Isolation and characterization of highly thermophilic xylanolytic Thermus thermophilus strains from hot composts

This is the first detailed report of xylanolytic activity in Thermus strains. Two highly thermophilic xylanolytic bacteria, very closely related to non-xylanolytic T. thermophilus strains, have been isolated from the hottest zones of compost piles. Strain X6 was investigated in more detail. The growth rate (optical density monitoring) on xylan was 0.404.h-1 at 75 degrees C. Maximal growth temperature was 81 degrees C. Xylanase activity was mainly cell-bound, but was solubilized into the medium by sonication. It was induced by xylan or xylose in the culture medium. The temperature and pH optima of the xylanases were determined to be around 100 degrees C and pH 6, respectively. Xylanase activity was fairly thermostable; only 39% of activity was lost after an incubation period of 48 h at 90 degrees C in the absence of substrate. Xylanolytic T. thermophilus strains could contribute to the degradation of hemicellulose during the thermogenic phase of industrial composting.     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

嗜热菌的耐热L—乳酸脱氢酶的研究

About 200 strains of extreme thermophilic bacteria were isolated from hot springs in Guandong province. A strain, HG25, was found to produce thermostable intracellular L-lactate dehydrogenase (EC. 1.1.1.27). It has the characteristic of Thermus sp. The cells were gram-negative, non-sporulating, nonmotile, aerobic rods containing yellow pigment. The optimum temperature for growth was between 65 degrees C to 75 degrees C, the maximum 85 degrees C, and minimum 40 degrees C. The generation time at the optimum was about 80 min. Starch was not hydrolyzed. Acid was not produced from glucose. The G+C content in DNA was 62-65 mol% (Tm). As the properties of strain HG25 is similar to those of Thermus aquaticus and T. thermophilus HB 8 belonging to the genus Thermus. The thermostable L-lactate dehydrogenase was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. For pyruvate reduction, the optimum temperature of the enzyme was 60 degrees C and pH 8.0. After incubation in 0.1 mol/L phosphate buffer pH 7.4 at 70 degrees C for 10 min, the enzyme retained about 85% of its original activity. The half-live time (t1/2) at 85 degrees C was 10 min.     

Isolation and Characterization of Extremely Thermophilic Bacteria from Hot Springs

In order to investigate the mechanism of microbial growth at elevated temperatures, it was tried to isolate different thermophilic microorganisms from wide origins, such as soils, composts, manure piles and hot spring waters. As the result, 5 strains of extremely thermophilic bacteria, the maximum, the optimum and the minimum temperatures for growth of which were 80, 70~75, and 40°C, respectively, were isolated from Izu-Atagawa hot spring and Beppu hot springs. These bacteria were gram-negative, yellow-pigmented, non-motile and non-sporulating rods of 0.5~0.7 μ in diameter and 2~5 μ in length. They were heterotrophs requiring several amino acids (such as glutamate, aspartate, et al.) and vitamins (such as biotin, folic acid and p-aminobenzoic acid) and grew well at neutral to slight alkali pH. The content of GC pairs of DNAs from the 5 strains was 69~70%, and this seemed to be one of the highest values in bacteria so far known. Among the 5 strains, strain AT–62 was named as Thermus flavus sp. n. AT–62 from its morphological and physiological characteristics. Comparison between Thermus flavus and other extremely thermophilic bacteria as Thermus aquaticus and Flavobacterium thermophilum is described and discussed in reference to classification of extremely thermophilic bacteria.     

Thermophilic and thermotolerant bacteria that assimilate methane]

Microorganisms assimilating methane at temperatures above 40 degrees C were isolated from various natural sources: ooze, mud, waste water of coal pits. The bacteria are obligate methylotrophs and are represented by two groups: (a) thermotolerant, growing at 37 to 45 degrees C; and (b) thermophilic, growing at 50 to 62 degrees C. The selective factor used to isolate various physiological forms of methylotrophs is corresponding temperatures of growth which allow to isolate from the same substrate meso-, thermotolerant, and thermophilic forms. Morphological and physiological properties of the strains are described. The thermotolerant cultures of methylotrophs are similar to Methylobacter vinelandii, though differ from it by some characteristics. The thermophilic microorganisms should be classed as a separate species Methylococcus thermophilus.     

Effect of temperature on bacterial species diversity in thermophilic solid-waste composting

Continuously thermophilic composting was examined with a 4.5-liter reactor placed in an incubator maintained at representative temperatures. Feed was a mixture of dried table scraps and shredded newspaper wetted to 55% moisture. One run at 49 degrees C (run A) employed a 1:4 feed-to-compost ratio, while the other runs used a 10:1 ratio and were incubated at 50, 55, 60, or 65 degrees C. Due to self-heating, internal temperatures of the composting mass were 0 to 7 degrees C hotter than the incubator. Two full-scale composting plants (at Altoona, Pa., and Leicester, England) were also examined. Plate counts per gram (dry weight) on Trypticase soy broth (BBL Microbiology Systems) with 2% agar ranged from 0.7 X 10(9) to 5.3 X 10(9) for laboratory composting and 0.02 X 10(9) to 7.4 X 10(9) for field composting. Fifteen taxa were isolated, including 10 of genus Bacillus, which dominated all samples except that from run A. Species diversity decreased markedly in laboratory composting at 60 degrees C and above, but was similar for the three runs incubated at 49, 50, and 55 degrees C. The maximum desirable composting temperature based on species diversity is thus 60 degrees C, the same as that previously recommended based on measures of the rate of decomposition.     

Effect of temperature on bacterial species diversity in thermophilic solid-waste composting.

Continuously thermophilic composting was examined with a 4.5-liter reactor placed in an incubator maintained at representative temperatures. Feed was a mixture of dried table scraps and shredded newspaper wetted to 55% moisture. One run at 49 degrees C (run A) employed a 1:4 feed-to-compost ratio, while the other runs used a 10:1 ratio and were incubated at 50, 55, 60, or 65 degrees C. Due to self-heating, internal temperatures of the composting mass were 0 to 7 degrees C hotter than the incubator. Two full-scale composting plants (at Altoona, Pa., and Leicester, England) were also examined. Plate counts per gram (dry weight) on Trypticase soy broth (BBL Microbiology Systems) with 2% agar ranged from 0.7 X 10(9) to 5.3 X 10(9) for laboratory composting and 0.02 X 10(9) to 7.4 X 10(9) for field composting. Fifteen taxa were isolated, including 10 of genus Bacillus, which dominated all samples except that from run A. Species diversity decreased markedly in laboratory composting at 60 degrees C and above, but was similar for the three runs incubated at 49, 50, and 55 degrees C. The maximum desirable composting temperature based on species diversity is thus 60 degrees C, the same as that previously recommended based on measures of the rate of decomposition.     

Temperature effect on Fusarium oxysporum f.sp. melonis survival during horticultural waste composting

AIMS: The aim of this work was to study the effect of high temperatures generated during composting process, on the phytopathogen fungus Fusarium oxysporum f.sp. melonis. This investigation was achieved by both in vivo (semipilot-scale composting of horticultural wastes) and in vitro (lab-scale thermal treatments) assays. METHODS AND RESULTS: Vegetable residues infected with F. oxysporum f.sp. melonis were included in compost piles. Studies were conducted in several compost windrows subjected to different treatments. Results showed an effective suppression of persistence and infective capacity, as this process caused complete fungal elimination after 2-3 days of composting. In order to confirm the effect of high temperature during this process, in vitro experiments were carried out. Temperature values of 45, 55 and 65 degrees C were tested. All three treatments caused the elimination of fungal persistence. Treatment at 65 degrees C was especially effective, whereas 45 degrees C eliminated fungal persistence only after 10 days. CONCLUSIONS: The composting process is an excellent alternative for the management of plant wastes after harvesting, as this procedure is able to suppress infective capacity of several harmful phytopathogens such as F. oxysporum f.sp. melonis. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium oxysporum f.sp. melonis is a plant pathogen fungus specially important in the province of Almería (south-east Spain), where intensive greenhouse horticulture is very extended. High temperatures reached during composting of horticultural plant wastes ensure the elimination of phytopathogen microorganisms such as F. oxysporum f.sp. melonis from vegetable material, providing an adequate hygienic quality in composts obtained.     

Phenotypic and genotypic characterization of esterase-producing Ureibacillus thermosphaericus isolated from an aerobic digestor of swine waste.

Eight closely related thermophilic strains were isolated from an aerobic and thermophilic treatment of swine wastes. The pleomorphic cells (short and long rods; cocci) showed peritrichous flagella, terminally swollen sporangium, and liberated spores exhibiting hairy appendages. The Gram reaction was negative for both young (4 h) and old (48 h) cultures. Several features, such as colonial morphology, growth between 35 degrees C and 65 degrees C, presence of catalase, presence of spores, and strictly aerobic metabolism (except for one strain), are similar to those found for the genus Bacillus. The inability of the strains to use sugars, except esculin, as source of carbon and energy and the whole cell fatty acid composition are similar to those found in Bacillus thermosphaericus DSM 10633. Sequence analysis of the 16S rRNA gene revealed 99.8%-99.9% identity for seven of the thermophilic strains with this species. A new genus, Ureibacillus, was recently proposed for type strain B. thermosphaericus DSM 10633 The last strain exhibits 97.8% and 97.3% identity with Ureibacillus terrenus DSM12654 and Bacillus sp. TP-84, respectively. Esterase activities were detected for all strains, and assays on p-nitrophenyl butyrate and p-nitrophenyl caprylate revealed that strains were more active on the shorter substrate.     

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.     

The proline biosynthesis gene proA from the thermophilic bacterium Thermus ruber: its cloning, sequencing and heterologous expression]

The proA proline biosynthesis gene of thermophilic bacterium Thermus ruber was cloned and sequenced, and several properties of the encoded enzyme, gamma-glutamylphosphate reductase (GPR) were studied. The proA open reading frame (ORF) was of 1286 bp. Nucleotide sequence analysis revealed the ATG initiation codon in position 36 and the TTA termination codon in position 1304. A deduced protein product of the gene was shown to be of 44,919 Da in molecular weight. The GC content was 66%, as is characteristic of various bacteria of the genus Thermus. An amino acid sequence encoded by the cloned gene showed the highest homology (up to 64%) with GPR of T. thermophilus. The maximum activity of GPR (8.2 x 10(-2) units/ml) was observed at 55 degrees C. A weak enzymatic activity was also detected at 70 degrees C. The enzyme can be used in biotechnological studies.     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

Respiration profiles in monitoring the composting of by-products from the olive oil agro-industry

The composting of olive press cake (OPC) repeatedly mixed either with olive mill wastewater (OPC+OMW) or with tap water (OPC+W) was studied using the thermogradient respirometer, an apparatus that determines the respiration rates from a substrate over a wide range of different temperatures (respiratory profile). The composting processes took place over a period of five months during which nine moistenings of the OPC were performed with the respective liquids. The composting resulted in detoxification of the materials used in both treatments, as indicated by seed germination tests. However, the repeated applications of OMW resulted in recurring thermophilic phases (following each application) and in greater pH and conductivity increases in the final product, as compared to water applications. Respiration measurements performed at 35 degrees C were good indicators of the mean metabolic potential in the compost piles (the mean respiration derived from the whole respiration profile over a wide range of environmental temperatures). However, respiration measurements at higher temperatures (48.5 degrees C) were better indicators of the respiration activity occurring in situ. Following the initial thermophilic phase, the respiration potential of the composts at high temperatures (42-63 degrees C) increased drastically compared to their respiration potential at lower temperatures (17-42 degrees C) indicating the establishment of a thermophilic microflora. Subsequently, only the periodic new substrate-C applications in the form of OMW resulted in increased ratios of low temperature-to-high temperature respiration potential. These ratios decreased again following the respective thermophilic phase that each new OMW application had induced.     

Effect of high compost temperature on enzymatic activity and species diversity of culturable bacteria in cattle manure compost

To clarify the characteristics of thermophilic bacteria in cattle manure compost, enzymatic activity and species diversity of cultivated bacteria were investigated at 54, 60, 63, 66 and 70 degrees C, which were dependent on composting temperature. The highest level of thermophilic bacterial activity was observed at 54 degrees C. Following an increase in temperature to 63 degrees C, a reduction in bacterial diversity was observed. At 66 degrees C, bacterial diversity increased again, and diverse bacteria including Thermus spp. and thermophilic Bacillus spp. appeared to adapt to the higher temperature. At 70 degrees C, bacterial activity measured as superoxide dismutase and catalase activity was significantly higher than at 66 degrees C. However, the decomposition rate of protein in the compost was lower than the rate at 66 degrees C due to the higher compost temperature.     

Species Diversity and Substrate Utilization Patterns of Thermophilic Bacterial Communities in Hot Aerobic Poultry and Cattle Manure Composts

This study investigated the species diversity and substrate utilization patterns of culturable thermophilic bacterial communities in hot aerobic poultry and cattle manure composts by coupling 16S rDNA analysis with Biolog data. Based on the phylogenetic relationships of 16S rDNA sequences, 34 thermophilic (grown at 60 degrees C) bacteria isolated during aerobic composting of poultry manure and cattle manure were classified as Bacillus licheniformis, B. atrophaeus, Geobacillus stearothermophilus, G. thermodenitrificans, Brevibacillus thermoruber, Ureibacillus terrenus, U. thermosphaericus, and Paenibacillus cookii. In this study, B. atrophaeus, Br. thermoruber, and P. cookii were recorded for the first time in hot compost. Physiological profiles of these bacteria, obtained from the Biolog Gram-positive (GP) microplate system, were subjected to principal component analysis (PCA). All isolates were categorized into eight different PCA groups based on their substrate utilization patterns. The bacterial community from poultry manure compost comprised more divergent species (21 isolates, seven species) and utilized more diverse substrates (eight PCA groups) than that from cattle manure compost (13 isolates, five species, and four PCA groups). Many thermophilic bacteria isolated in this study could use a variety of carboxylic acids. Isolate B110 (from poultry manure compost), which is 97.6% similar to U. terrenus in its 16S rDNA sequence, possesses particularly high activity in utilizing a broad spectrum of substrates. This isolate may have potential applications in industry.     

云南温泉高温菌的研究Ⅲ保山县摆洛塘温泉中的极端嗜热细菌

用1~#、7~#PPY三种培养基,从保山摆洛塘温泉(86℃,pH7.0)分离到生长温度范围为35—75℃的极端嗜热细菌4株,即“YN8617”、“YN8618”、“YN86290”,YN86291”。经鉴定,“YN3617”和“YN86291”为Bacillus stearother mophilus,“YN86290”为Becillus caldolyticus;“YN3618”暂定为“Extremely thermophilic strains of B.Coagulans”。没有分离到水生栖热菌属(Thermus)的细菌。     

Comparative genomics of Thermus thermophilus: Plasticity of the megaplasmid and its contribution to a thermophilic lifestyle

The bacterium Thermus thermophilus grows at temperatures up to 85 degrees C and is equipped with thermostable enzymes of biotechnological interest. The recently decoded genomes of two strains of T. thermophilus, HB27 and HB8, each composed of a chromosome and a megaplasmid, must certainly encode specific strategies to encounter the thermophile challenge. Here, a genome comparison was undertaken to distinguish common functions from the flexible gene pool, which gave some clues about the biological traits involved in a thermophile lifestyle. The chromosomes were highly conserved, with about 100 strain-specific genes probably reflecting adaptations to the corresponding biological niche, such as metabolic specialities and distinct cell surface determinates including type IV pili. The two megaplasmids showed an elevated plasticity. Upon comparison and re-examination of their gene content, both megaplasmids seem to be implicated in assisting thermophilic growth: a large portion of their genes are apparently involved in DNA repair functions. About 30 plasmid-encoded genes exhibit sequence and domain composition similarity to a predicted DNA repair system specific for thermophilic Archaea and bacteria. Moreover, the plasmid-encoded carotenoid biosynthesis gene cluster is interlocked with genes involved in UV-induced DNA damage repair. This illustrates the importance of DNA protection and repair at elevated growth temperatures.     

Thermus thermophilus TMY isolated from silica scale taken from a geothermal power plant

Aims:  To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains.
Materials and Results:  The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting.
Conclusions:  Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus . The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered.
Significance and Impact of the Study:  The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.     

Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively. The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins. The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state. Circular dichroic measurements indicated that this protein had an alpha-helical content of approx. 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C. The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1). Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.