Mechanism and Application of Anaerobic Oxidation of Methane Coupled to Various Electron Acceptors Reduction


Student thesis: Doctoral Thesis

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Awarding Institution
  • T C LAU (Supervisor)
  • Jianxiong ZENG (External person) (External Supervisor)
Award date9 Mar 2020


Anaerobic oxidation of methane (AOM) plays a significant role in controlling the flux of methane from anoxic environments. Sulfate-, nitrate/nitrite-, iron-, and manganese-dependent AOM processes have been studied in depth; their reactions are important in biogeochemical methane cycles, and have revealed the potential of using methane as a substrate in wastewater treatment. However, much remains to be discovered about AOM coupled to the reduction of other electron acceptors (soluble and insoluble) and pollutants (organic and inorganic). This study may provide more information about the mechanism of AOM coupled to various electron acceptors reduction and their potential applications in practical industrial and environmental fields.

In this study, we first explored the feasibility of AOM coupled to the reduction of a soluble and highly toxic electron acceptor, selenite, using a denitrifying anaerobic methane oxidation (DAMO) culture. Isotopic 13CH4 and long-term experiments indicated that selenite reduction was coupled to methane oxidation, and selenite was ultimately reduced to Se (0) based on the results of scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The introduction of nitrate, the original electron acceptor in the DAMO culture, inhibited selenite reduction. Furthermore, the microbial community of the DAMO culture changed substantially when the electron acceptor changed from nitrate to selenite after long-term selenite reduction. High-throughput 16S rRNA gene sequencing indicated that Methylococcus (26%) became the predominant microbe performing selenite reduction and methane oxidation, and possible pathways of AOM coupled to selenite reduction were then proposed. These results revealed additional potential associations during the biogeochemical cycle of carbon, nitrogen, and selenium.

Next, we determined that extracellular and organic electron acceptor humics could also serve as terminal electron acceptors for AOM using enriched DAMO microorganisms. AOM coupled to humics reduction was demonstrated based on the production of 13C-labelled carbon dioxide, and AOM activity was evaluated under different methane partial pressures and electron acceptor concentrations. After three-cycle reduction, both AOM activity and copy numbers of the archaea 16S rRNA and mcrA genes were the highest when anthraquinone-2,6-disulfonic acid and anthraquinone-2-sulfonic acid served as electron acceptors. The high-throughput sequencing results suggested that ANME-2d were the dominant methane oxidation archaea after humics reduction; although the partner bacteria NC 10 trended downward, other reported humics reduction bacteria (Geobactor and Anammox) appeared. Potential electron transfer models from ANME-2d to humics were thus proposed. These findings promote a better understanding of available electron acceptors for AOM in natural environments and broaden insight into the significant role of ANME-2d.

Moreover, we further investigated the electron transfer process from AOM to an insoluble solid electrode. We constructed a conductive fiber membrane (CFM) bioanode to improve the methane mass transfer and microbial electron transfer efficiency of methane-driven MFCs. After biofilm formation on the surface of the CFM bioanode, a voltage output of 0.6 to 0.7 V was recorded. CFM-MFCs also reached a maximum power density of 63.52 ± 2.37 mW/m2. Accumulation of intermediate acetate was observed during methane oxidation to carbon dioxide. High-throughput 16S rRNA gene sequencing revealed that the microbial community changed greatly after electricity generation, methane-related archaea (Methanobrevibacter and Methanobacterium) formed a synthetic consortium with characterized electroactive species (Rhodopseudomonas and Syntrophomonas). We thus identified intermediate (acetate)-dependent interspecies electron transfer as the proposed electrogenic mechanism.

Finally, we explored the feasibility of applying a methane-based hollow fiber membrane bioreactor (HfMBR) for high-rate decolorization of the organic pollutant methyl orange (MO). An MO decolorization efficiency of ~100% (hydraulic retention time [HRT]=1.5 to 2 days) and maximum decolorization rate of 883 mg/L/day (HRT = 0.5 days) were obtained by a stepwise increase in the MO loading rate in the methane-based HfMBR. SEM and fluorescence in situ hybridization (FISH) analyses revealed that archaea clusters formed synergistic consortia with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic, and high-throughput sequencing analysis results further confirmed biological consortia formation of methane-related archaea and partner bacteria, which played a synergistic role in MO decolorization. The high removal efficiency and stable microbial structure in HfMBR suggest that it is a potentially effective technique for high-toxicity azo dyes removal from textile wastewater.

    Research areas

  • anaerobic oxidation of methane, electron acceptors, electron transfer, selenite, humics, methyl orange, decolorization, hollow fiber membrane bioreactor, microbial fuel cells, electricity generation, ANME-2d, microbial community