Different sulfide to arsenic ratios driving arsenic speciation and microbial community interactions in two alkaline hot springs.

Arsenic (As) is ubiquitous in geothermal fluids, which threatens both water supply safety and local ecology. The co-occurrence of sulfur (S) and As increases the complexity of As migration and transformation in hot springs. Microorganisms play important roles in As-S transformation processes. In the present study, two Tibetan alkaline hot springs (designated Gulu [GL] and Daba [DB]) with different total As concentrations (0.88 mg/L and 12.42 mg/L, respectively) and different sulfide/As ratios (3.97 and 0.008, respectively) were selected for investigating interactions between As-S geochemistry and microbial communities along the outflow channels. The results showed that As-S transformation processes were similar, although concentrations and percentages of As and S species differed between the two hot springs. Thioarsenates were detected at the vents of the hot springs (18% and 0.32%, respectively), and were desulfurized to arsenite along the drainage channel. Arsenite was finally oxidized to arsenate (532 µg/L and 12,700 µg/L, respectively). Monothioarsenate, total As, and sulfate were the key factors shaping the changes in microbial communities with geochemical gradients. The relative abundances of sulfur reduction genes (dsrAB) and arsenate reduction genes (arsC) were higher in upstream portions of GL explaining high thiolation. Arsenite oxidation genes (aoxAB) were relatively abundant in downstream parts of GL and at the vent of DB explaining low thiolation. Sulfur oxidation genes (soxABXYZ) were abundant in GL and DB. Putative sulfate-reducing bacteria (SRB), such as Desulfuromusa and Clostridium, might be involved in forming thioarsenates by producing reduced S for chemical reactions with arsenite. Sulfur-oxidizing bacteria (SOB), such as Elioraea, Pseudoxanthomonas and Pseudomonas, and arsenite-oxidizing bacteria (AsOB) such as Thermus, Sulfurihydrogenibium and Hydrogenophaga, may be responsible for the oxidation of As-bound S, thereby desulfurizing thioarsenates, forming arsenite and, by further abiotic or microbial oxidation, arsenate. This study improves our understanding of As and S biogeochemistry in hot springs.

Tibeticola sediminis gen. nov., sp. nov., a thermophilic bacterium isolated from a hot spring.

Two closely related thermophilic bacterial strains, designated YIM 73013T and YIM 73008, were isolated from a sediment sample collected from a hot spring in Tibet, western Tibet province, China. The taxonomic positions of the two isolates were investigated using a polyphasic approach. The novel isolates were Gram-stain-negative, aerobic, short-rod-shaped and motile by means of a polar flagellum. They were oxidase- and catalase-positive and were able to grow at 30-55 °C (optimum, 37-45 °C), at pH 6.0-8.0 (optimum, pH 7.0) and with NaCl tolerance up to 1 % (w/v). Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 73013T and YIM 73008 formed a distinct lineage with respect to closely related genera in the family Comamonadaceae and shared highest 16S rRNA gene sequences similarities with Acidovorax caeni R-24608T (96.3 and 96.4 %, respectively). The respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids observed were C17 : 1ω6c, C16 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The genomic DNA G+C contents of strains YIM 73013T and YIM 73008 were 68.7 and 68.3 mol%, respectively. Based on the morphological, phylogenetic and chemotaxonomic results, the two isolates represent a novel species in a new genus, for which the name Tibeticola sediminis gen. nov., sp. nov. is proposed. The type strain of Tibeticola sediminis is YIM 73013T (=DSM 101684T=KCTC 42873T).

Altererythrobacter lauratis sp. nov. and Altererythrobacter palmitatis sp. nov., isolated from a Tibetan hot spring.

Two Gram-negative, moderately thermophilic, yellow-pigmented, rod-shaped and motile bacterial strains, designated YIM 75003T and YIM 75004T, were isolated from sediment samples collected from the Tagejia hot spring in Tibet, western China. The taxonomic affiliation of the two strains was investigated by a polyphasic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strains YIM 75003T and YIM 75004T are closely related to Altererythrobacter buctense M0322T (97.2 and 97.1% respectively), while sharing 99.9% sequence similarity against each other. Optimum growth of the two strains was observed at 37-45 °C, pH 8.0 and in the presence of 1-6% NaCl (w/v). Their predominant respiratory quinone was found to be ubiquinone 10. The major fatty acids in both the strains were identified as summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and summed feature 4 (C17:1 anteiso B and/or iso I). Their major polar lipid profiles were found to be diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. The DNA G+C contents of strains YIM 75003T and YIM 75004T were determined to be 61.3 and 60.1 mol%, respectively. The DNA-DNA hybridization values between strains YIM 75003T and YIM 75004T, and between the two strains and their closely related phylogenetic neighbour A. buctense M0322T (=CGMCC 1.12871T) were less than 70%. Based on the morphological and physiological properties, phylogenetic analyses, chemotaxonomic characteristics and DNA-DNA relatedness values, the two strains can be distinguished from each other and from their phylogenetically closely related strain. Strains YIM 75003T and YIM 75004T are, therefore, considered to represent two novel species of the genus Altererythrobacter, for which the names Altererythrobacter lauratis sp. nov. (type strain YIM 75003T = KCTC 52606T = CCTCC AB2016268T) and Altererythrobacter palmitatis sp. nov. (type strain YIM 75004T = KCTC 52607T = CCTCC AB2016270T) are proposed.

Brevibacillus sediminis sp. nov., isolated from a hot spring.

Strain YIM 78300T, a novel Gram-stain-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacterium, was recovered from the sediment of a hot spring in the Tagejia Geothermal Field, Angren, Tibet province, western China. Optimum growth was observed at 50-55 °C, at pH 7.0 and with 0-1.5 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene sequence of strain YIM 78300T indicated that it belongs to the genus Brevibacillus. Similarity levels between the 16S rRNA gene sequences of the new isolate and those of the type strains of Brevibacillus members were 96.9-96.3 %; highest sequence similarity was with Brevibacillus thermoruber DSM 7064T. The predominant menaquinone was MK-7 and the major cellular fatty acids were iso-C15 : 0 and iso-C17 : 0. The major polar lipids were phosphatidyl-N-methylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two unidentified phospholipids, an unidentified aminophospholipid and two unidentified polar lipids. The G+C content of the genomic DNA of strain YIM 78300T was 57.9 mol%. Based on phylogenetic analyses, and physiological and biochemical characteristics, strain YIM 78300T is considered to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus sediminis sp. nov. is proposed. The type strain is YIM 78300T ( = DSM 29928T = CPCC 100738T).

Mesorhizobium sediminum sp. nov., isolated from deep-sea sediment.

A novel Gram-staining-negative, short rod, non-motile, non-spore-forming, aerobic bacterium, designated strain YIM M12096T, was isolated from deep-sea sediment collected from the Indian Ocean. Optimal growth conditions of the strain were observed at 25-30 °C, pH 6.0 and in the presence of 3-5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM M12096T was closely related to Nitratireductor indicus C115T (97.4 % 16S rRNA gene sequence similarity) and Mesorhizobium thiogangeticum SJTT (97.3 %). The strain, however, formed a robust clade with members of the genus Mesorhizobium in phylogenetic dendrograms generated with neighbour-joining, maximum-likelihood and maximum-parsimony trees. Analysis based on the sequence of housekeeping gene recA also gave a similar phylogenetic relationship, indicating that strain YIM M12096T is a member of the genus Mesorhizobium. DNA-DNA relatedness values between strain YIM M12096T and related type strains N. indicus CCTCC AB209298T and M. thiogangeticum DSM 17097T were 40.5 % and 36.7 %, respectively. Chemotaxonomic features of the isolate included phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminophospholipid and an unidentified phospholipid as its characteristic polar lipids and Q-10 as respiratory ubiquinone. Major fatty acids (>10 %) detected were C18 : 1ω7c and/or C18 : 1ω6c, 11-methyl-C18 : 1ω7c and cyclo-C19 : 0ω8c. Based on the chemotaxonomic properties and phylogenetic analyses, strain YIM M12096T is determined to be a member of the genus Mesorhizobium. The strain could be differentiated from the closely related species by the differences in physiological and biochemical properties supported by low DNA-DNA relatedness values. It is therefore concluded that strain YIM M12096T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobiumsediminum sp. nov. is proposed. The type strain is YIM M12096T (=CCTCC AB 2014219T=KCTC 42205T).

Abyssicoccus albus gen. nov., sp. nov., a novel member of the family Staphylococcaceae isolated from marine sediment of the Indian Ocean.

A Gram-stain positive, aerobic, non-motile, asporogenous, coccoid shaped bacterium, designated YIM M12140(T), was isolated from a marine sediment sample collected from the Indian Ocean. Phylogenetic analysis showed that strain YIM M12140(T) forms a separate clade within the family Staphylococcaceae. Strain YIM M12140(T) shares high 16S rRNA gene sequence similarity with Macrococcus brunensis DSM 19358(T) (92.9 %). The isolate was found to grow at 0-10 % (w/v) NaCl (optimum, 2-3 %), pH 6.0-10.0 (optimum, pH 8.0) and temperature 5-40 °C (optimum, 28 °C). The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, one unidentified aminophospholipid and two unidentified polar lipids. The major cellular fatty acids of the strain were identified as anteiso-C15:0, -C17:0, iso-C16:0, anteiso-C19:0 and C20:0. The respiratory menaquinones were found to be MK-6 (94 %) and MK-7 (6 %). The cell wall amino acids were found to contain Lys, Ala, Glu, Gly, Asp, Ser and Thr. Whole cell sugars were identified as mannose, ribose, rhamnose, glucose, galactose and xylose. The G+C content of the genomic DNA of strain YIM M12140(T) was determined to be 42.4 mol %. Based on phenotypic, chemotaxonomic data and phylogenetic analysis, it is proposed that strain YIM M12140(T) represents a novel species of a new genus in the family Staphylococcaceae, for which the name Abyssicoccus albus gen. nov., sp. nov. is proposed. The type strain is YIM M12140(T) (= DSM 29158(T) = CCTCC AB 2014213(T)).

Kocuria subflava sp. nov., isolated from marine sediment from the Indian Ocean.

A novel Gram-staining positive, catalase-positive, oxidase-negative, aerobic, non-motile coccus, designated strain YIM 13062(T), was isolated from a marine sediment sample collected from the Indian Ocean. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 13062(T) belongs to the genus Kocuria, and is closely related to Kocuria polaris NBRC 103063(T) (97.8 % similarity), Kocuria rosea NBRC 3768(T) (97.6 % similarity) and Kocuria carniphila JCM 14118(T) (97.4 % similarity). The strain grew optimally at 28 °C, pH 8.0 and in the presence of 2-4 % (w/v) NaCl. Cell-wall peptidoglycan type was Lys-Ala3 (type A3α). The major isoprenoid quinones were MK-6(H2) and MK-7(H2). The polar lipids of strain YIM 13062(T) consisted of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), one unidentified phospholipid (PL), one unidentified aminophospholipid (APL), two unidentified aminolipids (AL) and four unidentified lipids (L). Major fatty acids of the novel isolate were anteiso-C15:0, iso-C14:0 and C18:1 2OH. The genomic DNA G+C content of strain YIM 13062(T) was 68.0 mol%. The level of DNA-DNA relatedness between strain YIM 13062(T) and K. polaris NBRC 103063(T), K. rosea NBRC 3768(T), K. carniphila JCM 14118(T) were 53.2, 48.8 and 42.6 %, respectively. On the basis of genotypic and phenotypic data, it is apparent that strain YIM 13062(T) represents a novel species of the genus Kocuria, for which the name Kocuria subflava sp. nov. is proposed. The type strain is YIM 13062(T) (=CGMCC 4.7252(T)=KCTC 39547(T)).

Actinomadura amylolytica sp. nov. and Actinomadura cellulosilytica sp. nov., isolated from geothermally heated soil.

Two aerobic, Gram-positive actinomycetes, designated YIM 77502(T) and YIM 77510(T), were isolated from geothermally heated soil of Tengchong county, Yunnan province, south-west China. The taxonomic position of strains YIM 77502(T) and YIM 77510(T) were investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains YIM 77502(T) and YIM 77510(T) belong to the genus Actinomadura. Both strains form extensively-branched substrate and aerial mycelia which differentiated into short spore chains. The cell wall of the two strains contained meso-diaminopimelic acid, while the whole-cell sugars detected were glucose, madurose, mannose and rhamnose. The polar lipid profile of strain YIM 77502(T) was found to consist of diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unidentified phospholipids and an unidentified polar lipid, while strain YIM 77510(T) consisted of diphosphatidylglycerol, phosphatidylinositol mannoside and phosphatidylinositol. The respiratory quinones of strains YIM 77502(T) and YIM 77510(T) were MK-9(H6) and MK-9(H8). The major fatty acids (>10 %) of strain YIM 77502(T) were C17:0, iso-C16:0, C17:010-methyl and iso-C18:0, and those of strain YIM 77510(T) were iso-C16:0, C17:010-methyl and iso-C18:0. The G+C contents of strains YIM 77502(T) and YIM 77510(T) were determined to be 71.3 and 70.2 mol%, respectively. The DNA-DNA hybridization values of strains YIM 77502(T), YIM 77510(T) and their closest phylogenetic neighbours Actinomadura echinospora BCRC 12547(T) and Actinomadura umbrina KCTC 9343(T) were less than 70 %. Based on the morphological and physiological properties, and phylogenetic analyses, strains YIM 77502(T) and YIM 77510(T) are considered to represent two novel species of the genus Actinomadura, for which the names Actinomadura amylolytica sp. nov. (type strain YIM 77502(T) = DSM 45822(T) = CCTCC AA 2012024(T)) and Actinomadura cellulosilytica sp. nov. (type strain YIM 77510(T) = DSM 45823(T) = CCTCC AA 2012023(T)) are proposed.

Different Regulatory Strategies of Arsenite Oxidation by Two Isolated Thermus tengchongensis Strains From Hot Springs.

Arsenic is a ubiquitous constituent in geothermal fluids. Thermophiles represented by Thermus play vital roles in its transformation in geothermal fluids. In this study, two Thermus tengchongensis strains, named as 15Y and 15W, were isolated from arsenic-rich geothermal springs and found different arsenite oxidation behaviors with different oxidation strategies. Arsenite oxidation of both strains occurred at different growth stages, and two enzyme-catalyzed reaction kinetic models were observed. The arsenite oxidase of Thermus strain 15W performed better oxidation activity, exhibiting typical Michaelis-Menten kinetics. The kinetic parameter of arsenite oxidation in whole cell showed a V max of 18.48 µM min-1 and K M of 343 µM. Both of them possessed the arsenite oxidase-coding genes aioB and aioA. However, the expression of gene aioBA was constitutive in strain 15W, whereas it was induced by arsenite in strain 15Y. Furthermore, strain 15Y harbored an intact aio operon including the regulatory gene of the ArsR family, whereas a genetic inversion of an around 128-kbp fragment produced the inactivation of this regulator in strain 15W, leading to the constitutive expression of aioBA genes. This study provides a valuable insight into the adaption of thermophiles to extreme environments.

Arsenic mobilization in a high arsenic groundwater revealed by metagenomic and Geochip analyses.

Microbial metabolisms of arsenic, iron, sulfur, nitrogen and organic matter play important roles in arsenic mobilization in aquifer. In this study, microbial community composition and functional potentials in a high arsenic groundwater were investigated using integrated techniques of RNA- and DNA-based 16S rRNA gene sequencing, metagenomic sequencing and functional gene arrays. 16S rRNA gene sequencing showed the sample was dominated by members of Proteobacteria (62.3-75.2%), such as genera of Simplicispira (5.7-6.7%), Pseudomonas (3.3-5.7%), Ferribacterium (1.6-4.4%), Solimonas (1.8-3.2%), Geobacter (0.8-2.2%) and Sediminibacterium (0.6-2.4%). Functional potential analyses indicated that organics degradation, assimilatory sulfate reduction, As-resistant pathway, iron reduction, ammonification, nitrogen fixation, denitrification and dissimilatory nitrate reduction to ammonia were prevalent. The composition and function of microbial community and reconstructed genome bins suggest that high level of arsenite in the groundwater may be attributed to arsenate release from iron oxides reductive dissolution by the iron-reducing bacteria, and subsequent arsenate reduction by ammonia-producing bacteria featuring ars operon. This study highlights the relationship between biogeochemical cycling of arsenic and nitrogen in groundwater, which potentially occur in other aquifers with high levels of ammonia and arsenic.