Genomic profiling of Antarctic geothermal microbiomes using long-read, Hi-C, and single-cell techniques.

Geothermal features in Antarctica provide favorable conditions for diverse microorganisms, yet their genomic diversity remains poorly understood. Here, we present an integrated dataset comprising PacBio HiFi and Hi-C metagenomic sequencing, along with single-cell amplified genomes (SAGs) from two high-altitude geothermal sites, Mount Melbourne and Mount Rittmann, in Antarctica. The long-read HiFi sequencing, coupled with Hi-C, enhances the understanding of microbiome diversity and functionality in this unique ecosystem by providing more complete and accurate genomic information. SAGs complement this by recovering rare microbial taxa and offering a strain-resolved perspective. This dataset aims to deepen our understanding of microbial evolution and ecology in Antarctic geothermal environments, and facilitate cross-comparison with other geothermal environments globally.

Living to Lithified: Construction and Preservation of Silicified Biomarkers.

Whole microorganisms are rarely preserved in the fossil record but actively silicifying environments like hot springs provide an opportunity for microbial preservation, making silicifying environments critical for the study of microbial life through time on Earth and possibly other planetary bodies. Yet, the changes that biosignatures may undergo through lithification and burial remain unconstrained. At Steep Cone Geyser in Yellowstone National Park, we collected microbial material from (1) the living system across the active outflows, (2) the silicified areas adjacent to flows, and (3) lithified and buried material to assess the preservation of biosignatures and their changes across the lithification transect. Five biofabrics, built predominantly by Cyanobacteria Geitlerinema, Pseudanabaenaceae, and Leptolyngbya with some filamentous anoxygenic phototrophs contributions, were identified and tracked from the living system through the process of silicification/lithification. In the living systems, δ30Si values decrease from +0.13‰ in surficial waters to -2‰ in biomat samples, indicating a kinetic isotope effect potentially induced by increased association with actively growing biofabrics. The fatty acids C16:1 and iso-C14:0 and the hydrocarbon C17:0 were disentangled from confounding signals and determined to be reliable lipid biosignatures for living biofabric builders and tenant microorganisms. Builder and tenant microbial biosignatures were linked to specific Cyanobacteria, anoxygenic phototrophs, and heterotrophs, which are prominent members of the living communities. Upon lithification and burial, silicon isotopes of silicified biomass began to re-equilibrate, increasing from δ30Si -2‰ in living biomats to -0.55‰ in lithified samples. Active endolithic microbial communities were identified in lithified samples and were dominated by Cyanobacteria, heterotrophic bacteria, and fungi. Results indicate that distinct microbial communities build and inhabit silicified biofabrics through time and that microbial biosignatures shift over the course of lithification. These findings improve our understanding of how microbial communities silicify, the biomarkers they retain, and transitionary impacts that may occur through lithification and burial.

Fossil Geyserite and Testate Amoebae in Geothermal Spring Vent Pools: Paleoecology and Variable Preservation Quality in Jurassic Sinter of Patagonia (Deseado Massif, Argentina).

Geyserite is a type of terrestrial siliceous hot spring deposit (sinter) formed subaerially in proximal vent areas, with near-neutral pH, alkali chloride discharge fluids characterized by initial high temperatures (~73°C to up to 100°C) that fluctuate rapidly in relation to dynamic hydrology, seasonality, wind, and other environmental parameters. We analyzed sinters at the Claudia paleogeothermal field from the Late Jurassic (~150 Ma) Deseado Massif geological province, Argentinean Patagonia. The geyserite samples-with spicular to columnar to nodular morphologies-contain abundant microfossils in monotypic assemblages that occur in three diagenetic states of preservation. The best-preserved microfossils consist of vesicle-like structures with radial heteropolar symmetry (~35 µm average diameter), circular apertures, smooth walls lacking ornamentation, and disk- or beret-like shapes. Comparisons with extant, morphologically similar organisms suggest an affinity with the testate amoebae of the Arcella hemisphaerica-Arcella rotundata complex and Centropyxis aculeata strain discoides. These species occur in active geothermal pools between 22°C and 45°C, inconsistent with the temperature of formation of modern geyserites. We propose that the testate amoebae may have colonized the geyserite during cooler phases in between spring-vent eruptive cycles to prey on biofilms. Silica precipitation through intermittent bathing and splashing of fluctuating thermal fluid discharge could have led to their entrapment and fossilization. Petrographic analysis supports cyclicity in paleovent water eruptions and later diagenesis that transformed the opal into quartz. Spatially patchy degradation and modification of the silicified microorganisms resulted in variable preservation quality of the microfossils. This contribution illustrates the importance of microscale analysis to locate early silicification and identify high-quality preservation of fossil remains in siliceous hot spring deposits, which are important in early life studies on Earth and potentially Mars.

Metabolic interdependencies in thermophilic communities are revealed using co-occurrence and complementarity networks.

Microbial communities exhibit intricate interactions underpinned by metabolic dependencies. To elucidate these dependencies, we present a workflow utilizing random matrix theory on metagenome-assembled genomes to construct co-occurrence and metabolic complementarity networks. We apply this approach to a temperature gradient hot spring, unraveling the interplay between thermal stress and metabolic cooperation. Our analysis reveals an increase in the frequency of metabolic interactions with rising temperatures. Amino acids, coenzyme A derivatives, and carbohydrates emerge as key exchange metabolites, forming the foundation for syntrophic dependencies, in which commensalistic interactions take a greater proportion than mutualistic ones. These metabolic exchanges are most prevalent between phylogenetically distant species, especially archaea-bacteria collaborations, as a crucial adaptation to harsh environments. Furthermore, we identify a significant positive correlation between basal metabolite exchange and genome size disparity, potentially signifying a means for streamlined genomes to leverage cooperation with metabolically richer partners. This phenomenon is also confirmed by another composting system which has a similar wide range of temperature fluctuations. Our workflow provides a feasible way to decipher the metabolic complementarity mechanisms underlying microbial interactions, and our findings suggested environmental stress regulates the cooperative strategies of thermophiles, while these dependencies have been potentially hardwired into their genomes during co-evolutions.

Thermophilic Hadarchaeota grow on long-chain alkanes in syntrophy with methanogens.

Methanogenic hydrocarbon degradation can be carried out by archaea that couple alkane oxidation directly to methanogenesis, or by syntrophic associations of bacteria with methanogenic archaea. However, metagenomic analyses of methanogenic environments have revealed other archaea with potential for alkane degradation but apparent inability to form methane, suggesting the existence of other modes of syntrophic hydrocarbon degradation. Here, we provide experimental evidence supporting the existence of a third mode of methanogenic degradation of hydrocarbons, mediated by syntrophic cooperation between archaeal partners. We collected sediment samples from a hot spring sediment in Tengchong, China, and enriched Hadarchaeota under methanogenic conditions at 60 °C, using hexadecane as substrate. We named the enriched archaeon Candidatus Melinoarchaeum fermentans DL9YTT1. We used 13C-substrate incubations, metagenomic, metatranscriptomic and metabolomic analyses to show that Ca. Melinoarchaeum uses alkyl-coenzyme M reductases (ACRs) to activate hexadecane via alkyl-CoM formation. Ca. Melinoarchaeum likely degrades alkanes to carbon dioxide, hydrogen and acetate, which can be used as substrates by hydrogenotrophic and acetoclastic methanogens such as Methanothermobacter and Methanothrix.

Acquisition of elemental sulfur by sulfur-oxidising Sulfolobales.

Elemental sulfur (S8 0)-oxidising Sulfolobales (Archaea) dominate high-temperature acidic hot springs (>80°C, pH <4). However, genomic analyses of S8 0-oxidising members of the Sulfolobales reveal a patchy distribution of genes encoding sulfur oxygenase reductase (SOR), an S8 0 disproportionating enzyme attributed to S8 0 oxidation. Here, we report the S8 0-dependent growth of two Sulfolobales strains previously isolated from acidic hot springs in Yellowstone National Park, one of which associated with bulk S8 0 during growth and one that did not. The genomes of each strain encoded different sulfur metabolism enzymes, with only one encoding SOR. Dialysis membrane experiments showed that direct contact is not required for S8 0 oxidation in the SOR-encoding strain. This is attributed to the generation of hydrogen sulfide (H2S) from S8 0 disproportionation that can diffuse out of the cell to solubilise bulk S8 0 to form soluble polysulfides (Sx 2-) and/or S8 0 nanoparticles that readily diffuse across dialysis membranes. The Sulfolobales strain lacking SOR required direct contact to oxidise S8 0, which could be overcome by the addition of H2S. High concentrations of S8 0 inhibited the growth of both strains. These results implicate alternative strategies to acquire and metabolise sulfur in Sulfolobales and have implications for their distribution and ecology in their hot spring habitats.

Covariation of hot spring geochemistry with microbial genomic diversity, function, and evolution.

The geosphere and the microbial biosphere have co-evolved for ~3.8 Ga, with many lines of evidence suggesting a hydrothermal habitat for life's origin. However, the extent that contemporary thermophiles and their hydrothermal habitats reflect those that likely existed on early Earth remains unknown. To address this knowledge gap, 64 geochemical analytes were measured and 1022 metagenome-assembled-genomes (MAGs) were generated from 34 chemosynthetic high-temperature springs in Yellowstone National Park and analysed alongside 444 MAGs from 35 published metagenomes. We used these data to evaluate co-variation in MAG taxonomy, metabolism, and phylogeny as a function of hot spring geochemistry. We found that cohorts of MAGs and their functions are discretely distributed across pH gradients that reflect different geochemical provinces. Acidic or circumneutral/alkaline springs harbor MAGs that branched later and are enriched in sulfur- and arsenic-based O2-dependent metabolic pathways that are inconsistent with early Earth conditions. In contrast, moderately acidic springs sourced by volcanic gas harbor earlier-branching MAGs that are enriched in anaerobic, gas-dependent metabolisms (e.g. H2, CO2, CH4 metabolism) that have been hypothesized to support early microbial life. Our results provide insight into the influence of redox state in the eco-evolutionary feedbacks between thermophiles and their habitats and suggest moderately acidic springs as early Earth analogs.

Molecular diversity of green-colored microbial mats from hot springs of northern Japan.

We acquired and analyzed metagenome and 16S/18S rRNA gene amplicon data of green-colored microbial mats from two hot springs within the Onikobe geothermal region (Miyagi Prefecture, Japan). The two collection sites-Tamago and Warabi-were in proximity and had the same temperature (40 °C), but the Tamago site was connected to a nearby stream, whereas the Warabi site was isolated. Both the amplicon and metagenome data suggest the bacterial, especially cyanobacterial, dominance of the mats; other abundant groups include Chloroflexota, Pseudomonadota, Bacteroidota/Chlorobiota, and Deinococcota. At finer resolution, however, the taxonomic composition entirely differed between the mats. A total of 5 and 21 abundant bacterial 16S rRNA gene OTUs were identified for Tamago and Warabi, respectively; of these, 12 are putative chlorophyll- or rhodopsin-based phototrophs. The presence of phylogenetically diverse microbial eukaryotes was noted, with ciliates and amoebozoans being the most abundant eukaryote groups for Tamago and Warabi, respectively. Fifteen metagenome-assembled genomes (MAGs) were obtained, represented by 13 bacteria, one ciliate (mitochondrion), and one giant virus. A total of 15 novel taxa, including a new deeply branching Chlorobiota species, is noted from the amplicon and MAG data, highlighting the importance of environmental sequencing in uncovering hidden microorganisms.

A Novel Species of the Genus Thermanaerothrix Isolated from a Kamchatka Hot Spring Possesses Hydrolytic Capabilities.

Hot springs are inhabited by specific microbial communities which are reservoirs of novel taxa. In this work strain 4228-RoLT was isolated from the Solnechny hot spring, Uzon Caldera, Kamchatka. Cells of the strain 4228-RoLT were Gram-negative rods forming multicellular filaments. The strain grew optimally at 60 °C and pH 7.0 and fermented various organic compounds including polysaccharides (microcrystalline cellulose, xylan, chitin, starch, dextrin, dextran, beta-glucan, galactomannan, glucomannan, mannan). Major fatty acids were iso-C17:0, C16:0, C18:0, C20:0, iso-C19:0, anteiso-C17:0 and C22:0. Genome of the strain was of 3.25 Mbp with GC content of 54.2%. Based on the whole genome comparisons and phylogenomic analysis the new isolate was affiliated to a novel species of Thermanaerothrix genus within Anaerolineae class of phylum Chloroflexota, for which the name T. solaris sp. nov. was proposed with 4228-RoLT (= VKM B-3776 T = UQM 41594 T = BIM B-2058 T) as the type strain. 114 CAZymes including 43 glycoside hydrolases were found to be encoded in the genome of strain 4228-RoLT. Cell-bound and extracellular enzymes of strain 4228-RoLT were active against starch, dextran, mannan, xylan and various kinds of celluloses, with the highest activity against beta-glucan. Altogether, growth experiments, enzymatic activities determination and genomic analysis suggested that T. solaris strain 4228-RoLT could serve as a source of glycosidases suitable for plant biomass hydrolysis.

Rosettibacter primus gen. nov., sp. nov., and Rosettibacter firmus sp. nov., facultatively anaerobic moderately thermophilic bacteria of the class Ignavibacteria from hot springs of North Ossetia.

A novel facultatively anaerobic moderately thermophilic bacteria, strains 4137-MeT and 4148-MeT, were isolated from hot springs of Karmadon and Ursdon, respectively (North Ossetia, Russian Federation). Gram-negative, motile rods were present singly, in pairs, rosettes, and aggregates, or formed biofilms. Both strains grew optimally at 50-55 °C, pH 7.0 and did not require sodium chloride or yeast extract for growth. They were chemoorganoheterotrophs, growing on mono-, di- and polysaccharides (cellulose, starch, xylan, lichenan, galactan, xyloglucan, mannan, xanthan gum, guar gum) as well as proteinaceous substrates (gelatin, peptone, beef and yeast extract). Growth under anaerobic conditions was observed in presence and absence of external electron acceptors. Sulfur, thiosulfate, arsenate, Fe-citrate, and ferrihydrite were reduced with acetate, starch, or yeast extract as electron donors. The respiratory quinone was MK-7. Major cellular fatty acids of both strains were iso-C15:0, anteiso-C17:0, C15:0, iso-C16:0 and additionally iso-C17:0 for strain 4137-MeT. The size of the genome and genomic DNA G + C content of strain 4137-MeT were 3.24 Mb. and 29.9 %, respectively; for strain 4148-MeT - 3.33 Mb and 30.7 %. According to the 16S rRNA gene sequence and conserved protein sequences phylogenies, strains 4137-MeT and 4148-MeT represented a distinct lineage of the family Melioribacteraceae within the class Ignavibacteria. Based on phylogenetic analysis and phenotypic features, the novel isolates were assigned to a novel genus, for which the name Rosettibacter gen. nov. is proposed. Strain 4148-MeT represents its type species Rosettibacter primus sp. nov., while strain 4137-MeT represents a new species Rosettibacter firmus sp. nov.

Ignisphaera cupida sp. nov., a hyperthermophilic hydrolytic archaeon from a hot spring of Uzon (Kamchatka), and emended description of the genus Ignisphaera.

A novel strictly anaerobic hyperthermophilic archaeon, strain 4213-coT, was isolated from a terrestrial hot spring in the Uzon Caldera, Kamchatka (Russian Federation). Coccoid cells were present singly, in pairs, or aggregates, and occasionally were motile. The strain grew at 75-100 °C and within a pH range of 5.4-8.2 with the optimum at 92 °C and pH 6.4-6.7. Strain 4213-coT was a chemoorganoheterotroph, growing on proteinaceous substrates and mono-, di- and polysaccharides (starch, guar gum, xanthan gum). It did not require sodium chloride for growth. The complete genome of strain 4213-coT was 1.74 Mbp in size; its G+C content was 36.18 %. Genome analysis allowed to identify 25 genes encoding glycosidases involved in polysaccharide hydrolysis as well as genes of ADP-forming acetate-CoA ligase, lactate dehydrogenase and two [NiFe] hydrogenases responsible for acetate, lactate and hydrogen formation during fermentation. Moreover gene cluster encoding archaellum subunits was found. According to the phylogenomic analysis strain 4213-coT formed a species-level phylogenetic lineage within Ignisphaera genus. Our phylogenomic analysis also supports the delineation of the Ignisphaera genus into a separate family Ignisphaeraceae, as recently published. Here we propose a novel species Ignisphaera cupida, sp. nov. with type strain 4213-coT (=JCM 39446T=VKM B-3715T=UQM 41593T). Ecogenomic analysis showed that representatives of the Ignisphaera are thermophilic archaea, the majority of them were found in terrestrial hot springs and deep-sea hydrothermal vents. This study allowed a better understanding of physiology and ecology of Ignisphaeraceae - a rather understudied archaeal group.

Deciphering the geochemical influences on bacterial diversity and communities among two Algerian hot springs.

Northeastern Algeria boasts numerous hot springs, yet these hydrothermal sites remain largely unexplored for their microbial ecology. The present study explores the bacterial abundance and diversity within two distinct Algerian hot springs (Hammam Saïda and Hammam Debagh) and investigates the link between the prevailing bacteria with geochemical parameters. High-throughput 16S rRNA gene sequencing of water and sediment samples revealed a bacterial dominance of 99.85-91.16% compared to Archaea (0.14-0.66%) in both springs. Interestingly, Saïda hot spring, characterized by higher temperatures and sodium content, harbored a community dominated by Pseudomonadota (51.13%), whereas Debagh, a Ca-Cl-SO4 type spring, was primarily populated by Bacillota with 55.33%. Bacteroidota displayed even distribution across both sites. Additional phyla, including Chloroflexota, Deinococcota, Cyanobacteriota, and Chlorobiota, were also present. Environmental factors, particularly temperature, sodium, potassium, and alkalinity, significantly influenced bacterial diversity and composition. These findings shed light on the interplay between distinct microbial communities and their associated geochemical properties, providing valuable insights for future research on biogeochemical processes in these unique ecosystems driven by distinct environmental conditions, including potential applications in bioremediation and enzyme discovery.

Microbial Ecology of an Arctic Travertine Geothermal Spring: Implications for Biosignature Preservation and Astrobiology.

Jotun springs in Svalbard, Norway, is a rare warm environment in the Arctic that actively forms travertine. In this study, we assessed the microbial ecology of Jotun's active (aquatic) spring and dry spring transects. We evaluated the microbial preservation potential and mode, as well as the astrobiological relevance of the travertines to marginal carbonates mapped at Jezero Crater on Mars (the Mars 2020 landing site). Our results revealed that microbial communities exhibited spatial dynamics controlled by temperature, fluid availability, and geochemistry. Amorphous carbonates and silica precipitated within biofilm and on the surface of filamentous microorganisms. The water discharged at the source is warm, with near neutral pH, and undersaturated in silica. Hence, silicification possibly occurred through cooling, dehydration, and partially by a microbial presence or activities that promote silica precipitation. CO2 degassing and possible microbial contributions induced calcite precipitation and travertine formation. Jotun revealed that warm systems that are not very productive in carbonate formation may still produce significant carbonate buildups and provide settings favorable for fossilization through silicification and calcification. Our findings suggest that the potential for amorphous silica precipitation may be essential for Jezero Crater's marginal carbonates because it significantly increases the preservation potential of putative martian organisms.

Identifying the genetic mechanism of medium-low temperature fluoride-enriched geothermal groundwater by the self-organizing map and evaluating health risk in the Wugongshan area, southeast China.

Fluoride-enriched groundwater is a serious threat for groundwater supply around the world. The medium-low temperature fluoride-enriched geothermal groundwater resource is widely distributed in the circum-Wugongshan area. And the fluoride concentration of all geothermal samples exceeds the WHO permissible limit of 1.5 mg/L. The Self-Organizing Map method, hydrochemical and isotopic analysis are used to decipher the driving factors and genetic mechanism of fluoride-enriched geothermal groundwater. A total of 19 samples collected from the circum-Wugongshan geothermal belt are divided into four clusters by the self-organizing map. Cluster I, Cluster II, Cluster III, and Cluster IV represent the geothermal groundwater with the different degree of fluoride concentration pollution, the different hydrochemical type, and the physicochemical characteristic. The high F- concentration geothermal groundwater is characterized by HCO3-Na with alkalinity environment. The δD and δ18O values indicate that the geothermal groundwater origins from the atmospheric precipitation with the recharge elevation of 1000-2100 m. The dissolution of fluoride-bearing minerals is the main source of fluoride ions in geothermal water. Moreover, groundwater fluoride enrichment is also facilitated by water-rock interaction, cation exchange and alkaline environment. Additionally, the health risk assessment result reveals that the fluorine-enriched geothermal groundwater in the western part of Wugongshan area poses a more serious threat to human health than that of eastern part. The fluoride health risks of geothermal groundwater for different group show differentiation, 100% for children, 94.74% for adult females, and 68.42% for adult males, respectively. Compared with adult females and adult males, children faced the greatest health risks. The results of this study provide scientific evaluation for the utilization of geothermal groundwater and the protection of human health around the Wugongshan area.

Characterization of thermostable carboxypeptidase from high-altitude hot spring metagenome.

This study explored the metagenome of the Pir Panjal Hot Spring (PPHS) to identify thermostable hydrolases. The carboxypeptidase (CarP) gene was successfully amplified and cloned into Escherichia coli DH5-α cells, followed by expression in E. coli BL21-DE3 cells. The CarP enzyme was comprehensively characterized in vitro. Sequencing analysis revealed an open reading frame encoding a functional protein of 504 amino acids, with a molecular weight of 58.65 kDa and an isoelectric point of 4.81. The CarP protein was purified using Ni-His affinity chromatography, and the experimental molecular weight matched in silico predictions. The enzyme exhibited significant thermostability and alkaliphilic properties, with optimal activity at 70 °C and pH 10.0. Additionally, the presence of Zn+2 ions at concentrations of 5 and 10 mmol/L enhanced protease activity by 1.4 and 1.5-fold, respectively. This study reports the discovery of a novel, multifunctional, and thermostable CarP from hot-spring metagenomes. The enzyme's stability against high temperatures, metal ions, surfactants, and inhibitors, along with its specific substrate interactions, highlights its potential for various biotechnological applications.

Phylogenomic analyses and comparative genomic studies of Thermus strains isolated from Tengchong and Tibet Hot Springs, China.

Genus Thermus is the main focus of researcher among the thermophiles. Members of this genus are the inhabitants of both natural and artificial thermal environments. We performed phylogenomic analyses and comparative genomic studies to unravel the genomic diversity among the strains belonging to the genus Thermus in geographically different thermal springs. Sixteen Thermus strains were isolated and sequenced from hot springs, Qucai hot springs in Tibet and Tengchong hot springs in Yunnan, China. 16S rRNA gene based phylogeny and phylogenomic analyses based on concatenated set of 971 Orthologous Protein Families (supermatrix and gene content methods) revealed a mixed distribution of the Thermus strains. Whole genome based phylogenetic analysis showed, all 16 Thermus strains belong to five species; Thermus oshimai (YIM QC-2-109, YIM 1640, YIM 1627, 77359, 77923, 77838), Thermus antranikianii (YIM 73052, 77412, 77311, 71206), Thermus brokianus (YIM 73518, 71318, 72351), Thermus hydrothermalis (YIM 730264 and 77927) and one potential novel species 77420 forming clade with Thermus thalpophilus SYSU G00506T. Although the genomes of different strains of Thermus of same species were highly similar in their metabolic pathways, but subtle differences were found. CRISPR loci were detected through genome-wide screening, which showed that Thermus isolates from two different thermal locations had well developed defense system against viruses and adopt similar strategy for survival. Additionally, comparative genome analysis screened competence loci across all the Thermus genomes which could be helpful to acquire DNA from environment. In the present study it was found that Thermus isolates use two mechanism of incomplete denitrification pathway, some Thermus strains produces nitric oxide while others nitrious oxide (dinitrogen oxide), which show the heterotrophic lifestyle of Thermus genus. All isolated organisms encoded complete pathways for glycolysis, tricarboxylic acid and pentose phosphate. Calvin Benson Bassham cycle genes were identified in genomes of T. oshimai and T. antranikianii strains, while genomes of all T. brokianus strains and organism 77420 were lacking. Arsenic, cadmium and cobalt-zinc-cadmium resistant genes were detected in genomes of all sequenced Thermus strains. Strains 77,420, 77,311, 73,518, 77,412 and 72,351 genomes were found harboring genes for siderophores production. Sox gene clusters were identified in all sequenced genomes, except strain YIM 730264, suggesting a mode of chemolithotrophy. Through the comparative genomic analysis, we also identified 77420 as the genome type species and its validity as novel organism was confirmed by whole genome sequences comparison. Although isolate 77420 had 99.0% 16S rRNA gene sequence similarity with T. thalpophilus SYSU G00506T but based on ANI 95.86% (Jspecies) and digital DDH 68.80% (GGDC) values differentiate it as a potential novel species. Similarly, in the phylogenomic tree, the novel isolate 77,420 forming a separate branch with their closest reference type strain T. thalpophilus SYSU G00506T.

Optimization and Characterization of an Ultra-Thermostable, Acidophilic, Cellulase-Free Xylanase from a New Obligate Thermophilic Geobacillus thermoleovorans AKNT10 and its Application in Saccharification of Wheat Bran.

Microbial xylanases are enzymes of great importance due to their wide industrial applications, especially in the degradation of lignocellulosic biomass into fermentable sugars. This study aimed to describe the production optimization and partial characterization of an ultra-thermostable, acidophilic, cellulase-free xylanase from an obligate thermophilic eubacterium Geobacillus thermoleovorans strain-AKNT10 (Ac.No. LT158229) isolated from a hot-spring of Puga Valley located at an altitude of 4419 m in Ladakh, India. The optimization of cultural conditions improved enzyme yield by 10.49-fold under submerged fermentation. The addition of 1% (w/v) xylose induced the enzyme synthesis by ~ 165 and 371% when supplemented in the fermentation medium containing wheat bran (WB) 1 and 3%, respectively. The supplementation of sucrose reduced the xylanase production by ~ 25%. Results of partial characterization exhibited that xylanase was optimally active at pH 6.0 and 100 °C. Enzyme retained > 75%, > 83%, and > 84% of activity at 4 °C for 28 days, 100 °C for 60 min, and pHs 3-8 for 60 min, respectively. An outstanding property of AKNT10-xylanase, was the retention of > 71% residual activity at extreme conditions (121 °C and 15 psi pressure) for 15 min. Enzymatic saccharification showed that enzyme was also capable to liberate maximum reducing sugars within 4-8 h under optimized conditions thus it could be a potential candidate for the bioconversion of lignocellulosic biomass as well as other industrial purposes. To the best of our knowledge, this is the first report on such an ultra-thermo-pressure-tolerant xylanase optimally active at pH 6 and 100 °C from the genus Geobacillus.

Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).

Cultivation and visualization of a methanogen of the phylum Thermoproteota.

Methane is the second most abundant climate-active gas, and understanding its sources and sinks is an important endeavour in microbiology, biogeochemistry, and climate sciences1,2. For decades, it was thought that methanogenesis, the ability to conserve energy coupled to methane production, was taxonomically restricted to a metabolically specialized group of archaea, the Euryarchaeota1. The discovery of marker genes for anaerobic alkane cycling in metagenome-assembled genomes obtained from diverse habitats has led to the hypothesis that archaeal lineages outside the Euryarchaeota are also involved in methanogenesis3-6. Here we cultured Candidatus Methanosuratincola verstraetei strain LCB70, a member of the archaeal class Methanomethylicia (formerly Verstraetearchaeota) within the phylum Thermoproteota, from a terrestrial hot spring. Growth experiments combined with activity assays, stable isotope tracing, and genomic and transcriptomic analyses demonstrated that this thermophilic archaeon grows by means of methyl-reducing hydrogenotrophic methanogenesis. Cryo-electron tomography revealed that Ca. M. verstraetei are coccoid cells with archaella and chemoreceptor arrays, and that they can form intercellular bridges connecting two to three cells with continuous cytoplasm and S-layer. The wide environmental distribution of Ca. M. verstraetei suggests that they might play important and hitherto overlooked roles in carbon cycling within diverse anoxic habitats.