RESULTS:

Three highly alkaliphilic bacterial strains designated as A1T H1T and B1T were isolated from two highly alkaline springs at The Cedars a terrestrial serpentinizing site. Cells from all strains were motile Gram-negative and rod-shaped. Strains A1T H1T and B1T were mesophilic (optimum 30 °C) highly alkaliphilic (optimum pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1T H1T and B1T was 2 574 013 2 475 906 and 2 623 236 bp and the G+C content was 66.0 66.2 and 66.1 mol% respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera Malikia (95.1–96.4 %) Macromonas (93.0–93.6 %) and Hydrogenophaga (93.0–96.6 %) in the family Comamonadaceae . Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species forming a distinct branch. Average amino acid identity values of strains A1T H1T and B1T against the genomes of related members in this family were below 67 % which is below the suggested threshold for genera boundaries. Average nucleotide identity by blast values and digital DNA–DNA hybridization among the three strains were below 92.0 and 46.6 % respectively which are below the suggested thresholds for species boundaries. Based on phylogenetic genomic and phenotypic characterization we propose Serpentinimonas gen. nov. Serpentinimonas raichei sp. nov. (type strain A1T=NBRC 111848T=DSM 103917T) Serpentinimonas barnesii sp. nov. (type strain H1T= NBRC 111849T=DSM 103920T) and Serpentinimonas maccroryi sp. nov. (type strain B1T=NBRC 111850T=DSM 103919T) belonging to the family Comamonadaceae . We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs.

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 10MeC16 : 0 and C16 : 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 19T=DSM 15699T=UNIQEM U223T).

A previously unknown ecotype of obligately chemolithoautotrophic sulfur-oxidizing bacteria was discovered in sediments of various inland hypersaline lakes and a solar saltern. The salt requirements for these bacteria were similar to those of haloarchaea representing the first example of extreme halophiles occurring among the chemolithoautotrophs. They were enriched and isolated at 4 M NaCl under aerobic conditions with thiosulfate or tetrathionate as the electron donor or under micro-oxic conditions with sulfide. In total 20 strains were obtained from hypersaline inland lakes in central Asia central Russia and Crimea and a sea saltern of the Adriatic Sea. The isolates were thin motile spirilla some of which possessed a yellow membrane-bound pigment. They were obligately aerobic chemolithoautotrophic sulfur-oxidizing bacteria that used thiosulfate sulfide sulfur and tetrathionate as electron donors. The characteristic feature of the group was the production of large amounts of tetrathionate as an intermediate during the oxidation of thiosulfate to sulfate. All but one of the strains grew within the pH range 6.5–8.2 (optimally at pH 7.3–7.8) and at NaCl concentrations from 2.0 to 5 M (optimally at 3.0 M). A single strain designated ALgr 6spT obtained (by enrichment) from the hypersaline alkaline lakes of the Wadi Natrun valley was found to be moderately halophilic and facultatively alkaliphilic (capable of growth at pH 10). The predominant cellular fatty acids were quite unusual with 10-methyl C16 : 0 and C16 : 0 predominating. Cells grown at 4 M NaCl accumulated extremely high concentrations of glycine betaine as a compatible solute. The 20 neutrophilic isolates contained three genospecies (on the basis of DNA–DNA relatedness data) but could not be discriminated phenotypically. On the basis of the phenotypic and genotypic analyses the isolates constitute a novel genus and species for which the name Thiohalospira halophila gen. nov. sp. nov. is proposed. The type strain of Thiohalospira halophila is HL 3T (=DSM 15071T=UNIQEM U219T). The haloalkaliphilic strain ALgr 6spT represents a second species of the new genus for which the name Thiohalospira alkaliphila sp. nov. is proposed. The type strain of Thiohalospira alkaliphila is ALgr 6spT (=DSM 17116T=UNIQEM U372T).

Strain ALEN 2T 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 2T (=DSM 14787T=UNIQEM 213T) is proposed as the type strain of a novel species of the genus Thialkalivibrio Thialkalivibrio nitratireducens.

Two strains of haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria were isolated from the oxygen-sulfide interface water layer of stratified alkaline and saline Mono Lake California USA. Strain ALM 1T was a dominant species in enrichment on moderate-saline carbonate-buffered medium (0.6 M total Na+ pH 10) with thiosulfate as an energy source and nitrate as a nitrogen source. Cells of ALM 1T are open ring-shaped and are non-motile. It has a high growth rate and activity of thiosulfate and sulfide oxidation and very low sulfur-oxidizing activity. Genetic comparison and phylogenetic analysis suggested that ALM 1T (= DSM 14477T = JCM 11371T) represents a new species of the genus Thioalkalimicrobium in the gamma-Proteobacteria for which the name Thioalkalimicrobium cyclicum sp. nov. is proposed. Another Mono Lake isolate strain ALM 2T dominated in enrichment on a medium containing 2 M total Na+ (pH 10). It is a motile vibrio which tolerates up to 4 M Na+ and produces a membrane-bound yellow pigment. Phylogenetic analysis placed ALM 2T as a member of genus Thioalkalivibrio in the gamma-Proteobacteria although its DNA hybridization with the representative strains of this genus was only about 30%. On the basis of genetic and phenotypic properties strain ALM 2T (= DSM 14478T = JCM 11372T) is proposed as Thioalkalivibrio jannaschii sp. nov..

Summary: A novel metabolic pathway for anaerobic ammonium oxidation with nitrite as the electron acceptor has been elucidated using 15N-Iabelled nitrogen compounds. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor. The hydroxylamine itself is most likely derived from nitrite. Batch experiments in which ammonium was oxidized with hydroxylamine transiently accumulated hydrazine. The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating electron equivalents for the reduction of nitrite to hydroxylamine. During the conversion of ammonium a small amount of nitrate was formed from some of the nitrite. The addition of NH2OH to an operating fluidized bed system caused a stoichiometric increase in the ammonium conversion rate (1 mmol I−1 h−1) and a decrease in the nitrate production rate (0.5 mmol I−1 h−1). Addition of hydrazine also caused a decrease in nitrate production. On the basis of these findings it is postulated that the oxidation of nitrite to nitrate could provide the anaerobic ammonium-oxidizing bacteria with the reducing equivalents necessary for CO2 fixation.

An autotrophic synthetic medium for the enrichment of anaerobic ammonium-oxidizing (Anammox) micro-organisms was developed. This medium contained ammonium and nitrite as the only electron donor and electron acceptor respectively while carbonate was the only carbon source provided. Preliminary studies showed that the presence of nitrite and the absence of organic electron donors were essential for Anammox activity. The conversion rate of the enrichment culture in a fluidized bed reactor was 3 kg NH4 + m−3 d−1 when fed with 30 mM NH4 +. This is equivalent to a specific anaerobic ammonium oxidation rate of 1000–1100 nmol NH4 +h−1 (mg volatile solids)−1. The maximum specific oxidation rate obtained was 1500 nmol NH4 +h−1 (mg volatile solids)−1. Per mol NH4 + oxidized 0.041mol CO2 were incorporated resulting in a estimated growth rate of 0.001 h−1. The main product of the Anammox reaction is N2 but about 10% of the N-feed is converted to NO3 −. The overall nitrogen balance gave a ratio of NH4 −-conversion to NO2 −-conversion and NO3 −-production of 1:1·31±0·06:2·02±0·02. During the conversion of NH4 + with NO2 − no other intermediates or end-products such as hydroxylamine NO and N2O could be detected. Acetylene phosphate and oxygen were shown to be strong inhibitors of the Anammox activity. The dominant type of micro-organism in the enrichment culture was an irregularly shaped cell with an unusual morphology. During the enrichment for Anammox micro-organisms on synthetic medium an increase in ether lipids was observed. The colour of the biomass changed from brownish to red which was accompanied by an increase in the cytochrome content. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.

Thiosphaera pantotropha is a heterotrophic nitrifying bacterium which reduces nitrite produced from ammonia to nitrogen gas regardless of the ambient dissolved O2 concentration. Under certain growth conditions nitrous oxide may be produced. The ammonia oxygenase showed a number of similarities with that of autotrophic nitrifiers [e.g. light sensitivity Mg2+ requirement NAD(P)H utilization] as did the hydroxylamine oxidoreductase (cytochrome c oxidation hydrazine inhibition). However there were also differences (e.g. hydroxylamine inhibition of ammonia oxidation) and this apparent similarity may be superficial. Control experiments with a strain of Paracoccus denitrificans (which does not nitrify) did not show the presence of either enzyme.

The successive steps in the isolation of mitochondria from chemostat-grown Candida utilis have systematically been investigated for their effects on organelle integrity.Growth rate had a profound effect on the susceptibility of carbon-limited cells towards Zymolyase whereas the nature of the carbon source had no effect. Stabilization of spheroplasts with at least 2m-sorbitol was required to prevent premature lysis. This was concluded from the amounts of glucose-6-phosphate dehydrogenase liberated during Zymolyase treatments. The influence of the method for disruption of spheroplasts on the quality of the mitochondria was analysed with particular emphasis on respiratory control values and the distribution of marker enzymes among the cell fractions. Disruption by osmotic shock resulted in mitochondria without respiratory control and a high degree of solubilization of NADH and NADPH dehydrogenase activities. Only a gradual decrease of the osmotic value of the medium preferably by dialysis against a hypotonic buffer in combination with mechanical disruption with a Potter–Elvehjem homogenizer yielded mitochondria with high respiratory control values and a high retention of NADH dehydrogenase in the organelle. It is concluded that for the quality of mitochondrial preparations from yeasts the distribution of NADH dehydrogenase among the cell fractions is a more reliable measure than that of the usual marker enzymes.

A chemostat culture of Thiobacillus A2 was grown under alternate limitation of acetate and thiosulphate at a dilution rate of 0·05 h−1. With a substrate alternation of 4 h acetate/4 h thiosulphate or 8 h acetate/16 h thiosulphate uninterrupted growth of the culture was obtained. However with 16 h acetate/8 h thiosulphate Thiobacillus A2 required several hours before it attained autotrophic growth at a rate of 0·05 h−11 following the transition from acetate to thiosulphate supply. In a two-membered mixed culture with the heterotrophic spirillum G7 Thiobacillus A2 outcompeted the heterotroph when grown under alternating limitation of acetate (4 h) and thiosulphate (4 h). Under the same growth conditions Thiobacillus A2 coexisted in equal numbers with Thiobacillus neapolitanus from which it was concluded that Thiobacillus A2 grew only heterotrophically on acetate in this case. Analogous competition experiments with three-membered cultures grown under the same conditions resulted in complete elimination of Thiobacillus A2 and coexistence of T. neapolitanus and spirillum G7 in equal numbers. In an enrichment culture again grown under the same regime a facultatively chemolithotrophic spirillum-shaped Thiobacillus or Thiomicrospira became dominant; this organism was subsequently isolated in pure culture.