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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.
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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).
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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)
seawater + heavy oil n.d. 0.054 1.61
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 + heavy oil n.d. 0.1 2.44
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.
S.K. Chaerun et al. / Environment International 30 (2004) 911922 921
8/13/2019 Aislamiento Degradadoras_TRATABILIDAD_Chaerun y Col (2004)
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.
S.K. Chaerun et al. / Environment International 30 (2004) 911922922