Rahalkar Monali C, Khatri Kumal, Pandit Pranitha, Bahulikar Rahul A, Mohite Jyoti A
C2, Bioenergy Group, MACS Agharkar Research Institute, Pune, India.
Department of Microbiology, Savitribai Phule Pune University, Pune, India.
Front Microbiol. 2021 Sep 3;12:669244. doi: 10.3389/fmicb.2021.669244. eCollection 2021.
Methanotrophs are aerobic to micro-aerophilic bacteria, which oxidize and utilize methane, the second most important greenhouse gas. The community structure of the methanotrophs in rice fields worldwide has been studied mainly using culture-independent methods. Very few studies have focused on culturing methanotrophs from rice fields. We developed a unique method for the cultivation of methanotrophs from rice field samples. Here, we used a modified dilute nitrate mineral salts (dNMS) medium, with two cycles of dilution till extinction series cultivation with prolonged incubation time, and used agarose in the solid medium. The cultivation approach resulted in the isolation of methanotrophs from seven genera from the three major groups: Type Ia (, , and ), Type Ib ( and ), and Type II ( and ). Growth was obtained till 10-10 dilutions in the first dilution series, indicating the culturing of dominant methanotrophs. Our study was supported by 16S rRNA gene-based next-generation sequencing (NGS) of three of the rice samples. Our analyses and comparison with the global scenario suggested that the cultured members represented the major detected taxa. Strain RS1, representing a putative novel species of , was cultured; and the draft genome sequence was obtained. Genome analysis indicated that RS1 represented a new putative species. has been detected globally in rice fields as a dominant genus, although no strains have been isolated from rice fields worldwide. Ours is one of the first extensive studies on cultured methanotrophs from Indian rice fields focusing on the tropical region, and a unique method was developed. A total of 29 strains were obtained, which could be used as models for studying methane mitigation from rice fields and for environmental and biotechnological applications.
甲烷氧化菌是需氧至微需氧细菌，可氧化并利用甲烷（第二重要的温室气体）。全球稻田中甲烷氧化菌的群落结构主要通过非培养方法进行研究。很少有研究专注于从稻田中培养甲烷氧化菌。我们开发了一种从稻田样本中培养甲烷氧化菌的独特方法。在此，我们使用改良的稀释硝酸盐矿物盐（dNMS）培养基，经过两个稀释至灭绝系列培养周期并延长孵育时间，并在固体培养基中使用琼脂糖。这种培养方法从三个主要类群的七个属中分离出了甲烷氧化菌：I型（、和）、Ib型（和）以及II型（和）。在第一个稀释系列中，稀释至10-10时仍有生长，表明培养出了优势甲烷氧化菌。我们的研究得到了对三个水稻样本基于16S rRNA基因的下一代测序（NGS）的支持。我们的分析以及与全球情况的比较表明，培养出的成员代表了主要检测到的分类群。培养出了代表一个假定新物种的菌株RS1，并获得了基因组草图序列。基因组分析表明RS1代表一个新的假定物种。作为优势属，在全球稻田中均有检测到，尽管在全球稻田中尚未分离出菌株。我们的研究是首批针对印度稻田（聚焦热带地区）培养甲烷氧化菌的广泛研究之一，并开发了一种独特方法。共获得29株菌株，可作为研究稻田甲烷减排以及用于环境和生物技术应用的模型。
