Vaksmaa Annika, Guerrero-Cruz Simon, van Alen Theo A, Cremers Geert, Ettwig Katharina F, Lüke Claudia, Jetten Mike S M
Department of Microbiology, IWWR, Radboud University Nijmegen, Nijmegen, The Netherlands.
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
Appl Microbiol Biotechnol. 2017 Sep;101(18):7075-7084. doi: 10.1007/s00253-017-8416-0. Epub 2017 Aug 4.
Paddy fields are a significant source of methane and contribute up to 20% of total methane emissions from wetland ecosystems. These inundated, anoxic soils featuring abundant nitrogen compounds and methane are an ideal niche for nitrate-dependent anaerobic methanotrophs. After 2 years of enrichment with a continuous supply of methane and nitrate as the sole electron donor and acceptor, a stable enrichment dominated by 'Candidatus Methanoperedens nitroreducens' archaea and 'Candidatus Methylomirabilis oxyfera' NC10 phylum bacteria was achieved. In this community, the methanotrophic archaea supplied the NC10 phylum bacteria with the necessary nitrite through nitrate reduction coupled to methane oxidation. The results of qPCR quantification of 16S ribosomal RNA (rRNA) gene copies, analysis of metagenomic 16S rRNA reads, and fluorescence in situ hybridization (FISH) correlated well and showed that after 2 years, 'Candidatus Methanoperedens nitroreducens' had the highest abundance of (2.2 ± 0.4 × 10) 16S rRNA copies per milliliter and constituted approximately 22% of the total microbial community. Phylogenetic analysis showed that the 16S rRNA genes of the dominant microorganisms clustered with previously described 'Candidatus Methanoperedens nitroreducens ANME2D' (96% identity) and 'Candidatus Methylomirabilis oxyfera' (99% identity) strains. The pooled metagenomic sequences resulted in a high-quality draft genome assembly of 'Candidatus Methanoperedens nitroreducens Vercelli' that contained all key functional genes for the reverse methanogenesis pathway and nitrate reduction. The diagnostic mcrA gene was 96% similar to 'Candidatus Methanoperedens nitroreducens ANME2D' (WP_048089615.1) at the protein level. The 'Candidatus Methylomirabilis oxyfera' draft genome contained the marker genes pmoCAB, mdh, and nirS and putative NO dismutase genes. Whole-reactor anaerobic activity measurements with methane and nitrate revealed an average methane oxidation rate of 0.012 mmol/h/L, with cell-specific methane oxidation rates up to 0.57 fmol/cell/day for 'Candidatus Methanoperedens nitroreducens'. In summary, this study describes the first enrichment and draft genome of methanotrophic archaea from paddy field soil, where these organisms can contribute significantly to the mitigation of methane emissions.
稻田是甲烷的重要来源，其甲烷排放量占湿地生态系统总甲烷排放量的20%。这些被水淹没的缺氧土壤富含氮化合物和甲烷，是依赖硝酸盐的厌氧甲烷氧化菌的理想生存环境。在以甲烷和硝酸盐作为唯一电子供体和受体持续供应的条件下进行2年的富集培养后，获得了一个稳定的富集培养物，其中以“硝化还原甲烷菌属（Candidatus Methanoperedens nitroreducens）”古菌和“嗜氧甲基菌属（Candidatus Methylomirabilis oxyfera）”NC10门细菌为主。在这个群落中，甲烷营养型古菌通过与甲烷氧化偶联的硝酸盐还原为NC10门细菌提供必需的亚硝酸盐。16S核糖体RNA（rRNA）基因拷贝的qPCR定量结果、宏基因组16S rRNA读数分析以及荧光原位杂交（FISH）结果相关性良好，显示2年后，“硝化还原甲烷菌属（Candidatus Methanoperedens nitroreducens）”的丰度最高，每毫升有（2.2±0.4×10）个16S rRNA拷贝，约占微生物群落总量的22%。系统发育分析表明，优势微生物的16S rRNA基因与先前描述的“硝化还原甲烷菌属（Candidatus Methanoperedens nitroreducens ANME2D）”（同一性为96%）和“嗜氧甲基菌属（Candidatus Methylomirabilis oxyfera）”（同一性为99%）菌株聚类。汇总的宏基因组序列产生了“维切利硝化还原甲烷菌属（Candidatus Methanoperedens nitroreducens Vercelli）”的高质量基因组草图组装，其中包含反向甲烷生成途径和硝酸盐还原的所有关键功能基因。诊断性mcrA基因在蛋白质水平上与“硝化还原甲烷菌属（Candidatus Methanoperedens nitroreducens ANME2D）”（WP_048089615.1）的相似性为96%。“嗜氧甲基菌属（Candidatus Methylomirabilis oxyfera）”的基因组草图包含标记基因pmoCAB、mdh和nirS以及假定的NO歧化酶基因。用甲烷和硝酸盐进行的全反应器厌氧活性测量显示，平均甲烷氧化速率为0.012 mmol/h/L，“硝化还原甲烷菌属（Candidatus Methanoperedens nitroreducens）”的细胞特异性甲烷氧化速率高达0.57 fmol/细胞/天。总之，本研究描述了从稻田土壤中首次富集培养甲烷营养型古菌及其基因组草图，这些微生物对减少甲烷排放有重要作用。
