Tatjana N. Zhilina

Alkaliphilic anaerobic community at pH 10

Relict or ancient microbial communities in extreme environment might be analogous to the centers of origin of bacterial diversity. A bacterial community of an alkaline lake was investigated, and the diversity of bacteria found there indicates that both conditions of autonomy and phylogenetic variety are fulfilled for anaerobic bacteria developing at pH 10±0.2. Major functional groups in the trophic network were present. Representatives of proteolytic, bacteriolytic, cellulolytic, saccharolytic, dissipotrophic, acetogenic, sulfate-reducing, methanogenic bacteria were isolated.

Tindallia magadii gen. nov., sp. nov.: An Alkaliphilic Anaerobic Ammonifier from Soda Lake Deposits

Abstract. Strain Z-7934, an alkaliphilic, obligately anaerobic, fermentative, asporogenous bacterium with Gram-positive cell wall structure, was isolated from soda deposits in Lake Magadi, Kenya. The organism ferments only a few amino acids, preferentially arginine and ornithine, with production of acetate, propionate, and ammonia. It is a true alkaliphile, with pH range for growth ranging from 7.5 to 10.5 (optimum pH 8.5), and growth is dependent on the presence of sodium ions. The G+C content of the genomic DNA is 37.6 mol%. 16S rDNA sequence analysis of strain Z-7934 shows that it belongs phylogenetically to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and unique physiological properties, we propose a new genus and new species, Tindallia magadii, for this strain. The type strain is Z-7934T (=DSM 10318).

Natroniella acetigena gen. nov. sp. nov., an extremely haloalkaliphilic, homoacetic bacterium : A new member of Haloanaerobiales

Abstract. A new extremely haloalkaliphilic, chemoorganotrophic, homoacetogenic bacterium strain Z-7937T(T-type strain) was isolated from the bottom mud of the soda-depositing Lake Magadi, Kenya. It is an obligately anaerobic, motile, Gram-negative, spore-forming rod growing in the pH range pH 8.1 to 10.7 and optimally in the range pH 9.7 to 10.0 under conditions of high alkalinity caused by saturation with trona. It has an obligate requirement for sodium carbonate and chloride ions. The optimum salt concentration for growth is in the range 12–15% wt/vol, and growth occurs within the range from 10% to 26%. Strain Z-7937T is a mesophile with an optimal temperature for growth of 37°C, and a maximum of 42°C. The G + C content of strain Z-7937T is 31.9 mol%. A limited number of compounds are utilized, including lactate, ethanol, pyruvate, glutamate, and propanol. Acetate is the main end product. 16S rDNA sequence analysis shows strain Z-7937T to be a member of the order Haloanaerobiales and to represent a new branch within the family Halobacteroidaceae. On the basis of its novel physiology and phylogenetic position, we propose strain Z-7937 as a new species of a new genus, Natroniella acetigena gen. nov. sp. nov. The type strain is Z-7937T (= DSM 9952).

Natronoincola histidinovorans gen. nov., sp. nov., A new alkaliphilic acetogenic anaerobe.

Abstract. Two strains, asporogenous Z-7940 and sporogenous Z-7939, of a moderately haloalkaliphilic, obligately anaerobic, fermentative bacteria, motile, with Gram-positive cell wall structure, were isolated from soda deposits in Lake Magadi, Kenya. Both strains are mesophilic and utilize only two amino acids, histidine and glutamate, with formation of acetate and ammonium as the main end products. Strain Z-7939 in addition is able to utilize pyruvate. DNA-DNA homology between strains Z-7940 and Z-7939 was 94%, indicating that in spite of phenotypic differences they belong to the same species. They are true alkaliphiles with a pH range for growth of the type strain Z-7940 from pH 8.0 to pH 10.5, optimum at pH 9.4. Both strains obligately depend on sodium and bicarbonate ions. The optimum salt concentration for growth of the type strain is 8–10% wt/vol and the range from 4% to 16%. The G+C content of strain Z-7940 is 31.9 mol% and the strain Z-7939 is 32.3 mol%. Analysis of 16S rDNA sequence of the type strain shows it to belong to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and physiological properties, we propose a new genus and new species Natronoincola histidinovorans for these strains. The type strain is Z-7940 (=DSM 11416).

Ecology, Physiology and Taxonomy Studies on a New Taxon of Haloanaerobiaceae, Haloincola saccharolytica gen. nov., sp. nov.

Summary Four strains of new moderately halophilic, Gram-negative anaerobic saccharolytic bacteria were isolated from Lake Sivash and its lagoons on Arabat Strait (eastern Crimea, USSR). The cells are non-sporeforming, motile rods 0.5-0.7 × 1-1.5 μm, that occur single or in pairs. Carbohydrates (preferably disaccharides, such as sucrose and trehalose) but not amino acids serve as energy sources. The end products of glucose fermentation are acetate, H 2 and CO 2 . The biomass yield is 3.8 g of protein per mol of glucose consumed. Elemental sulphur when present is reduced to H 2 S. All isolates are mesophiles and grow at salt concentrations from 3 to 30% NaCl (w/v) and at a pH range from 6.0 to 8.0. Doubling time of strain Z-7787 at optimal conditions (37°C., 10% NaCl, pH 7.5) is 3.9 h. The G+C content of this isolate is 31.3 mol%; that of the other strains varies from 30 to 33 mol%. All the isolates appear to be members of the same taxon but according to 5S rRNA sequencing do not belong to the genus Halobacteroides . They are considered to represent a new genus for which the name Haloincola is proposed with Haloincola saccharolytica as the type species. The type strain is Z-7787, DSM 6643. The taxonomic relationships of H. saccharolytica to other haloanaerobes of the family Haloanaerobiaceae is discussed.

Anoxynatronum sibiricum gen.nov., sp.nov. alkaliphilic saccharolytic anaerobe from cellulolytic community of Nizhnee Beloe (Transbaikal region).

New alkaliphilic anaerobic fermentative bacteria, strains Z-7981 and Z-7981′, with Gram-positive cell walls, were isolated from the cellulolytic community from the soda lake Nizhnee Beloye, south-east of Baikal. Cells were motile rods, which differed in dimensions but, according to 98% DNA/DNA homology, belonged to the same species. Strain Z-7981 was chosen as the type and studied in detail. It did not produce spores and its cells were non-thermoresistant. It was a true alkaliphile with a growth range from pHxa07.1 to pHxa010.1 and optimal pH for growth at pHxa09.1. It was obligately dependent on Na+ and carbonate ions but not on Cl−. Growth occurred in media with total sodium content from 0.076xa0M to 1.27xa0M Na+ with a broad optimum from 0.25 to 0.86xa0M Na+. Growth showed an optimum at 35°C, with absence of growth above 46°C. The organism was aerotolerant and was capable of fermentation in non-reducing medium at less than 4.75% O2 in the gas phase. Strain Z-7981 fermented mono- and disaccharides, sugar alcohols, but only glutamate and cysteine among the amino acids, and the proteinaceous substrates, chitin and dried Spirulina biomass. Fermentation products were acetate and ethanol. Fe3+ was reduced in a process that yielded no energy. Phylogenetically the new organism belonged to cluster XI of the Gram-positive bacteria with low G+C content and its closest neighboring taxon was Tindallia magadiensis. However, according to its phenotypic and genotypic characters it did not belong to any known genus from this group. We suggest a new genus and species with the name Anoxynatronum sibiricum and strain Z-7981 as its type (=DSM15060).

Anaerobic, alkaliphilic, saccharolytic bacterium Alkalibacter saccharofermentans gen. nov., sp. nov. from a soda lake in the Transbaikal region of Russia.

Three strains of new obligately anaerobic alkaliphilic bacteria have been isolated as a saccharolytic component from the cellulolytic community of alkaline Lake Nizhnee Beloe (Transbaikal region, Russia), a lake with low salt concentration. DNA analysis of these strains showed an interspecies level of DNA similarity of 96–100%. Strain Z-79820 was selected for further investigations. Cells were Gram-positive, asporogenous, nonmotile short rods with pointed ends. The strain was a true alkaliphile: growth occurred from pHxa07.2 to 10.2 with the optimum at pHxa09.0. Strain Z-79820 was halotolerant and could grow in medium with up to 10% (w/v) NaCl, with the optimum between 0 and 4% NaCl. The new isolate obligately depended on Na+ ions in the form of carbonates or chlorides. Total Na+ content needed for optimal growth was 0.46xa0M Na+, with a wide range from 0.023–0.9xa0M Na+ at which growth also occurred. The isolate was a mesophile and grew at temperatures from 6 to 50°C (slow growth at 6 and 15°C) with an optimum at 35°C. The organotrophic organism fermented ribose, xylose, glucose, mannose, fructose, sucrose, mannitol, and peptone. The products of glucose fermentation were acetate, ethanol, formate, H2, and CO2. Yeast extract was required for some anabolic needs. The DNA G+C content of the type strain Z-79820 was 42.1xa0mol%. The new bacterium fell into the 16S rRNA gene cluster XV of the Gram-positive bacteria with low G+C content, where it formed an individual branch. Based on its growth characteristics and genotype traits, we propose the new genus and species named Alkalibacter saccharofermentans with the type strain Z-79820 (=DSM14828), Uniqem-218 (Institute Microbiology, RAS; http://inmi.da.ru).

Halophilic Acetogenic Bacteria

As outlined in preceding chapters of this book, acetogenic bacteria have a specialized physiological potential for the conservation of energy via the reduction of CO2 to acetate. They also harbor diverse catabolic processes and are found in unusual habitats. Although their role in nature was initially viewed somewhat restrictively, it is now evident that they might have a large impact on carbon and energy flow in some environments, in particular that of certain gastrointestinal ecosystems (Breznak et al., 1988; Kane and Breznak, 1991a, 1991b). However, specialized (or extreme) terrestrial environments are largely unexplored relative to the involvement of acetogenesis and associated organisms.

Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum-Alkalilimnicola group

Four new isolates were obtained from denitrifying enrichments with various electron donors using sediment samples from hypersaline soda lakes. Based on 16S rRNA gene analysis and DNA-DNA hybridization results, they were all identified as members of the Gammaproteobacteria closely associated with the Alkalispirillum–Alkalilimnicola group. Two isolates were obtained from samples enriched with nitrate as electron acceptor and H2 or polysulfide as electron donors, and another two strains were obtained with N2O as the electron acceptor and sulfide or acetate as electron donors. All four new isolates, together with the type strains of the genera Alkalispirillum and Alkalilimnicola originally described as obligate aerobes, were capable of anaerobic growth with acetate using either nitrate or N2O as electron acceptors. Their denitrification pathway, however, was disrupted at the level of nitrite. RuBisCO form I gene was detected and sequenced in the new isolates and in Alkalilimnicola halodurans but not in Alkalispirillum mobile. These data, together with the evidence of Oremland et al. (Appl Environ Microbiol 68:4795–4802, 2002) on the potential of Alkalilimnicola sp. MLHE-1 for autotrophic growth with arsenite as electron donor and nitrate as electron acceptor, demonstrate much higher metabolic diversity of this specific group of haloalkaliphilic Gammaproteobacteria than was originally anticipated.

Cellulase activity of a haloalkaliphilic anaerobic bacterium, strain Z-7026

SummaryThe cellulolytic activity of an alkaliphilic obligate anaerobic bacterium, Z-7026, which was isolated from the microbial community of soda-lake sediments and belongs to the cluster III of Clostridia with low G+C content, was studied. The bacterium was capable of growing in media with cellulose or cellobiose as the sole energy sources. Its maximal growth rate on cellobiose (0.042–0.046xa0h−1) was observed at an initial pH value of 8.5–9.0, whereas the maximal rate of cellulase synthesis, assayed by using a novel fluorimetric approach, was found to be 0.1xa0h−1 at pHxa08–8.5. Secreted proteins revealed high affinity for cellulose and were represented by two major forms of molecular masses of 75 and 84xa0kDa, whereas the general protein composition of the precipitated and cellulose-bound preparations was similar to cellulosome subunits of Clostridium thermocellum. The optimum pH of the partially purified enzyme preparation towards both amorphous and crystalline cellulose was in the range 6–9, with more than 70% and less than 50% of maximal activity being retained at pHxa09.2 and 5.0, respectively.