Environment International 30 (2004) 911 922 www.elsevier.

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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 Chaerun a,*, Kazue Tazaki b, Ryuji Asada b, Kazuhiro Kogure c
a

Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan b Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan c Ocean 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 Nakhodka oil spill in the Sea of Japan, seven bacterial strains capable of utilizing the heavy oil spilled from the Nakhodka Russian oil tanker were isolated from three coastal areas (namely Katano Seashore of Fukui Prefecture, Osawa and Atake seashores of Ishikawa Prefecture) and the Nakhodka Russian 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 to Pseudomonas 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 hydrocarbondegrading bacteria still existed in the sites consisting of a variety of morphological forms of bacteria, such as coccus (Streptococcus and 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 inside building and the outside building) were established for bioremediation of heavy oil to investigate the significance of the role of hydrocarbondegrading 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: 1 6 mg/l; Eh: 12 300 mV) and neutral-alkaline conditions (pH 6.4 8) with NaCl concentrations of 3 15% (ECs of 45 200 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 anthropogenic origins (particularly via leak of coastal oil refineries) 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
* Corresponding author. Tel.: +81-76-264-5732; fax: +81-76-2645746. E-mail addresses: kchaerun@earth.s.kanazawa-u.ac.jp, kchaerun@yahoo.com (S.K. Chaerun). 0160-4120/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2004.02.007

is that of the Nakhodka Russian 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 microorganisms 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 hydrocarbon degraders have been isolated from heavily contaminated coastal areas under this study (Itagaki and Ishida, 1999; Kasai et al., 2001; Tazaki, 2003). However, no data exist on

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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 Nakhodka oil spill were 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 bioremediation 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 Nakhodka oil 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). In Nakhodka heavy 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 the Nakhodka Russian oil tanker. Investigation of the role of these bacteria in the bioremediation of heavy oil polluted sites, particularly coastal areas, was carried out as well. Bacterial strains capable of growing on 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 (0 30 cm) about 3 4 m outside the shoreline, and at the top layer (0 60 cm) about 5 6 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 the Nakhodka oil spill. This paper described the results of efforts of isolation and characterization of the seven aerobic, halotolerant hydrocarbondegrading indigenous bacterial strains from seashores of Mikuni, Fukui Prefecture to Noto Peninsula, Ishikawa Prefecture (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) aerobically 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 5 years of bioremediation processes) were evaluated. In addition, 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 Prefecture, 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 (0 30

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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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).

cm) about 3 4 m outside the shoreline and from the top layer (0 60 cm) about 5 6 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-degrading 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. Field parameters including seawater temperature, pH, dissolved oxygen (DO), electrode potential versus the standard hydrogen 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
Table 1 Elemental composition of seawater used for ZOBELL medium analyzed by ED-XRF Element Na Mg Si S Cl K Ca Co Br Sr wt.% 8.27 1.49 0.26 2.94 79.94 2.99 3.57 0.05 0.42 0.06

medium in isolating hydrocarbon-degrading bacteria. Elemental 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 biodegradation of hydrocarbons. Additionally, the initial composition 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 Nakhodka oil spill, namely Katano seashoreFukui Prefecture, Osawa and Atake seashoresIshikawa Prefecture, and
Table 2 Average elemental composition (wt.%) of beach sands collected from Katano seashore and Atake seashore analyzed by ED-XRF Element Mg Al Si K Ca Ti Mn Fe Sr Katano seashore 0.34 6.55 73.16 8.77 2.45 0.77 0.15 7.73 0.07 Atake seashore 0.55 9.62 59.19 11.42 7.00 1.27 0.13 10.61 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 (Al Si), and very few clay minerals.

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Table 3 The initial composition of heavy oil spilled from the Nakhodka Russian oil tanker analyzed by CHNS chemical analyzer (FISONS) Chemical composition of heavy oil Carbon Hydrogen Nitrogen Sulfur Oxygen Sampei et al., 2003. wt.% 62.2 10.1 0.246 < 0.1 27.4

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 FeCl3 60%. The pH was adjusted to 7 7.8 (neutral-alkaline condition). The bacteria were isolated by using an enrichment culture and a single-colony isolation technique. Seawater, 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) was preserved 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 aromatic compounds) were added directly to BHMS medium before autoclaving. Test tubes were agitated for 15 days at room temperature. Growth was then evaluated by comparison 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 0.25 Al 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 PTC100 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 Biosystems). 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 experiment 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 determined 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 Model 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. Laboratory microcosms contained seawater, heavy oil, and beach

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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 observe the bioremediation process taking place. Laboratory experiment at the inside building was conducted at room temperature (22 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, microbiological parameters were measured to investigate microbial 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 JEM2000EX transmission electron microscope.

Table 4 Physical characteristics of seawater for each sampling site Sampling site Osawa seashore Atake seashore Sample source seawater seawater seawater + heavy oil pH 7.9 8.6 8.3 Eh (mV) 20 75 118 EC (mS/cm) 10.4 48.6 19 DO (mg/l) 11.1 9.6 4.8 WT (jC) 16 16.5 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 the Nakhodka tanker and Katano seashore, these parameters were not measured. Only samples of heavy oil and sand were collected.

3. Results 3.1. Physical analysis of seawater Physical analysis was performed to provide a detailed description of sampling site condition. The physical characteristics of seawater are listed in Table 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 bacteria 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 7 7.8), 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 seawater and heavy oil samples, 9 strains and 14 strains resulted from sand samples from the top layer (0 30 cm) and (0 60 cm), respectively. All bacterial strains were able to consume either heavy oil or peptone and yeast extract as a sole 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. However, of the 39 bacterial strains isolated, only 7 bacterial strains could grow well on heavy oil when grown aerobically 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 affiliated 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%) or Paracoccus marcusii (97%) (Table 5).

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Table 5 Strains isolated able to grow well on and use heavy oil as carbon source No. Sampling site Sample source heavy oil Strain Species Sequence similarity (%) 99 99

Nakhodka tanker

A1 A2

2 3 4

Katano seashore Osawa seashore Atake seashore

sand + heavy oil heavy oil heavy oil

A3 A4 A5

Pseudomonas aeruginosa Bacillus thuringiensis or Bacillus cereus Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Paracoccus seriniphilus Paracoccus marcusii

98 99 98

(outside seashore) (inshore) seawater + A6 heavy oil (sand of 30 cm sand A7 deep at 3rd layer)

99 97

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

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 Nakhodka oil
Fig. 3. Epifluorescence photomicrograph (A) and scanning electron micrograph (B) of strain A3 (Katano) at the exponential phase after 44 h 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.

Table 6 Growth and ability of strains isolated to use different organic compounds as carbon source Organic Strain compounds 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 A1 + + ++ ++ ++ + + + + + A2 + + ++ ++ ++ + + + + + A3 A4 + + + + ++ ++ + + + + + + ++ ++ ++ ++ + + + + A5 + + +++ +++ ++ ++ + + + ++ + + A6 + + ++ ++ +++ ++ + + + + + A7 + + ++ ++ ++ ++ + + + + ++

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

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

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

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 the Nakhodka oil 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 phase was observed. All strains were capable of utilizing concentrations as great as 3030 mg/l COD without an appreciable 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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S.K. Chaerun et al. / Environment International 30 (2004) 911922 Table 7 Chemical composition of heavy oil in N, C, S (wt.%) for each sampling site analyzed by NCS analyzer Sampling site Nakhodka tanker Laboratory assay (inside building) Laboratory assay (outside building) Osawa seashore Atake seshore Sample source seawater + heavy oil seawater + heavy oil seawater + heavy oil heavy oil heavy oil (outside seashore) heavy oil (inshore) heavy oil (nearshore) seawater + heavy oil seawater sand (1st layer) sand (2nd layer) sand (3rd layer) sand (1st layer) sand (2nd layer) sand (3rd layer) sand (4th layer) sand (5th layer) N (%) n.d. n.d. n.d. 0.19 n.d. n.d. 0.22 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. C (%) 87.2 59.68 0.054 83.08 83.4 24.43 82.9 0.1 0.03 0.14 0.1 0.14 0.2 0.16 0.26 0.08 0.06 S (%) 1.44 1.83 1.61 1.12 1.38 0.18 1.2 2.44 2.27 0.01 n.d. 0.01 n.d. n.d. 0.004 n.d. n.d.

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 firstorder reaction (Fig. 5) with the following growth kinetic parameter: for strain A1 (Nakhodka), l = 0.063 h 1; strain A3 (Katano), l = 0.01 h 1; strain A4 (Osawa), l = 0.014 h 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

Atake seashore (sand of 30 cm in depth) Atake seashore (sand of 60 cm in depth)

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

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 indicated 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 condition (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.

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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 described not only bioremediation process on the laboratory scale (ex situ bioremediation) but also in situ bioremediation processes 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, 0 0.22 wt.% N, and 0 2.44 wt.% S. The Nakhodka tanker 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 at Nakhodka tanker site was relatively low if compared with those of other sites. Compared with inside building treatment, 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; B1 3, 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 here between the amount of organic matter (i.e., carbon content) and the abundance of hydrocarbon-degrading bacteria. Kennish (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 hydrocarbondegrading bacteria between year 1997 and year 2001 The enumeration of bacterial cell number, SEM and TEM observations were undertaken to investigate the microbial activity of hydrocarbon-degrading bacteria in heavy oil samples collected from Wajima seashores, Noto Peninsula, Ishikawa Prefecture, Japan. The population size of hydrocarbon-degrading bacteria in 2001 (after 5 years of bioremediation) was in the range of 104 106 cells ml 1, while the population size in 1997 (at the beginning of bioremediation process) was in the range of 105 106 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 population size of greater than 105 bacteria ml 1 indicating a relative abundance of hydrocarbon-utilizing bacteria representing 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 of hydrocarbon-degrading bacteria during the 5-year bioremediation process (comparison between year 1997 and year 2001). Hydrocarbon-degrading bacteria occurred in two basic shapes, namely cocci (spherical) and bacilli (rodshaped), 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 0.5 1 Am in width for rod-shaped bacteria, and 0.3 1.5 Am in diameter for spherical bacteria, illustrating similarities from 1997 to 2001. Thus, the findings 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 (Streptococcus and Staphylococcus), 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 isolated from marine coastal areas polluted by Nakhodka Russian oil tanker spill in the Sea of Japan (mainly from Wajima seashore, Ishikawa Prefecture, Japan). Aerobic culture conditions 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 to P. aeruginosa. The discovery of a majority of Pseudomonas 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; Heitkamp and Cerniglia, 1988; Kastner et al., 1994; TrzesickaMlynarz and Ward, 1995). Bacteria degrading alkanes commonly 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 consisted mainly 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 hydrocarbon 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 denitrification, sulfate reduction, and metanogenesis has been described (Aeckersberg et al., 1991, 1998; Rueter et al., 1994; So and Young, 1999a,b). Moreover, aliphatic hydrocarbons 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 showing that the bioremediation processes mostly ensued under aerobic condition confirmed the bacterial role in the degradation of aliphatic hydrocarbons of the Nakhodka oil spill during the 5-year bioremediation (Fig. 6). It is most likely for the indigenous bacteria to degrade and clean up sites contaminated 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-degradation 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 substrate consumed that is a variable which depends on the balance between: (a) how much of the available growthsubstrate 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 the more 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 processes 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.

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 aromatics, 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 also been shown that bacteria are the most predominant microorganism 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 hydrocarbonoclastic bacteria are present in the hydrocarbon-polluted sites. The bacterial strains isolated from the Nakhodka oil spillcontaminated 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 Nakhodka oil spill and indigenous to the Nakhodka oil 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 bioremediation of the Nakhodka oil spill (being performed by our group).

Acknowledgements The authors thank Darius Greenidge, PhD, for correcting the 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.

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