLAND

Cooling system in power plant is based on recycling Baltic Seawater between sheets of

the lamellar heat exchangers. Several marine bacteria have capability to oxidize soluble

manganese (Mn2+) to insoluble form (MnO2). MnO2 deposits on heat exchanger surface

may decrease cooling capacity of heat exchangers and expose to biocorrosion.

We screened for manganese-oxidizing bacteria from deposits persisting on

pressure washed heat exchanger surfaces. The deposits contained high concentration of

manganese and microorganisms. Manganese-metabolizing bacteria were isolated from

the deposits using selective media and identified based on the 16S rRNA gene

sequences. We investigated the ability of manganese-oxidizing bacteria to form Mn-

nodules on stainless steel (AISI 316) and titanium coupons. Effect of anti-fouling

treatments on accumulated Mn-nodules on stainless steel was investigated in laboratory

scale.

We acknowledge support of TEKES PINTA-program to Biofouling-project

(484/31/03) and the industrial partners Fortum, Kemira, M-real, Savcor, StoraEnso and

UPM-Kymmene.

Keywords: Cooling System, Biocorrosion, Manganese-Oxidizing Bacteria, 16S rRNA

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Applying molecular techniques to the study of marine biofouling in heat exchangers

L. J. Taracido1*, R. Solera1, J.M. González2, E. Nebot

1 Department of Chemical Engeenering, Food Tecnology and Environmental Technologies,

Faculty of Environmental and Marine Science, CASEM. University of Cádiz. Campus Río San Pedro s/n, 11510 Puerto Real, Cádiz, SPAIN

2 Departament of Biogeochemestry, Instituto de Recursos Naturales y Agrobiología, Spanish Council of Scientific Research (CSIC), P.O. Box 1052, 41080 Seville, SPAIN


Biofouling is a global problem affecting many industrial processes specially those using

sea water in their cooling systems. Deposits of biological origin builds up in the

conductions of heat-exchangers/condensers generating highly significant economic and

energetic losses. Common antibiofouling strategies require the use of biocides. At

present environmental legislation is becoming increasingly restrictive on the use of

biocides in natural environments. Our initiative focuses on the understanding of the

colonization process and the microbial communities involved in order to design

alternative, highly efficient, antifouling strategies. With this aim in mind, a portable

pilot plant has been designed to carry out in situ experimental studies of biofilm

formation under controlled thermal and hydrodynamic conditions. In the present study,

we use molecular fingerprinting techniques (Denaturing Gradient Gel Electrophoresis,

DGGE) to investigate the bacterial diversity deposited inside the pipes of heat

exchangers untreated and treated with chlorine and peracetic acid, in spring and summer

during a period corresponding to six month. Biofilms developed in the pipes were

sampled, DNA was extracted and 16S rRNA fragments were amplified by PCR. DGGE

allow the visualization of the individual amplified products and the comparison of

different treatments. To characterize the bacterial communities constituting the biofilms,

DNA libraries were prepared, screened and the selected clones sequenced. A mong the

obtained results, we anticipate the finding of bacteria belonging to the Roseobacter-

group (Alpha-Proteobacteria) as a major member of the bacterial community forming

the biofilms during the heat-exchange processes.

Keywords: Sea Water, Cooling System, Antifouling, Denaturing Gradient Gel

Electrophoresis (DGGE), 16S rRNS, Alpha-Proteobacteria

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Microfouling on metal surfaces exposed to atmosphere: development of a method for isolation of aggressive microorganisms – corrosion agents

Dalia Pečiulytė*

Biodestructors’ Research Laboratory, Institute of Botany Žaliųjų ežerų 49, LT-08406 Vilnius, LITHUANIA

Corresponding author e-mail: [email protected]

The aim of this study was to develop an effective method of screening microorganisms

that induce metals’, exposed to the atmosphere, corrosion processes. Organisms

ubiquitous in the natural environment on metal surface are able to convert insoluble

metal compounds into soluble forms by excretion of protons and/ or various

metabolites, including organic acids. Fungi especially can act by protonation of the

anions on the metal surface, making it less available to the metal cations. Protonation

occurs in several ways. Protons can be pumped into the external medium by the ATPase

of the plasma membrane. This is the main source of protons and serves to set up ionic

gradients for the acquisition of nutrients or for the resistance to high metal

concentration. Organic acids may also be produced which provide another source of

protons. In addition, the organic acid anion is usually capable of forming a complex

with the metal cation, thus affecting its mobility and toxicity.

The method developed is based on observing clear zones, or haloes, around

colonies growing on agar medium amended with the selected insoluble metal

compounds. The incorporation of compounds which were readily solubilized into solid

medium provided an easy means of screening for cultures aggressive in the corrosive

layer. In a comparative study of fungal, bacteria yeast and yeast-like fungi flora growing

on the metals’ samples exposed at five different places, we observed that the species

after one or three months exposure were not different from those found in the

surrounding natural environment (on vegetation and in the air), but their abundance

correlated with the ability excrete acid metabolites and withstand high concentrations of

metal compounds in the corrosion layer.

Keywords: Fungi, Metal, Protonation, Organic Acids

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Electrochemical behavior of stainless steels in media containing iron-oxidizing bacteria (IOB)

Starosvetsky J. 1*, Starosvetsky D. 2, Hilel T., Armon R. 1

1 Division of Environmental, Water and Agricultural Engineering, Faculty of Civil and Environmental Engineering

2 Laboratory of Electrochemistry and Corrosion, Faculty of Materials Engineering, Technion-Israel Institute of Technology, Haifa 32000, ISRAEL Corresponding author e-mail: [email protected]

The corrosion behavior of two stainless steel (SS) types UNS S30403 and UNS 31603

in solutions containing iron oxidizing bacteria Sphaerotilus spp. isolated from rust

deposits was studied. Corrosion and electrochemical measurements were conducted

through the exposure of tested steels to 3% NaCl solution supplemented with a

Sphaerotilus culture for 30 days. The corrosion test consisted of three periods with

different exposure conditions: 1st period - 5-day exposure of tested specimen in sterile

3% NaCl solution; 2nd period - addition of 3 days-old Sphaerotilus culture to 3% NaCl

at 3:2 v/v ratio and further 11 days exposure of tested specimens up to complete

sedimentation of ferric oxides; 3rd period –exposure of specimens in solution after

sedimentation. During 3rd period exposure one portion of specimens was positioned in

upper part of the cell (above sediment layer) and second portion-at the bottom part

(among sediment layer). In order to determine the effect of IOB on the electrochemical

behavior of tested SS and the initial stages of pit nucleation the electrochemical

characteristics of specimen were measured periodically during exposure.

No pitting corrosion was detected during SS exposure in sterile 3% NaCl

solution and corrosion potential (ECORR) of specimen gradually increased indicating

steel passivity. Pitting corrosion was initiated on both tested SS in solution containing

IOB culture. After culture addition corrosion potential of SS rapidly decreased from -0.1

V to values below -0.4 V due to gradual degradation of protective characteristics of

passive film up to pit nucleation which is the result of crevice effect caused by biogenic

ferric oxides forming biofilm on SS surface.

Keywords: Stainless Steel, Sphaerotilus, Pitting Corrosion, Passivity

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The role biofilms play in microbially induced corrosion. A new non-thermal plasma –based approach for inhibition of corrosion.

Kholodenko V.P.1, Chugunov V.A.1, Irkhina I.A1., Kobzev E.N 1., Zhirkova N.A.1,

Yermolenko Z.M.1, Trushkin N.I.2, Grushin M.E.2, Akishev Yu.S2.

1State Research Center for Applied Microbiology, Obolensk, Moscow Region, 142279 RUSSIA 2State Research Center of Russian Federation TRINITI, Troitsk, Moscow region, 142190, RUSSIA

Presenting author e-mail: [email protected] Corresponding author e-mail: [email protected]

The dynamics of development of biofilms and biocorrosion of mild steel depending on

culture media was studied. Two culture media were used: modified medium API and

medium supplemented with kerosene as a sole source of carbon. A microbial association

isolated from oil-polluted soil was used. Scenarios of development of the biofilms were

similar for both media. The content of sulfate-reducing bacteria (SRB) increased and the

number of aerobic heterotrophs, including acid-producing bacteria decreased. Corrosion

under biofilms took place at different rates. The biofilm in medium API stimulated

corrosion. Corrosion losses for control coupons cultured in medium added with

kerosene increased proportionally to time of incubation. Corrosion of experimental

coupons markedly inhibited as the biofilm progressed. The extent of corrosion by the

end of the experiment was 10.2 % vs control. Influence of aeration on parameters of

biofilm, as well as on corrosion itself was also investigated. It was shown that SRB

grew faster, and corrosion enhanced when culture conditions were changed from

microaerophilic to anaerobic ones. Currently, different biocides are used to control MIC.

However, many microorganisms of biofilms are known to be highly resistant to these

agents. Therefore, the application of cold plasma procedure to control biocorrosion may

be perspective. Two methods of generation of cold plasma were tested. It was shown

that treatment of liquid and the surface of a metallic plate with discharge killed 99.9%

highly resistant spores of Bacillus sp. within a few minutes. Vegetative cells of

Pseudomonas sp, Escherichia coli, Bacillus subtilis and microorganisms constituting

model and natural biofilms (SRB and aerobic heterotrophs) were also inactivated.

This research was carried out in the frame of ISTC Projects ##2245p and 1344-2.

Keywords: Mild Steel, Sulfate-Reducing Bacteria (SRB), Oil-Polluted Soil, Biocide

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Electrochemical behaviour of stainless steel alloys in wastewater under the influence of biofilms

Montero MJ1*, Bethencourt M1, Botana J1, Corzo A2, Matres V3, González J4,

Andrades JA5, Garcia de Lomas J2.

1Depto. Ciencia de los Materiales, Ingeniería Metalúrgica y Química Inorgánica Facultad de Ciencias del Mar y Ambientales

2Depto. Biología, Facultad de Ciencias del Mar y Ambientales Pol. Río San Pedro s/n. 11510, Puerto Real, Cádiz, SPAIN

3Acerinox S.A. 11379, Palmones-Los Barrios, Cádiz, SPAIN 4IRNAS-CSIC. Avenida de Reina Mercedes, 10. 41012, Sevilla. SPAIN

5Aguas de Jerez, EMSA. C/ Cádiz, 1. 11402, Jerez de la Frontera, Cádiz, SPAIN *Corresponding author. e-mail: [email protected]

Microorganisms play an important role in the localized corrosion process of metals,

causing important damages in many industries and motivating the search of more

resistant materials. In this study, the corrosion behaviour of three stainless steel alloys

were tested (AISI-304, AISI-304LS, AISI-316) in order to assess its possible use in a

wastewater treatment plant. Three sets of analysis were developed: (i) open circuit

analysis in which corrosion potential (Ecorr) of sterile and non-sterile wastewater was

compared, (ii) impedance analysis under the presence of Pectinatus portalensis, and (iii)

weight loss and pits with and without the presence of P. portalensis and Desulfovibrio

sp after 45 days. In the first experiment, AISI-316 underwent relatively lower corrosion

potentials indicating the highest resistance. Impedance analysis also showed a higher

polarization resistance in AISI-304LS and AISI-316. In the third experiment, P.

portalensis formed relatively dense biofilms when compared to Desulfovibrio sp. The

highest biomass of biofilm was observed when both bacteria were cultivated together,

suggesting the importance of consortia in biofilm establishment. AISI-304 showed the

lowest weight-loss after 45 days with and without bacteria. No pits were observed under

ESEM in the three different alloys. Weight-loss analysis showed some discrepancies

with electrochemical assays. Ecorr and impedance analysis give overall information

about the corrosion processes involved in MIC, making difficult to distinguish local

processes occurring in the metal-biofilm interface. Despite electrochemical assays

showed differences between alloys, the absence of pits recommend longer experiments.

Keywords: Stainless Steel, Corrosion Potential, Impedance, Pit, Pectinatus portalensis,

Desulfovibrio sp

- 18 -

The role of the Microbes in Marine Water on the Corrosion of the 316L Stainless Steel

Y. Miyano1*, M. Yamamoto2, K.Watanabe3, Y. Kikuchi4

1 National Institute of Science and Technology

1-8-31 Midorioka, Ikeda-city, Osaka 563-8577, JAPAN 2 Hitachi, Ltd. Power & Industrial Systems, Nuclear Systems Division

3-1-1 Miyuki-cyo, Hitachi, Ibaragi 317-8511, JAPAN 3 Marine Biotechnology Institute Co.Ltd. Kamaishi Laboratories,

3-75-1 Heita, Kamaishi, Iwate 026-0001, JAPAN 4 Osaka University, 11-1 Mihogaoka, Ibaraki, Osaka 567-0047, JAPAN

*Corresponding author e-mail: y.miyano @aist.go.jp

Microbiologically Influenced Corrosion (MIC) was suspected in the corrosion failure of

a cooling system of stainless piping welded joint, carrying marine water. Marine water,

which is used for cooling water in a plant was transferred to the laboratory and used for

experiments. In the first experiment, weld metal samples were exposed to the test

solution for 56 days (marine water and sterilized marine water (control)). The surface

condition of experimental coupons was observed using a Scanning Electron Microscope

(SEM). In another experiment, the free corrosion potential of these materials was

monitored for 56 days. Pitting corrosion was found in the coupons exposed to marine

water. Free corrosion potential ennoblement was found to be significant compared to the

controls. It was suspected that this corrosion case was MIC. In the second experiment,

coupons were exposed to diluted nutrient medium containing single cultures of

microbes isolated from the MIC causing marine water sample used for the first

experiment. After the exposure test, surface condition of the experimental coupon was

observed using SEM. Pitting corrosion sites was found in coupons exposed to some of

the isolates. The results indicate that they contribute to the corrosive effect of the AISI

type 316L welds. In this study the role of microbes which were detected as isolates were

examined.

Keywords: Marine Water, Scanning Electron Microscope (SEM), Corrosion Potential,

Stainless Steel

- 19 -

Causes lowering effectiveness of cathode protection in conditions of microbial influence corrosion.

Andriy I. Pilyashenko-Novokhatny

Zabolotny Institute of Microbiology & Virology NAS of Ukraine 154 Zabolotny str., 03143 Kiev, UKRAINE

Corresponding autor e-mail:[email protected]

Being practical guidance regulating parameters of corrosion protection, recommend

buoying metal underground buildings potential equal -0,85V. This significance is based

on theoretical thermodynamic value computation of electrochemical iron oxidation

reaction potential by oxygen in neutral environment at water presence. However, at

present, it is widely admitted that by basic cause of soil corrosion are the condition

changes of surrounding environment, nascent along of vital functions of sulphate-

reducing bacteria. Herewith oxidation of iron takes place without air oxygen

participation. Consequently, in real exploitation conditions of underground buildings,

the corrosion flow using in quality terminal acceptors of electrons other compounds,

for example - soil anions. On our look, nominally this is cause of low effectively

cathodic protection in cases when in soil surrounding underground buildings

microbiological processes actively flow. By us were taken theoretical computations of

possible values of electro-chemical potential of corrosive reaction with use of diverse

anions in quality of terminal acceptors of electrons. The computations allow us to put

together an original row of biocorrosion activity of soil anions:

Rightness of our computations was confirmed in model laboratory experiments

on study of change corrosion rate in dependence on electron acceptor nature. Analogous

approach was applied for study of dominance chloride ion on activity of soil microbial

induced corrosion. For explanation of got results is offered an original hypothesis

permissive to account for exceptional danger chloride ion for underground metallic

constructions.

Keywords: Biocorrosion, Sulphate-Reducing Bacteria, Soil Anions, Thermodynamic

Computations.

- 20 -

The role of sulphate reducing bacteria in corrosion of potable water mains

Seth A.D., Edyvean R.G.J.

Department of Chemical and Process Engineering, University of Sheffield,

Newcastle Street, Sheffield S1 3JD, UK Corresponding Author e-mail: [email protected]

Growing awareness of the presence of biofilms in potable drinking water systems has

lead to increasing research due to significantly deteriorated water mains resulting from

corrosion in conjunction with bacterial activity. In particular, sulfate-reducing bacteria

(SRB) that are found underneath the main corrosion shell favoring the anaerobic

conditions are noted for their effects in promoting corrosion by reducing sulfate to

sulphide, which may in turn react to give hydrogen sulphide, ferrous sulfides and/or

sulphuric acid in iron pipes.

This study investigates the presence of sulfate reducing bacteria with corrosion

in established cast iron water mains; and in order to understand their origin, coupon

tests using a Robbins Device installed in the water mains, and in laboratory tanks were

carried out. Swabs taken from the interface between the metal pipe surface and the

corrosion product showed a 78% frequency of SRB in the WDS. The Robbins device

holding cast iron coupons gave positive results for sulfate reducing bacteria though

negative for total viable count (TVC), coliforms, faecal coliforms, and non-lactose

fermenting bacteria. Laboratory coupon tests indicated no bacteria present by these

methods, even after 10 months in static anaerobic water. However, it has been shown

that a high proportion of anaerobic biofilm bacteria in WDS are non-culturable by

standard methods. Results from corrosion weight loss tests showed increased weight

loss of the coupons contained in the Robbins Device in comparison to the static

laboratory tanks, this maybe partly due to the effect of shear rates, but also due to

microbiologically induced corrosion (MIC).

Keywords: Sulphate-Reducing Bacteria (SRB), Iron Water Mains, Robbins Device,

- 21 -

Effect of time exposure in THPS biocide efficacy on sessile microorganisms

Eliana Flavia Camporese Servulo, Mariana de Oliveira Souza

Escola de Química, Universidade Federal do Rio de Janeiro, Centro de Tecnologia Bloco E, Ilha do Fundão, 21949-900, Rio de Janeiro, BRAZIL Corresponding authoer e-mail: [email protected]

The efficacy of shock treatment using tetrakis hydroxymethyl phosphonium sulphate

(THPS) commercial biocide was evaluated in a dynamic system (looping) for the

control of biofilms formed on the surface of AISI 1020 carbon steel coupons previously

exposed to seawater for 7 days were treated intermittently with 500 ppm of the biocide.

The treatment consisted of four applications at three different times (1, 2 and 3 hours).

After each contact time, the reservoir content of the looping - water with the biocide -

was substituted with in natura seawater. Control tests were also carried out without

adding the biocide. A decrease in the number of aerobic, anaerobic and sulfate-reducing

(SRB) bacteria and filamentous fungi was observed in the presence of biocide at the

different tested exposure times. Whereas, the efficacy of the biocide was proportional to

the exposure time the aerobic microorganisms are more resistant than the anaerobes. A

considerable reduction of the SRB population was detected soon after the first 1-hour

biocide application, while a pronounced reduction of the other microbial groups

examined was observed only after four consecutive 3-hour applications. The obtained

results confirm the broad spectrum of THPS action, especially with regard to its efficacy

on BRS, which demonstrates the capacity of this biocide of diffusing into the

polysaccharide matrix of the biofilm without losing its antimicriobial properties; thus

guaranteeing control over the microorganisms which are the main cause of

microbiologically influenced corrosion process.

Keywords: Tetrakis Hydroxymethyl Phosphonium Sulphate (THPS), Biocide, Carbon

Steel, Seawater, Sulfate-Reducing (SRB) Bacteria, Filamentous Fungi

- 22 -

Microbial and electrochemical evaluation of carbon steel exposed to pseudomonas and sulfate-reducing bacteria in aerobic and anaerobic

condition

Simone Louise Delarue Cezar Brasil, Mariana Machado Galvão, Rayane Rodrigues Fonseca, Eliana Flavia Camporese Sérvulo

Escola de Química, Universidade Federal do Rio de Janeiro, Centro de Tecnologia Bloco E,

Ilha do Fundão, 21949-900, Rio de Janeiro, BRAZIL Corresponding autor e-mail:[email protected]

The corrosivity of Pseudomonas aeruginosa and sulfate-reducing bacteria (SRB) on

1020 carbon steel coupons, immersed in seawater, was evaluated in a 1L reactor at room

temperature and dynamic conditions.

The initial population of planktonic bacteria inoculated was about 108 cells/mL.

Nutrients were added in order to guarantee cellular feasibility and biofilm formation on

the metal surfaces. The total time of each experiment was 28 days and electrochemical,

physical and microbiological analyses were carried out weekly.

After 7 days of exposure, the colonization of the surfaces was evident in both

conditions. A variation in the behavior of the microbial sessile population was observed

in the presence and the absence of oxygen. In aerobic condition a more pronounced

corrosion process was evidenced; the value of corrosion rate determined was equivalent

to a severe corrosion. These results indicate that the weight loss is more related to the

Pseudomonas activity, although the SRB are known as producers of more corrosive

substances.

Keywords: Pseudomonas aeruginosa, Sulfate-Reducing Bacteria (SRB), Carbon Steel,

Seawater

- 23 -

Evaluation of some factors that can influence the composition of extracellular polymeric substances produced by D. desulfuricans

ATCC 27774 batch cultures

I. Martinsa,c, J. M. F. Nogueirab, J. J. G. Mourac, A. R. Linoa*

a CQB-DQB, Faculdade de Ciências da Universidade de Lisboa b CCMM-DQB, Faculdade de Ciências da Universidade de Lisboa

Campo Grande, 1749-016 Lisboa, PORTUGAL c CQFB-DQ, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa,

2829-516 Caparica, PORTUGAL Presenting author e-mail: [email protected] * Corresponding author. e-mail: [email protected]

Sulfate-reducing bacteria (SRB) are unequivocally related with biocorrosion and

biofouling phenomena in industrial plants. However, the mechanism by which they

influence these processes still remains uncertain. SRB-related corrosion is probably a

conjunction of several phenomena: activity of hydrogenase enzymes, production of

hydrogen sulfide, with the consequent precipitation of metal sulfides, and liberation of

volatile phosphorous compounds. Nevertheless, it seems to be univocally stated the key-

role played by extracellular polymeric substances (EPS) produced by SRB either in the

deterioration of metals and in bioremediation processes due to their ability to selectively

bind metal ions.

EPS from D. desulfuricans ATCC 27774, grown as planktonic and biofilm cells,

at different growth times, were isolated and characterized by several analytical

techniques, i.e. GC-MS, SDS-PAGE, FTIR and ICP, in respect to their chemical

composition: neutral hexoses, protein, uronic acids and metals. Microscopic techniques

(SEM) allowed the visualization of the SRB biofilms formed in the mild steel coupons.

Acknowlegments: This work was supported by 6th FP-NEST 508866 EA-BIOFILMS

and CQB-FCUL.

Keywords: Sulfate-Reducing Bacteria (SRB), Biofouling, Extracellular Polymeric

Substances (EPS), Bioremediation, D. desulfuricans

- 24 -

Effect of quinizarin dye on the aluminium corrosion by Pseudomonas

aeruginosa

P. Norouzia, J. Hamedib, M. R. Ganjali.a, A. Ahmadic

a Department of Chemistry, Tehran University, Tehran, IRAN b Department of Biology, Tehran University, Tehran, IRAN

c Department of Chemistry, Islamic Azad University, Karaj Branch, IRAN Corresponding author e-mail: [email protected]

Formation of a passive oxide film on aluminum enhances its corrosion resistance.

However, this can be damaged by microbiologically species. Pseudomonas aeruginosa

and Cladosporium sp. have been commonly associated with the microbiologically

influenced corrosion (MIC) of aluminum and its alloys. In this study, the effect of an

organic dye (Quinizarin), on resistance of aluminum plates to MIC was studied. An

overnight culture of Pseudomonas aeruginosa in a chemically defined medium was

inoculated in each test tubes containing artificial seawater (ASW) plus various prepared

aluminum plates; a) polished, b) anodized, c) anodized and colored, d) anodized,

colored and sealed. The inoculated test tubes were incubated at 25oC for 30 days. Then,

the Aluminum plates tested were removed and investigated by various methods

consisting: FFT impedance spectroscopy, cyclic voltametry and scanning electron

microscopy (SEM). The results obtained showed that the growth of the P. aeruginosa in

the medium containing Quinizarin dye had not significantly difference with that of the

control medium (ASW). The SEM micrographs and electrochemical impedance

measurements showed that although the amounts of the bacteria on the surface of

colored plate were more than that of on polished plates, however, the corrosion rate in

the later plates was more than colored plates. It seems that Quinizarin dye is useful for

inhibiting of aerobic corrosion of Aluminum materials by P. aeruginosa.

Keywords: Aluminum, Quinizarin, Scanning, Electron Microscopy (SEM),

Pseudomonas aeruginosa

- 25 -

Microbial diversity of marine biofilms from carbon steel in port environment

Pineau Samuel (1), Janvier Monique (2), Cabassut Gaël (1), Dupont Isabelle (1)

(1) Corrodys, 55 rue de Beuzeville, BP9, 50120 Equeudreville, FRANCE

(2) Unité Biodiversité des Bactéries Pathogènes émergentes, Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex 15, FRANCE Corresponding autor e-mail: [email protected]

The degradation of carbon steel structures, especially in the case of Accelerated Low

Water Corrosion (ALWC) in port environment, can be induced by the presence of

microorganisms, or the products of their metabolic activities including enzymes,

exopolymers substances, organic and inorganic acids. Bacteria most frequently studied

in Microbiologically Induced Corrosion (MIC) are mainly the Sulphate-Reducing

Bacteria (SRB). To study bacterial consortia in MIC, a research program is purposed for

in situ experimentation in three French ports. Carbon steel samples have been placed in

several different configuration sites. New molecular technologies have been developed

and used to investigate the composition of microbial communities. However, successful

application of molecular techniques relies on effective recovery of nucleic acids from

environmental samples. The objective of this study is to develop a rapid method to

remove biofilm from marine samples and further a robust, simple and effective total

DNA extraction protocol. We applied the molecular approach of PCR-amplified 16S

rDNA fragments separated by Denaturing Gradient Gel Electrophoresis (DGGE) to

analyze the microbial community. The comparison of DGGE patterns from different

samples gives us information about similarities and differences in their community

structures. This approach can be used to compare bacterial diversity from different

sampling sites. This molecular approach is combined to classic microbiological methods

(cultivable strains).

Keywords: Carbon Steel, Accelerated Low Water Corrosion (ALWC), Sulphate-

Reducing Bacteria (SRB), 16S rDNA, Denaturing Gradient Gel Electrophoresis

(DGGE)

- 26 -

Biofilm formation of pure and mixed culture of Thiobacillus thioparus

M. Protasova, V. Sobko, I. Kozlova

Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine

154, Zabolotny str., 03143, Kyiv, UKRAINE Corresponding author e-mail: [email protected]

Previous studies shown that on the surface of the corroding underground structures

aggressive microbial community had been formed. Its composition was constant and

included besides sulfate-reducing bacteria thionic acidofobic and heterotrophic gram-

negative bacteria required in methionine. In such mixed biofilms, bisides sulphides it

was revealed elemental sulfur that is very aggressive. The purpose of the given work

was to execute the comparative studying of the initial biofilm stages of pure and mixed

culture of T. thioparus .

T. thioparus and its natural sattelite Stenotrofomonas maltophilia have been

isolated from the soil, adjacent to the surface of Kyiv underground tonnel.

As a model surface it was used the sterile glass, which imitates the surface of

well grinded steel. Beijerinck nutrition media was innoculated by pure and mixed

culture of T .thioparus. The prepared glasses were filled by such suspension. The

number of the attached cells was calculated after 5, 15, 30 minutes and 1, 2, 4, 6....168

hours. The number of the attachech cells of S. maltophilia was exceeded the number of

T. thioparus. The formation of biofilm of T. Thioparus mixed culture takes place more

active in comparison with the pure culture.

Keywords: Sulfate-Reducing Bacteria (SRB), T. thioparus, Stenotrofomonas

maltophilia, Biofilm

- 27 -

Production of exopolymer complex by sulfate-reducing bacteria at presence of corrosion inhibitor

Purish L.M., Asaulenko L.G.

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine

154, Zabolotny str., 03143 Kyiv, UKRAINE Corresponding author e-mail: [email protected]

Use of inhibitors is one of the most widespread methods of metal protection from

corrosion. The effective inhibitor of corrosion, induced by sulfate-reducing bacteria is

high-molecular nitrogen-containing PAV cationic of a type. Adsorption on a surface of a

metal, inhibitor forms a protective film. SRB aspire to formation of a biofilm on a

metal, as only in adsorbed condition, in a biofilm, they can take part in a

bioelectrochemical process of corrosion. The question of a correlation and interaction

between a biofilm formed on a surface of metal by SRB and adsorbed by a protective

film inhibitor for today remains not investigated. One of the factors of a biofilm

formation and its attachments to a film surface are extracellular polymeric substances

(EPS). As it was shown earlier, the more agressive strain Desulfovibrio sp. Kiev-10

form on metal a more powerful biofilm than Desulfovibrio desulfuricans Kiev-45.

We investigated the influence inhibitor of corrosion on a bacteria number and

EPS production SRB in a biofilm and in plankton. It was shown, that inhibitor of

corrosion stimulated the EPS production by plankton cells of sulfate-reducing bacteria.

At inhibitor concentration of 0.5 g/l the EPS production by plankton cells of aggressive

strain Desulfovibrio sp. Kiev-10 was increased in 5.8 times and by Desulfovibrio

desulfuricans Kiev-45 was increased in 2.5 times in comparison with the control.

From our point of view the increase of EPS production under the inhibitor

influence displays the protective action of bacteria cells in adverse conditions of

environment. The inhibitor of corrosion blocked the EPS synthesis in a biofilm.

Keywords: Sulfate-Reducing Bacteria (SRB), Extracellular Polymeric Substances

(EPS), Corrosion Inhibitor, Desulfovibrio

- 28 -

Determinant factors for the establishment of cyanobacterial biofilms and crustose lichens on upright structures

Jacob Garty

Department of Plant Sciences, Tel Aviv University

Tel Aviv 69978, ISRAEL Corresponding author e-mail: [email protected]

White mortar is common as a protective layer applied on upright structures in Israel.

Blackish biofilms consisting mainly of epilithic cyanobacteria thrive on these structures.

The greater intensity of colonization is observed on north- and west-facing walls rather

than on south- and east-facing walls depending on the amount of moisture contained by

the substrate. The expansion of these blackish lithobionts is from the upper edge

towards the basic part of structures following the vertical direction of precipitation

pouring down from the roof. In the opposite direction, rain water hits the soil and/or

horizontal lithic surfaces adjoining basic parts of structures, thus producing an upward

extension of humidity. The blackening of upright structures poses an aesthetic problem.

The most effective way to avoid the colonization of structures by lithobiontic

microorganisms is to protect the walls by roof tiles or other protruding elements, thus

keeping the walls as dry as possible. In urban areas, the greyish-brown coverage of

structures is the possible result of the presence of air pollutants deriving from the

combusion of fossil fuel. In air-polluted sites the colonization of structures by

lithobionts occurs on bare surfaces cleaned by downpouring streams of rainwater. The

present paper demonstrates the rate of colonization on upright structures in rural areas

along 20 years and the characteristic pattern produced on walls by anthropogenic soot

and blackish biofilms in urban sites. Factors of relevance to the above mentioned

pattern are, consequently, precipitation and pollution derived soot.

Keywords: Mortar, Cyanobacteria, Lichen, Lithobiont, Biofilm

- 29 -

Wood biodegradation and current problems of preventive protection

José A. Rodríguez-Barreal

Dep. de Silvopascicultura, Escuela Técnica Superior de Ingenieros Industriales Universidad Politécnica de Madrid

Ciudad Universitaria, s/n, E-28040 Madrid, SPAIN Corresponding author e-mail: [email protected]

Continuous use of wood by people, during centuries, let know that this renewable raw

material can be damaged. This damage is due to an abiotic and biotic mix of factors,

which they generate wood biodegradation.

During the last ten years the most important preventive and protection systems

against biodegradation are changing; active matters and formulated elements used in

products are specially affected. This situation involves a problem, in the use of these

products, because it is difficult to know the best way to protect the wood. All these facts

and their short time and medium time effects are explained in this communication.

Keywords: Wood, Biodegradation, Preventive Protection

- 30 -

Biodegradation of asbestos fibres by lichen apomycobionts

Sergio E.Favero-Longo, Mariangela Girlanda, Rosanna Piervittori

Dipartimento di Biologia Vegetale, University of Torino and Centre of Excellence CEBIOVEM,

V.le Mattioli 25, 10125 Torino, ITALY


Although asbestos is currently banned in most western countries due to its carcinogenic

potential, the presence of asbestos fibres in the environment is still an issue. Serpentine

fibres (chrysotile) were the most commercially used asbestos during the last century and

for this reason they are often considered as the main cause of mesothelioma in the

world. Thanks to the secretion of acidic and chelating compounds, lichenized fungi are

well known weathering agents on a wide substrate range and are thus worth of

consideration as potential biodegradators of asbestos. Interactions between chrysotile

and isolated mycobionts of three saxicolous lichen species (Candelariella vitellina,

Xanthoparmelia tinctina, Lecanora rupicola) were reproduced in controlled conditions

and compared to those observed in the field. Wind of apomycobiont hyphae around

chrysotile fibres was observed for all species in vitro. Significant modification of fibre

chemical composition, with selective depletion in Mg content, was found with

apomycobionts of the first two species. Oxalic acid and a secondary metabolite

(pulvinic acid) production are suggested to be involved in the weathering action of X.

tinctina and C. vitellina respectively. Since similar physical and chemical interactions

were observed in field conditions, our findings suggest that isolation and culturing of

lichen apomycobionts can represent a useful tool for bioweathering studies.

Keywords: Asbestos, Chrysotile, Biodegradation, Lichen, Apomycobionts

- 31 -

Biocidal properties of an antimicrobial concrete

Ana M. García1, Víctor M. Rivalta1, M. Isabel Sarró1, Diego A. Moreno1*, Patricia García2, Joaquín Rodríguez2

1 Dep. Ingeniería y Ciencia de los Materiales

Escuela Técnica Superior de Ingenieros Industriales, Universidad Politécnica de Madrid C/José Gutiérrez Abascal, 2. E-28006, Madrid, SPAIN

2 CEMEX España, S.A. C/ Hernández de Tejada, 1. E-28027, Madrid, SPAIN


Concrete is a very usual building material due to its properties. However, it can be

damaged by a lot of physical and chemical agents. Microorganisms or higher biological

organisms growing on its surface can accelerate this process, known in this case as

biodeterioration. Such biodeterioration processes have been documented, especially on

submerged and/or buried structures, like wastewater pipes, pools, reservoirs, hydraulic

stations, etc. Controlling water and nutrients availability for microorganisms is essential

to avoid or, at least, slow down the biodeterioration process. When it is not possible,

biocides can be used directly, in those cases allowed, or as an additive in the material

manufacturing. In this study the biocidal properties of an antimicrobial concrete

manufactured by CEMEX has been evaluated. Agar diffusion tests against Escherichia

coli, Staphylococcus aureus, and the fungus Aspergillus niger, and liquid medium

susceptibility assays against Desulfovibrio desulfuricans were carried out. Control

concrete coupons as well as coupons with the biocide in the form of a fibre and as a

powder and at different concentrations were used. The results show the inhibiting effect

of the antimicrobial concrete, which is higher in the case of the powder form and against

E. coli. S. aureus and A. niger were also affected by the antimicrobial concrete. With

respect to D. desulfuricans, epifluorescence analysis were carried out showing that the

bacterial colonization of the coupons was difficult at the beginning of the study, but in

48 hours and after 21 days the biocide effect of the concrete was depreciable. So the

development of different antimicrobial concretes and the study of their properties

depending on the environment and on the microbial populations inhabiting them is the

special concern to avoid its biodeterioration.

Keywords: Concrete, Biocide, Fungi, Agar Diffusion Test, Epifluorescence

- 32 -

Microscopic fungi damaging inner surfaces of buildings

Kondratyuk Tetiana Oleksiivna1, Zakharchenko Valentyna Oleksiivna2,Nakonechna Lidia Teodorivna2, Artyshkova Larysa Viktorivna2

1 Taras Shevchenko Kyiv National University, Volodymyrsjka str. 64, 01017 Kyiv, UKRAINE

2 D.K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences, 154 Zabolotnogo str., 03143 Kyiv, UKRAINE

Corresponding author e-mail: [email protected], [email protected]

Microscopic fungi of damaged inner surfaces of various buildings (offices, living rooms

and depositories of libraries and museums) caused by exploitation and building

infringements (out of order hydro isolation, infringements of conditioner work and

system of heating, accidents of last and system of provision with water etc.) stimulating

increase of water capacity of wall materials are studied.

Fungi were sampled in places of inner surfaces of walls and ceilings damaged

(swellings; divided into layers or fallen plaster, whitewash, or wall-paper; presence of

spots and thin layer of various size, color and consistence). 95 species of 35 genera of

filamentous fungi were found. Among them representatives of the class Hyphomycetes

have the highest species diversity of fungi, which are essential threat as biodestructors.

The highest indices of frequency were registered for the following species: Acremonium

strictum, Alternaria alternata, Apsergillus niger, A. ustus, A. versicolor, Cladosporium

cladosporioides, Penicillium aurantiogriseum var. viridicatum, Rhizopus oryzae.

Data on Shannon index of species diversity and Simpson index of dominance, as

well as comparison with species diversity of fungi found earlier as damagers of painting

objects of museums of Ukraine are provided. Materials of painting objects are more

available as source for nutrition of fungi. Indices of dominance are much higher on

hardly accessible materials of walls than in case of painting objects. Shannon index of

species diversity found to be lower in fungi of walls.

26 species, namely Aspergillus fumigatus, A. flavus, Geotrichum candidum,

Rhizopus oryzae, Stachybotrys chartarum, form potential threat for human health.

Keywords: Fungi, Building, Painting Objects

- 33 -

Deteriogenic fungi on historic buildings in southern Brazil

Paulo Tadeu Campos Lopes1; Ana Maria Pujol Vieira dos Santos1; Christine Gaylarde2

1Universidade Luterana do Brasil (ULBRA), Canoas, BRAZIL

2 UFRGS, Porto Alegre, BRAZIL Corresponding author e-mail: [email protected]

Presenting author e-mail: [email protected]

Filamentous fungi have long been recognized as the main deteriogens of constructional

materials, growing on the surfaces of buildings and penetrating into the structure.

Although some information is available about fungi in biofilms on buildings in some

Brazilian cities, there is no published literature on fungi present on buildings in the

south of the country, where the climate is more variable. Samples of biofilms were

taken from various buildings of historic importance in Porto Alegre, capital of the

southernmost state in Brazil, and plated on mineral medium containing 5 or 10% NaCl

to select organisms able to grow under dry and nutrient-poor conditions. Fungi were

identified by traditional methods and DNA was extracted from the most common

isolates for amplification with fungal-specific primers. The PCR products were used to

characterize the fungal populations on various historic buildings, using total DNA

extracted from the biofilms, PCR and DGGE. Main genera isolated, on both 5 and 10%

salt, were Botrytis, Ulocladium and Paecilomyces. Species of Aspergillus and

Penicillium were also present, but were considered unimportant aerial contaminants.

Larger numbers of fungi were detected on internal than on external surfaces and the

internal population was less resistant to salt, with higher numbers of colonies growing

on plates with 5% than with 10% NaCl. This situation was reversed for external

surfaces. DGGE was found to be a rapid method for determining the deteriorating

potential of a biofilm, in terms of its fungal composition

Keywords: Filamentous Fungi, Biofilm, Building, Denaturing Gradient Gel

Electrophoresis (DGGE)

- 34 -

The microbial discoloration of oil paintings. A laboratory investigation

T.V. Khijniak1, N.N. Medvedeva-Lyalikova1, V.P. Golikov2, J. Dick3

1 Winogradsky Institute of Microbiology of RAS, 7/2 Prospect 60-letiya Oktyabrya, Moscow 117312, RUSSIA 2 Research Institute for Cultural and Natural Heritage of RAS,

2 Kosmonavtov’s st., Moscow 129366, RUSSIA 3 Delft University of Technology, Rotterdamseweg 137, 2628 AL Delft, THE NETHERLANDS


Yellow natural and artificial inorganic pigments containing chromate-ions have been

used in painting in Europe and Russia since the second part of the 19th century and they

are still being used (Impressionists V. van Gogh, P. Gauguin and Russian avant-garde

artists K. Malevich, V. Kandinskii, M. Chagall). On some historical artworks lead

chromate shows signs of discoloration - a change toward green and brown colors.

However, the exact circumstances under which these discolorations occur are unknown.

Our study demonstrated that lead chromate (crokoite) discoloration occurs under

bacterial influence. Vanadate-reducing, chromate reducing and sulphate-reducing

bacteria influenced on both pure crokoite and prepared painted canvases under

anaerobic conditions during different time. Intensity of darkening process was

depending on bacterial species and time of incubation. Amorphous chromium products

were formed in all cases of bacterial reduction. Furthermore, the experimental evidence

was found that discoloration of lead chromate is accompanied with a diminishing of the

amount of oil (binding agent) in the paint layer. A better knowledge of the degradation

of prepared canvases may help to produce painting substrates less prone to microbial

invasion and to protect existing works of art.

Keywords: Painting, Pigment, Chromate-Ions, Vanadate-Reducing Bacteria, Chromate

Reducing Bacteria, Sulphate-Reducing Bacteria

- 35 -

Calcium oxalate patinas’ formation on laboratory specimens

M. Monte*, L. Bianchini**

* CNR-ICVBC sez. di Roma, via Monte d’Oro n. 28, 00186 Rome, ITALY ** Gabinetto Ricerche Scientifiche, Vatican Museums, Vatican City, 00120 Rome, ITALY


This work provides data on the formation of calcium oxalates, acquired through tests

carried out under controlled conditions. It is well known that up to now, various

hypotheses concerning oxalate origins have been put forward. Such hypotheses have,

however, been formulated deductively, based exclusively on the observation of the

patinas found on the surfaces of monuments and works of art. In our experiments, on

the contrary, calcium oxalates have been produced on stone specimens placed in fungus

cultures.

In three years of work, we have analyzed the activity of about 40 fungus strains

isolated from outdoor stone structures, from plaster on the internal walls of monuments,

and from the air. After a suitable incubation period, the formation of oxalate deposits

was observed on the stone specimens subjected to fungus action from about 20% of the

strains analyzed. Observation and chemical analysis of the neo-formed deposits was

carried out using SEM-EDS, XRD, FT-IR, TGA.

These results confirm the hypothesis of a biological origin of the oxalate patinas

and suggest the mechanisms through which they are formed.

Keywords: Calcium Oxalates, Patinas, Fungus, Stone

- 36 -

Lichen-like colonies of pure Trentepohlia on limestone monuments

Peter Gaylarde1, Gelsa Englert2, Otto Ortega-Morales3, Christine Gaylarde4

1Praca Julio Bozzano, Porto Alegre – RS, 90040-240 BRAZIL

2Dept. Metallurgy, Biocorrosion and Biofilms Lab., UFRGS, Porto Alegre – RS, 90035-190, BRAZIL 3Departamento de Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche,

Av. Agustín Melgar, Col. Lindavista, C.P.24030, Campeche, Camp., MÉXICO 4Dept. Biophysics, Federal University of Rio Grande do Sul,

Av. Bento Goncalves 9500, Porto Alegre – RS, 91500-970, BRAZIL. Corresponding autor e-mail: [email protected]

Trentepohlia has long been recognized as an algal biodeteriogen of buildings and is well

known for its production of pink/brown discoloration on painted and unpainted concrete

structures in the tropics. It is also a common photobiont in lichenised fungi. It is

relatively scarce in biofilms on modern painted surfaces in Brazil and Mexico.

Small samples of limestone and adhesive tape samples were collected from

black, grey and pink areas of historic buildings at the Mayan site of Edzna, Campeche,

Mexico. Samples were observed microscopically (light and scanning electron

microscopy), cultured on solid algal medium and extracted for pigment determination.

Light intensity approximately 2cm above the biofilms was measured and moisture

content of the limestone substratum was determined gravimetrically.

Non-pink areas were colonized predominantly by coccoid and colonial

cyanobacteria, also detected as endoliths. The major biomass on the pink stone was

Trentepohlia. At low power, surface micropits containing crustose-lichen-like black

colonies were seen within these pink areas. These were almost pure cultures of

Trentepohlia, probable species umbrina; no filamentous fungi were detected here. The

black colour was produced by dense, amorphous pigment deposits in the colony centres.

Sampling sites were exposed to high levels of insolation (~ 400 mphotons.m-2.s-1) and

dry conditions (0.7 % moisture content). These data, together with the high carotenoid:

chlorophyll α ratio of Trentepohlia biofilms (3.9:1), suggest that the carotenoids are

important for protection against environmental stress. Trentepohlia caused severe local

erosion (pitting) and, when present as a more uniform biofilm, the well-known pink

surface discoloration.

Keywords: Limestone, Lichen, Cyanobacteria, Trentepohlia, Biofilm

- 37 -

Microbial diversity in archaeology wood

Anne Christine Helms

Conservation Department, The National Museum of Denmark, Brede, DK-2800, Lyngby, DENMARK


Archaeological waterlogged wood does, under certain circumstances, survive for

hundreds and even thousands of years. However, it does eventually degrade and the

reason for this is that certain microorganisms have the ability to survive and thrive

under hostile and mostly anaerobic conditions. Recently we have initiated research

which aims to identify and characterise the microbial diversity in conditions where

wood is known to have survived for two thousand years: the Nydam bog. The diversity

found under these conditions is compared with the diversity of microorganisms found in

storage tanks at the National Museum of Denmark. Characterisation of these

microorganisms has revealed several hundred species, some of which can potentially

degrade cellulose and humus. A summary of these results, together with the

methodology that has been used for the characterisation will be presented.

Keywords: Wood, Cellulose, Humus

- 38 -

Prevention against airborne fungi biodeterioration at the French National Library

Brigitte Leclerc, Caroline Laffont

Bibliothèque Nationale de France- Laboratoire 58 rue de Richelieu 75002 Paris, FRANCE

Corresponding author e-mail caroline.laffont @bnf.fr

In order to avoid a widespread biocontamination and therefore to limit the use of

biocidal treatments which are time and staff consuming and sometimes detrimental for

collections, the French National Library implements a policy of prevention against

biodeterioration of its collections. This on-going project has been carried out for fifteen

years by microbiologists from the French National Library Laboratory trained in preventive

conservation. Relevant monitoring of aerobiology and climatic conditions is

conducted in storage areas because collections are likely to be deteriorated by viable

airborne micro-organisms (mainly fungi) if climatic conditions are suitable to their

development. To evaluate biological risk, micro-organisms collected from air and

surface sampling are enumerated and identified. Since no standards have been defined so

far, thresholds of alert specific to our institution have been determined. When these levels

are reached, specifically adapted actions are undertaken such as reinforced control,

prospecting for causes of contamination, and cleaning procedures.Methods for

monitoring have been improved with the growing understanding of fungi

development conditions and of the most damaging species.

The aim of our presentation would be to introduce within Biodeterioration of

Works of Art and Cultural Heritage topic our latest risk assessment methods in terms of

culture media, sampling, according to libraries specificities. We will also demonstrate the

importance of close collaboration between conservators, climaticians, and scientists for

the success of such a prevention policy.

Keywords: Fungi, Biodeterioration, Library, Conservation

- 39 -

Biodeterioration and archaeology: the role of biological agents in formation processes, sites and post-excavation treatments.

D. Allsopp, P. Tubb

Archaeologists have long recognized that artifacts and sites are subject to changes due

to natural and man-made conditions prior to excavation (formation processes). Artifacts

are also vulnerable to changes in environmental conditions post-excavation. Physical

and chemical agencies causing such changes are already recognized, but biological

factors, especially microbial ones, are not often acknowledged. This paper illustrates a

range of examples of biodeterioration for the interest of archaeologists, and advocates

the recognition of biological agents as part of formation processes (or transforms,) by

introducing the concept of NB (Natural Biological) transforms.

Keywords: Biodeterioration, Excavation, Archaeology

- 40 -

Microalgae and their effects on stone fountains: a comparison between two different environments

Peraza Zurita, Yaiza1; Cultrone, Giuseppe2; Fanés Treviño, Ingrid3, Sánchez

Castillo, Pedro3

1 Dpt. Painting, University of Granada,

Avda. Andalucía s/n 18071 Granada, SPAIN 2 Dpt. Mineralogy and Petrology, University of Granada, Campus de Fuentenueva 18071 Granada, SPAIN

3 Dpt. Botanics, University of Granada,

Campus de Fuentenueva, 18071 Granada, SPAIN Corresponding autor e-mail: [email protected]

Deterioration of outdoor works of art is subject to environmental conditions. When it

comes to stone fountains these conditions are determined by the continuous presence of

freshwater, which offers the perfect ambience for the growth of microalgae.

Microalgae produce several weathering phenomena on the fountain, mainly due

to the formation of biofilms and biogenic carbonate crusts. Mineral crusts, also known

as microstromatolites, provoke physical damages because of their different behaviour

related to temperature and humidity changes. Furthermore they enhance chemical

deterioration of the stone because of their water content. Finally, microalgae’s

physiological processes and development may actively damage the substrate.

In this study we have described the microflora and the related biodeterioration

phenomena within two similar stone fountains placed in different environments. One of

them is located in Granada, Southern Spain, where the climate is continental (dry cold

winters and hot summers); the other is in Garachico, the former capital of the Canary

Island, where the climate is oceanic (template and humid winters, soft fresh summers).

The aim of this work is to make a comparison between the genera identified and their

effects in both locations, in order to establish the influence of climatic conditions in

microalgal biodeterioration processes.

Keywords: Microalgae, Stone, Fountain, Freshwater, Microstromatolites

- 41 -

Methodology approaches to diagnose and monitore the biodeterioration processes in the stone of historical buildings

Julia A. Petushkova*, Julia P. Petushkova*, Yoshikazu Hoshi**

* Physiology of Microorganisms Department, Biology faculty,

M. V. Lomonosov Moscow State University, ul. Lobachevskogo, 2-5. Moscow 119415, RUSSIA ** Department of Chemical Science and Engineering, Ariake National College of Technology,

150 Omuta City, Fukuoka, 836-8585, Fukuoka, JAPAN Corresponding autor e-mail: [email protected]

The device of complex methodology approach to diagnose the stone biodeterioration

was developed for monitoring of historical buildings. The first step of investigations

consisted in employment of non-destructive methods in situ, such as determination of

damaged area, using digital techniques and special software, measurement the

photosynthetic activity of algae biofilms as the mark of intensity of deterioration. The

method of cartography of masonry moisture content was developed with the purpose of

detecting microbial contamination in the deep of stone masonry and optimizating the

sampling of stone material. The subsequent research was connected with

microbiological analysises of samples. Primarily the different samples of limestone,

brick, and mortar were investigated by SEM and then were selective plated for

cultivation of organothrophic, lithotrophic bacteria, fungi and algae. The predominant

species were isolated and identified by determination the partial sequence of 16S rDNA.

In parallel, the chemical composition of samples was examined to study its impact on

the diversity of physiological groups of microorganisms and their quantitative

correlations. The elemental composition of samples was determined using atomic

absorption and non-destructive X-rays fluorescence methods. The stability of

microorganisms, isolated from the stone of architectural monuments, to the influence of

heavy metals was investigated using simulation the growth of algae-bacterial complexes

on limestone.

Keywords: Biodeterioration, Stone, Historical buildings, 16S rDNA, SEM

- 42 -

Microbial Control of Museum Archaeological Textile and Leather Objects in the Course of Conservation and Restoration

Julia P. Petushkova*, Julia A. Petushkova*, Yoshikazu Hoshi**

* Physiology of Microorganisms Department, Biology faculty,

M. V. Lomonosov Moscow State University, ul. Lobachevskogo, 2-5. Moscow 119415, RUSSIA ** Department of Chemical Science and Engineering, Ariake National College of Technology,

150 Omuta City, Fukuoka, 836-8585, Fukuoka, JAPAN Corresponding autor e-mail: [email protected]

UNDER REVISION

- 43 -

Analysis of bacterial populations diversity in air and associated with rock walls in Covalanas and La Haza Paleolithic caves

Víctor M. Rivalta a, M. Isabel Sarró a,b, Ana M. García a, Diego A. Moreno a

a Universidad Politécnica de Madrid, Dep. de Ingeniería y Ciencia de los Materiales,

Escuela Técnica Superior de Ingenieros Industriales. C/ José Gutiérrez Abascal, 2. E-28006 Madrid, SPAIN

b Ministerio de Cultura, Dep. Científico, Instituto del Patrimonio Histórico Español. C/ El Greco, 4. E-28040 Madrid, SPAIN

Corresponding autor e-mail: [email protected]

The studies of bacterial population in caves are scarce, specially in Paleolithic paintings

caves. Karstic caves are special environments characterized by a relative stable

microclimate. However, caves are no uniform environments from a geochemical or

geological point of view and could be influenced by the maximum number of daily

visitors and artificial lighting.

The present report is the first study in two Cantabrian caves (northern of Spain)

with rock art, and located in the same karstic system (Asón Valley). One of them

(Covalanas) with a regulated number of daily visitors and the other (La Haza) closed to

visitors except for researchers.

The aim of this study is to investigate and compare the bacterial diversity in both

caves, studying the species present in microbial populations along one year, to evaluate

the effect of visitors in these populations. In order to do it, dry and humid rock wall

areas and air were sampled in different zones of the caves, with and without visitors.

Results revealed complex bacterial communities in all sampled points, with a larger

number of microorganisms in no visited zones and in humid areas. The biodiversity is

greater in humidity zones than in dry ones. Moreover, a minor biodiversity in La Haza

and in no visitor’s area of Covalanas have been documented. Bacillus species were

dominant in these areas, while in the visiting zones of Covalanas the bacterial

populations were more complex, belonging to Bacillus, Streptomyces, Arthrobacter and

Rhodococcus groups.

Keywords: Caves, Rock Art, Painting,

- 44 -

Isolation and identification of microorganisms associated to the biodeterioro of san felipe castle, cartagena colombia and evaluation of

substances biocidas for their control

Escobar D.M.1; Rodríguez L.C.1; Martínez M.M.2, Gaylarde C.3; Giudice C.A4.; Videla H.A.5

1,2 Facultad de Ciencias-PUJA- Cra 7a # 43-82 Ofi. 111

3 Rio Grande do sul 4 Ingeniero de Materiales-CIDEPINT-La Plata-Arg,

5 Coordinador Internacional Red Temática XV-E-CYTED In order to isolate and identify associated microorganisms in the biodeterioration

process of San Felipe's Castle, located in Cartagena-Colombia and to evaluate

substances biocide for their control; 6 sampling points were determined, this was carried

out the taking of sample for scraping and it was sowed in means for aerobic bacteria,

anaerobic, mushrooms, sulfur-reducing bacteria, nitrifying bacteria, algae and

cyanobacteria. Corynebacterium sp are inside the representative goods of bacteria.,

Bacillus sp., Micrococcus sp., Flavobacterium sp., Azotobacter sp., Xanthobacter sp.,

Criptococcus sp., Pseudonocardia sp., Stomatococcus sp., Planococcus sp.,

Brevibacterium sp., Rothia sp., Rhodotorula sp. and Streptococcus sp.; of mushrooms

Aspergillus sp., Penicillium sp., Phoma sp., Botrytis sp., Tricothecium sp., it would

Alternaria sp., Mucor sp., Trichoderma sp. and Phytium sp.; and of algae and

cianobacteria Gloeocapsa sp., Synechocystis sp., Xenococcus sp., Stanieria sp., and

Chlorella sp. After to isolate and to identify the microorganisms five biocides they were

evaluated: TIMSEN, TEGO 51 *, GLD (glutaraldehyde), CLOROX and

KEMDAZINto concentrations of: 0.5%, 1%, 2%, 3% and 4%. With base in this

evaluation, the biocide TIMSEN Is effective to eliminate all the isolated

microorganisms at one time minimum of 24 hours, without affecting the material of the

Castle.

Keywords: Biodeterioration, San Felipe Castle- Cartagena- Microorganims, Biocides

- 45 -

Biodeterioration in Historical Building in the South of Brazil

Denise de Souza Saad1, Gelsa Edith Englert2, Christine Gaylarde3

1 Department of Structures and Construction Building, Federal University of Santa Maria, Faixa da Camobi, km9 Campus Camobi, BRAZIL

2 Laboratory of Microbial Corrosion Federal University of Rio Grande do Sul, BRAZIL

3 Biophysics Department Federal University of Rio Grande do Sul, BRAZIL

In this work, samples from biodeteriorated building were collected in different cities in

the South of Brazil. They were analyzed by traditional techniques; SEM and Molecular

Biology (PCR) were also used.

The samples were deteriorated by different fungi, such as Aspergillus sp. and

Penicillium sp., the commonest environmental fungi. Cephalosporium sp.and Hypocrea

sp. were also detected among others. The latter one was not common in biodeteriorated

old buildings. SEM analysis demonstrated that fungal contamination in external surface

was very frequent and intense due to tropical climate and very high humidity.

Keywords: Biodeterioration, Building, Fungi, SEM. PCR

- 46 -

Biodeterioration of cinematographic films:

C. Abrusci 1; D.Marquina1; A. Del Amo2; F. Catalina3

1 Departamento de Microbiología III, Facultad de Biología, Universidad Complutense de Madrid,

C/ José Antonio Novais, 2, 28040-Madrid, SPAIN 2 Filmoteca Española, Instituto de la Cinematografía y de las Artes Audiovisuales,

C/Magdalena 10, 28012-Madrid, SPAIN 3 Departamento de Fotoquímica, Instituto de Ciencia y Tecnología de Polímeros, CSIC,

C/Juan de la Cierva 3, 28006-Madrid, SPAIN Corresponding autor e-mail: [email protected]

Cinematographic films are one important part of the historic and cultural Heritage and

these materials require adequate conservation conditions in order to prevent their

deterioration. Bacteria and fungi present in black and white cinematographic film were

isolated and identified from samples collected in Spanish archives of Madrid, Barcelona

and Gran Canaria. Sixteen strains of filamentous fungi and one yeast, Cryptococcus

albidu were isolated; four species of Aspergillus i.e. A.ustus, A.nidulans var.nidulans,

A.versicolor, seven Penicillium chrysogenum strains, as well as, Cladosporium

cladosporioides, Alternaria alternata, Mucor racemosus, Phoma glomerata and

Trichoderma longibrachiatum.

In this work the biodegradation of photographic gelatine (Bloom 225) has been

studied by viscometry in aqueous solution (at 37º, 6.67%w/w) using the fungal strains.

From viscosity data, different variables such us molecular weight and chain scission

were calculated. The viscosimetry of gelatine solutions inoculated with fungi and its

evolution with time can be used as a quantitative method to evaluate their gelatinase

activity and the biodegradation of the material. All the fungal isolates showed capacity

to liquefy gelatine, with the exception of Cryptococcus albidus. Also, the bioassay-

temperature dependence allowed to establish the influence of this important variable in

the biodegradation and subsequently to gain information on the interval of metabolic

efficiency of the fungi.

Keywords: Cinematographic Films, Viscosimetry, Fungi, Bacteria, Yeast

- 47 -

Biodeterioration of limestone by two microbial isolates in the archaeological site of palenque, mexico.

Sylvie le Borgne1, and Carlos Angeles Chavez1, Liz Karen Herrera2,

Héctor A.Videla3

1 Instituto Mexicano del Petróleo, Mexico D.F. MÉXICO 2 Institutode Ciencias de Materiales de Sevilla, Sevilla, ESPAÑA

3 INIFTA- Universidad Tecnológica Nacional, La Plata. ARGENTINA *Corresponding autor e-mail: [email protected]

The archaeological site of Palenque belongs to the Mayan classic and late classic

periods (300-900 AD) and its main constructional material is limestone, the most

common stone in Mayan buildings. Due to the high temperatures and relative humidity

an abundant microflora is growing on the walls of the buildings causing discoloration,

mechanical damage and multiple undesirable effects. This work reports about some of

the organisms isolated and identified from 6 different sites of Palenque by using

traditional microbiological techniques and ESEM observations. A preliminary

assessment of the epilithic macroflora and its possible effects on limestone decay,

complemented with limestone characterization and surface analysis by using SEM and

EDS is summarized in this paper.

Keywords: Limestone, Microflora, Discoloration, ESEM, Epilithic Macroflora

- 48 -

Test of algaecides for the treatment of fountains and sculptures.

Blanca Uceda Reyes, Fernando Bolívar Galiano.

Departamento de Pintura, Facultad de Bellas Artes. Universidad de Granada, Avda. Andalucía s/n. 18071, Granada, SPAIN Corresponding author e-mail: [email protected]

The tests to evaluate the effectiveness of biocides treatments are considered to have a

great importance for the selection of suitable products that will be applied in the

conservation of the cultural heritage. For that reason an experiment has been conducted,

for the study of the colonization of microalgae communities on test stones of diverse

materials (Macael marble, limestone and brick) constituent of fountains, monuments

and sculptures of stony materials to evaluate the effectiveness of different biocide

products (Algochene, Newdes and Biotin-N).

In the laboratory were obtained three different cultures containing the main

groups of microalgae communities (green algae, cyanobacteria and diatoms), from the

samples taken coming from stone works of artistic interest. On a sufficient growth on

the test stones, biocides were applied incorporated in small discs of filter paper , placed

on the test stones and with two different concentrations (5 and 100%), to measure the

resistance of the photosynthetic communities to such, to determine the effectiveness of

the concentrations recommended by the manufacturers.

After the experiment, the algaecidal action of Biotin - N (quaternary ammonium

salts) stands out, as opposed to a small inhibited haul of Algochene (derived from the

pyridine) during the first days, showing a good delayed effectiveness observed 21 days

after its application.

The behavior of algaecides could advise the combination of two products to

prolong their effectiveness and their action over time, but possible combinations would

have to be tested before their application to avoid detrimental reactions.

Keywords: Algaecida, Fountain, Sculpture, Cyanobacteria, Diatom, Conservation

- 49 -

Microorganisms involved in the deterioration of works of art

Calvo, M.A., Shiva, C., Adelantado, C.

Facultat de Veterinària. Microbiologia. Universitat Autónoma de Barcelona 08193 Bellaterra, Barcelona, SPAIN


The diverse and varied elements used by the authors in creating art works, can be in

many occasions, a good substratum for the development of a wide gamma of

microorganisms.

With the aim of proving which are the most common microorganisms in our

environment, with the ability of altering and damaging art works, a systematic study of

several works deposited in Museums of the Mediterranean Area have been carried out.

Samples were obtained using sterile swabs and were inoculated in culture media to

determine the possible presence of bacteria and fungi. Enzymatic capabilities of the

isolated strains were evaluated to show the possible cause-effect relation among the

presence of specific strains and the observed degradations. At the same time, a study of

the sensibility of the isolated microbial strains against several products traditionally

used to control the presence of microorganisms in these substrata has been carried out,

in order to select the least aggressive and most effective product to eliminate and control

the biotic agents that cause the observed alterations.

Among the microbial strains more frequently isolated we may cite: Bacillus

subtilis, Penicillium rugulosum, Alternaria tenuis, Aspergillus niger and Cladosporium

herbarum.

In this study, prevention measures are shown as well as treatment measures in

case they were needed, with the aim of controlling the alterations with biotic origin

demonstrated in the evaluated art work.

Keywords: Biodeterioration, Prevention,

- 50 -

Biodeterioration of historic stained glasses at the Cartuja de Miraflores (Spain)

N. Carmona1, L. Laiz2, J.M. Gonzalez2*, M. García-Heras1, M.A. Villegas1,

C. Saiz-Jimenez2

1 Centro Nacional de Investigaciones Metalúrgicas, CSIC, Avda. Gregorio del Amo, 8. 28040 Madrid, SPAIN.

2 Instituto de Recursos Naturales y Agrobiología, CSIC, Apartado 1052. 41080, Sevilla, SPAIN.

In this paper the colonization by different microorganisms on a representative historic

glass sample set was analysed. The glass samples were obtained from five stained glass

window panels located in the north side of the “Cartuja de Miraflores” Monastery

(Burgos, Spain). Glass samples were characterised using: UV/Vis spectrophotometry,

X-ray fluorescence (XRF), optical microscopy (OM), field emission scanning electron

microscopy (FESEM) and energy dispersive X-ray microanalysis (EDX). Microbial

populations (both bacteria and fungi) were characterized by molecular methods using

PCR-DGGE (PCR and Denaturing Gradient Gel Electrophoresis) complemented with

cloning and sequencing of the PCR amplified products. All of the samples presented

evidences of a strong superficial deterioration. In general, the external sides of the glass

fragments showed a worse state of conservation than the internal ones. The superficial

morphology of the glasses exhibited numerous alterations forming strongly adhered

crusts of variable extension and colour depending on the sample. On the external sides

of the glasses, heterogeneous, dark brown and very interconnected crusts, covering most

of the glass surfaces, appeared together with craters and pits filled with whitish deposits.

The internal only showed small craters and pits and a scarce accumulation of corrosion

deposits. Molecular characterization of the microflora colonizing these glasses showed

the presence of a fungus from the genus Aspergillus and an associated bacterial

community. The resulting data will be useful to design a preservation strategy in the

recovery of this valuable legacy of 15th century A.D. stained glass windows.

Keywords: Glass, UV/Vis Spectrophotometry, X-Ray Fluorescence (XRF), Optical

Microscopy (OM), Field Emission Scanning Electron Microscopy (FESEM), Energy

Dispersive X-Ray Microanalysis (EDX), PCR-DGGE

- 51 -

Biodeterioration on fountains and sculptures in the historical garden of La Concepcion, Málaga

Ana Corral, Fernando Bolívar-Galiano

Departamento de Pintura, Facultad de Bellas Artes. Universidad de Granada,

Avda. Andalucía s/n. 18071, Granada, SPAIN Corresponding author e-mail: [email protected]

UNDER REVISION

- 52 -

Identification by PCR of pigment-producers Actinomycetales isolated from mural painting of Basilica de Nuestra Señora de los

Desamparados

G. Cuesta, R. M. Montes, E. Hernández

Dpto. de Biotecnología-Microbiología. Universidad Politécnica de Valencia Camino de Vera s/n. 46022, Valencia, SPAIN


Actinomycete grows on low nutrients culture medium and in a wide rank of

environmental conditions. They are able to degrade compounds like cellulose, lignin

and chitin. Filamentous actinomycetes contribute to the mechanic destruction of murals

painting as a result of the growth inside the paint. Several strains are able to dissolve

calcium carbonate and produce soluble pigments. These pigments have been identified

like a stain over some monuments. In this work we have isolated and identified by PCR

pigment-producers actinomycetes from mural painting.

Material from the frescoes were diluted and plated onto Modified Czapeck

supplemented with cycloheximide. Actinomycete strains were subcultured on the ISP

medium to study its macroscopic morphology, mycelium colour and spores and

production of pigments. For 16S DNA amplification primers 243F-A3R targeted to

Actinomycetales and Sm6F-Sm5R targeted to Streptomyces were used. Each

amplification program was initiated by denaturation for 5 min at 95º C and finished with

an extension for 10 min at 72º C. Cycling parameters for Streptomyces were 94º C for 1

min, 65º C for 45 s and 72º C for 1 min and for Actinomycetales were 94º C for 45 s, 68º

C for 2 min and 72º C for 2 min. As a positive control Streptomyces coelicolor was

used.

Four Actinomycetales strains were isolated from two samples of mural painting.

With primers 243F-A3R a 1.25 Kb product was obtained in all isolated strains and with

primers Sm6F-Sm5R a 600 pb fragment was amplified in three of these four strains.

Two of these Streptomyces strains were pigment-producers when growing in plaster

plates.

Keywords: Actinomycete, Pigment, Mural Painting, PCR, 16S DNA

- 53 -

Microorganisms involved in the deterioration of documentary archives in the Mediterranean area

Adelantado, C., Shiva, C., Calvo, M.A.

Facultat de Veterinària. Microbiologia. Universitat Autónoma de Barcelona

08193 Bellaterra, Barcelona, SPAIN Corresponding author e-mail: [email protected]

The cities of the Mediterranean Area generally have varied Documentary Archives

which represent the history of their identity as well as the warranty of their historical

memory and material for research in these fields. This Patrimony is exposed to biotic

and abiotic processes that determine a danger for its maintenance and conservation as

well as for the health for everyone who enters into contact with the contaminated

material. With the aim of showing the main biotic agents able to produce problems in

this environment and to establish control systems, in the last few years, environmental

studies have been carried out, as well as for documents deposited in several Archives of

the Mediterranean Area.

The methodology for sample collection divides into two parts: by sterile swabs

and document pieces (when possible to obtain them), and environmental

microorganisms collected by gravimetric and volumetric methods. The culture media

used were: Triptone Soya Agar, to determine bacteria, and Sabouraud Glucose Agar

with addition of 30 ppm of tetracycline clorhydrate to show the presence of fungi.

With the results obtained we can emphasize the presence in the studied Archives

of: Bacillus subtilis, Micrococcus luteus, Chaetomium globosum, Penicillium

rugulosum, Aspergillus niger, Aspergillus fumigatus, Cladosporium herbarum,

Alternaria tenuis and Saccharomyces cerevisiae. In the study, the possible implication

of the isolated microorganisms in the degradation of the analysed substrata and in the

health of everyone who comes into contact with them is taken into account.

Keywords: Documentary Archives, Bacteria, Fungi

- 54 -

Two cases of biodeterioration of cultural heritage in argentina

S. G. Gómez de Saravia 1,2 P. S. Guiamet 1,3 Gabriela Küppers4

1 Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, UNLP-CONICET, C.C. 16, Suc.4, (1900), La Plata., ARGENTINA

2CICBA 3CONICET

4Cátedra Zoología Invertebrados I, Facultad de Ciencias Naturales y Museo, Paseo del Bosque s/n, La Plata CONICET.


Biodeterioration of the cultural heritage is the physical or chemical damage caused by

biological agents on objects, monuments or buildings belonging to the cultural heritage.

A comparative study of biodeterioration on two sites of the Argentina cultural

heritage was made in this paper, comparing the biodeterioration of buildings located in

i) the Cathedral of La Plata, Buenos Aires, and ii) the Ruins of San Ignacio, Misiones.

Samples were taken aseptically from the surfaces of the buildings and a non-

destructive technique of sampling was employed. Different isolation methods and

biochemical assays complemented with microscopic observation were used to identify

the microbial contaminants. On the Ruins of San Ignacio copious dark greenish and

black biofilms were observed according to the high relative humidity and elevate

temperature of these areas. Cyanobacteria, algae, lichens and protozoan were identified.

In the area of the Cathedral of La Plata, the climate is moderate, but due to the

proximity of an oil distillery and petrochemical plants, its atmosphere is heavily

polluted. Aerobic heterotrophic mesophilic, acidophilic and sulphate reducing bacteria

were also found in several samples and taxonomically classified. A synergistic

relationship between biological and abiotic factors on deterioration of the two studied

sites was observed.

Keywords: Biodeterioration, Building, Biofilm, Sulphate Reducing Bacteria,

Cyanobacteria, Algae, Lichen, Protozoa

- 55 -

Biodeterioration of selected cultural monuments in the Republic of Macedonia

1Mitko Karadelev, 2Ilze Irbe, 2Bruno Andersons

1Institute of Biology, Faculty of Natural Sciences Gazi Baba bb, P.O.Box 162

Skopje, 1000 REPUBLIC OF MACEDONIA 2Latvian State Institute of Wood Chemistry, 27 Dzerbenes str., Riga LV 1006, LATVIA

Corresponding e-mail: [email protected]

The investigations of biological damages caused by wood decay fungi and insects of

cultural monuments in the Republic of Macedonia were carried out. Monasteries were

inspected in the areas of Skopje (Sv. Andrea, Sv. Pantelejmon), Prespa (Sv. Petka, Sv.

Bogorodica, Sv. Nikola and Sv. Dimitrij) Ohrid (Sv. Naum), Demir Hisar (Sv. Jovan),

Debar (Sv. Jovan Bigorski), Strumica (Sv. Bogorodica), Stip (Sv. Nikola), Zletovo (Sv.

Gavril Lesnovski) etc. Most of the monasteries are under state protection. Our attention

was paid mainly on wooden items inside the monasteries (iconostases, icons, chairs,

stairs, doors, window frames, roof constructions), as well as on the frescoes on walls.

The wooden items mainly were attacked by Anobiidae wood boring insects, and in one

case there was Cerambycidae attack (Sv. Petka). Mostly there were old damages, but in

some cases (Sv. Nikola in the area of Stip and Sv. Petka) we found fresh damages. Some

wooden details like outer doors, stairs and roof constructions were attacked by

lignicolous fungi such as: Gloeocystidiellum porosum, Hyphoderma praetermissum,

Hyphodontia aspera, Hyphodontia crustosa, and Athelia decipiens. All these species are

white rot formers and belong to the family Corticiaceae from the order Aphyllophorales.

In some cases on the wall frescoes mould attacks were registered, mainly Cladosporium

sp., Aspergillus sp., and Fusarium sp. The fungal and insects damages to the inspected

monasteries and churches are dangerous both for construction materials and wooden

items inside monasteries as well as to the wall frescoes. Therefore, in the nearest future

it is necessary to initiate measures for cultural heritage protection.

Keywords: Wood, Fungi, Insect, Wall Frescoes

- 56 -

Polyene macrolide antibiotics as fungal damage control agents

I.L.Kuzikova, V.I.Sukharevich

Scientific Research Centre of Ecological Safety, Russian Academy of Sciences, Korpusnaja 18, Saint-Petersburg 197110, RUSSIA Corresponding author e-mail: [email protected]

A great demand for efficient environmentally clean fungicides is met by continuous

active search for and creation of new fungicidal agents. An important performance

criterion for fungicides is their ability, when in sub-biocidal concentrations, to affect

synthesis of biodamage-inflicting metabolites by fungi. Such metabolites include

enzymes, organic acids, and pigments. Pigments not only represent a biodamage factor

but, like cellular polysaccharides, make fungi more resistant to fungicides.

This study showed that efficient natural agents for protecting industrial materials

against fungal damage agents can be found in polyene antifungal antibiotics heptaenes,

tetraenes. By contrast to numerous well-known chemical fungicides increasing

biosynthesis of pigments by a factor of 1.3-2.5, polyene antifungal antibiotics inhibit

synthesis of pigments by the fungi biomass by a factor of 1.4-2.2; they reduce the

activity of the complex of amilolythic enzymes and do not affect acid formation and

activity of proteolythic enzymes. When these antibiotics are combined with EDTA,

which inhibits synthesis of polysaccharides and pigments by fungi, the sensitivity of

fungi to the antibiotics is enhanced. In this case, the fungicidal concentration of the

antibiotics is reduced by a factor of 2.3-10, depending on the culture and antibiotic

properties. Antibiotic Nystatin, alone and in combination with EDTA, was tested in

protecting cellulose-containing materials against fungal damage. Upon treatment with

the fungicides studied, these materials remained fungi-resistant for a long time.

The above-mentioned properties of macrolide polyene antibiotics make

promising the search for and study of this group of antibiotics with the aim to develop

efficient ecologically safe biodamage-protecting agents thereof.

Keywords: Fungicide, Biodamage-Inflicting Metabolites, Fungi, Antibiotic

- 57 -

Biodeterioration of the walls of a Roman tomb by Streptomyces sp.: Eight years follow-up of microbial colonization.

L. Laiz, J.M. Gonzalez, V. Jurado, M.C. Portillo and C. Saiz-Jimenez*

Instituto deRecursos Naturales y Agrobiologia,

Apartado 1052, 41080 Sevilla, SPAIN Corresponding autor e-mail: [email protected]

The Roman Necropolis located in Carmona (Sevilla, Spain) has been dated from the 1st

and 2nd centuries A.D. A long-term study on the ability on a Streptomyces sp. to colonize

the walls was carried out along eight years. The increase of the area occupied by the

colonies was easily measured due to their violaceous colour. A preliminary assessment

of the culturable microbial communities provided with the isolation of several bacterial

groups dominated by the Class Actinobacteria, mainly by species of the genus

Streptomyces. Among the isolates, a strain, c48, producing violaceous-pigmented

colonies was selected by its similarity to the coloration observed on the tombs and its

high abundance among the cultured microorganisms. Coloration produced by the

growth of this strain and the observed on biodeteriorated areas were highly similar.

Following a polyphasic taxonomical approach, considering both phenotypic and

genotypic characteristics, this strain was classified as a member of the genus

Streptomyces, strain c48.

Keywords: Streptomyces sp, Actinobacteria, Tomb, Biodeterioration

- 58 -

Microbiological colonisation of paper and parchment

Astrid Michaelsen 1*, Flavia Pinzari 2, Katrin Ripka 1, Werner Lubitz 1, Guadalupe Pinar 1

1 Department of Medical/Pharmaceutical Chemistry, University of Vienna, AG Prof. Lubitz, UZAII, Geozentrum, Zi. 2B522, Althanstr. 14, 1090 Vienna, AUSTRIA

2 Istituto Centrale per la Patologia del Libro, Laboratorio di Biologia, Via Milano 76, I-00184 Rome, ITALY


Besides environmental reasons, microorganisms and especially fungi are responsible for

the biodeterioration of cultural objects of art and writs. Archives and libraries from all

over the world suffer from those microorganisms, but with the hither to used traditional

culture-dependent methods, only a small amount of effectively colonising organisms is

detected. Restoration and maintenance of written Cultural Heritage is therefore

problematic due to the incomplete knowledge of deterioration agents.

In this project, for the first time culture-independent molecular methods are used

to identify fungal communities on paper and parchment from different time periods and

quality. As already applied for environmental fungal communities, nucleic-acid-based

strategies targeting rRNA-encoding regions are selected for essays studying the

community structure of fungi on biodeteriorated paper. We focus on the ITS region,

which is nested in the rDNA repeat, due to the high variation between taxonomically

distinct fungal species and even within the species. We here report on the optimisation

of protocols, as for DNA extraction from paper materials and PCR conditions, for

fingerprinting techniques as DGGE (Denaturing Gradient Gel Electrophoresis) analysis.

Different methods are tested on pure fungal cultures for their efficiency in amplification

of either ITS1 or ITS2 region, comparing different fungi specific primer sets. An

amplification of bacterial or herbal DNA is to be excluded. Furthermore, the conditions

for DGGE analysis like gradient, voltage and running time are established to fit for the

accuracy of discrimination of fungi in complex communities and to constitute a marker

for further investigation of historical paper.

Keywords: Written Cultural Heritage, Fungal Communities, ITS Region, DGGE

(Denaturing Gradient Gel Electrophoresis)

- 59 -

Control of biodeterioration process in documents of colombian general archive

Jenny Mateus1, Diana Peña1, Gigiola Peña1, Angela Rojas1, Jeimy Rojas1, Sonia Zambrano1, Maria Mercedes Martinez1*, Claudia Florez2, Mario Santander2

1Grupo de Biotecnología Ambiental e Industrial. Laboratorio Microbiología Ambiental, Lab. 125,

Pontificia Universidad Javeriana, Carrera 7ª # 43-82, Bogotá, COLOMBIA 2Archivo General de la Nación. Laboratorio Biología y Química. Carrera 6ª # 6-91, Bogotá, COLOMBIA


Starting from the supports paper (manual, bond and newspaper), photography with

gelatin and silver emulsion and planes with support in textile fiber of Colombia’s

General Archive there were carried out different isolation tests and identification of

fungi determining the stains with highest appearance frequencies and the effect of the

disinfectant Tego51®, Timsen® to 4000 ppm (1.6 g of n-alquil dimetil bencil), Ethanol

to 70% and Newger® to 0.7% (N-Duopropenida) it has more than enough affected

paper and their form of application nebulization and aspersion. 231 photographs with

gelatin emulsion of the bottom of the Ministry of Works Publish (MOP) that presented

biodeterioration indicators, were evaluated and 48 fungal stains were isolated and

identificated, being Penicillum sp and Cladosporium sp the stains with highest

appearance frequencies and highest levels of aggression to the applied supporting

substances Rhizopus sp, Cladosporium sp, Aspergillus sp 1 and Fusarium sp with

biodeterioration effect in 25 days. Penicillum sp, Cladosporium sp, Rhizopus sp and

Aspergillus sp 1 were selected to evaluate three disinfectants at three different

concentration levels and at four forms of application (aspersion, immersion, punctual

and direct application) and determine the fungicidal effect and their action on the

photographic support. Immersion in the disinfectants used was the best application

method since doesn't affect the physical characteristics of the image. Finally 66 planes

with support in textile fiber were also evaluated and 37 fungal stains were identified

being Penicillum sp and Cladosporium sp the stains with highest appearance

frequencies which also demostrated high aggressive in the support together with

Bipolaris sp.

Keywords: Biodeterioration, Photographs, Filamentous Fungi, Paper, Planes

- 60 -

Definition of a bioreceptivity index for granitic rock

Prieto B.; Silva B.; Aira N.; Alvarez L.

Dpto. Edafología y Química agrícola. Fac. Farmacia. Universidad de Santiago de Compostela. SPAIN Corresponding autor e-mail: [email protected]

Even though the concept of bioreceptivity (susceptibility of a material to be colonised)

and its usefulness have been defined some years ago, up to now no attempts have been

made in defining a bioreceptivity index.

In previous works we defined an equation wich allow to calculate in a rapid and

easy way the potential bioreceptivity of granite. This ecuation express bioreceptivity as

µg chlorophyll a/cm2. This way of expressing bioreceptivity is useful but it is not easy

to understand to people working in conservation or restauration since they don’t know

the meaning of an amount of chlorophyll in the change of the appearance of the rock.

This problem is easy to sort out if a bioreceptivity index which related amount of

chlorophyll with change in appearance is defined. After carrying out some laboratory

experiment consisting of 1) inoculating block of different varieties of granite with serial

dilutions of a mixed culture of three cyanobacteria so that each block has a different

(and known) amount of chlorophyll, 2) characterising the change in appearance

undergone by each block as change in colour, and 3) giving an understandable name to

that change after asking to a significant number of people for the term they would use to

express that change, we were able to defined a bioreceptivity index.

Keywords: Bioreceptivity, Granite, Chlorophyll A, Cyanobacteria

- 61 -

Lichenological study on brick wall: the mineral deposits, the orientation and the humidity importance

Rafael Portero de la Torre, Fernando Bolívar-Galiano, Manuel Casares-Porcel, Giuseppe Cultrone

Departamento de Pintura, Universidad de Granada Avenida de Andalucía s\n, 18.071 Granada. SPAIN


In this paper it has been studied the lichen´s comunities that colonize the facade of the

“Colegio Máximo” of Granada (Spain), a building of the XIX century. The lichen

colonization appears three of the four facades of the building, in the upper part of the

skirting board, where there is a series of horizontals and verticals planes in on

adornment mode. There had been two parts in this paper. In the first place the inventory

realizations of the existing species and in second place the identification of the

sustrate´s mineralogy.

For the identification of lichen species, thirty three samples had been taken

previously from eleven different points of the facades (inventory 1-11) and six different

planes of the skirting board upper part (zone A-F). Each sample has been classified by

these parameters. Seventeen species of lichens had been identified, all of them

calciolaus, and the Caloplaca and Verrucaria genus are the ones that predominate.

The brick sustrate´s mineralogy has been study from the X-ray difracction. The

CaCO3 only appears limitedly and comes from the secondary genesis by the dust and

soil deposit.

As this, the lichen’s colonization came motivated by these sediments of dust and

soils that enrichment the CaCO3 sustrate. From this come, that only exists colonization

in the skirting board upper part, in which there are several horizontal planes. Capillarity

humidity also influence because of its proximity to the ground.

Keywords: Lichen, Brick, Building, Calciolaus, X-Ray Diffraction

- 62 -

Studies of the efficiency of the biocide “new des” incorporated in artificial stone composed of ground natural stone and a commercial

acrylic resin concerning the growth of algae

Jana Petermann1, Fernando Bolívar2, Javier Mas3, Blanca Uceda2, Rafael Portero2

1 Fachhochschule für Technik und Gestaltung Mannheim

2 Dept. Pintura. Fac. Bellas Artes. Univ. Granada, Avda. Andalucía s/n 18071, Granada, SPAIN

3 Dept. Conservació i Restauració. Fac. Bellas Artes. Univ. Politécnica Valencia. Corresponding author e-mail: [email protected]

The efficiency of the biocide New Des at concentrations of 2.5% and 5% incorporated

into artificial stone consisting of a natural stone and an acrylic resin was tested

regarding the growth of algae. The mineral part was mixed with the resin in 3 different

proportions (1:1, 1.5:1 and 2:1). The used natural stones were Crema Bateig or Tosca de

Rocafort which play an important role in the patrimony of Valencia. The stone was

ground using different sieve sizes of 0.42mm and 0.80mm. The samples were inoculated

in the laboratory with 3 different algae communities which were grown in a camera

consisting mainly of the most important types of algae (diatoms, cyanobacteria, green

algae). The algae growth was monitored and examines after 1, 4 and 7 weeks and was

judged by giving indexes for the amount of growth on the surface area of the samples.

Further the present species on the samples were examined using a microscope. It results

that a stone composition containing the natural stone either Tosca de Rocafort or Crema

Bateig ground with the small sieve (0.42mm) and incorporated biocide is less affected

by algae growth. The majority of the leading compositions contain the fine ground

particles. Less importance was observed regarding the mixing proportion. Regarding the

biocide New Des it results that in general samples with incorporated biocides seem to

be less affected by algae growth. Incorporated biocide with a higher concentration 5%

showed slightly more effectiveness than the lower concentration 2.5% although the

difference is not very large. It seems that the stone composition and its surface play an

important roll as well as the addition of a biocide.

Keywords: Biocide, Stone, Cyanobacteria, Diatom, Green Algae

- 63 -

Analysis of biodeterioration in pictorial works of art using electronic microscopy (FESEM)

Poyatos-Jiménez, F 1, Romero-Noguera, J1, Martín-Sánchez, I2,

Fernández Vivas, MA2, Bolívar-Galiano, F1

1 Dept. Painting. Fac. Fine Arts. University of Granada,Avd. Andalucía s/n, 18071, Granada, SPAIN 2 Dept. Microbiology. Fac. Sciences. University of Granada,

Avd. Severo Ochoa s/n, 18071, Granada, SPAIN Corresponding author e-mail: [email protected]

During the early beginnings of what would later become the Granada Museum of Fine

Arts, the pieces that compose the collection suffered through various and often poor

storage situations, changes of location and transfers, etc. In reference to the origin of the

works, we are dealing with a series of five linen canvases belonging to a larger group of

sixty-seven works found in old deposits, where they were stored after the confiscations

of 1835-37. To determine if the causes of the deterioration are of biological origin and

to identify what type of bioagent is acting we must make use of diverse microscopic,

physiological and biochemical techniques. For a correct identification it may be

necessary to grow anexic cultures enriched with specific additions that will enable the

favourable development of certain species. Once the samples have been introduced, the

cultures are maintained in growing units with controlled temperature, humidity and

light. The microorganisms identified were fungi (Alternaria solani, Aspergillus níger,

Rizopus, Cladosporium, Acremonium and Mucor cf) and bacteria (Bacillus,

Micrococcus, Staphylococcus, Nocardia and Corinebacterium). The existing nutritive

substances, the air temperature, humidity and the capacity of certain microorganisms to

produce substances that prevent or impede the development of others are the decisive

factors in the nature of the microflora and in the invasions that a picture then suffers.

Techniques of microscopic observation, notably studies using FESEM (field emission

scanning electronic microscope), are particularly useful in demonstrating the action of

these microorganisms, reflecting the changes suffered by the materials, such as the

disappearance of the adhesives in the preparatory coats on the canvas or the physical

effects caused on the surface of the works.

Keywords: Canvases, Picture, Field Emission Scanning Electronic Microscope

- 64 -

Effective biocide to prevent microbiological contamination during peg impregnation of wet archaeological iron-wood artefacts

M.S. Rakotonirainy1, K. Tran2, J.B. Memet3, C. Héraud1 and L. Caillat2

1. Centre de Recherche sur la Conservation des Documents Graphiques, CNRS-MCC-MNHN,

36 rue Geoffroy Saint-Hilaire, 75005 Paris, FRANCE 2 ARC-Nucléart Laboratory, 17 rue des martyrs, 38 054 Grenoble, FRANCE 3 ARC’Antique Laboratory; 26 rue de la Haute Forêt, 44 300 Nantes, FRANCE


A new conservation process was developed for archaeological iron-wood artefacts.

Metal part protection was achieved by adding a corrosion inhibitor (Hostacor IT®) in

the polyethylene glycol (PEG) soaking solutions. Nevertheless, a significant

microbiological growth was observed in the baths and around the objects. From the

different soaking solutions, 25 bacterial strains and 12 strains of filamentous fungi were

isolated. Adding compatible biocide has been absolutely essential to allow the PEG and

Hostacor to be effective. Among biocides tested, Dowicide A® that contains sodium o-

phenylphenate was the most efficient. As shown by electrochemical measurements

(corrosion potential and intensity and polarization resistance), this biocide does not

destabilize the formation of protective film created by Hostacor IT®. There is no

competition of adsorption between Hostacor and Dowicide. In the presence of

Dowicide, Hostacor IT acts more quickly and in a significant way.

At the concentration of 3000 ppm, Dowicide is effective to protect the aqueous

solutions of PEG 400 + Hostacor IT® from the development of the selected

microorganisms. Adding the biocide at the beginning of the object impregnation was the

most effective way to prevent bacterial and fungal growth. Tests in real conditions

showed that after two months of impregnation, solutions reminded clear and no biofilm

was observed around artefacts.

Keywords: Biocide, Iron-Wood Artefacts, Corrosion Inhibitor, Corrosion Potential,

Polarization Resistance

- 65 -

Study of fungal biodeterioration of terpenic varnishes

J. Romero1, M.A. Fernández2, F.C. Bolívar1, I. Martín2

1 Facultad de Bellas Artes. Dpto Pintura. Universidad de Granada.

Avda. Andalucía s/n, 18071, Granada, SPAIN 2 Facultad de Ciencias. Dpto Microbiología. Universidad de Granada.

Avda. Fuentenueva, 18071, Granada, SPAIN Corresponding autor e-mail: [email protected]

Natural terpenic varnishes have been widely used in art works, such as paintings and

sculptures, and it confer important properties from the aesthetic and preservation point

of view. Many of this material are biodeteriorable, and process of degradation causes

very important changes in its mechanic, optical and chemist properties, affecting deeply

the enjoyment and visual appearance of art works.

This article attempts to illustrate the development of fungal colonization of

almaciga, sandaraca and colofonia varnishes, widely used in traditional art practice, and

the chemical changes brought about by the fungal growth.

Keywords: Terpenic Varnishes, Almaciga, Sandaraca, Colofonia, Fungi

- 66 -

Halobacillus sp. nov. isolated from medieval wall paintings and building materials: a taxonomic characterization

Katrin Ripka1, Astrid Michaelsen1, Ewald Denner2, Werner Lubitz1,

Guadalupe Pinar1

1 Department of Medical/Pharmaceutical Chemistry, University of Vienna UZAII, Geozentrum, Zi 2B522, Althanstr.14, 1090 Vienna, AUSTRIA

2 Institute of Bacteriology, Mycology and Hygiene, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Wien, AUSTRIA


Colonization of objects of art by microorganisms depends on the environmental

conditions like moisture, temperature and light, as well as the chemical nature of the

substratum. In monuments salt efflorescences are common due to changing in physical

parameters. They build a niche for extremely salt tolerant and moderate halophilic

microorganism, which are related with biodeterioration in Cultural Heritage.

Several moderately halophilic gram-positive, spore-forming bacteria have been

isolated by conventional enrichment cultures from damaged medieval wall paintings

and building materials located in the Catherine chapel of castle of Herberstein (Styria,

Austria). DNA-DNA reassociation experiments identified the isolates as a population of

hitherto unknown new Halobacillus species. Members of the genus Halobacillus could

anew be isolated from samples of another medieval building the chapel of St.Virgil

(Vienna, Austria) which gives an evidence for frequency of this genus in cultural assets.

In this study we report the characterization and classification of the new isolated

strains by a polyphasic taxonomic study. In addition, a comparison with the established

Halobacillus species is performed. These studies comprehend standard biochemical,

morphological and physiological methods as well as molecular analysis including 16S

rDNA sequencing, Enterobacterial Repetitive Intergenic Consensus - PCR (ERIC-PCR),

Random Amplified Polymorphic DNA - PCR (RAPD-PCR) and Internal Transcribed

Spacer-Homoduplex-Heteroduplex Polymorphism (ITS-HHP).

Keywords: Wall Painting, Halobacillus sp, 16S rDNA, ERIC-PCR, RAPD-PCR, ITS-

HHP

- 67 -

Romanesque Church of La Vera Cruz (Segovia). A diagnosis of its biodeterioration aimed at future conservation measures

Ascaso, C.1, Galvan, V. 2, García del Cura, M.A. 3, Cámara, B. 1, De los Ríos, A. 1

1 Centro de Ciencias Medioambientales (CSIC), C/ Serrano 115 bis, Madrid 28006, SPAIN

2 Universidad SEK, Segovia, SPAIN 3 Instituto de Geología Económica, Laboratorio de Petrología Aplicada, Unidad Asociada CSIC-UA,

Universidad de Alicante, A.P. 99, Alicante 03080, SPAIN Corresponding author e-mail: [email protected]

The Romanesque church of La Vera Cruz is presently under investigation as part of the

integrated project “Recovery and Revaluing of the Architectural Heritage of Medieval

and Modern Epochs of the Province of Segovia”. Built at the start of the XII Century,

La Vera Cruz lies on the outskirts of Segovia, in the Arab quarters of San Marcos. In

1919, it was declared a National Monument and Item of Cultural Interest. Samples of

the church stone were processed for scanning electron microscopy with backscattered

electron imaging (hereafter SEM-BSE). This technique allows observation of areas

several squared centimetres large and has facilitated the cytological study of epi- and

endolithic microorganisms. Using low temperature scanning electron microscopy, we

can identify sites bearing liquid water and hydration-dehydration processes associated

with polysaccharides in microbial biofilms, both playing a major role in the

bioalteration suffered by the stone. In addition, molecular biology techniques have been

used to identify the species of fungi present. A petrographic evaluation has identified the

rock as a sandy dolostone with medium to finely crystalline euhedral and subheudral

dolomite and abundant intercrystal porosity. Through SEM-BSE, it has been possible to

establish the significant contribution of lichen mycobionts to the substrate alteration

process. These mycobionts are accompanied by free-living fungi, which have a greater

capacity to penetrate the stone.

Acknowledgements: This study is a contribution to Projects JCL SEK 504 and

BOS2003-02418.

Keywords: Stone, Scanning Electron Microscopy (SEM), Fungi, Lichen

- 68 -

Inactivation of fungi from deteriorated paper materials by radiation in the Tropics

Manuela da Silva1,*, Moraes, A.M.L.2, Nishikawa, M.M.1, Gatti, M.J.2, Vallim, M.3,

Brandão, L.3, Nóbrega, A.1

1Department of Microbiology, INCQS

2Department of Mycology, IOC, Fundação Oswaldo Cruz, Av. Brasil, 4365 CEP: 21040-900, Manguinhos 3Department of Radioprotection/IEN/UFRJ, Cidade Universitária - Ilha do Fundão - Rio de Janeiro,

Brazil *Corresponding author e-mail: [email protected]

Libraries and archives sometimes have suitable conditions for fungi growth, which can

bring risks to human health by mould contamination of books and documents, besides

causing the decay of these publications that might be ancient and rare ones. The

elimination of moulds from deteriorated books and archive documents can be efficiently

achieved by gamma-ray radiation with no critical damages to the paper material neither

to the print itself. Therefore, experiments were conducted to find the lowest radiation

dose needed to completely inactivate fungi. Fungi, previously isolated from naturally

contaminated books from a Brazilian public library, Cladosporium sp. and Aspergillus

versicolor, were plated in malt agar media and treated in a Co60 irradiation unit with

doses ranging from 14 to 30 kGy. The minimum dose needed to kill these fungi was 16

kGy. Thus, this dose was applied directly to a severely attacked book and deteriorated

old documents. Pieces and particles of damaged paper from these materials were

incubated in culture media before and after the treatment. Several fungi were isolated

and identified before the treatment, such as representatives of the genera Acremonium,

Aspergillus, Cladosporium, Penicillium and Trichosporum. After the treatment no living

fungi were detected from the irradiated material. These results are very promising and

demonstrate the effectiveness of gamma-ray radiation for the recovery of severely

damaged books and old documents, leading to the preservation of our cultural heritage

and the prevention of diseases caused by moulds in libraries and archives.

Keywords: Paper, Moulds, Cladosporium sp., Aspergillus versicolor, Acremonium,

Penicillium, Trichosporum, Gamma-Ray Radiation

- 69 -

Biological contribution to the formation of dark patinas on granitic surfaces

Silva B.; Prieto B.; Aira N.

Dpto. Edafología y Química agrícola, Fac. Farmacia, Universidad de Santiago de Compostela, SPAIN


The formation of dark patinas on rocky buildings has been mainly related to the

deposition of gases and particles and to sulphation mechanisms particularly in the case

of calcareous rocks. However, in the case of granitic monuments located in Galicia

(North West of Spain), this origin is unlikely since granite is a very poor calcium rock

and the atmospheric pollution in this area is negligible. In these cases a biological origin

seems to be most probable.

Up to now only few attempts in characterizing dark patinas on granitic surfaces

have been made, all of them on monuments. In this work we analyse dark patinas from

monuments located in a scarce polluted town and from outcrops located in a non-

polluted area, in order to compare the importance of the biological factor in the

formation of dark patinas since biological growth is stimulated in a non-polluted area.

Samples were analysed from a petrographical, chemical, mineralogical and

biological points of view. Comparison of the patinas with the underlying rock allowed

us to define the composition of each patina, to differentiate between components

coming from the rock and coming from the atmosphere and to prove the importance of

the biological contribution to the formation of most of the analysed patinas.

Keywords: Patinas, Granitic Surface

- 70 -

Characterizing waterlogged burial environments for in situ

preservation of organic archaeological remains.

I. Douterelo Soler a*, R. Goulder b, M. Lilliec

a Department of Geography, University of Hull, Hull, HU6 7RX, UK. b Department of Biological Sciences, University of Hull, Hull, HU6 7RX, UK.

c Department of Geography, University of Hull, Hull, HU6 7RX, UK. *Corresponding author e-mail: [email protected].

The ongoing discovery of archaeological sites in wetlands is associated with a lack of

funds for excavation and preservation. This situation has led to the consideration of

preservation in situ as the preferred option for dealing with the majority of waterlogged

archaeological remains in England. To gain a better understanding of the burial

environment, we have studied changes in the environmental variables in field soils; such

as pH, temperature, redox conditions, moisture and organic content, along with

variation in water-table. In addition, quantitative and qualitative changes in the

composition of the soil microbial communities were monitored down the soil profile.

Soil cores were taken from two wetlands, Hatfield Moors and Sutton Common,

the latter being an important Iron Age enclosure. Cores were taken every two months

over 16 months. Each core was subsampled at 5 depths (10, 30, 50, 70 and 100 cm) to

allow examination of the vertical distribution of the variables being studied. Microbial

abundance and activity were monitored by; total counts of bacteria, extracellular-

enzyme activities, and 14 C-leucine assimilation. The physiological profile of the

microbial community was analysed using BIOLOG Ecomicroplates.

The composition of bacterial communities was also studied using molecular-

genomic techniques. Total DNA was extracted from the soil and then a fragment of 16S

rDNA was amplified by PCR, using primers that are specific to bacteria. The bacterial

communities were then characterized by a “genetic fingerprint” technique; denaturing

gradient gel electrophoresis (DGGE) which allow separation of the dominant bacterial

taxa.

Keywords: Wetlands, Burial Environment, Soil Microbial Communities, 16S Rdna,

Denaturing Gradient Gel Electrophoresis (DGGE)

- 71 -

Study of biodeterioration of paper in a 16th century book

Mariasanta Montanari*, Flavia Pinzari, Paola Valenti

Istituto Centrale per la Patologia del Libro - Dipartimento per la Ricerca, L’innovazione e L’organizzazione - Ministero per i Beni e le Attività Culturali, Via Milano, 76, 00184 Rome, ITALY


A copy of “Le Stanze del Bandello”, printed at the end of the 16th century in Italy and

conserved in the Biblioteca Baidense in Milan (Italy) was sent to the Istituto Centrale

per la Patologia del Libro in Rome for restoration of damaged parts. Before any

restorative operation, books, like other artistic objects, usually undergo to an evaluative

study of the damages from the chemical and biological point of view. In this paper, the

study of microbiological lesions found on the pages of “Le Stanze del Bandello” is

described. The book appeared affected by peculiar symptoms of biodeterioration. Most

of the pages close to the cover of the book showed a great fragility and a felted

consistency. Pinkish to purple spots were distributed trough pages with a repetitive

scheme, suggesting that the cause of those marks acted crossing the pages.

One of the main problems in biological diagnostics of cultural heritage is the

non-invasive sampling. The study of artistic objects should be done possibly without

modifying the objects themselves, especially if these are of small dimension. The aim of

this report is the description of procedures needed in a conservative approach to

restoration of valuable books and papery objects of art. Techniques utilised to evaluate

and describe the damages and the organisms responsible for biodeterioration are

reported, and discussed. Different sampling techniques and instruments were utilised,

like swabs and adhesive tape. Classic culturing methods have been coupled with the

direct microscopic observation of sampled material. Moreover, a variable pressure SEM

instrument was used together with electronic dispersion spectroscopy (EDS) for both

the chemical characterisation of inorganic constituents and the description of

biodeteriorated paper surface, without the necessity of sample preparation through

metallization.

Keywords: Book, Spots, Non-Invasive Sampling, SEM, Electronic Dispersion

Spectroscopy (EDS)

- 72 -

Initial attachment of bacteria to tipical medical device surfaces. Goodness of model proposals

Amparo M. Gallardo-Moreno1, M.L. González-Martín1, R. Calzado Montero1,

C. Pérez-Giraldo2, J.M. Bruque1

1Department of Physics

2Department of Microbiology University of Extremadura, Av. Elvas s/n 06071 Badajoz, SPAIN


In medicine, some opportunistic microorganisms have the ability of adhere to implanted

abiotic surfaces and develop a biofilm. In this sense, one of the most important is the

slime-producer Staphylococcus epidermidis. The eradication of biofilms with

antimicrobial agents is an inefficacy therapy and this yields to severe health problems.

Consequently, the actuation in the first stage of the attachment is the best way of

procedure. The initial adhesion of microorganisms to inert biomedical surfaces is

mediated by physico-chemical forces. The most used models that describe this

interaction usually calculate the interaction free energy at the closest approach between

the surfaces from the values of the surface tension and zeta potential of both interacting

surfaces. In some cases this analysis has provided misleading conclusions when

comparing with experiences in vitro. In this work we compare the initial adhesion rates

to glass and silicone of two strains of Staphylococcus epidermidis with the interaction

free energies provided by the DLVO and XDLVO theories as a function of the

interaction distance. The initial adhesion rate values and interaction free energies

indicate different approach distances for glass and silicone.

Keywords: Slime, Staphylococcus epidermidis, Antimicrobial Agents, Biomedical

Surfaces

- 73 -

Some improvements in the static adhesion methods to study the first stage in the formation of biofilms

M. Luisa González-Martín1, R. Calzado Montero1, A. M. Gallardo-Moreno1,

M.G. Donoso1, C. Pérez-Giraldo2 J.M. Bruque1

1Department of Physics,

2Department of Microbiology, University of Extremadura, Av. Elvas s/n 06071 Badajoz , SPAIN


Static adhesion-based methods are rapid and useful techniques for studying the in vitro

tendency of some microorganisms to approach and adhere to different surfaces. In

general the samples are brought into contact with the microbial suspension and after a

certain period of time the microbial cells are count. In the case of transparent materials

an optical microscope is enough for determining the exact number of adhered cells but

in the case of opaque materials the fluorescence technique or the sonication of the

sample to proceed with micro-dilutions are necessary. The fluorescence method requires

the staining of the cells and this can cause the detachment of some of the adhered cells.

The countless by serial dilutions does not only provide the number of the adhered cells

to the surface of the sample of interest but also to the edges and to the bottom part. This

work proposes some modifications in the adhesion protocols and in the way of counting

to improve the mentioned methods.

Keywords: Static adhesion method, Biofilm, Adhesion

- 74 -

Cytotoxicity assays with metal ions. Results obtained using different methodologies.

1 Miguel Reigosa, 23 Mónica Fernández Lorenzo de Mele

1 Instituto Multidisciplinario de Biologia Celular (IMBICE)

2 Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA),

C.C. 16, Sucursal 4, 1900 La Plata, ARGENTINA 3 Facultad de Ingeniería, Universidad Nacional de La Plata, ARGENTINA


The release of ions from metallic biomaterial deserves investigation due to its medical

and dental applications, among others. Particularly, copper ions release is worthy of

studying since copper is one of the main components of different dental alloys and of

the intra-uterine devices used for contraception. In this field, different experimental

methodologies were reported for cytotoxicity assays and the results seem to be

contradictory. The aim of this report is to compare the results obtained using

osteoblastic cell lines (UMR106) exposed to copper ions in the presence (MeCuP) and

in the absence (MeCuA) of the metal sample. In the case of MeCuP the cells were

cultured with a copper disk inside the well. The variation of the number of death cells

with the radial distance was analyzed at different exposure times. In the case of MeCuA

extracts with copper ions obtained subsequent to the immersion of the metal disks in a

sterile culture medium for different periods were added to the cell cultures. For MeCuP

the initial copper ions concentration (cCu) that was measured corresponded to the

average concentration value, because the ion concentration diminishes from the origin

of delivering (metal) to the wall of the well. A high number of dead cells were found

even at cCu bellow the threshold of toxicity previously reported. On the contrary, a

homogenous distribution of dead cells was found in the case of MeCuA. The results

show the importance of considering the concentration gradients and exposure periods in

the analysis of cytotoxicity assays.

Keywords: Metallic Biomaterial, Copper, Cytotoxicity Assays, Osteoblastic Cell Lines

- 75 -

Biodegradation of oil hydrocarbons by composting. Efficacy of biopiles inoculation with biosurfactant plus activated sludge

C Calvo1, Fernández Jurado MA1, Toledo FL1, Martínez Toledo V1,

García Fandiño C2, González-López1

1 Grupo de Microbiología Ambiental, Instituto del Agua Universidad de Granada. SPAIN

2 Repsol YPF. Puertollano. SPAIN Corresponding author e-mail:[email protected]

Composting of contaminated soil in biopiles is an ex situ technology, where organic

matter is added to contaminated soil as bulking agent. Many organic contaminants such

petroleum and polyaromatic hydrocarbons have successfully been bioremediated and

degraded in biopiles. Therefore, Composting technologies are able to enhance the

general microbial activity and to stimulate biodegradation amount. In this study, we

have investigated the effects of the addition of biosurfactant and activated sludge on

biopiles for composting of soil contaminated with oil hydrocarbons. Biosurfactant AD2

applied in our studies was an exopolymer synthesized by a gram negative bacterium

isolated in our laboratory from wastewater samples obtained from the waste water

treatment plant (WWTP) of a Repsol YPF Oil refinery in Puertollano (Spain). Activated

sludge was also obtained from the same WWTP. Composting was performed in four

biopiles (1 m height / 1.25 m3 total volume) built with 245 Kg of soil, 13.05 Kg of

hydrocarbons, 300 Kg of straw and activated sludge and/or biosurfactant. Composting

process was checked by measuring: Temperatures, pH, COT, Total nitrogen, DQO,

heterotrophic microorganisms and petroleum hydrocarbon. Hydrocarbon biodegradation

was high enhanced in pile C, with activated sludge and biosurfactant compared to piles

A, B and D. Our results shown a high up efficiency of petroleum hydrocarbon

biodegradation in composting inoculated with a specific activated sludge and surfactant

biopolymer, suggesting that application of these substances could be of importance on

oil hydrocarbons bioremediation technologies.

Keywords: Biopile, Petroleum, Polyaromatic Hydrocarbons, Composting Technologies,

Biosurfactant

- 76 -

Effects of Oil Sludge and Fertilizer Application on the Growth and Performance of Amaranthus cruentus (L) AND Celosia argentaa (L)

B. A. Senjobia , A. A. Akinpelua , G. E. Akinbola b, S. A. Ayanlaja, A. O. Ogunkunle b

a Department of Soil Science and Farm Mechanisation, Olabisi Onabanjo University, Ago-Iwoye, Ogun State

b Department of Agronomy, University of Ibadan, Ibadan.

The effect of oil sludge on the performance of Amaranthus cruentus (L) and Celosia

argentaa (L) were evaluated using four levels of pollution viz: 0%; 0.25%, 0.5% and

0.75% of the weight of the soil and three levels of N.P.K. fertilizer (20:10:10) viz: low

level (0.005kg), normal level (0.01kg) and excess level (0.015kg). These treatments

were imposed after the soil analysis was carried out on the site. The results ontained

show that pollution has a significant effect (p<0.05) on the performance of Amaranthus

cruentus (L) and Celosia argentaa (L), despite the different levels of fertilizer

application with second level of pollution (0.25%) being the critical point. Thus,

pollution tends to retard the germination rates as well as the growth of the crops

drastically beyond the critical level with Celosia argentaa showing more resistance to

pollution than Amaranthus cruentus.

Increasing the fertilizer application did not remove the injurious effect of

pollution on crops performance. Therefore, proper usage of the land for the purpose it is

best suited for is recommended as a means of preventing soil pollution and enhanced

optimal soil production.

Keywords: Oil Pollution, Stunted Growth, Food Insecurity, Soil Degradation.

- 77 -

Differential abilities of bacteria species to degrade refined petroleum products

S A Ayanlaja

Olabisi Onabanjo University PMB 2002 Ago-Iwoye, NIGERIA


UNDER REVISION

- 78 -

Degradation of naphthalene by thermophilic bacteria via a novel pathway

G. Giedraitytė, A. Bubinas, L. Kalėdienė

Vilnius university, Faculty of Natural Sciences,

M. K. Čiurlionio 21/27, LT-03001 Vilnius, LITHUANIA Corresponding author e-mail: [email protected]

Pathways by which mesophilic bacteria degrade aromatic hydrocarbons under aerobic

conditions as well as enzymes involved in the metabolism of these hydrocarbons are

well studied. On the other hand, little is known about the growth of thermophilic micro-

organisms on aromatic hydrocarbons, such as benzoic acid, cresols and phenols. These

micro-organisms contain enzymes that function at elevated temperatures. Such enzymes

are of great interest for industrial applications. The only enzyme described in regard to

aromatic hydrocarbon metabolism of thermophilic bacteria is catechol 2,3-dioxygenase

of thermophilic, phenol-degrading Bacillus thermoleovorans. Recently it has been

established that species utilizes naphthalene and that its degradation pathways differ

from those characteristic of mesophiles. But, as far as we know, there is no information

about the enzymes participating in naphthalene degradation by thermophilic bacteria.

Determination of enzyme activities in cell-free extracts revealed that last aromatic ring

fission enzyme in naphthalene biotransformation by thermophilic isolate was inducible

protocatechuate 3,4-dioxygenase. Intermediates such as o-phthalate and protocatechuate

detected in culture supernatant confirmed that the metabolism of naphthalene by

thermophilic isolate can proceed through protocatechuate via ortho-cleavage pathway

and, thus, differs from the pathways known for mesophilic bacteria.

Keywords: Mesophilic Bacteria, Thermophilic Bacteria, Aromatic Hydrocarbons,

Naphthalene

- 79 -

Microorganisms for oil decontamination in the north

Khabibullina F.M., Shubakov A.A., Archegova I.B., Ibatullina I.Z.

Institute of Biology, Komi Science Center UrD RAS Institute of Physiology, Komi Science Center UrD RAS Soil science faculty of Lomonosov Moscow University

Environmental contamination by oil and petroleum products is a world-scale

environmental problem. Different techniques are developed for decontamination of oil-

contaminated habitats, including microbiological methods based on using enzymes.

The present work aims at finding and isolating active strains destroying oil

products, which can serve a base for rearing microbial associations perspective for

usage in environmental decontamination biotechnologies. We isolated microorganisms

from oil-contaminated soils and oil residues by the method of accumulative cultures.

These microorganisms are able to use oil as an exclusive source of carbon and energy.

For selection of active strains destroying oil products, we checked 56 strains isolated

from accumulated cultures, and 10 strains of yeast cultures from the collection of Soil

Biology Chair, Soil Studies department, Moscow State University in the liquid mineral

rearing medium with admixture of oil. The conducted experiments allowed extracting

19 strains actively using petroleum products during growth process. These strains

registered under index NK (oil-oxidizing culture) and corresponding numbers enriched

the collection of oil-oxidizing microorganism cultures of the Institute of Biology. They

were identified till generic or specific taxon: NK-101, NK-102, NK-103 are

representatives of genus Trichoderma, Bacterium is represented by NK-15, NK-41, NK-

105; NK-16 corresponds to Rhodococcus erythropolis; NK-106 is a Rhodotorula; NK-

104, NK-202 are species of Mucor; NK-203 and NK-204 belong to Fusarium

lateritium, NK-205, NK-206 to Gliocladium deliquescens, NK-301 and NK-302 to

Candida guillermondii, NK-303, NK-304 to Pichia guillermondii and NK-305 to

Yarrowia lipolytica. On the base of above-mentioned microbial strains we created

various associations which efficiency is tested in the laboratory.

The work was conducted within the framework of project №2216 of International

Scientific-Technical Center.

Keywords: Oil-Contaminated Soils, Oil-Oxidizing Culture, Accumulative Cultures

- 80 -

Degradation of oil hydrocarbons by plant-growth promoting rhizobacterium of the Azospirillum genus

Anna Muratova, Anastasia Bondarenkova, Sergey Golubev, Olga Turkovskaya

Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of

Sciences. prosp. Entuziastov 13, Saratov 410049, RUSSIA Corresponding author e-mail: [email protected]

Plant-growth-promoting rhizobacteria (PGPR) are a group of soil microorganisms that

is actively studied in order to assess their potential application in phytoremediation of

contaminated soil. Associative rhizobacteria of the Azospirillum genus were the object

of present research, aimed at revealing their degradative potential towards oil

hydrocarbons and at studying the effect of oil pollution on plant-growth-promoting

activity through production of the phytohormone indole-3-acetic acid (IAA).

From the Collection of Nitrogen-Fixing Bacteria of the Institute of Biochemistry

and Physiology of Plants and Microorganisms of the Russian Academy of Sciences, A.

brasilense strain SR80 was chosen as an active degrader of crude oil at concentrations

of 0.1-20 g/L in malate-containing medium. During the biodegradation, almost all oil

fractions were reduced. In the A. brasilense strain SR80 was capable of degrading

saturated hydrocarbons but without catalyzing the aromatic ring cleavage. It was shown

that the strain had a plasmid which was similar in weight to the well known OCT-

plasmid, encoding microbial degradation of alkanes. In addition, A. brasilense strain

SR80 was capable of producing the phytohormone IAA at a concentration of 0.024

mg/ml, which was not affected by the oil concentration in the medium. Analysis of the

plant-growth-promoting activity of strain SR80 showed that the bacterial treatment of 2-

day-old wheat seedlings had a positive effect on the development of the plant-root

system in the oil-contaminated environment.

Thus, this is the first time that the degradative activity of rhizobacteria of the

Azospirillum genus towards oil hydrocarbons has been found and characterized. The

results obtained allow us to propose a promissing application of this strain as an

effecting ninoculant for phytoremediation of hydrocarbon-contaminated soil.

Keywords: Rhizobacteria, Phytoremediation, Azospirillum, Oil Hydrocarbons,

Phytohormone

- 81 -

Remediation of the Former Military Airport – TRIANGLE Zatec

Jan Vanek1, Jan Sixta2, Ivo Hlasensky1

1DEKONTA, a.s., Prague Office, Volutova 2523, 158 00 Prague 5, CZECH REPUBLIC

2 Regional Authority of the Usti Region, Velka hradebni 48, 400 02 Usti nad Labem, CZECH REPUBLIC Corresponding author e-mail: [email protected]

The paper refers to remediation of the former military airport premises, which has in

total the area of 363 hectares. At present the premises has been remedied and

reconstructed into a strategic industry zone called Triangle.

The military airport was introduced into operation before the World War Two.

During operation petroleum hydrocarbon substances were stored and used on its

premises, which caused massive pollution. Oil products contaminated approx. 120,000

m3 of soil above groundwater level (unsaturated zone) and approx. 100,000 m3 of soil

and underground water in zone of groundwater level and below it (saturated zone).

Systematic remediation of the premises started in Aug 2003. The objective of

remedial work is to remove contamination and to prevent further contamination

spreading via underground water flow. Remedial activities started by additional

remedial investigation (Aug to Sep 2003), sequentially was followed by ex-situ

remediation of unsaturated zone (Sep 2003 to Jun 2004). During the ex-situ phase

118,382 m3 of soil contaminated above the target limit were excavated, decontaminated

and backfilled. In total 411.4 tons of petroleum substances were removed. Furthermore

64,612 m3 of inert materials with contaminant content below the limit were excavated.

Presently the in-situ remediation of saturated zone is carried out together with

protective remedial pumping of underground water. This part of remediation work

should be carried out till the end of year 2008; post-remedial monitoring is planned till

2013.

The presented remediation of the Zatec military airport is one of the largest

remedial actions in the Central and Eastern Europe.

Keywords: Petroleum Hydrocarbon Substances, Groundwater, Soil, Remediation

- 82 -

Frequency of appearance of bacterias associated to a spill of light hydrocarbons in a “morichal” ecosystem in monagas state. Venezuela

Bastardo, H *; Bermúdez, D*.; Betancourt, L**; Ramírez, E.**, Ramos, L.*

*Universidad National Experimental de Guayana

**Complejo Muscar, PDVSA Corresponding autor e-mail: [email protected]

Taking into account the capacity of certain type of bacterias to produce tensoactive

compounds in presence of hydrocarbons in soils and water bodies, in the present work

we look for a place that was impacted with petroleum with a variable residence time

that allowed to establish a relationship among the presence of diverse functional groups

of bacteria in time and age of the crude oil spilled in soil and water object of the present

research. This investigation was carried out in the Complex “Muscar” of “Petroleos de

Venezuela S.A.” (PDVSA) located in the city of Punta de Mata, Monagas State, with the

purpose of establishing a relationship between the presence of different bacteria

communities and the impregnation of petroleum in water and soils that conform the

“Morichal” ecosystem. Water samples were taken in different surrounding places as

well as soils impacted by light oil spills, which has been happening in the study place.

We took eight samples at different places where oil spills haven been occurring.

After the bacterial isolation there were carried out a biochemical characterization. There

was performing a correlation between the analyzed samples of water and soils with the

purpose of establishing the relationship among the different bacteria communities

present in the “morichal”, as well as their activity to degrade of crude oil and their

capacity to produce surfactants. A significant group of functional groups was obtained,

which will allow us through statistical rehearsals to know its appearance frequency in

the different studied places and its relationship with the presence of oil, which was

determined by means of a S.A.R.A. analysis in the oil extracted from the water and

soils.

The obtained results point out that a narrow relationship exists among the

functional groups present in the soils as well as those coming from waters near to the

places with more oil spill activity in the space and time.

Keywords: Tensoactive Compounds, Oil Spills, Crude Oil, Surfactants

- 83 -

Water sanitation of an impacted “morichal” by an oil spill provenient from a tank of the “muscar” gas complex of “PDVSA”, Punta de Mata,

Monagas state, Venezuela.

Bastardo, H *; Bermúdez, D*.; Betancourt, L**; Ramírez, E.**, Chacare,J.C..*

Universidad National Experimental de Guayana **Complejo Muscar, PDVSA


During the gas gathering in the MUSCAR Gas Complex of “Petroleos de Venezuela

S.A. (PDVSA)” the fluids stored into a tank are in some occasions spilled in view of the

fact that the contention capacity of the tank is overcome. A particular spill of these

liquids, including oil, took place last year and affected an area of “morichales” adjacent

to the Gas Complex. In Venezuela, the term “Morichal” is used to denominate a

community in which the mature individuals of the arboreal palm Mauritia flexuosa,

conforms the most conspicuous element. The “morichal” polluted with petroleum was

treated in situ by sprinkling a total of 2,600 L with a weekly frequency and during two

months, using a derivative biosurfactant of the butyric acid. Two samplings were made,

one in May 2004, before beginning the sanitation (bio-treatment) in the affected sectors,

and other in July 2004, after having culminated the bio-treatment. The values of the

different parameters measured in situ after the application of the bio-surfactant such as:

Temperature, Dissolved Oxygen, pH and Conductivity, were very similar to the

obtained before its application, with the exception of the Conductivity. The results

indicate that the emulsifying capacity of the surfactant was very effective, since it

allowed the production of the emulsion, facilitating the extraction of the oil from the

“morichal” waters. The Oils and Fats content diminished between 50 and 95% after the

bio-treatment. The presence of functional groups of heterotrophic bacteria varied

between sampling what allows directly pointing out that many of these functional

groups were related with the presence of oil in the water. With regard to the fecal

contamination, the relationships FC/FS found in the present investigation (1.4 and 1.1)

indicate that the contamination comes from mixed sources (human and not human).

Keywords: Biosurfactant, Bio-Treatment, Heterotrophic Bacteria, Water Sanitation, Oil

Spill

- 84 -

Screening and selection of active psychotrophic oil degrading microorganisms

Filonov A.E.1,2, Pyrchenkova I.A.1,2, Puntus I.F.1, Gapharov A.B.1, Boronin A.M.1,2

1Institute of Biochemistry and Physiology of Microorganisms RAS, 142290 Prospekt Nauki 5, Pushchino, Moscow Region, RUSSIA

2Pushchino State University, 142290 Prospekt Nauki 3, Pushchino, Moscow Region, RUSSIA


The aim of this research was screening of microorganisms – oil degraders isolated from

different regions of Russia, and selection of active psychrotrophic oil-degrading

microorganisms. Screening of 96 microorganisms – oil degraders and 65

microorganisms –degraders of polycyclic aromatic hydrocarbons in according with their

ability to grow on diesel fuel and oil at 24ºС and 4-6ºС was carried out. The most active

degrader strains were: Rhodococcus erythropolis Sh5, Pseudomonas sp. 142 NF

(pNF142), Pseudomonas putida Sh1(4), Pseudomonas putida KT2442 (pNF:: TnMod-

OTc), Pseudomonas sp. 384 NF, Pseudomonas aureofaciens 1393 (pNF:: TnMod-OTc),

S22, Microbacterium liquefaciens Ash10(2), Pseudomonas sp. 382 NF, non-identified

X5, S26, X25, S25, S67, S41. For the strains selected the range of utilized substrates

was determined, antibiotic resistance, temperature range, halotolerance and ability to

synthesize biosurfactants. Genomic fingerprints of 11 oil degraders were obtained, using

rep-PCR and ARDRA that could provide monitoring of these strains in complex

microbial populations. Study of oil degrading level by the most active strains in liquid

mineral medium showed that individual strains could utilize from 15 to 25% of oil for 7

days. When cultivating association consisting of 6 stains, oil degradation level reached

46%.

This research was supported by the ISTC grant 2366, Russian Federal Scientific and

Technical Program (state contract 43.073.1.1.2502), RFBR grants 03-04-49145 and 04-

04-57807-AF2004.

Keywords: Oil Degraders, Psychrotrophic Microorganism, Polycyclic Aromatic

Hydrocarbons, Halotolerance, Biosurfactants

- 85 -

Quantification of petroleum biodegradation events offshore Brazil

Christian Hallmann1*, Luiz Trindade2, Lorenz Schwark1

1 Geological Institute, University of Cologne,

Zuelpicher Strasse 49a, 50674 Cologne, GERMANY 2 Petrobras/Cenpes, Av. 1, Quadra 7, Ilha do Fundão, Rio de Janeiro, BRAZIL

* Corresponding author. E-mail: [email protected]

Subsurface biological petroleum degradation is a major hassle to the petroleum industry

by lowering the value of oils and raising production costs. In particular, the asphaltene

content, viscosity and acidity of oils increase while saturated hydrocarbons are

preferentially degraded. As exploration currently protrudes into deep-water settings, the

risk of drilling degraded hydrocarbon accumulations increases since low sediment-water

interface temperatures allow degrading biota to be active at increasing depths compared

to continental settings. Establishing techniques to predict the extent of biodegradation

before or during early drilling phases is therefore of vital importance for efficient future

exploration.

Three sandstone cores from an oil column in a Mesozoic basin offshore Brazil

were sequentially extracted by SFTE to obtain residual oil fractions that correspond to

discrete pore-filling oil pulses. This allowed the reconstruction of a detailed petroleum

charging history. The extent of oil biodegradation was determined by the ratio of

trisnorhopanes versus regular hopanes, and by comparison of tricyclic/pentacyclic

triterpenoids versus the C29/C30αβ hopane-ratio. Furthermore, the susceptibility of

nitrogen-containing carbazole compounds to biodegradation was observed.

Results reveal two discrete oil charges that can be discriminated by their degree

of biodegradation. The latter charge (60% of total volume) is stronger biodegraded, and

mixed with the early charge (40% of total volume), which is only mildly degraded. We

can conclude that the study area either experienced two discrete petroleum degradation

events, or that biological degradation is taking place to different extents at separate oil-

to-water contacts (OWC) in stacked reservoirs.

Keywords: Biodegradation, Petroleum, Trisnorhopanes, Hopanes, Triterpenoids

- 86 -

Monitoring of the Evolving Microbial Diversity during the Remediation of Mineral Oil Contaminated Soil

Mphekgo P. Mailaa*, P Randimaa, K Drønenc, Thomas E Cloeteb

a Council for Scientific and Industrial Research, P O Box 395 Pretoria, 0001 SOUTH AFRICA b Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, SOUTH AFRICA

c Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, NORWAY *Corresponding author e-mail: [email protected]

The aim of the experiment is to test the hypothesis that microbial diversity in

bioaugmented and non-bioaugmented soil treatment (artificially contaminated with

mineral oil) changes with the dissipation of hydrocarbons. The evolving microbial

diversity during bioremediation of a mineral oil contaminated soil will be monitored

using culture based and non-culture based techniques. The removal efficiency of

mineral oil in the bioaugmented and non-bioaugmented soil treatment will be compared.

In addition, the changes in microbial diversity during the removal of oil will be

evaluated using community level physiological profiles and PCR-DGGE. Both

biostimulation and bioaugmentation approaches have been tested for their efficiency in

removing hydrocarbons from the soil. However, there is very little information about

the evolving microbial diversity during the removal of petroleum products from the soil.

The information about the evolving microbial diversity during bioremediation of

organic polluted soil environments is very important as it can help in understanding the

unravelling subsequent limiting factors during bioremediation of fuel contaminants.

This study aims to understand the changes in microbial diversity during bioremediation

of oil contaminated soil environments.

Keywords: Bioaugmented Soil Treatment, Hydrocarbons, Mineral Oil,

Bioaugmentation, PCR-DGGE

- 87 -

Biodegradation of hydrocarbon by fungi and bacteria isolated from two different polluted soils

Mancera López, M.E1; Rodríguez Casasola, M.T1; Esparza García, F1; E. Ríos

Leal1, Chávez Gómez, B2; Barrera Cortés, J1.

1CINVESTAV IPN Depto. Biotecnología y Bioingeniería, Av. IPN #2508 Col. San Pedro Zacatenco, C.P. 07360, MÉXICO D.F.

2Instituto Mexicano del Petróleo. Depto Biotecnología, Eje central Lázaro Cárdenas #152 Col. San Bartolo Atepehuacan C.P. 07730, MÉXICO D.F.


In this work the hydrocarbon degradation capacities of microbial strains

(hydrocarbonoclast strains) isolated from polluted soils was studied. Three types of soil

from different origin and pretreatment were examined: soil pretreated on composting

and containing 400,000 mg/kg of hydrocarbons (TPH’s); soil pretreated in a rotating

drum (slurry 50% of solids) and containing 40,000 mg/kg of TPH’s; and a non treated

soil with a TPH’s percentage of 50,000 mg/kg. The microorganisms were isolated by

serial dilution and agar plate count method, carried out a 30ºC for 7 days. Thirty three

bacteria were isolated from the rotating drums, 19 strains from the soil pretreated in

composting (6 fungi and 13 bacteria) and 22 strains from the non treated soil (16

bacteria and 6 fungi). The TPH’s degradation capacity of all isolates was determined in

liquid culture for 15 days at 30ºC and 130 rpm for bacteria and 150 rpm for fungi. It

was found that all the isolated strains had the capacity to degrade TPH’s. Aliphatic

hydrocarbons were eliminated in the range 20-90% and 10-90% by bacteria and fungi,

respectively. In the case of PAH’s, the percentage degradation was in the range of 20-

75% and 10-70%, for bacteria and fungi, respectively. The highest removal percentage

of aliphatic and PAH’s were obtained with 8 strains (5 bacteria and 3 fungi), 6 of which

were isolated from the pretreated soils (2 fungi from the soil containing 400,000 mg/kg

of TPH’s and 4 bacteria from the soil with 40,000 mg/kg of TPH’s).

It is concluded that active strains with high capacity to degrade TPH’s can be

found in pretreated and non-pretreated polluted soils. These strains could be used as

source of inoculum for bioaugmentation of other bioremediation processes.

Keywords: Hydrocarbonoclast Strains, Polluted Soils, Aliphatic Hydrocarbons,

Bioaugmentation

- 88 -

Bacterial composition “desna” for decision of some ecological problems

Nudga A.Yu.1, Dulgerov O.M.2, Kokina T.O.2, Boiko S.A.3

1 Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine,

154, Zabolotny Str., 03143, Kiyv, UKRAINE 2 Ukrainian Research Centre of Oil and Gas Ecology, LTD,

32/85, Glushkova Av., 03187, Kiyv, UKRAINE 3 National University of Food Technology, 68, Volodymirska Str., 01660, Kiyv, UKRAINE Te l . +380442669960 , fax +380442663296 , E -ma i l : u r c@onl i ne . com.ua

The researchers team of the Zabolotny Institute of Microbiology and Virology of the

National Academy of Sciences of Ukraine with the colleagues of the Ukrainian

Research Centre of Oil and Gas Ecology, Ltd. have developed and tested the bacterial

preparation “DESNA”. It can be used for rehabilitate soils in cases of hydrocarbons

pollution and purification of lakes and rivers from float (surface), emulsified and

dissolved petroleum products.

Bacterial composition “DESNA” consists of sorbent of hydrocarbons, some

inorganic stimulators of microorganisms and bacteria Bacillus megaterium sp.IMB B-

7003. Introduction of bacterial composition “DESNA” provides for increasing of

petroleum destruction rate. Biotechnologies were used in cases of accidents of pipelines

in different regions of Ukraine. As result of using of extensive biotechnology the

contents of hydrocarbons reducing to 50 mg per kg of soil during 6 months (start

contents – 12 000 mg per kg). The intensive biotechnology was used in case of pollution

of river and surface of soil by hydrocarbons. The content of hydrocarbons was reduced

to 100 times during 2 months. Microbiological control showed increasing of quantity of

main agricultural groups of soil’s microorganisms. Control area under crops of test

cultures provides for stimulating of plant’s growth.

As results of our investigations one can make the next conclusions: it has the

advantage analogues: possibility to connect of lability forms and to destruct of oil

products; possibility to destruct of C6-C27 hydrocarbons; it may be used in case of

surface and underground pollution and for detoxicating of polluted water; ecological

safety to mammalians and cold-blooded.

Keywords: DESNA, Bacillus megaterium, Sorbent, Soil, Polluted Water

- 89 -

Oxygen transfer in a slurry treated into a rotating drum implemented with helicoidal baffles

Miguel A. Rodríguez Meza1, David Taboada López1, Héctor Poggi Varaldo1,

Benjamín Chávez Gómez2, Josefina Barrera Cortés1

1 Departamento de Biotecnología y Bioingeniería. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional. (CINVESTAV-IPN)

Av. Instituto Politécnico Nacional 2508. Col. San Pedro Zacatenco. MÉXICO 07360, D.F. 2 Departamento de Biotecnología. Instituto Mexicano del Petróleo (IMP)

Lázaro Cárdenas 152. Col. San Bartolo Atepehuacan., MÉXICO 07730, D.F. Corresponding autor e-mail: [email protected]

The effect of baffle type and the variation of the rotating speed on the oxygen transfer in

a rotating drum (RD) operated at atmospheric pressure was studied. This work was

carried out with a non contaminated soil amended with mineral salts and a surfactant

(tergitol). The latter were included with the purpose to reproduce the remediation of soil

slurries, currently treated in those systems. The oxygen transfer phenomena were

analyzed by the sulphite method.

The RD was 50 cm large and 13 cm of diameter. It was fitted with two types of

baffles: 2 helicoidal and 4 trapezoidal baffles of 1x1/2 inch each end. The RD was

operated at three different rotating speeds: 5, 10 y 15 rpm.

Based on kLa values, the oxygen transfer increased an average of 32% when the

helicoidal baffles were used. Respecting rotating speed, an increase from 5 to 15 rpm

enhanced the kLa from 5 x10-5 to 9 x10-5 m/s when the helicoidal baffles are used. The

mixing efficiency of the helicoidal baffles was verified by a bioremediation treatment

using contaminated soil with 50,000 mg/kg of hydrocarbons (TPH’s). The removal

percentage of TPH’s was of 70% in 15 day and batch operation at 10 rpm. When the

same contaminated soil was treated in a RD implemented with trapezoidal baffles, the

removal percentage of TPH’s lowered to 60% in 21 days. It is concluded that the

helicoidal baffles improved the oxygen transfer and as a consequently, the treatment

time was reduced.

Keywords: Rotating Drum, Surfactant, Soil Slurries, Helicoidal Baffles

- 90 -

Solid-phase microextraction by gas chromatography and headspace applied to the aliphatic hydrocarbon analysis contained in wastewater

Elvira Ríos Leal 1, Rocío Castro Ríos 2

1 CINVESTAV IPN Depto. Biotecnología y Bioingeniería, Av. IPN #2508 Col. San Pedro Zacatenco, C.P. 07360, MÉXICO D.F.

2 UANL. Facultad de Medicina, Depto.Química Analítica Monterrey, Nuevo Leon, MÉXICO Corresponding author e-mail: [email protected]

The solid-phase microextraction has been successfully applied for the analysis of

volatile compound contained in gas, liquid and solid samples. The interest of this

technology is to eliminate the previous sample treatments such as the extraction and

purification. The advantages are the reduction of treatment cost and time, the increase of

result precision, due to the elimination of interferences added by the use of solvents, and

the reduction of sample lost.

This work presents an analytical method developed to determine the aliphatic

hydrocarbon concentration contained in wastewater. The method combines the solid-

phase microextraction, headspace and gas chromatography. The analytical procedure is

carried out in two stages: a) retention of analytes in the stationary phase, which in the

present case is a microextraction fiber; b) desorption of analytes by gas

chromatography. In the first stage, the fiber material was selected as well as the

adsorption conditions (temperature and time extraction). In the second stage, time and

temperature of desorption were determined. In this study, three microextraction fibers of

different materials were studied. For each fiber three-temperature and equilibrium time

were studied. The selection criteria were the aliphatic hydrocarbon recuperation and the

measurement reproducibility. The microextraction fiber is constructed on

polydimethylsiloxane (PDMS) of 100µm. The contaminant extraction demand to heat

the liquid sample 10 min at 80ºC and a fiber-headspace contact of 3 min. The

contaminant desorption demand 30 s of heating at 200 ºC. It is concluded that the

implemented method can be successfully and efficiently applied to the analysis of

wastewater samples with an efficiency recuperation of 90% and a precision of ±6.2.

Keywords: Solid-Phase Microextraction, Aliphatic Hydrocarbon, Wastewater, Gas

Chromatography

- 91 -

Identification of opportunistic oil-degrading microorganisms operating at the initial steps of oil extraction and handling

Olga Sánchez1*, Isabel Ferrera1, Tirso García de Oteyza2, Joan Grimalt2,

Jordi Mas1

1 Department of Genetics and Microbiology, Faculty of Sciences, Autonomous,

University of Barcelona, 08193 Bellaterra, SPAIN 2 Department of Enviromental Chemistry (IIQAB-CSIC),

Jordi Girona 18, 08034 Barcelona, SPAIN *Corresponding author e-mail: [email protected]

Many reports have focused on the isolation or identification of a variety of hydrocarbon

degrading microorganisms using culture-dependent or molecular techniques. However,

little research has been done on the occurrence of microorganisms incorporated into

crude oil in the initial steps of extraction and handling, which can start petroleum

biodegradation once an oil spill occurs. We have utilized a packed-column reactor filled

with perlite soaked with crude oil and subjected to a continuous flow of medium in

order to determine the presence of potential hydrocarbon degraders. The

microorganisms developed on the surface of the perlite, and although the experiment

lasted 73 days, we did not detect the presence of cells in the effluent until day 50,

indicating that the microbial activity and consequently the biofilm formation were slow.

DNA extraction of the biofilm and PCR amplification with 16S rRNA bacterial and

archaeal primers and 18S rRNA eukaryotic primers was performed. We did not find

amplification using archaeal primers. DGGE (Denaturing Gradient Gel Electrophoresis)

analysis revealed the presence of a unique band for bacterial and eukaryotic

amplification. Excision of these bands, sequencing, and subsequent BLAST search

showed that they correspond to Bacillus sp. and Aspergillus versicolor. The fungus was

later isolated from intact perlite in agar plates. A bacterial clone library was also carried

out, which confirmed the presence of a unique hydrocarbon degrading bacterium in the

biofilm, closely related to Bacillus sp. Analysis of petroleum components by gas

chromatography showed that there was degradation of n-alkanes, aromatic

hydrocarbons and carbazoles.

Keywords: Crude Oil, Packed-Column Reactor, Perlite, PCR, DGGE, Biofilm

- 92 -

Role of cyanobacteria in oil biodegradation by microbial mats

Olga Sánchez1*, Isabel Ferrera1, Tirso García de Oteyza2, Joan Grimalt2, Jordi Mas1

1 Department of Genetics and Microbiology, Faculty of Sciences, Autonomous,

University of Barcelona, 08193 Bellaterra, SPAIN 2 Department of Enviromental Chemistry (IIQAB-CSIC),

Jordi Girona 18, 08034 Barcelona, SPAIN *Corresponding author e-mail: [email protected]

There are evidences that microbial mats are able to degrade petroleum components in

polluted environments, but it is still a matter of discussion whether cyanobacteria from

these mats participate actively in hydrocarbon biodegradation. In this work, we attempt

to evaluate whether cyanobacteria can grow using crude oil as the only carbon source.

We used an illuminated continuous packed-column reactor filled with perlite soaked

with crude oil. Our reactor did not contain inorganic carbon, and to rule out possible

growth of cyanobacteria on CO2 produced from the degradation of oil by heterotrophic

bacteria, oxygen tension was kept low (5% air saturation). Different microorganisms

grew attached to the column filling at the expense of petroleum under microaerophilic

conditions. The experiment lasted 78 days, but cell numbers in the effluent increased

progressively during the first 10 days until reaching a stable value. We investigated the

diversity of the biofilm with molecular tools (DGGE, clone library) using 16S rRNA

bacterial and archaeal primers and 18S rRNA eukaryotic primers for amplification. No

archaeal amplification was observed. On the other hand, we detected the presence of

bacteria belonging to different groups, mainly to the Alpha and Gamma Proteobacteria,

the Chlorobi and to the low G+C Gram-positive bacteria. In addition, the biofilm

included an organism closely related to the algae Navicula cryptocephala. However,

cyanobacteria were not able to grow utilizing hydrocarbons as carbon source.

Measurements of aliphatic and aromatic hydrocarbons by gas chromatography showed

that microbial growth probably occurred at the expense of the fraction being lixiviated.

Keywords: Cyanobacteria, Biodegradation, Crude Oil, Continuous Packed-Column

Reactor, Perlite

- 93 -

Characterization of an oil-degrading cyanobacterial consortium by molecular techniques

Olga Sánchez*, Elia Diestra, Isabel Esteve, and Jordi Mas

Department of Genetics and Microbiology, Faculty of Sciences, Autonomous,

University of Barcelona, 08193 Bellaterra, SPAIN *Corresponding author e-mail: [email protected]

Recent studies have shown that the cyanobacterium Microcoleus chtonoplastes forms a

consortium with heterotrophic bacteria growing within the cyanobacterial sheath. This

consortium is able to grow in the presence of crude oil, degrading basically aliphatic

heterocyclic organo-sulphur compounds as well as alkylated monocyclic and polycyclic

aromatic hydrocarbons. In this work we characterize this oil-degrading consortium

through the analysis of the different 16S rRNA genes. We carried out the study in

cultures of Microcoleus grown in mineral medium and in cultures of the

cyanobacterium grown in mineral medium supplemented with crude oil. The results

indicate that most of the clones found in the polluted culture correspond to well-known

oil-degrading and nitrogen-fixing microorganisms, and comprise members of different

phylogenetic groups, such as the Alpha, Beta and Gamma subclasses of Proteobacteria,

and the CFB group. The control was dominated by one organism (88% of the clones)

closely affiliated to Pseudoxanthomonas mexicana (similarity of 99.8%).

Keywords: Crude Oil, Oil-Degrading Consortium, 16S rRNA, Microcoleus

chtonoplastes, Pseudoxanthomonas mexicana

- 94 -

Radial profiles of a TPH’s degradation process on a pilot scale biopile

Israel Espinosa Espinosa1, Benjamín Chávez Gómez2, Héctor Poggi Varaldo1, Josefina Barrera Cortés1

1 Departamento de Biotecnología y Bioingeniería. Centro de Investigación y de Estudios Avanzados del

Instituto Politécnico Nacional. (CINVESTAV-IPN) Av. Instituto Politécnico Nacional 2508. Col. San Pedro Zacatenco. MÉXICO 07360, D.F.

2 Departamento de Biotecnología. Instituto Mexicano del Petróleo (IMP). Lázaro Cárdenas 152. Col. San Bartolo Atepehuacan., MÉXICO 07730, D.F.


The objective of this work is the determination of the air flux on a pilot scale biopile

process for the bioremediation of an aged soil polluted with 500,000 mg/kg of TPH’s,

50% of which were recalcitrant (asphaltenes). The polluted soil amended with mineral

salts, cane bagasse, tergitol and water, was treated in a square-base reactor of

1mx1mx10cm, which was installed in a controlled temperature chamber (20-25ºC).

Temperature, pH, humidity, CO2 production and TPH’s residual variables were

monitored in 8 sample points, located over three equidistant circles between the center

and the periphery of the bioreactor. Air fluxes of 2, 4 and 6 L/min were fed at the

bioreactor through a central point performed over its upper cover. According to an

ANOVA of the experimental results, the air flux had a significant effect over the TPH’s

removal. The TPH’s percentage removal at 2, 4 y 6 L/min of air flux were 7.19%,

20.68% y 32%, respectively. An analysis of the pH, humidity, CO2 production and

TPH’s removal radial profiles showed that the air flux had a drastic effect over the CO2

production and TPH’s removal. The CO2 production was higher near the feed air point

that in the peripheral zone, where its value was very down. Concerning to the pH and

humidity, non significant effects were observed. An analysis of the TPH’s kinetics

shows that 2L/min of air is not enough to activate the native microorganisms

metabolism. At air fluxes of 4 and 6 L/min a very high initial degradation rate was

shown. At 6L/min of air flow rate, both the initial and the final removal speeds were

almost two fold the values of the correspondent rates at 4L/min flow rate. It is

concluded that central aeration of a model pilot biopile may develop significant

gradients of microbial activity and pollutant removal.

Keywords: Biopile, Recalcitrant, Polluted Soil, Bioreactor

- 95 -

Toxic and genotoxic effect of MTBE and TBA to selected microorganisms

Miluse Vosahlikova1, Jan Bartu1, Tomas Cajthaml2, Jarmila Pazlarova1,

Katerina Demnerova1

1 Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technicka 3, 166 28, Prague, Czech Republic

2 Department of Ecology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20, Prague, Czech Republic


Methyl tert-butyl ether (MTBE) has been used to improve engine performance and

enhance air quality in the U.S. and Europe for over 20 years, and it is currently used

around the world. Nowadays MTBE is frequently detected in surface water and

groundwater because MTBE is much more soluble in water than other components of

gasoline, and it is more resistant to biodegradation. The first aim of this study was to

evaluate toxicity and genotoxicity of MTBE and of its intermediate tert-butyl alcohol

(TBA) by two short-term test systems: toxicity – Microtox®, and genotoxicity – Ames

test. The second task was to find potential MTBE-degrading bacterial strain or

consortium. The potential for aerobic MTBE degradation was investigated with mixed

cultures isolated from contaminated soils in the Czech Republic. The growth of tested

bacterial strains was supported by n-alkanes and hexane as cosubstrates. The growth

rate of tested bacterial strains was measured and decrease of MTBE after biodegradation

was monitored by GC. The third aim was a monitoring of diversity of soil

microorganisms isolated from the same site but cultivated with different source of

carbon and energy: 1) n-alkanes and MTBE, 2) hexane and MTBE or 3) MTBE. From

each sample DNA was isolated, then PCR was performed with primers specific for 16S

rDNA and amplicons were analysed by temporal temperature gel electrophoresis

(TTGE).

Keywords: Methyl Tert-Butyl Ether (MTBE), Biodegradation, Genotoxicity,

Contaminated Soils, Temporal Temperature Gel Electrophoresis (TTGE)

- 96 -

Study of the structure of biodegraded wood using the water vapours sorption method

J.Chirkova, I.Irbe, B.Andersons, I.Andersone

Latvian State Institute of Wood Chemistry, 27 Dzerbenes St., LV-1006 Riga, LATVIA

Upon the action of brown-rot on wood, its depletion in carbohydrates occurs, which is

accompanied by a change in the wood structure. A study of the consequences of the

biodegradation of wood with different cultures of fungi can contribute to the elucidation

of the mechanism of their action.

One of the methods to investigate the wood microstructure is the water vapours

sorption method. Based from the sorption-desorption isotherms, it is possible to

determine the structural characteristics – specific surface, pore volumes and their

distribution in pore sizes, as well as to characterize the hydrophilic properties of the

wood surface.

In the present study, the change in the structural and hydrophilic characteristics

of Pinus sylvestris pine wood after the exposure on the brown-rots Coniophora puteana,

Poria placenta and Gloeophyllum trabeum is investigated. The study is carried out in a

wide range of exposure times – from several days (3-9) to several months (1-4). The

appearance of new pores 2-4 nm in size, whose volume depends on the fungus culture

and exposure time, in wood has been established. A possible mechanism of the

structural changes is discussed.

Procedure aspects of the investigation of biodegraded wood are considered. It

has been shown that the drying of the samples after exposure at the temperature 103 ±

2oC (in compliance with EN 113) is accompanied by a reversible collapse of the

structure, which should be taken into account in the interpretation of the results of the

study.

Keywords: Wood, Fungi, Biodegradation, Water Vapours Sorption Method

- 97 -

Phenolic removal of from olive mill dry residue by laccase activity of white rot fungi and its impact on tomato plants growth

E. Aranda, I. Sampedro, J.A. Ocampo, I. García-Romera

Dpto. Microbiología del suelo y sistemas simbióticos, Estación Experimental del Zaidín, C.S.I.C., Prof.

Albareda 1, Apdo. 419, 18008 Granada, SPAIN Corresponding author e-mail: [email protected]

Olive mill dry residue exhibits high phytotoxic properties, mainly due to its phenols

content. White-rot fungi possess an extracellular enzymes system capable of degrading

a wide variety of pollutants. The aim of this work was to study the influence of the

laccase activity of two white-rot fungi on the toxic effect that have the water-soluble

substances of the dry residues from olive (ADOR), on the growth of tomato plants.

Pycnoporus cinnabarinus and Coriolopsis rigida were incubated during 30 days and the

phenolic content, the laccase activity, and the effect of this extract in the growth of

plants after every 5 days of incubation were determined. Results showed that P.

cinnabarinus and C. rigida produced laccase activity after 5 and 15 days of incubation

respectively and the higest activity was detected at 20 days of incubation. P.

cinnabarinus and C. rigida were able to decrease the phenol content up to 73% after 15

days of incubation. The incubation of ADOR with P. cinnabarinus and C. rigida

decreased the phytotoxicity of this residue in tomato plants. A close relationship

between the laccase activity, the decrease in phenol content of ADOR by the saprobic

fungi and the increase in the dry weight of plants was found. These results show that the

phenols elimination by the lacasse activity of white-rot fungi can be important in the

elimination of the phytotoxicity of olive mill dry residues.

Keywords: White-Rot Fungi, Laccase Activity, Phenol, Phytotoxicity

- 98 -

Biodegradation of woods from caribbean pine (Pinus caribaea mor. var. hondurensis) and “melina” (Gmelina arborea roxb,), in natural and

induced conditions.

Castro-Medina, F. 1, Mohaly, S. 2; Encinas, O. 2

1 Centro Biotecnológico de Guayana (CEBIOTEG),

Universidad Nacional Experimental de Guyana (UNEG), Upata, Edo. Bolívar. VENEZUELA 2 Centro de Estudios Forestales y Ambientales de Postgrado, Facultad de Ciencias Forestales y

Ambientales, Universidad de Los Andes, Mérida, VENEZUELA Corresponding autor e-mail: [email protected].

The natural and induced durability of the woods from Pinus caribaea (Mor) var.

hondurensis Barrett and Golfari (Caribbean pine) and Gmelina arborea Roxb (melina),

were evaluated by using boxes with natural and non sterile soil. The evaluation take into

account the loss of weight of wood samples and the growth characteristics of the

microorganisms in the wood, for eight weeks of incubation period. Wood coming from

Caribbean pine without treatment showed the highest percentage of weight loss (25 %)

but when it was preserved the weight loss was the lowest (5 %), while wood coming

from Melina, also preserved, showed the highest percentage (8 %). Both woods were

treated with Tim-bor at 20 % of concentration (2. 4 kg/m3 in Caribbean pine and 2

kg/m3 in Melina) and copper sulphate at 9 % (1.9 kg/m3 in Caribbean pine and 1.2

kg/m3 in Melina). This was enough to reduce the action of the degraders

microorganisms of the wood present in the non sterile soil tested. Caribbean pine

allowed higher penetration and retention than Melina wood. The isolation of

microorganisms in wood in the tested soil, were mainly imperfect fungi, molds, soft rot

and decay fungi. The pathways of degradation by the microorganisms in the wood were

observed using microscope of light and scanning electronic microscope (SEM). The

transversal section of wood from Caribbean pine shows the presence of cavities in the

secondary wall (S2) of the late wood, which is characteristic of the decay by soft rot

fungi type I. In Melina wood erosion was also observed in the cell wall produced by

decay soft rot type II, additionally to the observed cavities.

Keywords: Caribbean Pine, Melina, Fungi, Molds, Scanning Electronic Microscope

(SEM)

- 99 -

White-rot basidiomycetes: bioconversion of plant raw materials and lignocellulolytic enzymes production

Vladimir Elisashvili a, Michel Penninckx b,*, Eva Kachlishvili a, Nino Tsiklauri a,

Eka Metreveli a, Nana Aladashvili a, Mikheil Asatiani a

a Durmishidze Institute of Biochemistry and Biotechnology, Academy of Sciences of Georgia,

10 km Agmashenebeli Alley, 0159 Tbilisi, GEORGIA bLaboratoire de Physiologie et Ecologie Microbienne, Faculte des Sciences,

Universite Libre de Bruxelles, c/o Institut Pasteur, 642 Rue Engeland, B-1180 Brussels, BELGIUM

White-rot basidiomycetes from various taxonomic groups and ecological niches were

compared for their ability to produce lignocellulolytic enzyme in SSF and SF of

different plant raw material. Fungi cultivation in identical culture conditions revealed

wide differences among both species and strains of the same species. The nature and

chemical composition of lignocellulosic material is factor determining the expression of

lignocellulolytic potential of fungi as well as the ratio of individual enzymes in enzyme

complexes. The study underlines that the expression of biosynthetic potential of

basidiomycetes is highly dependent on method of fungi cultivation. SSF of plant

substrates is favorable for MnP secretion, whereas SF decreases or inhibits this enzyme

production. The data obtained prove that only increased fungi growth at presence of

additional nitrogen accounts for the higher levels of laccase, MnP and ligninase activity.

The additive effect of simultaneous carbon and nitrogen supplementation to the medium

on ligninolytic enzymes production and lignin degradation is revealed. Role of

extractable from lignocellulosic substrates compounds in regulation of ligninolytic

enzymes production will be discussed.

Keywords: Basidiomycetes, Lignocellulolytic Enzyme, Laccase

- 100 -

Biological deterioration of “baraman” wood (Catostemma commune sandwith) caused by rot fungi white, brown and soft

Ramírez, Eliécer, Castro-Medina, Feraida; Velásquez, Jesús

Centro Biotecnológico de Guayana (CEBIOTEG),

Universidad Nacional Experimental de Guyana (UNEG), Upata, Estado Bolívar. VENEZUELA Corresponding autor e-mail: [email protected]

To determine the effect of white rot (Trametes versicolor), brown rot (Gloeophyllum

trabeum), and soft rot in the weight losses, variations in the proportions of the main

chemical components, and micro morphological structure in the baraman wood

(Catostemma commune Sandwith), samples were exposed using the American standard

ASTM D 2017 (soil block) for white and brown rot, and the standard EN 807 for soft

rot. There were used the standards ASTM D 1103, D 1104, D 1105, D 1106 for the

determination of the cellulose, hemicellulose, lignin and extractive proportion

respectively. The evaluations were made every month during four months for loss of

weight, and during three months for chemical proportions. The baraman was moderately

resistant to white and brown rot, with values of loss of weight superiors to 25%, and

resistant to soft rot where the weight loss of was 15.51%. The lignin proportion was the

more affected in white rot, although all the components were deteriorated

simultaneously. In brown rot the biggest effect was on the carbohydrates and very little

on the lignin, while in soft rot the cellulose was the most affected. In microscopy,

perforations of white and brown rot were observed, with enough deterioration of the

half sheet of cell wall in white rot. It was also observed general erosion of the lumen

toward the half sheet of cell wall in brown rot. In soft rot the series of oblique cavities

characteristics of soft rot type 1 were observed, as well as erosion channels in the lumen

corresponding to soft rot type 2.

Keywords: Baraman Wood, Rot, Fungi, ASTM

- 101 -

Degradation of chlorophenols in wood-chips by wood-rotting fungi

Hanna Oesch-Kuisma, Kari T. Steffen, Marja Tuomela, Annele Hatakka

Department of Applied Chemistry and Microbiology, P.O.Box 56, 00014 University of Helsinki, FINLAND Corresponding author e-mail: [email protected]

White-rot and litter-decomposing fungi secrete nonspesific extracellular enzymes,

which break down all components of lignocellulose and other recalcitrant compounds,

such as chlorophenols. A white-rot fungus efficiently mineralized pentachlorophenol

(PCP) in autoclaved soil. Here we used non-sterilized material because in field-scale

bioremediation applications, sterilizing the contaminated materials is not feasible. Thus,

the fungi used must be capable to compete with the native population.

Nine fungi were selected degradation experiments: Agrocybe praecox,

Gymnopilus penetrans, Irpex lacteus, Phlebia gigantea, Phlebia tremellosa,

Physisporinus rivulosus, Pleurotus ostreatus, Stropharia rugosoannulata and Trametes

ochracea. Wood material from old saw mills are a persistent problem. Non-sterilized

wood chips, which had been treated in a saw mill with chlorophenol- and dioxin-

containing commercial product against blue staining, were inoculated with the fungus

and 14C-labelled PCP was added. The bottles were aerated continuously for four

months, and the evolved 14CO2 was trapped during incubation. From parallel non-

radioactive cultures chlorophenols and their metabolites from wood were analysed by

GC-MS. Fungi used in our study mineralized up to 50% of added 14C-PCP while the

native population mineralized only 3%. The most efficient fungi were G. penetrans, P.

ostreatus, P. tremellosa and P. rivulosus. Some fungi significantly decreased the

concentration of chlorophenols in wood chips. The best decrease was achieved with S.

rugosoannulata for 2, 3, 4, 6-tetrachlorophenol and PCP, which were the most abundant

compounds in wood chips. Chloroanisoles were not formed during the incubation with

fungi.

Keywords: White-Rot Fungi, Litter-Decomposing Fungi, Wood, Pentachlorophenol

(PCP)

- 102 -

Biodegradation of cotton stalks into protein rich feed by fungi and nitrogen-fixing bacteria on solid state fermentation.

Bahaa T. Shawky

Microbial Chemistry Department, Genetic Engineering and Biotechnology Research Division, National Research Center, Tahrir Street, Dokki, Cairo, 12311, EGYPT


Lignocellulosic crop residues are generated as waste byproducts in huge quantities

every year, creating disposal problems in the absence of its proper utilization. Such

agricultural crop wastes can be recycled to produce several beneficial products. Solid

state fermentation (SSF) systems offer potential advantages in the development of

bioprocesses, such as biodegradation of crop residues for nutritional enrichment, and

production of value-added products such as protein rich feed. The potential of Pleurotus

ostreatus with and without combination of Azospirellum brasilense, for the degradation

of lignocellulosics and their ligninolytic enzyme activities under SSF, were carried out

for 5, 10, and 15 days at 30 °C using chopped cotton stalks as a substrate. After the

fermentation periods, the biodegradation of cotton stalks was evaluated for different

parameters. Samples were taken and analyzed for crude protein, cellulose,

hemicellulose, lignin, and in vitro DM digestibility. Activities of laccase, peroxidase,

cellulase, and ligninase were determined. The values for the different parameters were

changed with white-rot fungus along with the asymbiotic nitrogen-fixer. The proportion

of lignin, hemicellulose, and cellulose in untreated control substrate was 23, 27, and 40

%, respectively. Growth of P. ostreatus combined with A. brasilense on cotton stalks

resulted in the degradation of 20 % lignin, 15 % hemicellulose, and 17 % cellulose.

Results indicate that Pleurotus degrades lignocellulose with obvious selectivity for

lignin, resulting in lignin content significantly decreased and in vitro digestibility was

increased. The crude protein content of cotton stalks after fermentation was 15 %, this

was due to the synergistic growth of the basidiomycete and the dinitrogen-fixing

bacterium during the SSF.

Keywords: Solid State Fermentation (SSF), Cotton Stalks, White-Rot Fungus,

Dinitrogen-Fixing Bacterium

- 103 -

Role of fragmentation activity in cellulose hydrolysis

Abdul Aala Najmus Saqib, Philip John Whitney

School of Biomedical & Molecular Sciences, University of Surrey, Guildford, GU2 7XH, UK. Corresponding author e-mail: [email protected]

Despite its abundance, the use of cellulose as a source of sugars is very limited. Once

achieved economically, this process could be the basis of future sustainable energies.

Few organisms produce cellulases, which act only slowly. Most studies have been

carried on three components of the cellulolytic systems, viz, endoglucanases,

exoglucanases and cellobiases. Little attention has been paid to the fragmentation

activity of certain cellulolytic systems. We have noticed that despite being more

powerful towards degrading modified cellulose, the culture filtrate of Myrothecium

verrucaria is less efficient towards pure cellulose as compared to the culture filtrate of

Trichoderma reesei. SEM imaging showed that one distinguishing feature of the later is

its ability to macerate the cellulose. Cellulose particle size was decreased with time as it

was incubated in the culture filtrate of T. reesei at 37oC. Different particle sized

cellulose was obtained by incubation of cellulose with T. reesei culture filtrate for

different lengths of time (pre-treatment). Pre-treated cellulose was then incubated with

fresh T. reesei culture filtrate. Cellulose hydrolysis (liberation of reducing sugars) was

faster with small particles (pre-treated for 0-10hrs) but slower for the smallest particles

(pre-treated for 24hrs). However, upon extension of incubation to 96hrs, the hydrolysis

of cellulose was greater with the larger particle size (pre-treatment 0-10hrs). It was

hypothesized that fragmentation activity of the pre-treatment opens up attack sites for

other enzymes. T. reesei culture filtrate was fractionated on Sephadex G75 column and

the fragmentation activity was assigned to certain fractions.

Keywords: Cellulose, Myrothecium verrucaria, Trichoderma reesei, SEM

- 104 -

Degradation of synthetic dyes by litter-decomposing fungi

Petr Baldrian, Vendula Valášková, Jaroslav Šnajdr

Laboratory of Biochemistry of Wood-rotting Fungi, Institute of Microbiology ASCR, Videnska 1083, 14220 Praha 4, CZECH REPUBLIC Corresponding author e-mail: [email protected]

Some species of lignocellulose-degrading fungi produce a set of ligninolytic enzymes

(oxidases and peroxidases) that participate in the degradation of lignin contained in

wood. Due to low specificity, these enzymes can also degrade xenobiotic compounds

like PAH or synthetic dyes. Litter-decomposing fungi are an ecologically distinct group

of lignocellulose decomposers growing in forest soils and litter. Some of these fungi

have been demonstrated to produce ligninolytic enzymes laccase and Mn-peroxidase

that participate in the degradation of soil humic substances. Due to their natural

occurence in soils, these species are prospective for biodegradation in nonsterile

conditions. We studied the ability of selected litter-decomposing fungi from the genera

Agrocybe, Collybia, Mycena, and Stropharia as well as strains isolated from the oak

forest litter to degrade soil humic substances and synthetic dyes (Remazol Brilliant

Blue, Reactive Blue, Reactive Orange, Poly B-411) in liquid culture. The efficiency of

decolorization by litter-decomposing fungi was compared to the abilitites of typical

white-rot species Pleurotus ostreatus and Trametes versicolor. Most litter-decomposing

fungi produced laccase while some strains also produced peroxidases and thus turned

out to be promissing degraders of most of the synthetic dyes tested.

Keywords: Lignocellulose-Degrading Fungi, Xenobiotic Compounds, Soil Humic

Substances, Synthetic Dyes

- 105 -

Proteolysis in the white rot fungi: how proteinases help fungus to improve lignin metabolism

K. Grzywnowicz , J. Żuchowski, P. Zawadzki, K. Sobczyk, M. Jaszek

Department of Biochemistry, M. Curie- Skłodowska University, M.Curie- Skłodowska Square 3, 20-031 Lublin, POLAND Corresponding author e-mail: [email protected]

It is well known that fungi are heterotrophic microorganisms which use organic substances

as a source of carbon and of nitrogen (of inorganic and organic origin). Extracellular

proteases are, apparently, widely distributed among enzymes secreted by these organisms,

for feeding with nitrogen from peptides and proteins. White rot Basidiomycetes are mostly

known for their activities able to decompose wood constituents, mainly ligninocellulose.

These microorganisms have also been found to produce both intracellular and extracellular

proteolytic enzymes through their growth and development. The growth of wood-decaying

fungi, especially under natural conditions, requires nitrogen economy, involving regulation

of proteolytic activities for the intracellular protein turnover (including autolysis),

extracellular digestion of protein sources, and modification of fungus-own proteins

through limited. In our experiments in stress conditions (nitrogen and carbon starvation)

and in protein supplementation, changes in protease activities (altered patterns of

proteolytic activities also) and changes in levels and/or kinds of their natural inhibitors

were observed. We have observed also very interesting changes in properties of both

proteases and inhibitors. Because changes in activities of serine proteinases (mainly

intracellular) and aspartic proteinases (mainly extracellular), and in levels of their

inhibitors were correlated with type of source of nitrogen and with levels of ligninolytic

enzymes, the correlation of primary metabolism and ligninocellulose metabolism may be

strongly ascribed to proteinases. So it seems that wood rotting fungi work according

strategy – first feed with easy nutrients, then decay wood, metabolising ligninocellulose.

Keywords: White Rot Basidiomycetes, Protease, Serine Proteinases, Aspartic Proteinases

- 106 -

Production of extracellular metal chelating compounds by wood rotting fungi

Anna Jarosz-Wilkołazka, Marcin Grąz, Anna Olszewska

Department of Biochemistry, Maria Curie-Skłodowska University,

Skłodowska Place 3, 20-031 Lublin, POLAND Corresponding author e-mail: [email protected]

Several recent advances have been made in our knowledge of the mechanisms that

wood rotting fungi use to degrade wood and other organic compounds. They use low

molecular mass agents which may function in conjunction between lignolytic fungal

enzymes, substrates e.g. lignocellulose, phenolic compounds, dyes, and also metals in

the initiation of biodegradation reactions. On the other hand this low molecular weight

compounds are involved in the detoxification of heavy metals which are more common

in fungal natural environments.

In this report we tested 28 fungal strains from different physio-ecological groups

which could be broadly grouped into co-physiological groups: wood-decaying, litter

decomposing, and mycorrhiza-forming fungi. The main group of examined strains was

wood-decaying strains belonging to white rot fungi. We detected low molecular weight

compounds, with metal chelating capability, from the solid or liquid fungal cultures in

the presence or absence of various soluble or insoluble metal compounds. In this regard,

the presence of organic acids and iron-chelating compounds was tested using visual

solubilisation of insoluble metal compounds, capillary electrophoresis system or the

universal siderophore assay. In this paper, we report our results mainly from agar-

screening trials.

This work was partially supported the State Committee for Scientific Research

(139/E-339/SPB/6. PR UE/DIE 450/2004-2007).

Keywords: Wood-Decaying Fungi, Litter Decomposing Fungi, Mycorrhiza-Forming

Fungi, Heavy Metals, Siderophore

- 107 -

Ligninolytic enzymes can act as a part of multiple response system to oxidative stress in white rot Basidiomycetes Fomes fomentarius and

Tyromyces pubescens

M. Jaszek, J. Żuchowski, E. Dajczak, K. Cimek, M. Grąz, K. Grzywnowicz


Fungi, like other living organisms, are exposed to various types of stress, including high

and low temperature, nutrient starvation, water stress, exposure to heavy metals or

xenobiotics. Although the reactive oxygen species (ROS) are generated during the

normal cellular metabolism, when the level of ROS increase beyond antioxidant

capacity of the cell we can talk about oxidative stress conditions. It is known that white

rot basidiomycetes are the most efficient lignin degraders which can produce two kinds

of ligninolytic metaloproteins: laccase and peroxidases. Their ligninolytic system has

been very often described in the recent literature, but the question of the mechanisms of

their oxidative stress defense is still open and incomplete. Our earlier research showed

that the presence of some prooxidants can significantly increase the activities of laccase

and manganese dependent peroxidase in selected strains of wood degrading fungi. In the

present study idophasic cultures of Fomes fomentarius and Tyromyces pubescens were

treated with chosen doses of menadione - well known superoxide stress factor. It was

observed that the addition of prooxidant to the fungal cultures significantly changed the

extra- and intracellular activities of laccase and MnP. Basing on this fact we can suggest

that these enzymes can act as the antistress proteins in selected strains of white-rot

Basidiomycetes. The production of low molecular mediator - oxalic acid was estimated

using the capillary electrophoresis. The levels of typical antioxidants: phenols and GSH,

as well as activities of superoxide dismutase and catalase were also determined in both

investigated strains.

Keywords: Oxygen Species (ROS), White Rot Basidiomycetes, Oxidative Stress,

Fomes fomentarius, Tyromyces pubescens

- 108 -

Amino acid sequence and enzymatic activities similitude between Phanerochaete flavido alba laccase and P. chrysosporium MCO1

ferroxidase

Lucas M., de la Rubia T., Larrondo L.F.(1), Martinez J.

Dept. Microbiología, Fac. Famacia, Universidad de Granada, Campus Cartuja, 18071-Granada, Spain. (1)Dept. de Genetica Molecular y Microbiologia, Fac. Ciencias

Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile and Instituto Milenio de Biologia Fundamental y Aplicada, Santiago, Chile

Among the white rot fungi Phanerochaete chrysosporium has been by far the most

studied. In opposition to most white rot fungi, this basidiomycete seems unable to

produce laccases. Four genes encoding multicopper oxidases (MCOs) sharing properties

with Fet3 ferroxidases and laccases has been identified in P. chrysosporium. P. flavido-

alba, another fungus in the same genus, produces laccase (PFA-Lac) as well as

peroxidases. Neither PFA-Lac N-terminal amino acid sequence, nor those of internal

peptides matched significantly to any known laccase.This communication summarize

the comparison in amino acid sequence and enzymatic activity of PFA-Lac and P.

chrysosporium MCO1. The N-terminal amino acid sequence as well as those of six

oligopeptides obtained from PFA-Lac (Edman degradation or MADI-TOF mass

spectrometry) matched significantly to P. chrysosporium MCOs amino acid sequences.

Both P. chrysosporium MCO1 and PFA-Lac showed substrates range and enzymatic

efficiencies in the oxidation of 12 selected substrates different to those exhibited by a

fungal (T. versicolor) laccase. All together these data shown a greater similitude

between PFA-Lac and the newly described P. chrysosporium MCO1 than to the tested

fungal laccase.

This work was founded through the Project 30C0198700 from the European

Commission.

Keywords: White Rot Fungi, Phanerochaete chrysosporium, Laccase, Multicopper

Oxidases

- 109 -

Enzymatic degradation of oxalic acid in Bjerkandera fumosa cultures

Marcin Grąz, Anna Jarosz-Wilkołazka, Elżbieta Dernałowicz-Malarczyk

Department of Biochemistry, Maria Curie-Skłodowska University, Skłodowska Place 3, 20-031 Lublin, POLAND


Oxalic acid is widely distributed as calcium and magnesium salts and is catabolized by

two major pathways: oxidation to carbon dioxide and hydrogen peroxide or

decarboxylation to carbon dioxide and formic acid. Decarboxylases that catabolize this

latter process have been reported mostly from fungi such as Aspergillus niger,

Sclerotinia sclerotiorum, Flammulina velutipes, and Postia placenta. Oxalic acid is the

main carboxylic acid secreted by wood rotting fungi and as low-molecular weight

compound mediated fungal natural processes including lignocellulose degradation and

breakdown of xenobiotic pollutants. It acts mainly as protons and electrons source and

as strong metal chelator and stabilizator.

In our earlier study, oxalic acid was found to be the predominant organic acid

secreted by white rot fungi in the liquid cultures. These strains have to control the

quantity of oxalic acid in their surroundings very precisely and they secreted oxalate

decarboxylase (ODC). The aim of present work was the study of oxalic acid enzymatic

degradation in stationary and shaken cultures of Bjerkandera fumosa, white rotting

strain occurred as a saprophytic (and some times weakly parasitic) organism on trunks,

but also stubs and steams, of deciduous trees on moist, rich soils. We proved that

intracellular oxalate decarboxylase was responsible for decomposition of oxalate in

cultures of B. fumosa. The effect of exogenous oxalic acid and Mn2+ addition and pH

lowering to ODC activity was measured. We made an attempt to purify the ODC.

Degradation of oxalic acid was detected using capillary zone electrophoresis.

This work was partially supported by SOPHIED (NMP2-CT-2004-505899) and

the State Committee for Scientific Research (139/E-339/SPB/6. PR UE/DIE 450/2004-

2007).

Keywords: Oxalic Acid, Wood Rotting Fungi, Oxalate Decarboxylase (ODC),

Bjerkandera fumosa

- 110 -

Biodeterioration of Caribbean pine (Pinus caribaea mor. var. hondurensis), Melina (Gmelina arborea roxb,) and Carapa (Carapa

guianensis) wood, against attack of subterranean and drywood termites

Marquez Cabeza, Arlenys

Centro Biotecnológico de Guayana (CEBIOTEG), Ing. en Industrias Forestales,

Universidad Nacional Experimental de Guyana (UNEG), Upata, Edo. Bolívar. VENEZUELA Corresponding autor e-mail: [email protected]

To determine the resistance of three species wood Venezuelan, the conifer wood: Pinus

caribaea var. Hondurensis (Caribbean pine) and two broadleaf Gmelina arborea

(Melina) and Carapa guianensis (Carapa), against termites subterranean and drywood

termites laboratory testing was carried. The subterranean termite was Heterotermes

convexinotatus and the American Standard ASTM-3345 was used. A qualitative

evaluation and the loss in weight of each sample as well as the mortality of the termites

during 8 weeks were determined. The samples of Caribbean pine were strongly affected

with a losses in weight aroun 78% was obtained, in the qualitative evaluation after 8

weeks these samples failure. The Melina wood was affected with 25% of loss in weight

and they were alone lightly attacked in the external part. The Carapa wood went

relatively resistant to the attack of this species of termites without penetrating the wood,

reaching 5% of loss in weight. For the experiment of dry wood termites the

methodology suggested by the Institute of Technological investigations of Brazil was

used, using Cryptotermes brevis the damage caused to the wood and the penetration of

these organisms of biodeterioro in the samples was evaluated. The wood of Caribbean

pine was penetrated by these termites consuming its internal part and when the

penetration was not complete the termites built an external layer to be protected of the

light. The melina wood was lightly affected by the termites, penetration intent was

evidenced. In the Carapa wood were highly resistant to the degradation by Cryptotermes

brevis.

Keywords: Conifer Wood, Biodeterioration, Subterraneum Termites, Dry Wood

Termites

- 111 -

Post-treatment of a recalcitrant pretreated kraft effluent using hybrid pellets of a ligninolytic fungus

A. Ortega-Clemente(1), E. Ríos-Leal(1), M. T. Ponce-Noyola(1),

M. C. Montes-Horcasitas(1), N. Rinderknecht-Seijas(2), S. Caffarel- Méndez(3), J. Barrera-Cortés(1), Héctor M. Poggi-Varaldo(1)*

(1) CINVESTAV del IPN, Dept. Biotechnology and Bioengineering,

Environmental Biotechnology R&D Group, P.O.Box 14-740, Mexico D.F., 07000, MEXICO (2) ESIQIE-IPN, Division of Basic Sciences, MEXICO

(3) Tecnológico de Estudios Superiores de Ecatepec, Ecatepec, Edo. Mex., MEXICO Corresponding author e-mail: [email protected]

One of the most challenging issues in industrial wastewater treatment is the treatment of

effluents polluted with spent pulping liquors, which are quite toxic and contain amounts

of recalcitrant organic matter like lignin derivatives and fragments. The aim of this work

was (i) to develop a protocol for the formation of hybrid pellets of Trametes versicolor,

and (ii) to determine the extent of depuration of an anaerobically-treated weak black

liquor (AnE) that could be achieved by the hybrid pellets in semi-continuous flask tests.

Concerning the pelletization protocol, it was found that batch culturing 30 mg mycelium

and 30 mg fine sawdust, or 30 mg mycelium plus 15 mg fine sawdust and 15 mg fine

activated carbon in mycological medium supplemented with 5 g peptone/L per flask

gave 4 to 5 discrete dual and triple pellets, respectively. Scanning electron

microphotographs of pellet sections showed that they had a well formed external wall of

fungus approximately 1 mm thickness, whereas the support (sawdust or mixture

sawdust-activated carbon) formed an internal core with ramifications of fungal

mycelium intertwined. 50 mL of AnE effluent (pH 4.5) plus 2 to 3 hybrid pellets were

placed in flasks. Dual and triple pellets were tested for 10 cycles of 7 days each. At the

end of each cycle, treated effluent was removed and a new volume of AnE was fed to

the flaks while pellets remained. Units with triple pellets showed higher removals of

COD, color, and ligninoids than dual pellets, in terms of net, unit removal efficiency

analysis. Enzyme Mn-peroxidase activity seemed to be higher than that of lignin-

peroxidase and lacase, in average. Protease activity increased in selected cycles of

incubation and near the end of the test. No apparent anti-correlation was found between

activities of ligninolytic exo-enzymes and Protease, against expectations.

Keywords: Wastewater, Spent Pulping Liquors, Trametes versicolor, batch culturing

- 112 -

Fungal bioremediation of wood and soil contaminated by chlorinated phenols

Hanna Oesch-Kuisma, Marja Tuomela, Kari T. Steffen, Mika A. Kähkönen,

Annele Hatakka

Department of Applied Chemistry and Microbiology, P.O.Box 56, 00014 University of Helsinki, FINLAND Corresponding author e-mail: [email protected]

The suitability of white-rot and litter-decomposing fungi for bioremediation of wood

chips or soil contaminated by chlorinated phenols and dioxins was studied. The fungal

species were Agrocybe praecox, Bjerkandera adusta, Ceriporiopsis subvermispora,

Dichomitus squalens, Haploporus odorus, Irpex lacteus, Nematoloma frowardii,

Phanerochaete sordida, Phellinus ferrugineofuscus, Phlebia gigantea, Phlebia

tremellosa, Physisporinus rivulosus, Pleurotus ostreatus, Radulodon erikssonii,

Stropharia rugosoannulata, Trametes hirsuta, and Trametes versicolor. In addition

several fungi were isolated from an old saw-mill area in Southern Finland. Screening

tests included cultivation on sterile PCP-containing liquid media for testing the

tolerance towards chlorophenols, cultivation on sterile and non-sterile bark of Pinus

silvestris, cultivation on non-sterile wood chips contaminated with wood preservatives,

and growth into non-sterile contaminated soil. Bark, which is a residue of wood

industry, was selected as support material and substrate of the fungal inoculum. The key

feature in our studies was the use of non-sterilized material since in field-scale

applications fungi must survive and compete with the native microbial population. To

find out the impact of fungal bioremediation for contaminated soil, CH4 oxidation

potential, inhibition of bioluminescence of Vibrio fischeri, and endogenous CO2

evolution of soil were selected as tests for the toxicity, comparing the results with

toxicity of non-treated soil. Nine fungi proved to be tolerant to and capable to grow on

chlorophenols and dioxins containing material, two of which were isolated from saw

mill area and identified as G. penetrans and T. ochracea. These fungi were also able to

compete with the native microflora of the wood chips used.

Keywords. White-Rot Fungi ,Litter-Decomposing Fungi, Chlorinated Phenols, Dioxins,

Wood Chips, Soil

- 113 -

Si- and N-modified lignocellulosics: biodegradation in phytocenotic conditions and effects on soil microorganisms

Galina Telysheva, Tatiana Dizhbite, Galina Lebedeva, Galina Rossinskaja,

Galina Dobele, Vizma Nikolaeva

LS Institute of Wood Chemistry, 27 Dzerbenes st., LV-1006, Riga, LATVIA Corresponding author e-mail: [email protected]

At present lignocellulosic waste from phytomass chemical/biological processing are

considered among expedient products for agriculture. Therefore, study of

transformation of these residues, which structures differ significantly from natural

phytocomplexes, is important for elucidation of their role in keeping of soil fertility and

for design of advanced lignocellulosic-based products intended for this purpose. In this

work the transformations of several industrial lignocellulosic residues and products of

their modifications with silicon or quaternary ammonium compounds in soil during two

timothy vegetation periods (3 and 15 months) were investigated applying analytical

pyrolysis, thermal analysis, ESR-, FTIR- and UV-spectroscopy. The data obtained show

that dynamic of lignin transformation is controlled by the structure of lignin matrix and

the presence of carbohydrate admixtures. Si-modified lignocellulosics differed with the

highest rate of mineralization and, simultaneously, condensation of residual structure. In

opposite, biotransformation of N-modified lignocellulosic was restrained due to

preferable biodeterioration of long aliphatic chains of N-modifier, particularly during

the first 3 months of vegetation. The effects of the lignocellulosic products on soil

microorganisms were studied in 1-7 days experiments in vitro on pure cultures of

bacteria Bacillus subtilis and Burkholderia cepacia, plant pathogenic fungi Botrytis

cinerea, Cladosporium herbarum, Fusarium oxysporum, Microdochium nivale, and

Rhizoctonia solani. In long-term vegetation experiments, the increase of number of

fungal colony-forming units in timothy and ryegrass rhizosphere soil was detected. It

was estimated that lignin products have greater beneficial influence on the K-strategists

than on the r-strategists. This study suggests that Si- and N-modified products designed

have good potentials for in situ soil bioremediation.

Keywords: Phytomass, Phytocomplexes, Analytical Pyrolysis, Soil Bioremediation

- 114 -

Proteolysis in the white rot fungi: how natural proteinase inhibitors and their target proteinases control laccase activity

J. Żuchowski, K. Grzywnowicz, M. Jaszek


White rot Basidiomycetes are mostly known for their activities able to decompose wood

constituents (also ligninocellulosis). These microorganisms have been found to produce

both intracellular and extracellular proteolytic enzymes through their growth and

development. The growth of wood-decaying fungi, especially under natural conditions,

requires nitrogen economy, involving regulation of proteolysis and modification of fungus-

own proteins through limited proteolysis (including postranslational maturation,

proteolytic changes during secretion and 'superactivation' in 'biochemical emergency'

states). The control of proteinases is normally achieved by regulated expression (and/or

secretion) and/or activation of their pro-forms, by degradation of excess of mature

enzymes, and by inhibition of their proteolytic activity by naturally occurring inhibitors of

proteases. Detailed study on natural proteinase inhibitors from the fungi is still limited.

Changes in activities of laccase are coordinated with changes of activities of serine and

aspartic proteinases and of levels of proteinase inhibitors of these enzymes, and of this

reason the control of laccase activity may be strongly ascribed to these proteinases and

their inhibitors. Our results suggest that naturally occurring proteinase inhibitors, together

with their target proteinases, are likely to play a role in the control of level of laccase, both

in activation of prolaccase, and in ‘superactivation’ and/or degradation of an enzyme

involved in biotransformation of lignin by the fungi. Although performed for laboratory

conditions, these approaches can be applicable in biotechnological processes involving

laccase and can also be useful in theoretical and laboratory models of regulation of laccase

activity.

Keywords: White Rot Basidiomycetes, Proteolytic Enzymes, Laccase, Serine Proteinases,

Aspartic Proteinases

- 115 -

Inhibition of biogas production by substituted phenolic compounds in enriched methanogenic cultures

J.E. Hernandez, R.T. Bachmann, R.G. Edyvean

Department of Chemical and Process Engineering, University of Sheffield,

Mappin Street, Sheffield, S1 3JD, UNITED KINGDOM Correspopnding auhtor e-mail: [email protected]

Phenolic compounds are persistent pollutants in the environment. They can be

biodegradable under specific anaerobic conditions. These compounds, at different

concentrations, cause inhibition of anaerobes’ activity. The aim of this study is to

examine the toxic effects of tyrosol (TY); gallic (GA), protocatechuic (PTC) and 4-

Hydroxybenzoic (HBA) acids on the biodegradation of an organic substrate by enriched

methanogenic bacteria. Undiluted and non adapted, anaerobic sludge (20 g/L to 40 g/L

of total dry solids) and degradable medium were incubated alone and simultaneously

with a range of mass concentration of each phenolic compound in sealed serum bottles

for 3 days at 35 ºC. The amount and concentration of biogas produced in the headspace

was determined daily. The percentage inhibition of biogas and EC50 were calculated.

The concentration of phenolic compound corresponding to every EC50 was observed to

rise up as follows: HBA< PTC < GA < TY. This confirms a relationship between

polarity and toxicity under anaerobic conditions. In addition, the structures with more

substitutions were less toxic on the biogas production. Moreover, considering an

observation from GC analysis, it could be established that these compounds affect the

disintegration, hydrolysis and acetogenesis of the macromolecules used as a substrate.

These results suggest that, in order to avoid a biogas reduction below 50%, the

concentration in the sludge of HBA, PTC, GA and TY have to be lower than: 1200,

1750, 2800, 1500 mg/L.

Keywords: Phenolic Compounds, Methanogenic Bacteria, Anaerobic Sludge, Biogas

- 116 -

Degradation of the polycyclic aromatic hydrocarbon, anthracene, by the endomycorrhizal association, chicory roots-Glomus intraradices

grown in vitro

A. Verdin, A. Lounès-Hadj Sahraoui, J. Fontaine, A. Grandmougin-Ferjani, R. Durand

Laboratoire de Mycologie/Phytopathologie/Environnement, Université du Littoral-Côte d’Opale,

17 avenue Blériot, BP 699, 62228 Calais Cedex, FRANCE Corresponding author e-mail: [email protected]

UNDER REVISION

- 117 -

The characteristic of nitrogen-fixing eubacteria transforming of azocompounds

Natalia D. Wackerow-Kouzova1, Monique Janvier2, Patrick A.D. Grimont2

1 Agrophysical Res. Inst., Gragdansky pr.14, 195220 Saint-Petersburg, RUSSIA 2 Unité Biodiversité des Bactéries Pathogènes Emergentes, Unité INSERM 389

Institut Pasteur, F-75724 Paris Cedex 15, FRANCE Corresponding author e-mail: [email protected]

The transformation and degradation of synthetic organic compounds introduced into

environment largely depend on microbial activity. Increasing interest in xenobiotic

transformation by dinitrogen-fixing bacteria connects with their efficiency of

bioremediation and phytoremediation. The heterotrophic aerobic bacteria were isolated

from soil treated by acaricide azobenzene. Systematic study has resulted in the

identification of gram-positive and gram-negative species. The accumulation of

azocompounds (azobenzene, methyl orange and chrisoidin) by bacterial colonies was

observed. The morphological and biochemical properties of selected strains, including

catalase, oxidase, urease, arginine dehydrolase, denitrifying and nitrogenase activities

are investigated. The genetic diversity of bacteria was determined by rrs gene sequence

analysis. Some strains grew most actively on solid medium with xenobiotic addition

(1%): soil strains - Bacillus megaterium SNK4, Paenibacillus polymyxa SNK2,

Pseudomonas putida NK9, strains, isolated from glass slides placed into soil -

Ochrobactrum intermedium ANKI, Azotobacter chroococcum ANKII. It was shown that

two strains A. chroococcum ANKII and P. polymyxa SNK2 have the genes nifD and

nifH involved in process of N2-fixation. Mo- and V- dependent nitrogen-fixing activity

using acetylene-ethylene method was detected. Both strains developed on solid media

with 2% azobenzenes, fluid oil or sim-triazines. Azobenzene transformation by

A.chroococcum ANKII and P.polymyxa SNK2 was observed in pure culture in co-

metabolic conditions without nitrogen. These species were not described previously as

being capable of azobenzene degradation. We assume, that allocated nitrogen-fixing

strains can be used for biotransformation of a wide spectrum of xenobiotics.

Keywords: Xenobiotic, Dinitrogen-Fixing Bacteria, Phytoremediation, Azocompound

- 118 -

Biodegradation of malathion to malathion mono-and dicarboxylic acids by a Bacillus thuringiensis strain

Mohamed A. Ibrahim1, Nashwa Fetyan2, Zienat K. Mohamed3,

Sherif M. El-Nagdy4

1 Agricultural Genetics Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, EGYPT

2 Water Research Institute, Agricultural Research Center (ARC), Giza, EGYPT 3 Botany Dept., Faculty of Science, Cairo University, Giza, EGYPT

4 Dept. of Genetics, University of Cambridge, Downing street, CB2 3EH, Cambridge, UK Corresponding author e-mail: [email protected]

Biodegradation of malathion and its utilization as a sole carbon source was studied in

liquid salt media. Five bacterial isolates obtained from agricultural wastewater near

Berket El-sabaa Lake were investigated. These isolates were identified and compared

using biochemical and molecular methods, respectively. Our results revealed that four

isolates (MOS-1, MOS-2, MOS-3 and MOS-4) were indistinguishable but the fifth

isolate, MOS-5, was different. Isolate MOS-5 was identified as Bacillus thuringiensis

and selected for further analysis. The addition of MOS-5 to minimal salt media supplied

with 250 mg l-1 malathion resulted in 99.32% reduction of malathion after 30 days. The

ability of MOS-5 to degrade malathion was also investigated in the presence of glucose

and yeast extract. Addition of glucose (5 gm l-1) resulted in 104 fold increase in cfu/ml

after 12 days. Moreover, addition of yeast extract (0.5 gm l-1) resulted in 105 fold

increase compared to 1.4 x 103 in the presence of malathion alone. Results of HPLC,

gas chromatography/mass spectrometry (GC/MS) and infrared spectroscopic analysis

revealed that malathion was converted to mal-monocarboxylic acid after three days.

Two additional malathion derivatives, mal-dicarboxylic acid and unidentified mal-x

were detected after 7 days. Esterase activity involved in malathion degradation was also

determined in culture filtrate. Results indicated that esterase activity was increased two

folds in the presence of yeast extract compared to glucose. These results indicate that

strains belonging to Bt may consider as highly potential candidates to be used in the

clean up of organophosphorus contaminated environment.

Keywords: Malathion, Agricultural Wastewater, Bacillus thuringiensis, HPLC, Gas

Chromatography/Mass Spectrometry (GC/MS), Infrared Spectroscopic Analysis

- 119 -

Biodegradation of cyanide by white rot fungi

Ahmet Çabuk1, Arzu Taşpinar Ünal2, Nazif Kolankaya3

1 Osmangazi University Science & Art Faculty Department of Biology, 26480, Meşelik Campus, Eskişehir/ TURKEY

2 Republic of Turkey Ministry of Agriculture and Rural Affairs, General Directorate of Agriculture Research, Field Crops Research Department, Bağdat Street 38, 06171 Yenimahalle / Ankara / TURKEY

3 Hacettepe University, Science Faculty Department of Biology, Division of Biotechnology, 06532, Beytepe Campus, Ankara/TURKEY


Cyanide is released to the environment as a result of industrial activities natural

processes. Cyanide has been defined as a hazardous substance and toxic pollutant

according to EPA (Environmental Protection Agency). In nature, some microorganisms

are responsible for the degradation of cyanide, but there is limited information about the

degradation characteristics of Basidiomycetes for cyanide.

The aim of the present study was the optimization of cyanide degradation in

some Basidiomycetes strains including Trametes (Coriolus) versicolor (ATCC 200801),

Phanerochaete chhrysosporium (ME 446) and Pleurotus sajor-caju. The degradation

activity of Trametes versicolor (ATCC 200801) was found to be more than that of the

other fungi examined. The parameters including incubation time, amount of biomass,

initial cyanide concentration, temperature, pH and agitation rate were tested for

Trametes versicolor (ATCC 200801). The maximum cyanide degradation was obtained

after 42 hours of incubation at 30 ºC. The optimum pH and agitation rate was measured

as 10.5 and 150 rm-1, respectively. The biomass was found as 3.5g for the maximum

degradation with an initial cyanide concentration of 125 mg l-1.

We propose that, according to its ability to degrade cyanide, Trametes versicolor

(ATCC 200801) might be effective in the treatment of cyanide contaminated sites.

Acknowledgements: The study was supported by Osmangazi University Scientific

Research Projects Committee (Project number: 200419035). A. Çabuk thanks

TÜBİTAK-BAYG, the Scientific and Technical Research Council of Turkey-Directorate

of Human Resources Development, for grant.

Keywords: Cyanide, Basidiomycetes, Trametes versicolor, Phanerochaete

chhrysosporium, Pleurotus sajor-caju

- 120 -

Biodegradation of polycyclic aromatic hydrocarbons by a white rot fungus Irpex lacteus and involvement of ligninolytic system in

degradation

T. Cajthaml1, P. Erbanová1, P. Baborová2, Č. Novotný1, V. Šašek1

1 Department of Ecology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, CZECH REPUBLIC

2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-128 40 Prague 2, CZECH REPUBLIC Corresponding author e-mail: [email protected]

Polycyclic aromatic hydrocarbons (PAHs) and some of their derivatives are a class of

serious environmental pollutants. One way of removing PAHs from the environment is

bioremediation using different microorganisms. Microbial degradation by ligninolytic

fungi has been intensively studied during the past few years, however, the information

available about fungal degradation products is limited and there is still not a clear

connection between the activity of ligninolytic enzymes and the extent of degradation.

The following investigations into the fungal degradation of 8 (3-5 rings)

different representatives of PAHs by Irpex lacteus contributed to understanding the

whole process of degradation. The fungus has not yet been studied from this aspect,

although its ability to degrade PAHs has already been demonstrated. The fungus was

cultivated stationary in several liquid nutrient media with presence of various

combinations of PAHs. Activities of ligninolytic enzymes were determined applying

standard methods and intermediates were analyzed using gas chromatography coupled

with mass detection.New intermediates were detected that were formed by opening of

aromatic rings of PAHs and in some cases by the partial removal of aromatic rings. We

observed an induction and an inhibition effect on the activity of ligninolytic enzymes by

PAHs and also influence of degradation rates of PAHs according to the type of PAH

mixture. Correlation of the enzyme activities with degradation, probable mechanisms

and the involvement of the enzymes in the degradation will be discussed.

Acknowledgement: The research was supported by grant No. KJB6020308 of the

Grant Agency of Academy of Sciences of the Czech Republic.

Keywords: Polycyclic Aromatic Hydrocarbons (PAHs), Ligninolytic Fungi, Irpex

lacteus, Gas Chromatography

- 121 -

Agrochemical pesticides: biodegradation and bioremediation of the formulations and active ingredients

Russell J Grant1, W Bernard Betts2

1 Department of Industrial Microbiology, University College Dublin, Belfield, Dublin 4, IRELAND

2 Hillview, Back Road, Newton-upon-Derwent, York YO41 4DD, UK Corresponding author e-mail. [email protected])

The biodegradation of agrochemical pesticides and the bioremediation of contaminated

sites is becoming increasingly important as chemical complexity and legal regulation

increases. Agrochemical pesticides include insecticides, herbicides and fungicides as the

main groups, being used in both veterinary and botanical/arable applications. The fates

of these chemicals have been well studied, but almost entirely at the active ingredient

(AI) level and not at the formulation level. The formulations themselves may be

relatively non-toxic compared to the AI but they do increase the solubility, stability and

resistance to degradation of the AI.

The various ‘biodegradation’ termed mechanisms and results, such as those of

(but not exclusively) biotransformation, biomineralisation and biodetoxification of

agrochemical pesticides are examined at both the AI and formulation level, as is the

bioremediation of environments contaminated with these compounds. Biological and

biologically based controls in the agrochemical sector are not considered here.

The study shows many mechanisms of biodegradation of agrochemical

pesticides, resulting in detoxification, and the bioremediation of contaminated

environments. However there appears to be a lack of research at the formulation level,

although this would potentially require enormous volumes of research over AI studies,

due to the individual nature of the formulations.

Keywords: Agrochemical Pesticides, Active Ingredient (AI), Biotransformation,

Biomineralisation, Biodetoxification

- 122 -

Adsorption and biodegradation of phenanthrene in activated carbon permeable barrier, a column study

Leglize P. 1, Saada A. 2, Berthelin J. 1, Leyval C. 1

1 CNRS/LIMOS - Laboratoire des Interactions Microorganismes-Minéraux-Matière Organique dans les Sols FRE 2440, 17 Rue Notre Dame des Pauvres, BP 5, 54501 Vandoeuvre-Lès-Nancy cedex, FRANCE 2 BRGM - Environnement & Procédés - 3, av. C. Guillemin, BP 6009, 45060 Orléans cedex 2, FRANCE


Permeable Reactive Barrier (PRB) is a new cost effective technology for the

remediation of contaminated groundwater. PRBs have been rarely considered for PAH

contamination, and for combining sorption and biodegradation. Column experiments

were performed with activated carbon (AC) percolated with phenanthrene (PHE) and a

bacterial suspension. A 10 cm-high column packed with AC (0.85 – 1.1 mm) was

percolated with a PHE solution (1 mg.l-1) for 110 days. A PAH-degrading bacterial

inoculum (Burkholderia sp.) was added (6.109 cells) either at the beginning of the

experiment (before PHE percolation) or after 75 days. PHE concentration and bacterial

number in the outlet solution, and in the column at the end of the experiment were

estimated. Outlet PHE concentration in non-inoculated column was less than 10 µg.l-1

during the first 25 days and column saturation was reached within 75 days with an

outlet concentration of 600 µg.l-1. Bacterial inoculation of the column after 75 days

significantly reduced outlet PHE concentration (300 µg.l-1). When bacteria were added

in the column before PHE, no PHE was recovered in the outlet solution (<10 µg.l-1).

However, bacterial growth reduced permeability within the column and could lead to

clogg the column. The results showed a high PHE sorption by AC together with an

efficient biodegradation within the column. Further experiments should be performed

with other PAH, mixtures of PAH and bacterial consortia.

This research programme was supported by ADEME (Centre d’Angers France).

Keywords: Permeable Reactive Barrier (PRB), Groundwater, Activated Carbon (AC),

Phenanthrene (PHE), Burkholderia sp

- 123 -

Biodegradation of hepatotoxic cyanobacterial compounds, microcystins, by aquatic bacteria in estuarine

and marine waters from southern Brazil

G.A.F. Lemes1, A. Matthiensen1, W.A. Colvara1, R. Kersanach2, J.S. Yunes1

1 Unidade de Pesquisas em Cianobactérias 2Laboratório de Crustáceos Decápodes, FURG,

Av. Itália, Km 08 – Campus Carreiros, 96201-900 Rio Grande RS, BRAZIL Corresponding autor e-mail: [email protected]

The hepatotoxic microcystin (MC) group has been widely studied regarding their

molecular structure (cyclo D-Ala1-X2-D-Me-Asp3-Z4-Adda5-D-Glu6-Me-dh-Ala7, where

X and Z are L-amino acids), occurrence and production by different cyanobacterial

genera (e.g. Microcystis, Anabaena, Planktothrix, Nostoc), toxicity, and lately their

genetic biosynthesis. However, few studies have been carried out regarding the

biodegradability of these compounds by biological action. A biodegradation/

biotransformation experiment, involving crude cellular extracts of an indigenous

Microcystis strain (RST9501, UPC Culture Collection, FURG, Brazil) producing [D-

Leu1]MC-LR as its main variant and commercially acquired purified MC-LR, was

performed using aquatic bacteria population from surface water of two sites from the

southernmost part of Brazil. Concentrations of the toxic extracts and the purified MCs

were spiked in natural water samples and monitored by liquid chromatography during

approximately two months. The initial toxin concentrations decreased to complete

biodegradation/biotransformation after an average time of 15 days. Four MC-degrading

bacteria strains were isolated from the end of the experiment and kept growing in

minimal salt medium plus purified MC-LR as the only source of C. The identification of

these strains was performed by PCR amplification and genetic sequencing of the 16S

rDNA. Antibodies raised against MC-LR and [D-Leu1]MC-LR in an enzyme-linked

immuno sorbent assay performed at zero time and after complete biodegradation and/or

biotransformation had occurred do not cross-reacted with the compounds eventually

formed which appeared as new peaks at the final HPLC chromatograms.

Keywords: Hepatotoxic Microcystin (MC), Cyanobacteria, Microcystis, Liquid

Chromatography, 16S rDNA

- 124 -

The chemical destruction of the explosive hexahydro 1,3,5-trinitro 1,3,5-triazine and the biological denitrification of the explosive residue

Dr Jane Nicklin-Faull1, Stephen Wilkinson2

1 Birkbeck College, UK

2 Oil Plus Ltd UK Corresponding author e-mail: [email protected]

There is a requirement for the disposal of bulk explosives and contaminated soil, other

than using incineration. Modern explosives are toxic, highly recalcitrant and xenobiotic.

In terms of volume, RDX is the most common military explosive in use today.

The biodegradation of RDX in aqueous solutions has been investigated over the

last 20 years. However, the low water solubility (42.3mg/L at 20oC) of RDX means

biodegradation is inappropriate for the disposal of bulk explosives. However, RDX is

produced by acid nitration of hexamine and thus vulnerable to hydrolysis in the

presence of alkali. To this end, alkali hydrolysis (AH) was investigated to solublise and

partially degrade RDX prior to further biological degradation.

RDX was hydrolysed using potassium hydroxide at 80ºC. After cooling, nitric

acid neutralised the hydrolysate, which contained high concentrations of nitrate and

carbon species. Under anoxic denitrifying conditions, a bacterial consortium was able to

mineralise these. The nitrate was used as a terminal electron acceptor in the assimilation

of carbon from RDX fragments, as formate, acetate and formaldehyde. The carbon

demand was supplemented with ethanol. Potassium accumulated in the porous pot

bioreactor, but did not inhibit denitrification.

The biological treatment protected downstream environments from

eutrophication and served as a biological indicator of process efficiency and completion.

The water produced could be used in a closed loop for the AH process. Combining of

processes overcomes the intrinsic problems of mineralisation of explosives, notably

their chemical stability and water insolubility.

Keywords: Explosives, Xenobiotic, RDX, Alkali Hydrolysis(AH)

- 125 -

Comparison of the adsorption capacity of root cultures of Cyperus

elegans and Cyperus hermaphroditus to phenanthrene and their phenoloxidases response

Angélica Ma. Rodríguez Dorantes1, Angélica Guerrero Zúñiga2

1 Lab. Fisiología Vegetal, Depto. Botánica, Escuela Nacional de Ciencias Biológicas,

I.P.N. Prol. Carpio y Plan de Ayala s/n, 11340 México City, MÉXICO

2 Environmental Protection Management Office, Program of Environmental Solutions, Instituto Mexicano del Petróleo, Eje Central Lázaro Cárdenas 152, Col. San Bartolo Atepehuacán, 07730 México City, MÉXICO Corresponding author e-mail: [email protected]

The biodegradation of polycyclic aromatic hydrocarbons can take place with the

participation of the radical system of plants. These compounds are lipophilic and

hydrophobic, and can be concentrate first into the roots; and once immobilized they can

be transformed by the enzymes present in the root surface. This study evaluated the

capacity of the in vitro root cultures of Cyperus elegans and Cyperus hermaphroditus,

for the adsorption of phenanthrene and the measure of their phenoloxidases activity as a

physiological response to the presence of the contaminant.

Plants of Cyperus elegans and Cyperus hermaphroditus of 60 days were

employed and the root cultures were established in vitro in 250-mL Erlenmeyer’s flasks,

with 100-mL of mineral medium. The phenanthrene was added to the cultures as: 40,

60, and 90 ppm, and incubated in darkness at 30ºC for 10 days. Residual phenanthrene

was quantified by U.V. spectroscopy and HPLC chromatography and the activity of the

radical surface peroxidases and laccases was measured by the guaiacol and cathecol

oxidation, respectively.

The results showed that the phenathrene removal and the radical enzymatic

activity were higher in the root cultures of C. elegans than C. hermaphroditus. Although

the removal of phenanthrene of these cultures wasn’t high, the induction of both

phenoloxidases was evident as the contaminant concentration increase. This suggests

that these radical systems would be possible sources of enzymes that can participate in

the transformation of pollutants.

Keywords: Polycyclic Aromatic Hydrocarbons, Phenanthrene, Phenoloxidases, U.V.

Spectroscopy, HPLC Chromatography

- 126 -

Dye decolorization capacity of nonagitated and agitated cultures of white rot fungi

K. Svobodová, P. Erbanová, E. Musílková, J. Sklenář, Č. Novotný

Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, CZECH REPUBLIC Corresponding author e mail: [email protected]

Azo dyes represent a group of recalcitrant synthetic dyes that are frequently used in

textile industry for dyeing fibers. Capacity of the activated sludge process to decolorize

these compounds is rather limited, on the other hand, white rot fungi have been found to

be quite efficient in this respect. In this study, Irpex lacteus, Dichomitus squalens,

Phanerochaete chrysosporium and Pleurotus ostreatus were compared for their capacity

to decolorize azo dyes in liquid cultures. Compared to nonagitated cultures, the agitated

ones showed a lower ability to decolorize azo dyes Reactive Orange 16 and Naphtol

Blue Black. Similar effect was observed with some structurally different dyes. A

significant increase in the decolorization of Reactive Orange 16 in agitated cultures of I.

lacteus was obtained after the addition of 0.1% Tween 80, following a higher

manganese-dependent peroxidase (MnP) production. In vitro decolorization of Reactive

Orange 16 by purified MnP from I. lacteus was measured. The effect of agitation during

growth on the decolorization capacity of fungal cultures will be discussed.

The research was supported by the following projects: IAA6020411, 1P04OC847.002,

AV0Z50200510.

Keywords: Azo Dyes, White Rot Fungi, Irpex lacteus, Dichomitus squalens,

Phanerochaete chrysosporium, Pleurotus ostreatus

- 127 -

Biodegradation of alkanes, aromatic compounds and oils using Venezuelan thermophilic bacteria

Jeff Wilkesman G.1; Francelys Gómez1; Rudolf Müller2

1 Departamento de Química, Facultad de Ciencias y Tecnología, Universidad de Carabobo, 3336, Valencia, VENEZUELA

2 Biotechnologie 2, Technische Universität Hamburg-Harburg, Denickestrasse 15, 21073 Hamburg, GERMANY Corresponding author e-mail: [email protected]

Microorganisms that grow optimally between 40 and 70°C are designated as

thermophiles. Thermophiles posses a substantial potential for the biodegradation of

environmental pollutants, including all major classes like alkanes, phenols, PAH,

chlorinated hydrocarbons, etc. Thermophilic organisms isolated from hot water springs

in Las Trincheras, Venezuela, were used to study the feasibility to degrade alkanes,

aromatic and chlorinated compounds, and oils. A bacterial strain was isolated from solid

medium enriched with starch, and liquid mineral medium containing the substrate to be

tested was inoculated. Bacterial growth was followed by measuring the absorbance at

600 nm and by cell counting. Growth was detected in medium containing olive oil or

β-naphthol, and in less extent, with p-Cl-1-naphtol. Alkanes tested in this study (C10,

C16, C20 and C32) were not degraded. In order to study the possible enzymes

responsible for oil and for β-naphthol degradation, zymographic techniques were

applied. However, the presence of lipoxygenases, triacylglycerol lipases and catechol

oxidases were not detected using this technique. The detection of putative lipases

secreted into the bacterial medium was detected using specific substrates like p-

nitrophenyl caprylate. Degradation of this substrate was measured at 410 nm and

enzymatic activity was quantified. At pH 6.1 and 25°C, 0.7 ± 0.3 µmol.of substrate were

hydrolyzed per min, per mL of supernatant. Further studies are concerned with a better

characterization of the enzymatic activity found. Biotechnological applications using

thermoresistant enzyme complexes isolated from thermophiles are envisaged in order to

eliminate or diminish xenobiotic compounds in the Venezuelan industrial fields.

Keywords: Thermophiles, Hot Water Springs, Alkanes, Aromatic Compounds,

Chlorinated Compounds, Zymographic Techniques

- 128 -

Degradation of the plasticizer di-n-butyl phthalate by a bacterium isolated from mangrove sediment

Xiang-Rong Xu a, Xiao-Yan Li b, Ji-Dong Gu a, c,*

a Laboratory of Environmental Toxicology, Department of Ecology & Biodiversity, The University of

Hong Kong, Pokfulam Road, Hong Kong , CHINA b Laboratory of Environmental Engineering, Department of Civil Engineering, The University of Hong

Kong, Pokfulam Road, Hong Kong, CHINA c The Swire Institute of Marine Science, The University of Hong Kong,

Shek O, Cape d’Aguilar, Hong Kong, CHINA * Corresponding author e-mail: [email protected]

Phthalate esters (PAEs) are a class of chemical compounds primarily used as plasticizers

to improve mechanical properties of the plastic resin, particularly flexibility. Even at

very low concentrations they are suspected of interfering with reproductive systems and

behavior of humans and wildlife through disturbance of the endocrine system. Aerobic

degradation of di-n-butyl phthalate (DBP) was examined using a bacterium isolated

from mangrove sediment. This bacterium capable of utilizing DBP as the sole carbon

and energy sources was identified as Pseudomonas fluorescens B-1. DBP

biodegradation by P. fluorescens B-1 conformed to the first-order kinetic model. Effects

of salinity and temperature on DBP degradation were examined, respectively. Rate of

DBP degradation was faster at 35 °C than 10 °C. Highest efficiency of DBP degradation

was obtained at salinity of 15 parts per thousand. Biodegradation of DBP was monitored

by solid-phase extraction using reversed-phase high-performance liquid

chromatography with ultra-violet detection. The major metabolites of DBP degradation

were identified as mono-n-butyl phthalate and phthalic acid by gas chromatography-

mass spectrometry. P. fluorescens B-1 was also tested in mangrove water and complete

degradation of DBP was achieved in 144 h at an initial concentration of 100 g µg l-1 and

the microorganism may be used in the treatment of DBP-contaminated environment,

such as marine and saline waters.

Keywords: Phthalate Esters (PAEs), Biodegradation, Di-n-Butyl Phthalate (DBP),

Seawater, Pseudomonas fluorescens, Reversed-Phase High-Performance Liquid

Chromatography

- 129 -

Degrability of N-heterocyclic aromatic compound 3-methylindole by Pseudomonas aeruginosa Gs

BoYin 1, Ji-Dong Gu 1,2,3,*

1 Surface Biology and Environmental Microbiology Group, Key Laboratory of Tropical Marine Environment Dynamics (LED), South China Sea Institute of Oceanography, Chinese Academy of

Sciences, 164 West Xingang Road, Guangzhou 510301, P.R. CHINA 2 Department of Ecology & Biodiversity; The University of Hong Kong,

Pokfulam Road, Hong Kong, P.R. CHINA 3 The Swire Institute of Marine Science, The University of Hong Kong, Cpae d’Aguilar,

Shek O, Hong Kong, P.R. CHINA *Corresponding author e-mail: [email protected]

Indolic compounds such as N-heterocyclic aromatics are produced by many industrial

processes such as oil refinery, creosote and pharmaceuticals. Because of their

heterocyclic structure, these chemicals are more soluble in water than their homocyclic

analogs and can be more easily transported into groundwater and in the environment. 3-

Methylindole (3MI), a N-heterocyclic aromatic compound also known as skatole, is

associated with animal waste and industrial processing. A pure culture of bacterium

capable of using 3-methylindole as the sole source of carbon and energy was isolated

from mangrove sediment using #MI as the sole source of carbon and energy, and

identified as Pseudomonas aeruginosa Gs based on 16S rRNA gene sequence.

Microbial degradation of 3MI was studied in batch culture experiments for several

factors including initial substrate concentration, pH and salinity. The optimum pH and

salinity was 7.0 and 5‰, respectively. Degradation of 3MI by P. aeruginosa Gs was

quantified on a reversed-phase High-performance Liquid Chromatography. Two

metabolites of 3MI degradation were detected and proposed to be indoline-3-carboxylic

acid and indoline-3-ol by HPLC/MS. Our results suggest that 3-methylindole can be

rapidly degraded by indigenous microorganisms of the mangrove environment.

Keywords: Indolic Compounds, 3-methylindole (3MI), Biodegradation, Pseudomonas

aeruginosa, Reversed-Phase High-Performance Liquid Chromatography, Biochemical

Pathway

- 130 -

Tetrachloroetylene removal using simultaneous electron aceptors in continuous systems

Paola Zárate-Segura; Jaime García-Mena; and Héctor M. Poggi-Varaldo.

The aim of the present work was to evaluate the removal of PCE under methanogenic

conditions (M) and simultaneous methanogenic-denitrifying conditions (M-D) of a

mesophilic fluidized biological bed reactor (FBBR) and two lab-scale, complete mix

reactor (CMR1 and CMR2). The CMRs and the FBBR were operated at loading rates of

0.066 and 1 g COD/L.d, hydraulic retention times of 15 and 1 day, respectively. Three

operation phases can be distinguished: (i) methanogenic with 40 mg/L PCE in the feed

for both the FBBR and CM2, whereas the feed of CM1 was kept at 20 mg/L, and (ii)

operation with to 40 mg/L PCE for the FBBR, CM2 and 20 mg/L PCE only for CM1,

all of them operated whit 18 g COD/g N-NO3-; (iii) reactors were operated in the same

conditions than the last period except by the loading ratio that was set at 9 g COD/g N-

NO3-. All reactors received methanol as carbon source in all periods.

In period (i), the FBBR and the CMR1 showed similar PCE and COD removals

(98.8, 98.6% PCE and 94.66, 92.49% COD, respectively), whereas the CMR2 whose

feed kept with 40 mg PCE/L exhibited similar PCE removal (98.62%) but a lower COD

removal (73.66%). During period (ii), COD reductions of reactors increased with

respect to those of period (i) whereas PCE removals were similar. The reactor

performances in period (iii) indicated no change in organic matter reduction, although

the values of several parameters of methanogenesis in the CM reactors drastically

dropped with respect to corresponding values in both periods (i).

Overall, it seems that the FBBR performed best in all three periods of operation.

The advantage of M-D conditions over M operation was related to a more thoroguh

removal of PCE metabolites rather than PCE removal itself.

Keywords: PCE, Methanogenic Conditions, Methanogenic-Denitrifying Conditions,

Fluidized Biological Bed Reactor (FBBR)

- 131 -

Soil microbial community changes under the influence of Polycyclic Aromatic Hydrocarbons (PAHs) pollution

M. Byss1, D. Elhottova2, J. Triska3

1 Faculty of Biological Sciences, University of South Bohemia, Laboratory of Analytical Chemistry,

Institute of Landscape Ecology, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, CZECH REPUBLIC

2 Institute of Soil Biology, Academy of Sciences of the Czech Republic, Na Sádkách 7, 370 05 České Budějovice, CZECH REPUBLIC

3 Institute of Landscape Ecology, Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, CZECH REPUBLIC


Soil samples were taken from the site of the former wood-preserving plant extensively

polluted with creosote oil, which consists mostly of polycyclic aromatic hydrocarbons

(PAHs) and their derivatives, especially oxygen, nitrogen and sulphur containing

heterocyclic polyaromatic compounds. PLFA (phospholipid fatty acid) analysis was

selected for the characterization of the soil microbial communities, able to survive the

pollution. Samples contaminated with PAHs required special analytical procedures

including GC-MS/MS method for removing the large background signal caused by the

presence of different compounds from creosote oil.

Polluted soil samples show significantly smaller amount and limited microbial

community pattern than the control samples taken from unpolluted area nearby the

wood-preserving plant, which shows natural PLFA pattern. Most polluted samples have

strongly reduced PLFA pattern without any presence of fungal microorganisms

(18:2ω6,9), also the stress indication ratio (18:1ω9t/18:1ω9c) show stressfull

environment. Contaminated soil is being delivered for further remediation process,

where significant loss of polycyclic aromatic hydrocarbon pollution was taking place,

however some of higher PAH compounds (indeno[1,2,3,-c,d]pyrene,

benzo[g,h,i]perylene etc.) remain recalcitrant. Gradual return of microbial community

pattern to the nature-close composition was observed during the remediation process.

Soil samples taken from the remediation-finished dumps show similar PLFA pattern to

the control sample, however the amount of fatty acids is still significantly lower.

Keywords: Polycyclic Aromatic Hydrocarbons (PAHs), Phospholipid Fatty Acid

(PLFA), GC-MS/MS, Soil

- 132 -

Chlorobenzoate catabolic enzymes from Achromobacter xylosoxidans A8

Zdeněk Chodoraa, Věra Jenčováa, Hynek Strnadb, Miluše Hroudováa, Martina Chmelíkováa, Václav Pačesb

a Department of Biochemistry and Microbiology, Institute of Chemical Technology in Prague,

Technicka 5, CZ-166 28 Prague, CZECH REPUBLIC b Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,

Flemingovo sq. 2, CZ-166 37 Prague, CZECH REPUBLIC Corresponding author e-mail: [email protected]

Pollution with chloroaromatic compounds is a serious problem. Several bacterial strains

that degrade chloroaromatics under aerobic or anaerobic conditions have been isolated

from contaminated sites. It was shown that some of these strains transform

chloroaromatics by specific dioxygenases to chlorocatechols under aerobic conditions.

Chlorocatechols are mainly catabolized to 3-oxoadipate by modified ortho-cleavage

pathway that is formed by four enzymes: chlorocatechol-1,2-dioxygenase,

chloromuconate cycloisomerase, dienelactone hydrolase and maleylacetate reductase.

Genes encoding these enzymes are usually localized on plasmids. The knowledge of

these pathways could be useful in targeted bioremediation of contaminated areas by

chloroaromatic compounds.

The Achromobacter xylosoxidans strain A8 was isolated in the Czech Republic

from PCB contaminated soil. It is able to use chlorobenzoates as sole sources of carbon

and energy. The genome of this strain contains two large conjugative plasmids, namely

the pA81 and pA82 plasmids. Genes of ortho-halobenzoate dioxygenase complex and

modified ortho-cleavage pathway were identified in the pA81 sequencing project.

Individual genes were cloned and expressed in Escherichia coli BL21(DE3). Subunits

of ortho-halobenzoate dioxygenase OhbA, OhbB, ferredoxin, ferredoxin reductase and

all four enzymes of modified ortho-metabolic pathway were purified. Their

characteristics as molecule weight, subunit composition and isoelectric point were

determined. Optimal reaction conditions (temperature, pH and ionic strength) were

defined and kinetic parameters were confirmed by reactions with suitable substrates.

Keywords: Chloroaromatic Compounds, Dioxygenases, Achromobacter xylosoxidans,

Plasmids, Escherichia coli

- 133 -

Biodegradation of the herbicide diuron by streptomycetes isolated from soil

1 Castillo M.A., 1 Felis N., 2 Aragón P., 1 Cuesta G., 1 Sabater, C.

1 Biotechnology Department, and 2 Chemistry Department,

Politechnic University of Valencia, Camino de Vera 14, 46022 Valencia, SPAIN Corresponding author e-mail: [email protected]

There is growing concern over the potential for contamination of soils, surface and

groundwater by herbicides. One of them, diuron (3-(3,4-dichlorophenyl)-1, 1-

dimethylurea) is employed widely for weed control in non-crop areas and also in a

range of tree crops. This herbicide is applied to soil, where it tends to accumulate

because of its low solubility in water. Microbial degradation is considered to be the

primary mechanism for its dissipation from soil.

The ability of actinomycetes to transform pesticides has not been widely

investigated. The metabolic diversity of actinomycetes and their particular growth

characteristics (invasive mycelial, rapid colonization of selective substrates, etc.) make

them attractive organisms for the bioremediation. The aim of the present study was to

investigate the biodegradation potential of Streptomycetes isolated from soil, capable of

degrading diuron.

Streptomycetes strains were identified by morphologic characteristics and PCR-

based assays. Biodegradation assays were carried out in flasks containing ISP-2

medium + diuron at 4 mg/L, incubated at 25 ºC on an orbital shaker for 15 days. At

regular intervals aliquots of culture were removed and herbicide concentrations were

determined by HPLC. The results obtained indicated that all strains tested were able to

degrade diuron with variable percentages. Twelve strains degraded herbicide above 50%

at the end of assay; five strains achieved degradation percentages of 50% after five days

incubation. The strain A79 was the most efficient organism in the degradation of diuron,

achieved 95% after five days incubation. Given the high degradation percentages

observed here, Streptomycetes strains present a good potential for bioremediation of

soils contaminated with diuron.

Keywords: Herbicides, Diuron, Actinomycetes, Streptomycetes, PCR, HPLC

- 134 -

Stress effect of chlorophenols on the cell membrane of indigenous and bioaugmented bacteria

Katarína Dercová, Milan Čertík, Zuzana Sejáková

Slovak University of Technology, Faculty of Chemical and Food Technology, Department of Biochemical Technology, Bratislava, SLOVAK REPUBLIC


UNDER REVISION

- 135 -

Molecular and biochemical characterization of 2,4-dinitrophenol and picric acid degradation by a novel Rhodococcus sp.

Anuradha Ghosh, Debarati Paul, Dhan Prakash, Abhineet Goyal, Rakesh K. Jain*

Institute of Microbial Technology, Sector-39A, Chandigarh-160036, INDIA

Corresponding author e-mail:

The widespread use of nitrophenols as intermediate chemicals of large-scale synthesis

of N-substituted aromatic compounds has led to considerable environmental problems.

2,4-Dinitrophenol and 2,4,6-Trinitrophenol (picric acid) are major component of

pesticidal chemicals and explosives. Till date, very few bacterial strains such as

Rhodococcus erythropolis, Nocardioides sp. have been reported to to be able to degrade

these compounds. In their degradation pathway hydride Meisenheimer complex was

found to be the only well-characterized intermediate. Therefore, it is important to

understand the biochemistry, genetics and regulation of degradation of 2,4-dinitrophenol

and picric acid in the environment and long-term remediation strategies should be

established. Present work mainly focuses on Rhodococcus sp. RKJ300, isolated from a

contaminated soil and able to utilize 2,4-dinitrophenol as sole source of carbon and

energy. Growth pattern, nitrite release assay, induction and inhibition studies were

performed to elucidate the nature of the degradation pathway(s). In order to identify the

intermediates of the pathway biochemical techniques such as thin layer chromatography

and gas chromatography were performed. Hydride Meisenheimer complexes of 2,4-

dinitrophenol and picric acid have been identified by high performance liquid

chromatography analysis of the resting cell studies performed with the strain. For

detailed structural analysis of the intermediates nuclear magnetic resonance technique is

being carried out. To study the molecular basis of the pathway few upper pathway genes

known as nitrophenol degrading genes (npdC, npdG and npdI) have been amplified

using degenerate primers. Southern hybridization using npdG as radiolabeled probe has

highlighted an EcoRI digested genomic DNA fragment of ~ 8 kb and attempts are being

made to clone and annotate the gene cluster involved in the degradation pathway.

Keywords: Nitrophenols, Aromatic Compounds, Rhodococcus sp, Thin Layer

Chromatography, Gas Chromatography, Meisenheimer Complexes

- 136 -

Biodegradation and detoxification of 3-chlorobenzoate: effect of additional carbon sources and pH

Gallego, A.1, Gemini, V.1, Rossen, A.1, Rossi, S.1, Fortunato, M. S.1, Tripodi, V.2,

Corach, D.2, Planes, E.3, Korol, S.1

1 Cátedra de Higiene y Sanidad, y 2 Servicio de Huellas Digitales Genéticas. Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. Junín 956, 1113, Buenos Aires, ARGENTINA

3 Instituto Nacional de Tecnología Industrial. Parque Tecnológico Miguelete. Buenos Aires. ARGENTINA


Chlorinated benzoic acids are common metabolites in the degradation of many

chlorinated environmental pollutants such as polychlorinated biphenyls and herbicides.

Bacteria that co-metabolically attack polychlorinated biphenyls accumulate

chlorobenzoates, therefore chlorobenzoate degraders are essential for the complete

aerobic mineralization of polychlorinated biphenyls.

In a previous research we reported the isolation of 3-chlorobenzoate indigenous

degrading bacteria. The purposes of this investigation were: a) to study the

biodegradation of 3-chlorobenzoate by the isolated bacteria in aerobic conditions, b) to

assess the effect of additional carbon sources (glucose, acetate) and pH on the

biodegradation process and c) to evaluate detoxification using a toxicity test. All batch

experiments were performed with a 2-litre microfermentor at 28ºC with agitation (200

rpm). Biodegradation was estimated by ultraviolet spectrophotometry, gas

chromatography, chemical oxigen demand (COD) and microbial growth. Bioassays of

toxicity using Vibrio fischeri were performed to evaluate detoxification. The strain was

identified as Pseudomonas putida on the basis of its 16S rDNA sequence. The strain

metabolizes 100 mg/L of 3-chlorobenzoate within 14 hours and can degrade up to 1000

mg/L of the compound. COD removal was 92.3%. The presence of additional carbon

sources did not alter the biodegradation process, however, the strain showed a marked

delay in the biodegradation of 3-chlorobenzoate at pH 5. Toxicity was not detected at

the end of the biodegradation process. We suggest that our study provides a wider

perspective on the improvement of water and soil quality through bioremediation.

Keywords: Polychlorinated Biphenyls, Chlorobenzoates, Bioremediation, Vibrio

fischer,i Pseudomonas putida

- 137 -

Electro-optical properties of microbial suspensions at biodegradation of organophosphorus aromatic nitro insecticides

O.I. Guliy a, O.V. Ignatov a, V.D. Bunin b, V.V. Ignatov a

a Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences,

13 Prospekt Entuziastov, Saratov 410015, RUSSIAN FEDERATION b Institute of Applied Microbiology, Obolensk, Moscow region, RUSSIAN FEDERATION

Corresponding author e-mail: [email protected]; [email protected]

There were investigated the microbial cells of some strains with preliminary metabolic

enzyme system of toxic compounds. These processes conduce to the redistribution of

the charges in the microbial cells and may be register by electro-optical (EO) methods.

Electro-optical analysis is based on the recording of changes in optical characteristics of

cell suspensions under the orienting effect of an electric field. Redistribution of the

charges in the microbial cells may be used for determination of substrates of enzyme

reaction and finally for determination of substrate concentration. We examined the

effect of cellular metabolism of the organophosphorus aromatic nitro insecticides

metaphos and sumithion on the electro-physical properties of Pseudomonas putida C-

11, P. putida BA-11, and Acinetobacter calcoaceticum A-122 suspensions. We used the

dependences of cell-suspension optical-density changes induced by electric-field

orientation on the orienting-field frequency in the range of 10–10 000 kHz. We

investigated the possibility of developing a novel biosensor method for determining the

organophosphorus aromatic nitro insecticides metaphos and sumithion by measuring

orientational spectra of cell suspensions during the cellular metabolism of these

insecticides

This work was supported in part by Russian Science Support Foundation, and CRDF

grant N REC-006, grant Y1-P-06-09 program «Basic research and high education».

Keywords: Electro-Optical (EO) Analysis, Organophosphorus Aromatic Nitro

Insecticides, Pseudomonas putida, Acinetobacter calcoaceticum, Biosensor

- 138 -

Aerobic degradation of 2,4-dinitrophenol by an indigenous strain of Rhodococcus sp.

Gemini, V.1, Gallego, A.1, Tripodi, V.2, Corach, D.2, Planes, E.3, Korol, S.1

1 Cátedra de Higiene y Sanidad y 2 Servicio de Huellas Digitales Genéticas,

Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires, Junín 956, 1113, Buenos Aires, ARGENTINA

3 Instituto Nacional de Tecnología Industrial. Parque Tecnológico Miguelete. Buenos Aires, ARGENTINA Corresponding author e-mail: [email protected]

2,4-Dinitrophenol (2,4-DNP) and related nitroaromatics are frequently used in industry

as intermediates in the production of pharmaceuticals, dyes, plastics, pesticides,

fungicides, explosives and industrial solvents. Therefore nitrophenolic compounds are

often found as contaminants in industrial effluents, soils and waters. 2,4-DNP, which is

toxic to animals and humans, has been placed on the U.S. Environmental Protection

Agency (US.EPA) list of priority pollutants. The aims of this investigation were: a) to

isolate and identify 2,4-DNP degrading bacteria, b) to study the aerobic biodegradation

of 2,4-DNP and c) to assess detoxification using a toxicity test.

Aerobic biodegradation assays were performed with a 2-litre microfermentor at

28ºC with agitation (200 rpm). Biodegradation was evaluated by spectrophotometry, gas

chromatography and microbial growth. A Vibrio fischeri toxicity test was used to assess

detoxification. We isolated an indigenous bacterial strain that uses 2,4-DNP as its sole

carbon, nitrogen and energy source under aerobic conditions. The strain was identified

as Rhodococcus sp. on the basis of its 16S rRNA gene sequence. Rhodococcus sp.

degrades 0.27 mM and 0.54 mM within 22 hours and 28 hours, respectively, and

releases the nitro groups from 2,4-DNP as nitrites. Nitrites were removed by the

addition of an anoxic stage after the biodegradation process. Toxicity was not detected

after combining both aerobic biodegradation and denitrification.

The employment of this indigenous bacteria strain allows to carry out treatment

processes and actions of bioremediation of environments polluted by 2,4-DNP.

Keywords: 2,4-Dinitrophenol (2,4-DNP), Biodegradation, Vibrio fischeri, Rhodococcus

sp

- 139 -

Screening of PAH -degrading microorganisms by means of free-flow three-step system

Kobzev E.N.1, Schkidchenko A.N.2, Ivanova E.S.3, Zhirkova N.A.1,

Kholodenko V.P.1, Chugunov V.A.1

1 State Research Center for Applied Microbiology, Obolensk, Moscow Region, 142279 RUSSIA 2 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences,

Prospect Nauki, 5, Puschino, Moscow Region, 142290 RUSSIA 3 PSU, Prospect Nauki, 3, Puschino, Moscow Region, 142290 RUSSIA

Corresponding uthor e-mail: [email protected]

Screening of active microorganisms with wide spectrum of utilization of PAH is one of

main tasks of bioremediation of soils polluted with PAH.

PAH-degrading microorganisms were isolated by means of model flow-free

system with bioreactors sequentially connected. The system was a model of flow-free

biofilters with a carrier for immobilization of microorganisms. Mineral medium ( ¾ of

volume;1.5 L) was poured into bioreactors and mixed with air flow being supplied

through separators. Cultivation was performed in chemostatic carbon-starvation

regimen for 720 h. Strains capable for utilization of diezel fuel, oil and mazut were used

in the work. In the 1st reactor, microorganisms were cultured in the presence of diezel

fuel. The reactor was intended to grow biomass. The 2nd one used oil as substrate, a

portion of which was utilized and another portion was supplied to the 3rd reactor. Since

the 3rd bioreactor accepted non-utilized oil only (containing mainly PAHs and heavy

fractions of oil), microorganisms, which were capable for utilization of PAHs and tar

components, had to prevail there.

It was shown that the number of heterotrophs was 8.1⋅107 CFU/ml and 2.0⋅107

CFU/ml in the 1st and 2nd bioreactors, respectively. The content of heterotrophs on the

carrier in the 3rd reactor was 9.9⋅108 CFU/g. Clones of perspective strains specific for

some PAHs were selected. 14 clones grew in medium with 1% naphthalene, 18 clones

grew in medium added with 1% phenanthrene and 16 clones grew in the presence of 1%

pyrene. Using the system, it is possible to screen active PAH-degrading strains.

This research was carried out in the frame of ISTC Project #2366.

Keywords: PAH, Bioreactor, Biofilters, Diezel Fuel, Mazut, Heterotrophs

- 140 -

Modeling of composition of artificial association of microorganisms degrading PAHs

Kobzev E.N.1, Schkidchenko A.N.2, Petrikevich S.B.2, Kholodenko V.P.1

1 State Research Center for Applied Microbiology, Obolensk, Moscow Region, 142279 RUSSIA 2 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences,

Prospect Nauki, 5, Puschino, Moscow Region, 142290 RUSSIA Corresponding author e-mail: [email protected]

In recent years, bioformulations comprising two or more strains are widely used for

bioremediation of environments polluted with mixed PAHs. Any individual strain may

appear low effective because of the lack of enzymes required to utilize a PAH mix. One

approach to solve the problem is use of several strains differing in spectra of utilizable

substrates.

Microbial associations were completed on the basis of dendrograms of strain

identity. The dendrograms were plotted by using a database of properties of strains

degrading oil. One can expect that strains closely located on the dendrograms have

similar parameters of their growth in medium containing hydrocarbons and are likely

carry similar sets of enzymes for hydrocarbon degradation. And vice versa strains

remotely located differ in sets of enzymes. Therefore, to make up any artificial

association effective against hydrocarbons, strains remotely located on the dendrogram

should be selected. To verify the model, we degrade oil as substrate containing mixed

PAHs by means of model microbial associations.

We selected three closely located and three remotely located strains. Three

closely located strains degraded oil on average by 12 % lower than three remotely

located strains did. It was also shown that it is rather problematic to make up active

associations, if only activity of strains is taken into account. At the same time, the

replacement of strains in association for more active ones did not always improved

activity of associations themselves.

This research was partially carried out in the frame of ISTC Project #2366.

Keywords: PAH, Oil, Bioremediation, Dendrograms, Microbial Associations

- 141 -

Behaviour of naphthalene degraders and conjugational transfer of catabolic plasmids in flow bioreactors

Akhmetov L.I.1, Ivanova E.S.1,2, Esikova T.Z.1, Shkidchenko A.N.1,2,

Filonov A.E.1,2, Boronin A.M.1,2

1 Institute of Biochemistry and Physiology of Microorganisms RAS, 142290 Prospekt Nauki 5, Pushchino, Moscow Region, RUSSIA

2 Pushchino State University, 142290 Prospekt Nauki 3, Pushchino, Moscow Region, RUSSIA Corresponding author e-mail: [email protected]

The goal of the research was to study kinetics of naphthalene degraders’ number and

possibility of catabolic plasmids conjugational transfer in non-sterile flow system for

600 hours. Experimental system included three combined in series, open, aerated

bioreactors with mineral medium. Being a sole carbon and energy source, diesel fuel

came to the 1st reactor and 20% oil emulsion came to the 2nd one. The 3rd one was the

final oxidizer. The 1st reactor was inoculated with four strains utilizing oil compounds,

including naphthalene degrader Pseudomonas putida КТ2442 harboring the labeled

catabolic naphthalene plasmid pNF142::TnMod-OTc. Indigenious degrader strains were

also presented in the system. During the experiment total number of oil degraders was

107-109 CFU/ml, number of introduced Rhodococcus erythropolis Sh5 and

Microbacterium liquefaciens being at the level 50-80% from the total one. P. putida

КТ2442 was disappearing from the system, especially in the 3rd reactor. In the

beginning of the experiment number of aboriginal naphthalene degraders was at the

level of КТ2442 but, in contrast, increased and reached 106-107 CFU/ml of medium to

the end of process (9-56% and >99% in the beginning and final of the experiment,

respectively). Conjugational transfer of the labeled catabolic naphthalene plasmid

pNF142::TnMod-OTc occurred from P. putida КТ2442 to aboriginal microorganisms

that could enhance degradative potential of soil microbial association.

This research was supported by the ISTC grant 2366, Russian Federal scientific and

Technical Program (state contract 43.073.1.1.2502), RFBR grant 03-04-49145, CRDF

grant RB2-2377-PU02

Keywords: Naphthalene, Plasmids, Bioreactor, Pseudomonas putida, Rhodococcus

erythropolis, Microbacterium liquefaciens

- 142 -

Screening of strains of glyphosate-resistant rhizospheric microorganisms

Ye.V. Kryuchkova, O.E. Makarov, A.A. Shcherbakov

Institute of Biochemistry and Physiology of Plants and Microorganisms RAS,

13 Prospekt Entuziastov, Saratov 410049, Russia Corresponding author e-mail: [email protected]

Glyphosate is a promising modern organophosphorus pesticide that is widely used in

agriculture for selective and nonselective weed control. Aminomethylphosphonic acid

has been identified as the major product of glyphosate degradation in soil. This acid is

characterized by the presence o a direct C-P bond, which makes it resistant to chemical

hydrolysis, thermal decomposition, and photolysis, determining its persistence in the

environment.

The aim of this worke was (1) to select plant-growth-promoting microorganisms

with the highest resistance to the commercial preparation Roundup (a 36% aqueous

glyphosate solution), (2) to determine the minimum inhibitory and maximum tolerable

glyphosate concentrations for the microorganisms, and (3) to assess the abilities of the

chosen strains to utilize glyphosate as a P source by degrading the C-P bond and to

produce the phytohormone indol-3-acetic acid in a glyphosate-polluted environment.

The obtained results can be used in the development of biological methods for

the restoration of polluted land.

Keywords: Glyphosate, Plant-Growth-Promoting Microorganisms, Phytohormone,

Polluted Land

- 143 -

Anaerobic microbial communities degrading aminoaromatic substances

Linkova I.V., Dyakonova A.T., Esakova A.A., Kotova I.B., Netrusov A.I.

Microbiology Department, Moscow University, Moscow, 119992,Lenin’s Hills, 1/12

Corresponding author e-mail:[email protected], [email protected]

Amino-substituted aromatics play an important role in the production of pesticides,

polymers, pharmaceutics and appear in wastes generated by the corresponding

industries. They are dangerous for the environment due to mutagenic and cancerogenic

effects. The most aromatics as well as aminoaromatics can be degraded in aerobic and

in anaerobic conditions. But, in oxidative environment, several aminoaromatics can

polymerize into such compounds, which makes biodegradation difficult or impossible.

Moreover, aerobic biodegradation can lead to the production of toxic intermediates.

Therefore, the biodegradation in anaerobic conditions can be more effective.

The objects of our investigations are anaerobic microbial communities from

different sources. Obtained communities were quite stable in various cultivation

conditions and temperatures without loss of the biodegradation activities. Based on

amount of microbial species determined, communities from waste-water sludges

adapted to aminoaromatic biodegradation for a long time were more “spezialised”

compared to ones from unadapted sludges from brewery waste-water treatment station.

The process of aminoaromatic’s mineralization consisted of several stages

independently of source of inoculum. Benzoate was first intermediate in the process of

biomineralisation followed by volatile fatty acids and biogas at the end of the process.

We also observed the succession in microbial species, depending on biodegradation

stages. Initial conditions of incubation (the quantity of inoculum, the type and

concentration of substrate, temperature, pH, light) and hydrodynamic conditions

significantly influenced component’s composition and architecture of anaerobic

microbial communities.

Keywords: Amino-Substituted Aromatics, Waste-Water Sludges, Biomineralization,

Biogas, Microbial Communities

- 144 -

Influence of cultural condition on bacterial cell response to heavy metal ions presence

Jiri Mikes, Jan Masak, Alena Cejkova, Martina Siglova, Vladimir Jirku.

Department of Fermentation Chemistry and Bioengineering, Faculty of Food and Biochemistry

Technology of ICT Prague, Technicka 5, Prague 6 - 166 28, CZECH REPUBLIC Corresponding author e-mail: [email protected]

This study tries to find suitable condition of cultivation parameters for the possibility to

combine the utilisation of organic pollutants (phenol, glycol, acetone, aniline) with

heavy metal ions (cadmium and nickel) accumulation into bacterial cellular

compartments and bacterial extracellular polymeric substances (EPS).

The bacterial strains (Pseudomonas, Comamonas, Rhodococcus, Variovorax and

Gordonia - obtained of own culture collection or isolated from contaminated areas and

identified by accredited microbiology laboratory) were investigated during monitored

cultivation on minimal medium containing glucose or sucrose, respectively above

mentioned organic pollutants. In the first part of this work, growth characteristic and

production of EPS were measured. These outputs represented the much important piece

of knowledge which was compared to results of experiments with heavy metal ions

presence. Considerable differences in effect of heavy metal ions on bacterial strains

response to presence of heavy metal ions in growth medium have been observed. It was

shown that content of cadmium (50 – 80 mg.l-1) can induce the production of EPS by

some bacterial strains. In addition to the considerable amount of these ions in EPS over

their content accumulated in cells, the influenced production of EPS may present in this

case the defence mechanism of cell against cadmium lethal effect.

Keywords: Organic Pollutants, Heavy Metal, Extracellular Polymeric Substances

(EPS), Pseudomonas, Comamonas, Rhodococcus, Variovorax, Gordonia

- 145 -

Isolation and characterization of unique bphEGF genes of the thermophilic PCB degrader Bacillus sp. JF8

Gouri Mukerjee-Dhar1, Hatta Takashi2, Kazuhide Kimbara3, Minoru Shimura1*

1 Environmental Biotechnology Laboratory, Railway Technical Research Institute,

Kokubunji, Tokyo 185-8540, JAPAN 2 Research Institute of Technology, Okayama University of Science,

401-1 Seki, Okayama 703-8232, JAPAN 3 Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki,

Okayama 710-0046, JAPAN *Corresponding author e-mail: [email protected]

Bacillus sp. JF8 is the first thermophilic biphenyl (BP), polychlorinated biphenyl (PCB)

and naphthalene degrader to be isolated and studied. We have cloned and characterized

some of the genes involved in the BP/PCB and naphthalene pathway, notably the first

thermostable Mn(II)-dependent extradiol dioxygenases of the biphenyl (the plasmid

borne BphC_JF8) and the naphthalene (the chromosomally encoded NahC_JF8)

pathways.

Biphenyl is usually metabolized through the 2-hydroxypenta-2,4-dienoate

(HPD) and benzoate metabolic pathways. Three genes located downstream of the

Mn(II)-dependent BphC_JF8 were cloned and sequenced. Based on homology analysis

they were designated as genes of the HPD pathway, ie. HPD hydratase (bphE_JF8), 4-

hydroxy-2-oxovalerate aldolase (bphF_JF8) and acetaldehyde dehydrogenase

(bphG_JF8). Phylogenetic trees resulting from a comparison of the deduced amino acid

sequences of these plasmid borne BphEGF_JF8, the chromosomally encoded HPD

proteins (NahLOM_JF8) associated with the Mn(II)-dependent NahC_JF8 with other

HPD pathway proteins in the databases show BphG_JF8 and NahO_JF8 clustering

together as do BphF_JF8 and NahM_JF8. BphE_JF8 clusters with Group II hydratases,

while NahL_JF8 does not appear to cluster with any of the groups. The codon usage

pattern of BphEGF_JF8 and NahLOM_JF8 were also found to be slightly different.

Expression vectors of BphEGF_JF8 and NahL_JF8 have been constructed to study and

characterize the first HPD pathway products to be cloned from a thermophile.

Keywords: Bacillus sp., Plasmid, Biphenyl (BP), Polychlorinated Biphenyl (PCB),

Naphthalene

- 146 -

Detection of mutagenicity of synthetic dyes after their biodegradation

K. Malachová1, Z. Pavlíčková1, Č. Novotný2

1 University of Ostrava; Chittussiho 10, 70010 Slezská Ostrava, CZECH REPUBLIC

2 Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, CZECH REPUBLIC


The purpose was to study the genotoxic effect of selected synthetic dyes and find out

whether during biodegradation with activated sludge bacteria and ligninolytic fungus

Irpex lacteus mutagenic products are formed. Ames test was used to monitor the

production of mutagenic compounds during biodegradation and was also applicable to

detect whether, in the case of compounds exhibiting a certain mutagenic effect, the

biodegradation process was able to significantly reduce their mutagenic potential.

Another purpose was to assess whether the results of mutagenicity tests could be used

for the evaluation of efficiency of the biodegradation processes and/or whether the

individual biodegradation processes could be compared using the results obtained and

optimized on this basis. The study showed that the Ames test bacterial detection system

could be used to monitor the production of mutagenic compounds during

biodegradation and was also applicable to detect whether, in the case of compounds

exhibiting a certain mutagenic effect, the biodegradation process could or could not

significantly reduce their mutagenic potential. The activated sludge treatment did not

have an adverse effect on the subsequent application of the ligninolytic fungus, used to

decolorize the residual dye present after the exposure to the activated sludge. Such a

two-step biodegradation process might be advantageous for bioremediation of textile

wastewaters containing high amounts of the organic material of plant fiber origin that is

effectively removed by activated sludge bacteria.

Keywords: Synthetic Dyes, Activated Sludge, Ligninolytic Fungus, Irpex lacteus,

Ames Test

- 147 -

Degradation and detoxification of chlorophenolics by immobilized laccase enzyme

Arzu Ünal (Taşpınar), N. Kolankaya

Hacettepe University, Science Faculty, Dept.of Biology,Division of Biotechnology

06532 Beytepe ,Ankara,TURKEY Corresponding author e-mail: [email protected]

In this paper results of investigation on the detoxification of some chlorinated phenolics,

4-chlorophenol, 2,4-chlorophenol and 2,4,6-trichlorophenol, due to dechlorinative

degradation by immobilized laccase enzyme is reported. Aim of the study was to

examine the possibility of using laccase, which is one well known polyphenoloxidase,

for bioremediation of aquatic habitats contaminated with chlorinated phenolics. Laccase

enzyme used in experiments was produced from Trametes versicolor ATCC 200801 and

adsorbed on alginate beads before treatment with chlorophenolics. Release of chlorine

ions and oxygen consumption were detected in reaction medium by electrodes mounted

on the specially designed bioreactor during treatment of the substrates with immobilized

laccase. Reaction products of laccase treatment were analyzed by GC/MS (Gas

Chromatography & Mass Spectroscopy). Microtoxicity test system, which is working

being based on measurement of inactivation rate of β-galactocidase enzyme by

chlorinated toxic substances ,was used to determine efficiency of detoxification.

According data obtained in experiments, rate of chlorine removal from all

chlorinated phenolics due to laccase treatment seemed to be closely associated with rate

of oxygen consumption. GC/MS analysis of samples taken from reaction medium has

indicated that the release of chlorine ions during enzyme treatment is a result of

degradative action of laccase on chlorophenolic substrates. On the other hand, treatment

of chlorophenolics with immobilized laccase has caused loose of their toxicity in

remarkable extent.

Results of the experiments suggest that immobilized laccase can effectively be

used for bioremediation of media contaminated toxic chlorinated chemicals.

Keywords: Chlorinated Phenolics, Laccase, Aquatic Habitats, Trametes versicolor,

Alginate Beads, GC/MS (Gas Chromatography & Mass Spectroscopy)

- 148 -

Micromycetes from a PCBs contaminated field in Northern Italy: preliminary investigation

A. M. Picco1, D. Rodino1, M. Rodolfi1, O. Del Barba2

1 Section of Mycology, DET, University of Pavia, Via S. Epifanio 14, 27100-I-Pavia, ITALY

2 Unità Operativa Risorse Naturali ARPA Lombardia, Via Restelli 3/1, 20124-I-Milano, ITALY Corresponding autor e-mail: [email protected]

Fungal bioremediation is a recent technique that uses fungi to re-equilibrate soils

contaminated by different pollutants. In the project “Elimination of PCBs from the food

chain through bioremediation of agricultural superficies”(LIFE 03 ENV/IT/000321-

FREEPCB), our unit analysed a field located in Bergamo province (Lombardy)

contaminated by PCBs. Samples were collected in seven points, each one with different

concentration of PCB, as ascertained by chemical analysis. In laboratory, direct method

was firstly used: soil was plated on TWA (tap water agar). Moreover an indirect method

was also used: a 1:5.000 soil dilution was prepared and plated on PDA (potato dextrose

agar). Plates were maintained at room temperature and observed under the

stereomicroscope to look for mycelia. When observed, fungi were isolated in pure

culture and identified. The following fungi were isolated: Aspergillus niger, Curvularia

lunata, Fusarium spp., Gliocladium sp., Gliocladium virens, Metarrhizium anisopliae,

Mucor sp., Myrothecium verrucaria, Paecilomyces lilacinus, Penicillium spp.,

Trichoderma sp., Trichoderma viride, 2 mycelia sterilia and one nematophagous fungus.

Among the isolated genera Aspergillus, Fusarium and Penicillium are very well

known as producers of useful and toxic secondary metabolites, such as antibiotics and

mycotoxins. Gliocladium sp., Metarrhizium anisopliae and Myrothecium verrucaria are

already used in biological control as they are active against many plant pathogens either

fungi or insects. The nematophagous fungus was isolated from the most contaminated

point and its correct identification is under way.

Work is currently in progress to test other soil samples and verify a possible role

of the fungal strains as bioremediation agents.

Keywords: PCB, Fungi, Biodegradation, Mycotoxins

- 149 -

Sorption behaviour of HCH isomers in natural soils

B. Rodríguez Garrido, F. Macías, C. Monterroso

Departamento de Edafoloxía e Química Agrícola, Facultade de Bioloxía, Universidade de Santiago de Compostela, 15782-Santiago de Compostela, SPAIN


Hexachlorocyclohexane (HCH) isomers are persistent organic contaminants present in

the environment. Retention of these compounds in the soil, the extent of which depends

on the type and quantity of colloids present, inhibits their biodegradation because their

availability to microorganisms is reduced.

Sorption of the four most common isomers of HCH (α-, β-, γ- and δ-HCH) was

investigated in the present study in three natural soils, each with markedly different

colloidal properties. For this, the adsorption isotherms were obtained from "batch"

experiments carried out by adding different concentrations of aqueous solutions of each

of the isomers to each of the soils. The adsorption isotherms fitted well to the

Freundlich model, from which the coefficients of heterogeneity and the adsorption

constants were obtained. The type and quantity of organic matter were the main factors

accounting for the intensity of adsorption of the four isomers, with binding to inorganic

colloids being less important. The results obtained have practical implications in

predicting the environmental behaviour of these contaminants, especially as regards

their mobility and bioavailability.

Keywords: Hexachlorocyclohexane (HCH), Soil, Sorption, Freundlich Model

- 150 -

Aerobic sludge acclimation to formaldehyde biodegradation

Rossen, Ariana, Duran, Jorge

Sustainable Technologies Experimental Laboratory, National Institute of Water, AU Ezeiza Canuelas, Tr Newbery, Km 1,6 CP (1804). Buenos Aires, Argentina


The acclimation and biodegradation of formaldehyde in aerobic sludge was studied.

Formaldehyde is widely spread in the environment as a result of natural

processes and mainly from different industrial sources. It is used as a disinfectant,

preservative in consumer products, such as food, cosmetics, and mainly as urea-

formaldehyde and melamine-formaldehyde resins. Formaldehyde is classified as a

probable human carcinogen by USEPA and NTP. However, research made by IARC,

OSHA and NIOSH categorized it as carcinogenic.

The aim of this research is to enhance sludge potential to biodegrade

formaldehyde in industrial wastewater. The effectiveness of co-substrates to optimise

the process as a biostimulation methodology was evaluated.

A mix of different wastewaters treatment plant sludge was formed which was

divided in three, one was kept as control sludge, the second was acclimated with a fixed

concentration of formaldehyde (25 mg/L) and the third was incubated with an

increasing concentration of formaldehyde of 25 mg/L until a maximum concentration of

1000 mg/L. The incubation proceeded in batch systems with a weekly formaldehyde

supply.

Keywords: Formaldehyde, Biodegradation, Aerobic Sludge, Industrial Wastewater,

Biostimulation

- 151 -

Biodegradability of phenanthrene in model systems

F. Russo, R. Scelza, M. A. Rao, G. Iamarino, L. Gianfreda

Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II, Via Università 100, 80055 Portici, Napoli, ITALY Corresponding author e-mail: [email protected]

The biodegradability of persistent organic pollutants (POPs) in soil may be strongly

influenced by their interactions with organic, inorganic and biological soil components.

A polluted soil may exhibit an intrinsic ability to resist pollution damage if a microbial

population degrading the pollutant, the actual bioavailability of the pollutant, and

optimal environmental conditions simultaneously occur. If foreign microorganisms are

inoculated into the system, their survival and degrading ability will depend on the

physical-chemical properties of inoculated soils and several conditions such as pollutant

concentration, nutrients availability, aging processes, competition and predation

phenomena by active indigenous microorganisms.

The biodegradability of phenanthrene (PHE) was studied in model systems

simulating processes naturally occurring in soil. In the first system, PHE degradation by

a selected PHE -degrading mixed bacterial culture was studied upon its interaction with

synthetic organo-mineral colloids, formed by abiotic polymerization of catechol, a

common intermediate in several PHE microbial pathways. In the second system, PHE

degradation was followed in an agricultural soil with or without amendment with

compost from RSU and in the absence or presence of the degrading bacterial culture.

A reduced biodegradation, ascribable to a restricted bioavailability, was

measured when PHE was involved in catechol polymers. By contrast, PHE was rapidly

transformed by the cells added to the soil and the compost favored the process. A slow,

but detectable, PHE transformation, still enhanced by compost, also occurred in the

pristine soil, thus confirming its natural attenuation capability.

Keywords: Soil, Biodegradation, Phenanthrene (PHE), Compost, Catechol

- 152 -

Propanil biodegradation by an isolated fungus from rice rhizosphere

Camila Ortiz Martinez, Célia Maria Maganhotto de Souza Silva

Embrapa Environment, Rodovia SP-340, km 127,5 69, 13820-000, Jaguariúna, SP.

Corresponding author e-mail: [email protected].

The herbicide propanil belongs to acilanilids class and it has been largely use for rice

culture (Orzya sativa L.). It suffers hydrolytic cleavage and produces a 3,4

dicloroaniline (3,4-DCA) and propionic acid. Its application has caused a large number

of serious environmental problems, and this study aimed to find the fate of this

herbicide in the environment in order to propose alternatives to reduce its negative

effects. For that, it was used a fungi strain that produced lignolitic enzymes in the

presence of such herbicide, and then it was selected the P3SAIF strain to study the

biodegradation process of propanil. The fungus was tested to evaluate its potential of

degradation in a salt medium enrichement with different concentrations of propanil, and

also with different concentrations of its metabolite 3-4 dicloroaniline (2,5; 5,0;10,0;

15,0; 25,0; and 50,0 µg ml) added with sucrose’s. Single colonies that grew in these

herbicide concentrations, or in similar concentrations of its metabolites, were purified

and evaluated to verify the ability of degrading the herbicide in Czapeck mineral salt

medium. Czapeck medium was composed with 2,5 and 25 µg ml of propanil. Gas

chromatography was used to determine propanil and 3-4 dicloroaniline concentrations.

The fungi growth was affected by higher sub doses (25 and 50 µg ml), and for all the

treatments the growth was constant until 14 days. The growth rate was higher in the

absence of sucrose’s starting with a 10µg ml dosage. The percentage of herbicide

degradation on a liquid medium supplemented with 25 µg ml, was 62,34% after 18 days

of incubation, and it was not detected the presence of 3,4 DCA after 28 days of

incubation.

Keywords: Propanil, Herbicide, Fungi, Lignolitic Enzymes, Gas Chromatography

- 153 -

Biodegradation of pesticides in aquatic systems

1 Sabater, C., 1 Carrasco, R., 2 Castillo, M. A., 2 Palero, N., 1 Carrasco, J.M.

1, Pesticide Laboratory and 2 Microbiology Laboratory. Department of Biotechnology. Polytechnic University of Valencia.

Camino de Vera, 14; 46022-Valencia, SPAIN Corresponding author e-mail: [email protected]

A variety of synthetic chemicals are present in the human environment and in the

environments supporting natural populations of animals and plants. A very important

factor for a risk assessment of environmental contamination by xenobiotics is their

degradability. For that, the ability of selected microbial populations to degrade several

xenobiotics were investigated. The degradation rate of these compounds in

microrganisms cultures was determined by analyses with gas and liquid

chromatography (GC and HPLC). Degradation of pesticides atrazine, bensulfuron-

methyl, chlorsulfuron, cinosulfuron and pyridaphenthyon, in the biodegradation assays

with reactors of activated sludges has been low, in all the cases. Nevertheless, the

sulfonylurea herbicides, bensulfuron-methyl, cinosulfuron and chlorsulfuron, were

degraded by Streptomyces griseolus CECT 3325, in pure cultures, being the percentage

of degradation of 97.4, 89 and 82.5 % at 10, 4 and 15 days after assays incubation,

respectively. Aspergillus niger DBVPG 8540, also degraded bensulfuron-methyl but

slower than S. griseolus. Likewise, different strains of Fusarium degraded the herbicide

molinate, between 85 and 100 %, in pure cultures, at 15 days after the start of the assay,

but the same strains did not degrade the herbicide atrazine. Alcaligenes eutrophus H850

has been very effective in PCBs degradation reducing the concentration of these

compounds between 64 and 100 %, to 7 days, in pure cultures, after assays incubation.

Keywords: Xenobiotics, Gas Chromatography (GC), Liquid Chromatography (HPLC),

Pesticides, Herbicidas, Activated Sludges, Streptomyces griseolus, Aspergillus niger,

Alcaligenes eutrophus

- 154 -

Exogenous isolation of incp-9 naphthalene biodegradation plasmids from soil samples

Sergei Sokolov1, Irina Kosheleva1, Nina Kovalenko1, Ekaterina Akatova1,

Newton Gomes2, Kornelia Smalla2

1 Institute of Biochemistry and Physiology of Microorganisms RAS,

Pushchino, pr. Nauki, 5 142290, RUSSIAN FEDERATION 2 Federal Biological Research Centre for Agriculture and Forestry,

Messeweg 11-12, D-38104 Braunschweig, GERMANY Corresponding author e-mail: [email protected]

To study a large number of different plasmids that could potentially exist in

Pseudomonas naphthalene-degrading strains the isolation of degrader microorganisms

and exogenous isolation of catabolic plasmids were performed. The presence of key

genes nahAc and nahH of the naphthalene catabolism of fluorescent Pseudomonas spp.

in total soil DNA samples detected by PCR technique indicates on the plasmid

localization of these genes. Exogenous isolation of IncP-9 plasmids was carried out

from soil samples contaminated with oil hydrocarbons and break fluid. IncP-9 plasmids

were isolated by biparental mating with soil bacteria as plasmids donors and

Pseudomonas putida KT2442 as recipient. There were selected 35 plasmid-bearing

transconjugant KT2442 strains, transconjugants were confirmed by REP-PCR

fingerprinting. 23 isolated plasmids contain genes for the whole pathway of naphthalene

catabolism to tricarbonic cycle intermediates through salicylate and catechol. 12

plasmids are similar in size to naphthalene degrading plasmids but they do not possess

functional genes for naphthalene biodegradation. Testing with oligonucleotide primers

for the IncP-9 genes revealed that all isolated 35 plasmids belong to this incompatibility

group. There were isolated from the same soil samples by direct plating method and by

enrichment culture four aboriginal fluorescent naphthalene-degrading strains. Catabolic

plasmids found in these strains were different by their restriction patterns and unlike

those of exogenously isolated plasmids. It has been shown that exogenously isolated

plasmids fall into IncP-9δ subgroup when plasmids found in aboriginal strains mostly

belong to IncP-9β subgroup.

This work was supported by INTAS, grant No 01-2383 and by ISTC, grant No 2366.

Keywords: Plasmid, Pseudomonas sp, Naphthalene, Soil, PCR

- 155 -

Rhizogradation of nonylphenol by poplar-associated microfungi

Girlanda Mariangela*, Segreto Rossana, Favero-Longo Sergio E., Pitet Marta, Siniscalco Consolata, Perotto Silvia

Dipartimento di Biologia vegetale, University of Torino and Centre of Excellence CEBIOVEM,

V.le Mattioli 25, 10125 Torino, ITALY *Corresponding author e-mail: [email protected]

Nonylphenol is a recalcitrant contaminant with endocrine disrupting activity, that

mainly occurs in the environment as a degradation product of nonylphenol ethoxylates,

which are used widely as non-ionic surfactants in many industrial applications. We

examined nonylphenol degradation by six microfungal species (Aspergillus flavus,

Cunninghamella elegans, Paecilomyces lilacinus, Penicillium canescens, P. restrictum,

P. pinophilum) isolated from an aged nonylphenol-contaminated industrial soil. Growth

and degradative capacities of these fungi were tested in liquid mineral medium amended

with nonylphenol (200 or 2000 ppm). Growth of all species was significantly stimulated

by the higher nonylphenol concentration, with the greater stimulation observed for

Paecilomyces lilacinus, Penicillium canescens and for the consortium of the six species.

Up to 50% nonylphenol degradation was observed for the microfungal consortium at

the higher pollutant concentration. Growth and degradative capacities of the

microfungal consortium, tested alone or in association with Populus nigra or Pisum

sativum, were also examined in greenhouse experiments (sterile sand substrate amended

with 2000 ppm nonylphenol and nonsterile soil from the industrial estate). After three

months, all fungal species had survived, with higher abundances when in association

with poplars. Nonylphenol concentration was significantly reduced (50-85%) in both

substrates. The highest degradation was observed with the poplar-fungi combination;

however, plants exhibited a significant decrease in biomass and chlorophyll content.

These findings suggest that rhizodegradation by plant-associated microfungi may be a

tool for bioremediation of nonylphenol-contaminated soil, although phytotoxicity of

degradation products needs further investigation.

Keywords: Nonylphenol, Recalcitrant, Surfactants, Microfungal Consortium,

Rhizodegradation

- 156 -

Isolation and characterization of a soil bacterium isolated from a Greek creosote polluted site: Biodegradation of PAHs by free and

entrapped cells

Kallimanis A.1, Karabika E.1, Dados A.1, Pramateftaki P.3, Perysinakis A.1, Pilidis G.2, Drainas C.1, Koukkou A.I1*

1 Sector of Organic Chemistry and Biochemistry, Department of Chemistry,

2 Department of Biological Application and Tecchnologies, University of Ioannina, 45110 Ioannina, GREECE

3 Genetics Laboratory, Department of Biology , University of Athens, Athens, GREECE *Corresponding author e-mail: [email protected]

A PAH degrading bacterial strain Spyr1, was isolated from a creosote polluted soil from

Epirus, Greece, using pyrene as sole carbon and energy source. Spyr1 was identified as

Mycobacterium sp. based on 16S rDNA sequence, G+C % content and biochemical

tests. The results of gas chromatography showed that strain Spyr1 could degrade also

anthracene, phenanthrene, fluoranthene and acenapthene. The biodegradation of PAHs

was monitored using free and entrapped cells. An alginate-starch mixture was selected

as cell entrapment material because of its low cost and starch hydrolysis by the

microorganisms in the soil, hence increasing degrading effectiveness. Gel beads

maintained their viability and degrading ability for at least one month. Pyrene

degradation by free and entrapped cells was complete within 12 days. The ability of

Spyr1 to degrade a mixture of PAHs was also tested in spiked soil. Phenanthrene was

degraded completely after 14 of incubation while pyrene and fluoranthene were

degraded completely after 23 days of incubation with free cells. Degradation conferred

with entrapped cells although was slower resulted in complete degradation of

phenanthrene in soil within 23 days whereas only 8% of the rest of the PAHs remained

untouched. Plasmid analysis of Spyr1 strain revealed the existence of three plasmids

with size ranging from 4 to 70 kb. Since many catabolic genes are located in

megaplasmids, the contribution of the large plasmid in PAH degradation should be

examined.

Keywords: PAH, Creosote Polluted Soil, Mycobacterium sp., 16S rDNA, Gas

Chromatography

- 157 -

Isolation and characterization of indigenous thermophilic bacteria active in natural attenuation of bio-hazardous petrochemical

pollutants

Magdy El-Said Mohamed1,2*, Mona Al-Dousary1, Riyad Hamzah1, Georg Fuchs2

1 Biotechnology Program, Arabian Gulf University, Manama, KINGDOM OF BAHRAIN

2 Mikrobiologie, Institut Biologie II, Universität Freiburg, Freiburg GERMANY *Corresponding author e-mail: [email protected]

Common petrochemical compounds such as homocyclic polyaromatic hydrocarbons

(PAHs) and heterocyclic NOS-polyaromatics (NOS-compounds) were used as the sole

carbon and energy source to enrich indigenous bacteria harboring catabolic ability to

degrade these compounds from petroleum-contamination soils from Kuwait. Chemical

analysis of the extracted soil materials revealed residual amounts of oil (< 5% w/w),

presumably of heavy oil fractions with elevated S-contents. Aerobic culturable

mesophilic PAHs- and NOS-compounds degraders (expressed as CFU g-1 soil) were

abundant in these soils, whereas their moderately thermophilic counter part constituted

only a minor fraction. Glucose stimulated the growth of mesophiles and drastically

suppressed the number of thermophiles. 16S rDNA gene was amplified by PCR from

nine of the purified thermophilic strains using primers specific for eubacteria.

Sequencing of 900bp of 16S rDNA and database homology search tentatively aligned

these isolates to low G+C Gram positive bacteria from the family Bacillaceae. Electron

microscopy characterization revealed endospore forming bacilli varying in size (from

0.6 µm × 2.5 µm to 0.9 µm × 5.0 µm) with well-structured cell walls. GC/MS analysis

revealed enhanced catabolic ability of the pure as well as the mixed cultures to degrade

all tested compounds. The metabolism of the offered substrates does not involve co-

metabolism since all pure cultures consumed the offered substrates completely.

Keywords: Polyaromatic Hydrocarbons (Pahs), NOS-Compounds, Indigenous Bacteria,

Petroleum-Contamination Soils, 16S rDNA, GC/MS Analysis

- 158 -

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond of Baikalsk pulp and paper mill

(Russia)

G. G. Nimatsyrenova1*, G. S. Dabalaeva1, G.-W. Kohring2, V.B. Batoev1, F. Giffhorn2

1- Baikal Institute of Nature Management SB RAS, Sakhyanova St., 6, Ulan-Ude, 670047, RUSSIA

2- Institute for Applied Microbiology, University of Saarbrücken, PO Box 151150, 66041 Saarbrücken, GERMANY


Chlorophenols are toxic organic pollutants for the aquatic environment originating from

anthropogenic and natural sources. In the Lake Baikal ecosystem (World Heritage Site

since 1996), chlorophenols are formed when reactive chlorine is used for pulp bleaching

in the Baikal pulp and paper mill (BPPM) located directly on the southern shore of the

lake. Chlorophenols have been included in the list of «particularly hazardous

compounds» for the Lake Baikal ecosystem that must not be found in the lake and its

basin. In this work, a pure culture of 2,4-dichlorophenol (2,4-DCP) degrading bacteria

was isolated from a natural enrichment culture that had been adapted to chlorophenols

in the aeration pond of BPPM. The bacteria were identified by analysis of 16s rDNA

intergenic region, obtained by amplification of genomic DNA using PCR with universal

primers. Comparative analysis of the 16s rDNA sequence (1545 bp) in the GenBank

database revealed that these bacteria are related to the genus Bacillus. Degradation of

2,4-DCP has been studied using this culture in the liquid medium under aerobic

conditions at initial concentrations from 20 µM to 560 µM 2,4-DCP. The 2,4-DCP

degradation rates by Bacillus sp. could be determined at concentrations up to 400 µM.

However, higher concentrations of 2,4-DCP (560 µM) were inhibitive to the cell

growth.

Keywords: Chlorophenols, Pulp Bleaching, Pulp And Paper Mill (BPPM), 16s rDNA,

Bacillus sp.

- 159 -

Microbiological aspects of environmental fate studies of carbofuran and carbosulfan in irrigated rice in the “low lands’ agroecosystem

Maria Laura Turino Mattos, José Francisco da Silva Martins

Embrapa Clima Temperado, BR 392, km 78, 96001-970, Pelotas, BRASIL


The irrigate rice culture occupies more than 900 thousand cultivated hectares in the

states of Rio Grande do Sul, Brazil. Pesticides like carbofuran and carbosulfan

contribute for the maintenance of good productivity of the “orizicolas” crops,

guaranteeing what is expected from a cropland with environmental savings. The

insecticides are used for Oryzophagus oryzae control in paddy-rice but its effects on the

paddy-field microflora, of this region, is unknown. To study the environmental fate of

this pesticides, several diverse methods and procedures were used. Microbiological

approaches range from applied to basic, laboratory to field, qualitative to quantitative,

and from low to high technology. In the area of biodegradation, teams scientists are

needed to develop predictive models for the behavior of pesticides in the soil

environment. From our perspectives, we have documented the existing status of the

microbiology of environmental fate studies with carbofuran and carbosulfan.

Verification of data from laboratory studies to the field environmental is needed and

essential to advance the undersatanging of environmental fate studies with carbofuran

and carbosulfan.

Keywords: Pesticides, Carbofuran, Carbosulfan, Soil, Biodegradation

- 160 -

Occurrence of nah-like catabolic genes in fluorescent Pseudomonas

strains

T. Y. Izmalkova, O. I. Sazonova, S. L. Sokolov, I. A., Kosheleva, A. M. Boronin.

Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, pr. Nauki 5, IBPM RAS, Moscow region, 142290, Russian Federation, RUSSIA


Fifty-two naphthalene degrading fluorescent Pseudomonas strains were isolated from

different coal tar- and oil-contaminated sites of Russia, Ukraine and Belarus. Twenty-

five strains are P. putida, twenty-five – P. fluorescens and two – P. aeruginosa. PCR

amplification, restriction and sequence analysis of internal fragments for the key gene

encoding large subunit of naphthalene 1,2-dioxygenase (nahAc) of nah1-operon and the

key gene encoding salicylate hydroxylase (nahG) of nah2-operon revealed three nahAc

subgroups and six subgroups of nahG genes. One subgroup of nahAc (A88) and two

subgroups of nahG (A88 and NKNS) are new. Occurrence of the nahAc and nahG gene

subgroups is different for fluorescent Pseudomonas. Most diversity of nahAc and nahG

genes occurs in P. fluorescens strains. All studied P. putida strains contain only C18

subgroup of nahAc and pDTG1 subgroup of nahG. Analysis of plasmid profiles in

studied Pseudomonas strains revealed the presence of plasmids in 36 strains. Twenty-

two plasmids were placed into IncP-9 group, while eight plasmids – into IncP-7 group.

Plasmid pBS2 contains fused replicons of IncP-9 and IncP-7. Most of the P-7 and P-9

catabolic plasmids irrespective of host strain carry C18 subgroup of nahAc and pDTG1

subgroup of nahG.

This work was supported by INTAS, grant No 01-2383 and by ISTC, grant No 2366.

Keywords: Naphthalene, Pseudomonas sp, PCR, Restriction Analysis, Sequence

Analysis Plasmid

- 161 -

Comparative study on textile dyes removal using biotic and abiotic agents

Wafaa M. Abd El-Rahim

Agricultural Microbiology Department, National Research Centre, Dokki, Cairo, Egypt *Corresponding author e-mail: [email protected]

Experiments were carried out to assess the removal of Direct and Reactive dyes using

biotic and abiotic agents. Removal of dyestuffs from aqueous solutions by different

adsorbing agents such as sugarcane bagasse, sawdust, rice straws and charcoal as well

as fungal biomass as bioagent was investigated. This study included seven fungal strains

known to have high capacity in removing the textile dyes. Our results indicated that

three fungal strains namely: Penicillium commune (20), Penicillium Freii (31),

Penicillium allii (37) were highly efficient in removing the Direct violet dye. The

percentages of dye removal by these fungi reached to 96, 64 and 65% after two hours

respectively. The results indicated that the use of rice straws for dye removal as

compared to bagasse and sawdust was the more efficient. This abiotic agent recorded

approximately removal of about 72 % from Direct violet dye color through 24 hours

from incubation time. This percentage was reduced to 27 % with Reactive dye. Similar

trends were recorded with biotic agents where no fast removal of Reactive dye was

found (after 2, 4, 24 and 48 hours). This paper deals with two low-cost, renewable

biosorbents methods for textile dye removal.

Keywords: Fungal Strains, Biotic Agent, Abiotic Agent, Sugarcane Bagasse, Sawdust,

Rice Strews, Textile Dye

- 162 -

Removal of Cr(VI) from aqueous solution using wine processing waste sludge

Cheng-Chung Liu1, Ming-Kuang Wang2, Yuan-Shen Li1, Yu-An Lin3

1 Department of Environmental Engineering, National Ilan University, Ilan, TAIWAN, 260 2 Department of Agricultural Chemistry, National Taiwan University, Taipei, TAIWAN, 106 3 Department of Animal Science Engineering, National Ilan University, Ilan, TAIWAN, 260


The origin of wine processing waste sludge (WPWS) was a deposit mixture, including

considerable quantities of activated sludge, from final settling basin of the wastewater

treatment plants. From earlier reported, removal of Cr(VI) from wastewater using

biosorbents merely were focused on the adsorption between sorbents and sorbates but

neglected the chemical reaction. The objective of this research was to determine the

effects of initial concentration of Cr(VI), pH, temperature and reaction time on the

interaction between WPWS and dichromate ions. The Cr removal was favored at low

pH as well as high temperature, and the Cr removal ratio was less than 25% when initial

Cr(VI) concentration ranged from 10 to 500 mg L-1. The equilibrium times increased

with increasing in initial concentration, acidity and temperature. The WPWS sorption

isotherms of Cr(VI) are only well described by the Langmuir sorption isotherm under

the conditions of low initial concentration and weak acidity. The zero point charge

(pHzpc) of WPWS determined by titration method was 6.0, and therefore the sorption of

dichromate ions increased with decrease in pH because of the protonation of carboxylic

groups on WPWS. In addition, the dichromate ions also might be reduced and

transformed into chromium ions and then sorbed on WPWS firm. Thus, the chromium

species sorbed on WPWS were examined using extended X-ray absorption fine

structure (EXAFS) technique.

Keywords: Wine Processing Waste Sludge (WPWS), Cr(VI), Biosorbents, Langmuir

Sorption Isotherm, Extended X-Ray Absorption Fine Structure (EXAFS)

- 163 -

Microbial reduction of toxic and radioactive elements at alkaline thermal conditions.

Z.B. Namsaraev,T.V. Khijniak

Winogradsky Institute of Microbiology of RAS,

7/2 Prospect 60-letiya Oktyabrya, Moscow 117312, RUSSIA Corresponding author e-mail: [email protected]

Microbial reduction and concomitant precipitation of various toxic and radioactive

elements are commonly used in remediation of various waste waters. Many neutrophilic

thermophilic and mesophilic alkaliphilic bacteria are used for these purposes, but little

is known about possibility of use of alkalithermophilic bacteria. Recently we isolated

and described a new species of alkalithermophilic bacteria – Anaerobranca

californiensis capable to reduction of different sulphur compounds, Fe(III), Mn(IV), Cr

(VI), As(V) and Se(VI). Microbial transformation of iron, manganese, chromium, and

selenium oxides under alkalithermophilic conditions was demonstrated first time.

Here we present a new data on reduction of technetium by this organism. The

reduction occurred on the media with tryptone peptone as energy source. Optimum

conditions for this process is 5–25 g l-1 NaCl, pH 9.0 – 9.5 and temperature of 58°C.

Technetium (99Tc; half-life, 2.1 × 105 years), a fission product of 235U, is a

problematic component of some wastes from the nuclear fuel cycle. In its most stable

form, the highly soluble pertechnetate ion (TcO4), the element is very mobile in the

environment. For these reasons treatment is highly desirable.

Keywords: Radioactive Elements, Alkalithermophilic Bacteria, Anaerobranca

californiensis, Technetium, Nuclear Fuel Cycle

- 164 -

Selective biosorption of Cd(II) and Cr(VI) by Pseudomonas sp. and Staphylococcus xylosus

M. Ziagova, G. Dimitriadis, M. Liakopoulou-Kyriakides*

Faculty of Chemical Engineering, Aristotle University of Thessaloniki, Thessaloniki, GREECE, 54124


Biosorption has been proved to be one of the most fast and effective methods in

removing of toxic metals from wastewaters. In a previous study we have shown the

removal of cadmium and chromium ions using the microbial species of Pseudomonas

and Staphylococcus xylosus. Here, we have examined the selectivity of these two

species when both Cd(II) and Cr(VI) ions are present in the same aqueous solution. A

series of experiments were conducted at standard 5mg/L chromium concentration by

varying the concentrations of cadmium from 10 to 500mg/L and vice versa. Preliminary

kinetic studies that carried out, showed that the system of the metals and the

microorganisms needs only 15min, to reach the equilibrium state. At 5mg/L chromium

and 10mg/L cadmium concentrations the removal of Cd(II) ions was 83 and 89% for

Pseudomonas sp. and Staphylococcus xylosus respectively, whereas that of Cr(VI) ions

remained at very low levels (not significant adsorption). At higher cadmium

concentrations its removal from the mixture was lower but still to high percentages 65%

and 45% at 300mg/L for Pseudomonas sp. and Staphylococcus xylosus respectively,

indicating the selectivity of both species for cadmium.

In the opposite case, where concentrations of chromium varied from 10 to

500mg/L and cadmium kept constant at 5mg/L, high chromium removal (82%) was

observed at 10mg/L with both species. The percentage of chromium uptake remained at

good levels (50%) at higher concentrations, whereas cadmium percentage remained

stable at 35%, showing that its adsorption does not depend on the presence of chromium

in the medium.

Keywords: Biosorption, Toxic Metal, Cd(II), Cr(VI), Pseudomonas sp., Staphylococcus

xylosus

- 165 -

Biodegradation of trichloroethylene and enhancement of biodegradation pathways by indigenous bacterial communities

Jillian Brown*1,2 Max Coleman3, Stephen Dooley4, Ken Killham2, Graeme Paton2

Andrew Singer1 and Ian P. Thompson1

1 CEH Oxford, Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, 2 University Of Aberdeen

3 Jet Propulsion Lab – Caltech 4 Atomic Weapons Establishment (AWE) Corresponding author e-mail: [email protected]

Activities at a site formerly used as a military site resulted in contamination of

groundwater with trichloroethylene (TCE).Quarterly monitoring of boreholes from the

site suggest that the polluted habitat is predominantly aerobic and therefore reliance

cannot be placed on reductive dechlorination by natural attenuation. Samples collected

from the site were used to assess total cell counts, bacterial community structure and

diversity, toxicity, isotopic signatures, chemical analysis and microcosm establishment.

TCE degradation experiments were conducted in the laboratory with glucose, fructose

and toluene as carbon source then substituted with TCE as the primary source using

Burkholderia cepacia G4, Pseudomonas putida F1, Ralstonia eutropha, Methylosinus

trichosporium OB3b, Dehalobacter restrictus and Dehalospirilium multivorans grown

in chloride free and minimal medium. Isotopic signatures will be correlated with aerobic

and anaerobic pathways and secondary plant metabolites added to stimulate degradation

Preliminary results suggest low bacterial abundance, multiple contaminants are present,

varying degrees of toxicity which is not entirely due to TCE, and broad functional

bacterial diversity. Isotopic fractionation also suggests that some amount of degradation

of TCE is occurring.

Keywords: Trichloroethylene (TCE), Biodegradation, Groundwater, Burkholderia

cepacia, Pseudomonas putida, Ralstonia eutropha, Methylosinus trichosporium,

Dehalobacter restrictus, Dehalospirilium multivorans

- 166 -

Study of a diesel degrading bioremediation system: a molecular approach

Lena Ciric1, 2, Jim Philp2, Mark J. Bailey1, Andrew S. Whiteley1

1 Biodiversity and Ecosystem Function, CEH Oxford, Mansfield Rd, Oxford, OX1 3SR 2 School of Life Sciences, Napier University, 10 Colinton Rd, Edinburgh, EH10 5DT


Bioaugmentation, the addition of degrading organisms in order to promote contaminant

breakdown, has been used as a means of bioremediation for decades. However, our

understanding of in-situ biodegradation systems is still limited. Here, the remediation of

diesel contaminated groundwater at a former industrial site was investigated to establish

whether bioaugmentation really is a viable strategy.

The initial objective was to profile the microbial community at the site using

molecular biology tools and eventually determine which organisms were performing the

degradation of diesel fuel constituents. The communities at the site have been profiled

over the course of eight months. During this time, three different treatments have been

applied: bioaugmentation, biostimulation and intrinsic bioremediation.

The results obtained indicate that degrading organisms are present at the site,

however the community added to the site has had little influence on the indigenous

population and no colonisation occurred. The conclusion is that other environmental

biotic and abiotc factors have had a more marked effect on the population present at the

site. It is, therefore, of crucial importance to take these environmental factors into

account when constructing a degrading community.

Keywords: Bioremediation, Bioaugmentation, Biostimulation, Diesel Contaminated

Groundwater, Microbial Community

- 167 -

Trichlorophenol and phenol removal under two simultaneous electron aceptors conditions in a fluidized bed biorreactors

Claudio Garibay-Orijel, Jaime García-Mena, Héctor M. Poggi-Varaldo

CINVESTAV-IPN, Dept. Biotechnology, P.O.Box 14-740 México, D.F., 07000, MEXICO


Chlorophenolic compounds (2,4,6trichlorophenol and phenol) were removed by using

Methanogenic and Methanogenic-Aerated conditions in a fluidized bed bioreactor

(FBBR) more than 550 days. The bioreactor was fed with a synthetic wastewater

containing 80 mg/L of TCP and 20 mg/L of Phe. Sucrose (1g COD /L) was used as

cosubstrate in the two conditions. The hydraulic residence time was kept constant at 1 d.

Both conditions showed similar TCP and Phe removal (99.9+%); nevertheless, in

Methanogenic regime, the accumulation of 4-chlorophenol (4CP) up to 16 mg/L and

phenol up to 4 mg/L was observed, whereas in Methanogenic-Aerated condition 4CP

and other intermediates were not detected.

The advantage of the Methanogenic-Aerated condition over the Methanogenic

regime in this study relates more to the thorough removal of less substituted

chlorophenols produced by the reductive dehalogenation of TCP rather than the removal

of the parent compound itself.

Keywords: Chlorophenolic Compounds, Methanogenic Conditions, Methanogenic-

Aerated Conditions, Fluidized Bed Bioreactor (FBBR), Wastewater

- 168 -

Pharmaceutical and cosmetical residues in the environment: Identification and remediation

Ester Heath1, Tina Kosjek1, Boris Kompare2

1 Jožef Stefan Institute, Department of Environmental Sciences, Jamova 39, 1000 Ljubljana. SLOVENIA

2 University of Ljubljana, Faculty of Civil and Geodetic Engineering., Institute of Sanitary Eng., Hajdrihova 28, 1001 Ljubljana, SLOVENIA Corresponding author e-mail: [email protected]

In this paper we investigate the possible impact on the ecosystems of the active

ingredients in pharmaceutical and personal care products (PPCPs). These molecules are

in their nature biologically active substances whose effects, once released into the

environment, remain unclear. Commonly used drugs and cosmetics have been detected

in drinking and surface waters. Due to their possible bioaccumulation in the food chain

and their chronic toxicity it is necessary to better understand their environmental fate.

Pharmacologically and cosmetic active substances involved in our study were

chosen according to their wide application in central Europe and to their suspected

toxicity and bioaccumulation (nonsteroidal anti-inflammatory drugs-NSAIDs, sunscreen

agents, antiseptics). For isolation and identification of the selected compounds GC-MS

procedures were developed. To determine the presence of PPCP in the Slovene aquatic

environment, different water samples (river, potable, well, lake, sea, swimming pool and

waste waters) were analyzed. The results showed that the average PPCP contamination

in Slovenian waters is comparable to published results for Central and Western Europe.

To understand the environmental fate of these substances we studied the biodegradation

paths in controlled conditions using laboratory scale bioreactor facilities. Degradation

experiments were made with spiked water samples of different concentrations of

pollutants (1µg/L-1ng/L) starting with activated biomass from the active wastewater

treatment plant. After 6 months of continuous operation, a steady removal of all

observed compounds (NSAID representatives) was achieved (up to 90%). In the future

other PPCP representatives will be studied and the reactor configuration will be

optimized.

Keywords: Pharmaceutical And Personal Care Products (Ppcps), Bioaccumulation, GC-

MS, Biodegradation Paths, Scale Bioreactor

- 169 -

Aerobic continuous nitrotoluene mixture degradations from simulated waste water

J. Paca1, J. Barta1, M. Halecky1, A. Kosteckova1, Z. Pronayova1, R. Bajpai2

1 Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology, Prague,

Technicka 5, 166 28, CZECH REPUBLIC 2 Department of Chemical Engineering, University of Missouri, Columbia, MO 65211, USA


Despite having microorganisms capable of degradation of nitrotoluenes and known

metabolic pathways for the degradation by several pure cultures, there is still a lack of

knowledge describing the degradation in a mixture of nitrotoluenes, especially in

continuously operated reactors. This study was focused on continuous biodegradations

of various nitrotoluene (NT) mixtures under controlled operating conditions, such as

excess oxygen, temperature, and pH. Biodegradation of mixtures of 4-NT, 3-NT, and 2-

NT was carried out in a packed bed reactor with defined biocatalyst composition. Two

different types of loading were tested: (a) an increasing inlet concentration of one

nitrotoluene in presence of the constant concentrations of the other two NTs, and (b)

increasing the loading of NT to the reactor by lowering the hydraulic retention time in

the reactor while keeping ratio of concentrations of NT constant. The results show that

the rate and efficiency of degradation of individual nitrotoluenes is strongly dependent

on the nitro group position on the benzene ring. Nevertheless, keeping strongly aerobic

conditions no incompletely oxidized dead-end intermediates were found in the outflow.

Analyzing NO3-, NO2

-, and NH4+ ions in the outlet water enabled us to predict the

metabolic pathways of the individual NTs. Changes in biofilm-composition and its

surface structure were also measured during long-term experiments lasting 8 months,

and these too will be reported in this presentation.

Acknowledgement: The work was financially supported by the Czech Science

Foundation, Joint Project 104/04/0686 and Joint Project 104/03/0407.

Keywords: Nitrotoluene, Packed Bed Reactor, Biodegradation, Hydraulic Retention

Time, Benzene Ring, Biofilm

- 170 -

Biosorption of Cr(VI) and Cr(III) using silica gel immobilized Nostoc calcicola

S.Ramachandran a, MVRK Sarma a, T Coradin b, SK Verma a

a Center for Biotechnology, Biological Sciences Group, Birla Institute of Technology and Science,

Pilani, Rajasthan-333031, INDIA bChimie de la Matière Condensée, Université Pierre et Marie Curie,

4 place Jussieu, 75252 Paris, FRANCE Corresponding author e-mail: [email protected]

Chromium compounds are considered to be potential carcinogens, their use and release

into the environment is strictly regulated. The high cost and undesirable sludge

formation in conventional methods for chromium removal makes it imperative to look

for alternative technologies. Recently described biosorption technique utilizes the

biologically mediated metal biosorption process. The present investigation was aimed at

exploiting the cyanobacterium Nostoc calcicola for the removal of Cr(VI) and Cr(III).

The N. calcicola cells were immobilized on silica gel using sodium silicate and

colloidal silica. The silica-immobilized cells were dried, powdered and sieved to pass

20-40 mesh size, were used in the subsequent experiments. The nitrogen sorption

experiments showed the silica gel with very high surface area (200m2 g-1) with a pore

diameter of 100Å when compared to the conventional silica gel with a surface area of

5m2 g-1 and pore diameter of 250Å. The scanning electron microscopy revealed the

uniform macro porous structure of the silica gel. The optimum metal biosorption of

Cr(VI), Cr(III) by N.calcicola occurs at pH 2.0 Temp 25 ºC; and pH5.0 Temp 30 ºC,

respectively. The lyophilized N.calcicola cells could remove ca.160mg Cr(VI) g-1 of

cells within 12 hours of metal exposure, whereas the similar amount of Cr(III) could be

removed with a shorter span of 10 min by the same. The chromium biosorption follows

the Langmuir adsorption model. The silica-immobilized cells were found to be superior

in terms of metal removal, metal loading capacity and reusability in multiple cycles in

an up-flow columnar bioreactor than other biosorbents.

Keywords: Chromium Compounds, Biosorption, Nostoc Calcicola, Silica Gel,

Scanning Electron Microscopy, Langmuir Adsorption Model, Up-Flow Columnar

Bioreactor

- 171 -

Investigation of surface lipids of water plants used in bioremediation of contaminated waters

Shtemenko N.I., Shepelenko V.N.

Dnepropetrovsk National University, 49050 Dnepropetrovsk, 13 Naukoviy by-street,Ukraine


Surface lipids is a layer of lipid molecules (epicuticular lipids, surface waxes) the main

functions of which are protection of plants from UV-irradiation and formation of a

barrier for hydrophobic pollutants. Earlier we have shown an unusial composition of

surface lipids of some water plants studied as phytoremediants in constructed wetlands.

That concerned the length of hydrocarbon chain of dominating substances, unsaturation

of fatty acids, branch chained, cyclic and terpenoid compounds. We think that

degradation of some toxic organic pollutants in the rhizosphere of plants may be

followed by biochemical shifts in the surface lipids formation and changing of their

functions. IR- Spectra of some water plants were obtained and the area of absorbance of

carbonyl groups (1680 -1745 cm-1) was investigated. Some plants, as Coniferous, that

have not oxo-substances (aldehydes and ketones) in the surface lipids content, have two

bands of absorbance in this area. And some tropical plants, or wax-reach species, have a

significant quantity of oxo-substances and several bands. The investigated plants posess

a middle place, have three bands of absorbance and some quantity of oxo-substances.

Possible role of these components in the protective functions of plants is discussed.

Keywords: Surface Lipids, Rhizosphere, Phytoremediants, IR-Spectra, Oxo-Substances

- 172 -

Biodegradation of fluoxetine (Prozac) in riverwater using plant-derived chemicals as cometabolites

Andrew C Singer, Jana Helm, Xiaohong Yu, Ian P. Thompson

Centre for Ecology & Hydrology, Mansfield Road, Oxford OX1 3SR, UNITED KINGDOM


Fluoxetine hydrochloride, commercially known as Prozac®, is one of the world's most

widely prescribed antidepressants. It has recently been discovered as a potential

pollutant in riverine systems. There is no current understanding of the fate of fluoxetine

in the environment, nor are there any studies that indicate remediation strategies. Here

in this study we present a series of microcosm studies using riverwater, that aim to

demonstrate: (1) fluoxetine biodegradation by indigenous microorganisms; (2)

cometabolism of fluoxetine using plant-derived compounds; (3) the microbial ecology

of fluoxetine-degrading communities; (4) a mechanism for fluoxetine biodegradation.

A suite of 24 different potential plant-derived cometabolites, were screened for

their ability to stimulate fluoxetine biodegradation in coarsely filtered riverwater spiked

with 10 ppm fluoxetine. All cometabolites were provided at 20 ppm and re-introduced

at regular intervals over a 73 d incubation during which samples were analysed for

fluoxitine by HPLC. Subsamples from treatments demonstrating greater than 25%

fluoxitine loss over 58 d were transferred to fresh riverwater containing one of three

treatments: (1) 10 ppm fluoxitine; (2) 10 ppm fluoxitine and 20 ppm cometabolite; (3)

45 ppm fluoxitine. Fluoxetine was shown to degrade in six of the treatments; the

greatest degradation was achieved in 4-isopropylphenol amended mesocosms, where

90% had degraded in 73 d. No degradation was seen in fluoxetine-only or glucose

controls. Bacteria from mesocosms were isolated on R2A agar. Pure cultures were

tested for their ability to degrade fluoxetine and were subsequently identified by

sequencing 16s rRNA. Samples exhibiting fluoxetine degradation were subjected to

analysis by MaldiTOF, from which a degradation pathway was generated.

This work offers the first investigation into the fate of fluoxetine in a riverine-like

system and provides an ecologically-friendly solution for stimulating its cometabolism.

Keywords: Fluoxetine, Microcosm, Riverwater, Cometabolites, HPLC, MaldiTOF

- 173 -

Bioaugmentation and biological treatment of metal-working fluid wastewaters

Christopher van der Gast, Duane Agar, Ian Thompson

Environmental Biotechnology Section. NERC Centre for Ecology & Hydrology Oxford.

Mansfield Road, Oxford. OX1 3SR. UNITED KINGDOM Corresponding author e-mail: [email protected]

Metal-working fluids (MWF) are an essential component of heavy manufacturing

facilities (including automotive engine, transmission and stamping plants). Specifically,

they are used as coolants and lubricants for metal cutting, grinding, and drilling

operations. Once exhausted, MWF waste contributes to the vast majority of recalcitrant

and xenobiotic organic compounds in the wastewater produced by such manufacturing

plants. As EU legislation is tightened, producers of MWF wastewaters must now take

more responsibility for their effluent disposal. An environmentally and economically

viable solution is to biologically treat wastewater by bioaugmentation with bacterial

consortia comprised of strains originating from in-use MWF.

The main aims of this study where to 1) determine if a constructed bacterial

consortium could consistently reduce pollution load and toxicity of three different MWF

wastewaters under long term fixed film sequence (bench scale) batch reactor operation.

2) Investigate the fate of the bacterial consortium in the biofilm and suspension habitats

contained within the bioreactor system.

Over 219 days bioreactor operation, the bacterial consortium consistently

reduced pollution load (chemical oxygen demand) by 89.5 % ± 5.4 % per sequence

batch. Also, the resulting treated wastewaters were no longer toxic and pH was reduced

from approximately 9.5 to neutral. Using culture-independent techniques (denaturing

gradient gel electrophoresis and length heterogeneity PCR) revealed that the bacterial

consortium not only survived but was also dominant in the biofilm and suspension

habitats throughout the entire long-term bioreactor operation under different MWF

wastewater conditions.

Keywords: Metal-Working Fluids (MWF), Bioaugmentation, Bacterial Consortium,

Denaturing Gradient Gel Electrophoresis, Length Heterogeneity PCR

- 174 -

The biofiltration permeable reactive barrier – practical experience from synthesia

Lenka Vesela1, Jan Nemecek2, Martin Kubal3

1 DEKONTA, a.s., Prague Office, Volutova 2523, 158 00 Prague 5, CZECH REPUBLIC 2 ENACON, s.r.o., Marie Cibulkove 34/356, 140 21 Prague 4, CZECH REPUBLIC

3 Institute of Chemical Technology in Prague, Technicka 5, 166 28 Prague 6, CZECH REPUBLIC Corresponding author e-mail: [email protected]

The paper refers to utilization of biological elements within permeable reactive barriers.

The concept of a biofiltration permeable barrier has been tested in laboratory and in

pilot scale. Oxyhumolite (oxidised young lignite) was used as an absorption material

and a biofilm carrier.

During laboratory work it was found out that oxyhumolite adsorbs organic

pollutant at minimum but that it can be used for biofilm attachment. Laboratory batch

and column tests were used to study decomposition rates and mechanisms in order to

develop design parameters for pilot testing. An experimental barrier was built on

chemical factory premises contaminated by various organic pollutants (BTEX,

naphthalene, chlorobenzenes, phenols, nitro-derivatives, TPH). Before barrier

installation preliminary survey of unsaturated zone, hydrogeological investigation and

microbiological survey were performed. The barrier was designed as ‘trench-and-gate’;

it comprises a drainage trench, an underground bioreactor and recharge galleries. The

underground bioreactor has three chambers. The first one is equipped with an aeration

segment; the second and third chambers with a biofilter unit - one is filled with

oxyhumolite and the other with ‘keramzit’ (ceramic granulate).

Since January 2004 measurements of groundwater flux and retention time under

current hydrological conditions together with chemical and microbiological monitoring

has been carried out on the site. Up to now achieved results have showed high

effectiveness of organic contamination treatment. Average elimination degree varied

from 57.3 % (naphthalene) to 99.9 % (BTEX, nitro-derivatives); microbial density in

the bioreactor is sufficient; presently is approx. 105 to 106 CFU.ml-1.

Keywords: Biofiltration, Oxyhumolite, Biofilm, Bioreactor, Groundwater

- 175 -

Formulation of biologically treatable metalworking fluids

Helen Connolly 1,2, Chris van der Gast1, Doug Wylie3, Tom Stevenson2, Ian Thompson1

1 Environmental Biotechnology, Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR. (01865) 281650, UNITED KINGDOM

2 Cranfield University, Cranfield, Bedfordshire MK43 0AL, UNITED KINGDOM 3 Castrol Industrial, Wakefield House, Pipers Way, Swindon SN3 1RE, UNITED KINGDOM


Metalworking fluids (MWF) are used as coolants and lubricants for the manufacture of

metal components. Traditional MWF comprised of oil in water emulsions, but recently

synthetic MWF have been developed that contain a complex mixture of components

leading to enhanced fluid properties. Microbial contamination of in-use MWF is a

common problem, which leads to biodeterioration of the fluid emulsion and poorer

metal processing properties. Consequently, over 440000 tonnes of spent MWF are

produced annually in the UK. Waste fluids have a high pollution load, and cannot be

disposed of into conventional sewerage systems. Previous research in this area has

aimed to produce a bacterial consortium and bioreactor system to treat this waste,

leaving a fluid that can be safely disposed. This project aims to enhance the potential for

biodegradation, by investigating reformulation and pre-disposal treatment of a synthetic

metalworking fluid. The inherent bacterial diversity of a synthetic MWF was assessed

using FAME analysis, DGGE and LH-PCR. This study confirmed that samples taken

from sites worldwide were found to support convergent bacterial communities with

relative low diversity. Isolated bacteria were screened to determine the best degraders

and subsequently exploited to create a consortium capable of degrading the fluid. The

long-term degradation of the fluid was assessed in bioreactors, with recalcitrant

components identified using GC-MS, ICP and volumetric chemistry. Based on the

identification of recalcitrant components, alternative MWF formulations have been

suggested and will be tested to determine their biodegradability, with the aim of

producing a more biodegradable product without compromising performance.

Keywords: Metalworking Fluids (MWF), Bacterial Consortium, Bioreactor, DGGE,

LH-PCR, GC-MS, ICP, Volumetric Chemistry

- 176 -

Using biosorbents to remove pollutant metal ions from mine water drainage

S. Hartley1, N. Pearce1, W. Perkins1, E. Dinelli2, G. Priestman3, R. Edyvean3

1 Institute of Geography and Earth Sciences, Penglais Campus, The University of Wales,

Aberystwyth, SY23 3DB UK 2 Department of Earth and Geo-environmental Sciences, Bologna University,

Italy, I-40126, Bologna, ITALY 3 Department of Chemical and Process Engineering, The University of Sheffield, Sheffield S1 3JD UK


“BIOMAN” is an EU funded project (EU LIFE programme) to design and test a novel

biosorbent (seaweed waste) based system and apply this innovative technology in the

clean-up of mine water drainage.

Biosorption, the removal of dissolved metals from wastewaters by biomass, has

been demonstrated on a laboratory scale for a range of materials but there has been

almost no demonstration of scale-up to effective industrial systems. The biomasses

involved tend to be low cost, often waste materials and the potential of such systems is

very good. However, commercialisation requires demonstration to show a system

working on a large scale and thus attract industrial funding. The problems of scale-up

include aspects of engineering design, including monitoring and robustness, adequate

contacting and separation and ensuring ease of maintenance.

The BIOMAN project will build a large scale system using seaweed waste from

the alginate industry as the biosorbent, and to use this system in the treatment of

contaminated water draining from abandoned metal mines in Wales and Italy. The aim is

to demonstrating the clean-up of mine waters to EU Water Quality Directive (Good

Surface Water Chemical Status) for metals using a low cost, low maintenance and low

energy system.

This paper presents preliminary results from laboratory studies of the removal of

metals from real minewaters by the seaweed waste and from a pilot multi-stage system

working in the field at a minewater drainage site demonstrating the proof of concept.

Keywords: Biosorbent, Mine Water Drainage, Seaweed Waste, Metals

- 177 -

Textile dye decolourization by free and immobilized fungal materials

Anna Jarosz-Wilkołazka, Anna Olszewska

Department of Biochemistry, Maria Curie-Sklodowska University, Sklodowska Place 3, 20-031 Lublin, POLAND Corresponding author e-mail: [email protected]

Textile dyes are designed to resist fading upon exposure to sweat, light, water, oxidizing

agents and microbial attack. Therefore they are hardly removed from textile effluents by

conventional wastewater treatments. In recent years, there has been an intensive

research on wood-rotting fungal decolourization of dye wastewater and it is becoming a

promising alternative to replace or supplement present treatment processes. In fungal

dyes decolourization processes it is possible to use living biomass to biodegrade and

biosorb dyes or dead fungal biomass to adsorb dyes. In the case of living cells, the

major mechanism is biodegradation correlated with fungal ability to secrete

extracellular lignin-degrading enzymes such as lignin peroxidases, manganese

peroxidases or laccases. In addition to biodegradation, a biosorption mechanism might

also play an important role in the decolourization of dyes by living fungi.

The aim of the present work was to find a suitable technique as well as an

adequate material for the immobilization of selected strains of wood-rotting fungi.

These strains were previously shown to have potential in synthetic textile dye

biodegradation. Different carrier materials e.g. alginate beads, polyurethane foam, and

stainless steel sponge, were tested during decolourization experiments. We used both

simple dyes about different chemical structures and real raw coloured wastewater from

textile dye-producing plant. Real textile dye effluents contained not only dyes but also

chelating agents, precursors, by-products, and surfactants and above all salts, sometimes

at very high ionic strength and extreme pH values.

This work was partially supported by SOPHIED (NMP2-CT-2004-505899) and the

State Committee for Scientific Research (139/E-339/SPB/6. PR UE/DIE 450/2004-

2007).

Keywords: Textile Dyes, Wood-Rotting Fungi, Wastewater, Biodegradation,

Biosorption

- 178 -

Prevention of water deterioration in fish farms of Southern Asia (Consequences of tsunami)

Rashid A. Khaydarov, Renat R. Khaydarov

Institute of Nuclear Physics, 702132, Ulugbek, Tashkent, UZBEKISTAN Corresponding author e-mail: [email protected]

The after-effects of the tsunami have resulted in huge amounts of algae being washed to

the water surface of fish farms located in coast areas of South-East Asia. Usual methods

of water purification of fishponds (mechanical filter + UV treatment) was not successful

to supply fishponds with clean water.

To reduce risk of fish- and seafood-borne illnesses an additional method of water

treatment has been developed in the Institute of Nuclear Physics (Uzbekistan) and

successfully applied in certain fish farm of affected region of Malaysia. The method has

been based on (1) water disinfection using Electrolytically Generated Copper Ions with

following (2) water treatment by special sorbents having high selectivity and

distribution coefficient for Cu2+. First stage of treatment was performed in a small

special water pond constructed near fish farm, to which polluted fish farm water passed

through mechanical filter.

Experiments have shown that it was enough 50-minute electrolytically generated

copper ion disinfection for complete algae killing. Second stage of treatment provided

reducing copper concentrations in treated water down to 0,007 mg/L value. Experiments

have also demonstrated safety of low concentrations of dissolved copper to different

marine species of fish farm. After the second stage of treatment treated water passed

through standard UV-bulb device utilized in the fish farm before.

This year the developed method is expected to apply in several fish farms in the coast

regions of South-East Asia.

Keywords: Tsunami, Algae, Fishponds, Electrolytically Generated Copper Ions

- 179 -

Natural attenuation of chlorinated solvents by microbial habitat in groundwater in Yamagata prefecture

Yoshishige Kawabe1*, Keiko Ogawa1, Ariana Rossen2, Takeshi Komai1,

Yasuhide Sakamoto1

1 National Institute of Advanced Industrial Science and Technology (AIST), Institute for Geo-Resources and Environments, Geo-Environment Analysis and Evaluation Research

Group, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, JAPAN 2 Sustainable Technologies Experimental Laboratory National, Institute of Water AU. Ezeiza canuelas Tr.

Newbery Km 1.6 PC (1804) Pcia. Buenos Aires, ARGENTINA * Corresponding author e-mail:[email protected]

Natural Attenuation (NA) of chlorinated solvent such as tetrachloroethylene (PCE) and

trichloroethylene (TCE) in contaminated groundwater in the Yamagata Prefecture, Japan

was studied. The correlation between Natural Attenuation behavior and the microbial

habitat in groundwater was investigated in order to understand the mechanisms involved

in Natural Attenuation process and to forecast future conditions.

The chlorinated solvent degradation patterns were different among sampling

sites and were classified in three types: (A) degradation with by-products, (B)

degradation without by products and (C) non-degradation. The first one showed a lower

redox potential and dissolved oxygen, while a high concentration of heavy metals. This

anaerobic reducing condition is suitable for reductive bacteria. In site type A the number

of bacteria found was about 104-106 cells/ml; however in sites type B and C this number

was less than 103-104 cells/ml. Moreover, only in site (A) microbial activity with

degradative capacity was registered.

The most important intermediate in the degradation pathway of TCE and PCE is

the 1,2-cis-dicholoroethylene (cisDCE). Although in certain cases, vinyl chloride (VC)

or ethylene can be observed. In the site type (A) the higher degradation product was

cisDCE while VC and ethylene only appeared in a very low concentration. Those results

suggest that only a small proportion of bacteria have the ability to degrade cisDCE to

ethylene. To improve the biodegradation process toward the formation of ethylene the

effectiveness of different medium conditions were evaluated.

Keywords: Natural Attenuation (NA), Tetrachloroethylene (PCE), Trichloroethylene

(TCE), Groundwater, Microbial Habitat

- 180 -

Coriolopsis polyzona CBS 319.36 laccase oxidation of olive-mill wastewaters aromatic compounds

Lucas M., de la Rubia T., Martinez J.

Department of Microbiology, Faculty of Pharmacy, Campus Cartuja,

University of Granada, 18071 Granada, SPAIN E-mail:

White rot fungi (WRF) and their lignin modifying enzymes (LME) have been involved

in the bioremediation of olive mill wastewaters (OMW). We have found that the WRF

Coriolopsis polyzona was able to seriously decrease OMW toxicity measured as a

reduction in antibacterial activity. In OMW containing cultures laccase was dramatically

overproduced. In the semipurifed extracellular fluids from these cultures, laccase was

the only enzymatic activity detected at pIs lower than 2.80. Proteins with higher pI were

detected but have nor laccase nor peroxidase activities. The oxidation of OMW single

aromatic compounds by a commercial Trametes versicolor and by the semipurified C.

polyzona acidic laccases was compared. Fourteen single aromatic compounds were

selected because of their abundance and toxicity in OMW. Nine of the 14 compounds

were oxidized by both laccases after incubation for 5 hours at 30°C (pH 4.5) but the

oxidation of catechol and 3.4-dihydroxybenzoic acid was higher for C. polyzona

laccases. In addition C. polyzona laccases were more stable than T. versicolor laccase to

the inactivation observed in solutions containing 3,4,5-trimethoxybenzoic acid. The

above results suggest the importance of the C. polyzona acidic laccases induced in

OMW containing cultures for the bioremediation of these wastes

Acknowledgments: This work was founded through the Project 30C0198700 from the

European Commission.

Keywords: White Rot Fungi (WRF), Lignin Modifying Enzymes (LME), Olive Mill

Wastewaters (OMW), Coriolopsis polyzona, Trametes versicolor, Aromatic Compounds

- 181 -

Bioremediation of phenol polluted waters

G. Iamarino, R.Scelza, M. A. Rao, F. Russo, L. Gianfreda

Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II, Via Università 100, 80055 Portici, Napoli, Italy


In Mediterranean Countries, large quantities of Olive Mill Wastewaters (OMW)

annually derive from the olive extraction process. Waters are constituted mainly by

polyphenolic compounds, similar to lignine, but also by toxic monomeric phenols, thus

representing a serious environmental problem. An efficient OMW detoxification might

be achieved by using fungal or enzymatic activities, thus representing an useful

problem-solving strategy.

The efficiency of a laccase in the detoxification of either whole OMW or their

fractions, obtained from the Soxhlet extraction procedure, generally used for producing

valuable OMW components, was addressed. The procedure produces a phenolic extract

rich in lipophylic monomeric phenols and an exhausted fraction, rich of all the other

OMW components including polymeric phenols, amino acids, proteins and

carbohydrates. The residual phenol concentration, the toxicity toward Lycopersicum

esculentum and Lepidium sativum seeds germination and bacterial growth, referred to a

control lacking laccase, clearly indicate that the enzymatic treatment was more efficient

in reducing phenol content in crude OMW than in its phenolic extracts. A corresponding

decrease of phytotoxicity was observed whereas no decrease of bacterial toxicity was

observed. These results seem to suggest that either some OMW components improve or

enhance the activity of laccase or that some phenols, probably present in the phenolic

extract at higher concentrations, inhibit laccase activity. Moreover, they indicate that

other OMW components rather than monomeric phenols are responsible of OMW

toxicity.

Keywords: Olive Mill Wastewaters (OMW), Laccase, Lycopersicum esculentum,

Lepidium sativum, Polyphenolic Compounds, Monomeric Phenols

- 182 -

Olive oil waste as a biosorbent of heavy metals

G. Martinez-Garcia, Ceri J. Williams, Andrea Burgoyne, Robert G. J. Edyvean

Department of Chemical and Process Engineering, The University of Sheffield, S1 3JD Sheffield, UK Corresponding author e-mail: [email protected]

The sourcing of novel, inexpensive, bio-wastes, such as olive mill waste (OMW) from

the two-decanter olive oil production system, offers potential for the removal of metal

ion by biosorption. OMW can be used in repeated regeneration cycles for the adsorption

of heavy metals from aqueous solutions. The metal ions sequestered can be released in

an acid solution until the concentration of these metal ions reaches a level where

conventional methods can be used to provide economic metal recovery and potential

revenue generation. The ability of this biomass to adsorb more than one metal ion from

solution may increase its potential for application in the wastewater industry since the

majority of industrial effluents contain more than one metallic species. Metal ion uptake

was found to increase with the speed of agitation and at an optimum pH value of

between 4 and 7.

Keywords: Olive Mill Waste(OMW), Heavy Metals, Biosorption, Wastewater

- 183 -

Biotechnological application of halophiles in bioremediation of textile effluents

M. A. Amoozegar, S. Asad, A. A. Pourbabaii

Biology department (Microbiology Division), Faculty of Science, Tehran University, Tehran,IRAN


The textile industry produces large quantities of wastewater containing materials that

are becoming major environmental concern.

The aim of this work is to study the ability of halophiles to decolorize the azo

dyes. Since in the dyeing process for stabilizing of some dyes, a high amount of soluble

salts is used and also considerable amount of toxic metals is found in textile effluents,

application of halophilic strains is advantageous. This is the first report describing

decolorization of textile dyes by moderately halophilic bacteria.

In the present study, 36 halophilic strains isolated from textile effluents were

investigated to decolorize 10 different azo dyes. According to their ability to decolorize

different azo dyes and the rate of decolorization, three of them were chosen for further

studies. Remazol Black B was used as the representative of azo dyes for further studies.

It was shown that these strains were able to decolorize dyes at a wide range of pH(6-11).

It should be mentioned that a group of azo dyes, i.e. reactive azo dyes, bind to the cotton

fibers under alkaline conditions, consequently textile effluents usually have an alkaline

pH (8-9). These three strains also readily grow at high concentrations of dye (5000

ppm) and decolorize it. Further experiments have revealed that bacterial biomass

actively does the decolorization while no decolorization activity was observed by the

supernatant. Specterophotometric analysis has also shown complete disappearance of

the dye pics in the λ range of 200-800 nm after biodecolorization. This result and the

observation of colorless appearance of bacterial biomass after decolorization suggest

that decolorization is due to biodegradation rather than inactive surface adsorption.

Keywords: Halophiles, Azo Dyes, Textile Effluents, Biodecolorization,

Specterophotometric Analysis

- 184 -

Degradation of oxadiazon in a bioreactor integrated in the water close-circuit of a greenhouse

Pinilla P.1, Ruiz J.1, Garbi C.2, Martín M.2, Lobo M.C.1, Martínez-Iñigo, M.J.1

1Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA),

Finca El Encín. N-II Km 38,2. 28800 Madrid, SPAIN 2 Facultad de Veterinaria. Universidad Complutense de Madrid. 28040 Madrid, SPAIN


Ornamental conifer cultivation under greenhouse fertigation regime involves a rational

supply of nutrients all along the growth circle of plants. Nevertheless, the frequency of

irrigation enhances herbicide lixiviation and the consequent contamination hazards.

Pseudomonas fluorescens was found to be capable of degrading the herbicide

oxadiazon, the pre-post emergence herbicide applied to soil in pots. Bacterial cells were

immobilized on a ceramic support (sepiolite) contained in a 150L-bioreactor for

recirculating lixiviates while oxadiazon biodegradation occurs.

The levels of oxadiazon were determined in samples taken after each bioreactor

percolation cycle in order to establish the efficiency of the clean-up process. Water

samples were purified with C18 solid phase extraction columns and analysed by means

of gas chromatography with electron capture detection system.

Fifty eight percolation cycles resulted in a significant reduction of oxadiazon up

to just 5 ppb in lixiviates. Therefore, after the correction of nutrient concentration the

recycled lixiviates could be reused for fertilization without increasing the herbicides

doses recommended for conifers cultivation.

Keywords: Greenhouse, Pseudomonas fluorescens, Herbicide, Sepiolite, Gas

Chromatography, Lixiviates

- 185 -

Biotechnology for heavy metals removal from industrial wastewater

Eliza Pena Leonte1, Ciprian Dumitrescu1, Ileana Ghita1, Lucia Dumitru2, Ana Maria Faghi2

1 National Research and Development Institute for Industrial Ecology,

Panduri Road 90-92, sector 5, 050663, Bucharest, ROMANIA 2 Romanian Academy – The Biology Institute – Microbiology Center,

Splaiul Independentei 296, 060031, Bucharest, ROMANIA Corresponding author e-mail: [email protected]

The presence of heavy metals in the industrial and urban wastewater is a menace for

human and aquatic life forms. Due to this, an increase attention not only for knowledge

point of view but as an application potential is shown to bioremediation of heavy metals

polluted aquatic environment, at present.

The experiments were conducted aiming to remove heavy metals from general

wastewater collectors of nonferrous works. The wastewater had a Zn content above 4

mg/l, Mn above 1 mg/l, and Pb above 2,5 mg/l, contents which are over the discharge

limits imposed by Romanian legislation (Zn – 0,5 mg/l; Mn – 1 mg/l; Pb – 0,2 mg/l).

The experimental installation consisted of one submersed fixed bed bioreactor

with plastic support material and a secondary settler; technological parameters of

biofilter were: hydraulic retention time (HRT) – 12 and 24 hours, specific hydraulic load

(SHL) – 0,02-0,05 m3/h, Zn massic load (MLZn ) – 0,005-0,01 kg Zn/ m3⋅ d. The

bioreactor was inoculated with bacterial strains selected from natural media

(Pseudomonas sp. 4, Enterobacter sp. 16, Corynebacterium sp.17).

On HRT of 12 hours: Zn has been removed till 0,56 mg/l, remaining in a

concentration which exceeds the standardized Romanian limits. Mn and Pb have been

removed till concentrations below the standardized Romanian limits, or even below

limits of the detection methods. On HRT of 24 hours: Zn has been removed till

concentrations situated below the detection limits of the method. Mn and Pb

concentration in the effluent are below the detection limits of the methods.

Keywords: Wastewater, Heavy Metals, Biosorption, Fixed Bed Bioreactor, Hydraulic

Retention Time (HRT)

- 186 -

Heavy metals and temperature impact on physicochemical properties of lipids from Pseudomonas fluorescens (strain 296)

O.B. Popova, N.M. Sanina, I.P. Bezverbnaya, A.I. Mal`tsev

Far Eastern National University, 27 Oktiabrskaya str, R#423,690600 Vladivostok, RUSSIA


For more effective utilization of bacteria in bioremediation of the sea waters is essential

to understand the adaptive mechanisms of these strains to heavy metals impact.

Thermotropic behavior and lipid composition of marine Pseudomonas fluorescens

(strain 296), as a potential bioremediator, were studied by DSC, TLC and GLC,

respectively, to elucidate the efficacy of adaptation to cadmium and copper ions at 24

and 4°C. It was shown, that temperature of growth and ions of heavy metals did not

essentially influence temperature of phase transitions of the total lipids, while ions of

heavy metals induced the cleavage of DSC peaks. It may be connected with phase

separation of lipids that seems to cause the increased membrane permeability and

therefore inhibition of growth or death of bacteria. So, the bacterial biomass decreased

especially under influence of copper and cadmium ions in comparison with the

influence of low temperature only. The increased ratio between acid phospholipids (PG,

DPG and PI) and zwitterionic PE seems to induce phase separation of bacterial lipids.

At 24°C, the additional reasons can be the elevation of FA saturation and the level of

17:0cp FA. Heavy metals also didn’t influence UFA at low temperature, but the additive

effect of heavy metals and higher temperature occurred at 24°С. Percentage of the

monoene FA decreased simultaneously with the elevation in the level of the

cyclopropane FA. So, saturation and methylation of the UFA underlie adaptation of P.

fluorescens to high temperature, whereas heavy metals induce the late protective

mechanism only.

Keywords: Sea Waters, Heavy Metals, Cadmium, Copper, Pseudomonas fluorescens,

Bacterial Lipids

- 187 -

Preliminary mycological analysis of a contaminated lake in Northern Italy

D. Rodino, M. Rodolfi, N. Rizzini, A.M. Picco

Section of Mycology, DET, University of Pavia, Via S. Epifanio 14, 27100-I-Pavia, ITALY


Within the project “Eutrophic reduction through natural techniques of free little Italian

lakes waters” (LIFE02 ENV/IT/000079-TRELAGHI) our unit analysed the freshwater

hyphomycetes and the micromycetes associated to aquatic plants of the Endine lake

(Lombardy). Euthrophization that have taken place during these last decades in the lake

water, due to an excessive amount of nutrients coming from the drainpipes and the

agricultural techniques, have caused a progressive impoverishment of the present

biotypes, leading to a loss or a reduction of some life forms of the lake ecosystem.

During 2003 and 2004 we sampled plants and water seasonally, in 4 points at the bottom

of the lake. Filtration technique was used to look for aquatic hyphomycetes: we find

conidia of Alatospora sp. and Flagellospora sp. only in winter and at a very low

concentration (2.25 conidia/liter). Water was also plated directly, and some common

taxa (Aspergillus, Fusarium, Epicoccum, Penicillium) were observed. Regarding fungi

associated with plants, samples of Phragmityes communis L. and Nymphaea alba L.

were collected and plated onto TWA. P. communis samples resulted much more

colonized by fungi than N. alba and variation was also observed depending on the

season. Aspergillus flavus dominated in summer, Fusarium verticillioides and

Sclerotium sp. in autumn, while Phoma sp. in winter. Three species of Stagonospora (S.

caricis, S. macropicnidica, S. vitensis), Chaetospermum chaetosporum,

Cytoplacosphaeria rimosa and Mirandina corticola were isolated from P. Communis.

These records are not so common therefore work is in progress to test their potential

biodegradation activity.

Keywords: Hyphomycetes, Micromycetes, Aquatic Plants, Euthrophization, Biotypes

- 188 -

Passive bioremediation of groundwater contaminated with oil and derivates on the location of drinking water supply ''Ratno Ostrvo''

Srdjan Rončević1, Božo Dalmacija1, Olga Petrović2, Jelica Simeunović2, Milena Bečelić1, Ivana Ivančev-Tumbas1, Jasmina Agbaba1, Vesna Pešić1

1 Department of Chemistry, Faculty of Sciences, University of Novi Sad,

Trg Dositeja Obradovica 3, 21000 Novi Sad, SERBIA AND MONTENEGRO 2 Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, 21000 Novi Sad, SERBIA AND MONTENEGRO


In the vicinity of drinking water supply ''Ratno Ostrvo'' of Novi Sad (Serbia and

Montenegro) is situated the Novi Sad Oil Refinery, which was severely damaged during

the NATO bombing in 1999. Also, the site is traversed by the pipeline conducting oily

wastewaters from the Refinery to be discharged to the Danube River. As a consequence,

hydrocarbons and mineral oils have been detected in the groundwater. In order to

establish whether the groundwater possesses self-purification capacity, i.e. whether

passive bioremediation takes place, mineral oils and total hydrocarbons, total organic

carbon, as well as counting of some groups of bacteria were monitored from 2000 to

2004. Microbiological studies showed that groundwater at the location of water supply

contained hydrocarbon-oxidizing bacteria and lipolytic microorganisms. Taking into

account the complexity of processes ocurring when oil contaminants enter the

environment, the appearance of particular bacterial groups do undoubtedly accompany

fluctuations of relevant chemical components in space and time. It should be considered

that the microbiological status is a primary indicator of the quality state and give a

reliable picture of the environment prior to the measurement. This is probably the

reason for the lack of a linear relationship between chemical and microbiological

parameters that characterize the presence of hydrocarbon pollution in water. However,

in a certain number of piezometers, there has been established dependence between

chemical and microbiological quality parameters. Therefore, microbiological and

chemical parametres, are equally important for the overall characterization of passive

bioremediation processes.

Keywords: Drinking Water, Hydrocarbons, Mineral Oils, Passive Bioremediation,

Groundwater

- 189 -

Biosolids as amendment in a physically degraded cropland of the Rolling Pampas

Rodríguez, M. B 1 , R. Lavado 1, C. Quinteros 2, M. A. Tabeada 1

1 Soil Fertility and Fertilizers and 2 Inorganic Chemistry,Agronomy School - Buenos Aires University, Av San Martín 4453. Capital Federal, C1417 DSQ, ARGENTINA

Corresponding author e-mail:[email protected]

The destination of the residues coming from the municipal wastewater treatment plants

(biosolids) is a serious environmental problem. Their application of biosolids to the

croplands can restore soils physically degraded, but their contribution in potentially toxic

elements may increase the risk of contamination of soils and crops.

A field experiment was developed to determine the aptitude of biosolids to improve

the physical condition of a pampean Typic Argiudoll. Before sowing maize crop it was

amended with 100 and 200 Mg ha –1 of fresh biosolids (75 % water content). At harvest,

Superficial Resistance (SR), Total Carbon (TC), Soluble Carbon (SC) and pH H2O

(1:2,5) were determined in soil. Also contents of Cd, Cr, Cu, Mn, Ni, Pb and Zn were

extracted with DTPA and with hot water. Plant and grain material were acid extracted.

All extracts were analized by ICP-AES.

In soil samples, the SR decreased significantly (p<0.05) from 3.3 MPa (control) to

2,2 and 2,3 MPa (100 and 200 Mg ha–1 respectively). This improvement could be

explained by neither TC nor SC as they didn´t change with biosolids. The pH decreased

significantly but raised to the initial value in a year. The trace elements levels in soil, plant

and grain were below the maximum tolerable by the regulation. The only one to be

affected by biosolids was the Pb level; however it was not absorbed by maize. We consider

biosolids as an interesting alternative to improve the physical condition of degraded soils

without risk of contamination.

Keywords: Biosolids, Wastewater Treatment Plants, Soil, Crop, ICP-AES

- 190 -

Assessment of Sulphate-Reducing Bacteria isolates for the precipitation of heavy metals

M. Riesgo, A. Coppola, M. Viera and E. Donati

CINDEFI, CONICET y Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional

de La Plata, 47 y 115, 1900 La Plata, ARGENTINA Corresponding author e-mail: [email protected]

Several industries generate liquid effluents and solid wastes with a high concentration of

heavy metals which prevent their final disposal. Unlike organic contaminants, heavy

metals can not be degraded, and the remediation treatments are aimed for a change in

their mobility and/or bioavailability. Sulphate-Reducing Bacteria use sulfate as a

terminal electron acceptor and organic compounds as carbon/energy sources under

anaerobic conditions. These cells can contribute to the remediation of heavy metal

contamination due to the production of sulphide which gives dense and insoluble

compounds with most heavy metals.

The aim of this work was to evaluate the precipitation of different heavy metals

by Sulphate-Reducing Bacteria consortia. Five different isolates of Sulphate-Reducing

Bacteria from natural environments (some of them containing heavy metals) were tested

for their capacity to precipitate nickel, cadmium and chromium at different

concentrations (5 to 100 mg/l). The ability of growing at different pH (4.5 to 6.5) and

the possibility of metabolising different simple carbon sources (lactate, ethanol, acetate,

benzoate, propionate and glucose) were also evaluated.

Keywords: Heavy Metals, Nickel, Cadmium, Chromium, Bioavailability, Sulphate-

Reducing Bacteria, Precipitation

- 191 -

Bacterial community changes at TCE biodegradation detected in microcosm experiments

Sára Révész1, Rita Sipos1, Anikó Kende2, Tamás Rikker2, Éva Mészáros1,

Károly Márialigeti1

1 Eötvös Loránd University, Department of Microbiology, 1117 Pázmány P. sétány I/C., Budapest, HUNGARY

2 Dr. E. Wessling Chemical Laboratory Ltd. 1047 Fóti út 56., Budapest, HUNGARY


Chlorinated solvents such as trichloroethylene (TCE) have been widely used in the

industry. Conventional pump-and-treat technologies may contain and control spill

plumes however, these techniques have limited effectiveness in remediating

groundwater pollution. In situ reductive dechlorination is considered to be a promising

measure in the bioremediation of chloroethene contaminated sites.

Laboratory microcosm experiment was set up to model biodegradation of TCE.

Two contaminated groundwaters were selected, one of them with low SO42-

concentration (LS), the other with high (HS). Lactate, molasses and methanol were

added to the bottles, as supplementary substrates. At day 0, 24, 54, 98, 155 and 318

chemical and bacteriological parameters (e.g. Dehalococcoides test) were investigated.

TRFLP based diversity assessments were carried out to observe the bacterial community

changes. It was found, that lactate and molasses enhanced degradation at different rate.

In case of samples HS amended with lactate neither ethylene nor ethane or methane

generated. In case of sample LS with lactate both ethylene and methane were detected.

Molasses seems to increase degradation rate better in case of sample HS. The results of

Dehalococcoides tests were positive for all samples. The TRFLP patterns of HS samples

amended with lactate and molasses were most similar. There are two main groups

formed by cluster-analysis, one of them contains samples with methanol, control

samples and samples from the LC original groundwater. The other main group contains

samples with lactate and molasses. Community pattern of LS samples with lactate were

the most different among samples of the same microcosm.

Keywords: Trichloroethylene (TCE), Groundwater, Microcosm, Biodegradation,

Dehalococcoides Tests

- 192 -

Biodegradation of halogenated compounds in contaminated water from open waste landfill

Siglova M., Vesela L.*, Masak J., Cejkova A., Zachar P., Mikes J., Jirku V.

Institute of Chemical Tecnology in Prague, Technicka 5, Prague 6, 166 28, CZECH REPUBLIC

* DEKONTA a.s., Volutova 2523, Prague 5, 158 00, CZECH REPUBLIC Corresponding author e-mail: [email protected],

The European Union generates 1300 million tonnes of waste a year. The processing of

this waste within the EU is distributed as follows: disposal on waste deposits 57%;

incineration 16%; recycling 13%; composting 7% (figures from the European

Environmental Agency). Landfilling is therefore an essential element of waste

management. But, it raises many problems of compliance in Europe. Not only the

hazardous waste landfill can pose a health hazard, but we also have to consider the

impact of leachate from these sites. The goal of this project is to advance understanding

of anaerobic and aerobic bioremediation of halogenated compounds to a level when is

possible to decide whether the bioremediation approaches are suitable for the full-scale

treatment of contaminated leachates straight on the landfill.

This research was focused on the biodegradation of several halogenated

compounds (mainly aliphatic and aromatic, nonvolatile compounds) occurring in the

complicated mixture with other pollutants presenting in the water leaking from the

landfill. In order to accurately assess potential for anaerobic or aerobic bioremediation

technology, all experimental systems (cultivation flasks, laboratory fermentor) operated

under conditions close to those observed in contaminated place. Additionally, the real

water was used for the obtaining of indigenous microorganisms from contaminated

sites. These cultures (Pseudomonas stutzeri and Pseudomonas stutzeri – untypical)

proved to be as organisms perfectly suited for chlorinated compounds degradation.

During the 6 weeks experiments we observed the 50 % decrease of chlorinated

compound amount during the anaerobic cultivations and 70 % decrease under the

aerobic conditions.

Keywords: Landfill, Halogenated Compounds, Leachates, Indigenous Microorganisms,

Pseudomonas stutzeri

- 193 -

Microbial detoxication of sewage of explosive production

Smyrnova H.F. 1, Serpokrilov N.S. 2, Podgorsky V.S. 1

1 Zabolotny Institute of Microbiology & Virology, National Academy of Sciences of Ukraine, 154 Zabolotny str. 03142, Kyiv, UKRAINE

2 The State Building University, 162 Socialist street 343001 Rostov On Don, RUSSIA Corresponding author e-mail: [email protected]

Chlorine oxyanione salts are widely used by chemical, aerospace and defense industries

as oxidizers in propellant, explosive, and pyrotechnics. Sewage of explosive production,

besides of chlorate and perchlorate, contains chromate which is the most toxic and

mobile form of chromium. Remediation efforts of chlorine oxyanions have focused

primarily on microbial detoxication because of unique chemical stability and high

solubility of these compounds. Microorganisms can also reduced Cr (VI) and could be

used for cleaning Cr(VI) contaminated water.

A bacterium coded as strain TGS-463 was isolated from a match factory sewage

for its ability to reduce chlorate to chloride for energy and growth. The organism is also

capable for the dissimilatory reduction of chromate to chromium hydroxide and sulfate

to H2S. The rates of reduction of these compounds by TGS-463 are 62,8, 0,75 and 41,08

mg/h per gram, ccordingly. TGS-463 can use fatty acids, ethanol and organic

compounds of municipal and tannery sewage and organic compounds of match factory

waste waters as electron donors for chlorate- and chromate reduction. It was shown that

optimal C:N:P correlation for successful bioremediation is 402:50:5.

Pilot sewage treatment bioreactor on Cherepovez′ match factory demonstrate

stabile work and provide detoxication of 97,8% of chromate, 93,8-98,4% of chlorate

and 74,5% of sulfate.

Keywords: Chlorine Oxyanione Salts, Chromate, Microbial Detoxication, Sewage

Bioremediation

- 194 -

Comparison of anaerobic and aerobic bacterial communities in metal-working fluids

Ji-Inn Song 1,2, Ian P. Thompson 1, Simon A. Jackman 3, Christopher J. Knowles 3,

Christopher J. van der Gast 1*

1 Environmental Biotechnology Section. Centre for Ecology and Hydrology, Oxford OX1 3SR, UNITED KINGDOM

2 Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UNITED KINGDOM

3Department of Earth Science, University of Oxford, Parks Road, Oxford OX1 3PR, UNITED KINGDOM

*Corresponding author: [email protected]

Metal-working fluids (MWF) are used ubiquitously, as coolants and lubricants, in the

automotive and shipbuilding industries for processing metals. The treatment and

disposal of spent MWF is of increasing environmental concern as globally, over 2.2 x

1010 litres are used per annum. MWF pose an attractive nutrient resource to bacterial

communities and as a result are rapidly invaded by colonisers, leading to unwanted

biodeterioration and subsequent production of waste MWF. There has been a

considerable amount of research into the microbiology of MWF over the last few

decades, where the majority of these studies have concentrated exclusively on the

culturable aerobic communities in free suspension. However, it has been postulated that

anaerobic bacterial populations in biofilm communities play a significant role in MWF

biodeterioration. The main aim of this research was to study anaerobic bacterial

populations and understand their role in bacterial biofilms within MWF sump tank

reservoirs of in-use engineering machines (e.g. lathes, CNC and vertical mills). Using

culture-independent microbiological techniques, the biodeterioration of in-use MWF by

indigenous bacterial communities, we investigated the anaerobic and aerobic bacterial

populations compared. Traditionally, aerobic biodegradation / biodeterioration of

hydrocarbon has been understood to be more effective than the biodegradation /

biodeterioration under anaerobic conditions. However, this study revealed that under

anaerobic conditions MWF biodeterioration was greater when compared to aerobic

bacterial mediated deterioration.

Keywords: Metal-Working Fluids (MWF), Anaerobic Bacterial Populations, Biofilm,

Biodeterioration

- 195 -

Role of bacterial periphyton in bioremediation of oil contaminated water

Julia Tumaikina, Olga Turkovskaya, Vladimir Ignatov

Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of

Sciences. prosp. Entuziastov 13, Saratov 410049, RUSSIA Corresponding author e-mail: [email protected]

Bioremediation is based on the study and application of the mechanisms of natural self-

cleaning processes. The biological factor (including bacterial periphyton) has a

dominant role among all self-cleaning factor. In this study we present experimental date

on biodegradation of some compounds of water soluble fraction (WSF) of oil. The

microbial strains El-2.1, El-3.1 and El-4.3 were tested in order to reveal their ability to

grow at the presence of phenol (1 g/l), xylene (0,25 - 1 g/l), diethylbenzene (0,25 - 4

g/l), decalin (0,25 - 5 g/l), benzene (1 – 4 g/l), toluene (0,5 – 5 g/l), naphthalene and

phenanthrene (0,1-4 g/l). The quantitative assessment of destructive activity shown that

not all strains examined were active. It was detected that strain El-4.3, which capable of

growing at the presence of all chosen compounds (behind exception phenanthrene), was

not able to destroy of any of them even at their small concentration. The strains El-2.1

and El-3.1 shown high destructive activity towards phenol (100 and 81,8%),

naphthalene (97 and 94%), toluene (40,8 and 69,2%) and benzene (27,8 and 36,4%

respectively). Only strain El-2.1 was capable of degrading decalin (57,2 %).

Phenanthrene and diethylbenzene was not destroyed by any of strains examined. It was

also established that bacterial periphyton protect Elodea canadensis against the toxic

pollutants.

Keywords: Water Soluble Fraction (WSF), Oil, Bacterial Periphyton, Biodegradation,

Self-Cleaning Processes

- 196 -

Heavy metal bioadsoption on bacterial biofilms: use of submerged filters for decontamination

Vilchez, R.1*, Rodelas, B. 2, Pozo, C.1, Martínez-Toledo, M.V.1, González-López, J.1

1 Instituto del Agua, and 2Departamento de Microbiología, Facultad de Farmacia,

Universidad de Granada, C/ Ramón y Cajal, 4, 18071 Granada, SPAIN *Corresponding author e-mail: [email protected]

We designed a pilot-scale treatment plant using submerged bacterial biofilms to reduce

the concentration of Cu in subterranean water. The plant was fed with subterranean

water contaminated with Cu, and supplemented with sucrose (250 mg/l) as C source.

Control parameters studied at different levels along the system height during the

experiment were: biomass production, mesophilic microbiota, sucrose concentration,

alkaline phosphatase, and ATP levels. Cu concentration at the biofilter outlet and at all

depths of the biofilter was monitored daily. Exopolymer produced by bacteria was

examined by SEM, and its content of sugars, proteins, uronic acids and acetyl moieties

characterised. Periodically, total DNA was extracted from biofilm samples, and V3-

hypervariable region of 16S-rDNA was amplified with universal primers. Diversity

profiles of the bacterial communities were analyzed by TGGE.

When Cu was present in the inlet water, volatile compounds in the biofilm were

increased, and mesophile counts, ATP content/g biofilm, and total alkaline phosphatase

activity were significantly reduced, compared with the control system amended with

water lacking Cu. The present of Cu inhibited sucrose uptake by bacteria. Sugars and

protein contents of the EPS were higher in Cu biofilms, while uronic acids and acetyl

moieties remained unchanged. Biofilm morphology was significantly modified in the

presence of Cu, as shown by SEM. TGGE analysis demonstrated a high spatial and

temporal stability of the population composition during the cycle of plant functioning.

Reamplification and sequencing of isolated TGGE bands showed that most bacteria

characterized from the biofilm belong to the α-Proteobacteria.

Work by B. Rodelas was funded by Spanish MCYT through Ramón y Cajal

Programme.

Keywords: Pilot-Scale Treatment Plant, Biofilm, Copper, EPS, 16S-rDNA, TGGE,

SEM

- 197 -

Efficient textile dye decolorization by the white-rot fungus Trichophyton rubrum LSK-27

Koray S Yesiladali, Gulseren Pekin, Hakan Bermek*, IdilArslan-Alaton,

Derin Orhon, Candan Tamerler

Istanbul Technical University, Molecular Biology And Genetics, TURKEY * Corresponding author e-mail: [email protected]

The potential of a white-rot fungus, Trichopython rubrum LSK-27 was evaluated for

decolorization of various azo groups of textile dyes. The organism demonstrated fast

biodegradation capability as well as biosorption as dye removal mechanisms. In less

than two days, the fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of

Remazol Blue RR and 80% of Supranol Turquoise 6GL in the liquid cultures. The

reactive dyes were removed by fungal biodegradation. However, decolorization of the

acid dye Supranol Turquoise 6GL was accomplished mainly by bioadsorption. The

amounts of extracellular manganese peroxidase and laccase detected demonstrated that

these enzymes were associated with the biodegradation mechanisms.

Keywords: White-Rot Fungus, Trichopython Rubrum, Azo Dyes, Biosorption,

Extracellular Manganese Peroxidase, Laccase

- 198 -

Chromium removal by Sulphate-Reducing Bacteria from contaminated groundwater

Petra Zackova 1, Pavel Kuran 2

1 DEKONTA, a.s., Prague Office, Volutova 2523, 158 00 Prague 5, CZECH REPUBLIC 2 Research Institute of Inorganic Chemistry, Revolucni 84, 400 01 Usti nad Labem, CZECH REPUBLIC


This paper refers to chromium removal from contaminated groundwater by Sulphate

Reducing Bacteria (SRB). Treatment of underground water polluted by Cr(VI) was

tested in laboratory conditions.

Contamination of soil and groundwater by heavy metals is a widespread problem

due to former activities of metal processing, mining industry and uncontrolled dumping

of waste in landfills. The SRB generate hydrogen sulphide, which causes precipitation

of metals present in a solution as insoluble metal sulphides. The SRB growth under

anoxic, reducing conditions in medium containing dissolved organic compounds and

sulphate.

During laboratory experiments two sulphate-reducing strains, namely

Desulfovibrio desulfuricans subsp. desulfuricans DSM 642 and Desulfovibrio vulgaris

subsp. vulgaris DSM 644 were tested. These strains were cultivated under anaerobic

conditions in contaminated groundwater containing chromium(VI) in concentration of

10.03 mg.l-1 with an addition of sodium sulphate. The content of chromium(VI)

decreased under 0.1 mg.l-1 during three-month cultivation. The grey precipitate was

formed at the vessel bottom.

Keywords: Chromium(VI), Groundwater, Sulphate Reducing Bacteria (SRB), Insoluble

Metal Sulphides, Precipitation, Desulfovibrio desulfuricans, Desulfovibrio vulgaris

- 199 -

Managing polluted soils of the niger delta región of nigeria for sustainable food production and economic development

Eshett, Ebong Thomas

Depart of Soil Science & Technology, School Of Agriculture And Agricultural Technology, Federal

University of Technology P.M.B. 1526,Owerri, Nigeria.

UNDER REVISION

- 200 -

A combined biological treatment system for the biodegradation of sugar industry wastewater

Amit kumar1, D.B. Sapkal2, B.B. Gunjal2, Bram Sercu3 ,

Herman Van Langenhove3

1Department of Environmental Science and Engineering Guru Jambheshwar University,

Hisar, 125001, Haryana- INDIA 2Department of Environmental Science and Alcohol Technology, V.S.I. Manjari (B.K)

Pune-412307 MH- INDIA 3EnVOC Research group, Faculty of Bioscience Engineering, Ghent University,

Coupure Links 653, B- 9000 Ghent, BELGIUM E-mail:

In the present study the feasibility of a laboratory-scale sugar industry wastewater

treatment by using an down-flow anaerobic filter (DAF) followed by activated sludge

process (ASP) was tested. Physical-chemical pre-treatment was used to recover the oil

and grease and to remove suspended solids from wastewater. The pre-treated

wastewater having pH 4.6- 5.9, biochemical oxygen demand (BOD) 1500-2000 mg l-1

and chemical oxygen demand (COD) 3200-4500 mg l-1 was used to feed DAF reactor as

a principal step for COD elimination. The effect of hydraulic retention time (HRT) on

the extent of the degradation of this wastewater was examined. Varying the HRT from

2.5 d to 0.8 d at 27 ± 1°C, had no significant effect on COD removal efficiency and the

pH remained in the range of 6.6-7.4. Under these conditions the COD removal

efficiency was 85-90% after steady state at a organic loading rate (OLR) between 2.06-

8.54 kg m-³ d-1. At HRT < 0.5 d the COD removal efficiency was decreased and a

decreased pH and methane yield was also observed. The ASP reactor operated in

aerobic condition was able to remove 90% of the COD from the anaerobic effluents at

HRT < 4 h. The study shows that combining anaerobic pre-treatment with aerobic post

treatment integrates the advantages of both processes amongst which reduced energy

consumption (net energy production), reduced biological sludge production and limited

space requirements. Thus, can be used for the treatment of sugar industry wastewater.

Keywords: Sugar Industry Wastewater Treatment; Down Flow Anaerobic Filter;

Hydraulic Retention Time; Activated Sludge Process

- 201 -

Natural minerals for soil remediation

N. Zaimenko

M.M.Grishko National Botanical Gardens, Timiryazevska 1, Kyiv 14, 01014, UKRAINE Corresponding author e-mail: [email protected]

The development of structure and differentiation of biotic blocks of geocenosis were

studied. Material, energetic and information interaction among all the components of

plant community were examined to determine the mechanisms of self-regulation of

plant self-regulation under the conditions of radioactive pollution and soil fatigue. We

are first who used natural minerals in agriculture, the minerals with proper to increase

soil fertility, decrease soil fatigue and radionuclides accumulation in crops, and

stimulation the development of useful microbiota. Short and long prognostication were

carried out for plant development under the influence of environment factors to search

the mechanisms of rapid reaction and removing of negative anthropogenic effect. Our

investigations show that the best radiation protector is analcinite, new dispersive

mineral. Considering the indexes of sum capacity of cations this mineral can be used for

stimulation the root system development and improvement of soil. The effect of calcium

silicon on cultural biogeocenosis on radioactive polluted territories were studied. The

experiments proved the expediency of calcium silicon use for activation of

physiological-biochemical processes in plants, increase of their adaptation to damage by

Sphaerothece pannosa Lev. var rosea Weron. Microbiological analysis shows the

positive effect of silicon on useful microflora and decrease of soil fatigue in average

2.6-5.2 times. The data obtained allow determining the character of interaction among

the processes of microbiotic and fermentative activity and can be used for improving the

biological condition of soil. Application of threpel saves 47-52% of soil moisture. We

create a number of the models for prognostication soil system behavior under

anthropogenic pressure.

Keywords: Geocenosis, Radioactive Pollution, Soil Fatigue, Natural Minerals,

Microflora

- 202 -

Petroleum hydrocarbon biodegradation on contaminated soils enhancement by the addition of biosurfactant produced from agro

industrial waste

Benincasa, M.*, Fernandes, F.S., Latanze, R., Kronka, E. M, Coppede, J. S.

Centro Tecnológico de Desenvolvimento Agroindustrial, UNAERP/Ribeirão Preto-SP,

Av. Costábile Romano, 2201, 14096-380, Ribeirão Preto/SP, BRAZIL *Corresponding author e-mail: [email protected]

Petroleum hydrocarbons form an important class of pollutants on a global scale. These

compounds presence in the environment is of considerable public health and ecological

concern because of their persistence, toxicity and ability to bioaccumulate.

Bioremediation technology has been used for crude oil degradation in soil matrix, by

using microorganisms to transform these hazardous substances in less toxic compounds.

However, the hydrocarbons availability to microorganisms is limited mainly due its low

solubility and adsorption on soil matrix. The use of synthetic or biological surfactants

has been described as a strategy to enhance oil mobility and, thus improving the

biodegradation rates. Biosurfactants present some advantages over the chemically

produced counterparts as their biodegradability, lower toxicity and environment

compatibility.

The aim of this work is to evaluate the effect of the addition of a biosurfactant,

produced from agro industrial process waste, on hydrocarbon degradation by indigenous

microbial community. Two different concentrations of non sterile, crude rhamnolipid

biosurfactant, produced by Pseudomonas aeruginosa LBI, were added to a soil obtained

from a contaminated site in bench scale. The lower concentration was slightly above the

Critical Micelle Concentration (CMC) and resulted in the hydrocarbon concentration

decrease from 65 % (natural biodegradation) to 98%. When using a higher

concentration, the pollutant reduction it was not as significant as the natural

biodegradation.

Acknowledgments: Financial Support: FAPESP

Keywords: Petroleum Hydrocarbons, Crude Oil Degradation, Surfactants, Indigenous

Microbial Community, Pseudomonas aeruginosa, Critical Micelle Concentration

(CMC)

- 203 -

Use of Organo-Mineral Complexes (OMCs) for remediation of pentachlorophenol (PCP) in soil

Katarína Dercová1*, Gabriela Barančíková2, Zuzana Sejáková1

1 Slovak University of Technology, Faculty of Chemical and Food Technology,

Department of Biochemical Technology, Radlinského 9, 812 37 Bratislava, SLOVAK REPUBLIC 2 Soil Science and Conservation Research Institute Bratislava, Regional Station Prešov,

Reimannova 1, 08001 Prešov, SLOVAK REPUBLIC *Corresponding author e-mail: [email protected]

This contribution provides some data on use of OMCs to decontaminate chlorinated

organic pollutants. The key role in elimination of contaminants from the soil is played

by a quality of organic matter, mainly by humic acids (HA). It is important to monitor

binding of pentachlorophenol (PCP) to organic matter as it can reduce bioavailability of

PCP to degrading microorganisms. This may lead to a false conclusion about the

microbial contribution to PCP degradation if this process is not accounted for. It is

possible to use the sorption properties of PCP for microorganisms. High influent

concentrations can be decreased to less toxic levels by adsorption allowing maximum

degradation to take place. Fast and effective adsorption and low desorption may serve

for reducing PCP content in soil and thus reducing its toxicity in remediation

technology. Adsorption/desorption of PCP experiments were carried out for a series of

OMCs prepared on the basis of clay mineral (zeolite/kaolin) and HA. The preliminary

results indicate that OMCs have better retention abilities than the clay minerals alone. In

comparison to kaolin-based OMCs, those based on zeolite reveal a better sorption-

desorption ratio. Higher amount of HA coated on zeolite enhances its potential to adsorb

and retain PCP. OMC containing 5 mg HA/g of zeolite possessed the best retention

ability towards PCP. According to our preliminary results, both tested OMCs appeared

to be good traps of PCP with potential application in remediation technology. Both

components of OMCs are of natural origin and therefore suitable to be used in the

environment and support natural ability of soil to eliminate contaminants.

Acknowledgements: Ministry of Education (Grant No. 1/1309/04).

Keywords: Organo-Mineral Complexes (OMCs), Humic Acids (HA),

Pentachlorophenol (PCP), Bioavailability, Remediation Technology

- 204 -

Mathematical simulation of hydrocarbons biodegradation by composting

Rocío-De la Torre S.1*, E.Luna2, Josefina-Barrera C.1

1 Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Av. Instituto Politécnico Nacional, 2508, Col. San Pedro Zacatenco, C.P. 07360,

14-740, 07000 MÉXICO D.F. 2 Tecnología de Yacimientos. Instituto Mexicano del Petróleo,

Eje Central Lázaro Cárdenas, 152, Col. San Bartolo Atepehuacan. C.P. 07730, 14-805 MÉXICO D.F. *Corresponding author e-mail: [email protected]; [email protected]

In this work, a mathematical model was proposed in order to simulate biodegradation

by composting. In spite of the high percentages of hydrocarbons that might be removed

at relative short time by this technology, the main obstacle is the lack of knowledge

related with its operational conditions and design. It has been considered that a

mathematical model, which includes the biological and phenomenological variables

could facilitate as well as improve its application. In contrast with the majority of

stationary and “black box” existing models, a phenomenological and transitory model

was developed. The system under study consisted on hydrocarbons polluted soil,

contained at a cylinder. Soil’s endogenous microorganisms was estimulated by nutrients,

air and water addition. The model’s biological assumptions were made at a multiple

gradient to macroscopic scale, where biodegradation took place via multiplicative

Monod kinetics. Temperature’s influence was modeled by the Arrhenius equation. A

complete mineralisation occured, by which water, carbon dioxide and biomass increased

as a result of hydrocarbons and oxygen consumption. On the other hand, the transport

phenomena aspects were modeled by mass and energy conservation equations, as well

as species balance and Darcy’s law. The final model consisted on non linear partial

equations which were solved by finite differences and iterative methods. The model

predicted the evolution of velocity fields as well as species concentration profiles. The

obtained results suggested that the main variables that influenced the hydrocabon’s

biodegradation and biomass growth were temperature and humidity distribution, as well

as air supply position and system’s geometry.

Keywords: Mathematical Model, Composting, Hydrocarbons, Endogenous

Microorganisms, Mineralisation

- 205 -

Comparative study of biodegradation of crude oil in soil amended with chicken droppings and NPK fertilizer

U. J. J. Ijah*, H. Safiyanu

Department of Microbiology, Federal University of Technology

P. M. B. 65, Minna, NIGERIA Corresponding author e-mail: [email protected]

Analysis of soil samples treated with 10% (v/w) Escravos light crude oil and amended

with chicken droppings and NPK fertilizer revealed that the aerobic heterotrophic

bacterial counts were depressed while the proliferation of crude oil degrading bacteria

(CDB) in the soil was encouraged. The counts of CDB in oil free (control) soil ranged

from 20 x 108 CFU/g to 33 x1 08 CFU/g of soil, while that of oil polluted soil ranged

from 48 x108 CFU/g to 93 x 108 CFU/g soil after 10 weeks. In fertilizer amended soil,

the counts of CDB ranged from 40 x 108 CFU/g to 92 x 108 CFU/g of soil and from 53

x 108 CFU/g to 95 x 108 CFU/g in soil amended with chicken droppings. The crude oil

degrading bacteria were identified as Bacillus, Pseudomonas, Acetinobacter and

Micrococcus. One of the isolates, Bacillus sp. SOB-10 exhibited a high ability in

degrading the crude oil. The organism degraded 54.6% of crude oil in 14 days and

therefore, may be useful in seeding oil-polluted soil. Crude oil addition to soil raised the

pH from 6.75 to 7.65 and amendment of the oil-polluted soil with chicken droppings

further raised the soil pH (7.17 – 7.76). Amendment of the oil polluted soil with

fertilizer however, caused a remarkable decrease in the soil pH (5.91 – 6.26).

Biodegradation studies revealed that 56.3% of crude oil was degraded in the unamended

soil while 75% and 87.5% of crude oil was degraded in soil amended with chicken

droppings and fertilizer respectively after 10 weeks. The use chicken droppings in crude

oil biodegradation can be a solution to the problem of animal waste disposal

Keywords: Light Crude Oil, Chicken Droppings, NPK Fertilizer, Crude Oil Degrading

Bacteria (CDB)

- 206 -

Application of indigenous microorganisms in bioremediation of fuel oil contaminated soil

L. Kalėdienė*, G. Giedraitytė

Faculty of Natural Sciences, Vilnius University,

M. K. Čiurlionio 21/27, LT-03001 Vilnius, LITHUANIA Corresponding author e-mail: [email protected]

Among the most contaminated areas in Lithuania are those around Klaipėda Oil

Terminal, old dumping sites, including the small petroleum storages (50-100 t) of

former collective farmers, the small gas stations in rural areas, the gasoline stations, the

territories around railways and etc. The top level of soil and ground water are most

heavily polluted at former fuel storage sites. Maximum concentrations in the soil (at the

depth of 1,5 m) reach 47-52 g/kg. As many as 85% of surface soil samples taken in

different spots show oil pollution with values up to 70-100 times higher than the

background readings. Waste management in Lithuania is one of the most pressing

environmental problems. Their solution requires large funds and many efforts.

The present study was undertaken to evaluate the efficacy of introduced

indigenous bacterial isolates for ex situ bioremediation of fuel oil contaminated soil.

Study showed the introduced bacterial consortium effectively adapted to the local

environment of the soil at the bioremediation site. The disappearance of poly aromatic

hydrocarbons (PAH) from inoculated soil samples depended on the general soil

impurity, term of bacterial treatment, level of PAH contamination and individual

microorganism efficacy. With application of bacterial consortium and fertilizers, the

PAH level was reduced to 60 - 66% after three months. The results proved that

indigenous microbial populations of fuel oil contaminated soil were able to feed on

every hydrocarbon fraction and the application of a bioremediation technology could be

a very useful tool for the treatment of the hydrocarbon fractions present in the studied

waste.

Keywords: Indigenous Bacterial Isolates, Fuel Oil, Poly Aromatic Hydrocarbons

(PAH), Fertilizers

- 207 -

Transferability of plasmids by conjugation in soil and influence of exogenous plasmid-harboring bacteria on the bacterial community: a model study for risk assessment on the use of genetically modified

organisms

Daisuke Inoue*, Katsushi Murashige, Kazunari Sei, Michihiko Ike, Masanori Fujita

Department of Environmental Engineering, Osaka University

2-1 Yamadaoka, Suita, Osaka 565-0871, JAPAN * Corresponding author e-mail: [email protected]

Assessment of bio-risk is required in order to use any genetically modified organisms

(GMOs) on the purpose of bioremediation. We conducted laboratory investigations as a

basic study for risk assessment on GMOs use in soil. First, we investigated the

frequency of conjugative transfers of a model conjugative plasmid, RP4, from

Escherichia coli C600 to typical soil bacterial species in soils in order to determine the

maximal transferability of plasmids and the influence of soil factors on the transfer

frequency. Transfer of RP4 from E. coli C600 to most recipients occurred in various

soils, although the frequency varied depending on the combination of recipient strain

and soil type. Contents of water and metals such as zinc and iron seemed to affect the

transfer frequency. Secondly, we examined the influence of introduction of E. coli

C600(RP4) on soil bacterial community using T-RFLP analysis. E. coli C600(RP4)

introduced into the soil decreased to below the detection limit within 16 days, while

more than 48% of RP4 remained over 30 days, suggesting the survival of RP4 not in the

original host but in the secondary hosts (transconjugants). T-RFLP analyses of the

bacterial community did not show significant changes in dominant bacterial populations

by the introduction of E. coli C600(RP4) during the 30-day incubation period. These

results indicate that conjugative plasmids can disseminate from exogenous bacteria to

indigenous populations at a certain level in soil, but the augmentation/introduction of

exogenous bacteria and their plasmids do not make a severe impact on indigenous

bacterial community.

Keywords: Genetically Modified Organisms (GMOs), Conjugative Plasmid,

Escherichia coli, Soil Factors, T-RFLP Analysis

- 208 -

The use of PETROBRAS-REPAR landfarming inoculum for enhancing the bioremediation of soil oil spills

F.J. Pereira de Campos Carvalho1*, R. Azevedo Castro2, M. Cristine Krenczki2,

C. Martins Gonçalves2, E. Zamberlam3

1 Universidade Federal do Paraná, Departamento de Solos e Engenharia Agrícola Rua dos Funcionários s/n, CEP 80035050, Curitiba, Paraná, BRAZIL

2 ANTROPOSPHERA – Instituto para o Desenvolvimento do Meio Ambiente Rua Jacarezinho 215, CEP 80710150, Curitiba, Paraná, BRAZIL

3 PETROBRAS – REPAR, Rodovia do Xisto Km 16,5, CEP 83700970, Araucária, Paraná, BRAZIL *Corresponding autor e-mail: [email protected]

Since 1991 PETROBRAS Petróleo Brasileiro S.A., in collaboration with the

Universidade Federal do Paraná, has invested substantial effort in the development of

the landfarming biotreatment system by means of a specialized indigenous bacteria and

fungi population. The applied research results in a high efficiency landfarming system

capable of degrading refinery oil waste, as much as 4,700 ton/ha.year of petroleum-

hyadrocarbon additions. In the resulting used microbial population there was a

predominant multiplication of fungi, used to explain the excellent biodegradation

performance. Since 1993, inoculum from the landfarming has been used in

biorremediation areas with a biotreatment oil wastes over 30,000 ton in contaminated

soil. At July 16th of 2000, a major crude oil spill in soil occurred at REPAR. In order to

biorremediate the contaminated soil, the region was divided in 12 areas that were

inoculated with dry inculum, produced in a pilot unit at the refinery. After the inculation

procedure, the hydrocarbon biodegradation was followed by means of TPH analysis and

soil respiration. In addition, the soil was monitored by the ANTROPOSPHERA–

Instituto para o Desenvolvimento do Meio Ambiente, using the following parameters:

bacteria and fungi total population, hydrocarbonoclastic bacteria and fungi, pH,

microtox, soil electric conductivity, soil organic matter, soil fertility and heavy metals

analysis. In the present work the results of the overall evaluation of the results obtained

in area 1, where the initial TPH decreased from 24,338 µg/g to 3,723 µg/g in the first 99

days after inoculation, and the monitoring for a period of 3 years after the spill.

Keywords: Landfarming Biotreatment System, Indigenous Bacteria, Refinery Oil

Waste, Hydrocarbon Biodegradation

- 209 -

Eco-genomic probes for enhanced soil remediation of former gasworks sites

KT Ralebitso-Senior1*, JJC Dawson2, W Huang1, KS Killham2, W-T Liu3, GI Paton2, J-H Wu3, IP Thompson1

1 Environmental Biotechnology, NERC-CEH Oxford, Mansfield Road, Oxford, OX1 3SR, UK

2 School of Biological Sciences, University of Aberdeen, Aberdeen AB24 3UU, UK 3 Department of Civil Engineering, 10 Kent Ridge Crescent, Singapore 119260,


To measure the response of indigenous soil microbial communities to remediation

programmes, and to gain insight of the effectiveness of the approach, it is essential to

identify the genetic potentials and key limiters of decontamination. With the goal of in

situ and ex situ bioremediation of three former town gasworks sites, characterised by

different contaminant (PAHs, alkanes, BTEX, and coal tars) distributions and

hydrogeological profiles, an Aberdeen site was selected. Twenty kg (fresh weight)

samples of homogenised contaminated soil were placed in triplicate pots after single

additions of: mushroom compost (5% w/w); nutrients (N+P); surfactant (1% v/w); and

mushroom + nutrients + surfactant. Controls with no augmentation and/or regular

mixing were included. Initially, physiological and chemical analyses showed that

surfactant addition promoted respiration rates, MPN counts and total hydrocarbon

degradation rates. However, 16S rRNA gene-based DGGE probing recorded

comparable ‘universal’ community profiles for all of the treatments although numerical-

dominance changes were apparent for some components in the presence of the

chemical. Further analysis of the surfactant-augmented mesocosms revealed subtle

changes in the total communities while naphthalene degrader primers identified marked

temporal differences. Clone libraries facilitated identification of these components.

Preliminary microarray analysis also reflected phenotypic/genotypic variations

congruous with changes in the microbial catabolic potential. Further eco-genomic

analyses should elucidate the naphthalene/PAH/alkane degrading communities in the

different treatments and, in particular, in response to increased pollutant bioavailability

through surfactant addition.

Keywords: Gaswork, Mushroom Compost, Surfactant, 16S Rrna, DGGE, Microarray

- 210 -

Biodegradation of petroleum hydrocarbons and biosurfactant production of Pseudomonas fluorescens and Bacillus subtilis

T.X. Ortega-Trejo*, R. Edyvean

Biological and Environmental Systems Group, Department of Chemical and Process Engineering,

The University of Sheffield, Mappin Street, Sheffield, S1 3JD, UNITED KINGDOM *Corresponding author e-mail: [email protected]

The role of biosurfactants in the biodegradation of hydrocarbons has gained increasing

attention in recent years. One of the limiting factors affecting microbial growth and

subsequently biodegradation of petroleum hydrocarbons is the nature of the interface

between the cell, oil and water. This interface can be considerably modified by bacterial

biosurfactants which aid the formation of a hydrocarbon microemulsion and hence

biodegradation.

Data is presented on the extent, nature and kinetics of biosurfactants produced

by two strains of bacteria (Pseudomonas fluorescens and Bacillus subtilis) in order to

identify the relationships between bacterial growth, substrate utilization, and

biosurfactant production and to evaluate the environmental factors and growth

conditions such as pH, temperature and oxygen availability affecting biosurfactant

production.

Keywords: Biosurfactants, Hydrocarbons, Microemulsion, Pseudomonas fluorescens,

Bacillus subtilis

- 211 -

Isolation and characterization of heavy metal resistant chemolithotrophic iron oxidizing microbes from copper mine wastes:

potential application in biomining and bioremediation

Pinaki Sara*, Kazy Sufia Khannamb

a Department of Biotechnology and b Department of Agriculture and Food Engineering, Indian Institute of Technology, Kharagpur, 721302, India *Corresponding author e-mail: [email protected]

The use of acidophilic chemolithotrophic iron- and sulfur- oxidizing microbes in the

recovering of metals from minerals concentrates is now well established. Looking at the

great potential of these metal oxidizing microbes and their abilities to occupy

inhospitable habitats and conditions, the present work was undertaken to isolate and

characterize chemolithotrophic iron oxidizing microbes from copper ores and mine

wastes of Hindustan Copper Ltd. Khetri, Rajasthan, India. Among the isolated

organisms, a particular strain showing high rate of iron oxidation was selected for

further investigation. It was a Gram negative, autotrophic, acidophilic bacteria with

optimum growth pH 2.6 and temperature 37 ºC, able to use Fe2+ as a sole source of

energy. A time and concentration dependent ferrous iron oxidization ability was noted.

With a high Fe2+ concentration of 100 mM, 100% iron oxidation was achieved within 2

days. However, at lower initial iron concentrations, it was just 24 h or less to complete

the oxidation process. The selected bacteria was extremely resistant to several heavy

metals and radionuclides (100% growth at concentrations of 200 mM copper, nickel and

cobalt, 100 mM zinc, 20 mM cadmium, 5 mM uranium) and retained its 100 % iron

oxidizing efficiency even in presence of such elevated toxic metals. The oxidized iron

species (Fe3+) showed a inhibitory effect on Fe2+ oxidation, however, till a concentration

of 5 mM, a significantly high Fe2+ oxidation was noticed. Preliminary studies on the

biochemical aspect of ferrous oxidation indicated a coupling between protein synthesis

and iron oxidation. Investigations are being conducted to further characterize the strain

as well as the iron oxidation process and its possible use in biomining/bioleaching

technologies for recovery of metals from low grade ores and other metalliferrus wastes.

Keywords: Chemolithotrophic Iron Oxidizing Microbes, Copper Ores, Mine Wastes,

Heavy Metals, Biomining/Bioleaching

- 212 -

Investigations of some soil agents for plant debris biodegradation in undisturbed and phoshpogypsum polluted environments

T.Yatsunenko1*, E.Dolbneyva2, T.Poputnikova2, A.Rakhleeva2, T.Semenova3,

V.Terekhova3

1 Washington University in St.Louis, St.Louis, Missouri, 63130, USA 2 Moscow State University, Department of Soil Science, Leninskie Gory, Moscow 119992, RUSSIA

3 Laboratory of Ecological Soil Functions, Soil Science Institute MSU and Severtsov Institute of Ecology and Evolution RAS, Leninskie Gory, Moscow 119992, RUSSIA


The transformation of natural biota complexes and the rate of biodegradation plant

debris were investigated in undisturbed soils, and environments subjected to chemical

waste pollution – phosphogypsum in Russia. Species successions of microscopic fungi

and soil mesofauna were observed using wide spectrum of methods (synecological

description of communities, direct luminescent microscopy of biomass, chromatography

detection of CO2 emission, etc.) on model cellulose substrates (linden leaves and spruce

needles) during 24 months period.

For the first time it was shown with direct microscopy method that the

destruction of leaves (not needles) without soil mesofauna was higher than that with it.

Sharp distinction in species fungal communities and in morpho-biological biomass

structure in disturbed and undisturbed soils were also at the first stage of destruction of

plant debris. Standart biotesting with Daphnia magna and Paramecium caudatum

showed that the soils on phosphogypsum possess toxicity. Soil toxicity reveals itself in

the intensity of plant debris fungal decomposition. So, for the most part, the rate of plant

debris mineralization depends on micromycetes diversity and technogenic factor.

We confirm that the role of micromycetes at the destruction of plant material is

very important, but at the same time, the activity of fungi and rate of mineralization

mostly depend on the environmental conditions. We particularly have to stress the

usefulness of micromycetes not only as agents of soil bioremediation, but also as

bioindicators of soil pollutions. In spite of its variability, the fungi allowed to get

information for discrimination of biota in standard (undisturbed) and disturbed soils.

Keywords: Phosphogypsum, Micromycetes, Soil Mesofauna, Biomass, Plant Debris,

Mineralization

- 213 -

Evaluating biostimulation of petroleum hydrocarbons in clay soil

Sandro J. Baptista*, Magali C. Cammarota, Denize D. Carvalho

Departamento de Engenharia Bioquímica – Escola de Química– Universidade Federal do Rio de Janeiro – Ilha do Fundão – CEP: 21949-900 – Rio de Janeiro – RJ – BRAZIL


Brazil has intensified efforts on biotechnology research, especially on soil

bioremediation, aiming to solve some environmental impacts resulting from accidental

oil spills. Biostimulation has been used as a technique in order to increase the microbial

activity by adding inorganic nutrients and/or terminal electron acceptor to the

contaminated site. The greatest aim of the present work was to evaluate the

biodegradation of petroleum hydrocarbons in clay soil within a 45-days experiment,

supplemented with nitrogen and phosphorus, in order to stimulate native

microorganisms. Those essays have been conducted using an aerobic fixed bed reactor,

containing 300g of contaminated soil, at room temperature, with an air rate of 6 L/h.

Biodegradation of the crude oil, in the contaminated clay soil, has been monitored by

measuring CO2 production and removal of organic matter (OM), oil and grease (O&G),

and total petroleum hydrocarbons (TPH), together with total heterotrophic and

hydrocarbon-degrading bacteria count. The growth medium was supplemented with

2.5% (w/w) (NH4)2SO4 and 0.035% (w/w) KH2PO4. The best removals of OM (50%),

O&G (37%) and TPH (45%) were verified in the bioreactors in which the highest CO2

production had been achieved.

Keywords: Oil Spills, Biostimulation, Petroleum Hydrocarbons, Clay Soil,

Microorganisms, Aerobic Fixed Bed Reactor

- 214 -

Development of an effective immunochemical test-system for monitoring rhizospheric and soil bacterial degraders in a polluted-soil

environment

Burygin G.L.*, Krasov A.I., Matora L.Yu., Shchyogolev S.Yu

Institute Biochemistry and Physiology Plants and Microorganisms RAS, 13 Prospekt Entuziastov, Saratov, 410049, RUSSIA *Corresponding author e-mail: [email protected]

The plant-growth-promoting bacteria Azospirillum can have the potential to degrade oil

hydrocarbons and to reduce oil toxicity to plant seedling. Azospirillum has also been

shown to be resistant to other pollutants (e.g. pesticides, arsenic compounds). Whether

studies of bacteria as potential biodegraders are successful depends largely on the

availability of rapid and reliable methods for Azospirillum identification and

quantitative determination.

We showed that the method of obtaining strain-specific antibodies to

Azospirillum lipopolysaccharides (LPS), in combination with detecting antigen-

antibody complexes by the enzyme immunoassay, was a highly sensitive test for

enumeration of Azospirillum. The procedure developed by us allowed quantitative

evaluation of introduced bacteria per unit volume of soil over the course of a

remediation by the concentration of antigen excreted by the bacteria in an environment.

It also allowed us to study possible modifications of surface antigens, which reflect the

adaptation of the bacteria to the contaminated-soil environment. This approach was

successfully adopted in the development of an effective method for detection and

enumeration of Dietzia maris AM3, an oil-hydrocarbon-degrading strain introduced into

contaminated soil for remediation purposes.

Acknowledgment: This work was supported in part by grant NSh-1529.2003.4

(Support for Leading Scientific Schools of the Russian Federation)

Keywords: Azospirillum, Oil Hydrocarbons, Plant Seedling, Lipopolysaccharides

(LPS), Strain-Specific Antibodies, Antigen-Antibody Complexes

- 215 -

Mulching for a healthy landscape

N.Bassirani

Dean Science Faculty & General Director Office of International and Scientific Cooperation, Zabol University, Zabol, I.R., IRAN *Corresponding author e-mail:

A result of the geographical and ecological situation of Iran, as well as the blowing of

unfavourable winds in the Central Desert, more than 80% of the 164 million hectare

area of the country is subject to the conditions of arid and semi arid regions,

characterized by low precipitation. At present, the area of the country's deserts and

sandy soils is estimated to be 34 million hectares, and that of the poor and desertified

rangelands which 12 million hectares correspond to the sandy, soils 5 million of the

same shifting sand dunes. Historical evidences and works remained from the ancient

time testify the fact that these regions were covered by desert forests and plants, which

were annihilated because of the wrong and excessive exploitation, the result of which

has been the diminution of the vegetation resources and the conversion of the lands into

deserts. It is estimated that the trends of desertification in the country is about 1%

annually. For as long as trees have grown in forests, leaves and needles have fallen to

the ground and formed a natural protective layer over the soil. This same protection can

be given to the plants in our landscapes by mulching. Mulches conserve soil moisture,

allowing you to water less often; keep downweeds; reduce erosion; keep plant roots

cool; provide winter protection; and make your yard more attractive. Mulches also

simplify lawn mowing around trees and shrubs. A ring of mulch allows you to bring

your lawn mower right up to the edge of the mulch, eliminating the need for string

weeders. At the same time you are protecting tree trunks and surface roots from damage

by mowing and clipping equipment. All mulches have different characteristics, some of

which may be advantages in some situations and disadvantages in others. See the list of

mulches in this brochure for information on individual mulches. Before choosing a

mulch, however,consider how you will use it.

Keywords: Mulch, Dessert, Sandy Soils, Landscapes, Natural Protective Layer

- 216 -

Soil degradation in South Asia with special reference to Iran-A case study of natural hazard

N.Bassirani

Dean Science Faculty & General Director Office of International and Scientific Cooperation,

Zabol University, Zabol, I.R., IRAN *Corresponding author e-mail:

Land degradation causes $10 billion loss to South Asia annually The environmental

impact on the countries of the South by soil erosion and other forms of land degradation

is all too familiar. A recent United Nations study shows the high economic cost of such

land degradations to the South Asian countries. Soil is one of the most vital of our

natural resources. Together with water and air, it forms the very basis of life. Indeed,

there is a thesis, quite difficult to refute, that the rise and fall of civilisations in history

can be linked to the quality and management of their soil and land. For from the soil

comes food and other crops and plants that provide medicine, clothing and so many

other things we need for daily life. And the retention of soil in its natural state and

habitat prevents erosion, river silting and flooding. When land is disturbed or degraded,

the ecology is damaged. There can be rather serious effects in terms of soil erosion, loss

of soil fertility and thus reduced plant growth or crop productivity, clogging up of rivers

and drainage systems, extensive floods and water shortages.

Environmentalists are familiar with the above cause-and-effect scenario. City

planners and water and soil policy makers have to deal with the problem. And ordinary

people experience and live in dread of the bad effects. But soil degradation and erosion

is also an economic issue. The loss of soil and its effects cause tremendous losses to the

economy. So much so that these costs may well outweigh the benefits of many

development projects that give rise to the problem. If only the economic costs of soil

erosion (and other environmental ills) were easily calculable then planners and

politicians would think twice or thrice before allowing activities and projects that

damage the land.

Keywords: Erosion, Land Degradation, Flooding, River Silting, Fertility

- 217 -

Microbiological purification of oil-polluted soils

Chugunov V.A.*, Martovetskaya I.I., Yermolenko Z.M., Mironova R.I., Zhirkova N.A., Zhigletsova S.K, Kholodenko V.P.

State Research Center for Applied Microbiology, Obolensk, Moscow region, Russia *Corresponding author e-mail: [email protected]

To find hydrocarbon-oxidizing bacteria, we screened strains isolated from soil samples.

Such criteria as high oil-oxidizing activity of strains at 28 оС and rather high oil-

oxidizing activity at low temperatures (6-10 оС), as well as their ability to be

bioemulsifiers were taken into account. As a result, an association comprising 4 oil-

oxidizing beneficially complementary strains was produced. The stains also utilized

some polyaromatic hydrocarbons (naphthalene, anthracene, phenanthrene). Based on

the association, liquid and dry formulations with cells immobilized on a carrier were

developed Field trials of the products for 16 months were conducted. The effect of

mineral and organic fertilizers on the dynamics of biodegradation of oil and some

groups of hydrocarbons, as well as on the number of main ecology-trophy groups of soil

microflora was studied. The addition of the formulations along with mineral and organic

fertilizers produced the best effect. The rate of soil bioremediation increased by 2-3

times vs control. Efficacies of both formulations were practically equal for soils

containing oil in concentration of 8 L/m2 The dry product was more effective for soil

with level of oil pollution of 24 L/m2 .

Acknowledgment: The research was carried out in the frame of ISTC Project #2366.

Keywords: Hydrocarbon-Oxidizing Bacteria, Bioemulsifiers, Fertilizers, Ecology-

Trophy Groups Of Soil Microflora

- 218 -

Inoculation of soil microcosms with Sphingomonas paucimobilis. Impact of the inoculation protocol on phenanthrene degradation and

soil community

Del Panno, M.T. 1, Coppotelli, B.1, Morelli, I.S.1, Berthe-Corti, L.2*

1 CINDEFI, UNLP-CONICET, Calle 50 y 115, La Plata, ARGENTINA 2 ICBM, University of Oldenburg, Oldenburg, D 26111 GERMANY *Corresponding author e-mail: [email protected]

Bioaugmentation by inoculating microorganisms into soil is considered a valuable tool

for increasing rate and extent of polyaromatic hydrocarbon (PAH)-biodegradation.

However, the positive effect of inoculation is not always proved. In this study, soil

microcosms artificially contaminated with phenanthrene were inoculated with the PAH-

degrading bacterium Sphingomonas paucimobilis strain 20006FA. Phenanthrene

concentration, structure (DGGE) and activity (CO2-production) of the microbial soil

community were monitored. Microcosms containing garden soil were freshly

contaminated with phenanthrene or pre-contaminated for several weeks. Both systems

were inoculated with free-living or adsorbed cells of S. paucimobilis varying number

and frequency of inoculations. Freshly contaminated microcosms revealed a significant

increase in the population density of heterotrophic bacteria after the first inoculation;

subsequent inoculations did not produce further increase. Pre-contaminated microcosms

revealed no changes in the population density after inoculation. CO2-production in

inoculated microcosms was higher than in non-inoculated controls, indicating a higher

biological activity. Phenanthrene elimination was highest in microcosms inoculated

weekly during three weeks. Immediately after the first inoculation till the end of the

experiment, we observed a strong DNA band corresponding to S. paucimobilis in all

inoculated microcosms. A similar but weak band was detected in the 24day-old non-

inoculated microcosms indicating that S. paucimobilis may also be an indigenous

bacterium of the soil used. The results indicate that S. paucimobilis was able to survive

in freshly and pre-contaminated soil under different inoculation conditions. It enhanced

phenanthrene biodegradation significantly when successive inoculations were realized.

Keywords: Bioaugmentation, Polyaromatic Hydrocarbon (PAH), Phenanthrene, Soil

Microcosms, Sphingomonas paucimobilis

- 219 -

Phenanthrene degradation in soil bioremediation assays.

Díaz Puente, F.J.*, Rodríguez, V., Escolano, O., García, S., Pérez, R., García Frutos, F.J.

Environment Department, CIEMAT, Avda. Complutense 22, 28040 Madrid, SPAIN


We have studied optimal conditions to mineralise phenanthrene in soil, modifying the

following conditions: aeration, C/N/P ratio and pulp density. Mineralisation of freshly

spiked soil (1000 mg phenathrene/ Kg dry soil) has been followed by soil respirometry.

Results show that aeration is the most influent factor in phenanthrene mineralisation.

Phenanthrene has been used as C and energy source by soil microorganisms before the

own soil organic matter.

Attending CO2 production –and O2 consumption- up to 68% phenanthrene is

mineralised in 8 days. Nevertheless chemical analysis shows a higher loss of

phenanthrene than indicated by respirometry. When studying microbiological

parameters, a clear increase in CFU was found, not being so high the rise of

phenanthrene degraders, this happened during the most active phase of phenanthrene

mineralisation (9 days) and after a longer period of time (up to 30 days).

Chemical and microbiological analysis may indicate phenanthrene degradation

to any metabolite. In order to obtain a full balance of phenanthrene we are performing

new trials with [9-14C] phenanthrene. Up to now, we have confirmed mineralisation

kinetic observed by respirometry. Loss of phenanthrene could be due to volatilisation,

but analysis of volatile compounds in traps has not show yet the presence of any

metabolite.

Finally, in order to test the toxicity of the polluted soils, some Microtox assays

have been performed on samples before and after treatment (mineralisation), showing a

reduced toxicity for the potential metabolites.

Keywords: Phenanthrene, Mineralisation, Respirometry, Volatilisation, Microtox

Assays

- 220 -

Degradation potential of hydrocarbons by cold-adapted sea-ice bacteria isolated from oil contaminated enrichment cultures and oil-

biodegradation studies with Arctic sea-ice

Birte Gerdes, Gerhard Dieckmann, and Elisabeth Helmke

Alfred-Wegener-Institute for Polar and Marine Research, Bremerhaven, GERMANY

*Corresponding author e-mail:

The danger of a petroleum hydrocarbon spillage in the polar, ice-covered regions is

increasing due to oil exploration in Arctic offshore areas and a growing interest to use

the Northern Sea Route (NSR) as an alternative transportation route for Arctic oil and

gas. However, little is known about the potential impact of accidental oil spills on this

environment. The sensitive sea-ice ecosystem in particular is highly vulnerable. Up to

now there is very little information on the potential effects of hydrocarbon pollution. In

spite of the harsh conditions the bacterial sea-ice communities are highly productive and

closely adapted to the cold temperatures with a percentage of about 90% psychrophiles.

We have investigated the effect of oil on sea-ice bacteria and studied their

potential to degrade hydrocarbons at low temperatures. Bacteria from oil enrichment

cultures and bioremediation studies with Arctic sea-ice were isolated. One of the

dominating genera in the oil enrichment cultures was Marinobacter, which is ubiquitous

and found world-wide, at oil polluted locations. The sea-ice isolates belong to the

bacterial divisions of the γ-proteobacteria, α-proteobacteria, β-proteobacteria,

Cytophaga-Flavobacterium group, as well as to the gram-positive Actinobacteria. They

proved to be well adapted to the cold Arctic conditions and most of them grow still at –

5°C. The oil degrading capacity was measured by [14C]-labeled hexadecane,

naphthalene, and phenanthrene incubation. All three oil-components were degraded

relatively fast at +1°C. The experiments showed that the sea-ice bacteria adjust

relatively fast to hydrocarbon contamination and are capable to degrade oil albeit at a

slower rate.

Keywords: Petroleum Hydrocarbon, Oil Spill, Sea-Ice Ecosystem, Psychrophiles,

Enrichment Cultures

- 221 -

Does phytostimulation play a significant role in the biodegradation of hexachlorocyclohexane (HCH) isomers?

Kidd, P.S., Prieto-Fernández, A.*, Acea M.J.

Dpto. Bioquímica del Suelo, Instituto de Investigaciones Agrobiologicas de Galicia,

CSIC, Aptdo. 122, Santiago de Compostela 15780, SPAIN *Corresponding author e-mail: [email protected]

The persistent organic pollutant (POP), hexachlorocyclohexane (HCH), was used

extensively as a commercial insecticide in two formulations: technical HCH, which

includes multiple stereoisomers and is dominated by α-HCH (~80 %) and γ-HCH (10-

15 %), and lindane, which consists almost entirely of the γ-isomer. HCH isomers are

commonly present in the environment due to their agricultural use and the land disposal

of waste products obtained during lindane fabrication. The potential to use plants to

remediate polluted soils has attracted much interest, a technique known as

phytoremediation. Phytostimulation involves the combination of microbial

bioremediation and phytoremediation in a complementary manner by using select

vegetation and root-associated microorganisms to enhance biodegradation of organic

pollutants such as PCBs and PAHs. Here we investigate the possible stimulatory effects

of plant roots on the biodegradation of HCH isomers. Holcus lanatus and Cytisus

multiflorus were germinated from seed collected from plants growing on HCH-

contaminated soils in Porriño, Galicia (NW Spain). Soil from the same site, which is

expected to contain HCH-degrading microorganisms, was collected for inoculating

purposes (3% w/w). Plants were grown in the following treatments: uncontaminated soil

(control), soil + 0.01% HCH, soil + 0.01% HCH + inoculation, and soil + inoculation.

Plants were grown for five months in a greenhouse and soil solutions were sampled

every two weeks. At harvest rhizosphere soil was separated from bulk soils. The

following analyses were made: pH, EC, chloride concentration, HCH degradation and

plant HCH concentration. The importance of phytostimulation in HCH degradation will

be discussed.

Keywords: Persistent Organic Pollutant (POP), Hexachlorocyclohexane (HCH),

Phytoremediation. Phytostimulation, Root-Associated Microorganisms

- 222 -

Environmental impacts of cyclodextrins enhancing bioremediation efficiency

Bálint Oldal1*, Attila Anton1, Nikolett Uzinger1, Éva Fenyvesi2, Katalin Gruiz3,

Attila Murányi1

1 Research Institute for Soil Science and Agricultural Chemistry, Hungarian Academy of Sciences, H-1022 Budapest, Herman Ottó út 15, HUNGARY

2 CycloLab Cyclodextrin Research & Development Laboratory, Ltd., H-1097 Budapest, Illatos út 7, HUNGARY

3 Department of Agricultural Chemical Technology, Faculty of Chemical Engineering, Budapest University of Technology and Economics, H-1111 Budapest, Műegyetem rkp. 3., HUNGARY


Bioremediation efficiency can be enhanced by application of additives. Cyclodextrins

are among these agents due to their amphiphilic behaviour, improving organic pollutant

availability. However, as each additive, they may deal with promoting, neutral or

suppressive chemical and biological effects, whereas toxicity restricts their application.

Influence of four cyclodextrins (β-, random methylated β-, (2-hydroxy)propyl β-

and acetyl β- cyclodextrin) on the proliferation of two characteristic soil microbes

(Fusarium moniliforme and Pseudomonas syringae) was studied in model experiment.

Microorganisms were grown in optimised Czapek liquid culture medium. Biological

(CFU mL-1) and chemical parameters (e.g. pH, redox potential, dissolved C, -P) were

monitored simultaneously. Cyclodextrins promoted significantly the proliferation of F.

moniliforme in liquid culture, being acetyl β-cyclodextrin the most effective. Chemical

data showed that Fusarium monotonously acidified the medium and decreased

dissolved P, whereas dissolved C described a maximum curve for 48-hour incubation.

Interactions of cyclodextrin and saccharose on Fusarium mycelium growth were

assessed by biological test in solid culture medium. Biological test revealed that neither

cyclodextrin nor saccharose affect fungal mycelium growth. The growth of P. syringae

was only influenced by acetyl β-cyclodextrin,which substantially inhibited bacterial

growth. Natural β-cyclodextrin is poorly soluble in the soil water phase, thus we

propose environmental friendly (2-hydroxy)propyl β-cyclodextrin and random

methylated β-cyclodextrin for enhancement of bioremediation efficiency.

Keywords: Additive, Cyclodextrins, Fusarium moniliforme, Pseudomonas syringae,

Organic Pollutant

- 223 -

Soil reclamation in the vicinity of the Usinsk oil field (the Republic of Komi, Russia)

Likhanova Irina A., Archegova Inna B., Khabibbulina Flyuza M.

Dirección Email

Most technogenic objects of the Usinsk region of the Republic of Komi (66° N, 57° E)

are represented by sand-pits and boreholes, covered with 1-m thick sand layer. When

compared between different agro-technical restoration methods, most effective appeared

to be peat (20 t/ha) and mineral fertilizer (N60P60R60) application, and sowing perennial

local grasses. 11 years after the sowing procedure, the grasses ascertain formation of

soddy organic-accumulative soil layer 7-9 cm deep, organic carbon 4-5%, hydrolysable

nitrogen about 3 mg/100 g air dry soil, microorganisms 4 mill colony-forming units/1 g

air dry soil. The minimum soil reclamation rate belongs to the method of planting

spruce and pine seedlings without fertilizers. Even a pine tree 3 m high with 0.4 crown

density cannot provide for quick soil reclamation. Sand substratum does not reveal any

changes, any soil profile morphological differentiation to the 12th living year, organic

carbon 0.2-0.8%, hydrolysable nitrogen around 1 mg 100 g air dry soil, microorganisms

10 ths colony-forming units/1 g air dry soil.

Keywords: Soil Reclamation, Sand-Pits, Boreholes, Peat, Mineral Fertilizer, Sowing

Perennial Grasses

- 224 -

Evaluation of aliphatic hydrocarbons behavior on gasoline contaminated soil in natural attenuation and in bioremediation process

using biosurfactant

Latanze, R.1*, Kronka, E. M.1, Ferreira, J. C.2, Benincasa, M.1

1 Centro Tecnológico de Desenvolvimento Agroindustrial, UNAERP/Ribeirão Preto-SP, Av. Costábile Romano, 2201, CEP: 14096-380, Ribeirão Preto/SP, BRAZIL

2 Centro de Pesquisa e Desenvolvimento Leopoldo Américo M. de Mello-CENPES- PETROBRAS-RJ

*Corresponding autor e-mail: [email protected]

The contamination of soil and groundwater by leaks from the gas station tanks has

received great attention of the environment agencies and of population in general. In

São Paulo State (Brazil) there are approximately 9 thousands gas stations which are

reaching their lifetime, resulting in a fuel release due to the natural aging. The major

problem of this contamination is their high toxicity over alive organisms. In this way, an

effort in developing cleaning up technologies has been done.

The gasoline is composed by two fractions, aliphatic and aromatic hydrocarbons.

The aim of this work was to evaluate the aliphatic hydrocarbons behavior on gasoline

contaminated soil in two situations: natural attenuation and bioremediation process

using a biosurfactant produced by Pseudomonas aeruginosa LBI. Therefore, bench

experiments with native soil contaminated with a determined amount of gasoline were

carried out. The hydrocarbon behavior was evaluated in treatments with biosurfactant

addition (1 mg/g soil) and in its absence by a GC-FID. The preliminary results has

demonstrated that this technique is quite sensitive in the gasoline contamination

detection, what makes it an important tool on environment assessment. It was also

observed that the biosurfactant addition at this concentration seems to inhibit the

aliphatic hydrocarbons biodegradation.

Acknowledgment: Financial Support: FAPESP, UNAERP

Keywords: Gas Station Tanks, Aliphatic Hydrocarbons, Biosurfactant, Pseudomonas

aeruginosa, Bench Experiments, GC-FID

- 225 -

Use of the rhizospheric bacterium Azospirillum brasilense Sp245 in arsenic bioremediation

Yelena V. Lyubun1*, Andreas Fritzsche2, Marina P. Chernyshova1

1 Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences,

13 Prospekt Entuziastov, Saratov 410049, RUSSIA 2 Institute of General Ecology and Environmental Protection, University of Technology Dresden,

Pienner Strasse 19, D-01737 Tharandt, GERMANY *Corresponding author e-mail: [email protected].

In recent times, environmental pollution caused by arsenic (As) has become a serious

problem. Soils often contain high concentrations of various natural and manmade As

compounds (e.g., extensive agricultural and forestry lands treated with As-containing

pesticides or fertilized with waste sludges). The toxicity of As depends on the content of

its soluble forms in soil. Currently plants and their associated microflora are finding

increasing use for soil bioremediation. Members of the genera Pseudomonas and

Azospirillum are the predominant gram-negative bacteria in the rhizosphere.

We studied the biotransformation of sodium arsenite and sodium arsenate by the

rhizospheric bacterium Azospirillum brasilense Sp245, associated with wheat (Triticum

aestivum L. ‘Saratovskaya 29’), and the effect produced by this strain on the plant’s

morphological parameters in an As-polluted environment. Our laboratory experiments

used a hydroponic system, with glass beads serving as the substrate. The results showed

that inoculation of A. brasilense Sp245 influenced wheat root length, which can be

explained by the auxin-synthesizing capacity of this strain. Strain Sp245 was able to

effect reactions leading to a change in As valency and to an increase in As

bioavailability to plants. More specifically, Azospirillum inoculation of plants grown in

a model medium containing a mixture of As salts [50% As(V) and 50% As(III), v/v]

increased the content of plant-available sodium arsenate while decreasing considerably

the total As concentration. The results of these laboratory experiments suggest that A.

brasilense Sp245 can be used for plant inoculation as part of bioremediation strategies

for As-polluted soils.

Keywords: Arsenic (As), Microflora, Pseudomonas, Azospirillum, Rhizosphere, Wheat

- 226 -

Microbiological destruction of yperite in soil

Nadezda Medvedeva 1*, T. Zaytceva 1, S. Zinovieva 1, O.G. Orlova 1, G.A.Zharikov 2

1 Scientific Research Center of Ecological Safety, Russian Academy of Sciences, Korpusnaja 18, Saint-Petersburg, 197110, RUSSIA

2 Research Center for Toxicology and Hygienic Regulation of Biopreparations, Ul. Lenina, bld. 102А, Serpukhov, 142253, RUSSIA


Among xenobiotics getting into the environment as a result of human activities the most

dangerous are war gases including yperite. Yperite is poorly soluble in water and

sufficiently stable to hydrolysis. Its water-soluble part undergoes a two-stage hydrolysis

to give the low-toxic thiodiglycol. The process is reversible, and for its completion

stirring and boiling in a large excess of the low alkaline solution is necessary.

Natural degradation of yperite in soil is a result of chemical and biochemical

processes. The main product of the hydrolysis of yperite is thiodiglicol. The complete

degradation of yperite and its chlorine-containing derivatives does not take place. The

residual amounts of yperite are detected in soils even a year after its application. The

process of biodegradation of thiodiglycol is more long and it may take several years.

For the acceleration of purification of soil from yperite the saprophytic microorganisms

may be used. The active yperite-degrading microorganisms were isolated from soils. In

model experiments the dynamics of dechlorinating yperite in soils and consuming the

products of its transformation with selected cultures-destructors was studied. The

isolated degrading microorganisms are highly active while their application to yperite-

contaminated soils. Complete destruction of yperite and thiodiglycol, taking place with

the participation of these microorganisms is accompanied by the increase in soil

biogenity, that is the increase in numbers of microorganisms, their range of species, the

increase in the activity of soil enzymes, and sharp decrease in phytotoxicity.

Acknowledgment: Studies are financially supported by the ISTC project #2488.

Keywords: Xenobiotics, Yperite, Hydrolysis, Thiodiglycol, Saprophytic

Microorganisms

- 227 -

Peroxidases produced for Psilocybe castanella in soil contaminated with hexachlorobenzene

Moreira Neto, S.L.1, Machado, K.M.G.2, Matheus, D.R.3*

1 Mestrando, Instituto de Botânica, São Paulo

2 Fundação para o Desenvolvimento da Pesquisa Agropecuária/FUNDEPAG, São Paulo 3 Seção de Micologia e Liquenologia, Instituto de Botânica,

Av. Miguel Stéfano, 3687 - Água Funda, CEP 04301-012, São Paulo, SP, BRAZIL *Corresponding author e-mail: [email protected].

In São Paulo State, the most industrialized state of Brazil, mycoremediation has been

evaluated as an interesting alternative to reduce the contamination of soils contaminated

with industrial wastes containing organochlorine compounds. The effect of the

hexachlorobenzene (1104 mg HCB Kg-1 soil) on the production of peroxidase enzymes

by Psilocybe castanella in contaminated soil was evaluated. The fungal inoculum was

prepared using sugar cane bagasse supplied with soy flour incubated during 21 days at

28°C. The fungus was inoculated in the soil and incubated during 70 days. The moisture

content was adjusted weekly. Peroxidases and MnP activities were detected using ABTS

and phenol red as substrate, respectively. The enzymatic extract was obtained with

sodium acetate buffer (50 mM, pH 4.6). Fungal biomass was estimated by ergosterol.

HCB and Cl- were quantified by gas chromatography and potentiometric titration,

respectively. HCB had no effect on fungal growth and the stationary phase started at 30

days. After 70 days of incubation, depletion of 32.40% of HCB and increase of 53.34%

of inorganic chloride were observed. The presence of HCB influenced the production

profile of peroxidases. In the soil with HCB the highest activity of peroxidases was

observed at 12 days (103.71 UL-1) while in the soil without HCB, at 19 days (80.25 UL-

1). On the other hand the highest MnP activity was observed at 5 days in soil with HCB

(0.74 UL-1) and at 7 and 47 days in the soil without HCB (0.44 e 0.43 UL-1).

Acknowledgment: Rhodia do Brasil Ltda/FUNDEPAG and Secretaria do Meio

Ambiente do Estado de São Paulo.

Keywords: Mycoremediation, Organochlorine Compounds, Hexachlorobenzene,

Psilocybe castanella, Peroxidase Enzymes

- 228 -

Study of the bioremediation of a petroleum contaminated soil

Danielle Marins Pala1*, Denize Dias de Carvalho2, Geraldo Lippel Sant’Anna Jr.1

1 UFRJ, COPPE/Programa de Engenharia Química, Brazil 2 UFRJ, Escola de Química/Departamento de Engenharia Bioquímica, BRAZIL


A critical environmental impact of the petroleum industry is the spillage of oil and

related products that causes severe soil contamination. Although biodegradation of

petroleum hydrocarbons may be successfully conducted under controlled conditions, the

bioremediation of large volumes of contaminated soils still present some technical

challenges, particularly when contamination occurs in soils with high clay content.

The application of bioremediation processes in recuperating contaminated sites

is gaining ground, due to its efficiency in removing a number of different classes of

contaminants and its cost, which tends to be lower than that of other technologies. In

this work, the influence of some factors that are important for the biodegradation of the

petroleum present in clayed soil, contaminated accidentally, was studied. Experiments

were conducted using three types of reactor, a slurry-phase reactor, a fixed bed reactor

and a biopile. The processes employed were shown to be highly effective in

decontaminating the soil, achieving removal levels of around 45% for the slurry-phase

reactor and 80% for the fixed bed reactor. No removal of contaminants was observed for

the biopile process, which was probably due to the low water content and low soil

porosity, thereby making it difficult for oxygen to access the internal regions of the pile.

Keywords: Petroleum Hydrocarbons, Clayed Soil, Slurry-Phase Reactor, Fixed Bed

Reactor, Biopile

- 229 -

Effect of selenooxyanions on tellurite removal by a new moderately halophilic bacterium, Salinicoccus sp. strain QW6

M. A. Amoozegar*, M. Ashengrop, F. Malekzadeh

Biology Department (Microbiology Division), Faculty of Science, Tehran University, Tehran, IRAN


Tellurite is a rare-earth oxyanions that can be found in high concentrations in land and

water near sites of waste discharge of industrial manufacturing processes. Tellurite is

highly toxic to mammalian cells and microorganisms at concentrations as low as 1 µg

ml-1. Removal and bioreduction of tellurite (TeO3-2) by bacteria has been studied in

previous works, but little is known about uptake of this oxyanion in halophilic

microorganisms.

In the current study, high-level tellurite-resistant moderately halophilic coccus

was isolated and tellurite removal from the growth medium was determined. According

to Bergey's Manual of Systematic Bacteriology and the physiological and biochemical

tests formed, it was tentatively named as Salinicoccus sp.strain QW6. The quantitative

determination of tellurite was done using the reagent diethyldithiocarbamate (DDTC).

The influence of various concentration of selenooxyanions (2, 4, 6, 8, 10 mM)

on tellurite removal was determined. The isolate was capable of removal tellurite in the

presence of Na2SeO3 and Na2SeO4. The maximum removal of tellurite was showed in

the presence of 6 mM Na2SeO3. By highering the Na2SeO3 concentration from 2 mM to

6 mM, tellurite concentration in the supernatant of culture decreased to below 0.05 mM

(approximately 91% of tellurite was removed) within 3 days when 0.5 mM of tellurite

was added to the growth medium. At the same conditions, enhancing the Na2SeO4

concentrations from 2 to 10 mM, tellurite removal decreased and maximum removal

was shown in 2 mm Na2SeO4 (approximately 62% of tellurite was removed).

Keywords: Tellurite, Oxyanion, Bioreduction, Halophilic Microorganisms, Salinicoccus sp

- 230 -

Bioreduction of tellurite and selenite by a new moderately halophilic bacterium, Salinicoccus sp. strain QW6

M. A. Amoozegar*, M. Ashengrop, F. Malekzadeh



Tellurium and selenium oxyanions are found as trace components in natural

environments as well as in higher concentrations within polluted soils and water.

Tellurium compounds can be found in high concentrations near sites of waste discharge,

while selenium is present in the soluble form in a alkaline soils and water. Some of the

tellurium and selenium oxyanions are highly toxic and mutagenic for bacteria and

mammals.

In the current study, high-level tellurite and selenite resistant moderately

halophilic coccus was isolated and the reduction of tellurite and selenite and the effect

of a variety of parameters on this reduction was determined. According to Bergey's

Manual of Systematic Bacteriology and the physiological and biochemical tests formed,

it was tentatively named as Salinicoccus sp.strain QW6.

A microbiological process using Salinicoccus sp.strain QW6 was developed for

the reduction of tellurite and selenite. During the process tellurite and selenite have

undergone a transformation in the black metallic elemental tellurium and red metallic

elemental selenium. The deposits were observed with a transmission electron

microscope and confirmed by X-ray energy-dispersive spectrogram analysis. Studies

were carried out in Erlen-Meyer flasks (100 ml) containing 25 ml sterile nutrient

medium supplemented with 0.5 mM tellurite and 6 mM selenite. The media were

inoculated with cell suspensions (1.5 × 108 cfu/ml) and incubated under stationary

conditions (T: 35 ºC, pH 7.5, agitation: 150 rpm, for 72 h). After incubation the contents

of the flasks were analysed for residual tellurium and selenium by X-ray fluorescence

analysis (XRF).

Keywords: Tellurium Oxyanions, Selenium Oxyanions, Salinicoccus sp, Transmission

Electron Microscope, X-Ray Energy-Dispersive Spectrogram, X-Ray Fluorescence

Analysis (XRF)

- 231 -

Evalution of chromate resistance in halophilic and halotolerant bacteria and effect of salinity on this tolerace

M.A.Amoozegar*, A.Ghassemi



Hexavalant chromium is a toxic pollutant in soil and wastewaters that can be found in

high concentration near sites of industrial wastes. Chromate is a strong oxidizing agent

that is reduced intracellulary to Cr5+ and react with nucleic acid and other cell

compound to produce mutagenic and carcinogenic effect on biological systems.

Tolerance of bacteria to chromate has been studied in previous works, but little is

known about this tolerance in halophilic microorganisms. In our study, we isolated 110

strains of bacteria from different environments(saline soils, tannery wastes, wastewater

of paint industry and soils around of manufacture of metallic alloys) in Iran. Among

them 30 strains were moderately halophiles and 80 strains were halotolerant. Agar

dilution method was used to determine the bacteria tolerance to chromate in 30 strains

of moderately halophiles and 20 strains of halotolerants. Minimum inhibitory

concentration (MIC) for chromate was determined. The MIC was defined as the

minimum concentration of inhibitor preventing growth at 34º C, pH 7.3 ,10 %(w/v)

NaCl and one week incubation. Among strains, SW3, L3 and MF2, halophilic bacteria,

and A6 and L4, halotolerant bacteria, showed high-level resistance to chromate. The

strains SW3, L3 and MF2 could tolerate 700 mM chromate, while strains A6 and L4

showed tolerance to potassium chromate at 450 and 500 mM , respectively. The

influence of salinity on tolerance to chromate was determined. In halophilic strains

SW3, L3 and MF2, enhancing the salinity from 1 to 15 %(w/v), increased chromate

tolerance.

Keywords: Chromate, Halophilic Microorganisms, Halotolerant, Minimum Inhibitory

Concentration (MIC), Salinity

- 232 -

Monitoring of survival of phenanthrene utilizing Sphingobium sp. P2 in soil microosms by using green fluorescent protein as a marker

Onruthai Pinyakong1,2*, Nattachai Kengpipat1

1 Department of Microbiology, and 2 National Research Center for Environmental and Hazardous Waste

Management (NRC-EHWM), Faculty of Science, Chulalongkorn University, Bangkok 10330, THAILAND


Polycyclic aromatic hydrocarbons (PAHs) are one class of toxic environmental

pollutants that have accumulated in the environment due to variety of anthropogenic

activities. Bioaugmentation is the most common practice to enhance bioremediation of

the contaminated site if the effective pollutant-degrading microorganisms are not

naturally present at the site. For the successful bioaugmentation, survival, persistence,

and the metabolic activity must be assessed prior to release of particular

microorganisms to the environment.

Phenanthrene is a three ring PAH widely used as a model compound in PAH

biodegradation studies. In this study, the green fluorescent protein (encoded by gfp

gene) was used as a marker to estimate survival of a phenanthrene-degrading

Sphingobium sp. strain P2 in soil microcosms. The gfp was integrated via Tn5

transposition into the chromosome of strain P2. The gfp inserted strains were identified

by green fluorescence emission under UV light. The gfp was stably maintained in strain

P2 and it still retained the ability to utilize phenanthrene as sole carbon and energy

sources. In bioaugmentation studies, two types of soil were used to construct eight types

of soil microcosms. Effect of physical and chemical properties of soil microcosm

systems on the survival and the ability to degrade phenananthrene of strain P2 was

determined by using gfp as a monitoring reporter system.

Keywords: Polycyclic Aromatic Hydrocarbons (PAHs), Bioaugmentation, Pollutant-

Degrading Microorganisms, Phenanthrene, Sphingobium sp, Soil Microcosms

- 233 -

Bioremediation of cyclic chlorinated compound (1, 2-Dichlorobenzene) in contaminated soil

Mahasta Ranjbar1*, Soheila Yaghmaei2

1 Azad University of Tehran / Science & Research Branch – Department of Chemical Engineering and

Biotechnology 2 Sharif University of Technology – Department of Chemical Engineering and Petroleum Engineering


1,2-Dichlorobenzene (1,2-DCB) is one of the chlorinated cyclic compounds of the

transformer oil. They were used extensively in industry as insulating fluids for

transformers and other electrical equipment. The widespread use of these compounds in

industrial applications has resulted in a tremendous release of 1,2-DCB into the

environment. It happened when discarded devices deteriorated and 1,2-DCB leaked into

nearby soil and water. Ironically, the properties which make transformer oil as good

insulators due to their stability and lack of reactivity are also the properties that make

them long-lasting harm the environment.

Clean-up of 1,2-DCB-contaminated media is quite expensive in the existing

technologies. In addition, all these technologies result in the generation of undesirable

byproducts. Bioremediation of 1,2-DCB contaminated soils because of its relatively

worth is nowadays attracting attention and it permits the development of in situ

treatment scheme and usually does not result in the generation of additional hazardous

waste. The problem is that those microorganisms capable of transforming 1,2-DCB are

commonly found in soil previously contaminated with transformer oil. For many years

this great quantity of oil, has been stored in transformers in “Bandar Abbas Power

Station”, south of Iran, causing the soil of this area to be contaminated.

The first step of our consideration after collecting three samples of soils from

“Bandar Abbas Power Station”, was focusing on isolation and purification of 1,2-DCB

utilizing microorganism in the soil. As further steps were taken, the best microorganism

was chosen according to its ability of transforming 1.2-DCB and then the optimum

conditions of the chosen microorganism were recognized.

Keywords: 1,2-Dichlorobenzene (DCB), Bioremediation, Gas Chromatography,

Transformers Oil, Byproducts

- 234 -

Decomposition of chlorophenol pollutants in soil by microbial degraders

A. Samsonova*, Z. Aleschencova, N. Siomachkina N.F., A. Hlushen

Address

*Corresponding author e-mail:

Chlorophenols as extremely hazardous organic pollutants are distinguished by high

persistence to photolytic, chemical and biological decomposition. They may be retained

in environment for long periods. Physical-chemical processes of soil remediation are

costly and not always be applied for contaminated natural environmental objects.

Ecologically safe biotechnological methods are currently successfully introduced to

detoxify contaminated wastewater and soil. One way to intensify xenobiotic degradation

in soil is introduction of pure microbial cultures initiating break-down of the pollutants.

Acceleration of 2,3- and 3,4-dichlorophenols degradation in soil was achieved

by introducing Rhodococcus erythropolis 70F and Bacillus cereus 14 XF cultures. R.

erythropolis culture applied into soil contaminated with 2,3- and 3,4-dichlorophenols at

0.3% concentration promoted their decomposition by 22.95% and 35.8% respectively.

The same culture immobilized on peat carrier ensured stimulation of 2,3- and 3,4-

dichlorophenols degradation after 28 days by 91% and 95%, respectively, as compared

to the control. B. cereus culture introduced into polluted soil intensified degradation of

2,3- and 3,4-dichlorophenols. Upon 28 days 2,3- and 3,4-dichlorophenols at 0.3%

concentration were degraded in soil by 7.15% and 33% more effectively in presence of

introduced bacteria. Application into soil of B. cereus culture immobilized on peat

support promoted more rapid removal of dichlorophenols in comparison with native

microbial degraders. 2,3-dichlorophenol was decomposed more effectively by microbial

degrader immobilized on peat carrier by 3.8%, 58.9%, 85.2% and 88.6% after 7, 14, 21,

28 days, respectively. 3,4-dichlorophenol was removed by immobilized B. cereus

culture in the same period even more efficiently (38.9%, 73.8%, 90.4% and 94.2%) as

compared to native microorganisms.

Keywords: Chlorophenols, Xenobiotic, Rhodococcus erythropolis, Bacillus cereus,

Peat Carrier

- 235 -

Extraction of ligninolytic enzymes from solid substrates

Jaroslav Šnajdr, Vendula Valášková, Petr Baldrian*

Laboratory of Biochemistry of Wood-Rotting Fungi, Institute of Microbiology ASCR, Videnska 1083, 14220 Praha 4, CZECH REPUBLIC *Corresponding author e-mail: [email protected]

Ligninolytic enzymes of white-rot fungi (oxidases and peroxidases) participate in the

degradation of lignin contained in wood and can also degrade xenobiotic compounds of

similar structure like PAH, PCB or synthetic dyes. For the evaluation of bioremediation

efficiency, a reliable method for enzyme activity measurements is required. When

sampling soild substrates (soil, forest litter, or lignocellulose substrate), the enzyme

activity has to be recovered from the substrate before the measurment.

We studied the effect of different extraction protocols for the recovery of the

ligninolytic enzymes laccase and Mn-peroxidase from wheat straw, soil and forest litter

colonized by white-rot fungi. Wheat straw is conveniently used as a substrate for the

introduction of white-rot fungi into soil, whereas soil and forest litter are the common

substrates to be treated by mykoremediation. The most suitable extraction of wheat

straw colonized by Pleurotus ostreatus and Trametes versicolor was with acetate buffer

(pH 5.0) or distilled water, whereas phosphate buffer (pH 7.0) was the most suitable for

soil extraction and distilled water for forest litter extraction. In the case of forest litter,

we detected the presence of low molecular weight compounds that inhibited the activity

of laccase and Mn-peroxidase. These compounds can be removed from the sample by

desalting or dialysis prior to enzymatic measurements.

Keywords: White-Rot Fungi, Ligninolytic Enzymes, Xenobiotic Compounds, Wheat

Straw, Mykoremediation, Pleurotus ostreatus, Trametes versicolor

- 236 -

Influence of lignin products on efficiency of bioremediation of contaminated ecosystem objects

Galina Telysheva1*, Tatiana Dizhbite1, Galina Lebedeva1, Vizma Nikolaeva1,

Angela F. Danil de Namor2, Saulius Grigiskis3

1 LS Institute of Wood Chemistry, 27 Dzerbenes st., LV-1006, Riga, LATVIA 2 Thermochemistry Laboratory, Department of Chemistry,University of Surrey,

Guildford, Surrey, GU2 7XH, UK 3 Biocentras Ltd., 10 Graichuno st., LT-2028, Vilnius, LITHUANIA


Usage of plant biomass-processing waste for decontamination of ecosystem objects

(soil and water) is expedient due to the sorption and complexing properties of

lignocellulosic matrices.

The approach developed is based on application of Si-modified lignocellulose,

that is an effective agent promoting mineralization of organic pollutants by a native soil

microbial community (exemplified in the present work with 2,4-D mineralization and

oil products biodegradation in unplanted and planted soil in laboratory, microfield and

field trials) as well as products designed on the basis of lignin and biocide metals that

adsorb pathogenic microflora and inhibit their development. Two types of commercial

lignins: hydrolysis and kraft lignins, differed significantly by their chemical and porous

structure, were used as the raw-material (matrix) for obtaining of the products

developed. Non-modified lignins were also tested in the same conditions as products

designed on their basis. The optimal conditions for Cu2+ fixation were found. It was

shown that lignin modification via non-covalent interaction with Si-containing

oligomers before Cu introduction increased complexing ability in comparison with non-

modified lignin matrix, and provided formation of a novel uniform mesopores (diameter

of 4-5 nm) structure and more than threefold increase in the specific surface. The

products obtained on the basis of Si-premodified lignin are more effective in bacteria

sorption (exemplified with Escherichia coli and Staphylococcus aureus).

Keywords: Plant Biomass, Si-Modified Lignocellulose, Mineralization, Organic

Pollutants, Native Soil Microbial Community

- 237 -

Impacts of arbuscular mycorrhizal fungi and Pseudomonas putida on distribution of some heavy metals in soils and faba bean plants

M Attia

Agricultural Microbiology Dept. National Research Centre Dokki, Giza, EGYPT


Field experiment was carried out to examine the interactions between Ps. putida and

arbuscular mycorrhizal (AM) fungi and their effects on nodulation and elements

distribution in faba bean plants and soil in the presence of 25 or 50% mineral fertilizers

(NPK). The soil before cultivation have noticeable amounts of copper and zinc in total

form also Cu and Zn in available form. During the plant growth, the soil pollution rate

increased with the investigated elements except Cu which decreased. The best treatment

led to decrease the soil pollution lead is to inoculation plants with AM fungi plus Ps.

putida and in combination with 50% NPK. Stimulation of AM fungi colonization by a

Ps. putida was noted. AM fungi alone were significantly enhanced nodulation.

Significantly more nodules had developed on the root systems of faba bean plants

inoculation with Ps. putida + AM fungi compared to AM alone or uninoculated control.

Plant inoculation with both organisms increased the population of Pseudomonas spp.

The best treatments to decrease Cu pollution are those inoculated with AM fungi + 25%

NPK followed by AM fungi+Ps.+50%NPK in mature stage. All plant parts contained

Pb and in mature stage the beast treatment to decrease Pb pollution is to use 25% NPK+

AM fungi. The concentration of zinc in all young plant parts are higher than 400 mg/kg

then decreased when the plant growth reached mature stage. The use 50% NPK +AM

fungi is the best treatment to decrease Zn content in the tested plant.

Keywords: Arbuscular Mycorrhizal (AM) Fungi, Faba Bean Plant, Heavy Metals, P.

Putida, Soil Pollution

- 238 -

Effect of PAHs concentration in three white rot fungal strains

Valentín, L., Feijoo, G.*, Moreira, M.T., Lema, J.M.

Department of Chemical Engineering, University of Santiago de Compostela, 15782 – Santiago de Compostela, SPAIN

*Corresponding author. e-mail: [email protected]

Different studies confirm the ability of white rot fungi to degrade 3 to 6 ring-benzene

PAHs, making these microorganisms suitable to remediate contaminated sites. However,

these studies only consider concentrations of PAHs lower than those found in real

contaminated sites. Therefore, the analysis of a potential inhibitory effect of the PAHs

on fungal physiology is lacking.

In the present study, three white rot fungi (Bjerkandera adusta BOS55, Irpex

lacteus Fr. 238 617/93 and Lentinus tigrinus PW94-2) were assessed for their tolerance

to different concentrations of PAHs. The decolorization of the polyanthraquinone

ligninolytic indicator dye Poly R-478 was monitored to estimate the effect of PAHs

concentration on the enzymatic activity. The experiment was performed in 100-mL

Erlenmeyer flasks containing 20 mL of Kirk medium, 0.02 % Poly-R and a mixture of 4

PAHs to reach a concentration of 1, 5, 10 and 50 mg/L of each PAH. After sterilization,

2 agar-plugs of active mycelium were added as inoculum and the cultures were

incubated statically at 30 ºC for 22 d. Samples were harvested every 2 d and Poly R-478

dye decolorization rate was measured as A520/A350 ratio per day in a spectrophotometer.

Initially the liquid medium was violet due to the addition of Poly-R and turned into

yellow when the dye was degraded by the action of the enzymes. Under 50 mg/L the

liquid remained violet in all the fungal cultures and the spectrum data showed no

changes during the assay, suggesting that this PAHs concentration may affect the

production of enzymes. In contrast, PAHs concentrations of 1, 5 and 10 mg/L caused no

effect. All of the fungi achieved similar decolorization ratios (A520/A350). However,

decolorization appeared fastest in L. tigrinus and I. lacteus, which started the

decolorization after 10 days in medium containing 10 mg/L of PAHs, being 4 days for

B. adusa. Same results were obtained for 5 and 1 mg/L in all the strains.

Keywords: White Rot Fungi, Polycyclic Aromatic Hydrocarbons (PAHs), Bjerkandera

adusta, Irpex lacteus, Lentinus tigrinus, Indicator Dye

- 239 -

Effect of saline conditions on the growth of white rot fungi

Valentín, L., Feijoo, G.*, Moreira, M.T., Lema, J.M.

Department of Chemical Engineering, University of Santiago de Compostela, 15782 – Santiago de Compostela, SPAIN

*Corresponding author. e-mail: [email protected]

White rot fungi are extensively studied for bioremediation of contaminated soils with

polycyclic aromatic hydrocarbons (PAH). Non-anthropogenic sites, as marine

sediments, seawater and shorelines, may be damaged by oil spills. Bioremediation of

these marine sites using fungi is a feasible treatment. However, little is know about the

effect of saline conditions on growth and enzyme activity of white rot fungi.

To evaluate this effect, three white rot fungi, Bjerkandera adusta BOS55, Irpex

lacteus Fr. 238 617/93 and Lentinus tigrinus PW94-2, were incubated in a saline

medium, simulating a coastal area. Liquid-state cultures were performed in 100-mL

Erlenmeyer flasks containing 15 mL of Kirk medium with 0.02% of Poly-R478. The

medium was supplemented with 5 different solutions of seawater ranging from 100%

distilled water to 100% seawater and incubated at 30ºC during 22 d. The effect of saline

conditions was evaluated by the decolorization of the dye Poly-R 478, since it is a

ligninolytic indicator dye. Decolorization rate was measured as the change in the

A520/A350 ratio per day in a spectrophotometer and the spectrum data of Poly-R

harvested at days 1, 10 and 22 were analysed.

I. lacteus and L. tigrinus were able to decolorize the dye under all the seawater

solutions and they started to decolorize the dye after 5 days of incubation. Both strains

reached similar decolorization ratios (A520/A350) although I. lacteus achieved better

ratios in the more saline media. B. adusta also achieved decolorization, but not in all the

samples measured specially in the more saline media. Not only did I. lacteus and L.

tigrinus tolerate saline conditions, but also they grew better and achieved better

decolorization ratios. Significant effect of salinity was found in B. adusta cultures as the

fungal growth was slower and non decolorization occurred under more saline

conditions.

Keywords: Bjerkandera adusta, Irpex lacteus, Lentinus tigrinus, Polycyclic Aromatic

Hydrocarbons (PAH), Seawater

- 240 -

Bioremediation of cresote-contaminated soil: biodegradation of target PAHs, microbial community dynamics and bioavailability of PAHs

Viñas, M.1*, Sabaté, J.1, Espuny, M. J.2, Solanas, A.M.1

1 Department of Microbiology, University of Barcelona, Diagonal 645. Barcelona, SPAIN

2 Department of Microbiology and Parasitology, University of Barcelona,

Joan XXIII, s/n .Barcelona, SPAIN * Corresponding author e-mail address: [email protected]

Bacterial community dynamics and biodegradation processes were examined in a

creosote-contaminated soil during bioremediation treatments at laboratory level. Also,

the bioavailability of polycyclic aromatic hydrocarbons (PAHs) and their biodegradation

was studied. Denaturing gradient gel electrophoresis (DGGE), most probable number of

heterotrophs and PAH-degraders, total petroleum hydrocarbon (TPH), PAH

biodegradation, and a estimation of desorbing fraction were monitored. TPH and PAHs

were degraded significantly (70 % and 80% respectively), raising higher values without

the addition of nutrients. Moisture content and aeration were established as key factors.

All treatments showed a high degradation rate in the first 45 d, decreasing after 90 d. At

the beginning, desorbing fraction showed high values for three- and four-ringed PAHs.

These values did not change in the untreated microcosms during incubation, although

they were reduced in treated microcosms due to biodegradation of desorbing PAHs. At

the end of treatment (200 d), estimated rates of desorbing fraction corresponded to the

percentage of PAH biodegraded. A sharp increase in heterotrophic and PAH-degraders

was reported during the highest TPH and PAH biodegradation rates. DGGE and

Principal Component Analysis (PCA) corroborate a remarkable shift in bacterial

community, due to both the biodegradation process and the addition of nutrients.

This study shows that specific phylotypes of bacteria were related both to

different phases of PAH degradation and to the nutrient addition. Initial estimation of

bioavailability can provide the theoretical maximum rate of biodegradation that could

be obtained during a bioremediation process.

Keywords: Creosote-Contaminated Soil, Polycyclic Aromatic Hydrocarbon (PAHs),

Denaturing Gradient Gel Electrophoresis (DGGE), Total Petroleum Hydrocarbon

(TPH), Heterotrophs, Desorbing Fraction

- 241 -

Bioremediation of sewaged soils through dual inoculation with arbuscular mycorrhizal fungi and Pseudomonas putida

Namet M. Awad

Agricultural Microbiology Department, National Research Centre, Dokki, Cairo, EGYPT


One way to lessen the deleterious effects of heavy metals taken up from the

environment on some plants might involve the use of plant growth-promoting bacteria

or mycorrhizal fungi. The purpose of this study was to use dual inoculation with

arbuscular mycorrhizal AM fungus (Glomus spp.) and bacteria (Pseudomonas putida) as

a bioremediating agent for potential toxic elements in a sewage-amended soils. Results

admitted that, a synergistic effect between the two microorganisms on the growth of

tomato was visible. In sewage-amended soil, Pb concentrations were very low in all

mycorrhizal plant parts, while in those non-inoculated or inoculated with P. putida a

significant increase of Pb concentration was conspicuous as compared to mycorrhizal

plants. The increase in Zn concentration in roots was much less compared to that in

shoots. The Zn concentration in the shoots of mycorrhizal plants was also increased

significantly when the plants grown in sewage-amended soil compared to these grown

in an organic fertilizer amended soil. In sewage-amended soils, mycorrhizal plants had

lower root Cu concentrations than uninoculated plants. In soils amended with organic

fertilizers, the concentration of Cu in roots and shoots was lower in the uninoculated

plant than in mycorrhizal plants. Mycorrhizal plants significantly decreased the toxicity

of Ni. In the present study, the isolated AM fungi and bacterium P. putida were highly

effective in protected plants from inhibition originated from high concentrations of

potential toxic elements.

Keywords: Arbuscular Mycorrhizal (AM) Fungi, Pseudomonas putida, Glomus spp.,

Sewage-Amended Soil, Heavy Metals

- 242 -

Rhizobium-legume interaction as a new tool for bioremediation of heavy metals contaminated soils

E. Pajuelo 1* ; M. Dary 2 ; J.A. Carrasco 1 ; M.A. Chamber 1, J.A. Palomares 2

1 Department of Microbiology and Parasithology, Faculty of Pharmacy, University of Seville

41012, Seville, SPAIN 2 C.I.F.A. Las Torres, IFAPA, Junta de Andalucía, Apartado Oficial, 41200, Seville, SPAIN

UNDER REVISION

- 243 -

AFM study of the colonisation of stainless steel by Aquabacterium

commune

R.T.Bachmann*, R.G.J. Edyvean

Biological and Environmental Systems Group, Chemical and Process Engineering Department, Sheffield University, Sheffield, S1 3JD, UK.


Aquabacterium commune, a member of the beta proteobacteria family, is a recently

isolated, predominant member of various European drinking water distribution system

biofilms. It was therefore selected as a more representative test organism in this study.

Attachment of A. commune cells onto two increasingly popular pipe materials, stainless

steel AISI 304 and medium density polyethylene (MDPE), was studied at 15 ºC, 150

rpm and a hydraulic retention time of 10.5 h in a rotating annular biofilm reactor

(RAB). Planktonic and sessile growth was monitored by spread plate technique.

Tapping atomic force microscopy (AFM) was used to obtain information about surface

topography and biofilm formation pattern.

Our study has shown that (i) steady state conditions were reached after ca. 300 h

for both materials, (ii) biofilm density on MDPE slides is 4 times greater than on AISI

304, (iii) the colonization of the growth substrata does not occur randomly but at the

edge of the slide, and (iv) no preferential attachment to stainless steel grain boundaries

was observed.

Stainless steel manufacturer and suppliers, researchers and companies working

in the drinking water sector will benefit from this paper. It is suggested that polishing of

the steel is not necessary to remove specific biofilm formation sites (i.e. grain

boundaries). Furthermore, this paper provides, for the first time, some fundamental

information for the continuous cultivation of a recently isolated and more realistic

drinking water microorganism, Aquabacterium commune.

Keywords: Aquabacterium commune, Stainless Steel, Biofilm, Medium Density

Polyethylene (MDPE), Rotating Annular Biofilm Reactor (RAB), Tapping Atomic

Force Microscopy (AFM)

- 244 -

Rapid detection and enumeration of mycobacteria and total bacteria load in metalworking fluids

Shu-Chi Chang, Peter Adriaens*

Department of Civil and Environmental Engineering, University of Michigan, EWRE Bldg., 1351 Beal Avenue, Ann Arbor, MI 48109, Michigan, U.S.A.


Currently, in the United States, the annual metalworking fluids (MWFs) consumption

has exceeded 2 billion gallons. Biofouling of MWFs results in serious wear and

corrosion of machining tools, release of health threatening endotoxins and pathogens,

and more frequent discharging to wastewater treatment. Therefore, monitoring

microbial levels in MWFs systems is a common practice for metalworking processes.

However, currently, most still rely on off-line growth function methods to monitor the

microbial loads. These methods are slow and pose biases on slow-growing and

aggregated bacteria; and, thus, do not provide accurate data. In the past decade, more

than 80% of MWF-related out-breaks of hypersensitivity pneumonitis have been

strongly linked to a specific strain, Mycobacterium immunogenum. Lack of rapid,

sensitive, and accurate detection and enumeration methods has hindered effective

accumulation of insightful knowledge.

By employing PicoGreen® nucleic acid dye and a resolving agent, N, N-

dimethylformamide, a good correlation was observed between flow cytometric (FCM)

and traditional epifluorescent microscopic counts, with R2 over 0.99. By using

nanoimmunomagnetic particles, a satisfactory recovery rate of M. immunogenum has

been repeatedly achieved in a model semi-synthetic metalworking fluid. Further, with

Alexa Fluor® 647 secondary antibody and PicoGreen®, the sample can be stained

simultaneously and two-color FCM detection is feasible for mycobacteria in MWFs.

Three different rabbit anti-mycobacterium immunoglobulin G’s have been tested and

showed different specificity in such application. The results suggested this method can

significantly reduce assay time over 97%, and enable accurate and simultaneous

enumeration of total bacteria and mycobacteria in a single sample.

Keywords: Metalworking Fluids (Mwfs), Biofouling, Mycobacterium Immunogenum,

Flow Cytometry (FCM), Epifluorescent Microscopy

- 245 -

Methanogenic and partially-aerated methanogenic bacterial communities in fluidized bed reactors treating chlorophenolic

compounds

Claudio Garibay-Orijel1, Jaime García-Mena2, Héctor M. Poggi-Varaldo1*

1 Dept. Biotechnology and Bioengineering, and

2 Dept. Genetics,

CINVESTAV-IPN, P.O.Box 14-740 México, D.F., 07000, MEXICO *Corresponding autor e-mail: [email protected]

The objective of the present work was to monitor the bacterial communities inhabiting a

fluidized bed bioreactor fed with chlorophenolic compounds. A lab scale, mesophilic

fluidized bed bioreactor was operated with sucrose as the sole carbon source. The

biomass in the bioreactor was immobilized on granular activated carbon support. The

experimental program consisted of two periods: (i) M-period or methanogenic regime

with 80 mg/L of TCP and 20mg/L of Phe in the influent; and (ii) PAM period or

partially-aerated methanogenic conditions with 15 vvd aeration rate, receiving similar

amounts of TCP and Phe. The bacterial community was monitored by PCR-DGGE. This

molecular biology technique has an important advantage: it may detect and quantify

both culturable and unculturable microorganisms. The richness was evaluated in terms

of the Poggi Index of diversity and Jacquard Index of similarity. We found that: (i)

during the M period, there was a small variation in the number of bands (slight change

in richness); (ii) during the PAM period, there was a decrease in richness when

comparing the beginning and the end of the period; (iii) there was a significant

divergence between bacterial communities in M and PAM periods; bacterial richness

decreased when the bioreactor was changed from M conditions to PAM regime. Against

expectations, it seemed that the oxygen had a negative impact on the number of bands

characterizing the bacterial community, although it helped in achieving a complete

removal of intermediate chlorophenols that accumulated during the operation under M

conditions.

Keywords: Fluidized Bed Bioreactor, Chlorophenolic Compounds, PCR-DGGE,

Metanogenic Bacterial Communities, Granular Activated Carbon Support

- 246 -

Archeobacterial communities in anaerobic reactors supplemented with PCE

Paola Zárate-Segura1, Jaime García-Mena2, Héctor M. Poggi-Varaldo1

1 Dept. Biotechnology and Bioengineering, and 2 Dept. Genetics, CINVESTAV-IPN, P.O.Box 14-740 México, D.F., 07000, MEXICO


The aim of the present work was to evaluate the effect of PCE and the type of biomass

(suspended and immobilized) on the diversity of archaea populations by 16S rDNA

fragments amplified by PCR (and further separated by DGGE) in two types of

mesophilic lab scale methanogenic reactors (two complete mix reactors with suspended

biomass, CM1 and CM2, and one fluidized bed bioreactor with immobilized biomass,

AFBBR). The biomass in the AFBBR was immobilized onto activated carbon. Diversity

in restricted sense (or richness) was determined using discrete indices of Similarity such

as Dice (SD) and Jaccard (JI), and a suitable divergence index of Poggi-Varaldo ∆Poggi.

Regarding the comparison of diversity between initial and final conditions in period (i)

archeae community divergence in each reactor followed the order CM2 > CM1 ≅

AFBBR. Comparison between reactor communities showed that archeae populations

diverged in the order AFBBR-CM1 > CM1-CM2 ≅ AFBBR-CM2.

It seems that divergence could be related to biomass type, i.e., the bacterial

community of biofilm is more stable than that of suspended biomass. The divergence

for each reactor between periods followed CM2 > CM1 ≅ AFBBR, indicating that

archeae community in biofilm is less affected by PCE than suspended growth biomass

in reactors, when comparing reactors fed with 40 mg PCE/L (AFBBR and CM2).

Overall there is an association between steady state of biochemical performance of the

biofilm reactor (AFBBR) and stability of its archeae community, although such an

association was not evident for suspended growth biomass (CM). In fact, the CM

reactors could steadily remove COD and PCE whereas their archeae populations

fluctuated very much; fluctuation increased with the concentration of PCE in the

influent for CM reactors.

Keywords: PCE, Archaea Populations, 16S rDNA, PCR, DGGE, Mesophilic Lab Scale

Methanogenic Reactors, Biofilm, Suspended Biomass

- 247 -

Pentachlorophenol degradation ability increased in Amylomyces rouxii by heterologous expression of peroxidases from Phanerochaete

chrysosporium

Francisco José Fernández*, Alba Mónica Montiel, Araceli Tomasini

Universidad Autónoma Metropolitana. Unidad Iztapalapa. Departamento de Biotecnología. AP 55-535, México D.F. C.P. 09340, MEXICO

*Correspoding author e-mail: [email protected]

Amylomyces rouxii is a filamentous fungi isolated from a paper industry effluent. This

fungus is capable of degrading pentachlorophenol (PCP). It was shown that a tyrosinase

(a phenoloxidase) is one of the enzymes responsible of PCP degradation by A. rouxii

and, in the other hand, extracellular peroxidases were not found in cultures of this

fungus. In the present work, A. rouxii was complemented with genes of lignin and

manganese peroxidases from Phanerochaete chrysosporium, looking for increasing the

natural PCP degradation ability of the fungus. Overexpression was achieved by co-

transformation of plasmids with resistance to fleomicin or hygromycin with plasmids

pTAAMnP1 and/or pVE-LiP that includes, respectively, genes for Mn-P and Li-P

enzymes from P. chrysosporium. Transformants were selected by antibiotic resistance

and decoloration capacity on the Polymer R-478 (a polymer degraded by peroxidases,

but not by phenoloxidases). Transformants were able to degrade 100 % of initial PCP

(12.5 mg/L) in 144 hours, while the original strain only could degrade 68 % of the PCP

in the same time. These results indicates the importance of pathways complementation

in the genetic manipulation of microorganisms used for xenobiotic degradation.

Keywords: Amylomyces rouxii, Phanerochaete chrysosporium, Pentachlorophenol

(PCP), Lignin Peroxidases, Manganese Peroxidases, Plasmid, Xenobiotic Degradation

- 248 -

Rapid detection of bacterial Nitrilase and Nitrile Hydratase genes using PCR-technique

S.V. Kozlov*, V.A. Demakov, A.Yu. Maksimov, M.V. Kuznetsova

Institute of Ecology and Genetics of Microorganisms of Ural Branch of Russian Academy of Sciences,

614081, Perm, ul. Goleva, 13, RUSSIA *Corresponding author e-mail: [email protected]

Microorganisms actively utilizing of nitriles of carboxylic acids are search objects for

bioremediation and biocatalysis purposes. Screening and selection of new bacterial

strains capable to convert nitriles of carboxylic acids are of high ecological and

industrial importance.

We designed PCR protocols for nitrilase and nitrile hydratase genes detection

with application primers to nucleotide sequences of the known genes coding these

enzymes. Amplification of genomic DNA samples with primers to nitrilase gene

sequences resulted to detection of the nitrilase genes in new strains. The DNA

sequences which are conforming to nitrilase genes from early described bacterial strains

R. rhodochrous J1, R. rhodochrous K22, A. faecalis JM3 and A. facilis 72W, were

found. Several strains possess DNA fragments which are conservative sequences of α-

and β-subunits of Fe-dependent and Co-dependent nitrile hydratases and full sequences

of α-subunit of these genes. Genomic DNA samples of some new strains comprise both

nitrile hydratase and nitrilase genes. The designed PCR protocols enable to perform the

fast and effective screening research to detect new bacterial strains producing nitrile

metabolising enzymes.

Acknowledgment: This work was supported by “Scientific bases of conservation of a

biodiversity of Russia”, ”Fundamental bases of management by biological resources”

and joint integration researches Programs of Russian Academy of Science.

Keywords: Biocatalysis, Carboxylic Acids, Nitriles, PCR, Nitrilase, Nitrile Hydratase

- 249 -

AFM imaging of barnacle cement nanostructure in artificial seawater

Liedert, R.1*, Owen, R.J.2, Kesel, A.B.2

1 University of Applied Sciences Bremen, Dept. 7: Naval Architecture,

Ocean Engineering and Applied Sciences, Neustadtswall 30, 28199 Bremen, GERMANY 2 JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin, GERMANY *Corresponding author e-mail: [email protected]

Barnacle larvae adhere to surfaces by producing a liquid-curing protein cement. Recent

investigations on barnacles showed, that barnacle cement seems to consist out of

nanoscopic globular structures. Depending on the substrate’s topography, elasticity and

surface free-energy, these cement globuli form different kinds of fibrillar network

structures. This could be interpreted as an adaptation process to enhance the adhesion

strength on different types of natural (or artificial) substrata.

We present high resolution images of barnacle cement under physiological

conditions in artificial seawater, using atomic force microscopy (AFM). The barnacles

used for these experiments were allowed to settle on different surface types prior to the

experiments. Most other studies on barnacle cement nanostructure have been carried out

with scanning electron microscopy (SEM), which requires preparational steps before

imaging. Especially sample drying might induce crucial changes in the spacial

arrangement of cement globuli, hence leading to artifacts in SEM pictures and

interpretation. In contrast, the AFM is capable of imaging in liquid without the need for

fixing or drying. Images are formed by scanning a sharp tip over the sample to build up

a true three-dimensional map of the surface. Using AFM further enables to measure

quantitative forces for both adhesion and compression, to fully characterise the

properties of the cement. In-situ measurements of fresh cement production will extend

understanding of the properties of this material and how the structure changes during

curing.

Keywords: Barnacle Cement, Fibrillar Network Structures, Artificial Seawater, Atomic

Force Microscopy (AFM), Scanning Electron Microscopy (SEM)

- 250 -

Hydrocarbon-oxidizing bacteria in natural environments contaminated with oil – A simple screening method for their count and

activity

Petrović Olga1*, Knežević Petar1, Rončević Srdjan2, Simeunović Jelica1

Marković Jelena1, Dalmacija Božo2

1 Department of Biology and Ecology, and 2 Department of Chemistry, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, 21000 Novi Sad, SERBIA and MONTENEGRO


The work is concerned with the counts of hydrocarbon-oxidizing bacteria in natural

environments contaminated with oil and oil derivatives. Soil and groundwater from a

water supply endangered by accidental oil spill from a refinery were examined. Besides

a substantial sample of the contaminated refinery soil was subjected to bioremediation

in a laboratory bioreactor, whereby bacteria counts, dehydrogenase and phosphatase

activities, as well as total hydrocarbons and mineral oils were monitored.

Because of the complex behavior of oil in natural environments, characterization

and assessment of contamination is not easy. In addition to physico-chemical,

microbiological parameters are significant indicators of the environment quality.

Hydrocarbon-oxidizing bacteria are present in natural ecosystems, but their asseessment

is rather complex. Many researchers determine them on liquid media as a most probable

count, whereas others use agarized media. In this study, hydrocarbon-oxidizing bacteria

were detected on agarized substrate with the addition of paraphinic oil (as oil powder on

silica gel as carrier). Use was made of the substrates with easily-taken up organic matter

- MSWYE and mineral Tauson substrate. Both were used in modified forms – basic

substrate was enriched with TTC (2,3,5-Triphenyltetrazoliumchlorid) reagent. Bacteria

were not only easier discerbanable and countable (red colonies), but the procedure also

indicated enzymic activity. The application of the medium with TTC can serve as an

appropriate screening method for simultaneous detection and counting of hydrocarbon-

oxidizing bacteria in contaminated natural environments.

Keywords: Hydrocarbon-Oxidizing Bacteria, Mineral Oil, Screening Method,

Phosphatase, Dehydrogenase

- 251 -

Utility of Stable Isotope Probing (SIP) to investigate biodegradation of the biocide Polyhexamethylene Biguanide (PHMB)

O’Malley*, Leon Peter

Arch U.K. Biocides Ltd, P.O. Box 42, Hexagon House, Blackley, Manchester. U.K. M9 8ZS

*Corresponding author e-mail: LO’[email protected]

Due to the introduction of the Biocides Product Directive (BPD), there exists a need to

determine the biodegradability of biocides marketed in the European Union. Therefore,

we have attempted to determine biodegradation of the biocide Polyhexamethylene

Biguanide (PHMB). Isolation of potential PHMB-biodegrading microorganisms has

been conducted in enrichment cultures dosed with the biocide as the sole nitrogen

source. As PHMB is highly adsorptive, it has proved difficult to distinguish between

losses due to biotic or abiotic processes.

Therefore, in an attempt to circumvent analytical problems, the applicability of

Stable Isotope Probing (SIP) to biodegradation studies was investigated. SIP has been

used by molecular ecologists to link metabolism displayed by a microbial community to

the species contained within it. This is achieved by dosing the community with

isotopically labelled substrates, and following the isotope into cell components. Hence,

this methodology should also be applicable for quantifying biodegradation. We have

achieved this by dosing 15N-PHMB to microbial cultures, and extracting 15N-labelled

genomic DNA from cultures at the late exponential phase of growth. The percentage 15N

content of the isolated DNA was determined by Isotope Ratio Mass Spectrometry

(IRMS). By comparison of genomic DNA extracted from identical cultures dosed with

equimolar concentrations of 15N from PHMB and a simple nitrogen source (in this case 15NH4Cl), it is possible to estimate both the extent, and the key organisms involved in

biodegradation of the biocide.

Keywords: Biocides Product Directive (BPD), Biodegradability, Polyhexamethylene

Biguanide (PHMB), PHMB-Biodegrading Microorganisms Stable Isotope Probing

(SIP), Isotope Ratio Mass Spectrometry (IRMS)

- 252 -

Microbial communities and linA and linB genes in a soil polluted with hexachlorocyclohexane isomers

Prieto-Fernández, A.1,2*, Timmis, K.N.2, Acea, M.J.1, Osborn A.M.2,3

1 Dpto. Bioquímica del Suelo, Instituto de Investigaciones Agrobiologicas de Galicia, CSIC,

Aptdo. 122, E-15780 Santiago de Compostela, SPAIN. 2 Dpto. Biological Sciences. University of Essex. Wivenhoe Park. Colchester CO4 3SQ, UK.

3 Dpto. Animal and Plant Sciences. University of Sheffield. Western Bank, Sheffield S10 2TN, UK.


Hexachlorocyclohexane (HCH) is an organochlorine insecticide, which was produced in

considerable amounts by the pesticide industry and applied worldwide. HCH has

several stable stereoisomers of which, only γ-HCH (lindane) presents high insecticide

activity. Due to the cost and technical problems for separating these isomers, a product

constituted mainly by α-HCH (55%-80%) β and γ-HCH, (5-15%) was used for many

years as insecticide. α andβ-HCH are also abundant in waste products obtained during

lindane fabrication. All these isomers, particularly α andβ-, are environmentally very

persistent and there are numerous sites contaminated with these compounds throughout

the world.

Due to their metabolic versatility and genetic diversity, bacteria are essential

agents for the decontamination of polluted sites. In the case of HCH isomers

degradation by soil microorganisms, specifically bacteria, has been reported. For

complex environments, such as polluted soils, the prior characterisation of the diversity,

size, activity and degradative capacity of microbial populations present is essential for

selecting, monitoring and optimising a bioremediation process. To achieve these goals it

is essential the use of nucleic acid based techniques. The results, which will be

presented, include T-RFLP analysis of total (16S rDNA) and active (16S rRNA)

bacterial communities present in samples of contaminated soil and HCH residue, as well

as, of bacteria able to colonize pure α- HCH and γ-HCH. The presence, diversity and

expression of linA and linB genes (known to be involved in the bacterial degradation of

α-and γHCH) in polluted soil will also be analysed and discussed.

Keywords. Hexachlorocyclohexane (HCH), Lindane, Soil Microorganisms, T-RFLP,

16S rDNA

- 253 -

A GFP reporter in Azoarcus sp. (DSMZ9506) for monitoring of toluene transformation activity

Minoru Shimura1*, Kazuhide Kimbara2 and Toshio Hayakawa1

1 Environmental Biotechnology Laboratory, Railway Technical Research Institute,

Kokubunji, Tokyo 185-8540, JAPAN 2 Research Institute for Bioresources, Okayama University, 2-20-1 Chuo,

Kurashiki, Okayama 710-0046, JAPAN *Corresponding author e-mail: [email protected]

Azoarcus sp. (DMSZ9506) is a facultative miroorganism capable of mineralising

toluene and m-xylene anaerobically. To investigate the transformation activity during an

incubation period, a plasmid containing GFP as a reporter was introduced in to it. The

GFP was ligated with the promoter for the bbs operon of Azoarcus sp. It is designed to

be expressed when toluene/m-xylene transformation pathway is activated. Although the

bbs promoter-GFP fusion was cloned in to pUC18 and was used for the transformation

of Azoarcus sp., transformants were hardly obtained. To overcome this difficulty, the

bbs promoter-GFP fusion was cloned in to pBBR1MCS2, which is known as a broad

host range vector. The bbs promoter-GFP fusion in pBBR1MCS2 (pBBR1bssGFP) was

successfully introduced in to Azoarcus sp. using E. coli S17-1 as a mating host. In

Azoarcus sp., pBBR1bssGFP was stably maintained for generations and the expression

of GFP was observed when it was incubated under the anaerobic condition with toluene

or benzyl CoA. An addition of yeast extract to the medium diminished the expression of

GFP even in the presence of toluene. Azoarcus sp. harbouring pBBRbbsGFP was

inoculated in to a flowcell and was incubated with a constant flow of synthetic medium

with toluene as a carbon source. Compare to the cultivation in a sealed vial, Azoarcus

proliferated faster in the flowcell culture and a bright fluorescent was observed from

cells. A cultivation using flowcell is revealed to be useful for study of Azoarcus sp.

Keywords: Azoarcus sp., Facultative Miroorganism, Toluene, m-Xylene, Plasmid,

Flowcell

- 254 -

Proteomic analysis of biofilm formation of Deinococcus geothermalis

Christina Saarimaa1*, Steffen Ohlmeier2, Ulrich Bergmann2,

Mirja Salkioja-Salonen3, Peter Neubauer1

1 Dept. of Process and Environmental Engineering, Bioprocess Engineering Laboratory, University of Oulu, P.O Box 4300, FIN-4300, FINLAND

2 Dept. of Biochemistry, University of Oulu, P.O Box 3000, FIN-90014, FINLAND

3) Dept. of Applied Chemistry and Microbiology, University of Helsinki P.O. Box 56, FIN-00014 University of Helsinki, FINLAND *Corresponding author e-mail: [email protected]

Deinococcus geothermalis is an efficient primary biofilm former in paper machine

environment where it serves as an adhesion platform for other biofouling bacteria. D.

geothermalis forms thick biofilms on various abiotic surfaces, such as stainless steel,

polystyrene and glass, but the mechanism of attachment of this nonmotile bacterium is

not known.

We aim to analyse the proteome of D. geothermalis biofilm cells compared to

planktonic cells with standard two-dimensional gel electrophoresis. Additional

knowledge from protein synthesis level is gained with radioactive labelling

experiments. We also study the protein composition of adhesion threads which

according to microscopial analysis mediate cell-to-surface and cell-to-cell attachment of

D. geothermalis. Proteins of interest have been identified with N-terminal sequencing

and mass spectrometry (Q-ToF).

Keywords: Deinococcus geothermalis, Paper Machine, Biofilm, Planktonic Cells,

Proteome, Two-Dimensional Gel Electrophoresis, Mass Spectrometry (Q-ToF)

- 255 -

Antimicrobial activity and mechanism of nano sized silver particles

Jee Yeon Kim, Sungeun Kim, Jaeeun Kim, Jongchan Lee, Jeyong Yoon*

School of Chemical and Biological Eng., Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul, 151-742 KOREA * Corresponding author e-mail : [email protected] Presenting author e-mail : [email protected]

In recent days, there is much interest in nano sized silver particles since silver is known

to be safe and effective antimicrobial materials. In particular, preparation,

characterization and surface modification of nano sized silver particles received more

attention. Therefore, it is important to prepare nano sized silver particles in a cost-

effective manner and investigate antimicrobial activity and mechanism of this material.

In this study, nano sized silver particles were prepared via photoreduction of a

silver salt (AgNO3) in the bulk phase of PEO20-PPO70-PEO20 (Pluronic 123) block

copolymer. The antimicrobial efficacy of silver nano particles against E. coli was

investigated as the concentration of silver nano particles, pH (5.6~8.2), temperature

(4 ºC ~ 35ºC) varied in liquid system. The antimicrobial efficacy of nano sized silver

particles coated plate was evaluated with halo test method and scanning electron

microscopy (SEM) and transmission electron microscopy (TEM) was used to examine

the nature of damaged microorganism.

This study showed that silver nano particles coated on the surface are an

effective biocide and its antimicrobial efficacy was shown at higher pH and

temperature. In addition, it was demonstrated that nano silver particles disrupted the

outer membrane of E. coli, subsequently causing their aggregation.

Keywords: Nano Sized Silver Particles, Antimicrobial Activity, Photoreduction,

Escherichia coli, Scanning Electron Microscopy (SEM), Transmission Electron

Microscopy (TEM)

- 256 -

Antimicrobial properties of silver and copper nanoparticles supported on sepiolite

Ana M. García1, Víctor M. Rivalta1, Diego A. Moreno1*, Eduardo Aguilar2, Julio Santarén2

1 Dep. Ingeniería y Ciencia de los Materiales, Escuela Técnica Superior de Ingenieros Industriales,

Universidad Politécnica de Madrid, C/José Gutiérrez Abascal, 2. E-28006, Madrid, SPAIN 2 TOLSA, S.A., Ctra. Vallecas-Mejorada del Campo Km 1.600, E-28031, Madrid, SPAIN


Nanosized inorganic particles, of either simple or composite nature, display unique

physical and chemical properties and represent an increasingly important material in the

development of novel antimicrobial agents.

In the present study the antimicrobial activity of silver and copper nanoparticles

supported on sepiolite was evaluated using different forms and concentrations of such

elements. Agar diffusion tests against several bacterial and fungi strains were carried out

in order to analyse the sepiolite´s spectrum action. Results showed that sepiolite on its

own had no effect on the microbial growth, but when it contained silver or copper

nanosized particles differences could be observed depending on the microbial strain. In

this sense, silver nanosized particles had an inhibiting effect that was higher in the case

of Gram positive than in Gram negative bacteria and the same effect could be observed

for copper nanosized particles which only produced weakness growth of Gram negative

species. In the case of fungi, nano-silver particles had no effect and nano-copper

particles inhibited spores formation and production. In general, the evaluated forms of

both elements didn’t show significant differences.

This work, which integrates nanotechnology and microbiology, leads to possible

advances in the formulation of new types of antimicrobial agents and shows the

potential of the metallic nanoparticles supported on sepiolite as an additive in the

formulation of cement, concrete and mortar or in the production of antifouling paints.

Keywords: Nanosized Inorganic Particles, Antimicrobial Agents, Silver, Copper,

Sepiolite

- 257 -

Biofilms formation on nano-microstructured materials

M.C. Cortizo1, P.L. Schilardi1, S. Gomez de Saravia1,4*, M. Disiervi2, M. Fernández Lorenzo de Mele1,3

1 INIFTA, Fac. de Ciencias Exactas, Universidad Nacional de La Plata (UNLP),

Casilla de Correo 16, Sucursal 4, 1900 La Plata, ARGENTINA 2 Instituto de Limnología (ILPLA), Fac. de Ciencias Naturales y Museo,

UNLP, C.C. 712, 1900 La Plata, ARGENTINA 3 Fac. de Ingeniería, UNLP

4 CICBA


Several environments, such as pipes and cooling water systems in industry, and implants

and orthodontic appliances in human body, are affected by biofilms. In particular,

Streptococci and Pseudomonas fluorescens biofilms are related to severe infections and

biofouling, respectively.

During the early stages of biofilm growth bacterial attach to the surface. The

degree of microbial adherence to surfaces depends on the substratum composition,

topography and wettability. The aim of this work is to study the adhesion of

Streptococci consortia and P. fluorescens on metal substrata with modified surfaces.

Self-assembled monolayers (SAMs) chemisorbed on the substratum were chosen as an

alternative way to vary the wettability of the surfaces. Surfaces of silice and titanium

were modified with an octadecyltrichlorosilane (OTS) monolayer and micropatterned

by using UV radiation in order to generate micro-domains with different hydrophilic/

hydrophobic characteristics. Substrates with micropatterned topography and with micro-

domains of different metals (palladium and silver) were also used. The influence of the

surface characteristics on the bacterial attachment and survival during the early stages of

bacterial biofilms formation was investigated. The number of cells was enumerated

using epifluorescence microscopy. The adherent bacteria were vitally stained with

fluorescein diacetate and ethidium bromide to measure the percentage of living and

dead bacteria. Results showed that the effect of topography and the nature of the metal

on bacterial adherence and survival were more significant than that of wettability.

Keywords: Biofilms, Streptococci, Pseudomonas fluorescens, Metal Substrata, Self-

Assembled Monolayers (Sams), Wettability, Topography

- 258 -

Suggestion of standard testing method for antimicrobial efficacy on photocatalytic products

Changhun Park, Jee Yeon Kim, Jaeeun Kim, Jeyong Yoon1*

School of Chemical and Biological Eng., Seoul National University, San 56-1,

Sillim-dong, Gwanak-gu, Seoul, 151-742 KOREA * Corresponding author e-mail : [email protected]

There are many circumstances where it is necessary to sterilize or inactivate

microorganisms found in water, air, or surfaces. Since the photochemical sterilization of

E.coli using Pt-TiO2 was first reported, many photocatalytic disinfection studies using

TiO2 have been carried out. But, no standard testing method for photocatalytic

antimicrobial efficacy of products was suggested. For protection of consumers and

industrial development, proper standard testing method is strongly required.

In this study, the standard testing method for antimicrobial efficacy on

photocatalytic products and testing conditions are suggested.

Three antimicrobial testing methods (halo test, adhesion test, shaking flask test

with UV irradiation) for evaluating antimicrobial efficacy in photocatalytic products

were examined using E. coli, a well-known indicator microorganism. The proper

experimental conditions for light intensity, UV irradiation time, the type of bacteria

were sought for determining standard testing method. Finally, the adhesion test method

with UV-A irradiation (BLB lamp) as light source was suggested as the standard

method.

Keywords: Photochemical Sterilization, Photocatalytic Products, Escherichia coli,

Standard Testing Method, Antimicrobial Efficacy

- 259 -

Key role of biotesting in evaluation of solid wastes eco-toxicity in Russia

Vera A. Terekhova*, Alexander S. Yakovlev

Moscow Lomonosov State University, Soil Science Faculty,

Vorob’evy Gory, Moscow, 119998, RUSSIA *Corresponding author e-mail: [email protected]

Until recently in Russia, the evaluation of waste class damage to environment was

based mainly on the results of chemical analysis. This way wasn’t able to get the

objective information about the impact of waste disposal sites on natural ecosystems.

According to the new ecological normative documents (Act # 511 of Russian Ministry

of Natural Resources) several positions are determined, when using biotest is obligatory.

Most important is that the waste producer factory must confirm the absence of

biotoxicity of waste if they declare it based on a calculation of chemical components.

State institutions of ecological control recommend the list of biotest-methods for

eco-toxicity estimation of water extraction from wastes. The tests are carried out with

the aquatic organisms, and must include no less than two taxonomic different species.

We used Daphnia magna, Paramecium caudatum, Scenedesmus caudatum. But our

investigation showed that in many cases we should have had the combination of aquatic

and terrestrial test-organisms to get full results about toxicity for some kinds of wastes.

Soil microorganisms are useful for this purpose under oil hydrocarbons and some

mineral pollutants. We suppose that the situation with standard methods in ecological

legislation will be in progress more. The range of authorized aquatic test-systems will

supplement with terrestrial. The improvement bioassay the effect of the waste on

environment is in full accordance with the biotic conception of the environmental

control. Step by step this conception applies in common practice and replaces the

sanitary-hygienic control regulations in preservation of the environment in Russia.

Keywords: Biotest, Biotoxicity, Eco-Toxicity, Daphnia magna, Paramecium caudatum,

Scenedesmus caudatum

- 260 -

Bio-deterioration of marketed betel nut (Areca catechu L.) by some fungi

J. K. Misra

Mycological Research Unrt, Department of Botany, Sri J. N. P. G. College,

Lucknow-226 001 INDIA e-mail

Bio-deterioration of marketed betel nut (Areca catechu L) by fungi such as Aspergillus

fíavus, A. fumigatus, A. nidulans, A. niger, A. tamarii, A. férreas, Curvularia lunata,

Fusaríum roseum, and Penicillium funiculosum was assessed using weight loss method.

It was found that Aspergilli are more potent ones in deteriorating the nuts than other

fungal forms tested. However, among the Aspergilli tested, A.nidulans was the poorest

in causing weight loss to the nuts. Effect of A. fíavus and A. niger, the two common

Aspergilli, on the total lipids, acid value and free fatty acids was also assesses and it

became evident from the data obtained that the biochemical parameters tested show

significant degree of change over the control indicating thereby that common fungi

infesting the nuts adversely affect the quality of the nut in the market.

Keywords: Marketed Betel Nut, Fungi, Weight Loss Method, Aspergillus

- 261 -

Exploitation of essential oil of Mentha arvensis as botanical fungitoxicant to protect food commodities from fungal infestation

Rajesh Kumar1*, N K Dubey1, Y B Tripathi2

1 Centre of Advanced Studies in Botany , Faculty of Science, and

2 Department of Medicinal Chemistry, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi- 221005, INDIA *Corresponding author e-mail: [email protected]

Essential oil extracted from leaves of Mentha arvensis Linn. (Lamiaceae) was tested

against the toxigenic strain of the test fungus Aspergillus flavus. The oil completely

inhibited the radial mycelial growth of the fungus at 100 ppm. The oil exhibited broad

fungitoxic spectrum against A. niger, A. fumigatus, Botryodiploidea theobromae,

Fusarium oxysporum, Sclerotium sp. Macrophomina phaseolina, Cladosporium

cladosporoides, Helminthosporium oryzae and Pythium debaryanum at 100 ppm. The

oil showed inhibitory action against mycotoxin production as absolutely inhibited the

aflatoxin B1 production by the toxigenic strain of A. flavus. Moreover, the Mentha oil

also exhibited potent antioxidant activity during ABTS bioassay. The GLC of oil

showed four major and thirty minor components.

Keeping view in the side effects of synthetic pesticides and global importance of

some plant products (neem products) in plant protection, the Mentha oil may be

recommended as ideal botanical fungi toxicant to protect food commodities from fungal

infestation and enhancing shelf life of the stored commodities due to its antioxidant

activity.

Keywords: Mentha Oil, Aspergillus flavus, Mycelial Growth, Fungitoxic Spectrum,

Mycotoxin Production

- 262 -

Application of reuterin produced by Lactobacillus reuteri DSM 20016 to inhibit some food-born pathogens in uf-fata-cheese

A. Mohamadi Sani1*, M.R. Ehsani2, M. Mazaheri Asadi3

1 Department of Food Science & Technology, Islamic Azad University of Quchan 2 Department of Food Science & Technology, Tehran University, Karadj, IRAN

3 Biotechnology Center, Iranian Research Organization for science & Technology, Terhan, IRAN *Corresponding author e-mail. [email protected]

A broad-spectrum reuterin produced during anaerobic fermentation of glycerol by

Lactobacillus reuteri strain 20016 was found to be inhibitory and bactericidal for

Listeria monocytogenes, Escherichia coli, Salmonella typhimorium, Pseudomonas

aerogenes, Klebsiella penomoniae and Staphilococcus aureus. Reuterin was produced

by a two-step fermentation process. A batch fermentation in a 1.5 liter flask fermentor

was applied to produce a biomass of L. reuteri using a modified MRS broth at pH 5.5.

Further, harvested cells were used to ferment glycerol (250 mMol) under anaerobic

conditions (flushed with nitrogen). The MIC values of reuterin for Listeria

monocytogenes, Staphilococcus aureus, Escherichia coli, Salmonella typhimorium,

Pseudomonas aeroginosa and Klebsiella penomoniae were 10, 4, 2, 2, 1 and 1 AU/ml,

so the strain L. monocytogenes was more resistant to reuterin than the others. In

potassium phosphate buffer (pH=7.2) using 80 AU/ml reuterin the count of L.

monocytogenes and S. aureus decreased from 10 7 to 5.5* 10 6 and 2.5*10 6 respectively

and the counts of E. coli, S. typhimorium, P. aerogenes and K. penomoniae decreased to

undetectable level in 5 hours. Addition of reuterin (40 units per gr) to the UF-Feta-

Cheese reduced the viability of all organisms. The inactivation rate was more

pronounced with P. aeruginosa and less with L. monocytogenes.

Keywords: Glycerol, Lactobacillus reuteri, Two-Step Fermentation Process, Feta-

Cheese, Biopreservation

- 263 -

Formation of biofilm on the surface of traditional PA/PE laminates by facultative anaerobic micro-flora in model conditions and in the

presence of food

Izabela Anna Steinka1, Anita Kukułowicz1*, Maria Rutkowska2

1 Department of Commodity and Cargo Sciences, and 2 Department of Chemistry,

Gdynia Maritime University, Morska 83, 81-225 Gdynia, POLAND *Corresponding author e-mail: [email protected]

Aim of research was to assess the ability of facultative anaerobic microflora to form

biofilms on the surface of PA/PE laminates. The ability to form biofilms and their sizes

were determined on the surface of PA/PE bags in model conditions and after food

storage. Formation of biofilm was determined on the basis of changes in number of cells

occurring on 1 cm2 of tested packaging after storage of the curd for 14 days at

temperature 4 ± 2 ºC, in relation to count of cells prior to storage. Number of

microorganisms on the packaging surface was determined using the washings method.

For that purpose, two PA/PE laminate squares of areas equal to 50 cm2 each were placed

in 50 cm3 of physiologic saline with peptone. 1 cm3 of washings was inoculated into

appropriate media selective for adequate microorganisms being determined. The ability

of Lactococcus sp. bacteria to form biofilms on the PA/PE surface in model conditions

was stated. After 14 days of storage, more than 40% of bacteria were present on the

surface of laminates. Enterococcus and yeast bacteria did not form any biofilm.

Filamentous fungi showed the insignificant growth on the surface of laminates.

Adhesion of bacteria to PA/PE packaging showed a weak linear correlation with time of

curd storage in this packaging. On films coming from stored food, yeast showed a high

degree of affinity and ability to form biofilm. The significant influence of population

count prior to storage on the ability to form biofilm was stated, both in model

experimental conditions and on the surface of packaging coming form food. Obtained

differences in the nature of formed biofilm were probably caused by the different water

content in tested material used in model conditions and in food.

Keywords: Facultative Anaerobic Microflora, PA/PE Laminates, Biofilm, Lactococcus

sp., Enterococcus, Food

- 264 -

The influence of lactic acid on the structure of polymers used for packing lactic acid cheese

Izabela Anna Steinka1*, Magda Morawska2

1 Department of Commodity and Cargo Sciences, and 2 Department of Chemistry,

Gdynia Maritime University, Morska 83, 81-225 Gdynia, POLAND *Corresponding author e-mail: [email protected]

Our earlier research showed that presence of lactic acid of concentration of pH 4.3-5.2

inside PA/PE packaging resulted in changes of laminate permeability toward water

vapour and gases.

Aim of research was to assess the structure of PA/PE laminates after 14 days of

presence of lactic acid inside the packaging, depending on time of exposure. Bags made

of PA/PE laminate were subjected to microscopic assessment after 24 h and after 4, 7

and 14 days of storage at temperature ± 6 ºC with lactic acid solution of pH 4.7. Squares

of laminate from the side of the inner surface that was in contact with lactic acid

solution were observed under polarisation microscope Nicon Alphaphat 2 YS2 in

polarised light destined for observation of a section of a given object. Object lens of

magnification 40x was used. At obtained magnification 1:110 one centimetre was

corresponding to a segment of length of 91 µm. The great significance of lactic acid in

shaping the properties of PA/PE laminates during storage of lactic acid cheese was

confirmed by our own research comprising microscopic observations of the packaging

structure. Microscopic observations made using a polarisation microscope and

presented in photographs confirmed insignificant changes in penetration of light

through the laminate during storage. It was stated that size of a flux of light penetrating

through the packaging after 4 days of storage was different than the ones observed after

7 and 14 days. Diameters of penetrating luminous flux in the first period of packaging

seasoning were different than those obtained after 7 and 14 days by 18.4-26.3%. The

observed behaviour of light after 7 and 14 days suggested quite insignificant changes of

tested laminate structure.

Keywords: PA/PE Laminates, Packaging, Laminate Permeability, Lactic Acid,

Luminous Flux

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