一株高温产粘菌株的筛选及其产胞外聚合物分析

从油井采出水中分离到一株高温产胞外聚合物的细菌MS-1,经16S rDNA基因序列分析属于芽孢杆菌属(Bacillus sp.).该菌能在60℃生长并产生胞外聚合物,其中胞外多糖含量为48.3%～54.5%.主要由甘露糖、葡萄糖、半乳糖组成,摩尔比分别是2.04:1.00:0.89.胞外聚合物中蛋白含量为37.2%～42.4%,主要由甲硫氨酸、亮氨酸、天门冬氨酸、丙氨酸、组氨酸和丝氨酸组成.利用透射电镜和环境扫描电镜对胞外聚合物的形成进行了观察.该菌的分离和研究为高温油藏的微生物调剖和驱油奠定了生物学基础.     

Methanol utilization by a novel thermophilic homoacetogenic bacterium,Moorella mulderi sp. nov., isolated from a bioreactor

A thermophilic, anaerobic, spore-forming bacterium (strain TMS) was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as the energy source. Cells were gram-positive straight rods, 0.4-0.6 microm x 2-8 microm, growing as single cells or in pairs. The temperature range for growth was 40-70 degrees C with an optimum at 65 degrees C. Growth was observed from pH 5.5 to 8.5, and the optimum pH was around 7. The salinity range for growth was 0-45 g NaCl l(-1 )with an optimum at 10 g l(-1). The isolate was able to grow on methanol, H(2)-CO(2 )(80/20%, v/v), formate, lactate, pyruvate, glucose, fructose, cellobiose and pectin. The bacterium reduced thiosulfate to sulfide. The G+C content of the DNA was 53 mol%. Comparison of 16S rRNA genes revealed that strain TMS is related to Moorella glycerini (96%, sequence similarity), Moorella thermoacetica (92%) and Moorella thermoautotrophica (92%). On the basis of physiological and phylogenetic differences, strain TMS is proposed as a new species within the genus Moorella, Moorella mulderi sp. nov. (=DSM 14980, =ATCC BAA-608).     

Production of a high molecular weight levan by Bacillus paralicheniformis,an industrially and agriculturally important isolate from the buffalo grass rhizosphere

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having β(2?→?6) linked backbone with β(2?→?1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~?42 g/l) of levan having high weight average molecular weight (Mw) of 5.517?×?10⁷ Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

     

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+).     

Taxonomic Characteristics of a Thermophilic Acidophilic Strain of Bacillus Producing Thermophilic Acidophilic Amylase and Thermostable Xylanase

Taxonomic characteristics of a strain of thermophilic acidophilic bacillus, Bacillus sp. 11-1S, which had the ability to produce thermophilic acidophilic amylase and thermostable xylanase were examined. Cells of the organism were aerobic, heterotrophic, Gram-positive, spore-forming rods. It grew at temperatures between 45 and 70°C (optimum 65°C) in media of pHs ranging from 2.0 to 5.0 (optimum 3.5 ~ 4.0). Physiological and biochemical characteristics were identical with those of Bacillus acidocaldarius, and % GC of DNA (59%) was close to that of the latter (61 ~ 62%). From these results it was concluded that the organism belongs to B. acidocaldarius Darland and Brock.     

A Novel EPS-Producing Strain of Bacillus licheniformis Isolated from a Shallow Vent Off Panarea Island (Italy)

A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13^T and Bacillus sonorensis DSM 13779^T. Further DNA–DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13^T and 39.12 % with B. sonorensis DSM 13779^T. Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l^?1) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a β-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.     

Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus

The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996     

Thermophilic bacterial isolates producing lipase

Abstract A number of lipase-producing thermophilic bacteria were isolated from natural habitats. One isolate, obtained from a coal tip sample, was examined in some detail: it was a highly thermophilic Bacillus sp. (optimum growth temperature approx. 65°C) and at 55°C it produced the maximum level of lipase (about 4 U/ml) in a medium containing Tween-80 (polyoxyethylene sorbitan monooleate) as the principal carbon source when growth had virtually ceased. Lipase synthesis thus appears to be inducible, and since a very low level of lipase was observed when the isolate was grown in a medium containing a carbon source like glucose as well as Tween-80, lipase synthesis is apparently also subject to catabolite repression.     

Characterization and emulsifying property of a carbohydrate polymer produced by Bacillus pumilus UW-02 isolated from waste water irrigated agricultural soil

Bacillus pumilus UW-02, an isolate from agricultural soil irrigated with waste water was found to produce a carbohydrate polymer in the form of extracellular polysaccharide (EPS) in glucose mineral salts medium (GMSM). The recovery rates of EPS by ion-exchange and gel filtration chromatography were around 63% and 90%, respectively. As evident from HPLC and FT-IR analyses, the EPS was found to be a heteropolymer consisting glucose, mannose, xylose, arabinose, and N-acetyl glucosamine as monomer units. Different oligosaccharide combinations namely hexose(4), hexose(6) pentose(1) and hexose(10) pentose(1) are obtained after partial hydrolysis of EPS using MALDI-ToF-MS. Electron micrographs portrayed the intense affinity of the EPS molecules for each other, thereby justifying its viscosifying and thickening properties. The EPS with an average molecular weight of 218 kDa and thermal stability up to 180 °C showed pseudoplastic rheology and significant emulsifying activities.     

Polysaccharides from Extremophilic Microorganisms

Several marine thermophilic strains were analyzed for exopolysaccharide production. The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions. We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains. The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character. The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter(-1) was obtained with mannose as carbon and energy source. NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different alpha-D-mannoses and three different beta-D-glucoses. It seems to be close to some xantan polymers. EPS 2 was a mannan. Four different alpha-D-mannoses were found as the repeating unit. Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.     

Enumeration of thermophilic Bacillus species in composts and identification with a Random Amplification Polymorphic DNA (RAPD) protocol

The thermophilic microbial flora of general garden and domestic wastes composts, derived from thermogenic, post-thermogenic and maturation phases, was analysed using spore and total plate counts in combination with an optimised RAPD protocol. A total of 459 isolates were recovered obtained at 55 degrees C, and another 56 at 70 degrees C using tryptic soy-starch agar plates, with near-equal numbers being derived from total plate counts or spore preparations. The isolates were obtained from 11 compost samples and were assigned to eighteen different RAPD fingerprint types, with 76.1% of these ultimately being positively assigned by their RAPD profiles to just 2 species including Bacillus thermodenitrificans and B. licheniformis. Viable cell numbers ranged from 1.4 to 150 x 10(6) colony forming units per gram compost (wet weight), with the highest two counts being from 2 week and 4 week old compost samples with temperatures of 70 degrees C and 55 degrees C, respectively. B. thermodenitrificans was a dominant isolate (representing more than 50% of isolates from total plate counts) in 7 of the 11 individual compost total plate count samples between 30 degrees C to 73 degrees C, and accounted for 68.9% of all isolates overall. Another relatively common Bacillus species that was identified with RAPDs in significant numbers included B. licheniformis (7.2% of all isolates and dominant isolate in 1 sample). Three other relatively common RAPD profiles could not be identified by comparison with known species in a RAPD profile database but were tentatively identified using 16S rDNA sequence comparisons. These were B. sporothermodurans (4.9% of all isolates and dominant in 1 sample), B. thermosphaericus (7.4% and dominant in 1 sample) and Terrabacter tumescens (5.0%). Overall, based on the vegetative and spore count results and the subsequent RAPD-based identification, the data strongly support a significant role for B. thermodenitrificans in the composting process, and casts doubt on the notion that B. stearothermophilus sensu strictu (DSMZ 22) is a prominent member within compost ecology.     

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.     

嗜热芽孢杆菌噬菌体的分离及特征研究

目的：从腾冲热海温泉中分离嗜热芽孢杆菌噬菌体,并初步分析其特征。方法：采用双层平板法分离纯化嗜热芽孢杆菌噬菌体,对分离得到的噬菌体进行电镜形态观察,按照感染复数（MOI）分别为0．01、0．1、1．0、10和100加入噬菌体纯培养液和宿主菌,55℃、160r／min培养8h后测定噬菌体滴度,并进行噬菌体的热稳定性和pH稳定性分析。结果：从腾冲热海温泉中分离得到的噬菌体为二十面体型;其感染宿主菌NHH4形成清晰的噬菌斑,最适MOI为1．0,最适感染温度为55℃,最适感染pH值为7．5。将这株噬菌体命名为TBIP1。结论：从腾冲热海温泉中分离得到的噬菌体TBIP1为典型的二十面体型,当MOI为1．0时,TBIP1感染其宿主菌产生的子代噬菌体滴度最高。     

Characterization of Symbiobacterium toebii, an obligate commensal thermophile isolated from compost

A symbiotic thermophilic bacterium, strain SC-1, was isolated from hay compost (toebi) in Korea. The new isolate exhibited an obligate commensal interaction with a thermophilic Geobacillus strain and required crude extracts and/or culture supernatant from Geobacillus sp. SK-1 for axenic growth. The growth factors from Geobacillus sp. SK-1 were irreversibly inactivated by phenol or protease treatment, suggesting that they might be proteins. The cells of strain SC-1 were non-spore forming, nonmotile rods that were stained Gram-negatively. The isolate was a microaerophilic heterotroph. Growth was observed between 45 degrees and 70 degrees C (optimum: 60 degrees C; 2.4-h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5). The G+C content of the genomic DNA was 65 mol%, and the major quinones were MK-6 and MK-7. A phylogenetic analysis of its 16S rDNA sequence indicated that strain SC-1 is closely related to Symbiobacterium thermophilum and so was named Symbiobacterium toebii on the basis of its physiological and molecular properties.     

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 and characterization of Bacillus thioparus sp. nov., chemolithoautotrophic, thiosulfate-oxidizing bacterium

A novel bacterium, strain BMP-1(T), was isolated from a continuous wastewater treatment culture system operating with a bacterial consortium. Cells of the isolate were Gram-variable, aerobic, moderately halotolerant, motile and endospore-forming rods. Strain BMP-1(T) grew chemolithoautotrophically by oxidation of thiosulfate to sulfate with a growth yield of 1.07 g protein mol(-1) of thiosulfate consumed. DNA G+C content was 43.8 mol%. Its cell wall had peptidoglycan based on m-diaminopimelic acid, and the major component of fatty acid was C(15 : 0). The 16S rRNA gene analysis showed that strain belongs to the genus Bacillus, sharing a 99.5% of sequence similarity with Bacillus jeotgali CCM 7133(T). DNA-DNA hybridization between the isolate of this study and this strain was 44%. Thus, the inclusion of strain BMP-1(T) in the genus Bacillus is suggested as a novel species and the name Bacillus thioparus sp. nov. (Type strain BMP-1(T)=BM-B-436(T)=CECT 7196(T)) is proposed. The sequence of the 16S rRNA gene has been deposited in GenBank with accession number DQ371431.     

Prototrophic Thermophilic Bacillus: Isolation, Properties, and Kinetics of Growth

A thermophilic bacillus (minimal growth temperature 41 C, optimal 55 to 58 C, and maximal 65 C) was isolated from a manure pile. It is very similar to Bacillus stearothermophilus, but it differs in its inability to hydrolyze starch. The thermophilic isolate is a prototroph which grows in a minimal medium consisting of glucose, ammonium salt, phosphate buffer, and inorganic salts. At all temperatures studied (low to high), the same minimal nutritional requirements prevailed. The Arrhenius constant for growth was found to be 15,000 and 13,500 cal/mole in the minimal and rich media, respectively.     

Isolation and characterization of a Bacillus strain capable of degrading the extracellular glucan from Cellulomonas flavigena strain KU

P.A. BERTRAM, C.S. BULLER, G.C. STEWART AND J.M. AKAGI. 1993. Bacteria capable of utilizing the water-insoluble purified extracellular (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU by extracellular enzymes, were insolated and characterized. Enrichment cultures from a Winogradsky column were incubated anaerobically at 55C with curdlan as the sole source of carbon. Colonies surrounded by zones of clearing were selected from subcultures on solid curdlan media. One of the isolates was chosen for further study and identified by conventional methods, API-tests with calculation of similarity coefficients and ID-scores, estimation of mol% (G + C) and DNA-DNA liquid hybridization. The isolate is a facultatively anaerobic, facultatively thermophilic Bacillus sp. Identification at the species-level was not achieved. The isolate was characterized by some rare traits among bacilli, but it remains unresolved whether it defines a new taxon.     

A moderately thermophilic and mercaptan-degrading Bacillus licheniformis strain CAN55 isolated from gas-washing wastewaters of the phosphate industry,Tunisia

A moderately thermophilic bacterium, designated strain CAN55, was isolated from the gas-washing wastewaters of the phosphate industry, located in Sfax, Tunisia, after enrichment on 1-decanethiol, a mercaptan used as the sole carbon and energy source. Strain CAN55 belonged to the Bacillus genus and was closely related to Bacillus licheniformis, with 16S rRNA gene sequence similarity of 99.2%. It was able to degrade 83% of 1-decanethiol (3 mM), and 39% of decyl disulfide, the product of spontaneous autoxidation, after 14 days of incubation at 55 °C, and without yeast extract added. Strain CAN55 was also capable of degrading 70% of 1-dodecanethiol (3 mM) after 14 days of incubation at 55 °C. During the growth on decanethiol and dodecanethiol, the decrease of surface tensions of cell-free culture supernatants from 55.1 to 40 mN/m and from 56.1 to 41 mN/m, respectively, after 26 h of incubation at 55 °C, was an agreement with the production of biosurfactant(s). In addition, strain CAN55 degraded 1-heptanethiol and cyclohexylmercaptan thus suggesting that this isolate may be useful for the bioremediation of mercaptan contaminated sites.