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A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.

A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360kDa. Each TvNiR subunit with molecular mass of 64kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.

The existence of chemolithoautotrophic sulfur-oxidizing bacteria (SOB) capable of growth in an extremely alkaline and saline environment has not been recognized until recently. Extensive studies of saline, alkaline (soda) lakes located in Central Asia, Africa and North America have now revealed the presence, at relatively high numbers, of a new branch of obligately autotrophic SOB in these doubly extreme environments. Overall more than 100 strains were isolated in pure culture. All of them have the potential to grow optimally at around pH 10 in media strongly buffered with sodium carbonate/bicarbonate and cannot grow at pH < 7.5 and Na+ concentration

This paper summarizes recent data on the occurrence and properties of lithotrophic prokaryotes found in extremely alkaline, saline (soda) lakes. Among the chemolithotrophs found in these lakes the obligately autotrophic sulfur-oxidizing bacteria were the dominant, most diverse group, best adapted to haloalkaline conditions. The culturable forms are represented by three new genera, Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira in the Gammaproteobacteria. Among them, the genus Thioalkalivibrio was most metabolically diverse, including denitrifying, thiocyanate-oxidizing and facultatively alkaliphilic species. Culturable methane-oxidizing populations in the soda lakes belong to the type I methanotroph group in the Gammaproteobacteria, mostly in the genus Methylomicrobium. The nitrifying bacteria in hyposaline soda lakes were represented by a new species Nitrobacter alkalicus (Alphaproteobacteria), and by an alkaliphilic subspecies of Nitrosomonas halophila (Betaproteobacteria). Both belonged to the low salt-tolerant alkaliphiles. The facultatively autotrophic haloalkaliphilic isolates able to grow with hydrogen as electron donor were identified as representatives of the α-3 subclass of the Proteobacteria (aerobic) and of the Natronolimnicola – Alkalispirillum group in the gammaproteobacteria (nitrate-reducing). While all chemolithotrophic isolates from soda lakes belong to the alkaliphiles with a pH optimum for growth around 10, only the sulfur-oxidizing group included species able to grow under hypersaline conditions. This indicates that carbon and nitrogen cycles in the hypersaline alkaline lakes might not be closed.

Salt adaptation in chemolithotrophic alkaliphilic sulfur-oxidizing strains belonging to genera Thioalkalimicrobium and Thioalkalivibrio has been studied by determination of salt-dependent changes in fatty acid and compatible solute composition. In both alkaliphilic groups, represented by the low salt-tolerant Thioalkalimicrobium aerophilum strain AL 3T and the extremely salt-tolerant Thioalkalivibrio versutus strain ALJ 15, unsaturated fatty acids predominate over saturated fatty acids. In strain AL 3T, C18:1, C16:0 and C16:1 were the dominant fatty acids. In strain ALJ 15, the concentrations of C18:1 and C19cyclo were salt-regulated in an inverse proportional relationship, suggesting the stimulation of cyclopropyl-synthetase activity. Squalene has been found in substantial amounts only in strain ALJ 15. Ectoine and glycine betaine were found to be the main osmolytes in Thioalkalimicrobium aerophilum and Thioalkalivibrio versutus, respectively. The production of ectoine and glycine betaine was positively correlated with the salt concentration in the growth medium. A novel type of membrane-bound yellow pigments was uniformly detected in the extremely salt-tolerant strains of Thioalkalivibrio with a backbone consisting of C15-polyene, whose specific concentration correlated with increasing salinity of the growth medium. The results suggest that the mechanisms of haloalkaliphilic adaptation in Thioalkalimicrobium sp. and Thioalkalivibrio sp. involve the production of cyclopropane fatty acids, organic compatible solutes and, possibly specific pigments.

We have studied the activity and composition of several geochemically significant physiological groups of bacteria in more than twenty alkaline salt lakes of the north-east Mongolia steppe with water salinity from 3 to 390 g l−1 and pH values ranging from 9.0 to 10.6. Active and diverse microbial communities have been found in most of the lakes. The methanotrophic bacteria were represented by the Type I members. Among the culturable forms of sulfur-oxidizing bacteria obligately chemolithoautotrophic and haloalkaliphilic representatives of the genera Thioalkalimicrobium and Thioalkalivibrio were detected in the sediments at high numbers (up to 106 cells ml−1). The largest population of anaerobic phototrophic bacteria was represented by purple sulfur bacteria of the Ectothiorhodospiraceae family. Salinity was the key factor in determining the activity and the composition of the microbial communities. The most diverse and active prokaryotic populations, including aerobic and anaerobic phototrophic, methanogenic, methanotrophic, sulfur-oxidizing, sulfate-reducing and nitrifying bacteria, were found in lakes with salinity less than 60 g l−1. In hypersaline lakes with a salinity >100 g l−1, the sulfur cycle remained active due to the activity of extremely halotolerant and alkaliphilic sulfur bacteria, while other important functional groups responsible for nitrification and methane oxidation processes were not detected. Overall, the prokaryotic communities of the Mongolian alkaline salt lakes represent an interesting new example of a diverse community of haloalkaliphilic bacteria well adopted to a broad salinity range.

Two strains of obligate chemolithoautotrophic sulfur-oxidizing bacteria were isolated from soda-lake sediments by enrichment culture with thiocyanate and nitrate at pH 9·9. The isolates were capable of growth with thiocyanate or thiosulfate as electron donor, either aerobically or anaerobically, and with nitrate or nitrite as electron acceptor. Cyanate was identified as an intermediate of thiocyanate oxidation, while sulfate, ammonia and dinitrogen gas were the final products. The anaerobic growth on thiocyanate plus nitrate was much slower (μmax=0·006 h−1) than on thiosulfate plus nitrate (μmax=0·02 h−1), while growth yields were similar (4·8 and 5·1 g protein mol−1, respectively). On the basis of their phenotypic and genetic properties, strains ARhD 1Tand ARhD 2 are described as a novel species of the genusThialkalivibrio, with the highest similarity toThialkalivibrio denitrificans. The nameThialkalivibrio thiocyanodenitrificanssp. nov. is proposed for this novel species.

Strain ALEN 2(T) was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake. The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules. The bacterium was obligately chemolithoautotrophic. It grew with reduced sulfur compounds aerobically and anaerobically with nitrate as electron acceptor, nitrate being reduced to nitrite. It was moderately halophilic and obligately alkaliphilic. On the basis of genetic analysis and its unique phenotype, strain ALEN 2 T ( = DSM 14787(T) = UNIQEM 213(T)) is proposed as the type strain of a novel species of the genus Thialkalivibrio, Thialkalivibrio nitratireducens.

Eight anaerobic enrichment cultures with thiosulfate as electron donor and nitrate as electron acceptor were inoculated with sediment samples from hypersaline alkaline lakes of Wadi Natrun (Egypt) at pH 10; however, only one of the cultures showed stable growth with complete nitrate reduction to dinitrogen gas. The thiosulfate-oxidizing culture subsequently selected after serial dilution developed in two phases. Initially, nitrate was mostly reduced to nitrite, with a coccoid morphotype prevailing in the culture. During the second stage, nitrite was reduced to dinitrogen gas, accompanied by mass development of thin motile rods. Both morphotypes were isolated in pure culture and identified as representatives of the genus Thioalkalivibrio, which includes obligately autotrophic sulfur-oxidizing haloalkaliphilic species. Nitrate-reducing strain ALEN 2 consisted of large nonmotile coccoid cells that accumulated intracellular sulfur. Its anaerobic growth with thiosulfate, sulfide, or polysulfide as electron donor and nitrate as electron acceptor resulted in the formation of nitrite as the major product. The second isolate, strain ALED, was able to grow anaerobically with thiosulfate as electron donor and nitrite or nitrous oxide (but not nitrate) as electron acceptor. Overall, the action of two different sulfur-oxidizing autotrophs resulted in the complete, thiosulfate-dependent denitrification of nitrate under haloalkaliphilic conditions. This process has not yet been demonstrated for any single species of chemolithoautotrophic sulfur-oxidizing haloalkaliphiles.

A novel molybdenum-free nitrate reductase was isolated from the obligate chemolithoautotrophic and facultative anaerobic, (halo)alkaliphilic sulphur-oxidizing bacterium Thioalkalivibrio nitratireducens strain ALEN 2. The enzyme was found to contain vanadium and haem c as cofactors. Its native molecular mass was determined as 195kDa, and the enzyme consists of four identical subunits with apparent molecular masses of 57kDa. Apart from nitrate, the enzyme can utilize nitrite, chlorate, bromate, selenate and sulphite as electron acceptors. Moreover, it also has a haloperoxidase activity.

Nine strains of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria able to grow with thiocyanate (SCN-) as the sole energy and nitrogen source were isolated from soda lakes in South-East Siberia, Kenya and Egypt after enrichment on sodium carbonate minerals buffered at pH 10 with thiocyanate as the substrate. The isolates fell into two groups that were substantially different in terms of cell morphology, growth parameters and the ability to oxidize carbon disulfide. The bacteria were able to oxidize sulfide, polysulfide, sulfur and tetrathionate, as well as thiocyanate. Two isolates belonged to an extremely halotolerant type growing in the presence of up to 4 M Na+. Cyanate (CNO-) was the main nitrogen-containing intermediate during thiocyanate degradation in both groups. According to DNA-DNA hybridization data and phylogenetic analysis, both groups of isolates belong to a recently described genus of haloalkaliphilic sulfur-oxidizing bacteria, i.e. Thioalkalivibrio, belonging to the gamma-Proteobacteria, in which where they represent two new species. The species name Thioalkalivibrio paradoxus (type strain ARh 1T = DSM 13531T = JCM 11367T) is proposed for the group with barrel-shaped cells, and the species name Thioalkalivibrio thiocyanoxidans (type strain ARh 2T, DSM 13532T = JCM 11368T) is proposed for the group with vibrio-shaped cells. The diagnosis of the genus Thioalkalivibrio is amended according to the new data.

© 2017 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Thioalkalivibrio is a genus of obligate chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria. Their habitat are soda lakes which are dual extreme environments with a pH range from 9.5 to 11 and salt concentrations up to saturation. More than 100 strains of this genus have been isolated from various soda lakes all over the world, but only ten species have been effectively described yet. Therefore, the assignment of the remaining strains to either existing or novel species is important and will further elucidate their genomic diversity as well as give a better general understanding of this genus. Recently, the genomes of 76 Thioalkalivibrio strains were sequenced. On these, we applied different methods including (i) 16S rRNA gene sequence analysis, (ii) Multilocus Sequence Analysis (MLSA) based on eight housekeeping genes, (iii) Average Nucleotide Identity based on BLAST (ANIb) and MUMmer (ANIm), (iv) Tetranucleotide frequency correlation coefficients (TETRA), (v) digital DNA:DNA hybridization (dDDH) as well as (vi) nucleotide- and amino acid-based Genome BLAST Distance Phylogeny (GBDP) analyses. We detected a high genomic diversity by revealing 15 new "genomic" species and 16 new "genomic" subspecies in addition to the ten already described species. Phylogenetic and phylogenomic analyses showed that the genus is not monophyletic, because four strains were clearly separated from the other Thioalkalivibrio by type strains from other genera. Therefore, it is recommended to classify the latter group as a novel genus. The biogeographic distribution of Thioalkalivibrio suggested that the different "genomic" species can be classified as candidate disjunct or candidate endemic species. This study is a detailed genome-based classification and identification of members within the genus Thioalkalivibrio. However, future phenotypical and chemotaxonomical studies will be needed for a full species description of this genus.

So far, anaerobic sulfate-dependent acetate oxidation at high pH has only been demonstrated for a low-salt-tolerant syntrophic association of a clostridium ‘CandidatusContubernalis alkalaceticum’ and its hydrogenotrophic sulfate-reducing partnerDesulfonatronum cooperativum. Anaerobic enrichments at pH 10 inoculated with sediments from hypersaline soda lakes of the Kulunda Steppe (Altai, Russia) demonstrated the possibility of sulfate-dependent acetate oxidation at much higher salt concentrations (up to 3.5 M total Na+). The most salt-tolerant purified cultures contained two major components apparently working in syntrophy. The primary acetate-fermenting component was identified as a member of the orderClostridialesforming, together with ‘Ca.Contubernalis alkalaceticum’, an independent branch within the familySyntrophomonadaceae. A provisional name, ‘Ca.Syntrophonatronum acetioxidans’, is suggested for the novel haloalkaliphilic clostridium. Two phylotypes of extremely haloalkaliphilic sulfate-reducing bacteria of the genusDesulfonatronospirawere identified as sulfate-reducing partners in the acetate-oxidizing cultures under extreme salinity. The dominant phylotype differed from the two species ofDesulfonatronospiradescribed so far, whilst a minor component belonged toDesulfonatronumthiodismutans. The results proved that, contrary to previous beliefs, sulfate-dependent acetate oxidation is possible, albeit very slowly, in nearly saturated soda brines.

A novel nitrite-oxidizing bacterium (NOB), strain LbT, was isolated from a nitrifying bioreactor with a high loading of ammonium bicarbonate in a mineral medium with nitrite as the energy source. The cells were oval (lancet-shaped) rods with pointed edges, non-motile, Gram-positive (by staining and from the cell wall structure) and non-spore-forming. Strain LbTwas an obligately aerobic, chemolitoautotrophic NOB, utilizing nitrite or formate as the energy source and CO2as the carbon source. Ammonium served as the only source of assimilated nitrogen. Growth with nitrite was optimal at pH 6.8–7.5 and at 40 °C (maximum 46 °C). The membrane lipids consisted of C20alkyl 1,2-diols with the dominant fatty acids being 10MeC18and C18 : 1ω9. The peptidoglycan lackedmeso-DAP but contained ornithine and lysine. The dominant lipoquinone was MK-8. Phylogenetic analyses of the 16s rRNA gene sequence placed strain LbTinto the classThermomicrobiaof the phylumChloroflexiwithSphaerobacter thermophilusas the closest relative. On the basis of physiological and phylogenetic data, it is proposed that strain LbTrepresents a novel species of a new genus, with the suggested nameNitrolancea hollandicagen. nov., sp. nov. The type strain of the type species is LbT( = DSM 23161T = UNIQEM U798T).

Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8(2-)) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd1-like cytochrome and a cytochrome-aa3-like component in the membranes.

A number of strains of heterotrophic bacteria were isolated from various environments on the basis of their potential to oxidize inorganic sulfur compounds to tetrathionate. The isolates were screened for the ability to oxidize thiosulfate under denitrifying conditions. Many of them could grow anaerobically with acetate and nitrate, and eight strains could oxidize thiosulfate to tetrathionate under the same conditions. In batch cultures with acetate as carbon and energy source, most active anaerobic thiosulfate oxidation occurred with N2O as electron acceptor. The level of anaerobic thiosulfate-oxidizing activity in cultures and cell suspensions supplied with nitrate correlated with the activity of nitrite reductase in cell suspensions. Some strains converted thiosulfate to tetrathionate equally well with nitrite, nitrate and N2O as electron accepters. Others functioned best with N2O during anaerobic thiosulfate oxidation. The latter strains appeared to have a lower level of nitrite reductase activity. Thiosulfate oxidation under anaerobic conditions was much slower than in the presence of oxygen, and was obviously controlled by the availability of organic electron donor. The strains had DNA-DNA similarity levels higher than 30%. Sequence analysis of the 16S rRNA gene of four selected isolates showed their affiliation to specific genomovars of Pseudomonas stutzeri and the proposed new species, Pseudomonas balearica. As shown by 16S rRNA sequence analysis and DNA-DNA hybridization, the previously misnamed 'Flavobacterium lutescans' (ATCC 27951) is also a P. stutzeri strain which can oxidize thiosulfate to tetrathionate aerobically and anaerobically in the presence of N2O. The data suggest that tetrathionate-forming heterotrophic bacteria, in particular those belonging to the P. stutzeri 'superspecies', can play a much mole significant role in the biogeochemical cycles than was previously recognized. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A bacterial consortium that accumulated more than 90 % (w/w) polyhydroxybutyrate (PHB) from lactate was selected in a laboratory-scale bioreactor with a ‘feast–famine’ regime. Bacterial strain YDT, representing a dominant species in this enrichment, was isolated and characterized. Analysis of the 16S rRNA gene sequence revealed that the isolate is a member of the class Gammaproteobacteria , forming an independent phylogenetic lineage. The closest relative of the isolate was Plasticicumulans acidivorans TUD-YJ37T, with 94 % 16S rRNA gene sequence similarity. Strain YDT was an obligate aerobe with large, ovoid, Gram-negative cells, motile by means of a polar flagellum. It utilized a relatively broad spectrum of substrates (e.g. carbohydrates, fatty acids) as carbon and energy sources. The temperature range for growth was 20–45 °C, with an optimum at 40 °C; the pH range was pH 6.0–8.0, with an optimum at pH 7.0. The major respiratory lipoquinones were Q-8 (91 %) and Q-7 (9 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and an unidentified aminolipid. The predominant fatty acids in the membrane polar lipids were C16 : 1ω7c, C16 : 0 and C18 : 1ω7c. The G+C content of the genomic DNA was 68.5 mol%. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, the isolate is proposed to represent a novel species in the genus Plasticicumulans , for which the name Plasticicumulans lactativorans sp. nov. is proposed. The type strain is YDT ( = DSM 25287T = NCCB 100398T).

The complete genome sequence of a novel halophilic archaeon, Salinarchaeum sp. strain HArcht-Bsk1 T , was determined using next-generation sequencing. The genome comprises a 3,255,260-bp circular chromosome with a G+C content of 66.7%. Automatic annotation of the genome revealed a single rRNA operon, 45 tRNAs, and 3,013 protein-coding gene sequences.

Anaerobic enrichments from hypersaline soda lakes with chitin as substrate yielded five closely related anaerobic haloalkaliphilic isolates growing on insoluble chitin by fermentation at pH 10 and salinities up to 3.5 M. The chitinolytic activity was exclusively cell associated. To better understand the biology and evolutionary history of this novel bacterial lineage, the genome of the type strain ACht1 was sequenced. Analysis of the 2.6 Mb draft genome revealed enzymes of chitin-degradation pathways, including secreted cell-bound chitinases. The reconstructed central metabolism revealed pathways enabling the fermentation of polysaccharides, while it lacks the genes needed for aerobic or anaerobic respiration. The Rnf-type complex, oxaloacetate decarboxylase and sodium-transporting V-type adenosine triphosphatase were identified among putative membrane-bound ion pumps. According to 16S ribosomal RNA analysis, the isolates belong to the candidate phylum Termite Group 3, representing its first culturable members. Phylogenetic analysis using ribosomal proteins and taxonomic distribution analysis of the whole proteome supported a class-level classification of ACht1 most probably affiliated to the phylum Fibribacteres. Based on phylogenetic, phenotypic and genomic analyses, the novel bacteria are proposed to be classified as Chitinivibrio alkaliphilus gen. nov., sp. nov., within a novel class Chitinivibrione.

Haloalkaliphiles differ from natronophiles by their requirement for chloride ions in addition to high alkalinity. Natronophilic bacteria grow optimally in soda medium buffered at alkaline pH by a combination of NaHCO3 and Na2CO3. The majority of known haloalkaliphilic and natronophilic prokaryotes are isolated from saline–alkaline ecosystems such as soda lakes and saline–alkaline soils. A great taxonomic and metabolic biodiversity is found in soda systems, enabling the functioning of all the cycles of the essential elements. In spite of the increasing number of haloalkaliphilic and natronophilic isolates, scarce biochemical and functional information on simultaneous adaptation at high salinity and alkalinity is reported. Most of the available data on haloalkaline adaptation can be inferred from the functional characterization of alkaliphilic and halophilic bacterial models as well as from a few haloalkaliphilic and natronophilic genome sequences deposited in databases. At the level of cell envelopes (cell wall and cytoplasmic membrane), the salt and alkaline adaptation strategies are different and relatively conserved between Gram-positive and Gram-negative bacteria. The cell wall of the former group is characterized by the excessive presence of acidic polymers, while cell membranes abound in phospholipids with branched fatty acids. Cell membranes of salt- and alkaline-adapted Gram-negatives contain a large variety of fatty acids as well as significant amounts of nonpolar lipids. Osmotic adaptation mostly depends on the accumulation of organic compatible solutes either by active solute uptake or by combined strategies of importing osmolytes or osmolyte precursors and de novo synthesis of organic compatible solutes. Aerobic and anaerobic haloalkaliphiles are distinguished from each other by very different bioenergetics. Energy conservation in aerobic alkaliphiles and haloalkaliphiles is mainly based on functioning of H+-driven F-type ATP synthase. In spite of the low transmembrane electrochemical proton gradient (equivalent to proton-motive force, pmf) encountered in the alkali-exposed membrane, the energy metabolism remains highly efficient, supporting high growth rate and yield in many aerobic alkaliphiles and haloalkaliphiles. The energetics of haloalkaliphilic anaerobes is less understood, but it seems to involve a greater deal of Na dependency than in their aerobic counterpart. Na+-dependent ATPase activity is reported in a few anaerobic haloalkaliphiles and its role probably deals with active Na+ ejection from the cytoplasm. In haloalkaliphiles and natronophiles, the sodium-motive force (smf) is mainly driving the flagellar movement and sodium/solute symport. Cytoplasmic pH and ion homeostasis in haloalkaliphiles and natronophiles are most probably achieved by a concerted activity of a constellation of alkaline-activated ion transporters, among which Na+/H+ and Mrp-like antiporters have a major contribution.

Microbial disproportionation of elemental sulfur to sulfide and sulfate is a poorly characterized part of the anoxic sulfur cycle. So far, only a few bacterial strains have been described that can couple this reaction to cell growth. Continuous removal of the produced sulfide, for instance by oxidation and/or precipitation with metal ions such as iron, is essential to keep the reaction exergonic. Hitherto, the process has exclusively been reported for neutrophilic anaerobic bacteria. Here, we report for the first time disproportionation of elemental sulfur by three pure cultures of haloalkaliphilic bacteria isolated from soda lakes: the Deltaproteobacteria Desulfurivibrio alkaliphilus and Desulfurivibrio sp. AMeS2, and a member of the Clostridia, Dethiobacter alkaliphilus. All cultures grew in saline media at pH 10 by sulfur disproportionation in the absence of metals as sulfide scavengers. Our data indicate that polysulfides are the dominant sulfur species under highly alkaline conditions and that they might be disproportionated. Furthermore, we report the first organism (Dt. alkaliphilus) from the class Clostridia that is able to grow by sulfur disproportionation.

The diversity of soxB gene encoding a key enzyme of the Sox pathway sulfate thiohydrolase has been investigated in pure cultures of various halophilic and haloalkaliphilic sulfur-oxidizing bacteria (SOB) and in salt and soda lakes in southwestern Siberia and Egypt. The gene was detected in the majority of strains belonging to eleven SOB genera excluding members of genera Thiohalospira and Thioalkalimicrobium. The uncultured diversity of soxB in salt and soda lakes was low with a majority of detected sequences belonging to autotrophic SOB from the Gammaproteobacteria. In addition, the soxB analysis allowed detection of putative heterotrophic Gamma- and Alphaproteobacterial SOB yet unknown in culture. All clone libraries obtained from soda lakes contained soxB belonging to the genus Thioalkalivibrio in agreement with the cultivation results. Besides, representatives of the genera Halothiobacillus, Marinobacter, and Halochromatium and of the family Rhodobacteraceae have been detected in both type of saline lakes.

Since its isolation from marine volcanic areas, Catenococcus thiocyclus has been known to be able to oxidize thiosulfate to tetrathionate, but the benefits gained from the reaction were unknown. The energy to be gained from such a reaction is so small (1 electron per mol of thiosulfate, compared with 8 electrons if the thiosulfate is oxidized to sulfate) that it seemed unlikely to be a useful metabolic reaction. However, continuous culture experiments have now revealed that C. thiocyclus is able to gain metabolically useful energy from this oxidation (biomass yields increased by approximately 20% after the addition of 7.75 mM thiosulfate to medium containing 20 mM acetate) by combining it with the chemical reduction of the tetrathionate by sulfide. The enzymes for thiosulfate oxidation appear to be constitutive. Moreover, with a suitable primary energy source (e.g. glucose), C. thiocyclus can reduce sulfur (S°) to sulfide and Fe3+ to Fe2+. A chemical reaction then generates FeS. Such reactions may have important implications for the sulfur cycle at oxic:anoxic interfaces in marine and freshwater systems.

Two novel membrane-bound yellow pigments natronochrome (1) and chloronatronochrome (2) were isolated from the obligately chemolithoautotrophic sulfur-oxidizing natrono(alkali)philic bacterium Thialkalivibrio versutus strain ALJ 15. They were derivatives of fully unsaturated fatty acids with a phenyl group, and their structures were determined by spectral data.

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.

Oil and gas exploitation and downstream processing may generate large volumes of contaminated aqueous wastewaters. Without treatment, the discharge of these streams into the environment would lead to considerable environmental problems. A combination of physicochemical and biological treatment processes are applied to treat such polluted streams (e.g., in conventional water purification plants). However, the application of microorganisms in these streams is rather limited streams to salt levels well below those of seawater. The potential of microorganisms to degrade various organic and inorganic pollutants at higher salinities was surveyed in the open literature. The advantage of biological treatment processes that operate at elevated salinities is that no dilution water is needed, which is particularly relevant for arid regions. The available literature is scarce, especially concerning truly halophilic bacteria that thrive at salt concentrations significantly higher than in seawater. The obvious trend of most of the publications indicates a significant inhibition of the biodegradation activity at salt concentrations above 10 wt.%. However, a specialized group of extremophilic microorganisms, called halo(alkali)philes, could replace freshwater or marine organisms in the biodegradation processes at salinities above 1.5 M and pH values above 9. They are present in natural hypersaline habitats and can be activated either directly in situ by addition of micronutrients and aeration, or introduced into engineered water treatment systems. In a number of studies, the possibility of microbial HC degradation up to 5 M NaCl has been mentioned. Relatively little is known about the removal of toxic inorganic compounds, such as H2S, CO, and CN-, at halo(alkaline) conditions. These compounds are clearly associated with the processing of oil, coal, and gas. An exception is the application of sulfur-cycling bacteria, as these organisms have been intensively studied during the last decade. Sulfur-oxidizing and sulfidogenic bacteria can function efficiently at extremely saline and alkaline conditions. The general conclusion from this survey is that many biodegradation routs are, in principal, possible at high salt/pH but more research is needed to make a real progress in the development of engineered processes that can be applied to protect the environment.

The activity and culturable diversity of sulfidogens were investigated in anoxic sediments of four hypersaline lakes with pH 7.6–8.2 in the Kulunda Steppe (Altai, Russia). Sulfate reduction rates were low, varying from 0.1 to 6.0 nmol HS−/(cm3 h) with a maximum in the top 10 cm layer. Potential sulfidogenic rates with thiosulfate and sulfur as the e -acceptors were higher than with sulfate and were stimulated by formate, lactate, and acetate. Sulfidogenesis was optimal at salt concentrations below 2 M NaCl. Cultivation at 2 M NaCl resulted in the isolation of several strains of moderately halophilic SRB, but no growth of SRB was observed at 4 M NaCl. At lithotrophic conditions (i.e., with formate or H2 as e -donors), several closely related alkalitolerant strains belonging to the genus D esulfonatronovibrio were isolated. Enrichments at heterotrophic conditions with lactate, propionate, acetate, or butyrate using sulfate or thiosulfate as e -acceptors yielded isolates related to D esulfosalsimonas propionicica , D esulfohalobium utahense, and D esulfocella halophila . Sulfur-reducing enrichments at 2 M NaCl with ethanol produced a member of the genus H alanaerobium , while enrichments at 4 M NaCl with acetate were dominated by archaea, demonstrating for the first time such type of catabolism in haloarchaea.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, a major process of the biogeochemical nitrogen cycle, but the recognized diversity of this guild is surprisingly low and only two bacterial phyla contain known NOB. Here, we report on the discovery of a chemolithoautotrophic nitrite oxidizer that belongs to the widespread phylum Chloroflexi not previously known to contain any nitrifying organism. This organism, named Nitrolancetus hollandicus, was isolated from a nitrifying reactor. Its tolerance to a broad temperature range (25-63 degrees C) and low affinity for nitrite (K-s = 1 mM), a complex layered cell envelope that stains Gram positive, and uncommon membrane lipids composed of 1,2-diols distinguish N. hollandicus from all other known nitrite oxidizers. N. hollandicus grows on nitrite and CO2, and is able to use formate as a source of energy and carbon. Genome sequencing and analysis of N. hollandicus revealed the presence of all genes required for CO2 fixation by the Calvin cycle and a nitrite oxidoreductase (NXR) similar to the NXR forms of the proteobacterial nitrite oxidizers, Nitrobacter and Nitrococcus. Comparative genomic analysis of the nxr loci unexpectedly indicated functionally important lateral gene transfer events between Nitrolancetus and other NOB carrying a cytoplasmic NXR, suggesting that horizontal transfer of the NXR module was a major driver for the spread of the capability to gain energy from nitrite oxidation during bacterial evolution. The surprising discovery of N. hollandicus significantly extends the known diversity of nitrifying organisms and likely will have implications for future research on nitrification in natural and engineered ecosystems. The ISME Journal (2012) 6, 2245-2256; doi:10.1038/ismej.2012. 70; published online 5 July 2012

A moderately salt-tolerant and obligately alkaliphilic, chemolithoautotrophic sulfur-oxidizing bacterium, strain HL-EbGr7T, was isolated from a full-scale bioreactor removing H2S from biogas under oxygen-limited conditions. Another strain, ALJ17, closely related to HL-EbGr7T, was isolated from a Kenyan soda lake. Cells of the isolates were relatively long, slender rods, motile by a polar flagellum. Although both strains were obligately aerobic, micro-oxic conditions were preferred, especially at the beginning of growth. Chemolithoautotrophic growth was observed with sulfide and thiosulfate in a pH range of 8.0–10.5 (optimum at pH 10.0) and a salinity range of 0.2–1.5 M total Na+ (optimum at 0.4 M). The genome sequence of strain HL-EbGr7T demonstrated the presence of genes encoding the reverse Dsr pathway and a truncated Sox pathway for sulfur oxidation and enzymes of the Calvin–Benson cycle of autotrophic CO2 assimilation with ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) type I. The dominant cellular fatty acids were C18 : 1ω7, C16 : 0 and C19 : 0 cyclo. Based on 16S rRNA gene sequencing, the two strains belonged to a single phylotype within the genus Thioalkalivibrio in the Gammaproteobacteria . Despite being related most closely to Thioalkalivibrio denitrificans , the isolates were unable to grow by denitrification. On the basis of phenotypic and phylogenetic analysis, the novel isolates are proposed to represent a novel species, Thioalkalivibrio sulfidiphilus sp. nov., with the type strain HL-EbGr7T ( = NCCB 100376T = UNIQEM U246T).

The ammonia-oxidizing bacterial community (AOB) was investigated in two types of laboratory-scale bioreactors performing partial oxidation of ammonia to nitrite or nitrate at high (80 mM) to extremely high (428 mM) concentrations of ammonium bicarbonate. At all conditions, the dominant AOB was affiliated to the Nitrosomonas europaea lineage as was determined by fluorescence in situ hybridization and polymerase chain reaction in combination with denaturing gradient gel electrophoresis. Molecular analysis of the mixed populations, based on the 16S rRNA and cbbL genes, demonstrated the presence of two different phylotypes of Nitrosomonas, while microbiological analysis produced a single phylotype, represented by three different morphotypes. One of the most striking features of the AOB populations encountered in the bioreactors was the domination of highly aggregated obligate microaerophilic Nitrosomonas, with unusual cellular and colony morphology, commonly observed in nitrifying bioreactors but rarely investigated by cultural methods. The latter is probably not an adaptation to stressful conditions created by high ammonia or nitrite concentrations, but oxygen seems to be a stressful factor in these bioreactors.

Microbial sulfidogenesis is the main dissimilatory anaerobic process in anoxic sediments of extremely haloalkaline soda lakes. In soda lakes with a salinity2 M of the total Na(+) sulfate reduction is depressed, while thiosulfate- and sulfur-dependent sulfidogenesis may still be very active. Anaerobic enrichments at pH 10 and a salinity of 2-4 M total Na(+) from sediments of hypersaline soda lakes with thiosulfate and elemental sulfur as electron acceptors and simple nonfermentable electron donors resulted in the isolation of two groups of haloalkaliphilic bacteria capable of dissimilatory sulfidogenesis. Both were closely related to obligately heterotrophic fermentative homoacetogens from soda lakes. The salt-tolerant alkaliphilic thiosulfate-reducing isolates were identified as representatives of Tindallia magadiensis, while the extremely natronophilic obligate sulfur/polysulfide-respiring strains belonged to the genus Natroniella and are proposed here as a novel species Natroniella sulfidigena. Despite the close phylogenetic relation to Natroniella acetigena, it drastically differed from the type strain phenotypically (chemolithoautotrophic and acetate-dependent sulfur respiration, absence of acetate as the final metabolic product). Apparently, in the absence of specialized respiratory sulfidogens, primarily fermentative bacteria that are well adapted to extreme salinity may take over an uncharacteristic ecological function. This finding, once again, exemplifies the importance of isolation and phenotypic investigation of pure cultures.

In this study a diverse range of purified cobalt containing nitrile hydratases (NHases, EC 4.2.1.84) from Rhodopseudomonas palustris HaA2 (HaA2), Rhodopseudomonas palustris CGA009 (009), Sinorhizobium meliloti 1021 (1021), and Nitriliruptor alkaliphilus (iso2), were screened for the first time for their enantioselectivity towards a broad range of chiral nitriles. Enantiomeric ratios of >100 were found for the NHases from HaA2 and CGA009 on 2-phenylpropionitrile. In contrast, the Fe-containing NHase from the well-characterized Rhodococcus erythropolis AJ270 (AJ270) was practically aselective with a range of different α-phenylacetonitriles. In general, at least one bulky group in close proximity to the α-position of the chiral nitriles seemed to be necessary for enantioselectivity with all NHases tested. Nitrile groups attached to a quaternary carbon atom were only reluctantly accepted and showed no selectivity. Enantiomeric ratios of 80 and >100 for AJ270 and iso2, respectively, were found for the pharmaceutical intermediate naproxennitrile, and 3-(1-cyanoethyl)benzoic acid was hydrated to the corresponding amide by iso2 with an enantiomeric ratio of >100.

Soda lakes represent a unique ecosystem with extremely high pH (up to 11) and salinity (up to saturation) due to the presence of high concentrations of sodium carbonate in brines. Despite these double extreme conditions, most of the lakes are highly productive and contain a fully functional microbial system. The microbial sulfur cycle is among the most active in soda lakes. One of the explanations for that is high-energy efficiency of dissimilatory conversions of inorganic sulfur compounds, both oxidative and reductive, sufficient to cope with costly life at double extreme conditions. The oxidative part of the sulfur cycle is driven by chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria (SOB), which are unique for soda lakes. The haloalkaliphilic SOB are present in the surface sediment layer of various soda lakes at high numbers of up to 10(6) viable cells/cm(3). The culturable forms are so far represented by four novel genera within the Gammaproteobacteria, including the genera Thioalkalivibrio, Thioalkalimicrobium, Thioalkalispira, and Thioalkalibacter. The latter two were only found occasionally and each includes a single species, while the former two are widely distributed in various soda lakes over the world. The genus Thioalkalivibrio is the most physiologically diverse and covers the whole spectrum of salt/pH conditions present in soda lakes. Most importantly, the dominant subgroup of this genus is able to grow in saturated soda brines containing 4 M total Na(+) - a so far unique property for any known aerobic chemolithoautotroph. Furthermore, some species can use thiocyanate as a sole energy source and three out of nine species can grow anaerobically with nitrogen oxides as electron acceptor. The reductive part of the sulfur cycle is active in the anoxic layers of the sediments of soda lakes. The in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries using sulfate, thiosulfate, or elemental sulfur as electron acceptors demonstrated relatively high sulfate reduction rates only hampered by salt-saturated conditions. However, the highest rates of sulfidogenesis were observed not with sulfate, but with elemental sulfur followed by thiosulfate. Formate, but not hydrogen, was the most efficient electron donor with all three sulfur electron acceptors, while acetate was only utilized as an electron donor under sulfur-reducing conditions. The native sulfidogenic populations of soda lakes showed a typical obligately alkaliphilic pH response, which corresponded well to the in situ pH conditions. Microbiological analysis indicated a domination of three groups of haloalkaliphilic autotrophic sulfate-reducing bacteria belonging to the order Desulfovibrionales (genera Desulfonatronovibrio, Desulfonatronum, and Desulfonatronospira) with a clear tendency to grow by thiosulfate disproportionation in the absence of external electron donor even at salt-saturating conditions. Few novel representatives of the order Desulfobacterales capable of heterotrophic growth with volatile fatty acids and alcohols at high pH and moderate salinity have also been found, while acetate oxidation was a function of a specialized group of haloalkaliphilic sulfur-reducing bacteria, which belong to the phylum Chrysiogenetes.

Thioalkalivibrio sp. K90mix is an obligately chemolithoautotrophic, natronophilic sulfur-oxidizing bacterium (SOxB) belonging to the family Ectothiorhodospiraceae within the Gammaproteobacteria. The strain was isolated from a mixture of sediment samples obtained from different soda lakes located in the Kulunda Steppe (Altai, Russia) based of its extreme potassium carbonate tolerance as an enrichment method. Here we report the complete genome sequence of strain K90mix and its annotation. The genome was sequenced within the Joint Genome Institute Community Sequencing Program, because of its relevance to the sustainable removal of sulfide from wastewater and gas streams.

“Thioalkalivibrio sulfidophilus” HL-EbGr7 is an obligately chemolithoautotrophic, haloalkaliphilic sulfur-oxidizing bacterium (SOB) belonging to the Gammaproteobacteria. The strain was found to predominate a full-scale bioreactor, removing sulfide from biogas. Here we report the complete genome sequence of strain HL-EbGr7 and its annotation. The genome was sequenced within the Joint Genome Institute Community Sequencing Program, because of its relevance to the sustainable removal of sulfide from bio- and industrial waste gases.

Sulfidogenic activity (SA) in anoxic sediments of several soda lakes with variable salinity in south Kulunda Steppe (Altai, Russia) has been investigated. The study included in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries in which sulfate, thiosulfate or elemental sulfur were used as electron acceptors. Despite the extreme conditions (high salt concentrations and high pH), the SA values were relatively high (ranging from 0.02 to 1.20 micromol HS(-) cm(-3) h(-1)), and only hampered under salt-saturated conditions. The highest SA was observed with elemental sulfur, followed by thiosulfate, while the lowest SA was determined in the presence of sulfate. Of all the electron donors tested, the addition of formate resulted in the highest SA with all three sulfur electron acceptors. Surprisingly, hydrogen as an electron donor had very little effect. Acetate was utilized as an electron donor only under sulfur-reducing conditions. Indigenous populations of sulfidogens in soda lake sediments showed an obligately alkaliphilic pH response of SA, showing a pattern that corresponded well to the in situ pH conditions. Sulfate reduction was much more susceptible to salt inhibition than thiosulfate and sulfur reduction. Microbiological investigations indicated that sulfate-reducing bacteria belonging to the orders Desulfovibrionales and Desulfobacterales could very likely be responsible for the SA with sulfate and thiosulfate as electron acceptors at moderate salt concentrations. Sulfur reduction at moderate salinity was carried out by a specialized group of haloalkaliphilic sulfur-reducing bacteria that utilize volatile fatty acids. In saturated soda brine, extremely natronophilic representatives of the order Halanaerobiales were responsible for the sulfur-dependent respiration.

Sulfidogenic activity (SA) in anoxic sediments of several soda lakes with variable salinity in south Kulunda Steppe (Altai, Russia) has been investigated. The study included in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries in which sulfate, thiosulfate or elemental sulfur were used as electron acceptors. Despite the extreme conditions (high salt concentrations and high pH), the SA values were relatively high (ranging from 0.02 to 1.20 μmol HS− cm−3 h−1), and only hampered under salt-saturated conditions. The highest SA was observed with elemental sulfur, followed by thiosulfate, while the lowest SA was determined in the presence of sulfate. Of all the electron donors tested, the addition of formate resulted in the highest SA with all three sulfur electron acceptors. Surprisingly, hydrogen as an electron donor had very little effect. Acetate was utilized as an electron donor only under sulfur-reducing conditions. Indigenous populations of sulfidogens in soda lake sediments showed an obligately alkaliphilic pH response of SA, showing a pattern that corresponded well to the in situ pH conditions. Sulfate reduction was much more susceptible to salt inhibition than thiosulfate and sulfur reduction. Microbiological investigations indicated that sulfate-reducing bacteria belonging to the orders Desulfovibrionales and Desulfobacterales could very likely be responsible for the SA with sulfate and thiosulfate as electron acceptors at moderate salt concentrations. Sulfur reduction at moderate salinity was carried out by a specialized group of haloalkaliphilic sulfur-reducing bacteria that utilize volatile fatty acids. In saturated soda brine, extremely natronophilic representatives of the order Halanaerobiales were responsible for the sulfur-dependent respiration.

A new Gram-negative facultatively anaerobic eubacterium isolated from a near-shore sulphidic hydrothermal area is described. It differs from known genera of Gram-negative cocci, and is thus proposed to be a representative of a new genus. This isolate, Catenococcus thiocyclus, is able to oxidize thiosulphate to tetrathionate during batch growth in the presence of organic carbon and energy sources. That energy is gained from thiosulphate oxidation is indicated by an increase in the growth yield compared with similar cultures from which the thiosulphate had been omitted. Thiosulphate—cytochrome c oxidoreductase and endogenous cytochrome c 553 were shown to be involved in thiosulphate oxidation in C. thiocyclus.

Aerobic enrichments from soda lake sediments with CO as the only substrate resulted in the isolation of five bacterial strains capable of autotrophic growth with CO at extremely high pH and salinity. The strains belonged to theAlkalispirillum/Alkalilimnicolacluster in the Gammaproteobacteria, where the ability to oxidize CO, but not growth with CO, has been demonstrated previously. The growth with CO was possible only at an oxygen concentration below 5 % and CO concentration below 20 % in the gas phase. The isolates were also capable of growth with formate but not with H2. The carboxydotrophic growth occurred within a narrow pH range from 8 to 10.5 (optimum at 9.5) and a broad salt concentration from0.3 to 3.5 M total Na+(optimum at 1.0 M). Cells grown on CO had high respiration activity with CO and formate, while the cells grown on formate actively oxidized formate alone. In CO-grown cells, CO-dehydrogenase (CODH) activity was detectable both in soluble and membrane fractions, while the NAD-independent formate dehydrogenase (FDH) resided solely in membranes. The results of total protein profiling and the failure to detect CODH with conventional primers for thecoxLgene indicated that the CO-oxidizing enzyme in haloalkaliphilic isolates might differ from the classical aerobic CODH complex. A singlecbbLgene encoding the RuBisCO large subunit was detected in all strains, suggesting the presence of the Calvin cycle of inorganic carbon fixation. Overall, these results demonstrated the possibility of aerobic carboxydotrophy under extremely haloalkaline conditions.

The oxic-anoxic transition zone (OATZ) of freshwater sediments, where opposing gradients exist of reduced iron and sulfide with oxygen, creates a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide. Gradient microcosms incubated with freshwater sediment showed rapid microbial turnover of sulfide and oxygen compared with sterile systems. Microcosms with FeS as a substrate also showed growth at the OATZ and subsequent dilution series resulted in the isolation of three novel strains, of which strain J10 grows chemolithoautotrophically with reduced sulfur compounds under microaerobic conditions. All three strains are motile spirilla with bipolar flagella, related to the genera Magnetospirillum and Dechlorospirillum within the Alphaproteobacteria. Strain J10 is closely related to Magnetospirillum gryphiswaldense and is the first strain in this genus found to be capable of autotrophic growth. Thiosulfate was oxidized completely to sulfate, with a yield of 4 g protein mol(-1) thiosulfate, and autotrophic growth was evidenced by incorporation of (13)C derived from bicarbonate into biomass. A putative gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase type II was identified in strain J10, suggesting that the Calvin-Benson-Bassham cycle is used for autotrophic growth. Analogous genes are also present in other magnetospirilla, and in the autotrophically growing alphaproteobacterium magnetic vibrio MV-1.

A moderately halophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain HRh1T, was obtained from mixed sediment samples from hypersaline chloride–sulfate lakes in the Kulunda Steppe, in south-western Siberia (Russia), using aerobic enrichment culture at 1 M NaCl with thiocyanate as substrate. Cells of the isolate were short, non-motile rods with a Gram-negative type of cell wall. The bacterium was an obligate aerobe capable of chemolithoautotrophic growth with thiocyanate and thiosulfate. With thiosulfate, it grew at NaCl concentrations of 0.2–3.0 M (optimum 0.5 M) and at pH 6.3–8.0 (optimum pH 7.3–7.5). During growth on thiocyanate, cyanate was identified as an intermediate. The dominant cellular fatty acids were C16 : 0and C18 : 1ω7. Phylogenetic analysis based on 16S rRNA gene sequencing placed the isolate in the classGammaproteobacteriaas an independent lineage, with an unclassified marine sulfur-oxidizing bacterium as the closest culturable relative (93 % sequence similarity). A singlecbbLgene (coding for the key enzyme of the Calvin–Benson cycle of autotrophic CO2assimilation) with relatively low similarity to any homologous genes found in chemolithoautotrophs was detected in strain HRh1T. On the basis of our phenotypic and phylogenetic analysis, the halophilic isolate is proposed to represent a new genus and novel species,Thiohalobacter thiocyanaticusgen. nov., sp. nov. The type strain ofThiohalobacter thiocyanaticusis HRh1T(=DSM 21152T=UNIQEM U249T).

Seven strains of extremely halophilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) were enriched and isolated at 4 M NaCl from sediments of hypersaline inland lakes in south-eastern Siberia and a Mediterranean sea solar saltern. Cells of the novel isolates were spindle-like, long and non-motile rods with a Gram-negative type of cell wall. They were obligately chemolithoautotrophic SOB using thiosulfate and tetrathionate as electron donors and represent the first example of extremely halophilic chemolithoautotrophs that are able to grow anaerobically with nitrate as electron acceptor. The characteristic feature of the group was the production of large amounts of tetrathionate as an intermediate during the oxidation of thiosulfate to sulfate. With thiosulfate, the novel strains grew within the pH range from 6.5 to 8.2 (optimum at pH 7.5-7.8) and at NaCl concentrations from 1.5 to 4.0 M (optimum at 3.0 M). Cells grown at 4 M NaCl accumulated extremely high concentrations of glycine betaine as a compatible solute. The dominant cellular fatty acids were 10MeC(16 : 0) and C(16 : 0). Based on the DNA-DNA relatedness values, the isolates consisted of a single genomic species and had a similar phenotype. Phylogenetic analysis placed the novel bacteria in the class Gammaproteobacteria as an independent lineage with no significant relationship to any other genera in this class. On the basis of phenotypic and genotypic analysis, the group is proposed to represent a new genus, Thiohalorhabdus gen. nov., with Thiohalorhabdus denitrificans gen. nov., sp. nov. as the type species (type strain HL 19(T)=DSM 15699(T)=UNIQEM U223(T)).

Strain MS 6(T) was obtained from a microoxic enrichment with a soda soil sample from north-eastern Mongolia in nitrogen-free alkaline medium at pH 10. The isolate had clostridia-like motile cells and formed ellipsoid endospores. It was able to fix dinitrogen gas growing on nitrogen-free alkaline medium. Strain MS 6(T) was a strictly fermentative bacterium without a respiratory chain, although it had a high catalase activity and tolerated aerobic conditions. It was an obligate alkaliphile with a pH range for growth between 7.5 and 10.6 (optimum at 9.0-9.5). Growth and nitrogen fixation at pH 10 were possible at a total salt content of up to 1.2 M Na(+) (optimum at 0.2-0.3 M). The dominant cellular fatty acids included C(16 : 0), C(16 : 1)omega7, anteiso-C(15 : 0) and C(14 : 0). The dominant isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. 16S rRNA gene sequencing identified strain MS 6(T) as a member of the genus Bacillus. Its closest relative was Bacillus arseniciselenatis E1H(T). The key functional nitrogenase gene nifH was detected in both strain MS 6(T) and its close relative and these strains formed a novel lineage in the nifH gene family. On the basis of these phenotypic and genetic comparisons, strain MS 6(T) is proposed to represent a novel species of the genus Bacillus, Bacillus alkalidiazotrophicus sp. nov. with the type strain MS 6(T) (=NCCB 100213(T)=UNIQEM U377(T)).

High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na(+) and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H(2) and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5-10, and grew in soda brines containing 1-4 M total Na(+) (optimum at 1.0-2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1(T) and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6(T) from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. nov.

Thiopaq biotechnology for partial sulfide oxidation to elemental sulfur is an efficient way to remove H2S from biogases. However, its application for high-pressure natural gas desulfurization needs upgrading. Particularly, an increase in alkalinity of the scrubbing liquid is required. Therefore, the feasibility of sulfide oxidation into elemental sulfur under oxygen limitation was tested at extremely haloalkaline conditions in lab-scale bioreactors using mix sediments from hypersaline soda lakes as inoculum. The microbiological analysis, both culture dependent and independent, of the successfully operating bioreactors revealed a domination of obligately chemolithoautotrophic and extremely haloalkaliphilic sulfur-oxidizing bacteria belonging to the genus Thioalkalivibrio. Two subgroups were recognized among the isolates. The subgroup enriched from the reactors operating at pH 10 clustered with Thioalkalivibrio jannaschii–Thioalkalivibrio versutus core group of the genus Thioalkalivibrio. Another subgroup, obtained mostly with sulfide as substrate and at lower pH, belonged to the cluster of facultatively alkaliphilic Thioalkalivibrio halophilus. Overall, the results clearly indicate a large potential of the genus Thiolalkalivibrio to efficiently oxidize sulfide at extremely haloalkaline conditions, which makes it suitable for application in the natural gas desulfurization. Electronic supplementary material The online version of this article (doi:10.1007/s00253-008-1598-8) contains supplementary material, which is available to authorized users.