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 of a thermophilic bacterium capable of low-molecular-weight polyethylene degradation

A thermophilic bacterium capable of low-molecular-weight polyethylene (LMWPE) degradation was isolated from a compost sample, and was identified as Chelatococcus sp. E1, through sequencing of the 16S rRNA gene. LMWPE was prepared by thermal degradation of commercial PE in a strict nitrogen atmosphere. LMWPE with a weight-average-molecular-weight (Mw) in the range of 1,700–23,700 was noticeably mineralized into CO₂ by the bacterium. The biodegradability of LMWPE decreased as the Mw increased. The low molecular weight fraction of LMWPE decreased significantly as a result of the degradation process, and thereby both the number-average-molecular-weight and Mw increased after biodegradation. The polydispersity of LMWPE was either narrowed or widened, depending on the initial Mw of LMWPE, due to the preferential elimination of the low molecular weight fraction, in comparison to the high molecular weight portion. LMWPE free from an extremely low molecular weight fraction was also mineralized by the strain at a remarkable rate, and FTIR peaks assignable to C–O stretching appeared as a result of microbial action. The FTIR peaks corresponding to alkenes also became more intense, indicating that dehydrogenations occurred concomitantly with microbial induced oxidation.     

Isolation and characterization of a thermophilic, acetate-utilizing methanogenic bacterium

Abstract A thermophilic acetate-decarboxylating methanogenic bacterium was isolated from a laboratory-scale 60°C sludge digestor. Cells form straight filaments with flat to blunted ends normally consisting of 2–3 cells held together by a sheath-like outer cell wall. The organism uses acetate, H₂-CO₂ and formate for methanogenesis and growth. With acetate as the sole methanogenic substrate, almost all of the radioactivity from methyl-labelled acetate appeared as methane. Acetate was converted to methane in equimolar amounts with a doubling time of 3 days.     

Isolation and characterization of hydrogen peroxide producing Aerococcus sp. from soil samples

Genes involved in 4-methyl-o-phthalate and 4-hydroxy-iso-phthalate catabolism reside on a 226-232 kbp catabolic plasmid termed MOP. This was confirmed by transformation and conjugation into an isogenic heat-cured (MOP-) derivative of the wild-type isolate, identified and termed Pseudomonas cepacia Pc701. Transformation confirmed the presence of Tn1 in MOP derived from Pc704, a mutant deficient in 4-methyl-o-phthalate catabolism. pCS1, a recombinant plasmid bearing MOP DNA, complemented MOP::Tn1 restoring the ability of Pc704 to grow on 4-methyl-o-phthalate. DNA-DNA hybridization using pCS1 as probe confirmed that loss of 4-methyl-o-phthalate catabolism by Pc704 was the result of Tn1 insertion into a 2.1 kbp HindIII fragment of MOP.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

Isolation and characterization of a thermostable beta-xylosidase in the thermophilic bacterium Geobacillus pallidus

The isolation, purification, biochemical and biophysical characterization of the first reported beta-xylosidase from Geobacillus pallidus are described. The protein has an optimum pH close to 8 and an optimum temperature of 70 degrees C. These biochemical properties agree with those obtained by spectroscopic techniques, namely, circular dichroism (CD), infrared (FTIR) and fluorescence measurements. Thermal denaturation, followed by CD and FTIR, showed an apparent thermal denaturation midpoint close to 80 degrees C. The protein was probably a hydrated trimer in solution with, an elongated shape, as shown by gel filtration experiments. FTIR deconvolution spectra indicated that the protein contains a high percentage of alpha-helix (44%) and beta-sheet (40%). The sequencing of the N terminus and the biochemical features indicate that this new member of beta-xylosidases belongs to the GH52 family. Since there are no reported structural studies of any member of this family, our studies provide the first clue for the full conformational characterization of this protein family.     

嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

【目的】分离高效降解纤维素的嗜热厌氧菌,通过与嗜热产乙醇菌株联合培养的方式,为生产纤维素乙醇提供微生物资源。【方法】利用厌氧分离技术从降解纤维素的马粪富集物中分离到一株嗜热厌氧细菌HCp。采用形态学观察、生理生化鉴定、结合16S rDNA序列的系统发育学分析确定该菌株的分类地位,利用DNS酶活分析方法测定此分离菌株的酶学性质。【结果】分离菌株HCp革兰氏染色阴性,直杆,细胞单个或成对出现,菌体大小为(0.35-0.50)μm×(2.42-6.40)μm,严格厌氧,形成芽胞,能运动,对新霉素有一定的抗性。此菌能利用滤纸纤维素、纤维素粉、微晶纤维素、脱脂棉和水稻秸秆、明胶等,还可以利用葡萄糖、纤维二糖、木糖、木聚糖、果糖、蔗糖、核糖、半乳糖、麦芽糖、山梨糖、海藻糖、蜜二糖、甘露糖等。该菌株在pH6.5-8.5、温度35-70℃、盐浓度0%-1.0%范围内利用纤维素生长,最适pH为6.85,最适温度为60℃,最适NaCl浓度为0.2%,最佳生长条件下,在10 d内滤纸纤维素降解率可达90.40%。在HCp的纤维小体中,滤纸酶、羧甲基纤维素酶、β-葡萄糖苷酶、木聚糖酶的最适作用温度分别为70℃、70℃、70℃、60℃,并且羧甲基纤维素酶具有较高的热稳定性。部分长度的16S rDNA序列分析表明,分离菌株HCp与Acetivibrio cellulolyticus、A.cellulosolvens相似性为97.5%。【结论】分离菌株HCp是从马粪富集物中分离到的一株嗜热厌氧细菌,该菌具有较强的降解纤维素能力,生长温度范围广,酶的热稳定性好,纤维素底物利用广泛等特性,为纤维素降解产乙醇提供了良好的材料。     

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

Aims: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. Methods and Results: A solvent‐tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram‐positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p‐xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p‐xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron‐transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. Conclusions: The thermophilic, Gram‐positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. Significance and Impact of the Study: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.     

Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.     

Isolation and characterization of a novel gamma-hexachlorocyclohexane-degrading bacterium.

The natural biotic capacity of soils to degrade gamma-hexachlorocyclohexane (gamma-HCH, lindane) was estimated using an enrichment technique based on the ability of soil bacteria to develop on synthetic media and degrade the xenobiotic compound, used as the sole source of carbon and energy. Bacterial inocula from relatively highly contaminated soils (from wood treatment factories) were found to promote efficiently the degradation of gamma-HCH, which subsequently permitted isolation of a competent gamma-HCH-degrading microorganism. The decrease of gamma-HCH concurrently with the release of chloride ions and the production of CO2 demonstrated the complete mineralization of gamma-HCH mediated by the isolate. This was confirmed by gas chromatography-mass spectrometry analyses showing that degradation subproducts of gamma-HCH included an unidentified tetrachlorinated compound and subsequently 1,2,4-trichlorobenzene and 2,5-dichlorophenol. The two linA- and linB-like genes coding, respectively, for a gamma-HCH dehydrochlorinase and a dehalogenase were characterized by using a PCR strategy based on sequence homologies with previously published sequences from Sphingomonas paucimobilis UT26. Nucleotide sequence analysis of the linA-like region revealed the presence of a 472-bp open reading frame exhibiting high homology with the linA gene from S. paucimobilis, while a preliminary study also indicated strong homology among the two linB genes. All enzymes involved in the gamma-HCH degradative pathway appear to be extracellular and encoded by genes located on the chromosome, although numerous cryptic plasmids have been detected.     

Isolation of a Bacillus subtilis transformant producing thermostable alpha-amylase by DNA from a thermophilic bacterium

A Bacillus subtilis transformant producing thermostable α-amylase was isolated using DNA from a thermophilic bacterium, Thermophile V2. The extracellular α-amylase did not crossreact with a rabbit antiserum against B. subtilis α-amylase. The structural gene for the thermostable α-amylase was integrated at a different locus from B. subtilis α-amylase. It was linked to pyrA. The transformant was not thermophilic, and its upper temperature for growth was similar to that of the host bacterium.     

一株油藏嗜热厌氧杆菌的分离鉴定和代谢特征的分析

摘要：【目的】从高温油藏中发掘新的微生物种质资源。【方法】采用 Hungate 厌氧操作技术从大港油田采出水中分离到一株厌氧杆菌 HL-3。通过生理生化特征比较和16S rRNA序列比对,确定HL-3的分类地位。【结果】菌株HL-3为严格厌氧的革兰氏阴性杆菌。生长温度范围 40℃－75℃（最适温度 60℃）;pH 范围 5.0－8.0（最适 pH 6.5）;NaCl 浓度范围 0%－3.2%（最适NaCl浓度0.25%）。能够利用葡萄糖、核糖、甘露糖、木糖、纤维二糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、CO2及少量丙酸和丁醇。菌株HL-3的（G+C）mol%含量为 33.9%,与Thermoanaerobacter（嗜热厌氧杆菌属）中模式菌株T.uzonensis DSM18761T (EF530067)的16SrRNA 序列相似性为98.8%,与T.sulfurigignens DSM17917T (AF234164)的相似度次之为98.1％。菌株能够耐受浓度较高的亚硫酸根（0.1 mol/L）离子和浓度极高的硫代硫酸根（0.8 mol/L）。当硫代硫酸根浓度高于0.075 mol/L时,菌体内产生硫单质颗粒;同时,在培养血清瓶顶空中检测到硫化氢气体。菌株 HL-3与T.uzonensis DSM18761T对硫代硫酸根和亚硫酸根的耐受程度有很大不同。菌株HL-3对硫代硫酸根和亚硫酸根耐受程度及对硫代硫酸根的代谢机制与T.sulfurigignens DSM17917T(AF234164)极为相似,但二者代谢葡萄糖的产物却极不相同。【结论】所以菌株HL-3可能是Thermoanaerobacter属中的一个新种,其确切分类地位还有待用DNA分子杂交[1]的技术手段做进一步的鉴定。     

Isolation and characterization of a mildly thermophilic nonsulfur purple bacterium containing bacteriochlorophyll b

A method for transformation of whole Bacillus amyloliquefaciens cells by electroporation was developed. The procedure is as efficient as the protoplast transformation method, resulting in up to 10(5) transformants/micrograms plasmid DNA, but requires less effort and time. Cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 M sucrose, washed with and resuspended in 0.25 M sucrose, 1 mM HEPES, 1 mM MgCl2, 10% (v/v) glycerol, pH 7.0, at 3-5 x 10(10) cells/ml for storage at -80 degrees C. The highest transformation frequency was obtained at 7.5 kV/cm with a 25 microF capacitor. The transformation efficiency increased linearly with DNA concentration at least over the range 10 ng-12.5 micrograms/ml. Transformations with ligated DNA and of industrial strains were also successful. In addition, B. subtilis cells treated as above could be transformed by electroporation, resulting in 10(4) transformants/micrograms DNA at 12.5 kV/cm.     

一株嗜热厌氧杆菌的分离、鉴定及其代谢特征

【目的】分离、保护油藏嗜热微生物资源,解析其主要的代谢特征。【方法】利用Hungte厌氧分离技术从大港油田埕海一区油层采出液中分离出厌氧菌株BF1。通过生理生化特征分析、16S rRNA基因序列比对与电化学分析,确定BF1的分类地位及其S元素代谢对腐蚀电流的影响。【结果】菌株BF1为严格嗜热厌氧革兰氏阴性杆菌,顶端产芽孢、不运动,菌体大小为0.42μm×(1.6 5.4)μm,单生、成对或成串生长。其温度生长范围为45°C 75°C(最适温度60°C);pH生长范围在4.5 8.5(最适pH 6.5)之间,比生长速率(μm)0.99 h 1,倍增时间为42 min。能利用葡萄糖、松三糖、棉子糖、甘露糖、乳糖、纤维二糖、果糖、核糖等碳水化合物,利用葡萄糖发酵的产物是乙醇、乙酸、CO2及少量的H2。菌株BF1能还原亚硫酸盐与硫代硫酸盐产生H2S,其耐受上限分别为50 mmol/L和75 mmol/L;还原硫代硫酸钠(50 mmol/L)后其极化电阻由2 099/cm2降低至776/cm2,腐蚀电流由9.936e-006 A提高至3.25e-005 A。细胞膜脂肪酸主要由高级饱和脂肪酸组成,含量最丰富的为十五烷酸占70.6%。菌株BF1的DNA(G+C)mol%含量为34.0%,其16S rRNA与Thermoanaerobacter pseudethanolicus DSM 2355T相似性最高,为98.3%,与T.brockii subsp.brockii DSM 1457T次之,为98.0%。菌株BF1的许多生理、生化特征与T.pseudethanolicus DSM 2355T和T.brockii subsp.brockii DSM 1457T有着明显的差别,如倍增时间、最适生长温度及底物利用等;而菌株BF1的细胞膜脂肪酸组成与T.pseude-thanolicus DSM 2355T也不相同。【结论】菌株BF1可能是Thermoanaerobacter属中的一个新种,其确切分类地位还需要进一步进行DNA分子杂交;其代谢元素硫提高腐蚀电流密度,可能会对油田管道与设备造成腐蚀。     

Purification and characterization of a novel type of catalase from the bacterium Klebsiella pneumoniae

A novel type of catalase, designated KpA, was purified from the bacterium Klebsiella pneumoniae. The enzyme is unique in that it is a dimer with subunit molecular weight of 80,000, it bears a chlorine-type heme as prosthetic group, and is active over a very wide range of H+ concentrations, with a plateau from pH 2.8 to 11.8. Yet, some properties of KpA are characteristic of typical catalases: it is stable when treated with with ethanol/chloroform, cannot be reduced by dithionite and it is inhibited by 3-amino-1,2,4-triazole and by the conjugate acid forms of azide and cyanide. The protein of KpA is outstandingly resistant to denaturing conditions: it retains full activity when incubated with 8 M urea, at 30 degrees C for 4 days, it is stable for 1 h at 70 degrees C and at pH values 3.1 and 11.5 and, when dialyzed against 50 mM H2O2, it still retains 42% of its activity after 80 min.     

Isolation and functional characterization of lipase from the thermophilic alkali-tolerant bacterium Thermosyntropha lipolytica

As a result of sequencing the genome of the termophilic alkali-tolerant lipolytic bacterium Thermosyntropha lipolytica, the gene encoding a lipase secreted into the medium was identified. The recombinant enzyme was expressed in Escherichia coli. It was isolated, purified, and functionally characterized. The lipase exhibited hydrolytic activity toward para-nitrophenyl esters of various chain lengths, as well as triglycerides, including vegetable oils. The optimal reaction conditions were achieved at temperatures from 70 to 80°C and pH 8.0. This new thermostable lipase may be a promising biocatalyst for organic synthesis; it may find application in the food and detergent industry and biodiesel production.     

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov.

An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H₂/CO₂. Lactate and pyruvate were oxidized completely to CO₂ with sulfate as electron acceptor. Sulfate was required for growth on H₂/CO₂, and both acetate and sulfide were produced from H₂/CO₂-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H₂/CO₂, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min^–1 [mg protein]^–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ.