Microorganism in concrete

MICROORGANISMS IN CONCRETE

A THESIS SUBMITTED FOR THE DEGREE OF ENVIRONMENTAL ENGINEERING

BY

ÁNGELA MARCELA QUINTERO MARTÍNEZ

DIRECTOR

MAURICIO SÁNCHEZ SILVA, PH.D.

ADVISOR

AIDA JULIANA MARTÍNEZ LEÓN, MSC.

UNIVERSIDAD DE LOS ANDES

FACULTY OF ENGINEERING

DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING

BOGOTÁ, COLOMBIA

2011

Dedicated to my parents and my sister…

Acknowledgements The author would like to acknowledge the supervision, suggestions and ideas about the investigation,

provided by the engineer Mauricio Sánchez Silva and the collaboration, contributions, explanations and

advices of the microbiologist Juliana Martínez. Many thanks are extended to the proofreader, the

philologist Laura Ontibón; the people that collaborated in the field work (transportation and

photographs), Mr. José Manuel Quintero and Mrs. Rocío Martínez; the company which realized the

sequencing of DNA samples, Macrogen –Korea Inc.; and the persons of the laboratory of electrophoresis

of Universidad de los Andes

Abstract This study reports the use of culture dependent and independent techniques to identify indigenous and

naturally occurring bacteria in deteriorate concrete surfaces. In the field work was sampled a total of 6

bridges located in via Bogotá – Villeta. Genomic DNA was isolated to the samples in two different ways:

indirect and direct. The indirect way involves the cultivation of microorganisms, while the direct way

does not. PCR amplification of bacterial ribosomal DNA (16S rDNA) was conducted with subsequent DNA

sequencing to identify the microbes. Three bacteria genus was recognized Pseudomonas, Rahnella and

Buttiauxella corresponding to four distinct DNA samples (Pseudomonas was found twice) occurring at

two bridges. Of these genuses, only Pseudomonas was reported in the scientific articles related to

deterioration of concrete. But in this study, Rahnella and Buttiauxella were associated indirectly with

acid attack of concrete.

Key words: molecular techniques, cultivation of microorganisms, deterioration of concrete,

biodeterioration.

Resumen Este estudio presenta el uso de técnicas dependientes e independientes del cultivo de microorganismos

para identificar bacterias autóctonas que se encuentren naturalmente en superficies de concreto

deterioradas. En el trabajo de campo se muestrearon un total de 6 puentes ubicados en la vía Bogotá –

Villeta. El ADN de las muestras fue extraído en dos formas: indirecta y directa. La forma indirecta

involucra cultivo, mientras que la directa no. Se realizó PCR con amplificación del ADN ribosomal

bacteriano (16S rADN) y subsecuentemente se hizo secuenciación del AND para identificar los microbios.

Tres géneros bacterianos se reconocieron, Pseudomonas, Rahnella y Buttiauxella de cuatro muestras de

ADN (Pseudomonas se encontró dos veces) tomadas de dos puentes. De estos géneros, sólo las

Pseudomonas fueron reportadas en artículos científicos relacionados con el deterioro del concreto. Sin

embargo en este estudio, Rahnella y Buttiauxella se asociaron indirectamente con el ataque ácido en el

concreto.

Palabras claves: técnicas moleculares, cultivo de microorganismos, deterioro del concreto,

biodeterioro.

Content Acknowledgements ................................................................................................................................. 3

Abstract ................................................................................................................................................... 4

Resumen.................................................................................................................................................. 4

1. Introduction ..................................................................................................................................... 7

2. Field visits......................................................................................................................................... 8

2.1. Places visited ............................................................................................................................ 9

Railway bridge in the ‘Alto de la Tribuna’ .............................................................................. 10

Vehicular bridge located 580m (approx.) after ‘Alto de la Tribuna’ ........................................ 12

Vehicular bridge located at the end of the ‘Bogotá – Facatativá – Los Alpes’ concession. ..... 13

Vehicular bridge located in landslide and collapse in ‘Albán’ (Guayacundo Alto) ................... 14

Concrete structures located near to weighing scale for heavy vehicles ................................. 15

Vehicular bridge located at 200 m (approx.) after ‘Jalisco’ Toll Road ..................................... 17

Railway bridge located at the entrance of the Vereda ‘Santa Ana’ ........................................ 19

Vehicular bridge at the edge of the ‘Seco’ River .................................................................... 21

2.2. Bridges were to be sampled.................................................................................................... 21

3. Materials and methods................................................................................................................... 21

3.1. Sampling technique in bridges – removal of microorganisms .................................................. 21

3.2. Culture mediums .................................................................................................................... 22

PYGV medium....................................................................................................................... 22

Brain Heart Infusion Broth .................................................................................................... 24

3.3. Culture techniques ................................................................................................................. 24

3.4. DNA isolation.......................................................................................................................... 25

3.5. PCR (Polymerase Chain Reaction) ........................................................................................... 27

3.6. Electrophoresis ....................................................................................................................... 29

3.7. Spectrophotometric measurement of DNA concentration. ..................................................... 30

3.8. DNA sequencing and purification ............................................................................................ 30

3.9. Gram staining ......................................................................................................................... 30

3.10. Culture collection of isolated microorganisms......................................................................... 32

3.11. Assembly of DNA sequences ................................................................................................... 32

4. Results ........................................................................................................................................... 33

4.1. DNA isolation and classic microbiology (culture mediums) ...................................................... 33

DNA isolation and PCR .......................................................................................................... 34

Classic microbiology and samples surfaces............................................................................ 35

4.2. DNA sequences and NCBI blast search .................................................................................... 45

Blast results of DNA sequences ............................................................................................. 45

Blast results of consensus sequences .................................................................................... 46

5. Discussion ...................................................................................................................................... 47

6. Conclusions and recommendations ................................................................................................ 49

7. References ..................................................................................................................................... 50

8. Annexes ......................................................................................................................................... 54

Annex A: Classification scheme of weathering forms .......................................................................... 54

Annex B: DNA indirect isolation .......................................................................................................... 62

Annex C: Bacteria morphology ........................................................................................................... 63

Cultural characteristics ......................................................................................................... 63

Annex D: Results of DNA sequencing .................................................................................................. 64

Quintero-Martínez, A.M. [2011] IAMB 201120 30

Microorganisms in concrete 7.

1. Introduction Concrete surfaces are susceptible to some kind of microbiological attack, called biodeterioration. All

microorganisms may be of importance in biodeterioration – phototrophic, chemolithotrophic and

chemoorganotrophic bacteria, cyanobacteria, algae, fungi and lichens. The higher plants may contribute

to weathering of concrete due to root growth and indirectly by the excretion of substrates for

microorganisms (Sand, Jozsa & Mansch, 2002). The diversity of microorganisms that inhabit particular

concrete surfaces dependent on environmental factors as well as the specific composition of the surface

(Gaylarde & Galarde, 2005).

Biological mediated deterioration of concrete occurs mainly by the action of inorganic and organic acids,

salt attack, the role of biofilms, discoloration, noxious compounds, and physical damage.

The acid attack consists in dissolution of susceptible material (e.g. concrete) causing weakening of the

structure and loss of material (Scheerer et al., 2009). Several groups of bacteria produce strong inorganic

acids like nitric acid (Bock & Sand, 1993; Mansch & Bock, 1998; Sand & Bock, 1991) and sulfuric acid

(Sand & Bock, 1991; Milde, Sand & Wolff, 1983). Regarded to organic acids, all microbes may excrete

organic acids in the course of their metabolism (Sand, Jozsa & Mansch, 2002).

On the other hand, the salt attack begins with the reaction between a cation of concrete and an anion of

metabolic compound of microbe that produces a salt. When the salt is hydrated induced tensile stresses

into the concrete matrix causing cracks. Biofilms promote the salt attack and increase the susceptibility

of the concrete to freeze – thaw attack, among other things (Sand, 1997).

For its part, discoloration is an aesthetic problem but if the concrete surfaces are black colored by

microorganisms other difficulties arise. Black surfaces increased the absorption of solar light, then a

gradient of temperature appear between colored and uncolored surfaces. That gradient induces

additional physical stress in concrete structures.

Apropos of noxious compounds, they are found in atmosphere naturally or by air pollution. These

compounds serve as substrates or energy source of undesirable microorganisms, such as nitrifying

bacteria or sulfate reducing bacteria. The last way of biodeterioration is physical damage. It is caused by

the penetration of microbes (especially filamentous organisms) in the concrete matrix.

If microorganisms that damage concrete surfaces are to be identified and mechanisms of deterioration

characterized, culture – based techniques are frequently used. This approach is valuable in obtaining

isolates but it can only identify a small fraction of microbial community (Staley & Konopka, 1985).

Molecular techniques such as 16S ribosomal DNA gene analysis could be used to identify microbial

communities.

The aim of this work is to identify bacteria of deteriorated concrete surfaces of structures located in via

Bogotá – Villeta. To achieve this aim, it is necessary to use culture dependent and independent

IAMB 201120 30 Quintero-Martínez, A.M. [2011]

8. Microorganisms in concrete

techniques for isolate DNA samples. It also requires amplification of 16S rDNA, purification and

sequencing of DNA samples.

The followings chapters describe the field visit, materials and methods, and results. At the end of the

document, it is presented discussion of the results and the respectively conclusions and recomendations.

Additionally, there are annexes that contain information about weathering forms in rocks, DNA isolation

protocol, bacterial morphology and the sequences of DNA of the identified microbes.

2. Field visits The aims of the field visits were to identify the structures suitable to be sampled and do the sampling

process.

The chosen route to identify deteriorated concrete structures corresponds to one of the routes selected

by Claudia Soler in her study about deterioration of concrete structures by biological agents. The route is

one of the alternatives to go to Villeta from Bogotá; passing through Mosquera, Madrid, Facatativá and

Sasaima. On that route, Soler found the largest number of deteriorated bridges.

The route and the number of structures visited in a specific place are shown in Figure 1.

Conventions

Number of structures visited

Places visited

Figure 1. Route and places visited

4

Albán 1

4

1

1 Sasaima

Bogotá Funza Mosquera

Madrid

Facatativá

Villeta

1



In most of the cases, the visited structures were vehicular bridges, but also there were observed two

railway bridges, one channel and one rectangular shaped structure. The structures were on the road

which facilitated the observation and the future sampling process. The number of structures visited was

eleven, located in eight points of the road. The buildings were found near to Los Andes, Albán, El Doran,

Taboga y Sasaima like it is show in the Figure 2.

Names assigned and approximate location of places visited

Place Coordinates

1. Railway bridge in the ‘Alto de la

Tribuna’

Latitude: 4°51'29.00"N

Longitude: 74°24'37.35"W

2. Vehicular bridge located 580m at

(approx.) after ‘Alto de la Tribuna’



3. Vehicular bridge located at the end of

the ‘Bogotá – Facatativá – Los Alpes’

concession.



4. Vehicular bridge located in landslide

and collapse in ‘Albán’ (Guayacundo

Alto)



5. Concrete structures located near to

weighing scale for heavy vehicles



6. Vehicular bridge located at 200m

(approx.) after ‘Jalisco’ Toll Road



7. Railway bridge located at the entrance

of the Vereda ‘Santa Ana’



8. Vehicular bridge at the edge of the

‘Seco’ River



Figure 2. Specific points visited and their coordinates.

2.1. Places visited This section contains the places visited with images of the structure and their surroundings and

description of the deteriorated surfaces.

The description of surfaces was done in concordance with the classification scheme of weathering forms

of Fitzner & Heinrichs (2002). All categories of weathering forms proposed by these authors are found in

Annex A.

Los Andes

Albán

El Dorán

La María

Taboga Sasaima



Railway bridge in the ‘Alto de la Tribuna’

Figure 3. Structure and surroundings of railway bridge in the ‘Alto de la Tribuna’

Table 1. Weathering forms of railway bridge in the ‘Alto de la Tribuna’

Deteriorated concrete surfaces – loss of concrete material

Back weathering

Relief

Relief

These concrete surfaces have loss of material. In one case the loss is parallel to original concrete surface because of the crumbly disintegration. In the other cases, it is due to partial weathering.

Deteriorated concrete surfaces – biological colonization and crust

Biological colonization of mosses

Black crust and biological colonization

Biological colonization of mosses (dark green) and lichens (white spots)

Micro- and biological colonization

Concrete surfaces of this bridge have biological colonization of different types. It was observed the presence of mosses, lichens and biofilms. Additionally, there was a surface with biological colonization and black crust at the same time.



Deteriorated concrete surfaces – biofilms and/or soiling

In the photographs it is showed dirty surfaces of black color. The coloration of the surfaces owing to particular matter, which is a byproduct of the combustion of fossil fuels; the formation of black biofilms or both.

Deteriorated concrete surfaces – Peeling

This surface has a loss of material parallel to concrete surface due to detachment of crusts with adherent concrete material. In addition, it was observed some biological colonization of mosses in that surface.

Damaged rails – corrosion

The photographs show corrosion of the Bridge rails, weathered wood and biological colonization of rusty rail. The superstructure of the bridge has an advance degree of deterioration.

Deteriorated gabions – bioweathering

The principal sign of weathering of the gabions is biological colonization. The presence of biofilms, mosses, lichens and grass is also showed.



Vehicular bridge located 580m (approx.) after ‘Alto de la Tribuna’

Figure 4. Structure and surroundings of vehicular bridge located 580m (approx.) after ‘Alto de la Tribuna’

Table 2. Weathering forms of vehicular bridge located 580m (approx.) after ‘Alto de la Tribuna’

Deteriorated concrete surfaces – crust and soiling

Black crust

Soiling of anthropogenic source


Soiling of anthropogenic source(graffiti)



The deteriorated surfaces presented coloration changes mainly by anthropogenic causes. With exception of graffiti, the other soiling of anthropogenic sources are due to carbon transportation. The black crust observed in one photograph may be a result of air pollution or biological action.

Deteriorated concrete surfaces – biological colonization

In the images it is observed a significant biological colonization of higher plants.



Deteriorated concrete surface – back weathering

In the bottom of the superstructure of the bridge there is shedding material parallel to the original surface. This form of deterioration can be classified in “back weathering due to loss of crusts” according to the categories of Fitzner & Heinrichs (2002).

Deteriorated concrete surface - fissures

In the image is surveyed concrete failure by tension stress. The fissure extends along entire retaining wall that is part of the bridge.

Vehicular bridge located at the end of the ‘Bogotá – Facatativá – Los Alpes’ concession.

Figure 5. Surroundings of vehicular bridge located at the end of the ‘Bogotá – Facatativá – Los Alpes’ concession

Table 3. Weathering forms of vehicular bridge located at the end of the ‘Bogotá – Facatativá – Los Alpes’ concession

Deteriorated concrete surfaces – soiling, crusts and biological colonization

Back crust and yellowish green and light green biological colonization.

Biological colonization (mosses, lichens and other plants)

Soiling of water source (mud)

In this vehicular bridge were detected three different types of deterioration forms associated with changes in the original coloration of the concrete surface: soiling, crust and biological colonization. Soiling is water source, the crust is just forming and the biological colonization in some surfaces is a critical problem.



Deteriorated concrete surfaces - fractures

In the image it is shown several fractures in concrete. The possible reason of this damage are the flaws in the design of the slab.

Vehicular bridge located in landslide and collapse in ‘Albán’ (Guayacundo Alto)

Figure 6. Structure and surroundings of vehicular bridge located in landslide and collapse in ‘Albán’ (Guayacundo Alto)

Table 4. Weathering forms of vehicular bridge located in landslide and collapse in ‘Albán’ (Guayacundo Alto)

Deteriorated concrete surfaces – soiling and biological colonization

Biological colonization (moss and microorganisms

Soiling of water source (brown colored)

Soiling of water source (brown and brown – green colored)

Soiling of atmosphere source

Soiling and biological colonization in the first instance cause aesthetic changes as it is observed in the photographs. In the presented cases soiling are water and atmosphere source. Water soiling brings produces brown and brown – green colorations on the concrete surface; while atmosphere soiling originates grey to black colorations. On the other hand, biological colonization causes green colorations, among other things.



Deteriorated concrete surfaces - clearing out of stone components

The deterioration form observed in these images is “clearing out of stone components”, according with Fitzner & Heinrichs (2002). In that deterioration form, the selective weathering produces the protruding of the aggregates of the concrete.

Deteriorated concrete surfaces - detachment

In these photographs it is observed the significant loss of large pieces of concrete. The lost material is detached in form of the crumbs and splinters.

Concrete structures located near to weighing scale for heavy vehicles

Figure 7. Surroundings of concrete structures located near to weighing scale for heavy vehicles

In “weighing scale for heavy vehicles” location were visited three different concrete structures: vehicular

bridge, channel and rectangular shaped structure (Figure 8).

Vehicular bridge

Channel

Rectangular shaped structure

Figure 8. Visited structures in “weighing scale for heavy vehicles” location



Vehicular bridge and channel are in contact with reddish-brown water, which are occurring iron

oxidation processes.

Figure 9. Water with oxide of iron. (Left) bridge, (right) channel

In the iron oxidation, bivalent ferrous ion is oxidized to trivalent ferric ion (B. Tapias, n.d.)

Later, ferric ion reacts with water hydroxide ion to produce iron (III) hydroxide. This reagent is poorly

soluble, highly encrusting and reddish brown colored (B. Tapias, n.d.)

In the following table it is presented the identified deterioration forms of the concrete structures visited.

Table 5. Weathering forms of concrete structures located near to weighing scale for heavy vehicles

Deteriorated concrete surfaces – coloration, soiling and biological colonization

Surfaces of vehicular bridge

Black microorganisms or soiling of atmosphere source

Soiling of water source (reddish brown colored)

Biological colonization (plants)



Surfaces of channel Surface of rectangular shaped structure

Soiling of water source (soil) and biological colonization (moss)

Light olive green microorganism, higher plans and soiling of atmosphere source.

Soiling of atmosphere and water source and biological colonization (possibly moss).

In these images it is possible to observe different colorations by diverse causes. In all of the visited structures the causes of coloration of surfaces are biological colonization and soiling. The most dangerous coloration form is biological colonization because induce other mechanisms of deterioration.

Deteriorated concrete surfaces of the channel – weathering out of stone components

According to Fitzner & Heinrichs (2002) the weathering form shown in the images are “weathering out of stone components”. The concrete surface loses material that is susceptible to erodible agents.

Deteriorated concrete surfaces – crumbly disintegration

Vehicular bridge

Rectangular shape structure

In the vehicular bridge and the rectangular shaped structure is observed the detachment of concrete material in the form of crumbs.

Vehicular bridge located at 200 m (approx.) after ‘Jalisco’ Toll Road

Figure 10. Overview of vehicular bridge located at 200 m (approx.) after ‘Jalisco’ Toll Road



Table 6. Weathering forms of vehicular bridge located at 200 m (approx.) after ‘Jalisco’ Toll Road

Deteriorated concrete surface – crust and biological colonization

Black crust and moss

Black crust and plants (especially moss)

Black crust and plants (mainly mosses)

Black crust

Most of the photographs show an important and massive biological colonization of mosses. Also, it is observed black strongly adhesive deposits, “crusts”, on the concrete surface. The sources of those deposits are not recognizable. In all cases, the crust and biological colonization trace the morphology of the concrete surface.

Deteriorated concrete surface – coloration and biological colonization

Areas of high humidity, uncontrolled release of water

Microorganisms (black and brown colored) and lichens (pale green colored)

Microorganisms

Microorganisms

Microorganisms and moss

Microorganisms, moss and other higher plants

Photographs show coloration by biological means. In most of cases, the biological colonization is promoted by the humidity of the surfaces. The deterioration of the concrete surface by biological colonization is not only esthetical, but also the metabolic activities produce corrosion and increase porosity of the concrete, among other things.

Deteriorated concrete surface – relief

The concrete surfaces have morphological changes due to partial and selective deterioration. The eroded agents were focus on sensible concrete components caused the protruding of aggregates and/or loss of concrete particles.



Railway bridge located at the entrance of the Vereda ‘Santa Ana’

Figure 11. Structure and surroundings of railway bridge located at the entrance of the Vereda ‘Santa Ana’

Table 7. Weathering forms of railway bridge located at the entrance of the Vereda ‘Santa Ana’

Weathering forms in rails

Moss and lichens

Moss

Lichens

Rails have biological colonization of moss and lichens. Also, rails show signs of corrosion.

Deteriorated concrete surface – discoloration and biological colonization

The discoloration of the concrete surface is by microbiological or inorganic causes. If discoloration is inorganic can be efflorescences or subflorescences. Additionally, it shows other forms of biological colonization (moss and lichens).

Deteriorated concrete surface – relief and biological colonization

In the photography, the concrete surface has morphological change due to selective weathering. The possible weathering forms are “weathering out of stone components” and “clearing out of stone components”, according to Fitzner and Heinrichs (2002). In the field visit was evident the loss of bearing capacity of the concrete in that zone because with a little applied force the concrete failed. Additionally, biological colonization (moss) is observed again.



Deteriorated concrete surface – severe loss of concrete material

The concrete in that zone has a severe loss of material, possibly by biological agents (higher plants and microorganisms). For example, the roots of the plant induce additional tensile stress in matrix of concrete causing loss of material. For its part, microorganisms induce corrosion of the concrete by action of acid products of subproducts of metabolism causing again loss of material.

Deteriorated concrete surface – carbonation

The weathering form shown in the previous images is carbonation. In the carbonation, the CO2 in air penetrates into de concrete and reduces the pH value from above 12 to less than 9; that cause the corrosion of embedded reinforce steel bars which may result in spall or split of concrete (Liang et al, 2001)

Deteriorated concrete surface – soiling and biological colonization

Overview 1

Overview 2

Soiling (water source)

Soiling (anthropogenic source) & dark colorization of the surface (non- recognizable cause)

Soiling (atmosphere source) & biological colonization (lychens)

Soiling (atmosphere source) & biological colonization (higher plants)

Soiling (soil particles- water source) & biological colonization (higer plants)

These images show the alteration of the original coloration of the bridge surfaces. The causes of alterations have different sources and can be divided into two group. The first group is soiling, that is dirt deposits on concrete surface. The identified dirt deposits are atmosphere (located mainly on bridge substructure, surfaces exposed to vehicular contamination <particular matter>), water (pipes, abutment) and anthropogenic (graffities) source. The other is related to grow of plant or eventually microorganism on concrete surface, called biological colonization.



Vehicular bridge at the edge of the ‘Seco’ River

Figure 12. Overview of vehicular bridge at the edge of the ‘Seco’ River

Table 8. Weathering forms of vehicular bridge at the edge of the ‘Seco’ River

Deteriorated concrete surfaces – biological colonization

In that bridge, deterioration is due to biological colonization and soiling of water source. The identified biological matters are mainly mosses and grass.

2.2. Bridges were to be sampled After the field visit, it was decided to sample all the bridges except two of them, “Vehicular bridge

located at 580m (approx.) after Alto de la Tribuna” and “Vehicular bridge located at 580m (approx.) after

Alto de la Tribuna”. The channel and the rectangular shaped structure were not sampled.

3. Materials and methods

3.1. Sampling technique in bridges – removal of microorganisms The sampling process used in the bridges was one of the non – destructive methods mentioned by

Hirsch, Eckhardt and Palmer (1995). The idea of the technique is the removal of microorganisms using a

swab and saline solution.

First of all, the excess of vegetal matter was removed from the concrete surface. Then the swab was

immersed in a test tube containing 9mLt of sterile diluent (e.g. sodium pyrophosphate solution at 0.1%).

Afterwards, the swab was rubbed on the concrete surface and returned to the test tube.



a. Clean surface

b. Immerse swab into saline

solution

c. Rubb swab over surface

d. Return swab to test tube

Figure 13. Sampling process in bridges

The test tubes with concrete samples were storage in a Styrofoam cooler during the transportation.

3.2. Culture mediums

PYGV medium

Oligotrophic PYGV medium was used in the isolation of

microorganisms that growth in extreme environments. For

example, soils in Antarctica (Mevs et al., 2000), rocks (Hirsch et al.,

1995), groundwater (Filip & Demnerova, 2009; Marxsen, 1988),

concretions (Ghiorse & Hirsch, 1982) and hypersaline,

heliothermal & meromictic lakes (Labrenz et al., 1999). Thus, its

medium was selected for isolation of microorganisms removed

from concrete surface of the bridges.

The procedure and reagents needed for the preparation of the PYGV medium are presented below. The

information was taken from DSMZ1 and Stackebrandt & Schaal (2006).

Mineral salt solution ("Hutner/Cohen-Bazire") 20.00 ml Peptone (Bacto) 0.25 g Yeast extract (Bacto) 0.25 g Agar (Bacto) 15.00 g Distilled water 965.00 ml

Sterilize 20 min./121°C. After cooling to 60°C add to the medium:

Glucose solution (2.5%, sterile-filtered) 10.00 ml Vitamin solution (double conc.) 5.00 ml

Adjust pH to 7.5 (the medium is only weakly buffered; needs approx. 10 drops/l medium of 6 N KOH).

Mineral salt solution:

Nitrilotriacetic acid (NTA) 10.00 g

1 Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures)

Figure 14. Petri dishes with medium PYGV without vitamin and glucose solution



MgSO4 x 7 H2O 29.70 g CaCl2 x 2 H2O 3.34 g Na2MoO4 x 2 H2O 12.67 mg FeSO4 x 7 H2O 99.00 mg Metallic salt solution 50.00 ml Distilled water 900.00 ml

Dissolve NTA first by neutralizing with KOH, then add other salts. Adjust pH to 7.2 with KOH or H2SO4.

Adjust volume to 1000.0 ml with distilled water.

Metallic salt solution

Na-EDTA 250.000 mg ZnSO4 x 7H2O 1095.000 mg FeSO4 x 7H2O 500.000 mg MnSO4 x H2O 154.000 mg CuSO4 x 5H2O 39.200 mg Co(NO3)2 x 6H2O 24.800 mg Na2B4O7 x 10H2O 17.700 mg Distilled water 100.000 ml

Dissolve the EDTA and add a few drops of concentrated H2SO4 to retard precipitation of the heavy metal

ions.

Vitamin solution (double conc.):

Biotin 4.00 mg Folic acid 4.00 mg Pyridoxine-HCl 20.00 mg Riboflavine 10.00 mg Thiamine-HCl x 2 H2O 10.00 mg Nicotinamide 10.00 mg D-Ca-pantothenate 10.00 mg Vitamin B12 0.20 mg p-Aminobenzoic acid 10.00 mg Distilled water 1000.00 ml

Store in the dark and cold (5°C).

The culture media done in the laboratory does not have the vitamin and glucose solutions. The absence

of glucose solution is caused by the microorganisms in concrete have meager glucose source. With

respect to the vitamin solution, the two reasons for not including it are: [1] the impossibility to weigh the

small quantities of mass required in the medium with the lab equipment available; [2] the unlikely to find

that vitamins in the micro-environment of concrete where live the microbes.



Brain Heart Infusion Broth

Brain Heart Infusion Broth is an enriched non – selective broth medium that is used for the cultivation of

fastidious and non – fastidious microorganisms. This medium is nutritious and well buffered to support

the growth of wide variety of microbes (Neogen Corporation, 2010; Anaerobe systems, 2009)

Figure 15. Brain Heart Infusion Broth in test tubes.

In the laboratory, it was prepared 200 mL of the broth. The first step was to weigh 7.4 g of Brain Heart

Infusion Broth and add it to 200 mL of distilled water. After, the solution was sterilized with autoclave.

Finally, the broth was poured in 17 test tubes.

3.3. Culture techniques

Swab technique

The swab technique is used to transfer the microorganisms from a liquid medium to a solid medium.

The test tube containing saline solution and particles of the sampled bridges is vortex. The swab is

immersed in a test tube to take microorganisms suspended. Afterwards, the swab is rubbed on the

culture media for inoculation of microbes (see Figure 16). Once the growth medium in the petri dish is

inoculated with microorganisms, the plates are incubated at 25 °C for four weeks.

a. Vortex b. Take microbes suspended in saline solution [moisten the swab]

c. Inoculation

Figure 16. Main steps for inoculation of microbes with a swab.



The streak plate procedure

The streak plate procedure is a classic method for isolating individual strains of bacteria from a sample.

The first step of this procedure is to sterilize a wire loop in a flame. To cool the sterilized loop, it is put in

contact with a sterile agar plate. Afterwards, the loop is dipped into a

sample containing the microorganisms. The loop is moved back and forth

across the surface of the agar. Before continuing to streak the plate, the

remaining bacteria on the loop are killed in the flame. After cooling the

sterilized loop, it is dragged through the previous path, picking up a small

number of bacteria and spreading them into a new area of the plate. After

sterilizing and cooling the loop again, the procedure is repeated twice. With each new path, the loop

picks up a smaller number of microorganisms and, therefore, spread them farther and farther apart

(Perry et al., 2002). Finally, the plates are incubated at 25 °C for four weeks.

a. Sterilize a wire loop in a flame

b. Take microorganisms

c. Streak the plate

Figure 18. Main steps for streaked plate. Adapted from http://www.sumanasinc.com/webcontent/animations/content/streakplate.html

3.4. DNA isolation

Direct DNA isolation

Direct DNA extraction was done with culturable and non-culturable microorganisms removed from the

concrete surface. The microbes were in liquid phase (saline solution) before DNA isolation.

PowerSoil® DNA Isolation Kit was used to direct DNA isolation. The kit extracts microbial DNA from all

soil types and other environmental samples

(e.g.: concrete). The isolation of DNA is

achieved by means of cells lysis and purification

of genetic material. Cells are lysed by a

combination of chemical agents and

mechanical shaking. Microbial cells are

subjected to collision of beads by randomly

shaking and disruption agents that cause the

cells to break open. Similarly, the purification of

genetic material is done by chemical and

physical means. Some reagents precipitate non-

DNA organic and inorganic material including

cell debris, proteins, fatty acids, etc. Other substances bind DNA to silica membrane and/or allow the

Figure 17. Streaked plate

Figure 19. PowerSoil® DNA Isolation Kit. Taken from www.mobio.com



discard of contaminants, leaving only DNA bound to the membrane. At the end of the process, there is

other substance that releases the DNA from the silica membrane. All chemical purification process

involves centrifugation (MO BIO Laboratories, Inc, 2010).

The summary of the protocol for DNA isolation is presented in the Table 9. The substances C1, C2, C3, C4,

C5 and C6 are manufacturer's secrets. Solution C1 contains sodium dodecyl sulfate2 (SDS) and other

disruption agents required for cell lysis. Solution C2 and C3 contain reagents to precipitate non-DNA

organic and inorganic material. Solution C4 is a high concentration salt solution. C4 adjusts the DNA

solution salt concentration to allow binding of DNA to the silica filter membrane in the Spin Filter.

Solution C5 is an ethanol based wash solution used to further clean the DNA that is bound to the silica

filter membrane. Finally, solution C6 is a sterile elution buffer used for the same purpose as the solution

C5 (MO BIO Laboratories, Inc, 2010).

Table 9. Protocol Summary. MO BIO Laboratories, Inc (2010)

1. Prepare sample - Add sample to PowerSoil® Bead

Tube

- Add solution C1 - Vortex

Centrifuge

3. Cell lysis - Add solution C2 - Incubate at 4°C

Centrifuge

5. Inhibitor Removal Technology®

- Add solution C3 - Incubate at 4°C

Centrifuge

7. Bind DNA - Add solution C4 - Load into Spin Filter

Centrifuge

9. Wash - Wash with solution

C5

Centrifuge

11. Elute - Elute with solution C6

Indirect DNA isolation

Indirect DNA extraction was done with culturable microorganisms. The microbes were in liquid growth

medium (Brain Heart Infusion Broth) before DNA isolation.

DNA isolation was performed using the organic (phenol – chloroform) extraction. This extraction method

involves the serial addition of several chemicals. First of all, SDS and proteinase K is added to break open

the cell walls and to break down the proteins that protect the DNA molecules. Next a phenol/chloroform

solution is added to separate the protein from the DNA. When the sample was centrifuged, the

unwanted proteins and cellular debris are separated away from the aqueous phase and DNA molecules

2 Sodium dodecyl sulfate is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane of several organisms (MO BIO Laboratories, Inc, 2010).



can be cleanly transferred for analysis. Extracted DNA is resuspended in TE buffer that contains EDTA,

which chelates the magnesium ions needs as cofactors for DNase3. (Butler, 2005; Corkill & Rapley, 2008).

Add sample, SDS, TE buffer and Proteinase K

Incubate at 65°C

Centrifuge

Add NaCl, CTAB, CIA, FCIA, isopropanol and ethanol.

Vortex

Centrifuge

Transfer aqueous upper phase to new tube

Add TE buffer

Figure 20. Schematic of DNA extraction process. Adapted from Butler (2005)

Figure 21. General steps involved in extracting DNA. Adapted from Corkill & Rapley (2008)

The protocol steps are in Annex B.

3.5. PCR (Polymerase Chain Reaction) PCR is used to make large numbers of copies of a specific DNA fragment in a

test-tube. PCR involves DNA synthesis from two oligonucleotide primers that act

as sites for initiation by the DNA polymerase and define the region of the

template DNA that will be copied. The primer is extended in the presence of the

four deoxynucleotides (dATP, dGTP, dCTP and dTTP) and specific buffer

conditions so that the DNA between the two primer binding sites will become

amplified during each cycle of the PCR. The reaction tube is placed in a thermal

cycler and subjected to a series of heating and cooling reactions (McPherson &

Moller, 2000):

3 Enzymes that degrade DNA

Figure 22. Thermal cycler used in PCR.



95°C to pre-denature the template strands; then

95°C to denature the template strands; then

51 °C to allow the primers to anneal; then

72°C, the optimal temperature for thermostable DNA polymerase (extention);

repeat 30 times steps from second to fourth.

The temperature is maintained at 72°C in termination phase;

ultimately, in the cooling phase the temperature drops to 10°C.

Figure 23. Series of heating and cooling reactions in 16S amplification.

The previous heating and cooling reactions were used in the laboratory for amplification of 16S

ribosomal DNA. The reagents (quantities and concentrations) for positively PCR amplification are

presented below.

Table 10. Reagent used in PCR

Test No. 7 Test No. 9

Reagent 1x (µLt) Reagent 1x (µLt)

MgCl2 (1.5mM) 1.50 MgCl2 (1.5mM) 1.50

Buffer (1x) 2.50 Buffer (1x) 2.50

dNTPs (3mM) 0.75 dNTPs (3mM) 0.75

Primer F (1µM) 0.25 Primer F (0.8µM) 0.20

Primer R (1µM) 0.25 Primer R (0.8µM) 0.20

Taq (5u) 0.3 Taq (5u) 0.30

DNA (25ng/µL) 1.00 DNA (25ng/µL) 1.00

Deionized water 18.45 Deionized water 18.55

∑ = 25.00 ∑ = 25.00

Primer F and primer R used in DNA amplification were 27F and 1492R which sequence are 5’ AGA GTT TGA TCM TGG CTC AG 3’ and 5’ TAC GGY TAC CTT GTT ACG ACT T 3’, respectively.



3.6. Electrophoresis Electrophoresis is a separation technique based on the mobility of ions in

an electric field. Under the influence of the field charged molecules (e.g.

DNA) and particles migrate in the direction of the electrode bearing the

opposite charge. Because of their varying charges, masses, sizes and

shapes; different molecules and particles of a mixture will migrate at

different velocities and will be separated into single fractions

(Westermeier, 2005; Tissue, 1996).

The procedure followed in the laboratory for electrophoresis has three stages. The first is the

preparation of the gel, the second is the separation and the third is the visualization.

In the preparation of gel it was used agarose, buffer (TAE) at 1x and GelRed. The quantities of the

reagents were taken according to the size of the gel. For a small gel it was used 0.6 g of agarose, 40 mLt

of buffer and 2 μL of GelRed; while for a big gel it was used 1.05 g of agarose, 70 mLt of buffer and 3.5

μL. The preparation of the gel consists of mixing agarose and buffer in an Erlenmeyer flask and heated in

a microwave oven for one minute. Then add GelRed to the solution of agarose and buffer. The last step

is to pour the solution into a mold and allow the solidification.

On the other hand, in the separation, the gel was placed in an electrophoresis chamber and covered by a

buffer. Subsequently the DNA was put in the gel’s channels. Then the electric current was applied with

75 V and 400 mA during 80 minutes (or 40 minutes if the gel is partitioned).

Figure 25. Gel of electrophoresis

Figure 26. Schematic illustration of a typical gel electrophoresis setup for the separation of the nucleic acids. Adapted from Corkill & Rapley (2008).

In the visualization stage, the gel was placed in a transillumination device that used ultraviolet light to

show the migration of the DNA in the gel.

Figure 24. Electrophoresis apparatus



3.7. Spectrophotometric measurement of DNA concentration. The ability to quantify DNA concentration is a prerequisite for some

methods used in molecular biology. In the majority of situations this

is carried out using spectrophotometry, which is a non-destructive

technique that allows the sample to be recovered for further

analysis or manipulation. Spectrophotometry uses the fact that

there is a relationship between the absorption of ultraviolet light by

DNA and its concentration in a sample. DNA concentration can be

determined by measuring the absorbance at 260 nm (A260) in a

spectrophotometer (Heptinstall & Rapley, 2000; Tuffaha, 2008).

It is important to mention that spectrophotometric measurements

do not differentiate between DNA and RNA and contamination by RNA can lead to an overestimation of

the DNA concentration. Additionally, phenol has an absorbance maximum of 270 – 275 nm, which is

close to that of DNA (Tuffaha, 2008). Thus there are two types of contamination that affect the

measurement of DNA concentration in a sample, by phenol and RNA.

For measurement DNA concentration it was used a pedestal of NanoDrop 2000c spectrophotometer. 2µL

of DNA sample is pipetted onto the lower measurement pedestal. After, the sampling arm is lowered

and spectral measurement is initiated using software on the computer. When the measurement was

completed, the sampling arm is raised and the sample is wiped from both, the upper and lower pedestals

using a dry, lint – free laboratory wipe.

Pipette DNA sample

Spectral measurement

Wipe pedestal

Figure 28. Main steps for measurement of DNA concentration. Adapted from www.nanodrop.com

3.8. DNA sequencing and purification DNA sequencing4 and purification were given by Macrogen Inc. (Korea). For some samples that company

gave the amplification of DNA (PCR).

3.9. Gram staining When bacteria are stained with certain dyes and treated with iodine, some species can be easily

decolorized with organic solvents (e.g. ethanol or acetone) whereas others resist decolorization. Bacteria

4 “DNA sequencing is any process used to map out the sequence of the nucleotides that comprise a strand of DNA” (http://dnasequencing.com/)

Figure 27. NanoDrop 2000c Spectrophotometer.



that retain the stain have been said to be Gram – positive, those that are decolorized are called Gram –

negative. The capacity to retain the stain is mainly related to the high amount of peptidoglycan in the cell

wall of Gram – positive microorganisms. Thus the distinction between both groups depends on

differences in the structure of the cell wall (Claus, 1992; Encyclopedia of Astrobiology, 2011).

The steps for Gram staining are the followings:

Put some drops of broth with bacteria in a sterile microscope slide.

Fixation → Use the air drying and flame fixation.

Cover the microscope slide with crystal violet during 1 minute and wash in distilled water.

Cover the slide with lugol during 1 minute and wash in distilled water.

Discolor bacterial cells with alcohol – acetone during 30 seconds and wash in distilled water.

Counterstaining cells with fuchsin during 15 seconds and wash in distilled water.

Allow air dying and clean de bottom of the slime with a paper towel to facilitate subsequent

observation under the microscope

Table 11. Gram staining reactions. Adapted from Obed (2011).

Gram staining Time Gram – positive Gram – negative

Sample

Not applicable

Crystal violet

60 seconds

Wash in distilled water

Lugol

60 seconds




Alcohol - acetone

30 seconds


fuchsin

15 seconds

3.10. Culture collection of isolated microorganisms Culture collection consists of in keeping in the laboratory the isolated microorganisms for future

investigations.

In the elaboration of culture collection it was used 500 µLt of liquid culture growth with suspended

microorganisms and glycerin. At first, the glycerin was pipetted in 17 microtubes. Then microtubes were

put into the autoclave for sterilization. In presence of a flame, the corresponding culture medium was

pipetted into each microtube and the name of the tube was assigned. Finally, microtubes were kept in a

freezer at - 80°C.

3.11. Assembly of DNA sequences The sequencing of genomic DNA samples was done in two ways, one starting with the forward primer

(27F) and the other starting with the reverse primer (1492R); hence the assembly of DNA sequences was

required to obtain a consensus. For the assembly, it was used a software called “Sequencher 5.0”. The

software platform permits the importation of DNA sequences of file type AB1. The assembly of the

sequences was done by selecting the minimum match percentage (60 to 100) and the minimum overlap

of the bases (1 to 100). After obtain a consensus it was accomplished a NCBI Blast5 search to find the

microorganism that correspond to the sequence.

5 “The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families”. (http://blast.ncbi.nlm.nih.gov/Blast.cgi)



Figure 29. Sequencher 5.0 platform

4. Results

4.1. DNA isolation and classic microbiology (culture mediums) This section contains information about the results of DNA isolation, PCR (only for indirect DNA

isolation), the descriptions of cultivation of microorganisms (only the last obtained culture mediums) and

gram staining of bacteria of interest.



In the following table it is presented the summary of the results: the names assigned to DNA isolation

samples, the names of the sampled surfaces of each bridge and the obtained culture media of the

corresponding concrete surface.

Table 12. Summary of the results of DNA isolation and classic microbiology

Place Direct DNA isolation

Surface name Classic microbiology (last obtained culture media)

Indirect DNA isolation

Railway bridge in ‘Alto de la Tribuna’

1A, 2A, 3A

Green part

Peeling

Orange & green part X 7, 8, 14

Vehicular bridge located in the end of ‘Bogotá – Facatativá – Los Alpes’ concession

4A, 5A

Black moss X 11, 5

Green part X 10, 15*


6A Green part

Concrete structures located near to weighing scale for heavy vehicles

7A Vehicular bridge X 16*

Vehicular bridge located 200m (approx.) after ‘Jalisco’ Toll Road

8A, 9A, 10A*, 11A*

Upper black part X 1*, 6, 13*

Green part X 2*, 3, 4*, 9*, 12

Lower black part X 17

Green moss


12A*, 13A* 1

2

(*) DNA samples sent to Macrogen Inc. Korea for sequencing

DNA isolation and PCR

Electrophoresis was used to know whether positive results were obtained to DNA extraction and PCR. In

the Figure 30 is shown that the DNA bands are very faint in the case of direct DNA isolation; in the case

of indirect DNA isolation, the corresponding image clearly shows the DNA bands for each sample and

some impurities that need to be removed; while, the PCR’s gel does not show band for all of the PCR

products and evince primer dimers. The samples that had the best results in direct DNA extraction were



10A, 11A, 12A and 13A. Regarding PCR products, in the test No. 7 the best results were for the samples

1, 2, 4, 9, 13, 15 and 16; whereas, the samples 1, 2, 4, 9, 15 and 16 had the best results in the test No 9.

Gel – direct DNA isolation

Gel – indirect DNA isolation

Gel – PCR for samples of indirect DNA isolation

Figure 30. Results of electrophoresis

Classic microbiology and samples surfaces

Railway bridge in ‘Alto de la Tribuna’

In this bridge were taken three samples of the surfaces named ‘green part’, ‘peeling’ and ‘orange &

green part’. Only orange & green part had favorable results with classic microbiology.

Green part

Peeling

Orange & green part

Figure 31. Sampled surfaces in the railway bridge in the ‘Alto de la Tribuna’



In the Table 13 and Figure 32 were presented the Petri dishes and the Gram staining results of microbes

of orange & green part.

Table 13. Petri dishes with isolated microorganisms of orange & green part

Photographs Description

In the culture medium grew two types of bacteria colonies. The first type has a white color, round shape and smooth margins. The second one has a translucent color, punctiform shape and drop –like elevation.

In the photograph of the entire petri dish is seen some blank points in the culture medium. These points are fungi; the bacterial colonies are much smaller than fungi. The bacteria colonies have a white color, curved margins and ingrowing into medium.

The bacteria colonies of interest have a white color, punctiform shape and drop – like elevation.



Sample # 7: Bacilli and coccus gram positive



Figure 32. Gram staining of isolated microorganisms of orange & green part.

Vehicular bridge located at the end of the ‘Bogotá – Facatativá – Los Alpes’ concession.

In this vehicular bridge were taken two samples of the surfaces called ‘black mosses’ and ‘green part’.

Out of both surfaces were isolated cultivable microorganisms.

In the next subsections were presented the results of classic microbiology of the sampled surfaces.

BLACK MOSSES

Figure 33. Sampled surface – black mosses

Table 14. Petri dishes with isolated microorganisms of black mosses surface


The white bacteria colonies have punctiform and irregular shapes with hilly elevation.



The white bacteria colonies have round and irregular shapes with flat elevation.

Sample # 11: Bacilli gram positive


Figure 34. Gram staining of isolated microorganisms of black mosses surface

GREEN PART

Figure 35. Sampled surface – green part

Table 15. Petri dishes with isolated microorganisms of green part


There are two types of bacteria colonies with light pale orange color. Some colonies have a concentric configuration and drop – like elevation. The others ones have a punctiform shape and drop – like elevation.



In the petri dish, there are white bacteria colonies with round and punctiform shapes and drop – like elevation.

Sample # 10: Coccus gram positive


Figure 36. . Gram staining of isolated microorganisms of green part


Figure 37. Sampled surface in vehicular bridge located in landslide and collapse in ‘Albán’ (Guayacundo Alto)

In this vehicular bridge it was taken a sample in a surface with signs of biological colonization;

unfortunately, the sampled microorganisms were not cultivable or the culture medium was not the

indicated.



Vehicular bridge located near to weighing scale for heavy vehicles

In this bridge, the only sample was taken in the water with processes of oxidation of the iron. In this

case the microbes were cultivable.

Figure 38. Sampled surface [vehicular bridge]

Table 16. Petri dishes and Gram staining of the isolated microorganisms of vehicular bridge

Photographs and description Gram staining

The bacteria colonies have a light cream color, punctiform shape, smooth margins and drop – like elevation.


Vehicular bridge located 200m (approx.) after ‘Jalisco’ Toll Road

This bridge was the place with the largest number of samples collected. The names assigned to the

sampled surfaces are ‘upper black part’, ‘green part’, ‘lower black part’ and ‘green mosses’. All surfaces,

with the exception of the last one, had cultivable microorganisms.

UPPER BLACK PART

Figure 39. Sampled surface – upper black part



Table 17. Petri dishes with isolated microorganisms of upper black part


The bacteria colonies are white translucent. Additionally, colonies shapes are round with a lobaty margin and smooth surface.

Colonies of bacteria are white with a punctiform, round and irregular shape. Moreover, colonies margins are lobaty and the elevation of the colonies are umbonate.

The bacteria colonies of interest are light orange with punctiform shape and drop – like elevation.




Figure 40. Gram staining of isolated microorganisms of upper black part



GREEN PART

Figure 41. Sampled surface green part

Table 18. Petri dishes with isolated microorganisms of green part


The bacteria colonies are white with irregular shape and convex elevation.

In the petri dish, there are white bacteria colonies with irregular and round shapes. Also, colonies have a convex elevation.

The transparent bacteria colonies are punctiform shape and have a drop – like elevation.

The transparent bacteria colonies are punctiform shape, smooth margins and have a drop – like elevation.



The bacteria colonies of interest are white with irregular shape and have a raised elevation.

Sample # 2: Bacilli and coccus gram negative

Sample # 3: Bacilli gram negative




Figure 42. Gram staining of isolated microorganisms of bridge after ‘Jalisco’ toll road – green part

LOWER BLACK PART

Figure 43. Sampled surface lower black part



Table 19. Petri dishes with isolated microorganisms of lower black part


The color of the bacterial colonies is light yellow. Bacterial colonies are punticform shape with smooth margins and have drop – like elevation.

GREEN MOSSES

In this surface there are predominate biological colonization of higher plants, especially mosses of green

color.

Figure 44. Sampled surface green mosses


The sampled microbes of deteriorated surfaces in this railway bridge were uncultured or the culture

medium is not adequate.

Figure 45. Sampled surfaces 1 and 2.



4.2. DNA sequences and NCBI blast search Not all DNA samples sent to Macrogen Inc. Korea had good sequencing results. The results of PCR

products of indirect DNA isolation failed. According to Macrogen Team (personal communication,

December 25, 2011) “the failed reason would be low density of sample or primer mismatches with

template”. Therefore, DNA sequences and NCBI blast search presented in this section correspond to DNA

samples of direct DNA isolation.

The sequencing results with the respectively chromatograms provided by Macrogen Inc. Korea are found

in Annex D. The blast result realized by Macrogen Inc. Korea is presented bellow. Later in this section, are

exposed the obtained blast results of the consensus.

Blast results of DNA sequences

The overall Blast results reported in the Table 20Table 21 according with score, identities and the two

alignments for each sample indicate biological significance of the results. The fact that the E value is 0.0

for all samples indicate that there are very low possibilities to find different alignments with scores

equivalent to or better than the obtained. In other words, Blast results indicate a great significance of

the score and the alignment; namely, there are good possibilities that the microorganisms isolated

correspond to the found with Blast analysis.

Therefore, the samples 10A, 11A, 12A and 13A correspond to microorganisms: Pseudomonas,

Buttiauxella, Rahnella aquatilis strain and Pseudomonas, respectively.

Table 20. Blast results of DNA sequences.

Score Identities

Name Gene Bit Raw EValue Match Total Pct. (%)

10A-27F Uncultured Pseudomonas sp. clone A09-06E 16S ribosomal RNA gene, partial sequence

1883 950 0.0 1020 1042 97

10A-1492R Pseudomonas sp. VI3X 16S ribosomal RNA gene, partial sequence

1497 755 0.0 833 851 97

11A-27F Buttiauxella sp. GC21 from China 16S ribosomal RNA gene, partial sequence

771 389 0.0 785 913 85

11A-1492R Uncultured bacterium clone IC11 16S ribosomal RNA gene, partial sequence

1195 603 0.0 686 703 97

12A-27F Uncultured bacterium clone nbw502c10c1 16S ribosomal RNA gene, partial sequence

1665 840 0.0 866 872 99

12A-1492R Rahnella aquatilis strain 2B-CDF 16S ribosomal RNA gene, partial sequence

2016 1017 0.0 1088 1100 98

13A-27F Pseudomonas sp. 3-28(2010) 16S ribosomal RNA gene, partial sequence

1842 929 0.0 965 973 99

13A-1492R Pseudomonas sp. MW6 16S ribosomal RNA gene, partial sequence

1828 922 0.0 1005 1026 97



Blast results of consensus sequences

The assembly parameters are: minimum overlap of 20 for all sequences and the minimum match of 78%,

75% and 60% for the samples 10A, 11A and 12A, respectively. Unfortunately, assembly for the sample

13A was not possible.

In these cases, the scores related to identities and mismatches are high, the E values are low and the

alignments for the same sample, in general, are interrelated. Again the results of Blast analysis have

biological significance. Additionally, the results of consensus sequences agree with the DNA sequences

before the assembly.

In the following tables are presented the results of the blast analysis for consensus sequences.

Table 21. Blast results of consensus sequence of 10A

Description Max score

Total score

Query coverage

E value

Max ident

Uncultured Pseudomonas sp. clone A09-06E 16S ribosomal RNA gene, partial sequence

1793 2002 91% 0.0 96%

Pseudomonas sp. WPCB008 16S ribosomal RNA gene, partial sequence

1793 2002 91% 0.0 96%

Burkholderia cepacia strain N8 16S ribosomal RNA gene, partial sequence

1790 1998 91% 0.0 96%



Total score

Query coverage

E value

Max ident

Bacterium 8-gw1-1 16S ribosomal RNA gene, partial sequence

1817 1817 96% 0.0 87%

Buttiauxella brennerae strain S1/6-571 16S ribosomal RNA, partial sequence >gi|4581971|emb|AJ233401.1| Buttiauxella brennerae 16S rRNA gene (strain DSM 9396)

1817 1817 96% 0.0 87%

Buttiauxella agrestis ATCC 33320 strain DSM 4586 16S ribosomal RNA, partial sequence >gi|4581970|emb|AJ233400.1| Buttiauxella agrestis 16S rRNA gene (strain DSM 4586)

1810 1810 96% 0.0 87%



Total score

Query coverage

E value

Max ident

Uncultured Rahnella sp. clone DR-E12 16S ribosomal RNA gene, partial sequence

2028 2028 80% 0.0 96%



Uncultured Rahnella sp. clone DR-A3 16S ribosomal RNA gene, partial

2028 2028 80% 0.0 96%

Rahnella aquatilis strain 2B-CDF 16S ribosomal RNA gene, partial sequence

2028 2028 80% 0.0 96%

5. Discussion Cultivation of microorganisms was done in a low-nutrient medium; however it showed microbial growth.

The growth of microbes in PYGV agar indicates that some of the microorganisms of the sampled bridges

were cultivable. However, the distribution of the microbiota in the petri dishes was not the same of the

natural micro-environment. Otherwise, the rate of growth of the microorganisms in PYGV media was

slow; it took around ten days for colonies to become visible.

Through macroscopic observation of the mediums, there were identified colonies of bacteria and fungi

(including yeast). Generally, in PYGV medium it isolates bacteria of diverse types. In this media it was

cultivated gram negative α-proteobacteria from permafrost environment (Zhang et a., 2007);

Roseovarius tolerans from the Antarctic hypersaline lake –Ekha Lake (Labrenz et al., 1999); Sulfitobacter

a sulfur – oxidizing chemolithoheterotrophic bacteria from Black sea and Ekho Lake (Sorokin et al., 2005);

Pedomicrobium and Leutothrix (manganese – oxidizing bacteria) (Nealson, 2006) and; Arthrobacter,

Pseudomonas, Flavobacterium, and Alcaligenes species from soil and groundwater (Filip & Demnerova,

2009).

The importance of the cultivation of microorganisms for this study is related to the possibility of doing

several and different analysis of microbe colonies. The cultivation allows to do Gram staining, to try out

different forms of DNA isolation and to make culture collection of microorganisms for further studies.

On the other hand, according to the Blast analysis, the microorganisms identified through sequencing

are probably uncultured. In the results of DNA sequences and consensus sequences the alignments of

the samples indicate in the majority of cases the uncultivable condition of the microorganisms. Hence, it

is important to use techniques independent to cultivation because a lot of microorganisms of

environmental importance are not cultivable. Cultivation studies may fail to detect abundant or

dominate members of an environmental sample and are strongly biased (Liesack & Dunfield, 2002).

Additionally, there are major possibilities of contamination of the samples.

Microorganisms identified of direct DNA samples are Pseudomonas, Rahnella aquatilis and Buttiauxella;

all of them joint to the class Gammaproteobacteria. Rahnella and Buttiauxella also are of the same

family, Enterobacteriaceae (see Table 24. Taxonomy of the microorganisms identified ).



Table 24. Taxonomy of the microorganisms identified

Pseudomonas Rahnella aquatilis Buttiauxella

Superkingdom Bacteria Bacteria Bacteria

Phylum Proteobacteria Proteobacteria Proteobacteria

Class Gammaproteobacteria Gammaproteobacteria Gammaproteobacteria

Order Pseudomonadales Enterobacteriales Enterobacteriales

Family Pseudomonadaceae Enterobacteriaceae Enterobacteriaceae

Genus Pseudomonas Rahnella Buttiauxella

Species Rahnella aquatilis

Bacteria were found in concrete bridges in the worst state. Pseudomonas and Buttiauxella were

extracted from the vehicular bridge near to Jalisco toll road and Rahnella and Pseudomonas in the

railway bridge located at the entrance of vereda Santa Ana. Most of the time, the sampled surfaces

analyzed were enclosed by biological coverage or black crusts (see results of the bridges). The coverage

may favor the colonization of microorganisms in concrete surfaces. Mansch & Bock (1998) demonstrated

that in rock surfaces the presence of chemoorganotrophic6 bacteria (e.g. Pseudomonas, Buttiauxella and

Rahnella) were significantly higher in the occurrence of black crusts than without crusts. Regarded to

biological coverage, it is known that higher plants excrete substances that serve as substrates for

microorganisms (Sand, Jozsa & Mansch, 2002).

Another factor that permits the colonization of concrete surfaces by chemoorganotrophic bacteria is air

pollution. In scientific literature, it was reported the enrichment of building stone surfaces with

hydrocarbons, ammonium and reaction products of nitrogen oxides provided of industrial air pollution

(Schröder 1991; Saiz-Jimenez 1993; Bock & Fahrig 1993; Nord et al. 1994; Behlen et al. 1996). These

compounds may support the growth of microbes that use them as substrates (Mansch & Bock, 1998). In

this research, the contamination of vehicles (especially heavy) and train could allow the enrichment of

concrete surfaces with hydrocarbons and reaction products of nitrogen oxides from combustion of fossil

fuel. The hydrocarbons can be carbon source for chemoorganotrophic bacteria like the isolated in the

bridges.

The role of chemoorganotrophic bacteria as Rahnella and Buttiauxella in the biodeterioration of

concrete was not investigated. This type of microorganisms are likely to cause adverse effects on

building materials by carbon dioxide production, biofilm formation and discoloration (Sand, Jozsa &

Mansch, 2002). In this work, it was proposed the possibility that Rahnella and Buttiauxella are bio-

indicator of the presence of nitrifying bacteria by their capacity of reduction of NO3− to NO2

−. Nitrifying

bacteria have been associated to biodeterioration of concrete causing a biogenic nitric acid attack (Bock

6 Chemoorganotrophic bacteria are microorganisms that have organic electron donor. In other words, that bacteria gain their energy by oxidation of organic substances (Javaherdashti, 2008).



& Sand, 1993; Mansch & Bock, 1998; Wilimzing & Bock, 1996). Then the presence of Rahnella and

Buttiauxella could be associated indirectly to acid attack of concrete surfaces.

The idea of bio-indicator is in concordance of the findings of Mansch & Bock (1998). They found a

negative correlation between cell numbers of chemoorganotrophic bacteria, along with fungi and the

nitrate content. Thus nitrate in building stone may be consumed microbiologically (Mansch & Bock,

1998). This fact can be extrapolated to concrete buildings but further research is needed.

Regarded to Pseudomonas, Nica et al. (2000) and Cwalina (2008) postulated that may contribute to

deterioration of concrete. Pseudomonas are able to oxidize sulfur compounds (eg. thiosulfate)

(Mizoguchi et al., 1976; Schook & Berk, 1978; Starkey, 1935; Vitolins & Swaby, 1969) and it were found in

degraded concrete growing autrophically on reduced sulfur compounds with minimal acid production

(Coleman & Gaudet, 1992). Thus Pseudomonas could be neutrophilic sulfur oxidizing microorganisms

(NSOM) under the classification made by Islander (1991). These microorganisms can growth in a media

of pH up to 9 (eg. concrete) and by the biological activities reduce the pH around 5 (Nica et al., 2000).

Additionally, Cwalina (2008) proposed that Pseudomonas have a highly corrosive aggressiveness on

concrete derived to reduction of nitrate (NO3–) to nitrite (NO2

–), oxidation of manganese of Mn2+ to Mn4+

and fermentative processes.

6. Conclusions and recommendations The microorganisms identified are non- cultivable, thus it is very important to use independent

cultivation techniques.

Pseudomonas, Rahnella and Buttiauxella are likely associated to biocorrosion of concrete in a

direct or indirect way.

Air pollution and coverage (higher plants and black crusts) contribute to microbiological

colonization of concrete surfaces.

Diverse techniques of DNA extraction could be applied to the same sample to obtain better

results.

It is convenient to sequence samples using 454 technique and with smaller fragment of

oligonucleotides in order to obtain better results.

For further investigations, it will be interesting to research in the laboratory the mechanisms of

deterioration of microorganisms isolated to concrete structures. For that experiment, it is

important to establish the optimal pH and temperature, the nutrients required by microbes, etc.

The surfaces that are important to analyze are those with soiling, crusts, signs of corrosion,

colonization of higher plants, among others.

PCR amplification will be done with primers different from 27F and 1942R to avoid the formation

of dimers and to improve the annealing to DNA stands.

It is important to know that investigation about bideterioration of concrete is a multidisciplinary

works that involves engineers, microbiologists and chemists.



To obtain reliable results from the statistical and biological standpoints, it is necessary to take a

large number of samples of deteriorated surfaces.

Cultivation of microorganisms permits the analysis of the samples through different techniques

(i.e. Gram staining, DNA isolation). In addition, the culture collection of microorganisms allows

further analysis; it permits to apply technologies developed in the future to analyze

characteristics of interest. These characteristics should be related to biodeterioration.

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8. Annexes

Annex A: Classification scheme of weathering forms The working group “Natural stones and weathering” developed a detailed classification of weathering

forms of rocks. Weathering forms are used for precise description of deterioration phenomena at

mesoscale (cm to m). There are four groups of weathering forms (see Figure 46).

The level 1 consists of 4 groups named: loss of stone material, discoloration / deposits, detachment and

fissures / deformation. In level 2, each group of level 1 is subdivided into main weathering forms. For its

part, Level 3 consist of subcategories of level 2 called individual weathering forms. In the last level, level

4, each individual weathering form is differentiated according to its intensity. In summary, level 3 gives a

more detailed description of the weathering of rocks than level 2; this level in turn, gives a more detailed

description of deterioration of rocks than level 1; and, level 4 gives the severity degree of deterioration.

Figure 46. Classification scheme of weathering forms. Adapted from Fitzner & Heinrichs (2002)

In the following table it is presented the classification scheme of weathering form for rocks.

Table 25. Classification scheme of weathering forms. Taken form Fitzner & Heinrichs (2002)

Level 1 – Group of weathering forms Group 1 - Loss of stone material

Level 2 Level 3 Level 4

Main weathering forms Individual weathering forms Classification of intensities (parameters)

Back weathering Uniform loss of stone material parallel to the

W Back weathering due to loss of scales Uniform loss of stone material parallel to the stone surface due to contour scaling.

sW Depth of back weathering (mm, cm)

sW1

sWn

Back weathering due to loss of crumbs / splinters Uniform loss of stone material parallel to the

uW uW1

Level 1 • 4 groups of weathering forms

Level 2 • 25 main weathering forms

Level 3 •75 individual weathering forms

Level 4

•Differentiation of individual weathering forms according to intensities



original stone surface.

stone surface due to crumbly disintegration. uWn

Back weathering due to loss of stone layers dependent on stone structure Uniform loss of stone material parallel to the stone surface due to exfoliation.

xW xW1

xWn

Back weathering due to loss of crusts Uniform loss of stone material parallel to the original stone surface due to detachment of crusts with adherent stone material.

cW cW1

cWn

Back weathering due to loss of undefinable stone aggregates / pieces Uniform loss of stone material parallel to the original stone surface. The type of the preceding detachment of stone material cannot be characterized.

zW zW1

zWn

Relief Morphological change of the stone surface due to partial or selective weathering.

R Rounding / notching Relief by rounding of edges or notching / hollowing out. Concave or convex soft forms.

Ro Depth of relief (mm, cm)

Ro1

Ron

Alveolar weathering Relief in the form of alveolae. Form comparable to honeycombs.

Ra Ra1

Ran

Weathering out dependent on stone structure Relief dependent on structural features such as bedding, foliation, banding etc. Frequently striped pattern.

tR tR1

tRn

Weathering out of stone components Relief due to selective weathering of sensitive stone components (clay lenticles, nodes of limonite etc.) or due to break out of compact stone components (pebbles, fossil fragments etc.). Hole-shaped forms.

Rk Rk1

Rkn

Clearing out of stone components Relief in the form of protruding compact stone components (pebbles, fossil fragments, concretions) due to selective weathering.

Rh Rh1

Rhn

Roughening Finest relief / alteration of gloss due to corrosion or loss of smallest stone particles on smoothed stone surfaces.

Rr Rr1

Rrn

Microkarst Relief due to corrosion, especially on carbonate rocks.

Rm Rm1

Rmn

Pitting Relief in the form of small pits due to biogenically induced corrosion, esp. on carbonate rocks.

Rt Rt1

Rtn

Relief due to anthropogenic impact Relief in the form of scratches etc.

aR aR1

aRn

Level 1 – Group of weathering forms



Group 1 – Loss of stone material


Main weathering forms Individual weathering forms Classification of Intensities (parameters)

Break out Loss of compact stone fragments.

O Break out due to anthropogenic impact Break out due to war, vandalism etc.

aO Volume of break out (cm3, dm3) or depth of break out (cm)

aO1

aOn

Break out due to constructional cause Break out due to statics, wedge effect of rusting iron etc.

bO bO1

bOn

Break out due to natural cause Break out due to wedgework of roots, earthquakes, intersection of fractures etc.

nO nO1

nOn

Break out due to non-recognizable cause oO oO1

oOn

Level 1 – Group of weathering forms Group 2 – Discoloration / Deposits



Discoloration Alteration of the original stone color.

D Coloration Chromatic alteration / coloring due to chemical weathering of minerals (e.g. oxidation of iron and manganese compounds), due to intrusion / accumulation of coloring matter or due to staining by biogenic pigments.

Dc Degree – change of color

Dc1

Dcn

Bleaching Chromatic alteration / decolorization due to chemical weathering of minerals (e.g. reduction of iron and manganese compounds) or extraction of coloring matter (leaching, washing out).

Db Db1

Dbn

Soiling Dirt deposits on the stone surface.

I Soiling by particles from the atmosphere Poorly adhesive, mainly grey to black deposits of dust, soot, fly ash etc.

pI Mass of deposits or degree – covering of the surface

pI1

pIn

Soiling by particles from water Poorly adhesive, mainly grey to brown deposits of dust, soil or mud particles.

wI

wI1

wIn

Soiling by droppings Deposits of droppings from birds, e.g. from pigeons.

gI

gI1

gIn

Soiling due to anthropogenic impact Paint, graffities, posters etc.

aI

aI1

aIn


Group 2 – Discoloration / Deposits




Main weathering forms Individual weathering forms Classification of intensities

(parameters)

Loose salt deposits

Poorly adhesive

deposits of salt

aggregates.

E Efflorescences

Poorly adhesive deposits of salt aggregates on the

stone surface.

Ee Mass of deposits

or degree –

covering of the

surface

Ee1

Een

Subflorescences

Poorly adhesive deposits of salt aggregates below

the stone surface, e.g. in the zone of detachment

of scales.

Ef Mass of deposits Ef1

Efn

Crust

Strongly adhesive

deposits on the

stone surface.

C Dark-colored crust tracing the surface

Compact deposit, grey- to black-colored, tracing

the morphology of the stone surface. Mainly due

to deposition of pollutants from the atmosphere.

dkC For dkC, hkC and

fkC:

degree – covering

of the surface for

diC, hiC and fiC:

thickness of the

crust (mm)

dkC1

dkCn

Dark-colored crust changing the surface

Compact deposit, grey- to black-colored, changing

the morphology of the stone surface. Mainly due

to deposition of pollutants from the atmosphere.

E.g. gypsum crust with impurities.

diC diC1

diCn

Light-colored crust tracing the surface

Compact deposit, light-colored, tracing the

morphology of the stone surface. Mainly due to

precipitation processes. Light-colored crusts of

salt minerals, calc-sinter or silica.

hkC hkC1

hkCn

Light-colored crust changing the surface

Compact deposit, light-colored, changing the


precipation processes. Light-colored crusts of salt,

calc-sinter or silica.

hiC hiC1

hiCn

Colored crust tracing the surface

Compact deposit, colored, tracing the morphology

of the stone surface. Mainly due to precipation

processes. E.g. colored crusts of salt minerals or

iron/manganese crusts.

fkC fkC1

fkCn

Colored crust changing the surface

Compact deposit, colored, changing the


precipation processes. Eg. colored crusts of salt

minerals or iron/manganese crusts.

fiC fiC1

fiCn

Biological

colonization

Colonization by

B Microbiological colonization

Colonization by microflora (fungi, algae, lichen)

and bacteria. Biofilms.

Bi Degree –

covering of the

surface

Bi1

Bin

Colonization by higher plants Bh Bh1



microorganisms or

higher plants.

Bhn

LEVEL 1 – Group of weathering forms Group 2 – Discoloration / Deposits



Discoloration to crust Transitional form between discoloration (D) and crust (C).

D-C Coloration to dark-colored crust tracing the surface Transitional form between coloration (Dc) and dark-colored crust tracing the surface (dkC).

Dc-dkC Degree – covering of the surface

Dc-dkC1

Dc-dkCn

Coloration to colored crust tracing the surface Transitional form between coloration (Dc) and colored crust tracing the surface (fkC).

Dc-fkC Dc-fkC1

Dc-fkCn

Soiling to crust Transitional form between soiling (I) and crust (C).

I–C Soiling by particles from the atmosphere to dark-colored crust tracing the surface Transitional form between soiling by particles from the atmosphere (pI) and dark-colored crust tracing the surface (dkC).

pI-dkC Degree – covering of the surface

pI-dkC1

pI-dkCn

Soiling by particles from the atmosphere to dark-colored crust changing the surface Transitional form between soiling by particles from the atmosphere (pI) and dark-colored crust changing the surface (diC).

pI-diC Thickness of the deposit (mm)

pI-diC1

pI-diCn

Loose salt deposits to crust Transitional form between loose salt deposits (E) and crust (C).

E–C Efflorescences to light-colored crust tracing the surface Transitional form between efflorescences (Ee) and light-colored crust tracing the surface (hkC).

Ee-hkC Degree – covering of the surface

Ee-hkC1

Ee-hkCn

Efflorescences to light-colored crust changing the surface Transitional form between efflorescences (Ee) and light-colored crust changing the surface (hiC).

Ee-hiC Thickness of the deposit (mm)

Ee-hiC1

Ee-hiCn

Biological colonization to crust Transitional form between biological colonization (B) and crust (C).

B-C Microbiological colonization to dark-colored crust tracing the surface Transitional form between microbiological colonization (Bi) and dark-colored crust tracing the surface (dkC).

Bi-dkC Degree – covering of the surface

Bi-dkC1

Bi-dkCn

Microbiological colonization to dark-colored crust changing the surface Transitional form between microbiological colonization (Bi) and dark-colored crust changing the surface (diC).

Bi-diC Thickness of the deposit (mm)

Bi-diC1

Bi-diCn

Level 1 – Group of weathering forms Group 3 – Detachment





Granular disintegration Detachment of individual grains or small grain aggregates.

G Granular disintegration into powder Detachment of smallest stone particles (stone powder).

Gp Mass of detaching stone material

Gp1

Gpn

Granular disintegration into sand Detachment of small grains as individual grains or small grain aggregates (stone sand).

Gs Gs1

Gsn

Granular disintegration into grus Detachment of larger grains as individual grains or small grain aggregates (stone grus). Especially on granites.

Gg Gg1

Ggn

Crumbly disintegration Detachment of larger compact stone pieces of irregular shape.

P Crumbling Detachment of larger compact stone pieces in the form of crumbs.

Pu Volume of detaching stone pieces (cm3, dm3) or mass of detaching stone material

Pu1

Pun

Splintering Detachment of larger compact stone pieces in the form of splinters. E.g. on compact carbonate rocks and quartzites.

Pn Pn1

Pnn

Crumbling to splintering Transitional form between crumbling (Pu) and splintering (Pn).

Pu-Pn Pu-Pn1

Pu-Pnn

Flaking Detachment of small, thin stone pieces (flakes) parallel to the stone surface.

F Single flakes Detachment of one layer of flakes parallel to the stone surface.

eF Mass of detaching stone material

eF1

eFn

Multiple flakes Detachment of a stack of flakes parallel to the stone surface.

mF mF1

mFn

Contour scaling Detachment of larger, platy stone pieces parallel to the stone surface, but not following any stone structure.

S Scale due to tooling of the stone surface Detachment of mainly thin scales due to tooling of the stone surface.

qS Thickness of the scales resp. stack of scales (mm, cm) or mass of detaching stone material

qS1

qSn

Single scale Detachment of one layer of scales.

eS eS1

eSn

Multiple scales Detachment of a stack of scales.

mS mS1

mSn


Group 3 – Detachment



(parameters)



Detachment of

stone layers

dependent on

stone structure

Detachment of

larger stone

sheets or plates

following the

stone structure.

X Exfoliation

Detachment of larger stone layers (sheets, plates)

following any stone structure (bedding, banding

etc.) and the stone surface. Structural feature is

oriented parallel to the stone surface.

Xl Thickness of

detaching stone

layers resp. stack

of layers

(mm, cm)

Xl1

Xln

Splitting up

Detachment of larger stone layers (sheets, plates)

following any stone structure (bedding, banding

etc.), but not the stone surface. Structural feature

is not oriented parallel to the stone surface.

Xv Number of

detaching stone

layers resp. splits

Xv1

Xvn

Detachment of

crusts with stone

material

Detachment of

crusts with stone

material sticking

to the crust.

K Detachment of a dark-colored crust tracing the

stone surface

dkK Mass of detaching

material

or

thickness of

detaching

layers

(mm)

dkK1

dkKn

Detachment of a dark-colored crust changing the

stone surface

diK diK1

diKn

Detachment of a light-colored crust tracing the

stone surface

hkK hkK1

hkKn

Detachment of a light-colored crust changing the

stone surface

hiK hiK1

hiKn

Detachment of a colored crust tracing the stone

surface

fkK fkK1

fkKn

Detachment of a colored crust changing the stone

surface

fiK fiK1

fiKn

Granular

disintegration to

flaking

Transitional form

between granular

disintegration (G)

and flaking (F).

G-F Granular disintegration into sand to single flakes

Transitional form between granular disintegration

into sand (Gs) and single flakes (eF).

Gs-eF Mass of detaching

stone material

Gs-eF1

Gs-eFn

Granular disintegration into grus to single flakes


into grus (Gg) and

single flakes (eF).

Gg-eF Gg-eF1

Gg-eFn


Group 3 – Detachment



(parameters)



Granular

disintegration to

crumbly

disintegration

Transitional form

between granular

disintegration (G)

and crumbly

disintegration (P).

G-P Granular disintegration into sand to single flakes

Transitional form between granular disinte-gration

into sand (Gs) and crumbling (Pu).

Gs-Pu Mass of detaching

stone material

Gs-Pu1

Gs-Pun

Granular disintegration into grus to crumbling


into grus (Gg) and crumbling (Pu).

Gg-Pu Gg-Pu1

Gg-Pun

Flaking to

crumbly

disintegration

Transitional form

between flaking

(F) and crumbly

disintegration (P).

F-P Single flakes to crumbling

Transitional form between single flakes (eF) and

crumbling (Pu).

eF-Pu Mass of detaching

stone material

eF-Pu1

eF-Pun

Single flakes to splintering


splintering (Pn).

eF-Pn eF-Pn1

eF-Pnn

Crumbly

disintegration to

contour scaling

Transitional form

between crumbly

disintegration (P)

and contour

scaling (S).

P-S Crumbling to single scale

Transitional form between crumbling (Pu) and

single scale (eS).

Pu-eS Mass of detaching

stone material or

volume of

detaching stone

pieces (cm3, dm3)

Pu-eS1

Pu-eSn

Splintering to single scale

Transitional form between splintering (Pn) and

single scale (eS).

Pn-eS Pn-eS1

Pn-eSn

Flaking to

contour scaling

Transitional form

between flaking

(F) and contour

scaling (S).

F-S Single flakes to single scale


single scale (eS).

eF-eS Mass of detaching

stone material

eF-eS1

eF-eSn

Multiple flakes to multiple scales

Transitional form between multiple flakes (mF)

and multiple scales (mS).

mF-

mS

mF-mS1

mF-mSn

Level 1 – Group of weathering forms Group 4 – Fissures / deformation



Fissures Individual fissures or systems of

L Fissures independent of stone structure Individual fissures or systems of fissures independent of structural features such as bedding, foliation, banding etc..

vL Number of fissures and dimension of fissures –length,

vL1

vLn



fissures due to natural or constructional causes.

Fissures dependent on stone structure Individual fissures or systems of fissures dependent on structural features such as bedding, foliation, banding etc.

tL width (mm, cm) tL1

tLn

Deformation Bending / buckling of mainly thin stone slabs due to plastic deformation. Especially on marble slabs.

V Deformation, convex lV Amplitude of bending / buckling

lV1

lVn

Deformation, concave rV rV1

rVn

For further information about weathering forms of rock, consult the web page of the working group

“Natural stones and weathering” (http://www.stone.rwth-aachen.de/)

Annex B: DNA indirect isolation The procedure and reagents needed for DNA extraction are presented below. The information was taken

from protocols of the “Centro de investigaciones en ingeniería ambiental – CIIA” of the Universidad de

los Andes.

a. Transfer 1.5 mLt of the culture media with

microorganisms to a microtube of 1.5 mLt

b. Centrifuge at 12,000 x g for 2 minutes at

room temperature

c. Discard supernatant by inverting

d. Resuspend pellet in 567 µLt of buffer TE

e. Add 30 µLt of SDS7 10% and 3 µLt of

Proteinase K (20 mg mLt–1)

f. Mix and incubate at 37°C during 1 hour in

water bath

g. Add 100 µLt of NaCl 5M and gently vortex to

mix

h. Add 80 µLt of CTAB/NaCl and gently vortex

i. Incubate at 65°C for 10 minutes in water

bath

j. Add approximately 780 µLt of CIA

k. Centrifuge at 12,000 x g for 5 minutes at 4°C

7 Sodium Dodecyl Sulfate detergent

l. Transfer the supernatant to a clean

microtube

m. Add an equal volume of supernatant

transferred of FCIA

n. Centrifuge at 12,000 x g for 5 minutes at 4°C

o. Transfer the supernatant to a clean

microtube

p. Add 600 µLt of isopropanol and gently

vortex.

q. Centrifuge at 4,000 x g for 1 minutes at room

temperature

r. Discard the supernatant

s. Wash the pellet in 250 µLt of ethanol (70%)

and centrifuge at 13,000 x g for 1 minute at

room temperature. Perform this step twice.

t. Discard the supernatant and allow air drying.

u. Resuspend DNA in buffer TE



Annex C: Bacteria morphology

Cultural characteristics

Macroscopic appearance of bacteria sometimes depends on the culture medium, conditions, age and

subculturing. Visual observations of the culture growing on solid medium in a petri dish on a specific

media are noted: color, pigmentation, elevation, margins and surface (Kango, 2010)

The following table shows some of the shapes, margins, elevations and surfaces of the bacteria colonies

growing in solid medium.

Table 26. Cultural characteristics of bacteria. Adapted from Saddleback College (n.d.) and Kango (2010)

Bacteria colony shape

Punctiform

(under 1mm diameter)

Round

Round with scalloped

margin

Round with

raised margin

Wrinkled

Concentric

Irregular and

spreading

filamentous

L – form

Round with

radiating margin

Filiform

Rhizoid

Complex

Bacteria colony margins

Smooth (entire)

Curled

Wavy

(Undulate)

Lobate

Irregular (erose)

Ciliate

Branching

Wooly

Thread – like

‘Hair – lock’ – like

Bacteria colony elevation

Flat

Raised

Convex

Drop – like

Umbonate

Hilly

Ingrowing

into medium

Crateriform

Bacteria colony surface characteristics

Smooth

Concentric

Wrinkled

Contoured



Annex D: Results of DNA sequencing

Sample 10A, primer 27F Sample 10A, primer 1492R

Microorganism in concrete

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