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Evidence of Novel Phylogenetic Lineages of Methanogenic Archaea from Hypersaline Microbial Mats

Abstract

Methanogenesis in hypersaline and high-sulfate environments is typically dominated by methylotrophic methanogens because sulfate reduction is thermodynamically favored over hydrogenotrophic methanogenesis in these environments. We characterized the community composition of methanogenic archaea in both unmanipulated and incubated microbial mats from different hypersaline environments in Baja California Sur, Mexico. Clone libraries of methyl coenzyme-M reductase (mcrA) sequences and DGGE band patterns of 16S rRNA and mcrA sequences showed that the methanogen community in these microbial mats is dominated by methylotrophic methanogens of the genus Methanohalophilus. However, phylogenetic analyses of mcrA sequences from these mats also revealed two new lineages corresponding to putative hydrogenotrophic methanogens related with the strictly hydrogenotrophic order Methanomicrobiales. Stimulated methane production under decreased salinity and sulfate concentrations also suggested the presence of hydrogenotrophic methanogens in these samples. The relative abundance of mcrA gene and transcripts, estimated by SYBR green I qPCR assays, suggested the activity of different phylogenetic groups of methanogens, including the two novel clusters, in unmanipulated samples of hypersaline microbial mats. Using geochemical and molecular approaches, we show that substrate limitation and values of salinity and sulfate higher than 3 % and 25 mM (respectively) are potential environmental constraints for methanogenesis in these environments. Microcosm experiments with modifications of salinity and sulfate concentrations and TMA addition showed that upper salt and sulfate concentrations for occurrence of methylotrophic methanogenesis were 28 % and 263 mM, respectively. This study provides phylogenetic information about uncultivated and undescribed methanogenic archaea from hypersaline environments.

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Acknowledgments

This project was supported by CONACYT grant 105969-Z; 2008–2014, CIBNOR grant PC0.18-2010-2014 to A.L.C, and a grant from the NASA Exobiology Program to B.M.B. J.Q.G.M. is a recipient of a CONACYT doctoral fellowship (212242). R.C.E acknowledges the support of the NASA Postdoctoral Program, administered by Oak Ridge Associated Universities. We are grateful to Exportadora de Sal, S.A. de C.V. for access to the Guerrero Negro field site. We would like to thank Cheryl A. Kelley for assistance with the determination of methane production rates, Angela Detweiler and Santiago Cadena for technical support in the lab, Ignacio Leyva for assistance in phylogenetic analyses. Ira Fogel of CIBNOR provided editorial services. Berenice Celis provided suggestions that improved the manuscript.

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Correspondence to Alejandro López-Cortés.

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Supplementary Fig. 1
figure5

(A) Maximum-likelihood phylogenetic tree based on sequences of 16S rRNA gene phylotypes retrieved from DGGE bands. Branch nodes supported by phylogenetic analysis with both bootstrap values >95 by maximum-likelihood (ML), and posterior probabilities >0.95 by Bayesian analyses (BA) are indicated by filled circles. Open circles indicate >75 % bootstrap support by ML, or >0.75 posterior probabilities support by BA analysis. Branch nodes without circles were not supported (bootstrap value <75 and posterior probabilities <0.75). The tree is rooted using a sequence of Cenarchaeum symbiosum (U51469) as the outgroup. GenBank accession numbers of the representative sequences of each cluster are indicated in parenthesis. The scale bar represents 0.1 changes per nucleotide position. (B) Composition of the phylogenetic clusters based on the distribution of archaeal 16S rRNA sequences retrieved by DGGE bands. (GIF 115 kb)

High resolution image (EPS 8.25 mb)

Supplementary Table 1

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García-Maldonado, J.Q., Bebout, B.M., Everroad, R.C. et al. Evidence of Novel Phylogenetic Lineages of Methanogenic Archaea from Hypersaline Microbial Mats. Microb Ecol 69, 106–117 (2015). https://doi.org/10.1007/s00248-014-0473-7

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Keywords

  • Methanogenesis
  • Hypersaline Environment
  • Hydrogenotrophic Methanogen
  • Methanogenic Community
  • Methanomicrobiales