Aislamiento Degradadoras_TRATABILIDAD_Chaerun y Col (2004)

8/13/2019 Aislamiento Degradadoras_TRATABILIDAD_Chaerun y Col (2004)

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Bioremediation of coastal areas 5 years after the Nakhodka oil

spill in the Sea of Japan: isolation and characterization of

hydrocarbon-degrading bacteria

S. Khodijah Chaeruna,*, Kazue Tazakib, Ryuji Asadab, Kazuhiro Kogurec

aGraduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, JapanbDepartment of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan

cOcean Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164-8639, Japan

Received 3 December 2003; accepted 24 February 2004

Available online 23 April 2004

Abstract

Five years after the 1997 Nakhodkaoil spill in the Sea of Japan, seven bacterial strains capable of utilizing the heavy oil spilled from the

NakhodkaRussian oil tanker were isolated from three coastal areas (namely Katano Seashore of Fukui Prefecture, Osawa and Atake seashores

of Ishikawa Prefecture) and the NakhodkaRussian oil tanker after a 5-year bioremediation process. All bacterial strains isolated could utilize

long-chain-length alkanes efficiently, but not aromatic, and all of them were able to grow well on heavy oil. Using 16S rDNA sequencing,

most of the strains were affiliated toPseudomonas aeruginosa.Comparing between the year 1997 (at the beginning of bioremediation process)

and the year 2001 (after 5 years of bioremediation), there was no significant change in morphology and size of hydrocarbon-degrading bacteria

during the 5-year bioremediation. Scanning and transmission electron microscopic observations revealed that a large number of hydrocarbon-

degrading bacteria still existed in the sites consisting of a variety of morphological forms of bacteria, such as coccus ( Streptococcusand

Staphylococcus) and bacillus (Streptobacillus). On the application of bioremediation processes on the laboratory-scale, laboratory microcosm

experiments (containing seawater, beach sand, and heavy oil) under aerobic condition by two different treatments (i.e., placed the insidebuilding and the outside building) were established for bioremediation of heavy oil to investigate the significance of the role of hydrocarbon-

degrading bacteria on them. There was no significant bacterial activity differentiation in the two treatments, and removal of heavy oil by

hydrocarbon-degrading bacteria in the outside building was slightly greater than that in the inside building. The values of pH, Eh, EC, and

dissolved oxygen (DO) in two treatments indicated that the bioremediation process took place under aerobic conditions (DO: 16 mg/l; Eh:

12300 mV) and neutral-alkaline conditions (pH 6.48) with NaCl concentrations of 315% (ECs of 45200 mS/cm).

D 2004 Elsevier Ltd. All rights reserved.

Keywords: Nakhodka Russian oil tanker; Bioremediation; Heavy oil; Hydrocarbon-degrading bacteria; Pseudomonas aeruginosa

1. Introduction

During the past few decades the incidence and threat of

oil pollution has resulted in extensive research. The anthro-

pogenic origins (particularly via leak of coastal oil refiner-

ies) of petroleum have led to interest in their distribution and

fate in the environment, mainly the marine environment.

Tanker accidents are major causes of oil pollution of marine

environments. One of the recent examples of such accidents

is that of theNakhodkaRussian oil tanker which discharged

approximately 6240 kl of C-heavy oil into the Japan Sea on

January 2, 1997. The heavy oil spill led to a serious impact

to the surrounding environment, particularly the heavy oil

pollution of the shoreline of Mikuni, Fukui Prefecture to

Noto Peninsula, Ishikawa Prefecture(Tazaki, 2003).

Petroleum hydrocarbon can be degraded by microorgan-

isms such as bacteria, fungi, yeast, and microalgae (e.g.,

Atlas, 1981; Leahy and Colwell, 1990). Numerous studies

have been conducted on microbial consortia and enrichment

(e.g., Atlas, 1981), and most bacterial petroleum hydrocar-

bon degraders have been isolated from heavily contaminat-

ed coastal areas under this study (Itagaki and Ishida, 1999;

Kasai et al., 2001;Tazaki, 2003). However, no data exist on

0160-4120/$ - see front matterD 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envint.2004.02.007

* Corresponding author. Tel.: +81-76-264-5732; fax: +81-76-264-

5746.

E-mail addresses: [email protected],

[email protected] (S.K. Chaerun).

www.elsevier.com/locate/envint

Environment International 30 (2004) 911 922


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the findings of the superior hydrocarbon indigenous

degraders of bacterial isolates in degrading the Nakhodka

oil spill. Also, there was no preservation of those isolates as

pure bacterial cultures that can be used for more detailed

investigations on the degradation processes of the Nakhodka

oil spill. For these reasons, the isolation and characterization

of pure bacterial strains indigenous to the Nakhodkaoil spillwere carried out to obtain the superior pure bacterial strains

in degrading the Nakhodka oil spill, being further used for

future studies. Since some attempts to demonstrate the

potential for bioaugmentation, a technique used in bioreme-

diation by adding microorganisms to sites contaminated

with oil, in soils have resulted in successes and failures

(Vogel, 1996), biostimulation of the indigenous bacteria that

break down oil pollutants is considered a priority. The

isolation of bacteria was undertaken mainly after 5 years

of bioremediation with a reason: the strains isolated may

have adapted to the toxicity of the Nakhodkaoil spill, so that

they will be capable of utilizing heavy oil as substrate if

compared with that was undertaken at the beginning of

bioremediation process (e.g., year 1997).

InNakhodkaheavy oil spill along seashores in the Sea of

Japan (from Mikuni, Fukui Prefecture to Noto Peninsula,

Ishikawa Prefecture, Japan), a careful bioremediational study

has been performed by our group since 1997. After a 5-year

bioremediation process, the isolation of indigenous bacterial

strains was conducted in three coastal areas (namely Katano

seashore of Fukui Prefecture, Atake and Osawa seashores of

Ishikawa Prefecture), and theNakhodka Russian oil tanker.

Investigation of the role of these bacteria in the bioremedi-

ation of heavy oil polluted sites, particularly coastal areas,

was carried out as well. Bacterial strains capable of growingon heavy oil were isolated from the above four locations by

using enrichment and isolation techniques. Additionally, the

bacteria were isolated not only from seawater, but also from

beach sands polluted by heavy oil for over 5 years, namely at

the top layer (030 cm) about 34 m outside the shoreline,

and at the top layer (060 cm) about 56 m outside the

shoreline at Atake seashore, Ishikawa Prefecture, Japan.

Thirty-nine hydrocarbon-utilizing bacterial strains capable

of growing on and utilizing heavy oil as a sole carbon and

energy source were isolated from coastal areas. Of the 39

bacterial strains tested, only 7 bacterial strains could grow

very well on heavy oil (unpublished data).

Accordingly, the primary objective of this study was to

isolate bacterial strains capable of utilizing hydrocarbons

indigenous to the Nakhodka oil spill-polluted coastal areas

in order to obtain the most superior hydrocarbon degrader,

being further used for investigating the environmental factors

influencing the bioremediation of theNakhodka oil spill. This

paper described the results of efforts of isolation and charac-

terization of the seven aerobic, halotolerant hydrocarbon-

degrading indigenous bacterial strains from seashores of

Mikuni, Fukui Prefecture to Noto Peninsula, Ishikawa Pre-

fecture (mainly from Wajima seashore, Ishikawa Prefecture

and Katano seashore, Fukui Prefecture) 5 years after the

Nakhodka Russian oil tanker spill in the Sea of Japan in

relation to bioremediation process. The strains could use

alkanes and heavy oil (from the Nakhodka oil spill) aerobi-

cally as their sole carbon sources but not aromatics, and all of

them were able to grow well on heavy oil. The microbial

activity of hydrocarbon-degrading bacteria between year

1997 (after the Nakhodka oil spill) and year 2001 (after 5years of bioremediation processes) were evaluated. In addi-

tion, ex situ bioremediation process in laboratory microcosms

(containing seawater, beach sand, and heavy oil) under

aerobic condition by two different treatments, viz. treatments

outside building and inside building, was also investigated.

2. Materials and methods

2.1. Field sites and sample collection

The sampling sites are located in the Sea of Japan, i.e.,

from Mikuni, Fukui Prefecture to Noto Peninsula, Ishikawa

Prefecture, Japan (Fig. 1). Field sampling was performed

three times. Samples of heavy oil, seawater, and sand were

collected from Nakhodka tanker on February 21, 1997, on

December 10, 1999 from Katano seashore in Fukui Prefec-

ture, and on November 21, 2001 from Osawa and Atake at

Wajima seashore in Ishikawa Prefecture, Japan (Fig. 2).At

the Atake seashore, sands were sampled at two previous

heavily oil-polluted fields, that is, from the top layer (030

Fig. 1. Location of the sampling sites in the Sea of Japan. The arrow

indicates sites in which the photographs (Fig. 2)were taken.

S.K. Chaerun et al. / Environment International 30 (2004) 911922912


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cm) about 34 m outside the shoreline and from the top

layer (060 cm) about 56 m outside the shoreline. These

were undertaken to investigate the difference of microbial

activity of hydrocarbon-degrading bacteria, and also as

controlled sampling in the isolation of hydrocarbon-degrad-

ing bacteria. In addition, heavy oil samples were collected at

three differently polluted field sites namely on inshore,

nearshore, and away from outside shorelines. All samples

were stored at 4 jC until required for analysis. Fieldparameters including seawater temperature, pH, dissolved

oxygen (DO), electrode potential versus the standard hy-

drogen electrode (Eh), and electrical conductivity (EC) were

measured in situ using a portable meter.

2.2. Chemical analyses of seawater, heavy oil, and sand

Chemical composition of seawater and sand was analyzed

using the energy-dispersive X-ray fluorescence (ED-XRF)

analyzer. The seawater was furthermore used for ZOBELL

medium in isolating hydrocarbon-degrading bacteria. Ele-

mental composition of the seawater and sand is given in

Tables 1 and 2. The chemical composition of heavy oil in

N (nitrogen), C (carbon), and S (sulfur) was estimated by

using the NCS analyzer, in order to determine the biodeg-

radation of hydrocarbons. Additionally, the initial compo-

sition of heavy oil spilled from the Nakhodka Russian oil

tanker was analyzed by CHNS chemical analyzer

(FISONS) (Table 3).

2.3. Isolation, purification and enumeration of hydrocarbon

degrading bacteria

Bacteria were isolated from seawater, heavy oil, and sand

samples from three coastal areas contaminated with the

Nakhodkaoil spill, namely Katano seashoreFukui Prefec-

ture, Osawa and Atake seashoresIshikawa Prefecture, and

Table 1Elemental composition of seawater used for ZOBELL medium analyzed by

ED-XRF

Element wt.%

Na 8.27

Mg 1.49

Si 0.26

S 2.94

Cl 79.94

K 2.99

Ca 3.57

Co 0.05

Br 0.42

Sr 0.06

Table 2

Average elemental composition (wt.%) of beach sands collected from

Katano seashore and Atake seashore analyzed by ED-XRF

Element Katano seashore Atake seashore

Mg 0.34 0.55

Al 6.55 9.62

Si 73.16 59.19

K 8.77 11.42

Ca 2.45 7.00

Ti 0.77 1.27

Mn 0.15 0.13

Fe 7.73 10.61

Sr 0.07 0.21

Samples of beach sands were collected from Katano seashore on December

10, 1999, and from Atake seashore on November 21, 2003. The beach sand

is mainly composed of quartz (SiO2), feldspars (AlSi), and very few clay

minerals.

Fig. 2. Samples of heavy oil and sand were collected from each sampling site. (A) The Nakhodka tanker (on February 21, 1997), (B) Katano seashore (on

December 10, 1999), (C) Osawa seashore (on November 21, 2001), and (D) Atake seashore (on November 21, 2001).

S.K. Chaerun et al. / Environment International 30 (2004) 911922 913


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Nakhodka tanker by selective enrichment by using the

Bushnell Hass Mineral Salts (BHMS) medium comprising

heavy oil 1% (v/v). Bacteria were maintained on either liquid

or solid mineral salts medium as the sole carbon source. The

medium supplemented with 2% NaCl was used for the

growth of marine bacteria. BHMS contained (per liter of

distilled water) 0.2 g of MgSO47H2O, 0.02 g of CaCl2, 1 g

of KH2PO4, 1 g of K2HPO4, 1 g of NH4NO3, 2 drops of

FeCl360%. The pH was adjusted to 77.8 (neutral-alkaline

condition). The bacteria were isolated by using an enrich-

ment culture and a single-colony isolation technique. Sea-

water, heavy oil, and sand samples collected from seashores

in the Sea of Japan were inoculated to BHMS medium

supplemented with 2% NaCl, and incubated on a rotary

shaker (125 rpm) at room temperature. When the growth of

bacteria was detected in the medium, a sample of culture

medium was spread on ZOBELL agar plates supplemented

with 1% (v/v) heavy oil as a substrate and incubated for 2

weeks at 25 jC. Colonies appearing on the plate were

isolated by a single colony isolation procedure, which was

repeated several times. The final isolate (each strain) waspreserved in ZOBELL agar medium. Enumeration of the

number of viable cells in bacterial culture was determined by

a serial dilution-agar plating procedure.

2.4. Characterization of hydrocarbon degrading bacteria

All the colonies obtained on ZOBELL agar were purified

several times on the same medium. Pure strains were

conserved on ZOBELL agar/glycerol (v/v) at 20 jC.

Growth of bacterial isolates on individual hydrocarbons

and heavy oil (from the Nakhodka oil spill) was tested in

a tube containing 10 ml of BHMS medium supplemented

with 2% NaCl and 1 g/l of hydrocarbon. Hydrocarbons were

sterilized by filtration (membrane filter 0.2 Am). Solid

hydrocarbons (octadecane, eicosane, octacosane, and aro-

matic compounds) were added directly to BHMS medium

before autoclaving. Test tubes were agitated for 15 days at

room temperature. Growth was then evaluated by compa-

rison of optical density (OD600) against sterile control.

2.5. 16S rDNA sequencing analysis

Sequence analyses of 16S ribosomal DNA (16S rDNA)

were performed on the isolates by amplifying the 16S rRNA

genes by PCR using the bacterial universal primers 27f and

1492r (Lane, 1991). The PCR mixture contained 1 Al of

template DNA, 2.5 Al of 10 reaction buffer, 2 Al of 2.5

mM of deoxynucleoside triphosphate (dNTPs), 0.5 Al of 10

pmol of each primers, and 0.25Al of 2.5 U of Z-Taq DNA

polymerase (TaKaRa Bio, Shiga, Japan) in a final volume of

25 Al. DNA amplification was performed in a model PTC-100 Thermal cycler (MJ Research, Watertown, MA, USA)

with following profile: 30 cycles of 96 jC for 1 s, 55 jC for

10 s, and 72 jC for 20 s. The PCR products purified with

EXOSAP-IT kit (USB, USA) were sequenced directly using

an ABI PRISM BigDye Terminator cycle sequencing ready

reaction kit (Applied Biosystems, USA). After purification

of sequencing products by ethanol precipitation, they were

run on an ABI 3100 Genetic Analyser (Applied Biosys-

tems). The most closely related sequences were found using

the BLAST programs(Altschul et al., 1997).

2.6. Experimental growth of bacterial strains in an

enrichment liquid medium

Four selected strains were tested for the ability to grow

in an enrichment liquid medium. The laboratory experi-

ment was designed in a batch reactor under aerobic

condition by shaking on orbital shaker (rotation speed

of 150 rpm) at room temperature (25 jC) for 185 h. A

series of 500-ml Erlenmeyer flasks were used for this

experiment. Each flask contained 200 ml of 8 g/l of

nutrient broth containing beef extract and peptone (i.e.,

organic compound with very high nutrient content), 1 g/

l of yeast extract (as co-substrate), 0.85% (w/v) of NaCl

were inoculated by 10% (v/v) inocula of strain A1(Nakhodka), strain A3 (Katano), strain A4 (Osawa), strain

A5 (Atake), respectively. Flasks with killed cells were

used for controls. All treatments were performed in

triplicate, and samples were compared to the killed

controls. The growth kinetic of these strains was deter-

mined in cultures (200 ml, 150 rpm, 25 jC) containing

enrichment medium. Samples were removed periodically

to assess the concentration of the biomass (dry weight), as

calculated from the absorbance (optical density at 600

nm) of washed cells. Values for specific growth rates

(designated l) were obtained by fitting data by using

linear regression analysis, whereas the cell density was

measured by using a spectrophotometer UV (APEL Mod-

el PD-303). The initial substrate concentration was

detected as mg/l chemical oxygen demand (COD) for

each treatment (APHA, 1998).

2.7. Laboratory microcosm experiment of bioremediation

process

Laboratory microcosm experiments were undertaken to

investigate the role of hydrocarbon-degrading bacteria in

bioremediation processing of heavy oil since 1997. Labo-

ratory microcosms contained seawater, heavy oil, and beach

Table 3

The initial composition of heavy oil spilled from theNakhodkaRussian oil

tanker analyzed by CHNS chemical analyzer (FISONS)

Chemical composition of heavy oil wt.%

Carbon 62.2

Hydrogen 10.1

Nitrogen 0.246

Sulfur < 0.1Oxygen 27.4

Sampei et al., 2003.



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sand. The two different treatments were established by

placing microcosms outside building and inside building.

At the outside building, it can be assigned that not only

heavy oil is as a sole energy source but also another energy

source is sunlight that is directly used by phototrophs. For

chemical and physical parameters, pH, Eh, EC, and DO,

respectively, were measured during the sampling to observethe bioremediation process taking place. Laboratory exper-

iment at the inside building was conducted at room tem-

pe ra ture (2 2 25 jC). At the outside building, the

experimental temperature was not maintained depending

on the seasons (i.e., winter, spring, summer, and autumn).

However, the temperature measurement of both treatments

was not conducted. The chemical composition of heavy oil

in N, C, and S to determine biodegradation of hydrocarbon

was estimated by using NCS analyzer. Additionally, micro-

biological parameters were measured to investigate micro-

bial activity of hydrocarbon-degrading bacteria during the

process observed by scanning electron microscope (SEM;

JEOL JSM-5200LV) and transmission electron microscope

(TEM; JEOL JEM-2000EX).

2.8. Light, scanning and transmission electron microscopy

For light microscopy, bacterial cells were wet mounted on

glass slides, stained with 4V,6-diamidino-2-phenylindole

(DAPI), then observed under epifluorescence microscope

(Nikon NTF2A). For scanning electron microscopy, bacterial

cells were fixed with 2.5% (v/v) glutaraldehyde in phosphate

buffer at room temperature for 1 h, washed in the same buffer,

post-fixed in 1% (w/v) osmium tetroxide (OsO4) at room

temperature for 1 h, dehydrated in a graded ethanol series (50,75, 95, and 100%), mounted on carbon-coated copper stubs,

and viewed on a JEOL JSM-5200LV scanning electron

microscope. Samples for transmission electron microscopy

were fixed and dehydrated as described above, mounted on

copper specimen grids, and viewed using a JEOL JEM-

2000EX transmission electron microscope.

3. Results

3.1. Physical analysis of seawater

Physical analysis was performed to provide a detailed

description of sampling site condition. The physical charac-

teristics of seawater are listed inTable 4.These showed that

seawater temperatures ranged between 14.0 and 16.5 jC; pH

from 7.9 to 8.6; dissolved oxygen from 4.8 to 11.1 mg/l; EC

from10.4 to 48.6 mS/cm, and Eh between 20 and 118 mV.

3.2. Isolation of hydrocarbon-utilizing bacteria from

various coastal areas

Thirty-nine strains of hydrocarbon-utilizing marine bac-

teria were isolated from heavy oil, seawater, and sand

samples obtained at three coastal sites in the Sea of Japan

and the Nakhodka tanker (data not shown). When grown

aerobically in BHMS medium (pH 77.8), the isolates gave

cell yields ranging from 106 to 107 cells/ml. The strains

grew well not only in BHMS medium but also in ZOBELL

agar medium with the high NaCl concentration (Table 1). It

seems clear that any bacterial strains proliferating well in the

NaCl-supplied medium could withstand high concentrations

of salt (NaCl).

Isolation by agar dilution series was then successfully

attempted. Two strains originated from Nakhodka tanker,

three strains originated from Osawa seashore, two strains

originated from Katano seashore, and 32 strains originated

from Atake seashore, namely 9 strains resulted from sea-

water and heavy oil samples, 9 strains and 14 strains

resulted from sand samples from the top layer (030 cm)

and (060 cm), respectively. All bacterial strains were able

to consume either heavy oil or peptone and yeast extract as asole source of carbon as well as octadecane for their growth

with the different generation time. Our experimental results

on carbon source utilization thus suggested that the 39

isolates were hydrocarbon-degrading bacterial strains. How-

ever, of the 39 bacterial strains isolated, only 7 bacterial

strains could grow well on heavy oil when grown aerobi-

cally in BHMS medium supplemented with 2% NaCl and

1% (v/v) heavy oil as substrate. They were coded: A1, A2,

A3, A4, A5, A6, and A7(Table 5).Strains A1 and A2 were

isolated from the Nakhodka oil tanker, strain A3 was

isolated from Katano seashore, strain A4 was isolated from

Osawa seashore, and strains A5, A6, and A7 were isolated

from Atake seashore.

3.3. Phylogenetic analyses of the bacterial isolates

In order to identify the bacterial isolates, their 16S rDNA

genes were amplified and sequenced. Strain A1 was affil-

iated to Pseudomonas aeruginosa (99% similarity), strain

A2 to Bacillus cereus (99%) or Bacillus thuringiensis

(99%), strain A3 to P. aeruginosa (98%), strain A4 to P.

aeruginosa (99%), strain A5 to P. aeruginosa (98%), strain

A6 to P. aeruginosa (99%), and strain A7 to Paracoccus

seriniphilus (97%) orParacoccus marcusii(97%)(Table 5).

Table 4

Physical characteristics of seawater for each sampling site

Sampling site Sample

source

pH Eh

(mV)

EC

(mS/cm)

DO

(mg/l)

WT

(jC)

Osawa seashore seawater 7.9 20 10.4 11.1 16

Atake seashore seawater 8.6 75 48.6 9.6 16.5

seawater +

heavy oil

8.3 118 19 4.8 14

Eh: electrode potential versus standard hydrogen electrode; EC: electronic

conductivity; DO: dissolved oxygen; WT: water temperature. The

measurement of DO was performed on the oil water phase. These

parameters were measured in November 2001. In theNakhodkatanker and

Katano seashore, these parameters were not measured. Only samples of

heavy oil and sand were collected.



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3.4. Growth on hydrocarbons as the sole carbon source

Result of bacterial growth on pure hydrocarbons and

heavy oil is given in Table 6. The seven bacterial strains

were able to grow well on aliphatic hydrocarbons such as

tetradecane (C14), hexadecane (C16), octodecane (C18), and

eicosane (C20) as well as on heavy oil (from the Nakhodkaoil

spill). Also, they grew poorly on hexane (C6), octane (C8),

octacosane (C28), paraffin, pristane (branched C19), and

cyclohexane. However, these bacterial strains did not appear

to utilize aromatic hydrocarbons as carbon source, except that

Table 6

Growth and ability of strains isolated to use different organic compounds as

carbon source

Organic

compounds

Strain A1 A2 A3 A4 A5 A6 A7

Alkanes Hexane (C6) + + + + + + +

Octane (C8) + + + + + + +

Tetradecane (C14) + + + + + + + + + + + + + +

Hexadecane (C16) + + + + + + + + + + + + + +

Octadecane (C18) + + + + + + + + + + + + + + +

Eicosane (C20) + + + + + + + + + + + +

Octacosane (C28) + + + + + + +

Paraffin + + + + + + +

Pristane

(branched C19)

+ + + + + + +

Cyclohexane + + + + + + + +

Aromatics Toluene (1 ring) +

Biphenyl (2 rings)

Naphthalene

(2 rings)

Phenanthrene

(3 rings)

+

Fluoranthene

(4 rings)

+

Heavy oil the Nakhodka

oil spill

+ + + + + + + + + + + + + + + + + +

+ = low growth; + + = medium growth; +++ = strong growth; = no

growth.

Table 5

Strains isolated able to grow well on and use heavy oil as carbon source

No. Sampling site Sample

source

Strain Species Sequence

similarity

(%)

1 Nakhodka tanker heavy oil A1 Pseudomonas

aeruginosa

99

A2 Bacillusthuringiensis or

Bacillus cereus

99

2 Katano seashore sand+

heavy oil

A3 Pseudomonas

aeruginosa

98

3 Osawa seashore heavy oil A4 Pseudomonas

aeruginosa

99

4 Atake seashore heavy oil A5 Pseudomonas

aeruginosa

98

(outside seashore)

(inshore) seawater +

heavy oil

A6 Pseudomonas

aeruginosa

99

(sand of 30 cm

deep at 3rd layer)

sand A7 Paracoccus

seriniphilus

Paracoccus

marcusii

97

The name of strains was a result of 16S rDNA sequencing analysis.

Fig. 3. Epifluorescence photomicrograph (A) and scanning electron

micrograph (B) of strain A3 (Katano) at the exponential phase after 44h of incubation grown in an enrichment liquid medium containing 8 g/l of

nutrient broth and 1 g/l of yeast extract, and 0.85% (w/v) of NaCl.

Fig. 4. Growth curve in density of four bacterial strains of A1 (Nakhodka),

A3 (Katano), A4 (Osawa), and A5 (Atake) in an enrichment liquid medium.



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strain A5 grew poorly on phenantherene and fluoranthene,

and strain A6 utilized toluene poorly. Of the seven bacterial

strains tested in utilizing various hydrocarbons, strain A5 was

the most superior hydrocarbon degrader, therefore being

studied in more detail and used for future studies, especially

in investigations on the environmental factors influencing the

bioremediation of theNakhodkaoil spill.

3.5. Bacterial growth and growth kinetics in an enrichment

liquid medium

Growth of strains A1 (Nakhodka), A3 (Katano), A4

(Osawa), and A5 (Atake) on enriched liquid medium was

tested(Figs. 3 and 4). These strains grew well in enriched

liquid media containing 8 g/l of nutrient broth, 1 g/l of

yeast extract (as co-substrate), 0.85% (w/v) of NaCl, as

the sole source of carbon, nitrogen, and energy. When

growth was not limited by growth factor(s), i.e., at

enrichment culture, a classical growth response consisting

of an exponential phase followed by a stationary phasewas observed. All strains were capable of utilizing con-

centrations as great as 3030 mg/l COD without an appre-

ciable lag phase. This concentration was the initial

substrate concentration detected as COD (mg/l) for each

treatment. On the other hand, it was apparent that it was

difficult for all bacterial strains to reach an optical density

Fig. 5. Growth curve in biomass and determination of specific growth rate of four bacterial strains of A1 (Nakhodka), A3 (Katano), A4 (Osawa), and A5

(Atake) in an enrichment liquid medium.



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at 600 nm (OD600) of R 1, indicating the low cell yield of

growth on this substrate (Fig. 4).

The growth of strains A1 (Nakhodka), A3 (Katano), A4

(Osawa), and A5 (Atake) in liquid culture followed the first-

order reaction (Fig. 5) with the following growth kinetic

parameter: for strain A1 (Nakhodka), l = 0.063 h 1; strain

A3 (Katano),l =0.01h 1

; strain A4 (Osawa),l =0.014h 1

;and strain A5 (Atake), l = 0.0164 h 1. The values of the

growth kinetic parameters, l, can be determined so as to

fit the calculated profiles with corresponding experimental

ones by the least squares method, as in the case of nutrient

broth as substrate. An example of the good coincidence

between the experimental and calculated profiles is shown

in Fig. 5 with the regression coefficient: R2 = 0.9249 (for

strain A1 (Nakhodka)). The values of specific growth rate

increased with the following sequences as: (strain A4

(Katano) < strain A4 (Osawa) < strain A5 (Atake) < strain

A1 (Nakhodka)), indicating that strain A1 (Nakhodka) had

the highest capability in using this substrate for its growth

than those of three strains. This also proved that strain A1(Nakhodka) was capable of growing quickly by using this

substrate where transport of the substrate through the cell

wall might be a rate-determining factor.

3.6. Bioremediation process on the laboratory scale

associated with in situ bioremediation

3.6.1. Analysis of physical characteristics of seawater

Comparison of physical characteristics between inside

and outside building at laboratory microcosm assays for

bioremediation processes during 5 years of bioremediation

is given in Fig. 6. The graphs showed that seawater pH

ranged between 6.4 and 7.8 having the tendency to

neutral-alkaline condition; DO ranged from 0.6 to 5.5

mg/l; Eh ranged from 50 to 300 mV; and EC ranged

between 45 to 180 mS/cm. During the sampling at all

treatments, there were no significant changes in pH,

whereas EC increased rapidly. Conversely, Eh decreased

sharply for all treatments, while DO concentrations indi-

cated that bioremediation processes took place under

aerobic conditions. On the other hand, at inside building

treatment, at the second measurement (October 20, 1997)

DO concentration decreased abruptly to anaerobic condi-

tion (0.6 mg/l).

Fig. 6. The values of pH, Eh, EC, and DO in laboratory microcosm

experiments (i.e., the inside and outside building treatments) during the

5-year bioremediation.

Table 7

Chemical composition of heavy oil in N, C, S (wt.%) for each sampling site

analyzed by NCS analyzer

Sampling site Sample source N (%) C (%) S (%)

Nakhodka tanker seawater + h eavy oil n.d. 87.2 1.44

Laboratory assay

(inside building)

seawater + heavy oil n.d. 59.68 1.83

Laboratory assay(outside building)


Osawa seashore heavy oil 0.19 83.08 1.12

Atake seshore heavy oil

(outside seashore)

n.d. 83.4 1.38

heavy oil (inshore) n.d. 24.43 0.18

heavy oil (nearshore) 0.22 82.9 1.2


seawater n.d. 0.03 2.27

Atake seashore sand (1st layer) n.d. 0.14 0.01

(sand of 30 cm sand (2nd layer) n.d. 0.1 n.d.

in depth) sand (3rd layer) n.d. 0.14 0.01

Atake seashore sand (1st layer) n.d. 0.2 n.d.

(sand of 60 cm sand (2nd layer) n.d. 0.16 n.d.

in depth) sand (3rd layer) n.d. 0.26 0.004

sand (4th layer) n.d. 0.08 n.d.

sand (5th layer) n.d. 0.06 n.d.

N: nitrogen; C: carbon; S: sulfur; n.d.: not detected.



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3.6.2. NCS analysis of heavy oil contents

The chemical compositions of heavy oil for each sample

after 5 years of bioremediation (year 2001) detected in N, C,

and S are summarized in Table 7. The unit of contents is

shown in percentage of weight. These compositions de-

scribed not only bioremediation process on the laboratory

scale (ex situ bioremediation) but also in situ bioremediationprocesses in comparison. The contents of C ranged from

59.68 to 87.20 wt.% (for Nakhodka tanker, laboratory

microcosm assay of inside building, Osawa seashore, and

Atake seashore), 24.43 wt.% C (for heavy oil inshore in

Atake seashore), 0.10 wt.% C (for seawater with heavy oil

slick), 0.03 wt.% C (for seawater without heavy oil slick),

and 0.06 0.20 wt.% C (for sand at different depths).

Compared with the content of C, the contents of N and S

were relatively low, that is, 00.22 wt.% N, and 02.44

wt.% S. TheNakhodkatanker site was highest in proportion

to the C content of those of other sites (i.e., 87.20 wt.%),

while the lowest one was obtained from a seawater sample

without heavy oil in Atake seashore (i.e., 0.03 wt.%). These

provided that the biodegradation rate of heavy oil atNakhodka tanker site was relatively low if compared with

those of other sites. Compared with inside building treat-

ment, outside building treatment had a relatively high

biodegradation rate of heavy oil shown by the low C content

(0.054 wt.%). In Atake seashore, the biodegradation rate of

heavy oil climbed slightly where it reached a peak on the

inshore and gradually dropped into the nearshore and outside

Fig. 7. Comparison of scanning (A1 2, B1 2) and transmission (A3, B3) electron micrographs of hydrocarbon-utilizing bacteria inhabiting heavy oil during

bioremediation process between the year 1997 and the year 2001 at Wajima seashores, Noto Peninsula, Ishikawa Prefecture, Japan. Note: A1 3, the year 1997;

B13, the year 2001.



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seashore designated by declining the C contents (83.40,

82.90, and 24.40 wt.%, respectively). Whereas C content

of sands at all depths elucidated that there was no significant

difference between them, indicated by the low contents if

compared with those of other samples. It thus seems that

there was a sharp decrease in the content of carbon inshore in

Atake seashore. A strong positive correlation exists herebetween the amount of organic matter (i.e., carbon content)

and the abundance of hydrocarbon-degrading bacteria. Ken-

nish (1995)reported that the abundance of bacteria peaks in

the estuaries and coastal waters and gradually declines into

offshore areas and the open ocean.

3.6.3. Comparison of microbial activity of hydrocarbon-

degrading bacteria between year 1997 and year 2001

The enumeration of bacterial cell number, SEM and

TEM observations were undertaken to investigate the mi-

crobial activity of hydrocarbon-degrading bacteria in heavy

oil samples collected from Wajima seashores, Noto Penin-

sula, Ishikawa Prefecture, Japan. The population size of

hydrocarbon-degrading bacteria in 2001 (after 5 years of

bioremediation) was in the range of 104106 cells ml 1,

while the population size in 1997 (at the beginning of

bioremediation process) was in the range of 105106 cells

ml 1 (Kasai et al., 2001). There was the similarity in

population size of hydrocarbon-degrading bacteria between

year 1997 and year 2001. The bacteria showed the popula-

tion size of greater than 105 bacteria ml 1 indicating a

relative abundance of hydrocarbon-utilizing bacteria repre-

senting a population density of greater than 103 cells ml 1.

Furthermore, SEM and TEM observations revealed that

there was no significant change in morphology and size ofhydrocarbon-degrading bacteria during the 5-year bioreme-

diation process (comparison between year 1997 and year

2001). Hydrocarbon-degrading bacteria occurred in two

basic shapes, namely cocci (spherical) and bacilli (rod-

shaped), and one unusual shape namely filamentous (Fig.

7). Bacteria sizes at all sampling sites were in the range of

2 7 Am in length and 0.51 Am in width for rod-shaped

bacteria, and 0.3 1.5Am in diameter for spherical bacteria,

illustrating similarities from 1997 to 2001. Thus, the find-

ings revealed that after 5 years of bioremediation, a large

number of hydrocarbon-degrading bacteria still existed in

the sites, consisting of a variety of morphological form of

bacteria such as coccus (StreptococcusandStaphylococcus),

bacillus (Streptobacillus), and filamentous (Bitton, 1994;

Tortora et al., 1994; Chaerun and Tazaki, 2003). The

bacterial cells tended to remain together in a cluster, that

is, cocci (spherical) or bacilli (rod-shaped) occurred in long

chains (Fig. 7A1). One rod-shaped bacterium appeared to

harbor numerous hair-like appendages (assumed to be

fimbriae or flagella) distributed over the surface of the cell

(Fig. 7A3). Some cocci and bacilli formed a slime layer of

cells(Fig. 7B2),while others occurred in three-dimensional

cubes or irregular grape-shaped or irregular star-shaped

clusters (figure not shown).

4. Discussion

The capacity of bacteria, especially P. aeruginosa, to

metabolize aerobically heavy oil or aliphatic hydrocarbons

is well known since a long time. In this study, for the first

time to our knowledge, bacteria that are able to grow on

heavy oil as a carbon source and electron donor were isolatedfrom marine coastal areas polluted by NakhodkaRussian oil

tanker spill in the Sea of Japan (mainly from Wajima

seashore, Ishikawa Prefecture, Japan). Aerobic culture con-

ditions were chosen to select for an oxygen-dependent

degradation. In addition, aerobic growth of the isolated

bacteria on heavy oil demonstrated the presence of heavy

oil-degrading capacities in oxygen-respiring bacteria. Of the

seven bacterial strains isolated, most of them were affiliated

toP. aeruginosa.The discovery of a majority ofPseudomo-

nas is not surprising based on their frequency in the

hydrocarbon-contaminated sites(Rosenberg, 1992; Janiyani

et al., 1993). The seven bacterial isolates were able to grow

well on n-alkane hydrocarbons and heavy oil. However,

these bacteria did not appear to use aromatic hydrocarbons as

carbon source. Microorganisms capable of degrading PAHs

are often isolated from environments with a history of

contamination by PAHs (Herbes and Schwall, 1978; Heit-

kamp and Cerniglia, 1988; Kastner et al., 1994; Trzesicka-

Mlynarz and Ward, 1995). Bacteria degrading alkanes com-

monly do not degrade PAHs (Foght et al., 1990), although

metabolic pathway for both types of compounds is not

incompatible (Whyte et al., 1997). The incapacity of our

bacterial isolates to use PAHs as sole carbon source was not

surprising, since the strains were isolated from seashores

contaminated with the Nakhodka oil spill which consistedmainly of aliphatic hydrocarbons. That is, n-alkanes of C9

C30, in which n-eicosane (n-C20H42) was the most abundant

compound, while the contents of aromatic compounds were

low(Itagaki and Ishida, 1999; Tazaki, 2003).

The n-alkanes are the most biodegradable of the petroleum

hydrocarbons and are attacked by more microbial species

than aromatic or naphthenic compounds. However, normal

alkanes in the C5 C10 range are inhibitory to many hydro-

carbon degraders at high concentrations because as solvents

they disrupt lipid membrane and the cycloalkanes (alicyclic

hydrocarbons) are less degradable than their straight-chain

parent, the alkanes, but more degradable than the polycyclic

aromatics (PAHs)(Jeffrey, 1980; Trudgill, 1984).From some

years, the biodegradation of alkanes correlated with denitri-

fication, sulfate reduction, and metanogenesis has been

described (Aeckersberg et al., 1991, 1998; Rueter et al.,

1994; So and Young, 1999a,b). Moreover, aliphatic hydro-

carbons are not fermentable, and only in the presence of O2,significant aliphatic hydrocarbon degradation occurs. The

initial degradation of petroleum hydrocarbons often requires

the action of oxygenase enzymes and so is dependent on the

presence of molecular oxygen (Brock, 1970; Atlas, 1991).

Aerobic conditions are therefore necessary for the initial

breakdown of petroleum hydrocarbons and in subsequent



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steps, nitrate or sulfate may serve as a terminal electron

acceptor(Bartha, 1986),but oxygen is most commonly used.

The DO values of laboratory microcosm experiments show-

ing that the bioremediation processes mostly ensued under

aerobic condition confirmed the bacterial role in the degra-

dation of aliphatic hydrocarbons of the Nakhodka oil spill

during the 5-year bioremediation(Fig. 6).It is most likely forthe indigenous bacteria to degrade and clean up sites con-

taminated with petroleum hydrocarbons (mainly aliphatics)

as long as there are no major limitations of bioavailability,

oxygen, and temperature (Atlas, 1981; Leahy and Colwell,

1990). Based on the composition of the oil and seawater

(Tables 1, 3, and 7), their sulfur content may affect the

oxidation rate of oil during the 5-year bioremediation.

On the basis of bacterial growth in an enrichment liquid

medium, for bioremediation or biodegradation processes to

cleanup or remove pollutants or contaminants, it was required

that growth yield was low, expressing the low specific growth

rate (low biomass produced) and the high substrate-degrada-

tion rate (high substrate consumed). Under the circumstances,

we could not determine which strain was the best one in using

this substrate, because the degradation rate of substrate was

not observed (not measured). Furthermore, there is a close

relationship between biomass produced and amount of sub-

strate consumed that is a variable which depends on the

balance between: (a) how much of the available growth-

substrate is used for new biomass produced; and (b) how

much of the substrate is consumed by the growing cells for

energy to drive biosynthetic reactions (Shuler and Kargi,

1992). Clearly, the greater the efficiency of energy generation

or the less energy required for biomass production, then the

less substrate has to be dissimilated for this purpose and themore substrate is available for biomass production.

Furthermore, SEM and TEM observations of bacterial

community of hydrocarbon-degrading bacteria suggest that

the bacteria are interestingly well known for their metabolic

diversity and the capability to degrade many biohazardous

or persistent anthropogenic chemical compounds or various

xenobiotic substances(Fig. 7).For both in situ and ex situ

bioremediation process on the laboratory scale, bacteria

were among the dominant microorganisms in these process-

es during the 5-year bioremediation, indicating that the

bacteria had the ability to survive for the relatively long

periods even in the nutrient-poor environments (figure not

shown). It was thus believed that under circumstances,

bacteria may be ubiquitous in oligotrophic environments

and essential to maintain the bioremediation process in the

oligotrophic process as well. These results further support

the view that bacteria can exist in and dominate some

extreme environments.

5. Conclusions

After 5 years of bioremediation processes in the

Nakhodka heavy oil spill along seashores in the Sea of

Japan (from Mikuni, Fukui Prefecture to Noto Peninsula,

Ishikawa Prefecture, Japan), the bacterial strains isolated

show that they are able to utilize alkanes, but not aro-

matics, with the exception of one strain (i.e., strain 5

isolated from Atake seashore), which has the capability to

use both aliphatic and aromatic compounds. Most of the

bacterial isolates are affiliated to P. aeruginosa. It has alsobeen shown that bacteria are the most predominant micro-

organism among other microorganisms in either in situ or

ex situ bioremediation processes, indicating that bacteria

are the main agents responsible for degradation of heavy

oil and appear to be the prevalent hydrocarbon degraders

in coastal areas. It is not surprising that hydrocarbono-

clastic bacteria are present in the hydrocarbon-polluted

sites.

The bacterial strains isolated from the Nakhodkaoil spill-

contaminated sites (at the beginning of bioremediation

process) has been reported in the early literature; however,

these cultures were not preserved and have not been further

studied. Thus, the findings of bacterial isolates as pure

bacterial cultures capable of utilizing the Nakhodkaoil spill

and indigenous to the Nakhodkaoil spill-polluted areas here

will be helpful and will be used for more detailed future

investigations on the biodegradation processes, especially in

studies on the environmental factors influencing the biore-

mediation of theNakhodkaoil spill (being performed by our

group).

Acknowledgements

The authors thank Darius Greenidge, PhD, for correctingthe manuscript. One of us (S.K.C) would like to thank

Associate Professor Chiaki Imada, PhD, and Professor

Naoto Urano, PhD (Tokyo University of Fisheries), for

helpful and motivating discussions, and Professor Dr.

Bernhard Schink (Konstanz University, Germany) for

continuous support. We are also grateful for the cooperation

and assistance of all students of the Tazaki laboratory. We

greatly acknowledge the help of all students of the Kogure

laboratory (University of Tokyo) for help with 16S rDNA

sequencing analysis. We also thank anonymous reviewers

for constructive comments. This study was funded by a

grant from the Japanese Ministry of Education, Culture,Science and Technology to Dr. Kazue Tazaki.

References

Aeckersberg F, Bak F, Widdel F. Anaerobic oxidation of saturated hydro-

carbons to CO2 by a new type of sulfate-reducing bacterium. Arch

Microbiol 1991;156:514.

Aeckersberg F, Rainey FA, Widdel F. Growth, natural relationships, cellu-

lar fatty acids and metabolic adaptation of sulfate-reducing bacteria that

utilize long-chain alkanes under anoxic conditions. Arch Microbiol

1998;170:3619.

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al.



12/12

Gapped BLAST and PSI-BLAST: a new generation of protein database

search programs. Nucleic Acids Res 1997;25:3389402.

APHA (American Public Health Association). Standard methods for the

examination of water andwastewater. 20thed. Washington(DC): Author;

1998.

Atlas RM. Microbial hydrocarbon of petroleum hydrocarbon: an environ-

mental perspective. Microbiol Rev 1981;45:180 209.

Atlas RM. Microbial hydrocarbon degradationbioremediation of oil

spills. J Chem Technol Biotechnol 1991;52:14956.

Bartha R. Biotechnology of petroleum pollutant biodegradation. Microbiol

Ecol 1986;12:15572.

Bitton G. Wastewater microbiology. New York: Wiley; 1994. p. 157.

Brock TD. Biology of microorganisms. New Jersey: Prentice-Hall; 1970.

p. 575 674.

Chaerun SK, Tazaki K. Hydrocarbon-degrading bacteria in the heavy oil

polluted soil and seawater after 5 years of bioremediation. In: Tazaki K,

editor. Water and soil environments: microorganisms play an important

role. 21st century COE Kanazawa University. Kanazawa, Japan: Kana-

zawa University Press; 2003. p. 187204.

Foght JM, Fedorak PM, Westlake DW. Mineralization of [14C]hexadecane

and [14C]phenanthrene in crude oil: specificity among bacterial isolates.

Can J Microbiol 1990;36:169 75.

Heitkamp MA, Cerniglia CE. Mineralization of polycyclic aromatic hydro-

carbons by a bacterium isolated from sediment below an oil field. Appl

Environ Microbiol 1988;54:1612 4.

Herbes SE, Schwall LR. Microbial transformation of polycyclic aromatic

hydrocarbons in pristine and petroleum-contaminated sediments. Appl

Environ Microbiol 1978;35:306 16.

Itagaki E, Ishida H. Drift of C-heavy oil spilled from a Russian tanker

NAKHODKA on Kanazawa seashore and its bioremediation by

marine hydrocarbon degrading bacteria. Coast Eng Jpn 1999;41:

10719.

Janiyani KL, Wate SR, Joshi SR. Morphological and biochemical charac-

teristics of bacterial isolates degrading crude oil. Environ Sci Health

1993;A28:1185 204.

Jeffrey LM. Petroleum residues in the marine environment. In: Geyer RA,

editor. Marine environmental pollution: 1. Hydrocarbon. Amsterdam:

Elsevier; 1980. p. 16379.Kasai Y, Kishira H, Syutsubo K, Harayama S. Molecular detection of

marine bacterial populations on beaches contaminated by the Nakhodka

tanker oil-spill accident. Environ Microbiol 2001;3:24655.

Kastner M, Breuer-Jammali M, Mahro B. Enumeration and characteriza-

tion of the soil micro-flora from hydrocarbon-contaminated soil sites

able to mineralize polycyclic aromatic hydrocarbons (PAH). Appl

Microbiol Biotechnol 1994;41:26773.

Kennish MJ. Practical handbook of marine science. New Jersey: CRC

Press; 1995. p. 299301.

Lane DJ. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M,

editors. Nucleic acid techniques in bacterial systematics. New York:

Wiley; 1991. p. 11575.

Leahy JG, Colwell RR. Microbial degradation of hydrocarbons in the

environment. Microbiol Rev 1990;54:3059.

Rosenberg E. The hydrocarbon-oxidizing bacteria. Procaryotes. 2nd ed.

Berlin: Springer-Verlag; 1992. p. 446 59.

Rueter P, Rabus R, Wilkes H, Aeckersberg F, Rainey A, Jannasch HW,

et al. Anaerobic oxidation of hydrocarbons in crude oil by new types

of sulfate-reducing bacteria. Nature 1994;372:455 8.

Sampei Y, Tazaki K, Obara Y, Yoshimura T, Sawano N, Takayasu K, et al.

Compositional changes of heavy oil and aliphatic hydrocarbon from the

spilled Nakhodka-oil washed ashore at Fukui, Ishikawa and Niigata

Prefectures, Japan. In: Tazaki K, editor. Heavy oil spilled from Russian

tanker Nakhodka in 1997: towards eco-responsibility, earth sense. 21st

Century COE Kanazawa University. Kanazawa, Japan: Kanazawa Uni-

versity Press; 2003. p. 174191.

Shuler ML, Kargi F. Bioprocess engineering basic concepts. New Jersey:

Prentice-Hall; 1992. p. 148 71.

So CM, Young LY. Isolation and characterization of a sulfate-reducing

bacterium that anaerobically degrades alkanes. Appl Environ Microbiol

1999a;65:2969 76.

So CM, Young LY. Initial reactions in anaerobic alkane degradation by a

sulfate reducer, strain AK-01. Appl Environ Microbiol 1999b;65:

553240.

Tazaki K, editor. Heavy oil spilled from Russian tanker Nakhodka in

1997: towards eco-responsibility, earth sense. 21st Century COE Kana-

zawa University. Kanazawa, Japan; Kanazawa University Press; 2003.

Tortora CJ, Funke BR, Case CL. Microbiology: an introduction 5th edition.

New York: Benyamin/Cummings Publishing; 1994. p. 71 4.

Trudgill PW. Microbial degradation of the alicyclic ring: structural relation-

ships and metabolic pathways. In: Gibson DT, editor. Microbial

degradation of organic compounds. New York: Marcel Dekker; 1984.

p. 131 80.

Trzesicka-Mlynarz D, Ward OP. Degradation of polycyclic aromatic hydro-

carbons (PAHs) by a mixed culture and its components pure cultures,obtained from PAH-contaminated soil. Can J Microbiol 1995;41:470 6.

Vogel TM. Bioaugmentation as a soil bioremediation approach. Curr Opin

Biotechnol- 1996;7:311 6.

Whyte LG, Bourbonniere L, Greer CW. Biodegradation of petroleum

hydrocarbons by psychrotrophic Pseudomonas strains possessing both

alkane (alk) and naphthalene (nah) catabolic pathways. Appl Environ

Microbiol 1997;63:3719 23.


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