Revisiting Microbial Diversity in Hypersaline Microbial Mats from Guerrero Negro for a Better Understanding of Methanogenic Archaeal Communities

Abstract

Knowledge regarding the diversity of methanogenic archaeal communities in hypersaline environments is limited because of the lack of efficient cultivation efforts as well as their low abundance and metabolic activities. In this study, we explored the microbial communities in hypersaline microbial mats. Bioinformatic analyses showed significant differences among the archaeal community structures for each studied site. Taxonomic assignment based on 16S rRNA and methyl coenzyme-M reductase (mcrA) gene sequences, as well as metagenomic analysis, corroborated the presence of Methanosarcinales. Furthermore, this study also provided evidence for the presence of Methanobacteriales, Methanomicrobiales, Methanomassiliicoccales, Candidatus Methanofastidiosales, Methanocellales, Methanococcales and Methanopyrales, although some of these were found in extremely low relative abundances. Several mcrA environmental sequences were significantly different from those previously reported and did not match with any known methanogenic archaea, suggesting the presence of specific environmental clusters of methanogenic archaea in Guerrero Negro. Based on functional inference and the detection of specific genes in the metagenome, we hypothesised that all four methanogenic pathways were able to occur in these environments. This study allowed the detection of extremely low-abundance methanogenic archaea, which were highly diverse and with unknown physiology, evidencing the presence of all methanogenic metabolic pathways rather than the sheer existence of exclusively methylotrophic methanogenic archaea in hypersaline environments.

Highly diverse – low abundance methanogenic communities in hypersaline microbial mats of Guerrero Negro B.C.S., assessed through microcosm experiments

“…Among the methanogens favored by the different incubation conditions, the order Methanosarcinales, whose metabolism is mostly methylotrophic and acetoclastic [31], was the most abundant in all samples; probably due to its versatility regarding carbon sources utilized. Previous studies have yielded similar results [13,16,17], although this is the first report on the composition of the methanogenic community at the A4N5 site using next-generation sequencing. Notably, sequences of methyl-reducing methanogens of the order Methanomassiliicoccales were recovered from both sampled sites (Fig.…”

“…Therefore, it was thought that the co-occurrence of methanogenesis and sulfate reduction in hypersaline environments was only due to the use of non-competitive substrates such as methanol, methane thiol, and methylamines [8]. However, recent studies [16,43] have proposed that acetoclastic and hydrogenotrophic methanogens can coexist with SRB in environmental contexts similar to ours.…”

“…For molecular analyses, the mat portions collected were kept immersed in the pond brine from each site, stored with ice and transported to the laboratory. A total of five microbial mat cores (1.0 cm diameter x 0.5 cm deep) were sub-sampled as described previously (16) and preserved in 5 ml of RNAlater (Thermo Fisher, Carlsbad, CA, USA), contained in 15 ml tubes. These samples (hereafter named "environmental samples") were not submitted to any treatment.…”

“…Owed to the samples nature and in order to determine the efficiency of the PCR reactions, all the mcrA gene assays (25 µL) were initially performed with primers mlas-mod-F and mcrA-rev-R containing Illumina adapters (16), following the next conditions: 6.5 µL sterile water, 2.5 µL of each primer solution (10 µM), 12.5 µL GoTaq master mix (Promega, Madison, WI, USA) and 1 µL (10 ng µL −1 ) DNA. The thermocycling conditions were performed in a Thermocycler Touch C-1000 (Bio-Rad, Berkeley, CA, USA) and consisted in one cycle of 95 °C for 5 min, five touchdown cycles of 95 °C for 30 s, 60 °C for 45 s, diminishing 1 °C per cycle and an elongation step of 72 °C for 1 min, followed by 35 cycles of 95 °C for 30 s, 54.5 °C for 30 s, 72 °C for 1 min and a final elongation step at 72 °C for 5 min.…”

“…Several studies have addressed the diversity, abundance, and physiology of methanogens from hypersaline environments [9][10][11][12][13][14][15], indicating the predominance of methylotrophic methanogens of the order Methanosarcinales. Nonetheless, a recent survey [16] based on 16S rRNA and methyl coenzyme M reductase (mcrA) gene sequences of hypersaline microbial mats revealed lineages of methanogens whose taxonomic assignment relates them to other type of metabolisms, suggesting that methane production in this environment may be underestimated and hence, not be limited to a single methanogenic pathway. Considering this, the present work aimed to assess the stimulation of low abundance methanogenic groups by substrates and electron donor additions; and to elucidate the changes in methanogenic communities when these conditions are present in the environment.…”