Simultaneous detection of transcribed functional assA gene and the corresponding metabolites of linear alkanes (C4, C5, and C7) in production water of a low-temperature oil reservoir.

Methanogenic hydrocarbon degradation is an important biogeochemical process in oil reservoirs; however, genomic DNA-based analysis of microorganisms and metabolite detection are not conclusive for identification of the ongoing nature of this bioprocess. In this study, a suite of analyses, involving the study of microbial community and selective gene quantification of both genomic DNA and RNA together with signature metabolites, were performed to comprehensively advance the understanding of the methanogenic biodegradation of hydrocarbons in a low-temperature oilfield. The fumarate addition products for alkanes-C4, C5, and C7-alkylsuccinates-and transcribed assA and mcrA genes were simultaneously detected in the production water sample, providing robust and convincing evidence for both the initial activation of n-alkanes and methane metabolism in this oilfield. The clone library of assA gene transcripts showed that Smithella was active and most likely responsible for the addition of fumarate to n-alkanes, whereas Methanoculleus and Methanothrix were the dominant and active methane-producers via CO2 reduction and acetoclastic pathways, respectively. Additionally, qPCR results of assA and mcrA genes and their transcribed gene copy numbers revealed a roughly similar transcriptional activity in both n-alkanes-degraders and methane producers, implying that they were the major participants in the methanogenic degradation of n-alkanes in this oilfield. To the best of our knowledge, this is the first report presenting sufficient speculation, through detection of signature intermediates, corresponding gene quantification at transcriptional levels, and microbial community analysis, of methanogenic degradation of n-alkanes in production water of an oil reservoir.

Synthesis and mass spectra of rearrangement bio-signature metabolites of anaerobic alkane degradation via fumarate addition.

Metabolite profiling in anaerobic alkane biodegradation plays an important role in revealing activation mechanisms. Apart from alkylsuccinates, which are considered to be the usual biomarkers via fumarate addition, the downstream metabolites of C-skeleton rearrangement can also be regarded as biomarkers. However, it is difficult to detect intermediate metabolites in both environmental samples and enrichment cultures, resulting in lacking direct evidence to prove the occurrence of fumarate addition pathway. In this work, a synthetic method of rearrangement metabolites was established. Four compounds, namely, propylmalonic acid, 2-(2-methylbutyl)malonic acid, 2-(2-methylpentyl)malonic acid and 2-(2-methyloctyl)malonic acid, were synthesized and determined by four derivatization approaches. Besides, their mass spectra were obtained. Four characteristic ions were observed at m/z 133 + 14n, 160 + 28n, 173 + 28n and [M - (45 + 14n)]+ (n = 0 and 2 for ethyl and n-butyl esters, respectively). For methyl esterification, mass spectral features were m/z 132, 145 and [M - 31]+, while for silylation, fragments were m/z 73, 147, 217, 248, 261 and [M - 15]+. These data provide basis on identification of potential rearrangement metabolites in anaerobic alkane biodegradation via fumarate addition.

Long-chain n-alkane biodegradation coupling to methane production in an enriched culture from production water of a high-temperature oil reservoir.

Paraffinic n-alkanes (C22-C30), crucial portions of residual oil, are generally considered to be difficult to be biodegraded owing to their general solidity at ambient temperatures and low water solubility, rendering relatively little known about metabolic processes in different methanogenic hydrocarbon-contaminated environments. Here, we established a methanogenic C22-C30 n-alkane-degrading enrichment culture derived from a high-temperature oil reservoir production water. During two-year incubation (736 days), unexpectedly significant methane production was observed. The measured maximum methane yield rate (164.40 µmol L-1 d-1) occurred during the incubation period from day 351 to 513. The nearly complete consumption (> 97%) of paraffinic n-alkanes and the detection of dicarboxylic acids in n-alkane-amended cultures indicated the biotransformation of paraffin to methane under anoxic condition. 16S rRNA gene analysis suggested that the dominant methanogen in n-alkane-degrading cultures shifted from Methanothermobacter on day 322 to Thermoplasmatales on day 736. Bacterial community analysis based on high-throughput sequencing revealed that members of Proteobacteria and Firmicutes exhibiting predominant in control cultures, while microorganisms affiliated with Actinobacteria turned into the most dominant phylum in n-alkane-dependent cultures. Additionally, the relative abundance of mcrA gene based on genomic DNA significantly increased over the incubation time, suggesting an important role of methanogens in these consortia. This work extends our understanding of methanogenic paraffinic n-alkanes conversion and has biotechnological implications for microbial enhanced recovery of residual hydrocarbons and effective bioremediation of hydrocarbon-containing biospheres.

Genomic and Transcriptomic Evidence Supports Methane Metabolism in Archaeoglobi.

Euryarchaeal lineages have been believed to have a methanogenic last common ancestor. However, members of euryarchaeal Archaeoglobi have long been considered nonmethanogenic and their evolutionary history remains elusive. Here, three high-quality metagenomic-assembled genomes (MAGs) retrieved from high-temperature oil reservoir and hot springs, together with three newly assembled Archaeoglobi MAGs from previously reported hot spring metagenomes, are demonstrated to represent a novel genus of Archaeoglobaceae, "Candidatus Methanomixophus." All "Ca Methanomixophus" MAGs encode an M methyltransferase (MTR) complex and a traditional type of methyl-coenzyme M reductase (MCR) complex, which is different from the divergent MCR complexes found in "Ca Polytropus marinifundus." In addition, "Ca Methanomixophus dualitatem" MAGs preserve the genomic capacity for dissimilatory sulfate reduction. Comparative phylogenetic analysis supports a laterally transferred origin for an MCR complex and vertical heritage of the MTR complex in this lineage. Metatranscriptomic analysis revealed concomitant in situ activity of hydrogen-dependent methylotrophic methanogenesis and heterotrophic fermentation within populations of "Ca Methanomixophus hydrogenotrophicum" in a high-temperature oil reservoir.IMPORTANCE Current understanding of the diversity, biology, and ecology of Archaea is very limited, especially considering how few of the known phyla have been cultured or genomically explored. The reconstruction of "Ca Methanomixophus" MAGs not only expands the known range of metabolic versatility of the members of Archaeoglobi but also suggests that the phylogenetic distribution of MCR and MTR complexes is even wider than previously anticipated.