Michel Magot

Microbiology of petroleum reservoirs.

Although the importance of bacterial activities in oil reservoirs was recognized a long time ago, our knowledge of the nature and diversity of bacteria growing in these ecosystems is still poor, and their metabolic activities in situ largely ignored. This paper reviews our current knowledge about these bacteria and emphasises the importance of the petrochemical and geochemical characteristics in understanding their presence in such environments.

Thermotoga elfii sp. nov., a Novel Thermophilic Bacterium from an African Oil-producing Well

A thermophilic, glucose-fermenting, strictly anaerobic, rod-shaped bacterium, strain SEBR 6459T (T = type strain), was isolated from an African oil-producing well. This organism was identified as a member of the genus Thermotoga on the basis of the presence of the typical outer sheath-like structure (toga) and 16S rRNA signature sequences and its ability to grow on carbohydrates (glucose, arabinose, fructose, lactose, maltose, and xylose). Major differences in its 16S rRNA gene sequence, its lower optimum temperature for growth (66 degrees C), its sodium chloride range for growth (0 to 2.8%), its lack of lactate as an end product from glucose fermentation, and its peritrichous flagella indicate that strain SEBR 6459T is not similar to the three previously described Thermotoga species. Furthermore, this organism does not belong to any of the other genera related to the order Thermotogales that have been described. On the basis of these findings, we propose that this strain should be described as a new species, Thermotoga elfii. The type strain of T. elfii is SEBR 6459 (= DSM 9442).

Fusibacter paucivorans gen. nov., sp. nov., an anaerobic, thiosulfate-reducing bacterium from an oil-producing well

A strictly anaerobic, halotolerant, spindle-shaped rod, designated strain SEBR 4211T, was isolated from an African saline oil-producing well. Cells stain Gram-positive, which was confirmed by electron microscopy observations. Strain SEBR 4211T was motile by means of one to four peritrichous flagella, had a G+C content of 43 mol% and grew optimally at 37 degrees C, pH 7.3, with 0 to 3% (w/v) NaCl. It utilized a limited number of carbohydrates (cellobiose, glucose, fructose, mannitol and ribose) and produced acetate, butyrate, CO2 and H2 as end products from glucose fermentation. It reduced thiosulfate to sulfide. In the presence of thiosulfate, a decrease in butyrate and an increase in acetate production was observed. Phylogenetically, strain SEBR 4211T was related to members of the low G+C Clostridiales order with Clostridium halophilum as the closest relative (16S rDNA sequence similarity of 90%). On the basis of phenotypic, genotypic and phylogenetic characteristics of the isolate, it is proposed to designate it as a new species of a new genus, Fusibacter gen. nov., as Fusibacter paucivorans sp. nov. The type strain is SEBR 4211T (= DSM 12116T).

Methanocalculus halotolerans gen. nov., sp. nov., isolated from an oil-producing well

Two irregular coccoid methanogens designated SEBR 4845T and FR1T were isolated from an oilfield in Alsace, France. Strain SEBR 4845T (T = type strain) is a hydrogenotrophic halotolerant methanogen, which grows optimally at 5% NaCI (w/v) and tolerates up to 12% NaCI. It does not use methylated compounds and therefore cannot be ascribed to any of the known genera of the halophilic methylotrophic methanogens. It differs from hydrogenotrophic members of the orders Methanococcales and Methanomicrobia les in the NaCI growth range (0-12% NaCI), which is the widest reported to data for any hydrogenotrophic methanogen. 16S rRNA gene sequence analysis indicated that strain SEBR 4845T is a novel isolate for which a new genus is proposed, Methanocalculus halotolerans gen. nov., sp. nov. (= OCM470T) that might be indigenous to the oilfield ecosystem. Strain FR1T (=OCM 471) is a moderately halophilic methanogen which growths optimally at 10% NaCI and tolerates up to 20% NaCI. It grows on trimethylamine and methanol as carbon and energy sources. The G+C content of its DNA is 43 mol%. It is therefore phenotypically and genotypically related to members of the genus Methanohalophilus. This report provides evidence that methylotrophic and hydrogenotrophic, but not aceticlastic methanogens are present in a saline subsurface oilfield environment, as already observed in surface saline to hypersaline environments.

Thermoanaerobacter subterraneus sp. nov., a novel thermophile isolated from oilfield water.

A new thermophilic, anaerobic glucose-fermenting, Gram-positive, rod-shaped bacterium, designated strain SEBR 7858T, was isolated from an oilfield water sample. Under optimal conditions on a glucose-containing medium (3% NaCl, 65 degrees C and pH 7.5), the generation time was 2.5 h. No growth occurred at 35 or 80 degrees C, nor at pH 5..5 or 9.0. Strain SEBR 7858T possessed lateral flagella. Spores were undetected but heat-resistant forms were present. Strain SEBR 7858T fermented a range of carbohydrates to acetate, L-alanine, lactate, H2 and CO2. The isolate reduced thiosulfate and elemental sulfur, but not sulfate or sulfite to sulfide. In the presence of thiosulfate, the ratio of acetate produced per mole of glucose consumed increased, suggesting a shift in the use of electron acceptors during carbohydrate metabolism. The DNA G+C content was 41 mol%. Based on 16S rRNA gene sequence analysis, the strain was almost equidistantly related to all members of the genus Thermoanaerobacter (mean similarity 92%). Based on phenotypic, genomic and phylogenetic characteristics, strain SEBR 7858T was clearly different from all members of the genus Thermoanaerobacter and was therefore designated as a new species, Thermoanaerobacter subterraneus sp. nov. The type strain is SEBR 7858T (= CNCM 1-2383T, DSM 13054T).

Characterization by culture and molecular analysis of the microbial diversity of a deep subsurface gas storage aquifer.

The bacterial diversity of a subsurface water sample collected from a gas storage aquifer in an Upper Jurassic calcareous formation was investigated by culture of microorganisms and construction of a 16S rRNA gene library. Both culture and molecular techniques showed that members of the phyla Firmicutes and class delta-proteobacteria dominated the bacterial community. The presence of hydrogen-utilizing autotrophic bacteria including sulfate reducers (e.g. Desulfovibrio aespoeensis) and homoacetogens (e.g. Acetobacterium carbinolicum) suggested that CO(2) and H(2) are the main carbon and energy sources sustaining a nutrient-limited subsurface lithoautotrophic microbial ecosystem (SLiME). Gram-positive SRB belonging to the genus Desulfotomaculum, frequently observed in subsurface environments, represented 25% of the clone library and 4 distinct phylotypes. No Archaea were detected by both experimental approaches. Water samples were collected in an area of the rauracian geological formation located outside the maximum seasonal extension of underground gas storage. Considering the observed microbial diversity, there is no evidence of any influence on the microbial ecology of the aquifer in the surroundings of maximum extension reached by the gas bubble of the underground storage, which should have resulted from the introduction of exogenous carbon and energy sources in a nutrient-limited ecosystem.

Desulfotomaculum spp. and related gram-positive sulfate-reducing bacteria in deep subsurface environments.

Gram-positive spore-forming sulfate reducers and particularly members of the genus Desulfotomaculum are commonly found in the subsurface biosphere by culture based and molecular approaches. Due to their metabolic versatility and their ability to persist as endospores. Desulfotomaculum spp. are well-adapted for colonizing environments through a slow sedimentation process. Because of their ability to grow autotrophically (H2/CO2) and produce sulfide or acetate, these microorganisms may play key roles in deep lithoautotrophic microbial communities. Available data about Desulfotomaculum spp. and related species from studies carried out from deep freshwater lakes, marine sediments, oligotrophic and organic rich deep geological settings are discussed in this review.

Hydrogen oxidation abilities in the presence of thiosulfate as electron acceptor within the genusThermoanaerobacter

Thermoanaerobacter (T.) brockii, T. ethanolicus, andT. thermohydrosulfuricus were tested for their capacities to oxidize H2 in the presence of thiosulfate.T. brockii oxidized H2 actively, whileT. ethanolicus andT. thermohydrosulfuricus oxidized it poorly. At the end of the exponential growth, H2 was oxidized byT. brockii in the presence of an energy source and thiosulfate. This oxidative process improved the growth ofT. brockii. Thermoanaerobacter species could be divided into two groups with regard to their H2 metabolism in the presence of thiosulfate. Thiosulfate reduction by species of the genusThermoanaerobacter is of significance in mineralizing organic matter in thermophilic environments.

Emended description of Thermosipho africanus as a carbohydrate-fermenting species using thiosulfate as an electron acceptor

We found that Thermosipho africanus was able to ferment D-glucose, D-ribose, Maltose, and starch, while D-galactose, fructose, and sucrose were utilized poorly. Acetate, H2, and CO2, as well as small amounts of ethanol and lactate, were end products of glucose metabolism in this organism. The presence of thiosulfate as an electron acceptor greatly improved growth and increased acetate production from the sugars. The genus Thermosipho is the only genus in the order Thermotogales that has been described as a non-carbohydrate fermenter. We propose that the description of the genus Thermosipho be emended because the only species in this genus, T. africanus, is a carbohydrate fermenter that is able to utilize thiosulfate as an electron acceptor.

Desulfocurvus vexinensis gen. nov., sp. nov., a sulfate-reducing bacterium isolated from a deep subsurface aquifer.

A novel anaerobic, chemo-organotrophic bacterium, designated VNs36(T), was isolated from a well that collected water from a deep saline aquifer used for underground gas storage at a depth of 830 m in the Paris Basin, France. Cells were curved motile rods or vibrios (3.0-5.0x0.5 microm). Strain VNs36(T) grew at temperatures between 20 and 50 degrees C (optimum 37 degrees C) and at pH values between 5.0 and 9.0 (optimum 6.9). It did not require salt for growth, but tolerated up to 20 g NaCl l(-1) (optimum 2 g l(-1)). In the presence of sulfate, strain VNs36(T) used lactate, formate and pyruvate as carbon and energy sources. The main fermentation products from lactate were acetate, H(2) and CO(2). Sulfate, thiosulfate and sulfite were used as electron acceptors, but not sulfur. The genomic DNA G+C content of strain VNs36(T) was 67.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain VNs36(T) was affiliated with the family Desulfovibrionaceae within the class Deltaproteobacteria. On the basis of 16S rRNA gene sequence comparisons, DNA G+C content and the absence of desulfoviridin in cell extracts, it is proposed that strain VNs36(T) be assigned to a new genus, Desulfocurvus gen. nov., as a representative of a novel species, Desulfocurvus vexinensis sp. nov. The type species of this genus is Desulfocurvus vexinensis with the type strain VNs36(T) (=DSM 17965(T)=JCM 14038(T)).