Dimitry Yu. Sorokin

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota . The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales . Cells of strains AB-alg1 ^T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch, and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose, and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1 ^T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1 ^T (JCM 35393 ^T =UQM 41574 ^T ) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae . IMPORTANCE: Alkaline soda lakes and soils are extreme habitats dominated by obligate haloalkaliphic prokaryotes, some of which can produce alkali- and salt-stable polysaccharide-degrading exoenzymes useful for industrial and domestic applications. However, so far, little was known about the microbial potential for mineralization of acidic polysaccharides, such as alginate, in these habitats. The described isolates are the first representatives of a new family within the phylum Verrucomicrobiota specializing in the degradation of alginate and related polysaccharides. We present the key enzymatic machinery for alginate breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example, in washing powders and biomass waste recycling. Furthermore, the new family is one of the most abundant taxa in alkaline environments, and these environments are not known to harbor signature alginate producing biota, such as brown algae. This way, our study opens a new window on polysaccharide turnover in alkaline environments.

Chitin is the second most abundant polysaccharide and can serve as a carbon and nitrogen source for microorganisms in various ecosystems, including hypersaline environments. However, chitin metabolism in extremely halophilic archaea of the class Halobacteria has not been systematically analyzed, and the pathways of N-acetylglucosamine (GlcNAc) utilization in these organisms remain poorly understood. In this study, we performed a large-scale comparative genomic analysis of the class Halobacteria to assess prevalence and organization of genes, presumably involved in chitin utilization. Potential chitinolytic species are unevenly distributed across the class Halobacteria and tend to cluster within only 7 out of its 117 recognized genera. Glycoside hydrolases from GH18 and GH3 families were found as predominant endochitinases and β-hexosaminidases, respectively. Most putative chitinolytic haloarchaea lacked the genes involved in GlcNAc catabolism in bacteria and hyperthermophilic archaeon Thermococcus kodakaraensis. However, many of them harbor enzymes of pathways previously proposed for Natrarchaeobius and nanohaloarchaeon Ca. "Nanohalobium constans". Analysis of gene neighborhoods demonstrated a conserved organization of the gene clusters associated with chitin metabolism in some genomes, resembling the clusters previously described for Natrarchaeobius species. Overall, our results indicate that Halomicrobium, Natrarchaeobius, Natrialba, Saliphagus, Halocatena, Haladaptatus, and Salinarchaeum genera are enriched in species with chitinolytic potential.

In methanogenic communities, two main pathways drive methanogenesis: acetoclastic methanogenesis, which converts acetate into CH ₄ and CO ₂ , and hydrogenotrophic methanogenesis, which reduces CO ₂ with H ₂ to CH ₄ . Under high-pH conditions, a shift in dominance from acetoclastic to hydrogenotrophic methanogenesis is often observed. The goal of this work was to identify the pH tipping point for this metabolic shift and to elucidate the influence of alkalinity on this transition in a haloalkaliphilic methanogenic community enriched from anaerobic soda lake sediments. To this end, a haloalkaliphilic microbial community was cultivated across a pH range (8.20–10.00) at three different alkalinities (0.1, 0.6, 1.2 eq/L). Specific qPCR probes were developed to quantify the two dominant methanogens for each catabolism: “ Ca. Methanocrinis natronophilus ” (acetoclastic) and Methanocalculus alkaliphilus (hydrogenotrophic). Results showed that the relative abundance of Methanocalculus increased with the rise of pH for all alkalinities, with alkalinity exerting a stronger influence than pH. At low alkalinity (0.1 eq/L), Methanocalculus abundance doubled from 5.14 ± 1.95% to 9.15 ± 0.77% (pH 8.40–10.35). At moderate alkalinity (0.6 eq/L), it increased from 8.33 ± 1.34% to 47.92 ± 3.76% (pH 8.41–10.00), and at the highest alkalinity (1.2 eq/L), it increased from 6.78 ± 1.06% to 60.25 ± 2.00% (pH 8.26–9.68). 16S rRNA gene amplicon sequencing further identified “ Candidatus Contubernalis” as a putative syntrophic acetate-oxidizing bacterium likely partnering with Methanocalculus in indirect hydrogenotrophic methanogenesis. This work highlights that haloalkaliphilic hydrogenotrophic methanogens offer a promising strategy to integrate CO ₂ capture in alkaline solutions with biomethanation.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota . The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales . Cells of strains AB-alg1 ^T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1 ^T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1 ^T (JCM 35393 ^T =UQM 41574 ^T ) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae . Importance: The presented isolates are the first isolated representatives of an environmental family of Opitutales , part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

Saline and highly alkaline soda lakes are often characterized by a persistent nitrogen loss and high sulfide levels. Cyanobacteria are key aerobic diazotrophs in soda lakes, where light-dependent nitrogen fixation (NF) is crucial for sustaining ecosystem functioning. While sulfide is a well-known inhibitor of oxygenic photosynthesis, some cyanobacteria may tolerate it and utilize it via anoxygenic photosynthesis. In this study, we investigated the NF and anoxygenic photosynthesis in the genus Sodalinema, including non-heterocystous cyanobacteria widely distributed in soda and saline environments around the world and possessing an anaerobe-like nitrogenase. Our data suggest that their nif-operon could have been more likely acquired in soda or saline–alkaline lakes from natronophilic sulfate-reducing bacteria of the family Desulfonatronovibrionaceae than in the marine environment. It was shown that Sodalinema sp. P-1104, isolated from a southwestern Siberian soda lake, is capable of NF only in a light/dark switching mode, both in oxic and anoxic conditions. Sulfide did not suppress photosynthesis and stimulated NF up to threefold in oxygenic conditions. Anaerobic NF was obligately sulfide-dependent and supported by anoxygenic photosynthesis. However, removal of photosynthetic oxygen due to the high reducing potential of sulfide stimulated NF to a greater extent than does the use of sulfide through anoxygenic photosynthesis.

The draft genome of a chemolithoautotrophic ammonia-oxidizing bacterium of the genus Nitrosomonas is reported. Nitrosomonas sp. strain ANs5, previously classified as a strain of N. halophila , is an alkali-tolerant ammonia-oxidizing bacterium isolated from the soda lakes of northeast Mongolia.

Polysaccharide-degrading natronoarchaea have been poorly studied to date. However, over the past decade, significant progress has been made in understanding their diversity and metabolic potential. In this study, two natronoarchaeal strains, enriched from oxic sediment samples of the soda lakes of Wadi an Natrun in Egypt (AArcel7) and Kulunda steppe in Russia (A-rgal3), were characterized. Strain AArcel7 was enriched with amorphous cellulose, while strain A-rgal3 dominated an enrichment culture using rhamnogalacturonan. Cells of both strains are polymorphic, from motile flat rods to nonmotile cocci. They are aerobic heterotrophs that are able to grow on chitin and several other carbohydrates. Both strains thrive within a salinity range of 2.5 to 4.5 M total Na⁺, with optimal growth at 3.5–4 M, and are moderately alkaliphilic with an optimum pH at 8.5–9.0 (AArcel7) and 9.2–9.5 (A-rgal3). Genome-based phylogenetic analysis demonstrated that these isolates form a new species lineage in the chitin-specialized genus Natrarchaeobius. An in-depth study of Natrarchaeobius genomes allowed us to identify several genes that potentially enable them to hydrolyze chitin and to metabolize N-acetylglucosamine (GlcNAc), which has not been investigated previously in the chitin-utilizing natronoarchaea. Based on physiological, phylogenetic, and genomic analyses, strains AArcel7 and A-rgal3 are suggested to form a novel species, Natrarchaeobius versutus sp. nov., with AArcel7^T (DSM 119357 = UNIQEM U973) as the type strain. Furthermore, strain AArcht7^T, formerly classified as the type species of the genus Natrarchaeobius, is proposed to be reclassified as Natrarchaeobius oligotrophus (DSM 119677 = UNIQEM U967).