Anaerobic biodegradation of alkanes by sulfate-reducing bacteria: Characterization of a novel isolate and comparison of two degradation mechanisms

The recalcitrance of alkanes under anoxic conditions and our limited knowledge of anaerobic biodegradation of these compounds prompted the present study. Enrichments under denitrifying, sulfate-reducing, iron-reducing and methanogenic conditions were established by using alkanes (C8 to C16 as the substrates and a petroleum-contaminated estuarine sediment as the inoculum. Alkane was mineralized by the denitrifying and sulfate-reducing consortia as shown by the recovery of 14CO2 formed from [1-14C]hexadecane. Stoichiometric studies with these consortia indicate that degradation is coupled to denitrification and sulfate-reduction.;A pure culture of an alkane-utilizing bacterium was isolated from the sulfate-reducing consortium. The strain, named AK-01, is a Gram-negative, non-motile rod. It utilizes C13--C18 alkanes for growth and mineralizes them to CO2 by coupling to sulfate reduction. Phenotypic and phylogenetic characteristics of AK-01 indicate that it belongs to the delta subdivision of Proteobacteria and is most closely related to the genus Desulfosarcina.;The mechanism of the initial attack on alkane by strain AK-01 was studied. The bacterium transforms alkanes to 2-, 4- and 6-methylated fatty acids as shown by the presence of stable isotope labels on these fatty acids when perdeuterated pentadecane and [1,2-13C2]hexadecane were used as the substrates. Detailed analyses of these methylated fatty acids by mass spectrometry shows that their methyl groups are either 13C- or deuterium-labeled and thus originated from the terminal carbon of the respective labeled alkanes. The results suggest that AK-01 anaerobically transforms alkane to fatty acid with subterminal addition of exogenous carbon to the hydrocarbon chain at the C2 position as an initial reaction.;Strain Hxd3, a previously reported alkane-degrading sulfate-reducer, metabolizes alkanes with different initial reactions. Transformation of C-odd alkanes to C-even fatty acids and vice versa was confirmed with the use of stable isotope-labeled alkanes. When Hxd3 was cultured on unlabeled hexadecane in [13C]bicarbonate, the derived 15:0 fatty acid incorporated a 13C carbon to form its carboxyl group, suggesting that the initial attack involves a one-carbon addition to the hydrocarbon chain by carboxylation and an elimination of two terminal carbons from the alkane. The observation that the [1,2-13C2]hexadecane-derived fatty acids contained two 13C labels located exclusively at their acyl chain terminals but not the carboxyl ends further evidence the removal of two terminal carbons from the alkane. The results suggest that the initial attack involves subterminal carboxylation of the alkyl chain at C3 with concomitant removal of the two adjacent terminal carbons.