嗜树木甲烷短杆菌和甲酸甲烷杆菌的分离和特性

从我国沼气池污泥样品中,采用改良的亨氏操作技术,分离出TC713和TC708两株不运动、革兰氏染色阴性、不形成芽孢的杆菌。TC713菌株呈短杆状,往往两个相连成双杆菌,个别呈链杆状,菌落圆形透明。TC708菌株培养后期呈长杆状,弯曲,菌落圆形,周边呈现丝状。 两菌株均只能利用H2／CO2作为碳源和能源,不能利用CH,COONa、CH,NH。、CH,OH。利用甲酸较慢,且利用率不高。在培养液中分别加入酵母膏、瘤胃液或酪素水解物均能刺激1c713菌株的生长,但该菌株不需要外源辅酶M。Tc713和Tc708菌株生长最适温度分别为35℃和35—40℃在45℃生长不良。适宜生长的pH值分别为6．5—8．5和6．8—9．20在含有o．I％酵母膏的培养液中,以H,／∞,为碳源和能源,37℃下振荡培养,其菌数倍增时间分别为6—7小时和8—10．5小时。Tc713菌株经荧光免疫测定,仅对甲酸甲烷杆菌抗体有微弱的反应。DNA中G+c克分子含量为27．83士0．26％。根据Tc713和Tc708两菌株的性状,分别确定为嗜树木甲烷短杆菌(Methanobrevibacter arboriphilus)和甲酸甲烷杆菌(Methano-bacterium formicicum)。     

甲烷短杆菌HX菌株的分离和特性

本文描述了从上海沼气池污泥中分离出的一株产甲烷菌。细胞为柳叶刀状到卵圆状的短杆菌,革兰氏阳性,0．5～0.8×1．0—1．2μm,单个或威对存在。滚管菌落很小,半透明,灰白色,近似圆形,边缘完整。反射荧光显徽镜检查呈鲜艳的蓝绿色荧光。分离物利用H z,／CO或者甲酸生长和产甲烷。不需要外加2一甲基丁酸(2一mcthylbu‘yrate)、辅酶M或者氨基酸。细胞生长不需要酵母膏、胰解酪蛋白、癌胃蔽和乙酸。但是,酵母膏、胰解酪蛋白和瘤胃液对生长和产甲烷有刺激作用。分离物在有胆盐存在的情况下不生长。生长和产甲烷的虽适温度均为q0℃。最适pH为7．6,生长和产甲烷的pH范围为6．9—8．60在含o．2 5％甲酸、0．2％酵母膏和胰解酪蛋白的培养液中,最低倍增时间为Io小时·DNA的G+c含量为31．I 3mo／~o该菌株具有甲烷短杆菌所特有的形态和DNA的G+c含量。因此,暂将它定为史氏甲烷短杆菌L-IX菌株(Metha~obrembaeter smitl~ii strain HX)o其真正的分类地位,需根据与已确定的(代表不同物种的)不同菌株产甲烷菌的抗血清发生的间接免疫荧光反应来决定。     

一个甲烷氧化菌株的分离、鉴定及其特性研究

实验室自行设计方案分离多株以甲烷为唯一碳源的菌株, 对其中的1株QJ16进行了研究, 根据该菌株形态特征与16S rDNA序列的同源性分析, 证实该菌株是一个与其最近的甲基单胞菌属各成员都不相同的菌株。对该菌株的培养条件和利用甲烷的特性进行研究结果表明, 氮源以氯化铵和硝酸钾共同作用最好, 碳源以甲烷最佳, 最佳生长温度为30℃, 最佳生长pH为6~7; 在批式实验时菌株利用甲烷的最适pH为6.5左右, 微量元素Cu2+的浓度为15 mmol/ L。     

一株东祁连山耐低温纤维素降解菌株的分离、鉴定及产酶特性

从东祁连山高寒草甸土壤中分离得到一株纤维素酶高产菌株【3-2。根据菌体形态观察、革兰氏染色反应及16S rDNA序列测定以及序列同源性比较,确定鉴定该菌为芽孢杆菌属的成员(Bacillus sp.)。对该菌的产纤维素酶条件研究结果表明:该菌在5℃~45℃、初始pH 4.0~9.0的环境下均能产纤维素酶。在初始pH为8.0、盐浓度为2.0%~3.0%之间、培养温度为20℃培养条件下最适产酶。在5℃时,相对酶活仍能保持60%。     

猪源双歧杆菌的分离与鉴定

运用双歧杆菌选择性培养基,从１～４周龄乳猪的粪便中共分离纯化到５２个菌株。通过染色镜检、生化反应、代谢产物分析、抗生素敏感性试验研究表明这些菌株均与双歧杆菌属特征相符。根据糖发酵试验初步鉴定结果,其中４５个菌株为小猪双歧杆菌,５株为猪双歧杆菌,２株为嗜热双歧杆菌。部分菌株的急性毒性试验表明受试菌株对小白鼠无任何毒性副反应。     

一株甲烷氧化菌的分离鉴定与特性

甲烷氧化菌是一类能以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物。从若尔盖高原不同地点采集的样品中筛选得到一株名为XN1的甲烷氧化菌,根据此菌株的形态与16SrRNA序列同源性分析,证实该菌株属于Methylomonas属。对该菌株的培养条件进行研究的结果表明,以甲烷与甲醇共同作为碳源,硝酸钾和氯化铵共同为氮源时菌生长最好,最适生长温度为25℃,最适生长pH为6.5,培养基中CuSO4·5H2O和FeSO4·7H2O的浓度以0.03mg/L和0.4mg/L为宜。     

动物乳杆菌的分离鉴定及其抑菌蛋白的特性分析

通过滤纸片法从健康肉猪猪大肠、小肠中分离得到212株抗生物质产生菌, 以杯碟法复筛, 得到1株对溶壁微球菌(Micrococcus lysodeikticus)、金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)等革兰氏阳性菌和大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)等革兰氏阴性菌以及部分真菌如禾谷镰刀霉(Fusarium graminearum)均有强烈抑制作用的乳酸菌。经形态学、生理生化特征及16S rDNA序列同源性分析等手段鉴定该菌株为动物乳杆菌(Lactobacillus animalis)。排除酸和过氧化氢的干扰后, 该菌株的发酵上清液对指示菌仍有明显抑菌活性; 用蛋白酶处理该菌株的发酵上清液后, 抑菌活性丧失; 发酵液粗提物具有较好的热稳定性(经121°C处理20 min仍有较强抑菌活性)以及较宽的抑菌活性pH值范围(3.5~5.5), 因此初步认为该菌株产生一类具有广谱抑菌活性的类细菌素物质。     

双歧杆菌的分离驯化与鉴定

本文报道了从重庆市正常人群肠道中筛选出的５株双歧杆菌经厌氧→微氧→有氧的方式进行了数百次传代驯化,最后选送３株送国家认可的法定菌种鉴定单位认可为：８６３２１两歧杆菌、８６０４３青春双歧杆菌和８６２７４婴儿双歧菌。它的成功为我们进一步开发系列双歧产品奠定了很好基础。     

鲟源弗氏柠檬酸杆菌分离鉴定及药敏特性研究

鲟隶属于鲟形目(Acipenseriformes), 是中国近年来养殖发展较快的淡水养殖品种。虽然养殖技术及条件要求较高, 但是由于养殖鲟鱼具有很高的经济价值, 而且生长快、肉质好、全身是宝, 因而受到广大养殖户推崇[1]。但是近年来随着养殖量的迅速增大, 养殖集约化程度不断提高, 造成鲟养殖环境不断恶化, 使得鲟体质普遍下降, 发病率大增。鲟鱼多发细菌性疾病, 暴发性强, 死亡率高, 主要病原菌包括链球菌等革兰氏阳性菌和气单胞菌属几种[27], 因此疾病逐渐成为鲟鱼健康养殖的一大障碍。     

干旱土壤中产漆酶真菌的分离、鉴定与生长特性分析

本研究经过选择性培养基分离及特异性底物筛选从干旱区荒漠植物松叶猪毛菜和红砂灌丛土壤中分离出两株产漆酶的真菌Z45和H53,扩增其ITS序列进行分子鉴定,并分析温度、碳源、氮源、碳氮比和pH对菌株Z45生长的影响,在此基础上利用正交试验优化其培养条件.结果 表明,产漆酶真菌Z45和H53均隶属于端梗霉属(Acrophialophora sp.),Z45温度适应范围广且耐高温,最适生长温度为40℃.最优培养基组合为:分别以麦芽糖和硝酸铵为碳、氮源,按15∶1的碳源/氮源比配制,pH值6.0.本研究从干旱区荒漠土壤中获得了1株耐高温、产漆酶、应用价值高的子囊菌端梗霉,丰富了产漆酶微生物的种质资源库.     

一株河流污泥中产甲烷杆菌的生物学特性研究

目的:筛选产甲烷菌并对其进行鉴定和特性分析.方法:利用亨盖特厌氧操作技术从河流污泥中分离出的一株产甲烷杆菌.通过形态特征、生理生化、16S rRNA基因序列分析确定菌株的分类地位,利用气相色谱仪测定甲烷含量.结果:该菌株杆状,产芽孢,极端严格厌氧,宽约0.36μm,长约3.20μm,有时观察成链状,在液体培养的过程中,聚集成絮状沉积在管底.能够利用H2/CO2和甲钠盐生长,不利用甲醇、三甲胺、乙酸和二级醇类.最适生长温度范围为37℃,最适pH为7.5 ～8.0,能在0～1％盐浓度范围内生长.结论:菌株AG的最适温度较高,最适pH偏碱,在沼气发酵中具有重要应用前景.     

Nitrogen fixation by Methanobacterium formicicum

Abstract: Methanobacterium formicicum utilized molecular nitrogen as the sole nitrogen source for growth as monitored by methane production and culture turbidity measurements. The rate of methane production by the bacteria was correlated to nitrogen gas concentrations. In the absence of nitrogen gas or any other nitrogen source, the bacteria completely stopped growing. The presence of selenium and molybdenum in the culture medium was vital for the growth of the bacteria under nitrogen fixing conditions.     

Thermodynamic analysis of growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum, an anaerobic archaebacterium using methanogenesis as the catabolic pathway, is characterized by large heat production rates, up to 13 W g⁻¹, and low biomass yields, in the order of 0.02 C‐mol mol⁻¹ H₂ consumed. These values, indicating a possibly “inefficient” growth mechanism, warrant a thermodynamic analysis to obtain a better understanding of the growth process. The growth‐associated heat production (Δ_rH) and the growth‐associated Gibbs energy dissipation per mol biomass formed (Δ_rG) were −3730 kJ C‐mol⁻¹ and −802 kJ C‐mol⁻¹, respectively. The Gibbs energy change found in this study is indeed unusually high as compared to aerobic methylotrophes, but not untypical for methanogens grown on CO₂. It explains the low biomass yield. Based on the information available on the energetic metabolism and on an ATP balance, the biomass yield can be predicted to be approximately in the range of the experimentally determined value. The fact that the exothermicity exceeds vastly even the Gibbs energy change can be explained by a dramatic entropy decrease of the catabolic reaction. Microbial growth characterized by entropy reduction and correspondingly by unusually large heat production may be called entropy‐retarded growth. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 74–81, 1999.     

Evidence for a defective prophage on the chromosome of Methanobacterium wolfei

Abstract Evidence shows the presence on the chromosome of Methanobacterium wolfei of a defective prophage which, by DNA-DNA hybridization, is closely related to the virulent archaeophage ψM1 of Methanobacterium thermoautotrophicum Marburg. Partial sequencing of a M. wolfei 16S rRNA gene and phylogenetic analysis indicated that this organism is more closely related to other representatives of the genus Methanobacterium than to M. thermoautotrophicum Marburg. The chromosomal region of M. wolfei encoding the putative prophage was found to be deleted for two non-contiguous segments of the phage ψM1 genome and thus encompassed only 80 to 90% of the ψM1 DNA. The prophage region was mapped to a 30 kb restriction fragment on the physical map of the M. wolfei chromosome. A randomly chosen DNA fragment was cloned from phage ψM1 DNA, as was its homologous counterpart from the chromosome of M. wolfei . The 126-bp region present in both clones exhibited 100% sequence identity.     

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 of lipid activated pseudomurein precursors from Methanobacterium thermoautotrophicum

From cell extracts of the pseudomurein possessing methanogen Methanobacterium thermoautotrophicum two putative pseudomurein precursors were isolated and characterized: (1) an undecaprenyl pyrophosphate activated disaccharide pentapeptide composed of N-acetylglucosamine, N-acetyltalosaminuronic acid, alanine, glutamic acid and lysine in a molar ratio of 1:1:2:2:1 and (2) the corresponding undecaprenyl pyrophosphate activated tetrapeptide lacking one alanine residue. The isolation of these precursors show that the biosynthesis of the eubacterial murein and the methano-bacterial pseudomurein differs not only in the cytoplasmic step, as recently described, but also in the lipid stage.Abbreviations GlcNitol glucosaminitol - NAcTalNUA N-acetyltalosaminuronic acid - Udp undecaprenol - TLC thin layer chromatography     

Calcium ion uptake by Methanobacterium thermoautotrophicum: Evidence for a carrier-mediated uptake system

Abstract Cell suspensions of Methanobacterium thermoautotrophicum took up ⁴⁵Ca²⁺ in a temperature-dependent, Ca²⁺-saturable and Co²⁺-sensitive process. The accumulation of ⁴⁵Ca²⁺ was lower in the cells energized by CO₂+ H₂ than in those under non-energized conditions. The accumulated Ca²⁺ were, in part, released by the divalent cations ionophore A23187 in the presence of EGTA while the uptake of Ca²⁺ was accelerated by the addition of A23187 to the medium containing Ca²⁺. The results indicate the presence of a carrier-mediated Ca²⁺ uptake in the Methanobacterium thermoautotrophicum membrane which is compensated by an energy-dependent and outward-directed Ca²⁺ transport.     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Methanobacterium thermoalcaliphilum spec. nov., a new moderately alkaliphilic and thermophilic autotrophic methanogen

An autotrophic moderately alkaliphilic and thermophilic nonmotile rod-shaped methanogen was isolated from a biogas plant. The isolate grows only on H₂+CO₂ and requires yeast extract. Growth optimum is at 60°C with a generation time of 4 h. In the absence of substrates complete lysis occurs. The pH range for growth is 7.5–8.5. Growth was also observed at pH values above 9.0. The DNA base composition is 38.8 mol% G+C. According to its physiological properties the nameMethanobacterium thermoalcaliphilum is proposed.Abbreviations G+C Guanine+cytosine