Thermus ruber obligate thermophilic bacteria in the thermal springs of Kamchatka]

The new species of the obligate-thermophilic bacterium Thermus ruber is widely distributed in hot springs of Kamchatka with the temperature of water from 57 to 90 degrees C. The bacterium does not oxidize sulphur. It contains a red carotenoid pigment similar to neuro-sporaxanthine and retrodehydro-gamma-carotene.     

Association of bacteria and yeasts in hot springs.

The thermophilic bacterium Bacillus sp. strain TB-1 was isolated in association with the yeast Debaryomyces vanriji from hot springs at 46 degrees C. It was shown that TB-1 excreted thiamine into the culture broth, which not only promoted D. vanriji growth in mixed culture but also increased the maximal temperature for yeast growth.     

Radioisotopic assays of rates of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H2-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of 14CO by the bacterium was estimated at 38-56 micromol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm3 sediment per day. 85% of the utilized 14CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell bio mass production; and only just over 0.001% was converted to methane.     

Production of a New Acidic Polysaccharide,Succinoglucan by Alcaligenes faecalis var. myxogenes

A slimy non-spore-forming bacterium strain 10C3 isolated from soil was motile with peritrichous flagella and named Alcaligenes faecalis var. myxogenes. Studies were made on the conditions necessary for maximal production of a new acidic succinoglucan polysaccharide by this strain in shaken cultures. Much production was observed with sucrose, glucose, xylose, galactose, cellobiose, maltose, fructose, mannose and rhamnose. The yield was greatest with sucrose and decreased in order with the above sugars from about 36 to 23 per cent. The most suitable medium contained 4 per cent sugar, 0.5 per cent yeast extract and one per cent calcium carbonate in tap water. The optimum temperature was 28°C.     

Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum.

Thirteen strains of a strict anaerobic, extreme thermophilic bacterium were isolated from soil samples of moderate temperature, from a sewage plant in Georgia, and from hot springs in Utah and Wyoming. They were identified as strains of Clostridium thermohydrosulfuricum. The guanosine + cytosine content (moles percent) was 37.6 (determined by buoyant density) and 34.1 (determined by melting temperature). All strains required a factor present in yeast extract or tryptone growth. Growth characteristics were as follows: a pH range of 5 to 9, with the optimum between 6.9 to 7.5, in a temperature range of 40 to 78 degrees C, with the optimum at 68 degrees C. The doubling time, when grown on glucose at temperature and pH optima, was 1.2 h. The main products of glucose fermentation were ethanol, lactate, acetate, CO2, and H2. The fermentation was inhibited by H2. Formation of spores occurred easily on glucose-agar medium or when cultures growing at temperatures above 65 degrees C were allowed to cool to temperature below 55 degrees C. C. thermohydrosulfuricum occurs widely distributed in the natural environment.     

Nitrification in terrestrial hot springs of Iceland and Kamchatka

Archaea have been detected recently as a major and often dominant component of the microbial communities performing ammonia oxidation in terrestrial and marine environments. In a molecular survey of archaeal ammonia monooxygenase (AMO) genes in terrestrial hot springs of Iceland and Kamchatka, the amoA gene encoding the alpha-subunit of AMO was detected in a total of 14 hot springs out of the 22 investigated. Most of these amoA-positive hot springs had temperatures between 82 and 97 degrees C and pH range between 2.5 and 7. In phylogenetic analyses, these amoA genes formed three independent lineages within the known sequence clusters of marine or soil origin. Furthermore, in situ gross nitrification rates in Icelandic hot springs were estimated by the pool dilution technique directly on site. At temperatures above 80 degrees C, between 56 and 159 mumol NO(3)(-) L(-1) mud per day was produced. Furthermore, addition of ammonium to the hot spring samples before incubation yielded a more than twofold higher potential nitrification rate, indicating that the process was limited by ammonia supply. Our data provide evidence for an active role of archaea in nitrification of hot springs in a wide range of pH values and at a high temperature.     

Thermus aquaticus gen. n. and sp. n., a Nonsporulating Extreme Thermophile

The isolation of a new thermophilic bacterium, Thermus aquaticus gen. n. and sp. n., is described. Successful enrichment requires incubation at 70 to 75 C, and the use of nutrient media relatively dilute with respect to the organic components. Strains of T. aquaticus have been isolated from a variety of thermal springs in Yellowstone National Park and from a thermal spring in California. The organism has also been isolated from man-made thermal habitats, such as hot tap water, in geographical locations quite distant from thermal springs. Isolates of T. aquaticus are gram-negative nonsporulating nonmotile rods which frequently form long filaments at supraoptimal temperatures or in the stationary phase. All isolates form a yellow cellular pigment, probably a carotenoid. A characteristic structure formed by all isolates is a large sphere, considerably larger than a spheroplast. These large spheres, as well as lysozyme-induced spheroplasts, are resistant to osmotic lysis. Deoxyribonucleic acid base compositions of four strains were determined by CsCl density gradient ultracentrifugation and found to be between 65.4 and 67.4 moles per cent guanine plus cytosine. The growth of all isolates tested is inhibited by fairly low concentrations of cycloserine, streptomycin, penicillin, novobiocin, tetracycline, and chloramphenicol. Nutritional studies on one strain showed that it did not require vitamins or amino acids, although growth was considerably faster in enriched than in synthetic medium. Several sugars and organic acids served as carbon sources, and either NH(4) (+) or glutamate could serve as nitrogen source. The organism is an obligate aerobe and has a pH optimum of 7.5 to 7.8. The optimum temperature for growth is 70 C, the maximum 79 C, and the minimum about 40 C. The generation time at the optimum is about 50 min. The possible relationships of this new genus to the myxobacteria, flexibacteria, and flavobacteria are discussed.     

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H₂-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of ¹⁴CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm³ sediment per day. 85% of the utilized ¹⁴CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.     

Production of Microbial Biomass Protein from Potato Processing Wastes by Cephalosporium eichhorniae

The use of Cephalosporium eichhorniae 152 (ATCC 38255) (reclassified as Acremonium alabamense; see Addendum in Proof), a thermophilic, acidophilic, amylolytic fungus, for the conversion of potato processing wastes into microbial protein for use as animal feed was studied. The fungus was not inhibited by alpha-solanine or beta-2-chaconine, antimicrobial compounds in potatoes, or by morpholine or cyclohexylamine (additives to steam used in the peeling process) at levels likely to be encountered in this substrate. Mixed effluent from holding tanks at a potato-processing plant contained about 10 bacteria per ml and inhibited fungal growth. The fungus grew well on fresh potato wastes containing up to 5% total carbohydrate and utilized both starch and protein at 45 degrees C and pH 3.75. On potato homogenate medium containing 2% carbohydrate (about 14% fresh potato) supplemented with monoammonium phosphate (0.506 g/liter) and ferric iron (0.1 g/liter), with pH control (at 3.75) and additional nitrogen supplied by the automatic addition of ammonium hydroxide, typical yields were 0.61 g (dry weight) of product and 0.3 g of crude protein per g of carbohydrate supplied. An aerobic, spore-forming bacterium, related to Bacillus brevis, commonly contaminated nonsterilized batch cultures but was destroyed by heating for 15 min at 100 degrees C.     

Fe-Si biominerals in the Vilyuchinskie hot springs, Kamchatka Peninsula, Russia.

The micromorphological structure of microbial mats (biomats) from the hot springs of the Vilyuchinskaya hydrothermal system, Kamchatka Peninsula, Russia, were investigated. The Vilyuchinskie hot springs had a discharge temperature of 55-56 degrees C and Na-Ca-HCO3-type waters rich in silicic and boric acids. Water and biomats had high concentrations of Fe, Mn, Sr, and As. Enumeration of total bacterial abundance (TBA) demonstrated a low density of bacterial populations. However, the fractions of metabolically active bacteria and respiring iron-oxidizing bacteria in the hot-spring water were high, comprising 68 and 21% of TBA, respectively. Scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (SEM-EDX) showed that unicellular rod-shaped bacteria about 5-microm long predominated in the brown biomats. The mineral capsules of these bacteria contained large amounts of Fe and Si. Extracellular and intracellular particles were observed by transmission electron microscopy. Fe-oxidizing bacteria were isolated from the biomats on agar plates with selective medium. Therefore, it can be concluded that microorganisms inhabiting the biomats of the Vilyuchinskie hot springs are essential for the deposition of Fe-minerals at neutral pH.     

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.     

Biohydrogen production from glucose in upflow biofilm reactors with plastic carriers under extreme thermophilic conditions (70 degrees C)

Biohydrogen could efficiently be produced in glucose-fed biofilm reactors filled with plastic carriers and operated at 70 degrees C. Batch experiments were, in addition, conducted to enrich and cultivate glucose-fed extreme-thermophilic hydrogen producing microorganisms from a biohydrogen CSTR reactor fed with household solid waste. Kinetic analysis of the biohydrogen enrichment cultures show that substrate (glucose) likely inhibited hydrogen production when its concentration was higher than 1 g/L. Different start up strategies were applied for biohydrogen production in biofilm reactors operated at 70 degrees C, and fed with synthetic medium with glucose as the only carbon and energy source. A biofilm reactor, started up with plastic carriers, that were previously inoculated with the enrichment cultures, resulted in higher hydrogen yield (2.21 mol H(2)/mol glucose consumed) but required longer start up time (1 month), while a biofilm reactor directly inoculated with the enrichment cultures reached stable state much faster (8 days) but with very low hydrogen yield (0.69 mol H(2)/mol glucose consumed). These results indicate that hydraulic pressure is necessary for successful immobilization of bacteria on carriers, while there is the risk of washing out specific high yielding bacteria.     

Extremely thermophilic cellulolytic anaerobes from icelandic hot springs

Anaerobic enrichment cultures with Avicel as substrate and inoculated with biomat samples from Icelandic hot springs were cultured at 70 ° or 78 °C and examined for the presence of microorganisms that produce extracellular cellulolytic and xylanolytic enzymes. From four enrichments grown at 78 °C eighteen strains were isolated. Five of the strains were screened for their substrate utilization, and on the basis of differences in morphology and substrates used, the two most unique strains were selected for further characterization. All cellulolytic cultures were rod-shaped and non-sporeforming. Motility was not observed. Cells stained gram-negative at various stages of the growth phase. During growth on Avicel, most cultures produced acetate as the major fermentation product, with smaller amounts of lactic acid and ethanol. Carbon dioxide and hydrogen were also produced. The phenotypic characteristics of the enrichment cultures and of isolates are described and assessed in relation to temperature and pH in the hot spring environment. A comparison is made between Icelandic strains isolated in our laboratory and strains isolated from hot springs from other parts of the world. The biotechnological potential of this group of bacteria is briefly discussed.     

Demonstration of growth in porcine thyroid cell culture

Eagle's minimum essential medium supplemented with 20 per cent newborn calf serum (N.C.S.) allows porcine thyroid cell survival but not cell growth in vitro. In NCTC 109 medium supplemented with 20 per cent N.C.S. these cells actively grow and may be serially propagated. Cell population doubling time expressed as DNA doubling value is 3.5 days at 37 degrees C in 95 per cent air-5 per cent CO2. Thyrotropin does not affect porcine thyroid cell multiplication in vitro but stimulates the plating efficiency in primary cultures to about 130 per cent of controls. Cell selection was obtained by replacing media with Earle's balanced salt solution. This operation provoked death of nearly all cells by day 18 but subsequent addition of growth medium resulted in proliferation of epithelial cell clones. From generation 2 to generation 8, cells produce thyroglobulin but they do not actively trap iodide nor form follicles when thyrotropin is added to the media. Cell selection, demonstration of growth, as well as freeze-storage techniques described in this paper permit selection and storage of porcine thyroid cells and the potential constitution of cell collections.     

Association of Bacteria and Yeasts in Hot Springs

The thermophilic bacterium Bacillus sp. strain TB-1 was isolated in association with the yeast Debaryomyces vanriji from hot springs at 46°C. It was shown that TB-1 excreted thiamine into the culture broth, which not only promoted D. vanriji growth in mixed culture but also increased the maximal temperature for yeast growth.     

Tepidibaculum saccharolyticum gen. nov., sp. nov. a moderately thermophilic,anaerobic, spore-forming bacterium isolated from a terrestrial hot spring

Extremophiles - A novel moderately thermophilic, anaerobic bacterium, strain STR9T was isolated from terrestrial hot spring at Uzon Caldera, Kamchatka peninsula, Russia. Cells of strain STR9T were...     

聚乙烯醇降解酶产生菌的分离的发酵条件

A bacterium D8 strain which high efficiently degrading PVA was isolated from waste water of factory. The strain possesses the abilities of completely degrading 0.5 per cent of PVA (500, 1700) included in the culture medium for four days. It was identified Pseudomonas pseudoalcaligenes. Fermentation conditions of the strain have been investigated. The suitable medium consisted of PVA 1.5% (NH4)2SO40.1%, K2HPO4 0.24%, KH2PO4 0.04%, MgSO4.7H2O 0.035%, NaCl 0.01%, FeSO4 0.001%, yeast extract 0.15%, pH 7.5. The optimal condition for enzyme production are as follows: 250 ml shake filled with 30 ml medium, 30 degrees C, 160n/min incubation period 72 h. Under such conditions enzyme activity is highest.     

Marinobacter lutaoensis sp. nov., a thermotolerant marine bacterium isolated from a coastal hot spring in Lutao,Taiwan

A heterotrophic and thermotolerant marine bacterium, designated strain T5054, was isolated from a hot spring on the coast of Lutao, Taiwan. It was a strictly aerobic, Gram-negative rod. Cells grown in broth cultures were non-spore-forming and motile by means of one to several polar flagella. It seems that pilus-like structures were produced from both poles of the cells. Strain T5054 required Na+ for growth and exhibited optimal growth at about 45 degrees C, pH 7, and 3-5% NaCl. It contained iso-C15:0 as the most abundant fatty acid and ubiquinone-8 as the only isoprenoid quinone. Its genomic DNA G+C content was 63.5 mol%. The strain did not require vitamins or other organic growth factors, and it grew on glucose, mannitol, and a variety of organic acids and amino acids as sole carbon sources. Characterization data, together with the results of a 16S rDNA-based phylogenetic analysis, indicated that strain T5054 could be classified as a new species in the genus Marinobacter. The name Marinobacter lutaoensis sp. nov. is proposed for this new bacterium. The type strain is T5054 (CCRC 17087; JCM 11179).     

Actinobacterial Diversity in Hot Springs in Tengchong (China), Kamchatka (Russia), and Nevada (USA)

Actinobacterial diversity was investigated in three hot springs [Tengchong Frog Mouth (TFM) hot spring (in Tengchong, China), Kamchatka Robb Flag hot spring (in Kamchatka, Russia), and Nevada Boiling Spring (in Nevada, USA)], a Tengchong Frog Mouth hot spring-derived pond and a soil sample near the TFM hot spring. Culture-independent techniques were employed including denaturing gradient gel electrophoresis and actinobacterial 16S rRNA gene phylogenetic analysis. The three hot springs and the hot spring-derived pond had pH of 6.8 to 7.9 and temperature of 50 to 81°C. Actinobacteria were for the first time found to be very diverse in hot springs with temperature up to 81°C, indicating that they have an unexpected capability of adapting to hot spring environments. A total of 346 actinobacterial clones were obtained from the three hot springs, and these clones were identified as 31 RFLP types, falling into the Actinomycetales (only limit to the Frankineae suborder), the Rubrobacterales, uncultured Candidatus Microthrix, unclassified Actinobacteria, and uncultured Candidate Division OP10. Our data have implications for a better understanding of the distribution of Actinobacteria in hot springs.     

The thermophilic bacteria hydrolyzing agar: Characterization of thermostable agarase

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55–60°C and pH 6.5–7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80°C (optimum, 70°C). The preincubation of this enzyme at 90° C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg²⁺ and 25 mM Co²⁺.