Characterisation of 'Candidatus Methylobacter titanis' sp. nov., a putative novel species of Methylobacter clade 2 and their distribution in sediments of freshwater lakes in maritime Antarctica.

Global warming has a strong impact on the polar regions, in particular, the Antarctic Peninsula and nearby islands. Methane (CH4) is a major factor in climate change and mitigation of CH4 emissions can be accomplished through microbial oxidation by methanotrophic bacteria. Understanding this biological process is crucial given the shortage of research carried out in this geographical area. The aim of this study was to characterise psychrophilic enrichment cultures of aerobic methanotrophs obtained from lake sediments of the Fildes Peninsula (King George Island, South Shetland Islands) and revealing the distribution of the genus Methylobacter in different lake sediments of the peninsula. Four stable methanotrophic enrichment cultures were obtained and analysed by metagenome-assembled genomes (MAGs). The phylogeny of methanotroph MAGs recovered from these enrichment cultures based on the 16S rRNA gene showed that K-2018 MAG008 and D1-2020 MAG004Ts clustered within the Methylobacter clade 2, with high similarity to Methylobacter tundripaludum SV96T (97.88 and 98.56% respectively). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with M. tundripaludum were < 95% (84.8 and 85.0%, respectively) and < 70% (30.2 and 30.3%, respectively), suggesting that they represent a putative novel species for which the name 'Ca. Methylobacter titanis' is proposed. This is the first species of clade 2 of the genus Methylobacter obtained from Antarctica. The bacterial diversity assessed by 16S rRNA gene sequencing of 21 samples of different lakes (water column and sediments) revealed 54 ASVs associated with methanotrophs and the genus Methylobacter as the most abundant. These results suggest that aerobic methanotrophs belonging to the Methylobacter clade 2 would be the main responsible for CH4 oxidation in these sediments.

Oxygen availability is a major factor in determining the composition of microbial communities involved in methane oxidation.

We have previously observed that methane supplied to lake sediment microbial communities as a substrate not only causes a response by bona fide methanotrophic bacteria, but also by non-methane-oxidizing bacteria, especially by members of the family Methylophilaceae. This result suggested that methane oxidation in this environment likely involves communities composed of different functional guilds, rather than a single type of microbe. To obtain further support for this concept and to obtain further insights into the factors that may define such partnerships, we carried out microcosm incubations with sediment samples from Lake Washington at five different oxygen tensions, while methane was supplied at the same concentration in each. Community composition was determined through 16S rRNA gene amplicon sequencing after 10 and 16 weeks of incubation. We demonstrate that, in support of our prior observations, the methane-consuming communities were represented by two major types: the methanotrophs of the family Methylococcaceae and by non-methanotrophic methylotrophs of the family Methylophilaceae. However, different species persisted under different oxygen tensions. At high initial oxygen tensions (150 to 225 µM) the major players were, respectively, species of the genera Methylosarcina and Methylophilus, while at low initial oxygen tensions (15 to 75 µM) the major players were Methylobacter and Methylotenera. These data suggest that oxygen availability is at least one major factor determining specific partnerships in methane oxidation. The data also suggest that speciation within Methylococcaceae and Methylophilaceae may be driven by niche adaptation tailored toward specific placements within the oxygen gradient.