Prevalence of trace gas-oxidizing soil bacteria increases with radial distance from Polloquere hot spring within a high-elevation Andean cold desert.

High-elevation arid regions harbor microbial communities reliant on metabolic niches and flexibility to survive under biologically stressful conditions, including nutrient limitation that necessitates the utilization of atmospheric trace gases as electron donors. Geothermal springs present "oases" of microbial activity, diversity, and abundance by delivering water and substrates, including reduced gases. However, it is unknown whether these springs exhibit a gradient of effects, increasing their impact on trace gas-oxidizers in the surrounding soils. We assessed whether proximity to Polloquere, a high-altitude geothermal spring in an Andean salt flat, alters the diversity and metabolic structure of nearby soil bacterial populations compared to the surrounding cold desert. Recovered DNA and metagenomic analyses indicate that the spring represents an oasis for microbes in this challenging environment, supporting greater biomass with more diverse metabolic functions in proximal soils that declines sharply with radial distance from the spring. Despite the sharp decrease in biomass, potential rates of atmospheric hydrogen (H2) and carbon monoxide (CO) uptake increase away from the spring. Kinetic estimates suggest this activity is due to high-affinity trace gas consumption, likely as a survival strategy for energy/carbon acquisition. These results demonstrate that Polloquere regulates a gradient of diverse microbial communities and metabolisms, culminating in increased activity of trace gas-oxidizers as the influence of the spring yields to that of the regional salt flat environment. This suggests the spring holds local importance within the context of the broader salt flat and potentially represents a model ecosystem for other geothermal systems in high-altitude desert environments.

Chelatococcus albus sp. nov., a bacterium isolated from hot spring microbial mat.

A Gram-stain-negative, non-endospore-forming, motile, short rod-shaped strain, designated SYSU G07232T, was isolated from a hot spring microbial mat, sampled from Rehai National Park, Tengchong, Yunnan Province, south-western China. Strain SYSU G07232T grew at 25-50 °C (optimum, 37 °C), at pH 5.5-9.0 (optimum, pH 6.0) and tolerated NaCl concentrations up to 1.0 % (w/v). Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SYSU G07232T showed closest genetic affinity with Chelatococcus daeguensis K106T. The genomic features and taxonomic status of this strain were determined through whole-genome sequencing and a polyphasic approach. The predominant quinone of this strain was Q-10. Major cellular fatty acids comprised C19 : 0 cyclo ω8c and summed feature 8. The whole-genome length of strain SYSU G07232T was 4.02 Mbp, and the DNA G+C content was 69.26 mol%. The average nucleotide identity (ANIm ≤84.85 % and ANIb ≤76.08  %) and digital DNA-DNA hybridization (≤ 21.9 %) values between strain SYSU G07232T and the reference species were lower than the threshold values recommended for distinguishing novel prokaryotic species. Thus, based on the provided phenotypic, phylogenetic, and genetic data, it is proposed that strain SYSU G07232T (=KCTC 8141T=GDMCC 1.4178T) be designated as representing a novel species within the genus Chelatococcus, named Chelatococcus albus sp. nov.

Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov.: Novel Oculatellacean genera (Cyanobacteria) isolated from desert soils and hot spring.

To increase the understanding of simple thin filamentous cyanobacteria in harsh environmental areas, we previously isolated and identified four strains (XN101, XN102, GS121, NX122) from desert soils and hot spring in China. As a result, two new Oculatellacean genera of these four strains, Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov., are described based on a polyphasic approach. The ultrastructure of these strains showed a similar arrangement of peripheral thylakoids with three to four parallel layers, indicating that they belonged to the orders Nodosilineales, Oculatellales, or Leptolyngbyales. In the 16S rRNA gene phylogeny, two sequences of the Gansulinema strains and the two sequences of the Komarkovaeasiopsis strains formed two independent and robust clusters, within the order Oculatellales. The 16S rRNA gene sequences of strains of Komarkovaeasiopsis and Gansulinema showed low identity to each other (≤93.2%) and to other sequences of the Oculatellacean genera (≤94.5% and ≤93.3%, respectively). Furthermore, the 16S-23S internal transcribed spacer rRNA region secondary structures of strains of Komarkovaeasiopsis and Gansulinema were not consistent with all existing descriptions of Oculatellacean taxa. These data suggest that cyanobacterial communities are rich sources of new taxa in under-exploited areas, such as desert soils and hot spring in China.

Rhodococcus indonesiensis sp. nov. a new member of the Rhodococcus ruber lineage isolated from sediment of a neutral hot spring and reclassification of Rhodococcus electrodiphilus (Ramaprasad et al. 2018) as a later heterotypic synonym of Rhodococcus ruber (Kruse 1896) Goodfellow and Alderson 1977 (Approved Lists 1980).

A polyphasic study was designed to determine the taxonomic status of isolate CSLK01-03T, which was recovered from an Indonesian neutral hot spring and provisionally assigned to the genus Rhodococcus. The isolate was found to have chemotaxonomic, cultural and morphological properties typical of rhodococci. It has a rod-coccus lifecycle and grows from 10 to 39 °C, from pH 6.5 to 8.0 and in the presence of 0-10 % (w/v) sodium chloride. Whole-organism hydrolysates contain meso-diaminopimelic acid, arabinose and galactose, the predominant menaquinone is MK-8 (H2), the polar lipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylmethylethanolamine and two unidentified components, it produces mycolic acids, and C16:0 is the major fatty acid. Whole-genome analyses show that the isolate and Rhodococcus electrodiphilus LMG 29881T (GenBank accession: JAULCK000000000) have genome sizes of 5.5 and 5.1 Mbp, respectively. These strains and Rhodococcus aetherivorans DSM 44752T and Rhodococcus ruber DSM 43338T form well-supported lineages in 16S rRNA and whole-genome trees that are close to sister lineages composed of the type strains of Rhodococcus rhodochrous and related Rhodococcus species. The isolate can be distinguished from its closest evolutionary neighbours using combinations of cultural and phenotypic features, and by low DNA-DNA hybridization values. Based on these data it is proposed that isolate CSLK01-03T (=CCMM B1310T=ICEBB-06T=NCIMB 15214T) be classified in the genus Rhodococcus as Rhodococcus indonesiensis sp. nov. The genomes of the isolate and its closest phylogenomic relatives are rich in biosynthetic gene clusters with the potential to synthesize new natural products, notably antibiotics. In addition, whole-genome-based taxonomy revealed that Rhodococcus electrodiphilus LMG 29881T and Rhodococcus ruber DSM 43338T belong to a single species. It is, therefore, proposed that R. electrodiphilus be recognized as a heterotypic synonym of R. ruber.

Characterization of the Type 2 L-Asparaginase Gene in Thermohalophilic Bacterial from Wawolesea Hot Springs, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> Type 2 L-asparaginase enzyme can be used as a cancer therapy agent and prevent acrylamide formation in food products. Enzymes produced by thermohalophilic bacteria can provide high activity at high temperatures so they are needed on an industrial scale. Hence, this study aims to determine the characteristics of the gene encoding type 2 L-asparaginase enzyme in the thermohalophilic bacterial isolate CAT3.4. <b>Materials and Methods:</b> This research is a type of exploratory research. The characteristics of the gene encoding type 2 L-asparaginase were determined using the PCR technique using the primer pairs AsnBac2-F2 (5'-CTCACGGGAATCTCCATAACTC-3') and AsnBac2-R2 (5'CAGCGATGTAACAGACAGCATC-3'). The characterization process was carried out in stages: Isolation of genomic DNA using a modified alkali-lysis method, nucleotide and protein similarity analysis using BLASTn analysis on the NCBI website, construction of a phylogenetic tree using the MEGAX program, restriction enzyme mapping and amino acid analysis using the Bioedit program. <b>Results:</b> The characterization results showed that the PCR product has a size of 1594 bp with a CDS of 1128 bp, has a similarity value of 100% with <i>Bacillus subtilis</i>, has seven restriction enzymes as molecular markers for the type 2 L-asparaginase gene at the species level: <i>Bsr</i>GI, <i>Dra</i>I, <i>Eco</i>RV, <i>Hind</i>III, <i>Hpy</i>CH4IV , <i>Ssp</i>I and <i>Tai</i>I, have dominant hydrophilic regions and are in the same subclass as <i>Bacillus subtilis</i> strain GOT9. <b>Conclusion:</b> The target gene was similar to the gene encoding type 2 L-asparaginase from <i>Bacillus subtilis</i> with a max identity of 98.85%, query coverage value of 100% and E-value of 0.

Spatial distribution of radon contamination in hot springs water and its cancer and non-cancer risks in the Hunza-Nagar valley, Pakistan.

Radon (222Rn) is a ubiquitous radioactive gas and could threaten human life due to its potential for cancer and non-cancer risks. This study examined the measurement of 222Rn concentration and associated health risks in the hot springs of Hunza-Nagar valley. For this purpose, the hot springs water of Hunza and Nagar districts and the background sites were analyzed for 222Rn concentration using the RAD7 detector (Durridge Company, USA). The average concentrations of 222Rn were 46.1 ± 0.94, 65.3 ± 0.45, and 5.47 ± 0.25 Bq/L in the Hunza district, Nagar district, and background sites, respectively. Results showed that 222Rn concentrations of hot springs water were multifold higher than the background sites. 222Rn concentrations for hot springs water in Hunza-Nagar valley had surpassed the maximum contamination level set by the US environmental protection agency (USEPA). Humans' annual mean exposure dose rates of various age groups were calculated for the estimated lifetime cancer risk (ELCR) and non-cancer risks. The total annual mean exposure doses from 222Rn in water (EwTotal) values were (187 ± 3.80, 265 ± 1.84, and 22.2 ± 1.02 µSv/a) for infants (143 ± 2.92, 203 ± 1.40, and 17.0 ± 0.78 µSv/a) children, and (138 ± 2.80, 196 ± 1.35, and 16.4 ± 0.76 µSv/a) adults in the Hunza district, Nagar district, and background, respectively. Among the age groups of humans, infants showed a higher risk than others. Results showed that hot springs water consumption surpassed the world health organization threshold of 100 µSv/y for chronic or non-cancer and USEPA 0.1 × 10-3 for ELCR risks. The concentration of 222Rn showed a positive correlation (> 0.68) with hot springs' water temperature and pH suggesting a common origin.

Description of Brachybacterium sillae sp. nov., a thermophilic bacterium isolated from a hot spring.

The taxonomic position of strain EF45031T, isolated from the Neungam Carbonate hot spring, was examined using the polyphasic taxonomic approach. Strain EF45031T shared the highest percentage of 16S rRNA gene sequence with Brachybacterium nesterenkovii CIP 104813 T (97.7%). The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between strain EF45031T and the type strains B. nesterenkovii CIP 104813 T and B. phenoliresistens Phenol-AT were 77.0%, 69.15%, 21.9% and 75.73%, 68.81%, 20.5%, respectively. Phylogenomic analysis using an up-to-date bacterial core gene (UBCG) set revealed that strain EF45031T belonged to the genus Brachybacterium. Growth occurred between 25 and 50 ℃ at pH 6.0-9.0 and could tolerate salinity up to 5% (w/v). Strain had anteiso-C15:0 and anteiso-C17:0 as major fatty acids. Menaquinone-7 (MK-7) was the predominant respiratory menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, three aminolipids, and two unidentified glycolipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid as a diagnostic diamino acid. The genome comprised 2,663,796 bp, with a G + C content of 70.9%. Stress-responsive periplasmic chaperone/protease coding genes were identified in the genome of EF45031T and were not detected in other Brachybacterium species. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Brachybacterium, for which the name Brachybacterium sillae sp. nov. is proposed. The type strain is EF45031T (= KCTC 49702 T = NBRC 115869 T).

Prospects of a hot spring-originated novel cyanobacterium, Scytonema ambikapurensis, for wastewater treatment and exopolysaccharide-enriched biomass production.

The present work performs the polyphasic characterization of a novel cyanobacterial species Scytonema ambikapurensis isolated from an Indian hot spring and evaluates its wastewater bioremediation potential. While the physicochemical analyses of the wastewater indicated high load of nutrients and metals, the wastewater bioremediation experiment performed using the test cyanobacterium denoted the removal of 70 and 86% phosphate, 49 and 66% sulfate, 96 and 98% nitrate, 91 and 92% nitrite, 95 and 96% ammonia, 66 and 72% chloride, 79 and 81% zinc, 68 and 80% nickel, 81 and 90% calcium, and 80 and 90% potassium from the autoclaved and un-autoclaved wastewater, respectively, after 20 days of culturing. The kinetics study of zinc and nickel removal from wastewater revealed that the cyanobacterium employed sequential biosorption (by following pseudo-second-order kinetics model) and bioaccumulation methods to remove these two metals. The quality of the autoclaved and un-autoclaved wastewater was further improved by the cyanobacterium through reduction of hardness by 74 and 81%, respectively. In wastewater, the cyanobacterium not only enhanced its biomass, chlorophyll and carbohydrate contents, but also produced small amount of released and high capsular exopolysaccharide (EPS). The FTIR and TGA analyses of capsular EPS unraveled that it was a negatively charged sulfated biomolecule having thermostability up to 240 °C, which suggested its possible use as excellent emulsifying, viscosifying, and biosorption agent. The credibility of this EPS as biosorption agent was ascertained by evaluating its metal chelating ability. Finally, the experimental data denoting the ability of S. ambikapurensis to bioremediate wastewater and simultaneously produce EPS was statistically validated by PCA1-pollutant removal model and the PCA2-cellular constituent model, respectively. Briefly, the study discloses that the cyanobacterium has huge biotechnological and industrial importance as it bioremediates wastewater and simultaneously produces thermostable exopolysaccharide.

Panguiarchaeum symbiosum, a potential hyperthermophilic symbiont in the TACK superphylum.

The biology of Korarchaeia remains elusive due to the lack of genome representatives. Here, we reconstruct 10 closely related metagenome-assembled genomes from hot spring habitats and place them into a single species, proposed herein as Panguiarchaeum symbiosum. Functional investigation suggests that Panguiarchaeum symbiosum is strictly anaerobic and grows exclusively in thermal habitats by fermenting peptides coupled with sulfide and hydrogen production to dispose of electrons. Due to its inability to biosynthesize archaeal membranes, amino acids, and purines, this species likely exists in a symbiotic lifestyle similar to DPANN archaea. Population metagenomics and metatranscriptomic analyses demonstrated that genes associated with amino acid/peptide uptake and cell attachment exhibited positive selection and were highly expressed, supporting the proposed proteolytic catabolism and symbiotic lifestyle. Our study sheds light on the metabolism, evolution, and potential symbiotic lifestyle of Panguiarchaeum symbiosum, which may be a unique host-dependent archaeon within the TACK superphylum.

Age-dependent potential health risk assessment due to radioactive radon-222 in the environs of highly populated Durgapur industrial zone and nearby Bakreswar hot spring, India.

It is well known that exposure to a high concentration of radon-222 causes severe health effects, including cancer. The present article includes a survey on radon-222 in the water bodies of the city Durgapur [non-geothermal area] and nearby Bakreswar hot spring [geothermal province], India. The possible sources of radon from natural radionuclides and industries have been discussed in the article. Durgapur is a densely populated [~ 3680 persons/km2] industrial city with a population of 0.57 million. On the other hand, many tourists and pilgrims usually visit Bakreswar throughout the year. Age-dependent potential health risk assessments of the dwellers at Durgapur and Bakreswar due to radon exposure were performed for the first time. The present work is the first attempt to estimate the mean ingestion /and inhalation dose per annum, total effective dose [TED] per annum and the health risk assessment for cancer in adults, children and infants due to radon exposure at Durgapur and Bakreswar. In some cases, the values of TED exceed the permissible limit of 100 micro Sievert per year [µSv/y] as recommended by EUC and WHO. The radiation profile maps relating to radon concentration and associated contour maps of health risk factors [HRF] for the adults, children and infants were also prepared for the first time. Some areas were identified as high-risk zones, and the dwellers are prone to a high risk of cancer. The article also proposed several techniques to reduce radon in water and buildings. The authors also recommended banning some water sources to protect people from radon risk. This study will help scientists, policymakers, industrialists, farmers, government agencies and public health departments.

Paenibacillus hamazuiensis sp. nov., a bacterium isolated from Hamazui hot spring in Yunnan province, south-west China.

A bacterial strain, Gram-positive, aerobic, rod-shaped, motile, designated YIM B00624T which was isolated from a Hamazui hot spring in Tengchong, Yunnan province, south-west China. The strain grew well on International Streptomyces Project (ISP) 2 medium and colonies were creamy yellow, flat and circular. The results of 16S rRNA gene sequence similarity analysis showed that strain YIM B00624T was closely related to the type strain of Paenibacillus filicis S4T (95.9%). The main menaquinone of strain YIM B00624T was menaquinone-7 (MK-7) and major fatty acids were anteiso-C15:0, anteiso-C17:0 and C16:0. The isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and four unidentified glycolipids. The DNA G+C content of strain YIM B00624T was 53.4 mol%. Based on physiological, phenotypic and chemotaxonomic data, strain YIM B00624T belongs to a novel species of the genus Paenibacillus, for which the name Paenibacillus hamazuiensis sp. nov. is proposed. The type strain is YIM B00624T (= CGMCC 1.19245T = KCTC 43365T).

Thermal Endurance by a Hot-Spring-Dwelling Phylogenetic Relative of the Mesophilic Paracoccus.

High temperature growth/survival was revealed in a phylogenetic relative (SMMA_5) of the mesophilic Paracoccus isolated from the 78 to 85°C water of a Trans-Himalayan sulfur-borax spring. After 12 h at 50°C, or 45 min at 70°C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained ~2% colony forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50°C and 70°C, respectively. After 12 h at 50°C, the thermally conditioned sibling SMMA_5_TC exhibited an ~1.5 time increase in CFU count; after 45 min at 70°C, SMMA_5_TC had 7% of the initial CFU count. 1,000-times diluted Reasoner's 2A medium, and MST supplemented with lithium, boron, or glycine-betaine, supported higher CFU-retention/CFU-growth than MST. Furthermore, with or without lithium/boron/glycine-betaine, a higher percentage of cells always remained metabolically active, compared with what percentage formed single colonies. SMMA_5, compared with other Paracoccus, contained 335 unique genes: of these, 186 encoded hypothetical proteins, and 83 belonged to orthology groups, which again corresponded mostly to DNA replication/recombination/repair, transcription, secondary metabolism, and inorganic ion transport/metabolism. The SMMA_5 genome was relatively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells' energy and resources toward system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped inherit those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCE For a holistic understanding of microbial life's high-temperature adaptation, it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat-specific inorganic/organic solutes, and potential genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multifaceted.

Stygiolobus caldivivus sp. nov., a facultatively anaerobic hyperthermophilic archaeon isolated from the Unzen hot spring in Japan.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KN-1T, was isolated from Unzen hot spring in Japan and characterized. The cells of KN-1T were irregular cocci with a diameter of 1.0-3.0 µm that grew at 55-87.5 °C (optimum: 75 °C) and pH 1.0-5.5 (optimum: 3.0). Chemolithoautotrophic growth of KN-1T occurred in the presence of S0 or H2 under oxic conditions. Under anoxic conditions, KN-1T grew with S0, ferric citrate and FeCl3 as electron acceptors. A phylogenetic analysis of 16S rRNA gene sequences showed that the species most closely related to KN-1T was Stygiolobus azoricus JCM 9 021T, with 98.9 % sequence identity, indicating that strain KN-1T belongs to the genus Stygiolobus. This genus has been considered to consist of obligate anaerobes since its description in 1991. However, KN-1T grew under oxic, microoxic and anoxic conditions. Moreover, KN-1Tutilized various complex substrates and some sugars as carbon or energy sources, which is also different from S. azoricus JCM 9 021T. The average nucleotide identity and amino acid identity values between KN-1T and S. azoricus JCM 9 021T were 79.4 and 76.1 %, respectively, indicating that KN-1T represents a novel species. Its main polar lipids were calditoglycerocaldarchaeol and caldarchaeol, and its DNA G+C content was 40.1 mol%. We also found that S. azoricus JCM 9021T grew under microoxic conditions in the presence of H2 as an electron donor, indicating that this genus does not comprise obligate anaerobes. Based on this polyphasic taxonomic analysis, we propose the novel species, Stygiolobus caldivivus sp. nov., whose type strain is KN-1T (=JCM 34 622T=KCTC 4 293T).

Actinospica acidithermotolerans sp. nov., a novel actinomycete isolated from sediment from an Indonesian hot spring.

A polyphasic study was designed to resolve the taxonomic position of isolate MGRD01-02T which was recovered from an acidic hot spring in Indonesia and assigned to the genus Actinospica. Phylogenetic analyses based on 16S rRNA gene sequences show that the isolate is most closely related to the type strains of Actinospica acidiphila (98.5%), Actinospica robiniae (97.8%) and Actinospica durhamensis (96.8%). Morphological and chemotaxonomic data underpin the assignment of the isolate to the genus Actinospica as it forms an extensively branched substrate mycelium which carries tufts of white aerial hyphae that differentiate into straight to flexuous chains of cylindrical spores with faint rugose surfaces, contains 2,6-diamino-3-hydroxydiaminopimelic acid in the peptidoglycan, mixtures of hydrogenated menaquinones with nine isoprene units, iso-C 15:O and iso-C 16:O as major fatty acids and phosphatidylethanolamine as the diagnostic phospholipid. Whole-genome sequence analyses show that the isolate, A. durhamensis CSCA 57T and Actinocrinis puniceicyclus DSM 45168T have genome sizes of 7.9, 9.6 and 6.7 Mbp, respectively. A phylogenomic tree shows that they form distinct branches in a well-supported clade, a result supported by associated phenotypic data. Average nucleotide identity and digital DNA:DNA hybridization similarities are below the recommended thresholds for assigning strains to the same species; they also indicate that isolate MGRD01-02T is most closely related to the A. durhamensis and A. robiniae strains. Corresponding amino acid identity and conserved protein data not only support these relationships but also confirm the taxonomic integrity of the genus Actinocrinis. Based on these results, it is proposed that isolate MGRD01-02T (= CCMM B1308T = ICEBB-09T = NCIMB 15218T) be classified in the genus Actinospica as Actinospica acidithermotolerans sp. nov. The draft genome of the isolate and its closest phylogenomic neighbours contain biosynthetic gene clusters with the potential to produce new natural products, notably antibiotics.

Thermomonas flagellata sp. nov. and Thermomonas alba sp. nov., two novel members of the phylum Pseudomonadota isolated from hot spring sediments.

Two novel species, designated strains SYSU G04041T and SYSU G04536T, were isolated from hot spring sediments collected in Yunnan, PR China. Phenotypic and chemotaxonomic analyses, and whole-genome sequencing were used to determine the taxonomic positions of the candidate strains. Phylogenetic analysis using 16S rRNA gene sequence indicated that strain SYSU G04041T showed the highest sequence similarity to Thermomonas haemolytica A50-7-3T (97.5 %), and SYSU G04536T showed the highest sequence similarity to Thermomonas hydrothermalis SGM-6T (98.2 %). The strains could be differentiated from other species of the genus Thermomonas by their distinct phenotypic and genotypic characteristics. Cells of strains SYSU G04041T and SYSU G04536T were aerobic, motile and Gram-stain-negative. Growth both occurred optimally at 45 °C and pH 7.0 for SYSU G04041T and SYSU G04536T. In addition, the predominant respiratory quinone in both isolates was ubiquinone Q-8. The major fatty acids (>10 %) of strain SYSU G04041T were C16 : 0, iso-C15 : 0 and iso-C16 : 0, while the major fatty acids (>10 %) of strain SYSU G04536T were iso-C15 : 0 and iso-C16 : 0. The main detected polar lipids in strains SYSU G04041T and SYSU G04536T included phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The G+C contents of the genomic DNA of strains SYSU G04041T and SYSU G04536T based on draft genomic sequences were 72.5 and 68.3 %, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strains SYSU G04041T and SYSU G04536T represent two novel species of the genus Thermomonas, for which the names Thermomonas flagellata sp. nov. and Thermomonas alba sp. nov. are proposed, with the type strains SYSU G04041T (=CGMCC 1.19366T=KCTC 92228T) and SYSU G04536T (=CGMCC 1.19367T=KCTC 82839T), respectively.

Hydrogenophilus thiooxidans sp. nov., a moderately thermophilic chemotrophic bacterium unable to grow on hydrogen gas, isolated from hot spring microbial mats.

A novel thermophilic chemotrophic bacterium, strain SS56T, was isolated from Nakabusa Hot Spring, Japan. The isolate was a rod-shaped (1.5-2.1×0.6-0.8 µm), Gram-stain-negative bacterium. The cells of this strain grew chemoheterotrophically under aerobic and anaerobic conditions. Autotrophic growth was observed with thiosulphate and elemental sulphur under aerobic conditions but not with H2 as the electron donor. Heterotrophic growth in the presence of O2 occurred on yeast extract, tryptone, polypeptone and organic acids. Strain SS56T used nitrite as an alternative electron acceptor under anaerobic chemoheterotrophic conditions. The isolate grew between 35 and 65 °C, with the optimum at 55 °C. The pH range for growth was pH 6.0-9.0; optimal growth occurred at pH 7.0-8.0. The 16S rRNA gene sequence of strain SS56T was 98.9% identical to that of Hydrogenophilus thermoluteolus TH-1T. The draft genome sequence of 2401804 bp for strain SS56T gave values of 53.7% for digital DNA-DNA hybridization, 92.9% for average nucleotide identity and 93.6% for average amino acid identity compared with the genome sequence of 2223143 bp for H. thermoluteolus TH-1T. Based on the information described above, strain SS56T (=DSM 111892T=JCM 34254T) is proposed as the type strain of a novel species, Hydrogenophilus thiooxidans sp. nov.

Rhodoflexus caldus gen. nov., sp. nov., a new member of the phylum Bacteroidota isolated from a hot spring sediment.

A thermophilic bacterium, designated strain SYSU G04325T, was isolated from a hot spring sediment in Yunnan, China. Polyphasic taxonomic analyses and whole-genome sequencing were used to determine the taxonomic position of the strain. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain SYSU G04325T shows high sequence similarity to Thermoflexibacter ruber NBRC 16677T (86.2%). The strain can be differentiated from other species of the family Thermoflexibacteraceae by its distinct phenotypic and genotypic characteristics. Cells of the strain SYSU G04325T were observed to be aerobic, Gram-stain negative and filamentous. Growth was found to occur optimally at 45 ºC and pH 7.0. In addition, the respiratory quinone was identified as menaquinone-7, while the major fatty acids (> 10%) were identified as iso-C15:0, iso-C17:0 and Summed Feature 9 (iso-C17:1ω9c). The polar lipids detected included phosphatidylethanolamine, three unidentified phospholipids, one unidentified glycolipid, five unidentified aminolipids and four unidentified polar lipids. The G + C content of the genomic DNA was determined to be 47.6% based on the draft genome sequence. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU G04325T is concluded to represent a novel species of a novel genus in the family Thermoflexibacteraceae, for which the name Rhodoflexus caldus gen. nov., sp. nov. is proposed. The type strain of Rhodoflexus caldus is SYSU G04325T (= MCCC 1K06127T = KCTC 82848T).

Thermus brevis sp. nov., a moderately thermophilic bacterium isolated from a hot spring microbial mat.

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001T, SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001T. Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 and anteiso-C15 : 0. The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001T (=KCTC 82627T=MCCC 1K06118T) represents a novel species of the genus Thermus, for which the name Thermus brevis sp. nov. is proposed.

Phenotypic Characterization and Identification of Potential L-Asparaginase-Producing Thermohalophilic Bacteria from Wawolesea Hot Spring, North Konawe, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> L-asparaginase-producing thermohalophilic bacteria have the potential of producing an enzyme tolerant to high heat and salt levels. This enzyme, L-asparaginase, can be used as a biological agent for the cancer therapy of acute lymphoblastic leukemia and melanosarcoma as it has a specific ability to inhibit the formation of nutrients for cancer cells. This enzyme is also used effectively in food industries operating at high temperatures due to its ability to reduce acrylamide, a trigger of cancer cells. This study sought to figure out the phenotypic characters of and identify potential L-asparaginase-producing thermohalophilic bacteria from Wawolesea Hot Spring, North Konawe, Southeast Sulawesi. <b>Materials and Methods:</b> The characterization conducted on potential L-asparaginase-producing thermohalophilic bacterial isolates consisted of the following: Colony morphological characterization, covering the shapes, edges, internal structures, elevations and colours of the colonies, cell morphological characterization, covering gram staining, endospore formation and motility, biochemical characterization, covering catalase, Methyl Red and Voges Proskauer (MR-VP), gelatin hydrolysis, citrate, indole and carbohydrate fermentation tests and physiological characterization, covering pH effect, salinity, oxygen demand and temperature effect tests. Bacterial isolate identification was carried out in two stages, namely phenetic identification based on the phenotypic characterization data determine through a preliminary identification and numeric-phenetic identification. <b>Results:</b> The characterization results showed that the bacterial isolates AAT 1.4, AAT 3.2 and CAT 3.4 were <i>bacillus</i>-shaped, Gram-positive, motile, catalase-positive and aerobic. Based on the numeric-phenetic analysis results, the isolates AAT 1.4 and CAT 3.4 had a 92.9% similarity to <i>Bacillus subtilis</i>, while isolate AAT 3.2 had a 92.9% similarity to <i>Brevibacillus limnophilus</i>. <b>Conclusion:</b> According to the numeric-phenetic analysis results, the isolates AAT 1.4 and CAT 3.4 belong to the species <i>Bacillus subtilis</i>, while isolate AAT 3.2 belongs to the species <i>Brevibacillus limnophilus</i>.

Caldovatus aquaticus sp. nov., a moderately thermophilic bacterium isolated from hot spring microbial mat.

Two closely related, aerobic, Gram-stain-negative, motile, oval-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05006T and SYSU G05005, were isolated from a bioreactor enrichment and the original sample was collected from Rehai National Park, Tengchong, Yunnan Province, PR China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these two strains were closely related to Caldovatus sediminis YIM 72346T (96.75 and 96.89 % sequence similarity, respectively). The whole genome size of strain SYSU G05006T was 3.87 Mbp with a DNA G+C content of 75.33 mol%. The average nucleotide identity (ANI based on the MUMmer algorithm≤90.31 % and ANI based on blast≤89.36 %) and digital DNA-DNA hybridization (≤35.10 %) values between strain SYSU G05006T and other members of the family Acetobacteraceae were all lower than the threshold values recommended for distinguishing novel prokaryotic species. Optimal growth of the strain was observed at 55 °C and pH 6.0. Ubiquinone-10 was the predominant respiratory lipoquinone. The major cellular fatty acids included iso-C14 : 0, C16 : 1 ω5c, summed feature 5 and summed feature 7. The major polar lipids consisted of diphosphatidylglycerol, one unidentified aminolipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified phospholipids and two unidentified lipids. Based on results of phylogenetic analyses, comparative genomics and phenotypic characteristics, we describe a new species of the genus Caldovatus represented by strain SYSU G05006T (=KCTC 82831T=MCCC 1K06125T), for which we propose the name Caldovatus aquaticus sp. nov.