Microbial communities in two production waters of a low-temperature and low-salinity petroleum reservoir in Canada were examined using cultural and molecular approaches. The predominant cultivated microorganisms were homoacetogens but sulfate-reducers, acetoclastic methanogens and denitrifiers also gave significant counts. The dominant members of the culturable population were affiliated with the Firmicutes, the "Deltaproteobacteria", the "Epsilonproteobacteria", the Spirochaetes and the Euryarchaeota. 16S rRNA gene clone libraries were also constructed from the total DNA collected from production waters. The bacterial library was entirely composed by a single phylotype related to Arcobacter. The archaeal phylotypes were generally very closely related to members of the orders Methanosarcinales and Methanomicrobiales. Consistent with earlier observations, our data suggest that methanogenesis is a dominant terminal process in the reservoir. Moreover, the cross-evaluation of culture-dependent and -independent techniques also indicates that, contrary to most studies, both acetoclastic and lithotrophic methanogens may be involved in this process. This first investigation of the microbial diversity in a non water-flooded low-temperature and low-salinity petroleum reservoir expands substantially our knowledge of the extent of microbial diversity and highlights the complexity of microbial communities involved in the oil field food chain.

A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co 2؉ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE-or TBAgrown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE-or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE-and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.Methyl tert-butyl ether (MTBE) has been incorporated in reformulated gasoline at concentrations up to 15% (vol/vol) to replace lead tetraethyl in order to comply with the octane index and to reduce the polluting emissions in exhaust gases. Other oxygenates, such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) and their corresponding alcohols, tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA), can play the same role regarding the octane index. MTBE is the dominant fuel oxygenate (38), with a worldwide production capacity of around 25 million tons. Because of its widespread use and the high frequency of underground tank leakage (35), this compound is now the second most commonly detected contaminant in urban groundwater in the United States (23, 42). In Europe, detectable levels of MTBE in rivers have also been reported (1).The persistence of MTBE in the environment can be ascribed, on the one hand, to its physicochemical properties (i.e., its low adsorption on organic matter and its high solubility in water) and, on the other hand, to its molecular structure (it has both an ether bond and high steric hindrance, which makes it recalcitrant to microbial degradation).Early reports based on microcosms having different origins mentioned the recalcitrance of MTBE and TAME (22) MTBE cometabolism was demonstrated by using propanegrown bacteria (35), an n-butane-grown fungus (17), camphorgrown Pseudomonas putida CAM (36), pentane-grown Pseudomonas aeruginosa (14), ETBE-grown Rhodococcus ruber IFP 2007 (...

Rhodococcus ruber (formerly