Studies about the Metabolic Mechanism and Environmental Significance of Denitrifying Anaerobic Methane Oxidation Archaea


Student thesis: Doctoral Thesis

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  • Jing DING

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Award date10 Jul 2015


The anaerobic oxidation of methane (AOM) has great environmental significance for it can control methane emission from marine sediment to the atmosphere. This process can couple with the reduction of sulfate, nitrite, nitrate, iron and manganese. The process of AOM coupling with nitrate reduction was catalyzed together by ANME archaea and NC10 phylum bacteria. The process has great significance not only in global methane sink, but also in global carbon and nitrogen cycles. There are many unknowns about the ANME archaea functioning in this process. The research works in this study may provide us more information about the metabolic mechanisms of the ANME archaea and the environmental siginificance of the nitrate-dependent anaerobic methane oxidation process.

At first, we evaluated the anaerobic alkane oxidation pathway in DAMO system. One of the fuctional genes of alkanesuccinate synthase (ASS), assA gene, was detected in DAMO culture. And the culture supplied with ethane and nitrate indeed showed ethane oxidation coupling with nitrate reduction. However, the indicative metabolite of anaerobic ethane oxidation, ethylsuccinate, was not detected in the culture. Following detailed community analysis showed the decrease of ANME-2d archaea and NC10 bacteria in the culture, concurrently with the highly enriched Parvibaculum, uncultured Rhodocyclaceae, and Aquabacterium. In these bacteria, the Parvibaculum and Aquabacterium were candidates for ethane oxidation, and the uncultured Rhodocyclaceae may be candidate for nitrate reduction in the culture. These results showed that ANME-2d may not oxidize methane through the anaerobic ethane oxidation pathway.

After that, we further evaluated the “reverse methanogenesis” hypothesis in DAMO system. The PCR results showed that mcrA gene existed and expressed in the DAMO culture, which supported the “reverse methanogenesis” hypothesis in this system. Then we evaluated the metabolic reversibility of ANME-2d between AOM and methanogenesis with H2/CO2 and acetate as substrates. The results showed that the system produced methane from H2/CO2 but not from acetate. Additionally, the clone library and real-time PCR analysis of the culture showed that both the percentage and quantity of ANME-2d decreased significantly under this condition, while methanogen abundance increased. These results indicated that ANME-2d might not reverse its metabolism to produce methane from H2/CO2 or acetate. After the archaea were returned back to DAMO condition, the DAMO activity decreased and the amount of ANME-2d continued to fall, implying that the DAMO archaea has encountered harmful injury and required a long time to recover.

There have already been many evaluations about the M. oxyfera (the NC10 bacteria partner in DAMO process) bacteria in environments, and the significance of nitrite-dependent DAMO process was discussed. However, the existence of ANME-2d archaea in environments has not been studied, though the nitrate-dependent DAMO process may also contribute significantly to the global methane sink. In this study, the co-existence of the DAMO archaea and DAMO bacteria in a paddy field was studied. The two orders, Nitrospirales and Methanosarcinales, to which the ANME-2d archaea and M.oxyfera belong, were detected in the four paddy soil samples. The further laborotory acitivity studies confirmed the occurrence of DAMO activity in this paddy field. Additionally, the detection of Desulfobacterales, Syntrophobacterales, Desulfuromonadales and Nitrosomonadales orders which are sulfate-reducing or iron-reducing related showed the possibility of SAMO and iron-dependent AOM in this paddy field.

Besides, for better detecting and quantifying the ANME-2d archaea in enrichment and environmental samples, new specific primers were needed to be designed for ANME-2d archaea. In this paper, the primer pairs, DP397F/DP569R and DP142F/DP779R were designed to quantify and detect ANME-2d archaea. The specificity and amplification efficiency of thses two primer pairs were acceptable. However, PCR with the DP142F/DP779R primers generated faint, multiple bands from the environmental samples. Nested PCR was thus conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the 16s rRNA gene copies of ANME-2d in these samples ranged from 3.72×104 to 2.30×105 copies μg-1 DNA. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d, and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples. The latter finding suggests that ANME-2d may spread in environments, and the role of nitrate-dependent AOM to global methane sink may be overlooked.

Microbial fuel cell (MFC) is the device which can directly transform suitable chemical to electricity with the catalysis of relevant microorganisms. In this study, we evaluated the electrogenic capability of DAMO culture using a MFC device. The experimental cells inoculated with DAMO culture performed higher voltage than the control one, which indicated the electrogeneic capability of the DAMO culture, though the volatages were much lower comparing with some previous publications. The community analysis results showed that the DAMO archaea maintained a high percentage in the community on the electrode, and the amount of ANME-2d enriched much on the electrode. These results showed that ANME-2d may grow with methane as sole electron donor and electrode as sole electron acceptor. The information obtained in this study indicated that MFC may be an appropriate device to be used to recover the energy produced in AOM process, and it may make us knowing more about the physiology of ANME-2d archaea.

    Research areas

  • DAMO, ANME-2d archaea, anaerobic ethane oxidation, reverse methanogenesis, metabolic reversibility, new primer design, environmental evaluation, microbial fuel cell (MFC)