Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and 13 C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and 13 C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2-13 C 2 ]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [ 13 C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a 13 C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2-13 C 2 ]hexadecane-derived fatty acids contained either two 13 C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, was isolated from an estuarine sediment with a history of chronic petroleum contamination. The bacterium is a short, nonmotile, non-spore-forming, gram-negative rod. It is mesophilic and grows optimally at pH 6.9 to 7.0 and at an NaCl concentration of 1%. Formate, fatty acids (C4 to C16) and hydrogen were readily utilized as electron donors. Sulfate, sulfite, and thiosulfate were used as electron acceptors, but sulfur, nitrite, and nitrate were not. Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequence indicate that AK-01 is most closely related to the generaDesulfosarcina, Desulfonema, andDesulfococcus in the delta subdivision of the classProteobacteria. It is phenotypically and phylogenetically different from strains Hxd3 and TD3, two previously reported isolates of alkane-degrading, sulfate-reducing bacteria. The alkanes tested to support growth of AK-01 had chain lengths of C13 to C18. 1-Alkenes (C15 and C16) and 1-alkanols (C15 and C16) also supported growth. The doubling time for growth on hexadecane was 3 days, about four times longer than that for growth on hexadecanoate. Mineralization of hexadecane was indicated by the recovery of14CO2 from cultures grown on [1-14C]hexadecane. Degradation of hexadecane was dependent on sulfate reduction. The stoichiometric ratio (as moles of sulfate reduced per mole of hexadecane degraded) was 10.6, which is very close to the theoretical ratio of 12.25, assuming a complete oxidation to CO2. Anaerobic alkane degradation by sulfate reducers may be a more widespread phenomenon than was previously thought.

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-13C2]hexadecane or perdeuterated pentadecane was used as the growth substrate, 13C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the 13C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two 13C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1,2-13C2]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.

Enrichments of alkane-degrading microorganisms were established under sulfate-reducing, denitrifying, iron-reducing, and methanogenic conditions using an estuarine sediment. The sulfate-reducing and denitrifying enrichments mineralized [1-14C]hexadecane to 14CO2. Degradation was coupled to sulfate reduction and denitrification, as indicated by the ratios of hexadecane degraded to the electron acceptors reduced. Dependence of hexadecane degradation by the sulfate-reducing enrichments on sulfate reduction was also shown. The results indicate the existence of diverse microbial communities capable of alkane degradation in the sediment studied.

Enrichments of alkane-degrading microorganisms were established under sulfate-reducing, denitrifying, iron-reducing, and methanogenic conditions using an estuarine sediment. The sulfate-reducing and denitrifying enrichments mineralized [1-14C]hexadecane to 14CO2. Degradation was coupled to sulfate reduction and denitrification, as indicated by the ratios of hexadecane degraded to the electron acceptors reduced. Dependence of hexadecane degradation by the sulfate-reducing enrichments on sulfate reduction was also shown. The results indicate the existence of diverse microbial communities capable of alkane degradation in the sediment studied.