Metagenomic analysis of an anaerobic alkane-degrading microbial culture: potential hydrocarbon-activating pathways and inferred roles of community members

Abstract

A microbial community (short-chain alkane-degrading culture, SCADC) enriched from an oil sands tailings pond was shown to degrade C6-C10 alkanes under methanogenic conditions. Total genomic DNA from SCADC was subjected to 454 pyrosequencing, Illumina paired-end sequencing, and 16S rRNA amplicon pyrotag sequencing; the latter revealed 320 operational taxonomic units at 5% distance. Metagenomic sequences were subjected to in-house quality control and co-assembly, yielding 984 086 contigs, and annotation using MG-Rast and IMG. Substantial nucleotide and protein recruitment to Methanosaeta concilii, Syntrophus aciditrophicus, and Desulfobulbus propionicus reference genomes suggested the presence of closely related strains in SCADC; other genomes were not well mapped, reflecting the paucity of suitable reference sequences for such communities. Nonetheless, we detected numerous homologues of putative hydrocarbon succinate synthase genes (e.g., assA, bssA, and nmsA) implicated in anaerobic hydrocarbon degradation, suggesting the ability of the SCADC microbial community to initiate methanogenic alkane degradation by addition to fumarate. Annotation of a large contig revealed analogues of the ass operon 1 in the alkane-degrading sulphate-reducing bacterium Desulfatibacillum alkenivorans AK-01. Despite being enriched under methanogenic-fermentative conditions, additional metabolic functions inferred by COG profiling indicated multiple CO(2) fixation pathways, organic acid utilization, hydrogenase activity, and sulphate reduction.

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

“…These cultures were maintained on the same substrates since their initial enrichment. Some metabolic features of the TOLDC and SCADC cultures have been reported (Fowler et al, 2012;Tan et al, 2013), while NAPDC is newly described here. NAPDC and SCADC were enriched (see Supplementary Information) from methanogenic mature fine tailings collected from Mildred Lake Settling Basin (MLSB), an oil sands tailings pond in Alberta, Canada that contains diverse low and high molecular weight hydrocarbons from unrecovered bitumen and naphtha (Siddique et al, 2006).…”

“…64742-49-0), a petroleum product comprising monoaromatic hydrocarbons and C 6 -C 10 alkanes that is present in many oil sands tailings ponds; NAPDC cultivation methods are provided in Supplementary Information. SCADC was enriched on 0.1% v/v C 6 -C 10 n-alkanes with minor proportions of 2-methylpentane, 3-methylpentane and methylcyclopentane, as described by Tan et al (2013). TOLDC was established from aquifer sediments contaminated with gas condensate (containing predominantly C 5 -C 15 hydrocarbons, Gieg et al, 1999) that was enriched solely on 0.01% (v/v) toluene (Fowler et al, 2012).…”

“…DNA extraction for 16S rRNA gene pyrotag sequencing and metagenomic sequencing DNA extraction from TOLDC and SCADC for amplification and pyrotag sequencing of 16S rRNA gene amplicons has been described (Fowler et al, 2012;Tan et al, 2013). Total DNA from NAPDC used for 16S rRNA gene pyrosequencing was extracted and PCR amplified performed as described by Foght et al (2004) and Tan et al (2013).…”

“…Total DNA from NAPDC used for 16S rRNA gene pyrosequencing was extracted and PCR amplified performed as described by Foght et al (2004) and Tan et al (2013). All 16S rRNA gene pyrotag sequencing reads were generated using the same 'universal' primer set, 926 F and 1392 R, targeting the V6-V8 regions of the 16S rRNA gene of bacteria and archaea .…”

“…All metagenomic 454 pyrosequencing reads were subjected to quality control and de novo assembly using a Newbler v2.6-based pipeline developed inhouse, as previously described (Tan et al, 2013). All quality-controlled metagenomic reads were submitted to MG-RAST for automated gene calling and annotation.…”