Sci. Rep. Kanazawa Univ.
Vol.49, No.1,2, p.25-46, 2004

25

Alkane-degrading bacteria and heavy metals from the
Nakhodka oil spill-polluted seashores in the Sea of Japanafter five years of bioremediation
S. Khodijah CHAERUN1,＊, Kazue TAZAKI2,
Ryuji ASADA2 and Kazuhiro KOGURE3(1) Graduate School of Natural Science and Technology, Kanazawa University, Kakuma,
Kanazawa, 920-1192 Japan(2) Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma,
Kanazawa, 920-1192 Japan(3) Ocean Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164-8639,
Japan＊

Corresponding author.

Email : kchaerun@earth.s.kanazawa-u.ac.jp ; kchaerun@yahoo.com
Phone : +81-76-264-5732
Fax : +81-76-264-5746
Abstract This paper describes the isolation of alkane-degrading bacteria from the
Nakhodka oil spill-polluted seashores in the Sea of Japan. Seven representative strains
were identified using 16S rDNA sequence analysis as Bacillus spp., Pseudomonas sp.,and Paracoccus spp. All bacterial strains showed their ability to grow well on aliphatichydrocarbons, but not on aromatic hydrocarbons. In addition, elemental levels in heavy
oil showed wide ranges in all the heavy oil samples consisting of Si, S, Ti, Cr, Mn, Fe,
Co, Ni, Cu, Zn, and Pb. Compounds of Si, S, and Cr were observed at high levels, while
those of Ti, Mn, Fe, Co, Ni, Cu, Zn, and Pb were observed at low levels. Of all heavy
metals, Co appeared to be toxic for all bacterial growth at concentrations of >1 ppm,
while the presence of Ti, Cr, and Cu at 0.01 to 10 ppm were found not to inhibit growthof all bacterial strains. It is suspected that the presence of heavy metals may have a significant effect on the composition of the bacterial community, (i.e., alkane-degrading
bacterial isolates), as well as on the biodegradative processes of the Nakhodka oil spill
during the 5-year bioremediation.
Keywords : Alkane-degrading bacteria, Nakhodka oil spill, Bioremediation, Heavy oil,
Heavy metal.

1. Introduction
Pollution of petroleum hydrocarbons, in particular oil spills, has attracted much attention in the past and recent decades. In general, oil spills, no matter of their sizes, influence
natural microbial community, and physical and chemical properties of the affected sites (e.

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

g., Ijah and Antai, 2003 ; Tazaki, 2003). Oil spills have also created an interest in the rateand course of oil removal by microbial communities of oil spill-impacted areas under both
aerobic and anaerobic conditions. Many approaches to oil spill clean up attempted to accelerate natural biodegradation processes in promoting the activity of oil degrading microorganisms. For most heavily oiled sites, failure to remove the bulk oil will result in it remaining for a considerable length of time (Kingston, 2002). There are several instances where
oil spill has persisted for many years. One such spill was that of the Russian tanker the
Nakhodka that spilled heavy oil into the Sea of Japan on January 2, 1997. The impact of
the Nakhodka oil spill resulted in a viscous sticky fluid fouling the shores in the Sea of Japan and affected wildlife and marine and coastal ecosystems (Sawano, 2003 ; Tazaki,2003).
Studies on bacterial strains degrading aliphatic and aromatic hydrocarbons attributableto oil spills received much attention as well. However, only few studies have been conducted on the 1997 Nakhodka oil spill in relation to the long-term bioremediational study,
as well as hydrocarbon degraders (Shibata et al., 1997 ; Itagaki and Ishida, 1999 ; Ishiyama et al., 1999 ; Hozumi et al., 2000a ; Hozumi et al., 2000b ; Kasai et al, 2001).
Hence, our group has carried out a careful bioremediational research of the Nakhodka oil
spill along seashores in Sea of Japan since 1997 (Tazaki et al., 1997a ; Tazaki et al.,1997b ; Tazaki et al., 1997c ; Chaerun et al., 2002a ; Chaerun et al., 2002b ; Chaerun etal., 2002c ; Chaerun and Tazaki, 2003 ; Asada et al., 2003 ; Tazaki, 2003).
In general, both types of contamination (i.e., organics and heavy metals) are often present in the environment. Many metallic compounds occur in petroleum in extremely small
concentrations such as inorganic salts, metal soaps, and organic metal-complex compounds.
Petroleum also contains heavy metal complexes (Neumann et al., 1981). Major metal contaminants in petroleum oil are usually aluminium (Al), sodium (Na), iron (Fe), nickel (Ni),and vanadium (V), with, frequently, smaller amounts of magnesium (Mg), tin (Sn), barium(Ba), zinc (Zn), molybdenum (Mo), calcium (Ca), copper (Cu), manganese (Mn), lead (Pb),chromium (Cr), and titanium (Ti) (Wrightson, 1949). Metals in crude oil may enhance orinhibit the degradation of oil by microorganisms. Some metals may be toxic (Gadd andGriffiths, 1978 ; Kamel et al., 1989), and many metals are essential trace nutrients for microbial growth (Doelman, 1986). Long-term exposure to heavy metals (Zn, Cu, and Ni) has
been found to alter microbial structure as assessed from total soil PLFA (phospholipids
fatty acids) profiles (Baath et al., 1998). Heavy metal contamination of soil reduced fungal
biomass and altered the composition of the fungal community (Nordgren et al., 1983 ;Nordgren et al., 1985).
Therefore, it is hypothesized that the presence of metals in heavy oil may be the important factor in the bioremediation/biodegradation of the Nakhodka oil spill during thefive years of bioremediation. During that period, some metals may be essential for oildegrading microorganisms and promote the biodegradation processes, while others may
not be necessary for biological functions and were toxic. Some metals (e.g., Cu, Fe, Mn,

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

27

and Zn) are required in trace concentrations (micronutrients). Some metals (e.g., Cr andPb) are considered to be important pollutants (Kovalick, 1991). Correspondingly, little orno attention has been paid to the effect of environmental factors (e.g., heavy metals) on the
degradation of Nakhodka oil spill in contaminated sites. Since environmental contamination with oil is often accompanied by the presence of high levels of heavy metals that are
known to be toxic for organisms, the presence of heavy metals in the environment, cantherefore, negatively influence the effectiveness of bioremediation processes.
The major objective of this study was to isolate and describe hydrocarbon-degrading
bacteria from the Nakhodka oil spill-contaminated seashores in the Sea of Japan after five
years of bioremediation. Representative bacterial strains were identified by 16S rDNA sequence analysis and characterized for their use and ability to grow on the different hydrocarbons as the sole carbon and energy source. In addition, the metallic contents of heavy
oil and their effect on the growth of bacterial isolates were evaluated in comparison, since
the indigenous microorganisms at Nakhodka oil spill-polluted sites may be exposed to themetals-contaminated oil spill, which may affect their growth and ability to degrade heavy
oil during the 5-year bioremediation. To our knowledge, there are no previous reports on
the influence of heavy metals on bioremediation of Nakhodka oil spill.
2. Materials and methods
2.1. Field sites and sample collection
The study site was the Nakhodka oil spill-contaminated coastal areas in the Sea of Japan (Fig. 1). Heavy oil, sand, and seawater contaminated with the Nakhodka oil spill were
sampled in February 1997 at the Nakhodka tanker, in December 1999 at Katano coast in
Fukui Prefecture, and in November 2001 at Osawa and Atake coasts in Ishikawa Prefecture,
Japan. Samples were stored at 4℃ prior to use.
2.2. Isolation and screening of hydrocarbon-degrading bacteria
Bacteria capable of degrading hydrocarbons were isolated directly from samples ofheavy oil, sand, and seawater contaminated with Nakhodka oil spill by selective enrichment using Bushnell Hass Mineral Salts medium (BHMS) modified with 2% NaCl andsupplied with heavy oil 1% (v/v) from Nakhodka oil spill as the sole added carbon and energy source. BHMS contained (per liter of distilled water) 0.2 g of MgSO4.7H2O, 0.02 g of
CaCl2, 1 g of KH2PO4, 1 g of K2HPO4, 1 g of NH4NO3, 2 drop of FeCl3 60%. The pH wasadjusted to 7 to 7.8 with 1N NaOH. The BHMS medium was sterilized by autoclaving(121℃ for 20 min). Heavy oil was sterilized by filtration through 0.2 μm membrane filters.
Enrichment of bacterial culture was carried out in 300 ml Erlenmeyer flasks containing 100
ml of BHMS. Samples of heavy oil, sand, or seawater (10% w/v or v/v) were added to 100
ml of BHMS supplemented with 2% NaCl and 1% (v/v) heavy oil from the Nakhodka oil
spill. All flasks were incubated at room temperature (24-25℃) by shaking at 125 rpm. Bacteria were isolated by plating serial dilutions on ZOBELL agar. All plates were incubated

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

Fig. 1. Map of the study area and sampling stations. The map of Japan on the right shows the coastal
sites (as indicated by a solid black line) in the Sea of Japan contaminated with the Nakhodka oil
spill occurring on January 2, 1997. The magnified map of contaminated coastal areas (on the left)
shows the sampling stations indicated by arrows.

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation29

at 25℃ for 2-3 days. Colonies were purified by repeated transfer on the isolation medium,and then screened for growth on BHMS amended with 0.85% (w/v) NaCl and 0.5 g/l yeast
extract, and supplied with 1% (v/v) heavy oil as carbon source. The isolates that grew wellon heavy oil were selected for detailed study, whereas those failed to grow on heavy oil
were excluded from further screening. Of the 39 bacterial isolates obtained, seven isolates
were assayed for their use and ability to grow on different individual hydrocarbons otherthan heavy oil (Nakhodka oil spill).
2.3. Characterization of hydrocarbon degrading bacteria
Seven isolates were characterized for their use and ability to grow on the different hydrocarbons as the sole carbon and energy source. They were tested in test tube containing
10 ml of BHMS amended with 0.85% (w/v) NaCl which was sterilized by autoclaving(121℃ for 20 min), and supplied with the following hydrocarbons as sole carbon source :hexane, octane, tetradecane, hexadecane, octadecane, eicosane, octacosane, paraffin, pristine, cyclohexane, toluene, biphenyl, naphthalene, phenanthrene, and fluoranthene. Hydrocarbons were sterilized by filtration (membrane filter 0.2 μm), while solid hydrocarbons(octadecane, eicosane, octacosane, and aromatic hydrocarbons) were added directly to
BHMS medium before autoclaving. The pH of the medium was adjusted to 7.8 with 1N
NaOH. A loopful of each of the tested isolates from the purity plates was inoculated in test
tubes containing sterile BHMS supplemented with the sterile hydrocarbons mentioned
above. The tubes were incubated with agitation (150 rpm) at 25℃ for 15 days. After 15days of incubation, the bacterial growth response was determined immediately by the increase in both optical density (OD600) and colony forming units (CFU) in nutrient agar
plates supplemented with 0.85% (w/v) NaCl and 0.5 g/l yeast extract.
2.4. 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 μl of template DNA, 2.5 μl of 10 X
reaction buffer, 2 μl of 2.5 mM of deoxynucleoside triphosphate (dNTPs), 0.5 μl of 10
pmol of each primers, and 0.25 μl of 2.5 U of Z-Taq DNA polymerase (TaKaRa Bio Inc,
Shiga, Japan) in a final volume of 25 μl. DNA amplification was performed in a model
PTC-100 Thermal cycler (MJ Research Inc, Watertown, Mass. USA) with following profile : 30 cycles of 96℃ for 1 sec, 55℃ for 10 sec, and 72℃ for 20 sec. The PCR products
purified with EXOSAP-IT kit (USB corp., USA) were sequenced directly using an ABIPRISM 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 Biosystems, USA). The closest related sequenceswere found using the BLAST programs (Altschul et al., 1997).

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

2.5. Metallic content of heavy oil
Metallic content of heavy oil was estimated using the ED-XRF (Energy Dispersive-XRay-Fluorescence) analyzer according to Obiajunwa et al. (2002) with modifications. 0.5 gof heavy oil sample was dissolved in 5 ml of n-hexane, shaken vigorously, and allowed tostand for 20 min. 50 μl of the oil-solvent mixture was mounted on Mylar films, and the solvent was allowed to evaporate overnight. The heavy oil was then analyzed by an energy
dispersive X-ray fluorescence spectroscopy (ED-XRF ; JEOL JSX-3201), using Rh-Ka
generated at 30 kV under vacuum. Measurement was conducted in triplicate. Three replicates of each heavy oil sample were prepared, and the metal content was estimated in all
replicates.
2.6. Effects of heavy metals on the growth of bacterial strains
To determine whether the presence of metals in heavy oil affect the isolated strains ofalkane-degrading bacteria, four representative strains (selected based on sampling sites) as
well as P. aeruginosa PAO1 and E. coli K-12 MG 1655 (as controls) were tested for theirability to grow on nutrient broth (NB) medium plus 1g/l yeast extract and 0.85% w/v NaCl
(except for E. coli K-12, which was grown on LB medium) amended with various concentrations (0.01, 0.1, 0.5, 1, 2, 5, and 10 ppm) of metals (Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Pb,
Cd, and V). They were tested in test tube containing 10 ml of NB medium plus 1g/l yeast
extract amended with 0.85% (w/v) NaCl which was sterilized by autoclaving (121℃ for 20min), and supplied with the following sterile concentrated metal salt stock solutions of Ti,
Cr, Mn, Fe, Co, Ni, Cu, Zn, Pb, Cd, and V. pH of the medium was adjusted to 7.8 with 1N
NaOH. 10% v/v of stock culture of each of the tested isolates was inoculated in test tubes
containing sterile NB medium supplemented with the sterile metals mentioned above. The
tubes were incubated with agitation (150 rpm) at 25℃ for 24 hours. After 1 day of incubation, the bacterial growth response was determined immediately by the increase in opticaldensity (OD600). Test cultures, control cultures without added metals, and uninoculated control cultures were tested in duplicate.
2.7. Light and Scanning electron microscopy
For light microscopy, bacterial cells were wet mounted on glass slides, stained with
4’,6-diamidino-2-phenylindole (DAPI), then observed under epifluorescence microscope(Nikon NTF2A). For scanning electron microscopy, bacterial cells were fixed with 2% (vol/vol) glutaraldehyde in phosphate buffer at room temperature for 1 h, post-fixed in 1% osmium tetroxide 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-5200
LV scanning electron microscope.

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

31

3. Results and discussion
3.1. Isolation and identification of bacterial strains able to use the heavy oil as carbon
sourceSeven bacterial strains isolated showed an ability to use the heavy oil from the Nakhodka oil spill as the sole source of carbon and energy. They were coded as D1, D2, D3,
D4, D5, D6, and D7. Strains D1 and D2 were isolated from the Nakhodka oil tanker, strain
D3 from Katano seashore, strain D4 from Osawa seashore, and strains D5, D6, D7 fromAtake seashore. Using 16S rDNA sequence analysis, the strains D1, D2, D3, D4, D5, D6,
and D7 were affiliated to those of Pseudomonas aeruginosa (99% similarity), Bacillus
cereus or Bacillus thuringiensis (99%), Pseudomonas aeruginosa (98%), Pseudomonas
aeruginosa (99%), Pseudomonas aeruginosa (98%), Pseudomonas aeruginosa (99%), andParacoccus seniphilus or Paracoccus marcusii (97%), respectively.
Sequence analysis of 16S rDNA showed that the bacterial communities of the Nakhodka oil spill-contaminated coastal sites were dominated by genus Pseudomonas during
the 5-year bioremediation. The appearance of Pseudomonas strain as a major componentof the bacterial community was common to all samples and therefore related to thehydrocarbon-polluted sites (Rosenberg, 1992 ; Janiyani et al., 1993). It is likely that the
Pseudomonas sp. isolated from four locations, and shown to be abundant in those seashores, also became a predominant member of the bacterial community at the site under the
Nakhodka oil spill-polluted seashores. This organism appears to be well adapted to hydrocarbon biodegradation in seashores and survival during bioremediation process.
Hydrocarbon-degrading communities must adapt to the Nakhodka oil spill that consists
mainly of alkane hydrocarbons. To date, the majority of the PAH-degrading bacteria reported belongs to the genus Pseudomonas (Cerniglia, 1993). Microbial communities
within contaminated ecosystems tend to be dominated by those organisms capable of utilizing and/or surviving toxic contamination (Macnaughton et al., 1999). As a result, genus
Pseudomonas obtained as the most predominant bacteria in bacterial community of contaminated sites seems to be capable of utilizing the Nakhodka oil spill as well. The bacterial diversity may be influenced by the complexity of chemical mixtures present and theexposure (Macnaughton et al., 1999). Pseudomonas strain may be tolerant and resilient to
the metabolic products that are formed during degradation/bioremediation of the Nakhodka
oil spill, so that this species was predominant among the bacterial communities at all thethree coastal sites and Nakhodka tanker. Another study also showed that after exposure totoluene, Pseudomonas strain was the dominant community capable of toluene mineralization (Hubert et al., 1999).
3.2. Growth of bacterial isolates on different hydrocarbons
Seven selected isolates were assayed for their suitability and ability to grow on both
aliphatic and aromatic hydrocarbons, as well as the heavy oil (Table 1). All the tested isolates grew very well on the aliphatic hydrocarbons (C14-C28) and the heavy oil from Nak-

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

Table 1. Growth and ability of strains isolated to use different organic compounds as carbon source

hodka oil spill. They were also able to grow on other alkane hydrocarbons such as hexane(C6), octane (C8), paraffin (a mixture of alkanes), pristine (branched alkane C19) which israther recalcitrant in nature (Leisinger and Brunner, 1986), and cyclohexane (one of themost abundant natural cycloalkanes). Most of the strains appeared to be unable to use aromatic hydrocarbons as carbon source. Nevertheless, strains D5 and D6 were isolates which
had little ability to grow on aromatic hydrocarbons : strain D5 was able to grow on phenanthrene and fluoranthene, while strain D6 could use toluene as a carbon source. Alkanehydrocarbons (in particular C14-C28) and heavy oil appeared to be their good growth substrates. All strains could grow on linear n-alkanes ranging from at least 6 or 8 to above 28
carbon atoms in length, as well as branched alkanes. However, alkanes from 14 to 20 carbon atoms allowed all strains to grow abundantly. These results suggested that all thestrains tested could predominantly utilize alkane hydrocarbons as carbon source, but notaromatic hydrocarbons. In addition, SEM images showed the rod-shaped bacterial strain
isolated from Katano seashore (Fig. 2A), the spherical bacterial strain isolated from Osawa

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

33

Fig. 2. Scanning electron micrographs of bacterial strains isolated from Katano seashore (A) and Osawa
seashore (B).

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

Fig. 3. Scanning electron micrograph (A) and epifluorescence photomicrograph (B) of bacterial strain
isolated from the Nakhodka tanker.

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

35

seashore (Fig. 2B), and the rod-shaped bacterial strain isolated from the Nakhodka tanker(Fig. 3A). Epifluorescence microscopy showed DAPI-stained cells isolated from the Nakhodka tanker (Fig. 3B).
The incapability of our strains to use PAHs as sole carbon source is not surprising,since the strains were isolated from seashores contaminated with the Nakhodka oil spill
which consisted mainly of aliphatic hydrocarbons. That is, n-alkanes of C9-C30, in which neicosane (n-C20H42) was the most abundant compound, while the contents of aromatic compounds were low (Tazaki et al., 1997b ; Tazaki et al., 1997c ; Shibata et al.,1997 ; Itagaki
and Ishida, 1999 ; Sampei and Tazaki, 2003 ; Sampei et al., 2003 ; Chaerun et al., 2003 ;Chaerun and Tazaki, 2003 ; Tazaki, 2003). Microorganisms capable of degrading PAHs
are often isolated from environments with a history of contamination by PAHs (Herbes and
Schwall, 1978 ; Heitkamp and Cerniglia, 1988 ; Kastner et al., 1994 ; Trzesicka-Mlynarz
and Ward, 1995). Bacteria which are capable of degrading alkanes commonly do not degrade PAHs (Foght et al., 1990), although metabolic pathway for both types of compounds
is compatible (Whyte et al., 1997). Also, degradation of PAHs occurs much more efficiently by means of mixed microbial consortia than by single strains (Venkateswaran andHarayama, 1995 ; Komukai-Nakamura et al., 1996).
3.3. Metal content of heavy oil
Elemental levels in heavy oil showed wide ranges in all the heavy oil samples which
consisted of Si, S, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, and Pb (Table 2). Si, S, and Cr were obTable 2. Average elemental concentrations (wt%) in heavy oil for each sampling site

Note : mean±standard deviation ; n = 3, except for S concentration of Osawa seashore, which was
calculated using n = 2. Samples of heavy oil were taken on February 21, 1997 (for Nakhodka tanker),on December 10, 1999 (for Katano seashore), and on November 21, 2003 (for Osawa and Atake seashores). n.d. : not detected.

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

served at high levels, while Ti, Mn, Fe, Co, Ni, Cu, Zn, and Pb were at low levels. Averagelevels of all metals except Mn in heavy oil were highest in Katano seashore. Mn concentration was higher at Osawa seashore. Concentrations of S, Fe, Cu, and Pb were not detected
at Katano seashore, whereas the most predominant elements in heavy oil at all seashores
were Si, S, and Cr, except in heavy oil at Katano seashore, which did not contain S. It is
important to note, however, that the high S content in heavy oil might affect the oxidation
rate of oil by bacteria during the bioremediation processes (Brock, 1970 ; Jeffry, 1980).
Sulfur is the third most abundant element in crude oil (Speight, 1980), and sulfur heterocycles are common constituents of petroleum and liquid derived from coal (Blessler and Fedorak, 2001).
Elements originating from the Nakhodka oil spill were Si, S, Ti, Cr, Ni, Cu, and Zn(at Nakhodka tanker), and so it could be stated that the other metals (i.e., Mn, Fe, Co, andPb) which were found at Katano, Osawa, and Atake seashores did not originate from Nakhodka oil spill. Distinct metallic contents of heavy oil among three seashores (Katano,
Osawa, and Atake) may have been attributable to both natural (erosion, fires, leaching, volcanic activity, and microbial transformation) and anthropogenic (industrial waste, dumpingof sewage, burning of fossil, etc.) sources (Babich and Stotzky, 1980 ; Wong, 1993 ; Kuo
and Genthner, 1996 ; Benka-Coker and Ekundayo, 1998 ; Baldrian et al., 2000).
3.4. Effect of heavy metals on bacterial growth
Effects of added metals (i.e., Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Pb, Cd, and V) at 0.01 to
10 ppm on the growth of bacterial isolates are presented in Table 3. Strains D1 (isolated
from the Nakhodka oil tanker) and D5 (isolated from Atake seashore) were able to grow inthe presence of all added metals at concentrations from 0.01 to 10 ppm, except Co, which
inhibited growth at 2 ppm or greater. All bacterial strains were able to grow in the presenceof low concentrations of all these metals (0.01 to 1 ppm), with the exception of strains D3(isolated from Katano seashore) and D4 (isolated from Osawa seashore), which were very
sensitive even to low concentration of Cd at !0.5 and !0.1 ppm, respectively. These are
not surprising, since Cd is not an element found in heavy oil samples (Table 2). Comparedwith strains D1 and D5, strains D3 and D4 tended to be sensitive to all metals tested at 2
ppm or greater. The presence of Ti, Cr, and Cu at 0.01 to 10 ppm did not inhibit growth of
all bacterial strains, while Co was inhibitory at concentrations more than 1 ppm, the sublethal concentrations reducing growth by approximately !50%. Little or no growth wasobserved at >2 ppm Co for all bacterial strains, with the exception of E. coli K-12 control
strain. Other metals (i.e., V, Mn, Fe, Ni, Zn, Cd, and Pb) also appeared not to inhibit
growth of all bacterial strains, except strain D3 which was very sensitive to !5 ppm Fe,!5 ppm Zn, and !0.5 ppm Cd, as well as strain D4 sensitive to 10 ppm V, 10 ppm Ni, 10
ppm Zn，!0.1 ppm Cd, and !2 ppm Pb. However, there was no significant difference inresistance to metals tested between bacterial isolates and control strains such as P. aeruginosa PAO1 or E. Coli K-12. They showed almost similar sensitivities to added metals.

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

Table 3. Effect of added metals on the growth of four representative strains

37

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

Table 3. (Continued )Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

Table 3. (Continued )

39

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

Table 3. (Continued )%V = growth with added metals ÷ growth in controls lacking metals x 100
Control lacking added metal
E. coli K−12 and P. aeruginosa PAO1 were used as control bacterial strains.
Strains D1, D3, D4, and D5 were isolated from the Nakhodka tanker, Katano seashore, Osawa seashore,and Atake seashore, respectively.

ab

Furthermore, although V is not an element found in heavy oil samples but is the mostabundant heavy metal in oil residues (Sasaki et al., 1998), our bacterial strains were able togrow in the presence of V at 0.01 to 10 ppm. Since Cr was the most abundant heavy metalin heavy oil samples, capability of our strains to grow well in the presence of Cr was not
surprising, indicating that bacterial strains may have adapted to the toxicity of Cr. However,their ability to grow at 5 to 10 ppm was extraordinary. Thus, the ability of our some strains
to grow in the presence of somewhat higher concentration of some heavy metals (V, Cr, Ni,
Zn, Cd, and Pb) indicated that bacterial acclimation periods ensued in metal-containing
heavy oil, resulting from induction of a protein required for metal precipitation or detoxifi-

Alkane-degrading bacteria and heavy metals from the Nakhodka oil spill-polluted seashores
in the Sea of Japan after five years of bioremediation

41

cation, a genetic change, or selection of a tolerant or detoxifying bacterial species or population. These, thereby, resulted in precipitation and/or detoxification of added metals (Kuoand Genthner, 1996).
Our present results showed that metal contents of heavy oil occurred at low levels,
thereby enabling these metals as trace elements (micronutrients) needed for microbial
growth, in particular to alkane-degrading bacteria, and affected their growth and ability todegrade heavy oil. The results may be related to the findings of Kuo and Genthner (1996),
that the presence of metals can be a way of predicting the outcome of anaerobic bioremediation of an organic pollutant as well as the possibility of using anaerobic bacterial species
to bioremediate a site that was contaminated with heavy metals. Several anaerobic species
may be used to bioremediate both heavy metals and organic pollutants either individuallyor in combination. It is also possible, however, that the indigenous microorganisms inhabiting the Nakhodka oil spill (i.e., alkane-degrading bacteria) which are being exposed tometals may also become resistant to or capable of transforming and detoxifying heavy metals (Compeau and Bartha, 1985 ; Hughes and Poole, 1989 ; Hughes and Poole, 1991 ;Lovley, 1993). The presence of miscellaneous pollutants in any of the oil spills may present extreme challenges to the maintenance of a phylogenetically and functionally diverse
microbial community (Shi et al., 2002). However, it is suggested that both heavy metals
and hydrocarbons would influence the composition of microbial community which genus
Pseudomonas was dominant member at all the contaminated coastal sites in this study. Genus Pseudomonas is apparently well-equipped to cope with aliphatic hydrocarbon toxicity
and may, therefore, be a primary catalyst for aliphatic hydrocarbon degradation close to theorigin of the Nakhodka oil spill during the 5-year bioremediation.
4. Conclusion
This study has shown that after long-term exposure to both hydrocarbons and heavy
metals of the Nakhodka oil spill, alkane-degrading bacteria isolated from three seashores
and the Nakhodka tanker, identified as Bacillus spp., Pseudomonas sp., and Paracoccus
spp., may also tolerate heavy metals other than toxic levels of hydrocarbons. Results of thisstudy suggest that Pseudomonas species which was a dominant organism in the bacterial
community of the Nakhodka oil spill-contaminated coastal sites during the 5-year bioremediation may have been associated with the hydrocarbon degradation at those sites. In addition, the bacterial composition of community in those sites may have played a key role inhydrocarbon degradation and that Pseudomonas strain, in particular, was associated withhydrocarbon-degrading activity as well as the bioavailability of heavy metals.
Heavy metals obtained in all the heavy oil samples of three seashores in the Sea of Japan showed that there was a considerable heavy metal pollution which could be correlated
not only with the Nakhodka oil spill but also with biogenic and anthropogenic sources. Results indicated that Si, S, Ti, Cr, Ni, Cu, and Zn were predominantly indigenous to the
Nakhodka oil spill, whereas Mn, Fe, Co, and Pb were present as the result of contamina-

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S. Khodijah CHAERUN, Kazue TAZAKI, Ryuji ASADA and Kazuhiro KOGURE

tion associated with other sources. Our data also have a significant insight into how these
metals in the Nakhodka oil spill affect the biodegradation of heavy oil during the 5-year
bioremediation, thereby giving more fruitful information for such kinds of oil spills. In
contrast, these results also provided information that the Nakhodka oil spill resulted in not
only organic pollutants (i.e., hydrocarbons) but also heavy metal pollutants (i.e., Cr) along
the coastal areas of Sea of Japan.
Acknowledgements
We are grateful for the cooperation and assistance of all students of the Tazaki laboratory of Kanazawa University. We greatly acknowledge the help of all students of theKogure laboratory (University of Tokyo) for help with 16S rDNA sequencing analysis. We
are particularly indebted to Prof. Dr. Yoshihiro Fukumori and Associate Professor Dr. M.
Kanemori of Kanazawa University for providing the laboratory strains of P. aeruginosa
PAO1 and E. coli K-12 MG 1655 used in this study. This work was supported by a grantfrom the Japanese Ministry of Education, Culture, Science, and Technology to Dr. Kazue
Tazaki.
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