Do Oil-Degrading Rhodococci Contribute to the Genesis of Deep-Water Bitumen Mounds in Lake Baikal?

Abstract

This study sought to understand the origin and fate of one of the bitumen mounds found on the bottom of Lake Baikal. These mounds are located at a depth of 900 m beneath oil spots detected on the surface of Lake Baikal (53° 18′24^″, 108° 23′20^″). The two mounds were sampled with a manipulator from a “MIR” deep-water manned submersible. Mature mound No. 8 was subjected to chemical and microbiological studies. Mound No. 3 was subjected only to chemical studies; we failed to perform microbiological analyses of this mound for logistic reasons. Oil spots collected from the water surface, samples of mound No. 3 and No. 8, were subjected to GC/MS analysis. The water contained aliphatic hydrocarbons with chains between C₈ and C₂₃, with the most abundant chain length being C₁₈. Mound No. 3 with the most abundant chain length being C₁₈ actively released oil droplets into the water. It contained 770 mg/g of C₁₃-C₃₂ n-alkanes, with a maximum at C₂₃ (160 mg/g). Mound No. 8 was inactive and contained 148 mg/g of aliphatic C₂₂-C₃₄ n-alkanes, with a maximum at C₂₅. Mound No. 8 also consisted of 3% inorganic matter, 48% unresolved complex mixture (UCM) and less than 1% other compounds (polyaromatic hydrocarbons, isoprenoids, carotenoids, and hopanes). The core of this sample used as inoculate, yielded Rhodococci when cultivated on oil as the only source of carbon. Cultivation of the sample on agar-containing Raymond inorganic medium with crude West Siberian oil as the only source of carbon revealed colonies of these bacteria, which all appeared identical. PCR was performed with DNA isolated from 5 colonies, using primers for 16S rRNA genes. Comparison of the sequences of the 5 PCR products over a length of 714 bp revealed that they were almost identical. Phylogenetic analysis of these homologous sequences showed that they were similar to the corresponding sequences of the genus Rhodococcus. Substrate demands, the morphology of the colonies, and SEM and TEM data confirmed that the isolates obtained could indeed be Rhodococci. All of the isolates could grow in bulk cultures with inorganic medium supplemented with crude oil. Moreover, all of the isolates degraded aliphatic hydrocarbons with lengths between C₁₁ and C₂₉. C₂₃-C₂₉ hydrocarbons were degraded completely. The isolates could grow at 4–37°C. The most unexpected finding was that of the many microorganisms capable of consuming oil, only Rhodococci exhibited this ability in the inactive bitumen mound. The possible mechanisms of how crude oil is transformed into bitumen mounds and mature bitumen are discussed.

Keywords:

• biodegradation
• bitumen
• Lake Baikal
• oil degradation
• Rhodococcus

Acknowledgments

We express our appreciation to Drs. O. N. Pavlova, I. V. Klimenkov, N. L. Belkova, Ms. E. V. Sukhanova (Limnological Institute Siberian Branch of Russian Academy of Science, Irkutsk), Dr. I. V. Morozov (the Joint DNA Sequencing Centre, Siberian Branch of Russian Academy of Science, Novosibirsk) for valuable comments and assistance and to T. V. Kashik (A. E. Favorsky Institute of Chemistry Siberian Branch of Russian Academy of Science, Irkutsk) and to M. Slipenchuk, Head of the METROPOL Group of Companies – R. Nigmatulin, Director of P. P. Shirshov Institute of Oceanology, A. Sagalevich, P. P. Shirshov Institute of Oceanology, and the crew of the “MIR” manned submersible. The work was supported by SB RAS Interdisciplinary Project No. 27, Russian Foundation for Basic Research No. 10-05-00681 and Russian President Grant MK-1901.2010.5.