PhD thesis: Metagenomic characterization of hypersaline anaerobic sediments of s’avall solar salterns, and evaluation of salinity as selection force in the taxonomic structure of methanogenic consortia



Foto: (De izq. a dcha.) Bartomeu Viver, Francisca Font y Ramon Rosselló (Autora: Ana Suárez)



Esporles, March 21, 2022. Francisca Font Verdera has defended her doctoral thesis supervised by the doctors Ramon A. Rosselló Mora and Bartomeu A. Viver Pizà from IMEDEA (CSIC-UIB). The event took place on March 18 at the University of Balearic Islands and could be telematically followed on ZOOM.



The anaerobic hypersaline sediments of an ephemeral pond from the S’Avall solar salterns constituted an excellent study system because of their easy accessibility, as well as the analogy of their microbial assemblages with some known deep-sea hypersaline anaerobic brines. By means of shotgun metagenomics and 16S rRNA gene amplicon sequencing, the microbial composition of the sediment was shown to be stable in time and space. The communities were formed by prokaryote representatives with an inferred clear anaerobic metabolism, mainly related to the methane, sulfur and nitrate cycles. The most conspicuous finding was the inverted nature of the vertical stratification. Contrarily to what could be expected, a methanogenic archaeal metabolism was found to dominate in the upper layers, whereas Bacteria with fermentative and anaerobic respiration metabolisms increased with depth. We could demonstrate a putative methanogenic nature of the members of candidate lineages DHVE2 and MSBL1, which were present in high abundance in this system, and described, for the first time, viruses infecting these lineages. Members of the aerobic genera Salinibacter and Halorubrum putatively active were detected especially in the deepest layers for which we hypothesize that either oxygen could be sporadically available, or they could perform anaerobic metabolisms. We also report a novel repertoire of virus species thriving in these sediments, which had special relevance because of their lysogenic lifestyles.



Sediments underlying the solar salterns of S’Avall have been subjected to the evaluation of abiotic factors as salinity, substrate concentration (Posidonia oceanica) and antibiotic. The sediment composition was analysed by amplicon 16S rRNA gene sequencing. Salinity acted as the major selection force to structure microbial communities, influenced by ampicillin, that exerted a selective pressure, and the use of distinct concentrations of P. oceanica, which displayed a weaker effect. At high salinities, communities were dominated by extreme halophilic Archaea, whereas bacterial alpha diversity decreased with salinity, being highest at 5%. Besides, among the most competitive taxa and their metabolic role in the ecosystem, overall sulfate-reducers decreased with salinity and in turn methanogens increased as salt concentration did, especially at 25% were only methanogens dominated. Beta diversity between communities in microcosms with and without ampicillin displayed an increasing trend with dilution, although its real effect could be partially hidden by the salinity factor. However, no important effect was observed of carbon substrate concentration. Deterministic ecological factors did not affect microbial community assemblies, whereas stochastic processes predominantly prevailed on the microbial dynamics of the sediment as salinity increased. Dilution applied to the slurry (initial conditions), ranging from 30% to 5% of salinity (at which the highest compositional changes were detected) was the most relevant ecological factor.



Microbial communities from sediments of S’Avall demonstrated an understudied potential in methanogenesis, and halophilic methanogenic consortia from enriched microcosms were described in this study by means of metagenomics. With a broad range in cultivability requirements at eight different salinities, distinct substrate concentrations and ampicillin, the most efficient microcosms were selected in accordance to gas methane measurement through gas chromatography. These enrichments were sub-cultured and supplemented with organic compounds (acetate, formate and trimethylamine: ATF), which enhanced the methane production. The results showed the highest methane yield in microcosms at 5% of salinity with ampicillin and 10% w/w P. oceanica, and even more the supplemented with ATF, obtaining higher methane rates in comparison to reported until now. Among the prokaryotic diversity regarding the ATF supplement availability, Methanosarcina was more abundant in the no supplemented enrichment, whereas Methanoculleus with ATF. These genera within Methanomicrobia showed a taxonomic turnover in accordance to the supplement disposal. Besides, aceticlastic Methanoculleus genus exhibited high replication activity in comparison to the metabolically versatile Methanosarcina, which can produce methane using any of the methanogenesis pathways. Additionally, new members of Bathyarchaeia and Lokiarchaeia were also able to produce methane, specially Bathyarchaeia in the no-ATF supplemented enrichment and the putative hydrogenotrophic Lokiarchaeia class in the supplemented one. In fact, methanogenesis was performed mainly via aceticlastic route, with Methanomicrobia and Bathyarchaeia as the most representative, and the supplement based on the addition of acetate, trimethylamine and formate could be mainly metabolized by members affiliated with Methanomicrobia and Lokiarchaeia.