The Bioinformatic Analysis Of Unclutured Syntrophic Alkane-degrading Bacteria ’ Candidatus Smithella Cisternae’ Based On Metagenomic

It’s one of promising technologies for converting hard-to-use crude oil in petroleum reservoirs into methane using anaerobic microcosms. Syntrophic metabolism is an essential step in methanogenic degradation of petroleum hydrocarbon. We enriched a methanogenic hexadecane-degrading culture M82, and found a novel uncultured bacterial phylotypes of Family Syntrophaceae that plays a key role in hexadecane degradation. However, the pure culture of syntrophic hydrocarbon-degrading bacteria has not been isolated and characterized, which not only hampers the research of the physiological and biochemical properties of the syntrophic bacteria, but also hinders studies on the molecular mechanism of syntrophic hydrocarbon degradation.In this study, we characterized the microbial diversity of the methanogenic alkane-degrading consortium M82 using 454-pyrosequencing of the 16 S rRNA gene. The dominant species in the archaeal domain mainly belong to Methanoculleus（82%）, and uncultured Smithella（55%） dominated in the bacterial domain. A total of 40 Gb of sequence data was constructed from this consortium M82 using Illumina HiSeq platforms, which yielded 144,759 scaffolds with N50 size of 4,619 bp. Scaffold sequences with high coverage（>3000） and an average G + C content of 43% were binned into one cluster. Phylogenetic analysis revealed that the 16 S r RNA gene shared nearly identical sequence similarity（99.7%） to Clone HB1₁1（GenBank No. of JX088314）, which representing a key player hexadecane degradation in the methanogenic consortium M82 by DNA-based stable isotope probing. Low quality sequences（e.g representing low abundant scaffolds or belonging to archaeal domain） were removed after reassembly of reads in the primary bin. a total of unique 104 single-copy genes has been identified in this bin, indicating over 97% of the genome completeness. Principal component analysis（PCA） of tetranucleotide frequency of the scaffolds also confirmed the integrity of the assembled genome.The draft genome of this bin has a total size of 2,75 Mb with a GC content of 42.8%, it was proposed a novel species of ‘Candidatus Smithella cisternae’ and named the strain M82₁.It harbors 2815 ORFs, 47 tRNA and 3 rRNA, and over 50% of ORFs could be matched in the database of KEGG, COG, GO and Pfam. ‘Candidatus S. cisternae’ harbors three sets of genes encoding for alkylsuccinate synthase, methylmalonyl-Co A mutase and methylmalonyl-Co A decarboxylase, and contains multiple genes for β-oxidation of fatty acids including butyrate, indicating that alkane degradation was activated through fumarate addition. Incomplete oxidation pathways of propionate and acetate were also detected. it has an incomplete set of genes related to TCA cycle, EMP pathway and pentose phosphate pathway, which indicated the narrow substrate utilization range. The detection of nif genes indicates the nitrogen fixation potential of this uncultured strain. We obtained the draft genome of uncultured syntrophic alkane degraders, and characterized the genetic potential. These findings provide valuable insight into syntrophic alkane metabolism and give clues to the directed isolation of the syntrophic alkane-degrading strain.