Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost

Abstract During the first stage of the preparation of mushroom compost oxygen is believed to be readily available. However we measured methane in the evoking air above the compost piles and were able to isolate thermophilic methanogenic bacteria from this compost. The isolates grow only on H₂ and CO₂ as energy and carbon source and do not require complex factors for growth. On the basis of nutritional and morphological characteristics these methanogens were identified as strains of Methanobacterium thermoautotrophicum .     

Isolation and characterization of indigenous thermophilic bacteria active in natural attenuation of bio-hazardous petrochemical pollutants

Common petrochemical compounds, such as homocyclic polyaromatic hydrocarbons and heterocyclic NOS-polyaromatics (NOS-compounds), were used as the sole carbon and energy source to enrich indigenous bacteria harboring the catabolic ability to degrade these compounds from petroleum-contaminated soils from Kuwait. Chemical analysis of the extracted soil materials revealed residual amounts of oil (<5% w/w), presumably of heavy oil fractions with elevated S-content. Aerobic culturable mesophilic polyaromatic hydrocarbon- and NOS-degraders were abundant in these soils, whereas their moderately thermophilic counterparts constituted only a minor fraction. Glucose stimulated the growth of mesophiles and drastically suppressed the number of thermophiles. 16S rDNA was amplified by PCR from nine of the purified thermophilic strains, using primers specific for eubacteria. Sequencing of 900 bp of the 16S rDNA and database homology search tentatively aligned these isolates to low G+C Gram positive bacteria of the family Bacillaceae. Electron microscopy characterization revealed endospore-forming bacilli varying in size, with well-structured cell walls. Gas chromatography and mass spectrometry (GC/MS) analysis revealed a versatile catabolic ability of the pure and mixed cultures to degrade all tested compounds. The metabolism of the offered substrates does not involve co-metabolism, since all pure cultures consumed the offered substrates completely.     

Isolation and characterization of indigenous thermophilic bacteria active in natural attenuation of bio-hazardous petrochemical pollutants

Common petrochemical compounds, such as homocyclic polyaromatic hydrocarbons and heterocyclic NOS-polyaromatics (NOS-compounds), were used as the sole carbon and energy source to enrich indigenous bacteria harboring the catabolic ability to degrade these compounds from petroleum-contaminated soils from Kuwait. Chemical analysis of the extracted soil materials revealed residual amounts of oil (<5% w/w), presumably of heavy oil fractions with elevated S-content. Aerobic culturable mesophilic polyaromatic hydrocarbon- and NOS-degraders were abundant in these soils, whereas their moderately thermophilic counterparts constituted only a minor fraction. Glucose stimulated the growth of mesophiles and drastically suppressed the number of thermophiles. 16S rDNA was amplified by PCR from nine of the purified thermophilic strains, using primers specific for eubacteria. Sequencing of 900 bp of the 16S rDNA and database homology search tentatively aligned these isolates to low G+C Gram positive bacteria of the family Bacillaceae. Electron microscopy characterization revealed endospore-forming bacilli varying in size, with well-structured cell walls. Gas chromatography and mass spectrometry (GC/MS) analysis revealed a versatile catabolic ability of the pure and mixed cultures to degrade all tested compounds. The metabolism of the offered substrates does not involve co-metabolism, since all pure cultures consumed the offered substrates completely.     

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50–80°C and pH 6.0–8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA–DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542^T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

Isolation and properties of malate dehydrogenase from Meso-and thermophilic bacteria

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978^T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 ± 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 ± 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.     

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.     

Isolation of thermophilic iron-oxidising bacteria from sulfidic waste rock

Summary A thermophilic, rod-shaped, iron-oxidising bacterium was isolated by enrichment culture of rock samples from an overburden dump at the Rum Jungle mine site in Australia's Northern Territory. Oxidation of ferrous iron and sulfur occurred at 50–55°C, with a temperature maximum of 60°C. The isolate required yeast extract for growth. The pH optimum for iron oxidation at 50°C was 1.4. Rapid iron-oxidation occurred at a pH as low as 0.35, but little or no oxidation occurred at or above pH 2.2.     

Isolation and properties of malate dehydrogenase from meso- and thermophilic bacteria

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 +/- 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 +/- 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Isocitrate dehydrogenase from thermophilic and mesophilic bacteria. Isolation and some characteristics

1. Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria. 2. Several characteristics of the isocitrate dehydrogenases have been examined, viz. molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section.     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。     

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)₅, the (GTG)₅ and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)₅ and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.     

一株嗜热聚对苯二甲酸乙二醇酯降解菌的分离及其降解特性解析北大核心

【目的】大量聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)塑料作为废弃物被丢弃,严重危害生态健康。针对嗜热PET降解菌缺乏这一情况,本研究旨在获得能够降解PET的嗜热菌,并阐述其降解机制。【方法】采集云南腾冲热泉中的废弃PET瓶,分析其表面生物膜的微生物群落多样性,从中筛选能够以PET为营养源生长的嗜热菌,并基于16S rRNA基因序列加以鉴定;以菌株的定殖能力与生长曲线为指标,优选出降解能力较强的降解菌,并测定其最适pH、温度和NaCl浓度;降解能力较强的降解菌分别作用于PET及PET中间体双(羟乙基)对苯二甲酸酯[bis(hydroxyethyl)terephthalate,BHET]和对苯二甲酸单(2-羟乙基)酯[mono(2-hydroxyethyl)terephthalate,MHET],测定产物生成量与降解率;通过观察PET膜表面微观结构、活菌数、酯酶活性等探究降解菌与PET的互作过程。【结果】废弃PET瓶表面生物膜中的微生物群落多样性低;从生物膜中筛选出5株能够以PET为营养源生长的嗜热菌;其中,菌株JQ3以PET为唯一碳源生长最佳,作为降解能力较强的降解菌,被鉴定为嗜热淀粉芽孢杆菌(Bacillus thermoamylovorans),其最适生长pH为7.0、最适生长温度为50℃、最适生长NaCl浓度为0.5%;菌株JQ3以0.043 mg PET/d的速率降解PET,对苯二甲酸(terephthalic acid,TPA)产量在第7天达到峰值45.2 mmol/L;菌株JQ3对PET中间体降解效率显著,6 h可降解85.9%的BHET,60 h可降解50.1%的MHET。菌株JQ3能够定殖于PET表面并形成生物膜,侵蚀PET并造成开裂和剥落。【结论】B.thermoamylovorans JQ3作为一株嗜热PET降解菌,能够高温(60℃)降解PET及其中间体,为实现PET的有效降解提供了新策略。     

Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt

Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO₂ production. The isolates grew well on MRS broth at 30–37°C and a pH of 6.5–8.0. The optimum temperature and pH for growth are 37°C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0–6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.     

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).     

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp.

Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.     

Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli.

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.     

Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria

A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plamids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.