Anaerobic biodegradation of hexadecane: Elucidation of activation mechanisms and identification of novel genes
Alkanes comprise a large component of petroleum. Biotransformation is the most important process governing their environmental fate and transport. A denitrifying, hexadecane-degrading consortium was established with Onondaga Lake, NY sediment. Phylogenetic analyses, based on DGGE and construction of clone libraries, revealed a diverse community of denitrifiers. Both methods identified an Azoarcus sp.. Protonated, d17- and [1-13C]-pentadecanoic acids and protonated, d24- and d25-tridecanoic acids were identified via GC-MS analysis. These data support a mechanism that involves carboxylation at the C-3 carbon, with subsequent C2-elimination. Hexadecane was mineralized as demonstrated by recovery of 14CO2 from cultures incubated with [1-14C]-hexadecane. This is the first report of carboxylation of an alkane under denitrifying conditions. Sulfate-reducing strain AK-O1 was studied to elucidate the mechanism of alkane degradation. We hypothesized that AK-O1 activates alkanes via subterminal fumarate addition. We surveyed metabolites of strains AK-O1, Hxd3, and a sulfate-reducing consortium via GC-MS analysis. Several metabolites were identified in extracts of AK-01 and the consortium that support subterminal fumarate addition. They included: methylpentadecylsuccinic acid, 4-methyloctadecanoic acid, 4-methyloctadec-2,3-enoic acid, 2-methy1hexadecanoic acid and tetradecanoic acid. We also report mass spectral evidence of a C-skeleton rearrangement of methylpentadecylsuccinic acid. Additionally, several metabolites were identified in the consortium that are consistent with the carboxylation mechanism of strain Hxd3. These results suggest that both fumarate addition and carboxylation are important mechanisms in anaerobic environments among phylogenetically and physiologically distinct genera. Little is known about the enzymes involved in anaerobic alkane degradation. We hypothesized that strain AK-01 possessed an enzyme similar to Bss based on mechanistic similarities. Degenerate bssA primers were designed based on known bssA sequences. Traditional and inverse PCR were used to amplify and sequence two genes, assAl and assA2, that are 80.9% identical to each other at the amino acid level and 35% identical to the closest bssA sequence in GenBank. SDS-PAGE analysis of protein extracts of AK-O1 grown on hexadecane versus sodium hexadecanoate suggests that assAl is expressed when AK-O1 is grown on hexadecane. This is the first report of a gene involved in anaerobic n-alkane degradation and provides evidence of a previously unknown class of glycyl radical enzymes.