Abstract—

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.

中文翻译：

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甲基球菌连续培养中微生物群落发展的分子分析。天然气概念八

摘要—

微生物群落的组成是在长期（60天以上）专性甲烷氧化菌甲基球菌（Methylococcus sp。）的连续培养过程中形成的。在42°C和pH 5.6下，在50升生物反应器中监测天然气的Concept-8。稳定的生长和高光密度的培养物（平均OD ₅₄₀ = 13.5），比生长速度为0.2 h ^–1以及生物量产量为4.0–4.5 g干物质L ^–1持续50天。在随后的体制不稳定时期，光密度急剧下降，降至OD ₅₄₀ ＝ 4。在培养液中检测到甲酸盐，乙酸盐和许多源自甲烷同系物氧化的不确定的脂肪酸。显微镜显示，在稳定生长期间，卫星细菌的比例最高（占总细胞数的10％至15％），而在生长下降期间则下降至4.5％。基于16S rRNA基因序列分析的群落组成的分子谱分析显示，在负责利用代谢产物和甲烷氧化菌裂解产物的主要微生物卫星中，Cohnella，Brevibacillus， Azospirillim ，Thermomonas，Cupriavidus和Paenibacillus属的成员。在增长不稳定阶段，相对丰度短杆菌减少；这些生物很可能是乙酸盐氧化的原因。通过耕种获得的分离株仅代表了通过分子分析在社区中发现的微生物多样性的一小部分。分离代表该社区功能上重要组成部分的短杆菌和铜绿菌种的文化，为有针对性地构建与Concept-8菌株相关的协会开辟了道路，这将显示出高生产率和稳定的增长。