Progressive Degradation of Crude Oil <i>n</i>-Alkanes Coupled to Methane Production under Mesophilic and Thermophilic Conditions

<div><p>Although methanogenic degradation of hydrocarbons has become a well-known process, little is known about which crude oil tend to be degraded at different temperatures and how the microbial community is responded. In this study, we assessed the methanogenic crude oil degradation capacity of oily sludge microbes enriched from the Shengli oilfield under mesophilic and thermophilic conditions. The microbial communities were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes combined with cloning and sequencing. Enrichment incubation demonstrated the microbial oxidation of crude oil coupled to methane production at 35 and 55°C, which generated 3.7±0.3 and 2.8±0.3 mmol of methane per gram oil, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that crude oil <i>n</i>-alkanes were obviously degraded, and high molecular weight <i>n</i>-alkanes were preferentially removed over relatively shorter-chain <i>n</i>-alkanes. Phylogenetic analysis revealed the concurrence of acetoclastic <i>Methanosaeta</i> and hydrogenotrophic methanogens but different methanogenic community structures under the two temperature conditions. Candidate divisions of JS1 and WWE 1, <i>Proteobacteria</i> (mainly consisting of <i>Syntrophaceae</i>, <i>Desulfobacteraceae</i> and <i>Syntrophorhabdus</i>) and <i>Firmicutes</i> (mainly consisting of <i>Desulfotomaculum</i>) were supposed to be involved with <i>n</i>-alkane degradation in the mesophilic conditions. By contrast, the different bacterial phylotypes affiliated with <i>Caldisericales</i>, “Shengli Cluster” and <i>Synergistetes</i> dominated the thermophilic consortium, which was most likely to be associated with thermophilic crude oil degradation. This study revealed that the oily sludge in Shengli oilfield harbors diverse uncultured microbes with great potential in methanogenic crude oil degradation over a wide temperature range, which extend our previous understanding of methanogenic degradation of crude oil alkanes.</p></div

The archaeal T-RFLP profiles at 35°C (A) and 55°C (B).

<p>Crude oil -: consortium without crude oil addition; crude oil +: consortium with crude oil addition. Error bars represent the standard deviation, three replicates in crude oil-amended consortium and duplicates in crude oil-free consortium.</p

Gas chromatograms of the saturated hydrocarbon factions of crude oil.

<p>A: 35°C; B: 55°C. Pr: pristane; Ph: phytane; nC₁₇: <i>n</i>-heptadecane; nC₁₈: <i>n</i>-octadecane; IS: <i>d</i>₅₀-<i>n</i>-tetracosane. The crude oil samples were collected from the crude oil-degrading cultures at different time points, with the exception that “sterilized-330 days” crude oil was sampled from the second control after 330 days of incubation at 35°C, and “sterilized-303 days” crude oil was sampled from the second control after 303 days of incubation at 55°C.</p

Changes in group composition and biomarker ratios of crude oil during methanogenic degradation.

<p>Pr/<i>n</i>C₁₇: pristane/<i>n</i>-heptadecane; three replicates in each time points with exception of original day 0. a <i>n</i>-heptadecane was only detected in one of the three replicates; b: <i>n</i>-heptadecane was not detected above the detection limit; Sterilized-330: crude oil sampled from the sterilized control group after 330 days of incubation at 35°C, Sterilized-303: crude oil sampled from the sterilized control group after 303 days of incubation at 55°C.</p><p>Changes in group composition and biomarker ratios of crude oil during methanogenic degradation.</p

Bacterial T-RFLP profiles at 35°C (A) and 55°C (B).

<p>Crude oil -: consortium without crude oil addition; Crude oil +: crude oil amended consortium. Error bars represent the standard deviation, three replicates in crude oil-amended consortium and duplicates in crude oil-free consortium.</p

Phylogenetic affiliation of archaeal 16S rRNA gene sequences of the methanogenic crude oil-degrading consortia.

<p>A: 35°C; B: 55°C. The number in the column indicates the major OTUs represented by specific T-RFs, followed by the number in parentheses indicating the clone numbers of each OTU.</p

Phylogenetic affiliation of bacterial 16S rRNA gene sequences of the methanogenic crude oil-degrading consortia.

<p>A: 35°C; B: 55°C. The number in the column indicates the major OTUs represented by specific T-RFs, followed by the number in parentheses indicating the clone numbers of each OTU.</p