Molecular Analysis of the Microbial Community Developing in Continuous Culture of Methylococcus sp. Concept-8 on Natural Gas

The composition of the microbial community formed in the course of long-term (over 60 days) continuous cultivation of an obligate methanotroph Methylococcus sp. Concept-8 on natural gas in a 50-L bioreactor at 42°C and pH 5.6 was monitored. Stable growth with high optical density of the culture (average OD540 = 13.5), specific growth rate of 0.2 h–1, and biomass yield of 4.0–4.5 g dry matter L–1 continued for 50 days. During the subsequent period of regime instability, optical density decreased sharply, down to OD540 = 4. Formate, acetate, and a number of unidentified fatty acids derived from oxidation of methane homologs were detected in the culture liquid. Microscopy revealed the highest share of satellite bacteria (10–15% of the total cell number) during the period of stable growth, while it dropped to 4.5% during growth decline. Molecular profiling of the community composition based on 16S rRNA gene sequence analysis revealed members of the genera Cohnella, Brevibacillus, Azospirillim, Thermomonas, Cupriavidus, and Paenibacillus among the major microbial satellites responsible for utilization of metabolites and the products of lysis of the methanotroph cells. During the phase of growth instability, the relative abundance of Brevibacillus decreased drastically; these organisms were most likely responsible for acetate oxidation. Isolates obtained by means of cultivation represented only a minor part of microbial diversity revealed in the community by molecular analysis. Isolation of the cultures of Brevibacillus and Cupriavidus species representing the functionally important components of the community opens the way for targeted construction of associations with strain Concept-8, which will display high productivity and stable growth.