© 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved. LETTERS Community dynamics of anaerobic bacteria in deep petroleum reservoirs CHRISTIAN HALLMANN 1,2 *, LORENZ SCHWARK 1 AND KLITI GRICE 2 1 Department of Geology and Mineralogy, University of Cologne, Zuelpicher Strasse 49a, 50674 Cologne, Germany 2 Western Australia Organic and Isotope Geochemistry Centre, The Institute for Geoscience Research, Curtin University of Technology, GPO Box U1987, Perth, Western Australia 6845, Australia *e-mail: C.Hallmann@curtin.edu.au; Hallmann.C@gmx.net Published online: 10 August 2008; doi:10.1038/ngeo260 The nature, activity and metabolism of microbes that inhabit the deep subsurface environment are a matter of ongoing debate 1–7 . Primarily limited by temperature 8 , little is known about secondary factors that restrict or enhance microbial activity 9,10 or about the extent of a habitable environment deep below the surface. In particular, the degraders of chemically inert organic substrates remain elusive 9 . Petroleum reservoirs can be regarded as natural bioreactors and are ideally suited for the study of microbial metabolism in the deep subsurface. Here we analyse series of oil samples that were biodegraded to different degrees. We find fatty acids after hydrolysis of purified crude oil fractions, indicating the presence of intact phospholipids and suggesting that indigenous bacteria inhabit petroleum reservoirs in sediment depths of up to 2,000 m. A major change in bacterial community structure occurs after the removal of n-alkanes, indicating that more than one consortium is responsible for petroleum degradation 11 . Our results suggest that further study of petroleum fluids will help understand bacterial metabolism and diversity in this habitat of the deep subsurface. The study of a deep-subsurface microbiosphere has become a focus of intense research 1–6,12 . Microbial habitats have been shown to extend to large depths beneath the Earth’s surface by the presence of live bacterial cells 1 . At the same time, new extremes in the physicochemical boundaries for life are continually being reported 13 . Although cell counts rapidly decrease in sub-sea-floor sediments, small numbers of prokaryotes can persist for long periods by sporulation 14 until continuous sedimentary subsidence transports them across the temperature limit for life 8 . Shallow sediments are rapidly depleted in nutrients and labile organic compounds, forcing microbes to slow down metabolism 2 . At greater depths, microbes capable of catabolizing macromolecular organic compounds produce volatile fatty acids and molecular hydrogen, which provides energy for themselves and other bacteria 3 . Such low-molecular-weight substrates are capable of sustaining the sulphate-reducing bacteria (SRB) and acetoclastic/hydrogenotrophic methanogenic Archaea commonly found in deep sediments 9 . When petroleum is released from organic-rich rocks at greater depth and migrates into the habitable range of a sedimentary column, it represents a more abundant growth substrate for microorganisms than macromolecular organic matter and volatile fatty acids. The occurrence of biodegraded shallow petroleum accumulations 9 provides evidence that under certain circumstances this oil is used as a microbial growth substrate. Although microbial life in sub-sea-floor sediments has recently received much attention, little is known about deep-subsurface prokaryotic behaviour in the presence of oil 15 . The modes of petroleum degradation in deep reservoirs can yield useful information on the catabolic behaviour of microbes in other subsurface environments, where little substrate and low cell numbers preclude detailed analyses. We investigated microbial dynamics in petroleum reservoirs through the analysis of bacterial fatty acids indicative of intact phospholipids (phospholipid fatty acids, PLFA) and thus microbial cells, in oils. The relative amount and distribution of selected PLFA sheds light on microbial populations inhabiting deep-subsurface petroleum reservoirs and their dynamics during the progressive biodegradation of oil. Series of oil samples from three basins were analysed. Each basin contains biodegraded oils in shallow reservoirs and pristine oils in deeper reservoirs, where elevated temperatures inhibit microbial life 8 . The genetic relationship of biodegraded–pristine oil couples was verified using the distribution of triterpenoids and biodegradation-resistant aromatic steroids 16 , thereby attesting changes in oils to be caused by microbial action and not by initial variations in chemical composition. The extent of biodegradation was determined by assessing the quasi-stepwise disappearance of compound classes. On the basis hereof, oils were categorized on a scale from 0 to 10 as proposed by Peters and Moldowan (PM) 11 . Investigating microbes in oils by culturing methods carries the risk of a bias, because subsurface conditions cannot be effectively reproduced in the laboratory 9 . Molecular methods that involve complementary base pairing of oligonucleotides to DNA or RNA (such as polymerase chain reaction or fluorescence in situ hybridization) have been conducted on oil-associated waters but are generally inhibited in the presence of large amounts of polar organic matter 17 . Although these methods consequently have severe limitations when applied to oils, the analysis of oil-associated waters carries a high risk of contamination from drilling fluids 10 . We approached the question of microbial activity by searching for membrane lipid fragments from intact cells and here present results that indicate the presence of indigenous PLFA in crude oils from deep reservoirs. Phospholipids are the principal building blocks in the cell membrane of all prokaryotes and eukaryotes 18 . They are not preserved but turned over rapidly during metabolism 19 and decompose outside the cell. Phospholipids are therefore molecular indicators of live cells 20 and proxies for their biomass. In the laboratory, fatty-acid tails can be chemically released from the phospholipid parent molecule 588 nature geoscience VOL 1 SEPTEMBER 2008 www.nature.com/naturegeoscience