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


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

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

K. 2.. ex situ bioremediation process in laboratory microcosms (containing seawater. This paper described the results of efforts of isolation and characterization of the seven aerobic. Accordingly. was carried out as well. the bacteria were isolated not only from seawater. and heavy oil) under aerobic condition by two different treatments. beach sand. Fukui Prefecture) 5 years after the Nakhodka Russian oil tanker spill in the Sea of Japan in relation to bioremediation process. on December 10. in soils have resulted in successes and failures (Vogel. Location of the sampling sites in the Sea of Japan. Ishikawa Prefecture (mainly from Wajima seashore. Fukui Prefecture to Noto Peninsula. Fukui Prefecture to Noto Peninsula. 2). In Nakhodka heavy oil spill along seashores in the Sea of Japan (from Mikuni. Samples of heavy oil. Also. 1999 from Katano seashore in Fukui Prefecture.e. that is. Japan (Fig. year 1997). The strains could use alkanes and heavy oil (from the Nakhodka oil spill) aerobically as their sole carbon sources but not aromatics. Ishikawa Prefecture and Katano seashore. and on November 21. Bacterial strains capable of growing on heavy oil were isolated from the above four locations by using enrichment and isolation techniques. biostimulation of the indigenous bacteria that break down oil pollutants is considered a priority. a careful bioremediational study has been performed by our group since 1997. 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. . Japan. a technique used in bioremediation by adding microorganisms to sites contaminated with oil. / Environment International 30 (2004) 911–922 the findings of the superior hydrocarbon indigenous degraders of bacterial isolates in degrading the Nakhodka oil spill. Ishikawa Prefecture.g. 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. and the Nakhodka Russian oil tanker.1. 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. Japan (Fig. only 7 bacterial strains could grow very well on heavy oil (unpublished data). being further used for investigating the environmental factors influencing the bioremediation of the Nakhodka oil spill. For these reasons. Field sampling was performed three times. Japan). treatments outside building and inside building. particularly coastal areas. Atake and Osawa seashores of Ishikawa Prefecture). At the Atake seashore. Field sites and sample collection The sampling sites are located in the Sea of Japan. sands were sampled at two previous heavily oil-polluted fields. 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. the isolation of indigenous bacterial strains was conducted in three coastal areas (namely Katano seashore of Fukui Prefecture.912 S. In addition. and sand were collected from Nakhodka tanker on February 21. 2001 from Osawa and Atake at Wajima seashore in Ishikawa Prefecture. Fukui Prefecture to Noto Peninsula. Of the 39 bacterial strains tested. 2) were taken. 1996). was also investigated. 1. 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. being further used for future studies. Chaerun et al. Ishikawa Prefecture. from the top layer (0– 30 Fig. The arrow indicates sites in which the photographs (Fig. Investigation of the role of these bacteria in the bioremediation of heavy oil polluted sites. Materials and methods 2. but also from beach sands polluted by heavy oil for over 5 years. Additionally. 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.. 1). i. 1997. 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. namely at the top layer (0– 30 cm) about 3 – 4 m outside the shoreline. After a 5-year bioremediation process. halotolerant hydrocarbondegrading indigenous bacterial strains from seashores of Mikuni. Ishikawa Prefecture. from Mikuni. and all of them were able to grow well on heavy oil. viz. seawater. Since some attempts to demonstrate the potential for bioaugmentation. and at the top layer (0– 60 cm) about 5 –6 m outside the shoreline at Atake seashore.

pH. and S (sulfur) was estimated by using the NCS analyzer. 2001). heavy oil samples were collected at three differently polluted field sites namely on inshore.19 11.00 1. Field parameters including seawater temperature.55 9.94 2. (B) Katano seashore (on December 10.2. and sand Chemical composition of seawater and sand was analyzed using the energy-dispersive X-ray fluorescence (ED-XRF) analyzer. the initial composition of heavy oil spilled from the Nakhodka Russian oil tanker was analyzed by CHNS chemical analyzer (FISONS) (Table 3).% 8. 2.42 0. dissolved oxygen (DO). 2. 1999). (A) The Nakhodka tanker (on February 21.07 Atake seashore 0. purification and enumeration of hydrocarbon degrading bacteria Bacteria were isolated from seawater. Chemical analyses of seawater.45 0. electrode potential versus the standard hydrogen electrode (Eh). 1999.27 0. 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. 2001). The beach sand is mainly composed of quartz (SiO2).21 Samples of beach sands were collected from Katano seashore on December 10. .13 10. All samples were stored at 4 jC until required for analysis. and from Atake seashore on November 21.49 0. nearshore. heavy oil. and (D) Atake seashore (on November 21.99 3. and sand samples from three coastal areas contaminated with the Nakhodka oil spill.26 2. feldspars (Al – Si).61 0. Chaerun et al. 2003. Elemental composition of the seawater and sand is given in Tables 1 and 2. These were undertaken to investigate the difference of microbial activity of hydrocarbon-degrading bacteria.05 0.3.27 1. and away from outside shorelines. Osawa and Atake seashores—Ishikawa Prefecture.94 79.16 8.06 medium in isolating hydrocarbon-degrading bacteria. Isolation. 1997). and very few clay minerals. and also as controlled sampling in the isolation of hydrocarbon-degrading bacteria. The chemical composition of heavy oil in N (nitrogen).34 6. C (carbon).55 73. and Table 2 Average elemental composition (wt.42 7. in order to determine the biodegradation of hydrocarbons. 2.73 0.57 0. (C) Osawa seashore (on November 21.S. In addition.15 7.77 0.62 59. cm) about 3– 4 m outside the shoreline and from the top layer (0 –60 cm) about 5 –6 m outside the shoreline. Samples of heavy oil and sand were collected from each sampling site. heavy oil. / Environment International 30 (2004) 911–922 913 Fig. namely Katano seashore—Fukui Prefecture.77 2. and electrical conductivity (EC) were measured in situ using a portable meter.%) 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.K. Additionally.

eicosane. All treatments were performed in triplicate.4 Nakhodka tanker by selective enrichment by using the Bushnell Hass Mineral Salts (BHMS) medium comprising heavy oil 1% (v/v).25 Al of 2. Values for specific growth rates (designated l) were obtained by fitting data by using linear regression analysis.5 Al of 10 pmol of each primers. Laboratory microcosms contained seawater. 2.2 g of MgSO4Á7H2O. octacosane. 1997). Enumeration of the number of viable cells in bacterial culture was determined by a serial dilution-agar plating procedure. and aromatic compounds) were added directly to BHMS medium before autoclaving. DNA amplification was performed in a model PTC100 Thermal cycler (MJ Research. strain A3 (Katano). 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. 2. 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. strain A4 (Osawa).2 Am). Each flask contained 200 ml of 8 g/l of nutrient broth containing beef extract and peptone (i. and incubated on a rotary shaker (125 rpm) at room temperature. BHMS contained (per liter of distilled water) 0. 2 drops of FeCl3 60%. heavy oil.6. Samples were removed periodically to assess the concentration of the biomass (dry weight).1 27. Pure strains were conserved on ZOBELL agar/glycerol (v/v) at À 20 jC. 25 jC) containing enrichment medium. organic compound with very high nutrient content).02 g of CaCl2.5 mM of deoxynucleoside triphosphate (dNTPs). 1 g of NH4NO3.. MA. 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 1997.914 S.246 < 0. The growth kinetic of these strains was determined in cultures (200 ml. 0. The final isolate (each strain) was preserved in ZOBELL agar medium.5.85% (w/v) of NaCl were inoculated by 10% (v/v) inocula of strain A1 (Nakhodka). 2 Al of 2. Bacteria were maintained on either liquid or solid mineral salts medium as the sole carbon source. 150 rpm. and 0. 0. heavy oil. whereas the cell density was measured by using a spectrophotometer UV (APEL Model PD-303). USA) with following profile: 30 cycles of 96 jC for 1 s. After purification of sequencing products by ethanol precipitation. Japan) in a final volume of 25 Al. 1998). 2. Growth was then evaluated by comparison of optical density (OD600) against sterile control. 1 g of K2HPO4. Solid hydrocarbons (octadecane. Characterization of hydrocarbon degrading bacteria All the colonies obtained on ZOBELL agar were purified several times on the same medium. The PCR mixture contained 1 Al of template DNA. Test tubes were agitated for 15 days at room temperature. The pH was adjusted to 7– 7. respectively. When the growth of bacteria was detected in the medium. and samples were compared to the killed controls. which was repeated several times. strain A5 (Atake). 1 g of KH2PO4. wt. The PCR products purified with EXOSAP-IT kit (USB.% 62. 1991).7. and 72 jC for 20 s. USA).5 Al of 10 Â reaction buffer. as calculated from the absorbance (optical density at 600 nm) of washed cells. / Environment International 30 (2004) 911–922 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. Watertown. 55 jC for 10 s. 2. Colonies appearing on the plate were isolated by a single colony isolation procedure.e. Seawater. The initial substrate concentration was detected as mg/l chemical oxygen demand (COD) for each treatment (APHA. 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. Hydrocarbons were sterilized by filtration (membrane filter 0. and sand samples collected from seashores in the Sea of Japan were inoculated to BHMS medium supplemented with 2% NaCl. 2.. 2003. they were run on an ABI 3100 Genetic Analyser (Applied Biosystems). The bacteria were isolated by using an enrichment culture and a single-colony isolation technique.1 0.K. and beach . Chaerun et al. 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. 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. 1 g/ l of yeast extract (as co-substrate). The most closely related sequences were found using the BLAST programs (Altschul et al. A series of 500-ml Erlenmeyer flasks were used for this experiment. 0.4.2 10. The medium supplemented with 2% NaCl was used for the growth of marine bacteria.. Shiga. USA) were sequenced directly using an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems.8 (neutral-alkaline condition). Flasks with killed cells were used for controls.5 U of Z-Taq DNA polymerase (TaKaRa Bio.

pH. Only samples of heavy oil and sand were collected. summer. and S to determine biodegradation of hydrocarbon was estimated by using NCS analyzer. and strains A5. 95. post-fixed in 1% (w/v) osmium tetroxide (OsO4) at room temperature for 1 h. EC: electronic conductivity. aeruginosa (98%). The measurement of DO was performed on the oil – water phase. A5. EC.1 9. . and viewed using a JEOL JEM2000EX transmission electron microscope. 75. These showed that seawater temperatures ranged between 14.0 and 16. aeruginosa (99%). Additionally. 3. Eh. Laboratory experiment at the inside building was conducted at room temperature (22 – 25 jC). two strains originated from Katano seashore. Chaerun et al.K.8 to 11. However. bacterial cells were fixed with 2.. and 32 strains originated from Atake seashore. C. bacterial cells were wet mounted on glass slides. stained with 4V. strain A3 to P. JEOL JSM-5200LV) and transmission electron microscope (TEM. the experimental temperature was not maintained depending on the seasons (i. When grown aerobically in BHMS medium (pH 7 –7. and sand samples obtained at three coastal sites in the Sea of Japan and the Nakhodka tanker (data not shown). washed in the same buffer. strain A4 was isolated from Osawa seashore. three strains originated from Osawa seashore. The chemical composition of heavy oil in N. 2.2. These parameters were measured in November 2001.3.8 WT (jC) 16 16. A4. respectively.6.4 to 48.6 4. EC from10. / Environment International 30 (2004) 911–922 915 sand. dehydrated in a graded ethanol series (50. spring. then observed under epifluorescence microscope (Nikon NTF2A). mounted on copper specimen grids. strain A2 to Bacillus cereus (99%) or Bacillus thuringiensis (99%). Phylogenetic analyses of the bacterial isolates In order to identify the bacterial isolates.6 19 DO (mg/l) 11.6-diamidino-2-phenylindole (DAPI). seawater. 3. Samples for transmission electron microscopy were fixed and dehydrated as described above.5 jC.e. strain A6 to P.8). In the Nakhodka tanker and Katano seashore. A6. Results 3. Light. were measured during the sampling to observe the bioremediation process taking place. 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. A6. Physical analysis of seawater Physical analysis was performed to provide a detailed description of sampling site condition.3 Eh (mV) 20 75 118 EC (mS/cm) 10. Two strains originated from Nakhodka tanker.6 8. WT: water temperature. 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. and autumn). these parameters were not measured. respectively. of the 39 bacterial strains isolated. 3. DO: dissolved oxygen. mounted on carbon-coated copper stubs. Isolation by agar dilution series was then successfully attempted. and Eh between 20 and 118 mV. 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. A3. 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. strain A5 to P.S.1. A2.5% (v/v) glutaraldehyde in phosphate buffer at room temperature for 1 h. strain A4 to P.8. dissolved oxygen from 4. and 100%). scanning and transmission electron microscopy For light microscopy. Our experimental results on carbon source utilization thus suggested that the 39 isolates were hydrocarbon-degrading bacterial strains. 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). the temperature measurement of both treatments was not conducted. They were coded: A1. JEOL JEM-2000EX). At the outside building. strain A3 was isolated from Katano seashore. aeruginosa (98%). aeruginosa (99%). and A7 (Table 5). The physical characteristics of seawater are listed in Table 4. For chemical and physical parameters. winter.6 mS/cm.9 to 8. pH from 7. However. For scanning electron microscopy. and DO. The strains grew well not only in BHMS medium but also in ZOBELL agar medium with the high NaCl concentration (Table 1). Strains A1 and A2 were isolated from the Nakhodka oil tanker.1 mg/l. and A7 were isolated from Atake seashore.5 14 Eh: electrode potential versus standard hydrogen electrode. and viewed on a JEOL JSM-5200LV scanning electron microscope. microbiological parameters were measured to investigate microbial activity of hydrocarbon-degrading bacteria during the process observed by scanning electron microscope (SEM. The two different treatments were established by placing microcosms outside building and inside building. Isolation of hydrocarbon-utilizing bacteria from various coastal areas Thirty-nine strains of hydrocarbon-utilizing marine bacteria were isolated from heavy oil. and strain A7 to Paracoccus seriniphilus (97%) or Paracoccus marcusii (97%) (Table 5). It seems clear that any bacterial strains proliferating well in the NaCl-supplied medium could withstand high concentrations of salt (NaCl).9 8. the isolates gave cell yields ranging from 106 to 107 cells/ml. At the outside building. Strain A1 was affiliated to Pseudomonas aeruginosa (99% similarity).4 48. their 16S rDNA genes were amplified and sequenced.

and A5 (Atake) in an enrichment liquid medium. they grew poorly on hexane (C6). and cyclohexane. 3. A4 (Osawa). À = no growth. Fig.K. Growth curve in density of four bacterial strains of A1 (Nakhodka). 3. + + = medium growth.916 S. 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 + + ++ ++ ++ + + + + + À À À À À A2 + + ++ ++ ++ + + + + + À À À À À A3 A4 + + + + ++ ++ + + + + À À À À À + + ++ ++ ++ ++ + + + + À À À À À A5 + + +++ +++ ++ ++ + + + ++ À À À + + A6 + + ++ ++ +++ ++ + + + + + À À À À A7 + + ++ ++ ++ ++ + + + + À À À À À ++ spill).85% (w/v) of NaCl. pristane (branched C19). A3 (Katano). except that +++ +++ ++ +++ +++ ++ + = low growth. Also. 4. Growth on hydrocarbons as the sole carbon source Result of bacterial growth on pure hydrocarbons and heavy oil is given in Table 6.4. octacosane (C28). and eicosane (C20) as well as on heavy oil (from the Nakhodka oil Fig. hexadecane (C16). Sampling site Sample source heavy oil Strain Species Sequence similarity (%) 99 99 1 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. ++ + = strong growth. and 0. The seven bacterial strains were able to grow well on aliphatic hydrocarbons such as tetradecane (C14). However. / Environment International 30 (2004) 911–922 Table 5 Strains isolated able to grow well on and use heavy oil as carbon source No. Chaerun et al. . 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. octodecane (C18). these bacterial strains did not appear to utilize aromatic hydrocarbons as carbon source. octane (C8). paraffin.

0.85% (w/v) of NaCl. A4 (Osawa). A4 (Osawa).S. Chaerun et al. as the sole source of carbon. it was apparent that it was difficult for all bacterial strains to reach an optical density Fig. and strain A6 utilized toluene poorly. therefore being studied in more detail and used for future studies. On the other hand. and energy. All strains were capable of utilizing concentrations as great as 3030 mg/l COD without an appreciable lag phase.K. 3. A3 (Katano).5. especially in investigations on the environmental factors influencing the bioremediation of the Nakhodka oil spill. These strains grew well in enriched liquid media containing 8 g/l of nutrient broth. Bacterial growth and growth kinetics in an enrichment liquid medium Growth of strains A1 (Nakhodka). Growth curve in biomass and determination of specific growth rate of four bacterial strains of A1 (Nakhodka). 3 and 4).e. strain A5 was the most superior hydrocarbon degrader. and A5 (Atake) in an enrichment liquid medium. 1 g/l of yeast extract (as co-substrate). . This concentration was the initial substrate concentration detected as COD (mg/l) for each treatment. 5. and A5 (Atake) on enriched liquid medium was tested (Figs. at enrichment culture. nitrogen. When growth was not limited by growth factor(s). A3 (Katano). i. Of the seven bacterial strains tested in utilizing various hydrocarbons. / Environment International 30 (2004) 911–922 917 strain A5 grew poorly on phenantherene and fluoranthene. a classical growth response consisting of an exponential phase followed by a stationary phase was observed..

d.d. 0. 6.054 83.004 n. 5 with the regression coefficient: R2 = 0.918 S. S: sulfur. strain A3 (Katano).%) 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.68 0.: not detected. and EC ranged between 45 to 180 mS/cm.d. The graphs showed that seawater pH ranged between 6. C: carbon. strain A4 (Osawa). n.14 0.4 24. and strain A5 (Atake).19 n. 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 the low cell yield of growth on this substrate (Fig. A4 (Osawa). Eh. 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. n. . Eh ranged from 50 to 300 mV. Bioremediation process on the laboratory scale associated with in situ bioremediation 3.2 0.d.9249 (for strain A1 (Nakhodka)).d. 0.43 82.9 0.d.1 0.18 1. On the other hand.26 0. Conversely.d.03 0. indicating that strain A1 (Nakhodka) had the highest capability in using this substrate for its growth than those of three strains.2 59. n.27 0.d.d. 0.6 mg/l). DO ranged from 0. l.0164 hÀ 1. 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. l = 0. n.08 0. can be determined so as to fit the calculated profiles with corresponding experimental ones by the least squares method.d. 6.01 hÀ 1. l = 0. there were no significant changes in pH.16 0.K. n.12 1. The growth of strains A1 (Nakhodka). 1997) DO concentration decreased abruptly to anaerobic condition (0.83 1.06 S (%) 1. at inside building treatment.e.22 n.5 mg/l.44 1. n.01 n. 4).1 0.2 2.014 hÀ 1.01 n.d.d.61 1. n.d. C (%) 87.38 0. l = 0. Fig. n. Eh decreased sharply for all treatments.6.8 having the tendency to neutral-alkaline condition.d.14 0. 0. 3.d. / Environment International 30 (2004) 911–922 Table 7 Chemical composition of heavy oil in N. A3 (Katano).6 to 5.d. n. 5) with the following growth kinetic parameter: for strain A1 (Nakhodka). During the sampling at all treatments. n.d. and DO in laboratory microcosm experiments (i. An example of the good coincidence between the experimental and calculated profiles is shown in Fig. and A5 (Atake) in liquid culture followed the firstorder reaction (Fig. at 600 nm (OD600) of R 1. n. n.d. S (wt. l = 0.d. whereas EC increased rapidly. the inside and outside building treatments) during the 5-year bioremediation.1.4 and 7. n.44 2. while DO concentrations indicated that bioremediation processes took place under aerobic conditions. broth as substrate. Chaerun et al.08 83. at the second measurement (October 20.063 hÀ 1. EC.d. The values of pH.. 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.6. n. The values of the growth kinetic parameters. n.d.

Comparison of scanning (A1 – 2. the year 2001.. 0 –0. the year 1997..20 wt. Osawa seashore. and 0 – 2.e. Ishikawa Prefecture. 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. The Nakhodka tanker site was highest in proportion to the C content of those of other sites (i.06 – 0. . 87. 0.% (for Nakhodka tanker. B1 – 2) and transmission (A3.6. and 0.03 wt. the contents of N and S were relatively low. while the lowest one was obtained from a seawater sample without heavy oil in Atake seashore (i. Chaerun et al.% N. Compared with the content of C.S. 24.20 wt.K. Compared with inside building treatment.20 wt. that is. The contents of C ranged from 59. outside building treatment had a relatively high biodegradation rate of heavy oil shown by the low C content (0. Japan.054 wt.%).43 wt. laboratory microcosm assay of inside building. 0.%).68 to 87.% C (for seawater with heavy oil slick). 7.e. C. 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. Note: A1 – 3. The unit of contents is shown in percentage of weight.% S. and Atake seashore).% C (for seawater without heavy oil slick). In Atake seashore.% C (for heavy oil inshore in Atake seashore). / Environment International 30 (2004) 911–922 919 3.22 wt. Noto Peninsula.10 wt. and S are summarized in Table 7.2.%). B3) electron micrographs of hydrocarbon-utilizing bacteria inhabiting heavy oil during bioremediation process between the year 1997 and the year 2001 at Wajima seashores. These compositions described not only bioremediation process on the laboratory scale (ex situ bioremediation) but also in situ bioremediation processes in comparison. These provided that the biodegradation rate of heavy oil at Nakhodka tanker site was relatively low if compared with those of other sites.% C (for sand at different depths).44 wt. B1 – 3. 0.03 wt.

Bacteria degrading alkanes commonly do not degrade PAHs (Foght et al. in which n-eicosane (n-C20H42) was the most abundant compound.K. and only in the presence of O2..b). 7A1). n-alkanes of C9 – C30. Furthermore. Tazaki. 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. illustrating similarities from 1997 to 2001. Tortora et al.920 S. 1978.. 1993). The discovery of a majority of Pseudomonas is not surprising based on their frequency in the hydrocarbon-contaminated sites (Rosenberg. and 24. indicated by the low contents if compared with those of other samples. Aerobic culture conditions were chosen to select for an oxygen-dependent degradation. 1984). for the first time to our knowledge. Noto Peninsula. Ishikawa Prefecture. 7). and one unusual shape namely filamentous (Fig. Atlas.e. Kastner et al. aliphatic hydrocarbons are not fermentable. In this study. SEM and TEM observations were undertaken to investigate the microbial activity of hydrocarbon-degrading bacteria in heavy oil samples collected from Wajima seashores. 1994. 1980. 1994. 2001).3. a large number of hydrocarbon-degrading bacteria still existed in the sites.%. 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. and metanogenesis has been described (Aeckersberg et al. consisting of a variety of morphological form of bacteria such as coccus (Streptococcus and Staphylococcus). 2003). bacillus (Streptobacillus). The bacteria showed the population size of greater than 105 bacteria mlÀ 1 indicating a relative abundance of hydrocarbon-utilizing bacteria representing a population density of greater than 103 cells mlÀ 1. 1997).6. 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. 1988. The bacterial cells tended to remain together in a cluster. 1994. The seven bacterial isolates were able to grow well on n-alkane hydrocarbons and heavy oil. while the contents of aromatic compounds were low (Itagaki and Ishida. significant aliphatic hydrocarbon degradation occurs. the biodegradation of alkanes correlated with denitrification. 3. So and Young. The population size of hydrocarbon-degrading bacteria in 2001 (after 5 years of bioremediation) was in the range of 104 –106 cells mlÀ 1. to metabolize aerobically heavy oil or aliphatic hydrocarbons is well known since a long time. 1991. Japan). 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). A strong positive correlation exists here between the amount of organic matter (i. that is. There was the similarity in population size of hydrocarbon-degrading bacteria between year 1997 and year 2001.40. 7A3). 1999a. 4. 2003). these bacteria did not appear to use aromatic hydrocarbons as carbon source. aerobic growth of the isolated bacteria on heavy oil demonstrated the presence of heavy oil-degrading capacities in oxygen-respiring bacteria. respectively).3– 1. 1990). Chaerun and Tazaki. Heitkamp and Cerniglia. The initial degradation of petroleum hydrocarbons often requires the action of oxygenase enzymes and so is dependent on the presence of molecular oxygen (Brock. while others occurred in three-dimensional cubes or irregular grape-shaped or irregular star-shaped clusters (figure not shown). However.90. Japan.. especially P.. Ishikawa Prefecture. Moreover. Whereas C content of sands at all depths elucidated that there was no significant difference between them. Rueter et al.. cocci (spherical) or bacilli (rod-shaped) occurred in long chains (Fig.. TrzesickaMlynarz and Ward. since the strains were isolated from seashores contaminated with the Nakhodka oil spill which consisted mainly of aliphatic hydrocarbons. 1998. 1999. while the population size in 1997 (at the beginning of bioremediation process) was in the range of 105 – 106 cells mlÀ 1 (Kasai et al. namely cocci (spherical) and bacilli (rodshaped).. That is. aeruginosa. 1994. Trudgill. carbon content) and the abundance of hydrocarbon-degrading bacteria. 1991). In addition. and 0. 1995). the alkanes. aeruginosa. 1992. 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. Janiyani et al.5 –1 Am in width for rod-shaped bacteria. From some years.40 wt. Bacteria sizes at all sampling sites were in the range of 2 –7 Am in length and 0. and filamentous (Bitton. / Environment International 30 (2004) 911–922 seashore designated by declining the C contents (83..5 Am in diameter for spherical bacteria. Some cocci and bacilli formed a slime layer of cells (Fig. Thus. 1970. However. Comparison of microbial activity of hydrocarbondegrading bacteria between year 1997 and year 2001 The enumeration of bacterial cell number. the findings revealed that after 5 years of bioremediation. Discussion The capacity of bacteria. although metabolic pathway for both types of compounds is not incompatible (Whyte et al. Of the seven bacterial strains isolated. most of them were affiliated to P. The incapacity of our bacterial isolates to use PAHs as sole carbon source was not surprising.. Microorganisms capable of degrading PAHs are often isolated from environments with a history of contamination by PAHs (Herbes and Schwall. 82. but more degradable than the polycyclic aromatics (PAHs) (Jeffrey. Hydrocarbon-degrading bacteria occurred in two basic shapes. It thus seems that there was a sharp decrease in the content of carbon inshore in Atake seashore. 7B2). sulfate reduction. The n-alkanes are the most biodegradable of the petroleum hydrocarbons and are attacked by more microbial species than aromatic or naphthenic compounds. Chaerun et al. Aerobic conditions are therefore necessary for the initial breakdown of petroleum hydrocarbons and in subsequent .

Ishikawa Prefecture.K. Bernhard Schink (Konstanz University. 1981. it was required that growth yield was low. the greater the efficiency of energy generation or the less energy required for biomass production. Growth. Bak F. Most of the bacterial isolates are affiliated to P. Chaerun et al. PhD. and Professor Naoto Urano. and temperature (Atlas.. Arch Microbiol 1998. Conclusions After 5 years of bioremediation processes in the Nakhodka heavy oil spill along seashores in the Sea of . We greatly acknowledge the help of all students of the Kogure laboratory (University of Tokyo) for help with 16S rDNA sequencing analysis. and Professor Dr.156:5 – 14. 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. Germany) for continuous support. Based on the composition of the oil and seawater (Tables 1. Science and Technology to Dr. Furthermore. then the less substrate has to be dissimilated for this purpose and the more substrate is available for biomass production. 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. nitrate or sulfate may serve as a terminal electron acceptor (Bartha. Aeckersberg F. 5. however. It is not surprising that hydrocarbonoclastic bacteria are present in the hydrocarbon-polluted sites. bacteria may be ubiquitous in oligotrophic environments and essential to maintain the bioremediation process in the oligotrophic process as well. cellular fatty acids and metabolic adaptation of sulfate-reducing bacteria that utilize long-chain alkanes under anoxic conditions. / Environment International 30 (2004) 911–922 921 steps. 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. and (b) how much of the substrate is consumed by the growing cells for energy to drive biosynthetic reactions (Shuler and Kargi. oxygen. for bioremediation or biodegradation processes to cleanup or remove pollutants or contaminants. Fukui Prefecture to Noto Peninsula. On the basis of bacterial growth in an enrichment liquid medium. but oxygen is most commonly used.170:361 – 9. We also thank anonymous reviewers for constructive comments. the bacterial strains isolated show that they are able to utilize alkanes. Leahy and Colwell. Miller W. 1986). Arch Microbiol 1991. References Aeckersberg F. 1992). their sulfur content may affect the oxidation rate of oil during the 5-year bioremediation. Widdel F. 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. One of us (S.C) would like to thank Associate Professor Chiaki Imada. Rainey FA. Under the circumstances. Zhang Z. especially in studies on the environmental factors influencing the bioremediation of the Nakhodka oil spill (being performed by our group). indicating that the bacteria had the ability to survive for the relatively long periods even in the nutrient-poor environments (figure not shown). Altschul SF. Scha’’ffer AA. We are also grateful for the cooperation and assistance of all students of the Tazaki laboratory. indicating that bacteria are the main agents responsible for degradation of heavy oil and appear to be the prevalent hydrocarbon degraders in coastal areas. 3. Culture. Kazue Tazaki. Madden TL. The bacterial strains isolated from the Nakhodka oil spillcontaminated sites (at the beginning of bioremediation process) has been reported in the early literature. which has the capability to use both aliphatic and aromatic compounds. but not aromatics. 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. Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium. Clearly. This study was funded by a grant from the Japanese Ministry of Education. These results further support the view that bacteria can exist in and dominate some extreme environments. Zhang J. with the exception of one strain (i. Furthermore. Japan (from Mikuni. 6). PhD (Tokyo University of Fisheries). for helpful and motivating discussions. natural relationships. 7). Acknowledgements The authors thank Darius Greenidge.S. It was thus believed that under circumstances. Widdel F. It has also been shown that bacteria are the most predominant microorganism among other microorganisms in either in situ or ex situ bioremediation processes. for correcting the manuscript.e. because the degradation rate of substrate was not observed (not measured). 1990). bacteria were among the dominant microorganisms in these processes during the 5-year bioremediation. PhD. strain 5 isolated from Atake seashore). Japan). these cultures were not preserved and have not been further studied. aeruginosa.K. Thus. et al. and 7). we could not determine which strain was the best one in using this substrate. expressing the low specific growth rate (low biomass produced) and the high substrate-degradation rate (high substrate consumed). For both in situ and ex situ bioremediation process on the laboratory scale.

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