Microbial taxa related to natural hydrogen and methane emissions in serpentinite-hosted hyperalkaline springs of New Caledonia.

The southeastern part of New Caledonia main island (Grande Terre) is the location of a large ophiolitic formation that hosts several hyperalkaline springs discharging high pH (∼11) and warm (<40°C) fluids enriched in methane (CH4) and hydrogen (H2). These waters are produced by the serpentinization of the ultrabasic rock formations. Molecular surveys had previously revealed the prokaryotic diversity of some of these New Caledonian springs, especially from the submarine chimneys of Prony Bay hydrothermal field. Here we investigate the microbial community of hyperalkaline waters from on-land springs and their relationships with elevated concentrations of dissolved H2 (21.1-721.3 µmol/L) and CH4 (153.0-376.6 µmol/L). 16S rRNA gene analyses (metabarcoding and qPCR) provided evidence of abundant and diverse prokaryotic communities inhabiting hyperalkaline fluids at all the collected springs. The abundance of prokaryotes was positively correlated to the H2/CH4 ratio. Prokaryotes consisted mainly of bacteria that use H2 as an energy source, such as microaerophilic Hydrogenophaga/Serpentinimonas (detected in all sources on land) or anaerobic sulfate-reducing Desulfonatronum, which were exclusively found in the most reducing (Eh ref H2 ∼ -700 mV) and the most H2-enriched waters discharging at the intertidal spring of the Bain des Japonais. The relative abundance of a specific group of uncultured Methanosarcinales that thrive in serpentinization-driven ecosystems emitting H2, considered potential H2-consuming methanogens, was positively correlated with CH4 concentrations, and negatively correlated to the relative abundance of methylotrophic Gammaproteobacteria. Firmicutes were also numerous in hyperalkaline waters, and their relative abundance (e.g., Gracilibacter or Dethiobacter) was proportional to the dissolved H2 concentrations, but their role in the H2 budget remains to be assessed. The prokaryotic communities thriving in New Caledonia hyperalkaline waters are similar to those found in other serpentinite-hosted high-pH waters worldwide, such as Lost City (North Atlantic) and The Cedars (California).

Genome-Resolved Metagenomics and Metatranscriptomics Reveal that Aquificae Dominates Arsenate Reduction in Tengchong Geothermal Springs.

Elevated arsenic (As) is common in geothermal springs, shaping the evolution of As metabolism genes and As transforming microbes. Herein, genome-level microbial metabolisms and As cycling strategies in Tengchong geothermal springs were demonstrated for the first time based on metagenomic and metatranscriptomic analyses. Sulfur cycling was dominated by Aquificae oxidizing thiosulfate via the sox system, fueling the respiration and carbon dioxide fixation processes. Arsenate reduction via arsC [488.63 ± 271.60 transcripts per million (TPM)] and arsenite efflux via arsB (442.98 ± 284.81 TPM) were the primary detoxification pathway, with most genes and transcripts contributed by the members in phylum Aquificae. A complete arsenotrophic cycle was also transcriptionally active as evidenced by the detection of aioA transcripts and arrA transcript reads mapped onto metagenome-assembled genomes (MAGs) affiliated with Crenarchaeota. MAGs affiliated with Aquificae had great potential of reducing arsenate via arsC and fixing nitrogen and carbon dioxide via nifDHK and reductive tricarboxylic acid (rTCA) cycle, respectively. Aquificae's arsenate reduction potential via arsC was observed for the first time at the transcriptional level. This study expands the diversity of the arsC-based arsenate-reducing community and highlights the importance of Aquificae to As biogeochemistry.

Bacterial community structure in geothermal springs on the northern edge of Qinghai-Tibet plateau.

Introduction: In order to reveal the composition of the subsurface hydrothermal bacterial community in the zones of magmatic tectonics and their response to heat storage environments. Methods: In this study, we performed hydrochemical analysis and regional sequencing of the 16S rRNA microbial V4-V5 region in 7 Pleistocene and Lower Neogene hot water samples from the Gonghe basin. Results: Two geothermal hot spring reservoirs in the study area were found to be alkaline reducing environments with a mean temperature of 24.83°C and 69.28°C, respectively, and the major type of hydrochemistry was SO4-Cl·Na. The composition and structure of microorganisms in both types of geologic thermal storage were primarily controlled by temperature, reducing environment intensity, and hydrogeochemical processes. Only 195 ASVs were shared across different temperature environments, and the dominant bacterial genera in recent samples from temperate hot springs were Thermus and Hydrogenobacter, with both genera being typical of thermophiles. The correlation analysis showed that the overall level of relative abundance of the subsurface hot spring relied on a high temperature and a slightly alkaline reducing environment. Nearly all of the top 4 species in the abundance level (53.99% of total abundance) were positively correlated with temperature and pH, whereas they were negatively correlated with ORP (oxidation-reduction potential), nitrate, and bromine ions. Discussion: In general, the composition of bacteria in the groundwater in the study area was sensitive to the response of the thermal storage environment and also showed a relationship with geochemical processes, such as gypsum dissolution, mineral oxidation, etc.

Microbial Diversity of Terrestrial Geothermal Springs in Armenia and Nagorno-Karabakh: A Review.

The microbial diversity of high-altitude geothermal springs has been recently assessed to explore their biotechnological potential. However, little is known regarding the microbiota of similar ecosystems located on the Armenian Highland. This review summarizes the known information on the microbiota of nine high-altitude mineralized geothermal springs (temperature range 25.8-70 °C and pH range 6.0-7.5) in Armenia and Nagorno-Karabakh. All these geothermal springs are at altitudes ranging from 960-2090 m above sea level and are located on the Alpide (Alpine-Himalayan) orogenic belt, a seismically active region. A mixed-cation mixed-anion composition, with total mineralization of 0.5 mg/L, has been identified for these thermal springs. The taxonomic diversity of hot spring microbiomes has been examined using culture-independent approaches, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene library construction, 454 pyrosequencing, and Illumina HiSeq. The bacterial phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes are the predominant life forms in the studied springs. Archaea mainly include the phyla Euryarchaeota, Crenarchaeota, and Thaumarchaeota, and comprise less than 1% of the prokaryotic community. Comparison of microbial diversity in springs from Karvachar with that described for other terrestrial hot springs revealed that Proteobacteria, Bacteroidetes, Actinobacteria, and Deinococcus-Thermus are the common bacterial groups in terrestrial hot springs. Contemporaneously, specific bacterial and archaeal taxa were observed in different springs. Evaluation of the carbon, sulfur, and nitrogen metabolism in these hot spring communities has revealed diversity in terms of metabolic activity. Temperature seems to be an important factor in shaping the microbial communities of these springs. Overall, the diversity and richness of the microbiota are negatively affected by increasing temperature. Other abiotic factors, including pH, mineralization, and geological history, also impact the structure and function of the microbial community. More than 130 bacterial and archaeal strains (Bacillus, Geobacillus, Parageobacillus, Anoxybacillus, Paenibacillus, Brevibacillus Aeribacillus, Ureibacillus, Thermoactinomyces, Sporosarcina, Thermus, Rhodobacter, Thiospirillum, Thiocapsa, Rhodopseudomonas, Methylocaldum, Desulfomicrobium, Desulfovibrio, Treponema, Arcobacter, Nitropspira, and Methanoculleus) have been reported, some of which may be representative of novel species (sharing 91-97% sequence identity with their closest matches in GenBank) and producers of thermozymes and biomolecules with potential biotechnological applications. Whole-genome shotgun sequencing of T. scotoductus K1, as well as of the potentially new Treponema sp. J25 and Anoxybacillus sp. K1, were performed. Most of the phyla identified by 16S rRNA were also identified using metagenomic approaches. Detailed characterization of thermophilic isolates indicate the potential of the studied springs as a source of biotechnologically valuable microbes and biomolecules.

PCR-based identification of thermotolerant free-living amoebae in Italian hot springs.

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans.

Defining Culture Conditions for the Hidden Nitrite-Oxidizing Bacterium Nitrolancea.

Nitrification is a key process for N-removal in engineered and natural environments, but recent findings of novel nitrifying microorganisms with surprising features revealed that our knowledge of this functional guild is still incomplete. Especially nitrite oxidation - the second step of nitrification - is catalyzed by a phylogenetically diverse bacterial group, and only recently bacteria of the phylum Chloroflexi have been identified as thermophilic nitrite-oxidizing bacteria (NOB). Among these, Nitrolancea hollandica was isolated from a laboratory-scale nitrifying bioreactor operated at 35°C with a high load of ammonium bicarbonate. However, its distribution remains cryptic as very few closely related environmental 16S rRNA gene sequences have been retrieved so far. In this study, we demonstrate how such thermophilic NOB can be enriched using modified mineral media inoculated with samples from a wastewater side-stream reactor operated at 39.5°C. Distinct cultivation conditions resulted in quick and reproducible high enrichment of two different strains of Nitrolancea, closely related to Nl. hollandica. The same cultivation approach was applied to a complex nitrite-oxidizing pre-enrichment at 42°C inoculated with biomass from a geothermal spring in the Copahue volcano area in Neuquen, Argentina. Here, an additional distinct representative of the genus Nitrolancea was obtained. This novel species had 16S rRNA and nitrite oxidoreductase alpha subunit (nxrA) gene sequence identities to Nl. hollandica of 98.5% and 97.2%, respectively. A genomic average nucleotide identity between the Argentinian strain and Nl. hollandica of 91.9% indicates that it indeed represents a distinct species. All Nitrolancea cultures formed lancet-shaped cells identical to Nl. hollandica and revealed similar physiological features, including the capability to grow at high nitrite concentrations. Growth was optimal at temperatures of 35-37°C and was strongly enhanced by ammonium supplementation. Genomic comparisons revealed that the four Nitrolancea strains share 2399 out of 3387 orthologous gene clusters and encode similar key functions. Our results define general growth conditions that enable the selective enrichment of Nitrolancea from artificial and natural environments. In most natural habitats these NOB apparently are of low abundance and their proliferation depends on the balanced presence of nitrite and ammonium, with an optimal incubation temperature of 37°C.

Occurrence and Health-Risk Assessment of Trace Metals in Geothermal Springs within Soutpansberg, Limpopo Province, South Africa.

Geothermal springs are natural geological phenomena that occur throughout the world. South Africa is blessed with several springs of this nature. Limpopo province contains 31% of all geothermal springs in the country. The springs are classified according to the residing mountain: Soutpansberg, Waterberg and Drakensberg. This study focused on the geothermal springs within the Soutpansberg region; that is, Mphephu, Siloam, Sagole and Tshipise. The study was aimed at assessing the occurrence and potential health risk associated with drinking water from geothermal springs within Soutpansberg. Geothermal springs and boreholes were sampled for a period of 12 months (May 2017-May 2018) to accommodate two major seasons in the study areas. The physicochemical and trace metal compositions of the geothermal springs and boreholes (tepid and hot) were analyzed using ion chromatography (IC) (Dionex Model DX 500) and inductively coupled plasma-mass spectrometer (ICP-MS). Trace metal concentrations of the geothermal springs and boreholes were within permissible drinking water guidelines by the South African National Standards (SANS) and World Health Organisation (WHO), with exception of mercury (Hg), which is high in summer season. The bioaccumulation from regular consumption could, however, result in negative effects. Pearson's correlation revealed that there is a direct relationship between temperature and pH, and some of the trace metals (V, Zn, Hg, Pb). This implies dissolution of minerals (rock-water interaction) under slightly high temperature. Multivariate statistics further elucidate the relationship and possible sources of the trace metals. Therefore, it can be inferred that the rock-water interaction is the main geochemical process governing the release of trace metals in groundwater. Hazard Index values for both children and adults were higher than 1, and this implies that the communities are at high risk of non-cancer health effects. Further, As, Cr and Cd were found to be the highest contributors to the potential cancer risk in the study areas, with children having a higher risk than adults. Therefore, there is a need for clinical/epidemiological study, and regular monitoring and control measures, to verify actual prevalence of cancer and protect human health, particularly the children, within the study areas.

Geothermal springs in Armenia and Nagorno-Karabakh: potential sources of hydrolase-producing thermophilic bacilli.

In recent years, scientists have increasingly focused on the microbial diversity of high-altitude hot springs to explore the biotechnological applications of extremophiles. In this regard, a total of 107 thermophilic bacilli were isolated from 9 high-altitude mineralized geothermal springs (of temperatures ranging from 27.5 to 70 °C) located within the territory of Armenia and Nagorno-Karabakh. The isolated bacilli were phylogenetically profiled and studied for their potential to produce extracellular hydrolytic enzymes (protease, amylase, and lipase). The identification of isolates based on 16S rRNA gene sequences revealed their relationship to members of more than 22 distinct species, of 8 different genera, namely Aeribacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Parageobacillus, Paenibacillus and Ureibacillus. Bacillus licheniformis, Parageobacillus toebii and Anoxybacillus flavithermus were found to be the most abundant species in the springs that were studied. Some of the isolated bacilli shared less than 91-97% sequence identity with their closest match in GenBank, indicating that Armenian geothermal springs harbor novel bacilli, at least at the species level. 71% of the isolates actively produced at least one or more extracellular proteases, amylases, or lipases. In total, 22 strains (28.6%) were efficient producers of all three types of thermostable enzymes.

Phylogenetic Diversity of Nitrogenase Reductase Genes and Possible Nitrogen-Fixing Bacteria in Thermophilic Chemosynthetic Microbial Communities in Nakabusa Hot Springs.

Chemosynthetic microbial communities develop and form dense cell aggregates in slightly alkaline sulfidic hot springs in the temperature range of 70-86°C at Nakabusa, Japan. Nitrogenase activity has recently been detected in the microbial communities collected. To identify possible members capable of nitrogen fixation, we examined the diversities of 16S rRNA and nitrogenase reductase (NifH) gene sequences in four types of chemosynthetic communities with visually different colors and thicknesses. The results of a 16S rRNA gene analysis indicated that all four microbial communities had similar bacterial constituents; the phylum Aquificae was the dominant member, followed in abundance by Thermodesulfobacteria, Firmicutes, and Thermotogae. Most of the NifH sequences were related to sequences reported in hydrothermal vents and terrestrial hot springs. The results of a phylogenetic analysis of NifH sequences revealed diversity in this gene among the communities collected, distributed within 7 phylogenetic groups. NifH sequences affiliated with Aquificae (Hydrogenobacter/Thermocrinis) and Firmicutes (Caldicellulosiruptor) were abundant. At least two different energy metabolic pathways appeared to be related to nitrogen fixation in the communities analyzed; aerobic sulfur/hydrogen-oxidizing bacteria in Aquificae and fermentative bacteria in Firmicutes. The metabolic characteristics of these two dominant phyla differed from those previously inferred from nitrogenase activity assays on chemosynthetic communities, which were associated with hydrogen-dependent autotrophic sulfate reduction. These assays may correspond to the observed NifH sequences that are distantly related to the known species of Thermodesulfovibrio sp. (Nitrospirae) detected in the present study. The activities of nitrogen-fixing organisms in communities may depend on redox states as well as the availability of electron donors, acceptors, and carbon sources.

Thermus sediminis sp. nov., a thiosulfate-oxidizing and arsenate-reducing organism isolated from Little Hot Creek in the Long Valley Caldera, California.

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0-2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA-DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C15:0, iso-C17:0 and anteiso-C15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by ß-galactosidase and ß-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198T (= CGMCC 1.13590T = KCTC XXX).

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

BACKGROUND: Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. RESULTS: In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. CONCLUSIONS: The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

A Combination of Extreme Environmental Conditions Favor the Prevalence of Endospore-Forming Firmicutes.

Environmental conditions unsuitable for microbial growth are the rule rather than the exception in most habitats. In response to this, microorganisms have developed various strategies to withstand environmental conditions that limit active growth. Endospore-forming Firmicutes (EFF) deploy a myriad of survival strategies in order to resist adverse conditions. Like many bacterial groups, they can form biofilms and detect nutrient scarcity through chemotaxis. Moreover, within this paraphyletic group of Firmicutes, ecophysiological optima are diverse. Nonetheless, a response to adversity that delimits this group is the formation of wet-heat resistant spores. These strategies are energetically demanding and therefore might affect the biological success of EFF. Therefore, we hypothesize that abundance and diversity of EFF should be maximized in those environments in which the benefits of these survival strategies offsets the energetic cost. In order to address this hypothesis, geothermal and mineral springs and drillings were selected because in these environments of steep physicochemical gradients, diversified survival strategies may become a successful strategy.We collected 71 samples from geothermal and mineral environments characterized by none (null), single or multiple limiting environmental factors (temperature, pH, UV radiation, and specific mineral composition). To measure success, we quantified EFF gene copy numbers (GCN; spo0A gene) in relation to total bacterial GCN (16S rRNA gene), as well as the contribution of EFF to community composition. The quantification showed that relative GCN for EFF reached up to 20% at sites characterized by multiple limiting environmental factors, whereas it corresponded to less than 1% at sites with one or no limiting environmental factor. Pyrosequencing of the 16S rRNA gene supports a higher contribution of EFF at sites with multiple limiting factors. Community composition suggested a combination of phylotypes for which active growth could be expected, and phylotypes that are most likely in the state of endospores, in all the sites. In summary, our results suggest that diversified survival strategies, including sporulation and metabolic adaptations, explain the biological success of EFF in geothermal and natural springs, and that multiple extreme environmental factors favor the prevalence of EFF.

Wide distribution of autochthonous branched glycerol dialkyl glycerol tetraethers (bGDGTs) in U.S. Great Basin hot springs.

Branched glycerol dialkyl glycerol tetraethers (bGDGTs) are membrane-spanning lipids that likely stabilize membranes of some bacteria. Although bGDGTs have been reported previously in certain geothermal environments, it has been suggested that they may derive from surrounding soils since bGDGTs are known to be produced by soil bacteria. To test the hypothesis that bGDGTs can be produced by thermophiles in geothermal environments, we examined the distribution and abundance of bGDGTs, along with extensive geochemical data, in 40 sediment and mat samples collected from geothermal systems in the U.S. Great Basin (temperature: 31-95°C; pH: 6.8-10.7). bGDGTs were found in 38 out of 40 samples at concentrations up to 824 ng/g sample dry mass and comprised up to 99.5% of total GDGTs (branched plus isoprenoidal). The wide distribution of bGDGTs in hot springs, strong correlation between core and polar lipid abundances, distinctness of bGDGT profiles compared to nearby soils, and higher concentration of bGDGTs in hot springs compared to nearby soils provided evidence of in situ production, particularly for the minimally methylated bGDGTs I, Ib, and Ic. Polar bGDGTs were found almost exclusively in samples ≤70°C and the absolute abundance of polar bGDGTs correlated negatively with properties of chemically reduced, high temperature spring sources (temperature, H2S/HS(-)) and positively with properties of oxygenated, low temperature sites (O2, NO(-) 3). Two-way cluster analysis and nonmetric multidimensional scaling based on relative abundance of polar bGDGTs supported these relationships and showed a negative relationship between the degree of methylation and temperature, suggesting a higher abundance for minimally methylated bGDGTs at high temperature. This study presents evidence of the widespread production of bGDGTs in mats and sediments of natural geothermal springs in the U.S. Great Basin, especially in oxygenated, low-temperature sites (≤70°C).