Bioremediation of coastal areas 5 years after the Nakhodka oil spill in the Sea of Japan: isolation and characterization of hydrocarbon-degrading bacteria
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 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 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 hydrocarbon-degrading 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.
Section snippets
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
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 °C; 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.
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
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
Conclusions
After 5 years of bioremediation processes in the Nakhodka heavy oil spill along seashores in the Sea of Japan (from Mikuni, Fukui Prefecture to Noto Peninsula, Ishikawa Prefecture, Japan), the bacterial strains isolated show that they are able to utilize alkanes, but not 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.
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
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