Merkel, Alexander Y.

Highly purified cultures of alkaliphilic aceticlastic methanogens were collected for the first time using methanogenic enrichments with acetate from a soda lake and a terrestrial mud volcano. The cells of two strains were non-motile rods forming filaments. The mud volcano strain M04Ac was alkalitolerant, with the pH range for growth from 7.5 to 10.0 (optimum at 9.0), while the soda lake strain Mx was an obligate alkaliphile growing in the pH range 7.7–10.2 (optimum 9.3–9.5) in the presence of optimally 0.2–0.3 M total Na+. Genomes of both strains encoded all enzymes required for aceticlastic methanogenesis and different mechanisms of (halo)alkaline adaptations, including ectoine biosynthesis, which is the first evidence for the formation of this osmoprotectant in archaea. According to 16S rRNA gene phylogeny, the strains possessed 98.3–98.9% sequence identity and belonged to the obligately aceticlastic genus Methanothrix with M. harundinaceae as the most closely related species. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein-coding marker genes clearly indicated a polyphyletic origin of the species included in the genus Methanothrix. We propose to reclassify Methanothrix harrundinacea (type strain 8AcT) into a new genus, Methanocrinis gen. nov., with the type species Methanocrinis harrundinaceus comb. nov. We also propose under SeqCode the complete genome sequences of strain MxTs (GCA_029167045.1) and strain M04AcTs (GCA_029167205.1) as nomenclatural types of Methanocrinis natronophilus sp. nov. and Methanocrinis alkalitolerans sp. nov., respectively, which represent other species of the novel genus. This work demonstrates that the low energy aceticlastic methanogenesis may function at extreme conditions present in (halo)alkaline habitats.

AbstractNitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-reducing iron-oxidizing (NRFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with NRFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a newCandidateorder, named Siderophiliales. This new species, “CandidatusSiderophilus nitratireducens”, carries central genes to iron oxidation (cytochromec cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). To our knowledge, this is the first report ofnap-based lithoautotrophic growth, and we demonstrate that iron oxidation coupled to dissimilatory reduction of nitrate to nitrite is thermodynamically favourable under realistic Fe3+/Fe2+andconcentration ratios. Ultimately, by bridging the gap between laboratory investigations and real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NRFOs taxonomic diversity and ecological role.

Bathyarchaeia are widespread in various anoxic ecosystems and are considered one of the most abundant microbial groups on the earth. There are only a few reports of laboratory cultivation of Bathyarchaeia, and none of the representatives of this class has been isolated in pure culture. Here, we report a sustainable cultivation of the Bathyarchaeia archaeon (strain M17CTs) enriched from anaerobic sediment of a coastal lake. The cells of strain M17CTs were small non-motile cocci, 0.4–0.7 μm in diameter. The cytoplasmic membrane was surrounded by an S-layer and covered with an outermost electron-dense sheath. Strain M17CTs is strictly anaerobic mesophile. It grows at 10–45°C (optimum 37°C), at pH 6.0–10.0 (optimum 8.0), and at NaCl concentrations of 0–60 g l−1 (optimum 20 g l−1). Growth occurred in the presence of methoxylated aromatic compounds (3,4-dimethoxybenzoate and vanillate) together with complex proteinaceous substrates. Dimethyl sulfoxide and nitrate stimulated growth. The phylogenomic analysis placed strain M17CTs to BIN-L-1 genus-level lineage from the BA1 family-level lineage and B26-1 order-level lineage (former subgroup-8) within the class Bathyarchaeia. The complete genome of strain M17CTs had a size of 2.15 Mb with a DNA G + C content of 38.1%. Based on phylogenomic position and phenotypic and genomic properties, we propose to assign strain M17CTs to a new species of a novel genus Bathyarchaeum tardum gen. nov., sp. nov. within the class Bathyarchaeia. This is the first sustainably cultivated representative of Bathyarchaeia. We propose under SeqCode the complete genome sequence of strain M17CTs (CP122380) as a nomenclatural type of Bathyarchaeum tardum, which should be considered as a type for the genus Bathyarchaeum, which is proposed as a type for the family Bathyarchaeaceae, order Bathyarchaeales, and of the class Bathyarchaeia.

Abstract Thi.o.ha.lo.bac.ter.a.ce'ae N.L. masc. n. Thiohalobacter , the type genus of the family, ‐ aceae ending to denote a family; N.L. fem. pl. n. Thiohalobacteraceae the Thiohalobacter family. Proteobacteria / Gammaproteobacteria / Thiohalobacterales / Thiohalobacteraceae fam. nov. The family Thiohalobacteraceae consists of aerobic, chemolithoautotrophic, sulfur‐oxidizing bacteria from hypersaline salt lakes. They are moderately halophilic aerobic, bacteria utilizing reduced sulfur compounds as the energy source and assimilating CO 2 via the Calvin–Benson–Bassham cycle. The family is a member of the order Thiohalobacterales within the Gammaproteobacteria and consists of a single genus Thiohalobacter . The family‐level status was established by phylogenomic analysis based on the Genome Taxonomy DataBase classification. DNA G + C content (%) : 64.2 (genome sequence). Type genus : Thiohalobacter Sorokin et al. 2010 VP .

Abstract Thi.o.ha.lo.rhab.dal'es N.L. fem. n. Thiohalorhabdus , the type genus of the order; L. fem. pl. n. suff. ‐ ales ending to denote an order; N.L. fem. pl. n. Thiohalorhabdales , order of the genus Thiohalorhabdus . Proteobacteria / Gammaproteobacteria / Thiohalorhabdales ord. nov. According to the phylogenomic analysis, the order Thiohalorhabdales forms a deeply branching phylogenetic lineage at the base of the Gammaproteobacteria . It consists of a single family Thiohalorhabdaceae and genus Thiohalorhabdus . The cultured members of the order are extremely halophilic, facultatively anaerobic (denitrifying), chemolithoautotrophic, sulfur‐oxidizing bacteria from hypersaline habitats with neutral pH. Type genus : Thiohalorhabdus Sorokin et al. 2008 VP .

Abstract Thi.o.ha.lo.rhab.da.ce'ae N.L. fem. n. Thiohalorhabdus , the type genus of the family; L. fem. pl. n. suff.‐ aceae , ending to denote a family; N.L. fem. pl. n. Thiohalorhabdaceae , the Thiohalorhabdus family. Proteobacteria / Gammaproteobacteria / Thiohalorhabdales / Thiohalorhabdaceae fam. nov. The family Thiohalorhabdaceae incorporates facultatively anaerobic, chemolithoautotrophic, sulfur‐oxidizing bacteria from hypersaline habitats. They are extreme halophiles utilizing reduced sulfur compounds as energy sources with either O 2 or nitrate as the electron acceptor and assimilating CO 2 via the Calvin–Benson–Bassham cycle. The family is a member of the order Thiohalorhabdales in the Gammaproteobacteria, and it consists of a single genus Thiohalorhabdus . The family‐level status was established by phylogenomic analysis based on the Genome Taxonomy DataBase classification. DNA G  +  C content (%) : 68.9 (genome of the type strain). Type genus : Thiohalorhabdus Sorokin et al. 2008 VP .

Abstract Thi.o.ha.lo.bac.ter.al'es N.L. masc. n. Thiohalobacter , the type genus of the order; L. fem. pl. n. suff. ‐ ales , ending to denote an order; N.L. fem. pl. n. Thiohalobacterales , the order of the genus Thiohalobacter . Proteobacteria / Gammaproteobacteria / Thiohalobacterales ord. nov. The order Thiohalobacterales accommodates obligately chemolithoautotrophic aerobic bacteria utilizing inorganic sulfur compounds as the energy source and assimilating CO 2 via the Calvin–Benson–Bassham cycle. The order is a deeply branching member of the Gammaproteobacteria and consists of two families Thiohalobacteraceae and Thiogranaceae, both of which include one genus. The order‐level status was established by phylogenomic analysis based on the Genome Taxonomy DataBase classification. Type genus : Thiohalobacter Sorokin et al. 2010 VP .

SummaryAn anaerobic enrichment with CO from sediments of hypersaline soda lakes resulted in a methane‐forming binary culture, whereby CO was utilized by a bacterium and not the methanogenic partner. The bacterial isolate ANCO1 forms a deep‐branching phylogenetic lineage at the level of a new family within the class ‘Natranaerobiia’. It is an extreme haloalkaliphilic and moderate thermophilic acetogen utilizing CO, formate, pyruvate and lactate as electron donors and thiosulfate, nitrate (reduced to ammonia) and fumarate as electron acceptors. The genome of ANCO1 encodes a full Wood–Ljungdahl pathway allowing for CO oxidation and acetogenic conversion of pyruvate. A locus encoding Nap nitrate reductase/NrfA ammonifying nitrite reductase is also present. Thiosulfate respiration is encoded by a Phs/Psr‐like operon. The organism obviously relies on Na‐based bioenergetics, since the genome encodes for the Na+‐Rnf complex, Na+‐F1F0 ATPase and Na+‐translocating decarboxylase. Glycine betaine serves as a compatible solute. ANCO1 has an unusual membrane polar lipid composition dominated by diethers, more common among archaea, probably a result of adaptation to multiple extremophilic conditions. Overall, ANCO1 represents a unique example of a triple extremophilic CO‐oxidizing anaerobe and is classified as a novel genus and species Natranaerofaba carboxydovora in a novel family Natranaerofabacea.