Abstract:[Objective] To explore the feasibility of using Clostridium and Methanomassiliicoccus from human intestine to synergistically convert betaine and choline to methane. [Methods] Illumina sequencing of the 16S rRNA gene was performed to survey the diversity of archaea in the feces from healthy people of 20-40 years old and over 40 years old. The Methanomassiliicoccus-specific mtaB gene and Methanomassiliicoccus-specific 16S rRNA gene were quantitated by quantitative PCR (qPCR) to quantify the trimethylamine-utilizing methanogens in human intestine. Metagenome-assembled genomes (MAGs) were reconstructed from metagenome data for the identification of the intestinal bacteria carrying the betaine reductase gene grdH and choline trimethylamine-lyase gene cutC. The bacteria that reduced betaine and choline were isolated from feces and used to construct the coculture with Methanomassiliicoccus. The potential of the coculture for producing methane from betaine and choline was then determined. [Results] The main methanogenic archaea in the intestine of the 20-40 years old included Methanobrevibacter (49.18%) and Methanobacterium (33.34%) affiliating to Methanobacteriaceae (82.16%), Methanosarcina (5.70%) of Methanosarcinaceae (5.67%), and Methanomassiliicoccus (3.14%) of Methanosassilicoccaceae (3.13%). The methanogen diversity was lower in the feces from the people over 40 years old, from whom Methanosassilicoccaceae was not detected. Quantitative PCR determined that the total abundance of archaea and bacteria in the people of 20-40 years old was 3.11 and 2.90 folds, respectively, higher than in those over 40 years old. Specifically, the abundance of Methanomassiliicoccus and Methanosarcina was 6.53 and 5.52 folds higher, respectively. A total of 229 bacterial MAGs were obtained from the fecal specimens, in which 42 MAGs carried genes grdH and cutC and were affiliated to Lachnospiraceae, Enterobacteriaceae, and Clostridiaceae. Clostridium malenominatum B8 was isolated from the fecal specimens. The co-culture of this strain with Methanomassiliicoccus luminyensis B10 in the medium with 20 mmol/L betaine or choline degraded 47.03% betaine and 25.83% choline to produce methane, during which trimethylamine was detected as the intermediate. [Conclusion] The human intestinal Clostridium B8 and M. luminyensis B10 synergistically convert betaine and choline to methane. Therefore, we hypothesize that they play a role in reducing the trimethylamine in human intestine.
