Targeted isolation of H₂-dependent methylotrophic methanogens by a cocktail approach

Methanogenic archaea play a crucial role in the global carbon cycle and in climate change. Recent metagenomic sequencing has revealed a considerable number of (putative) H₂-dependent methylotrophic methanogens (HMMs) across the archaeal tree and in diverse environments. Traditional isolation methods, such as dilution-to-extinction and roll-tube techniques, fail to cultivate fastidious HMMs. Here, we describe a four-stage isolation strategy designed to selectively isolate HMMs by using a flexible combination of methods to systematically reduce microbial complexity to a pure culture. In the initial stage, the growth conditions for the target HMM were optimized through closed-batch cultivation encompassing >50 conditions. Second, HMM-containing cultures were serially diluted in 96-well plates combined with substrate limitation to eliminate non-target archaea. In stage 3, the bacterial diversity in the culture was further decreased to a single bacterium by treatment with antibiotics and lysozyme. Finally, a last bacterial contaminant was removed by repeated addition of antibiotic mixtures and successive dilution transfers, leading to the successful isolation of the first pure culture of Methanosuratincola petrocarbonis LWZ-6, an HMM of the phylum Thermoproteota. This protocol also describes molecular methods, including 16S rRNA gene amplicon sequencing, metagenome sequencing and quantitative PCR, to track microbial community shifts and assess the growth advantage of the target HMM, enabling monitoring of the stepwise elimination of non-target microorganisms and ultimately confirming the purification of the target HMM. The duration of the protocol will vary for different HMMs depending on their substrate utilization, growth rate and method selection.