| Microbial anaerobic oxidation of methane(AOM) has significant impact on global climate, geology, geomorphology and ecosystem. First, AOM is a critical step controlling the release of methane from ocean. Every year about 400 million tons of methane is consumed by AOM, which accounts for 85% methane generated from ocean, and therefore AOM is an essential part of marine carbon cycle and directly influences global climate change. Second, AOM enhances carbonate minerals formation, which contributes to the makeup of the seafloor landscape. Lastly, AOM fuel the ecosystem by converting methane into organic matters. Moreover, methane is not only important greenhouse gas, but also an important potential energy. Concerning the consequences of global warming, research on microorganisms involving in methane biogeochemical cycles(MBC), especially on the molecular factor regulating MBC will provide scientific and theoretical basis for the sustainable development and environmental protection.Anaerobic methane-oxidizing archaea(ANME) are the known microbial taxa that perform AOM, and they usually form cell aggregate of 2-20 μm with sulfate-reducing bacteria(SRB) and/or other bacteria. Since neither of ANME nor associated bacteria have been obtained as pure culture, traditional methods of micrology is not suitable for ANME and associated bacteria. Previous resreach using metagenome retrived only 20% coverage of ANME-1 genome, and much less genomic information were obtained from the associated bacteria such as SRB. Therefore the metabolisms and interactions among different members of the aggregate are not well understood.Previously our team has obtained an enrichment exhibiting high activity of methane oxidation, of which the dominant microbial population was ANME-2a and SRB. In this study, we introduced single-cell sequencing to the individual AOM cell aggregate within the enrichment. By using this method, 10 microbial ANME aggregates were obtained, of which their archaea population were all ANME-2a with >99.4% similarity in 16 s rRNA gene siquences, while their bacteria composition were more diverse, which contain Limnobacter of Betaproteobacteria, Acinetobacter of Gammaproteobacter, and unclassified bacteria candidate OP phyla(Obsidian Pool 1) and Acidobacter in addition to the well-known sulfate reducing bacteria of Deltaproteobacteria. Moreover a microbial aggregate containing ANME-2a as the sole microbial polulation was isolated.Six of ten aggregates were subjected to high-throughput genome sequencing. And an ANME-2a genome that covered about 90% of the total genome was obtained from the aggregate containing ANME-2a only. Analyses on this ANME-2a genome reveal a canonical 7-step methanogenesis pathway(including the Mer enzyme catalizing redox reaction between methyl and methylene, which is missing in ANME-1), and all the genes related to these 7-enzyme are active at transcription level. This finding not only supports the previous hypothesis that ANME may perform AOM through a reversed methanogenesis pathway, but also suggests ANME-1 and ANME-2 archaea employ different mechanisms for AOM. Moreover genes encoding tetrahydromethanopterin S-methyltransferase(Mtr) and Mer enzyme were identified as double-copy in the single aggregate genome, which is the first to report duo-copy mtr and mer gene present in a methanotrophy archaea. This duo-copy of mtr and mer gene exhibited different transcription features, which indicates they may serve for different biochemical reaction in ANME-2a.Meanwhile, genes for electron transportation and energy conservation were characterized from ANME-2a genome. Genes encoding F420H2 dehydrogenase(Fpo), the cytoplasmic and membrane-associated Coenzyme B–Coenzyme M heterodisulfide(CoB-S-SCoM) reductase(HdrABC, HdrDE), cytochrome C and the Rhodobacter nitrogen fixation(Rnf) complex were identified and expressed, whereas genes encoding canonical hydrogenases were absent. Additionaly the identification of multi-copy hdr gene indicate the elections generated from AOM could flow to versatile electron acceptor. We conclude this electron transportation and energy conservation would provide the organism with more flexibility in substrate utilization and capacity for rapid adjustment to fluctuating environments.The potential metabolism function of the microorganisms, which is associated with ANME-2a were partially resolved. The results showed SRB was autotrophy, and they employed a reductive acetyl-CoA pathway for carbon fixation; Limnobacter spp. was able to perform lithoheterotrophic growth on the reduced sulfur compounds and organic matters which coule be derived from AOM process.Finally we studied the AOM-related mineral formation. It is widely accepted that AOM increases the alkalinity and dissolved inorganic carbon concentration of the environment, which results in the formation of carbonate minerals. However the mechanistic link between ANME and biogenic minerals has not been resolved. In this research we found all the AOM aggregates were covered by a thick envelope structure. Given to the fact that cell surface and expolymers had important functions in mineral formation, we investigated the morphology and composition of this envelope structure. By using scan electron microscope and nano-scale secondary ion mass, fine resolution images of both inner and outside aggregate were obtained. The result showed this envelope structure was composed of siliceous minerals rather than carbonate, which was the first evidence indicating ANME facilitate the precipitation of siliceous minerals. And ANME may play a significant role in linking marine carbon and silicon cycle.This work focuses on the biochemical reactions and electron transport pathway of AOM. By using multi-disciplinary technics, we demonstrated the diversity of symbiotic bacteria involving in AOM, partially resolved their C/N/S cycle metabolic potential and elaborated a model for AOM mineral formation. This work may provide novel insights to AOM process. |
PDF Full Text Request