2株瘤胃纤维降解细菌的分离鉴定

从日粮精粗比为3:7的小尾寒羊×蒙古羊杂交一代绵羊的瘤胃内容物中分离到2株严格厌氧细菌,一株球菌WQ-1,1株弧菌WH-2,二者对滤纸有很好的降解能力。通过酶活力试验测得WQ-1的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.66,7.0 U/mL和15.3 U/mL;WH-2的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.52,6.9 U/mL和17.2 U/mL。经形态学、生理生化反应、生态特性和遗传型的鉴定,WQ-1归类为瘤胃球菌属(Ruminococcus)的黄色瘤胃球菌(Ruminococcus flavefaciens)。WH-2归类为丁酸弧菌属(Butyrivibrio)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。     

高效厌氧纤维素降解细菌的分离及酶特性研究

采用透明圈初筛和滤纸降解率复筛的方法从内蒙古绵羊瘤胃内容物中分离到高效厌氧纤维素降解细菌4株.通过形态学、生理生化反应、生态特性和遗传型的鉴定,所分离的4株菌WHQ、LYQ、LBG-1和NDF-3分别归为溶纤维丁酸弧菌(Butyrivibrio fibrisollvens)、黄色瘤胃球菌(Ruminococcus flavefaciens)、产琥珀酸丝状杆菌(Fibrobacter succinogenes)和解多糖梭菌(Clostridium polysaccharolyticum).测定了4株菌对滤纸的降解率,WHQ、LYQ、LBG-1和NDF-3的2周滤纸降解率分别为25.1%、14.3%、21.0%和20.6%.本研究同时对4株菌的滤纸酶活力、羧甲基纤维素酶活力和β-葡萄糖苷酶活力进行了测定.     

能源上的应用

植物结构聚合物水解成比较小的单位是生物产甲烷的最初步骤。有代表性的木聚糖分解细菌是从厌氧消化器中分离出来的。通过将系列稀释的样品直接植板在含木聚糖作唯一碳源的无机盐培养基上,从填装紫狼尾草的厌氧发酵器得到了6株分解木聚糖的菌株。已鉴定为溶纤维丁酸弧菌(Butyrivi-biro fibrisolvens),2株最活跃的降解木聚糖的分离菌(GS112和 GS113)与以前     

一株纤维素降解细菌的筛选、鉴定及产酶条件分析

目的筛选高活性的纤维素降解细菌,并进行初步鉴定和产纤维素酶条件分析。方法采集吉首旗帜山松树林的土壤样品,通过富集培养和刚果红平板染色法筛选分离纤维素降解细菌;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析对分离的菌株进行初步鉴定。利用单因素实验对产纤维素酶条件进行优化。结果分离获得1株高活性纤维素降解细菌JDM11,初步鉴定其为Bacillus velezensis;菌株JMD11产纤维素酶最佳培养温度、最适初始pH和培养时间分别为28℃、7.0～7.5和32h,在该条件下其滤纸酶(FPase)和羧甲基纤维素酶(CMCase)活力分别为260.32U/ml和651.75U/ml。结论菌株JDM11是1株高活性纤维素降解的Bacillus velezensis。     

蟋蟀后肠纤维素降解细菌的分离与鉴定

近年来,具有农业、能源和环保价值的昆虫微生物种类和基因得到了开发,昆虫肠道微生物展示了其巨大的应用潜力,本研究旨在从蟋蟀后肠分离和鉴定纤维素降解细菌。首先采用羧甲基纤维素钠液体培养基对蟋蟀后肠中的微生物进行富集培养,然后使用羧甲基纤维素钠固体培养基分离和筛选单菌落,再通过16S rRNA测序对纤维素降解细菌进行分子鉴定,最后通过刚果红染色来进一步分析细菌降解纤维素的能力。从蟋蟀后肠中共分离出20株纤维素降解细菌,16S rRNA基因测序结果显示来自肠杆菌属(Enterobacter)9株,不动杆菌属(Acinetobacter)7株,克雷伯氏菌属(Klebsiella)2株,鞘氨醇杆菌属(Sphingobacterium)1株和葡萄球菌属(Staphylococcus)1株。刚果红染色试验结果显示,克雷伯氏菌属两株PDSCDXS_2B和8B,鞘氨醇杆菌属PDSCDXS_7C和不动杆菌属PDSCDXS_12C具有较高的纤维素降解能力。这是首次从蟋蟀后肠分离和筛选出来具有纤维素降解能力的细菌,为昆虫源纤维素降解细菌的研究提供了微生物资源。     

三株高效秸秆纤维素降解真菌的筛选及其降解效果

【目的】利用多种筛选方法,获得高效秸秆纤维素降解真菌,并研究其秸秆纤维素的降解能力。【方法】采用滤纸片孔洞法、滤纸条降解法、羧甲基纤维素钠(CMC-Na)水解圈测定法、秸秆失重法、纤维素分解率测定法、胞外酶活测定法等常规秸秆纤维素降解菌的筛选方法。【结果】筛选到3株具有较强纤维素降解能力的真菌菌株,经初步鉴定菌株98MJ为草酸青霉(Penicillium oxalicum)、菌株W3为木霉(Trichoderma sp.)、菌株W4为扩张青霉(Penicillium expansum)。菌株W4具有非常强的秸秆纤维素降解能力,10d内对秸秆的降解率可达56.3%,对纤维素、半纤维素和木质素的分解率分别为59.06%、78.75%和33.79%。菌株W4的胞外纤维素酶活力在14.25-49.75U/mL之间。【结论】筛选获得3株高效秸秆纤维素降解真菌菌株,其中菌株W4的纤维素酶活高于已报道的菌株,是一株十分具有研究开发潜力的纤维素酶生产菌株。     

甘蔗渣纤维素降解菌株的筛选与鉴定

甘蔗渣是制糖工业的主要副产物。筛选甘蔗渣纤维素降解菌株对甘蔗渣乙醇产业具有重要的意义。以甘蔗渣为原料,通过分离和纯化得到14株菌株,对其进行纤维素刚果红平板染色实验和滤纸崩解实验,最终获得3株可以生产纤维素酶的菌株02-2-2、21-1-2和40-1-1。酶活性测定结果表明,菌株40-1-1的酶活力在培养3 d后达到最高,为27.26 U/mg。通过形态学和分子生物学鉴定,菌株02-2-2为枝顶孢属(Acremonium sp.),菌株21-1-2和40-1-1为光滑短梗霉属(Acrophialophora sp.)。研究筛选的菌株将为开展甘蔗渣纤维素降解利用提供参考。     

一株纤维素降解真菌的筛选及鉴定

[目的]分离筛选高效降解纤维素的真菌菌株,并研究其产酶能力.[方法]利用刚果红染色法从甘蔗地土壤中分离纤维素降解真菌,再通过测定滤纸的降解率及发酵酶活复筛.[结果]综合考虑水解圈,水解圈和菌株直径的比值(HC值),滤纸的降解率和复筛酶活,对试验真菌降解纤维素的能力进行综合评价,筛选到具有较强纤维素降解能力的真菌菌株SJ1,经形态学观察及分子生物学鉴定,该菌属于草酸青霉.其滤纸酶活、内切葡聚糖酶酶活(CMC酶活)、β-葡聚糖苷酶酶活和外切葡聚糖酶酶活(CBH酶活)分别为25.15、740.42、58.03和2.442 U/mL.[结论]菌株SJ1是一株十分具有研究开发潜力的纤维素酶生产菌株.     

青海高原降解纤维素微生物的调查、分离、鉴定

从青海高原林区分离筛选 3 0 0余株分解纤维素的细菌及 3 1株降解纤维素的真菌。测定纤维素分解菌含量土样为 2 6× 1 0 5 g。对纤维素酶水解圈较大的 1 1株真菌 ,根据其滤纸酶活筛选出一株分离自互助北山森林的高产纤维素酶的真菌No 0 1 43菌株 ,根据其形态学及培养特征鉴定为康氏木霉 (TrichodermakoningiiQudem) ,该菌湿固体发酵物含滤纸酶活力(FPA)为 1 5u g。该菌无毒副作用 ,可用于饲料业     

发酵床中纤维素降解菌的分离与鉴定

从发酵床垫料中初步分离出43株纤维素降解菌。采用刚果红鉴别培养基及滤纸条培养基初筛,得到5株透明圈较大且使滤纸条产生崩解的菌株,通过进一步液体发酵,测定其CMC酶活、FPA酶活和天然纤维素酶活,获得2株具有较高纤维素降解活性菌株,并分别命名为F7和F21。经16S rRNA基因序列分子生物学鉴定和系统发育分析表明,这2株纤维素降解菌分别归属为枯草芽孢杆菌(Bacillus subtilis)和链霉菌(Streptomyces sp.)。     

Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens.

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.     

Group-specific 16S rRNA hybridization probes for determinative and community structure studies of Butyrivibrio fibrisolvens in the rumen.

Oligonucleotide probes covering three phylogenetically defined groups of Butyrivibrio spp. were successfully designed and tested. The specificity of each probe was examined by hybridization to rRNAs from an assortment of B. fibrisolvens isolates as well as additional ruminal and nonruminal bacteria. The sensitivity of the hybridization method was determined by using one of the probes (probe 156). When RNA was extracted from a culture of OB156, the probe was able to detect target cells at densities as low as 10(4) cells/ml. When 10(4) or more target cells/ml were added to cattle rumen samples, detectable hybridization signals were obtained with 1,000 ng of total RNA loaded onto the nylon membrane. In contrast, the sensitivity was reduced to 10(6) target cells/ml at 100 ng of RNA per slot. The probes were used to type 19 novel Butyrivibrio isolates. The phylogenetic placement was confirmed by partial 16S rRNA gene sequencing. The use of the probes in community-based studies indicated that the Butyrivibrio groups examined in this paper did not represent a significant portion of the bacterial 16S rRNA pool in the rumen of the cattle, sheep, and deer examined.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

Analyses of the gene and amino acid sequence of thePrevotella (bacteroides) ruminicola 23 xylanase reveals unexpected homology with endoglucanases from other genera of bacteria

The DNA sequence for the xylanase gene fromPrevotella (Bacteroides) ruminicola 23 was determined. The xylanase gene encoded for a protein with a molecular weight of 65,740. An apparent leader sequence of 22 amino acids was observed. The promoter region for expression of the xylanase gene inBacteroides species was identified with a promoterless chloramphenicol acetyltransferase gene. A region of high amino acid homology was found with the proposed catalytic domain of endoglucanases from several organisms, includingButyrivibrio fibrisolvens, Ruminococcus flavefaciens, andClostridium thermocellum. The cloned xylanase was found to exhibit endoglucanase activity against carboxymethyl cellulose. Analysis of the codon usage for the xylanase gene found a bias towards G and C in the third position in 16 of 18 amino acids with degenerate codons.     

Molecular diversity of the foregut bacteria community in the dromedary camel (Camelus dromedarius)

The molecular diversity of the foregut bacterial community in the dromedary camel (Camelus dromedarius) in Central Australia was investigated through comparative analyses of 16S rRNA gene sequences prepared from the foregut contents of 12 adult feral camels fed on native vegetation. A total of 267 full-length 16S rRNA gene clones were examined, with 151 operational taxonomic units (OTUs) identified at a 99% species-level identity cut-off criterion. The prediction of actual diversity in the foregut of the dromedary camel using the Chaol approach was 238 OTUs, while the richness and evenness of the diversity estimated using Shannon index was 4.84. The majority of bacteria in the current study were affiliated with the bacterial phylum Firmicutes (67% of total clones) and were related to the classes Clostridia, Bacilli and Mollicutes, followed by the Bacteroidetes (25%) that were mostly represented by the family Prevotellaceae. The remaining phyla were represented by Actinobacteria, Chloroflexi, Cynophyta, Lentisphaerae, Planctomycetes, Proteobacteria and Sphirochaetes. Moreover, 11 clones of cultivated bacteria were identified as Brevundimonas sp., Butyrivibrio fibrisolvens, Prevotella sp. and Ruminococcus flavefaciens. The novelty in this foregut environment is remarkable where 97% of the OTUs were distantly related to any known sequence in the public database.     

Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens.

Forty-nine isolates of Butyrivibrio fibrisolvens and a single isolate of Butyrivibrio crossotus were screened for the production of inhibitors by a deferred plating procedure. Twenty-five isolates produced factors which, to various degrees, inhibited the growth of the other Butyrivibrio isolates. None of the inhibitory activity was due to bacteriophages. The inhibitory products from 18 of the producing strains were sensitive to protease digestion. Differences in the ranges of activity among the Butyrivibrio isolates and protease sensitivity profiles suggest that a number of different inhibitory compounds are produced. These findings suggest that the production of bacteriocin-like inhibitors may be a widespread characteristic throughout the genus Butyrivibrio. The bacteriocin-like activity from one isolate, B. fibrisolvens AR10, was purified and confirmed to reside in a single peptide. Crude bacteriocin extracts were prepared by ammonium sulfate and methanol precipitation of spent culture supernatants, followed by dialysis and high-speed centrifugation. The active component was isolated from the semicrude extract by reverse-phase chromatography. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity, having an estimated molecular mass of approximately 4,000 Da. The N terminus of the peptide was blocked. A cyanogen bromide cleavage fragment of the native peptide yielded a sequence of 20 amino acids [(M)GIQLAPAXYQDIVNXVAAG]. No homology with previously reported bacteriocins was found. Butyrivibriocin AR10 represents the first bacteriocin isolated from a ruminal anaerobe.     

16s rDNA analysis of Bufyrivibrio fibrisolvens: phylogenetic position and relation to butyrate-producing anaerobic bacteria from the rumen of white-tailed deer

R.J. FORSTER, R.M. TEATHER, J. GONG AND s.-J. DENG. 1996. Complete 16S rDNA sequences of six strains of Butyrivibrio fibrisolvens , including the type strain (ATCC 19171), were determined. The type strain was found to have less than 89% sequence similarity to the other isolates that were examined. The five plasmid-bearing strains formed a closely related cluster and three of these strains (OB156, OB157 and OB192) were very highly related (> 99%), indicating that they are isolates of the same genomic species. The phylogenetic position of Butyrivibrio was found to be within the subphylum Clostridzum, of Gram-positive bacteria. The closest relatives to the type strain were Eubacterium cellulosolvens and Cl. xylanolyticum and the closest relatives to the separately clustered strains were Roseburia cecicola, Lachnospira pectinoschiza and Eubacterium rectale .     

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.     

腾冲热海一株嗜酸热硫化叶菌的分离与鉴定

从云南腾冲热海酸性温泉中分离纯化出一株极端嗜酸热菌株K4-1,并对其进行形态观察、生长特征、碳源和能源利用及16S rRNA基因分析.该菌株细胞形态为不规则球形,有单生鞭毛,严格好氧,兼性自养,能利用元素硫作为能源,也能利用酵母膏、精氨酸或核糖作为碳源和能源.其最适生长温度为75℃,最适pH为3.5.通过16S rRNA基因序列相似性对比对该菌株进行鉴定,结果表明该菌株与硫化叶茵属标准菌株的相似性介于86.6%～94.3%之间,与分离自腾冲热海的腾冲硫化叶菌Sulfolobus tengchongensis RT8-4菌株序列相似性最高,达到98.9%,可将菌株K4-1鉴定为硫化叶菌属菌株.菌株K4-1的16S rRNA基因序列号为EU729124.     

Phenotypic and genetic data supporting reclassification of Butyrivibrio fibrisolvens isolates

Among 55 Butyrivibrio fibrisolvens strains five ribotypes of B. fibrisolvens were described on the basis of RFLP profiles of 16S rDNA regions obtained with restriction endonuclease HaeIII. In the phylogenetic tree, these ribotypes were located in the XIVa cluster of Gram-positive bacteria. Phenotypic differences of selected ribotype groups became the basis for further reclassification of B. fibrisolvens.