Culture-Dependent and Culture-Independent Characterization of Microbial Assemblages Associated with High-Temperature Petroleum Reservoirs

Abstract

Recent investigations of oil reservoirs in a variety of locales have indicated that these habitats may harbor active thermophilic prokaryotic assemblages. In this study, we used both molecular and culture-based methods to characterize prokaryotic consortia associated with high-temperature, sulfur-rich oil reservoirs in California. Enrichment cultures designed for anaerobic thermophiles, both autotrophic and heterotrophic, were successful at temperatures ranging from 60 to 90°C. Heterotrophic enrichments from all sites yielded sheathed rods (Thermotogales), pleomorphic rods resembling Thermoanaerobacter, and Thermococcus-like isolates. The predominant autotrophic microorganisms recovered from inorganic enrichments using H 2 , acetate, and CO 2 as energy and carbon sources were methanogens, including isolates closely related to Methanobacterium, Methanococcus, and Methanoculleus species. Two 16S rRNA gene (rDNA) libraries were generated from total community DNA collected from production wellheads, using either archaeal or universal oligonucleotide primer sets. Sequence analysis of the universal library indicated that a large percentage of clones were highly similar to known bacterial and archaeal isolates recovered from similar habitats. Represented genera in rDNA clone libraries included Thermoanaerobacter, Thermococcus, Desulfothiovibrio, Aminobacterium, Acidaminococcus, Pseudomonas, Halomonas, Acinetobacter, Sphingomonas, Methylobacterium, and Desulfomicrobium. The archaeal library was dominated by methanogen-like rDNAs, with a lower percentage of clones belonging to the Thermococcales. Our results strongly support the hypothesis that sulfur-utilizing and methane-producing thermophilic microorganisms have a widespread distribution in oil reservoirs and the potential to actively participate in the biogeochemical transformation of carbon, hydrogen, and sulfur in situ.Over the past decade, microbiological investigations of high temperature, petroleum-rich strata from a number of geographically distant sites have revealed physiologically diverse assemblages of thermophilic and hyperthermophilic anaerobic microorganisms. Physiological types isolated from these biotopes include sulfate reducers (54, 58), sulfidogens (31, 54), fermentative bacteria (12, 22), manganese and iron reducers (23), methanogens (42, 52), and acetogens (13). Despite the description of an increasing number of new thermophilic species, relatively little information is available on the composition of microbial assemblages in these unique subsurface environments. This is primarily due to the reliance on cultured-based methods for the recovery and identification of individual oil field isolates and the focus on specific physiological groups of microorganisms, such as sulfate-reducing and fermentative microorganisms, rather than the entire subsurface microbial community.Culture-based approaches, while extremely useful for understanding the physiological potential of isolated organisms, do not necessarily provide comprehensive information on the comp...

Thermodynamic constraints on methanogenic crude oil biodegradation

“…However, several lines of evidence suggest that methanogenic CO 2 reduction, may be more prevalent in petroleum systems. The majority of methanogens identified from oil field waters are CO 2 -reducing methanogens, and acetoclastic methanogens are apparently rare (Magot et al, 2000;Orphan et al, 2000Orphan et al, , 2003Grabowski et al, 2005). Furthermore, experimental measurements of methanogenic pathways in oil field waters also suggest that CO 2 reduction to methane may be more important than acetoclastic methanogenesis in petroleum reservoirs.…”

Molecular and isotopic partitioning of low-molecular-weight hydrocarbons during migration and gas hydrate precipitation in deposits of a high-flux seepage site

“…Nonetheless, individual proportions of microbial and thermogenic LMWHC might be generated in different depths prior to mixing. Assuming a geothermal gradient of 0.03°C m − 1 and given that the maximum temperature tolerated by prokaryotes including methanogenic archaea is about 90°C (Orphan et al, 2000;Valentine et al, 2004), microbial methane is formed at burial depths of less than about 2.7 km bsf. In contrast, LMWHC from type II kerogens or oil cracking are typically generated at temperatures exceeding 100°C (Clayton, 1991) implying a minimum formation depth of 3.0 km bsf for the thermogenic LMWHC present in gases from the Batumi seep area.…”

Deferribacter desulfuricans sp. nov., a novel sulfur-, nitrate- and arsenate-reducing thermophile isolated from a deep-sea hydrothermal vent

“…Recent investigations on an iron-reducing microbial population in the EPR deep-sea hydrothermal vent environment suggested the abundant occurrence of a thermophilic iron-reducing microbial population, predominantly represented by members of Thermococcus and Deferribacter, in relatively oxidative microhabitats (Slobodkin et al, 2001). A body of evidence has demonstrated that sulfidogenic, thermophilic bacteria and archaea, mainly consisting of sulfur-reducing members of Thermotogales and Thermococcales, are predominantly present in global subsurface oil-reservoir environments other than marine hydrothermal systems (L'Haridon et al, 1995;Orphan et al, 2000;Slobodkin et al, 1999;Stetter et al, 1993;Takahata et al, 2000). In addition to members of Thermotogales and Thermococcales, sulfur-reducing and/or metal-reducing members of Deferribacter such as D. desulfuricans SSM1 T and D. thermophilus BMA T may be widely distributed in global deep-sea hydrothermal systems and subsurface oil-reservoir environments and may represent significant microbial components.…”