农业有机废物与城市生活垃圾堆肥高温期微生物种群结构比较

通过PCR、克隆文库方法分析了农业有机废物和城市垃圾堆肥高温期间细菌和真菌种群的多样性.提取堆肥高温期的DNA,PCR扩增,构建各高温期的16S rDNA和18S rDNA克隆文库,结果表明:农业有机废物和城市生活垃圾16S rDNA克隆文库中分别共有18个、21个OTUs,分别属于细菌域的14个、15个不同属,其18S rDNA克隆文库中分别共有8个、9个OTUs,分别属于细菌域的8个、9个不同属,推断农业有机废物堆体的优势菌为Bacillus megaterium、Rhizobium sp.、Phanerochaete chrysosporium、Penicillium sp.同属或同种的菌株;城市生活垃圾堆体的优势菌为Bacillus megaterium、Azospirillum sp.、Phanerochaete chrysosporium同种或同属的菌株.     

芦笋老茎堆肥中嗜热细菌的分离与鉴定

利用稀释涂布法对芦笋老茎堆肥不同发酵阶段6个样品中的嗜热细菌进行分离,并采用16S rDNA序列分析方法对分离得到的菌落形态有明显区别的22株细菌进行鉴定.根据16S rDNA序列分析结果,22株细菌菌株中13株属于芽胞杆菌属(Bacillus),1株属于类芽胞杆菌属(Paenibacillus),4株属于假黄色单胞菌属(Pseudoxanthomonas),1株属于肠杆菌属(Enterobacter),1株属于副球菌属(Paracoccus),1株属于短芽胞杆菌属(Brevibacillus),菌株D-b2在GenBank数据库中未找到与其相似的已知细菌属的序列,分类地位待定.从以上鉴定结果可以看出,芦笋老茎堆肥中的优势嗜热细菌主要是芽胞杆菌(Bacillus spp.)和假黄色单胞菌(Pseudoxanthomonas spp.).     

双孢蘑菇堆肥中真菌群落多样性分析

以农作物玉米秸和稻草、牛粪为原料,分别设计双孢蘑菇Agaricus bisporus堆肥配方并进行堆肥发酵,研究二者堆肥过程中真菌多样性。在建堆、一次发酵结束和二次发酵结束3个时期分别采集堆肥样品,提取总DNA,以真菌18S rDNA基因通用引物,进行PCR-DGGE扩增和序列分析。累计获得39条特异条带18S rDNA基因信息,分属于真菌14个属、藻类7个属和原生动物3个属。子囊菌是两种配方堆肥过程中的优势菌群,建堆时期的优势类群为Pichia和Wickerhamomyces,一次和二次发酵时期的优势类群为Chaetomium和Lecythophora。多样性指数分析显示,稻草配方微生物多样性大于玉米秸配方;主成分分析（PCA）显示,玉米秸配方一次发酵结束时期与稻草和玉米二次发酵结束时期聚为一类,说明玉米秸配方堆肥提前腐熟。     

1株海洋真菌ZH2.1的鉴定及生物学特性初步研究

从南海海域白姑鱼消化道分离到1株海洋真菌ZH2.1,对其进行了系统鉴定和生物学研究。采用传统的形态学鉴定方法,并结合18S rDNA序列分析确定其归属。18S rDNA序列分析表明其与孔状短小茎点霉Phoma exigua var.exigua在进化位置上最为接近,其18S rDNA在GenBank的登录号为FJ450059。结合形态学观察结果,可认为菌株ZH2.1为茎点霉属真菌。对该菌株的部分生物学特性研究表明,ZH2.1为兼性海洋真菌,最适生长盐度为3%。此外,在微氧条件下也可生长。     

褐飞虱不同致害性种群体内共生菌18S rDNA部分序列比较

分离纯化了褐飞虱3种不同致害性种群体内类酵母共生菌 (yeast-like symbionts, YLS),并对其18S rDNA基因序列进行了比较。结果表明,褐飞虱3种不同致害性种群体内类酵母共生菌18S rDNA均扩增出600 bp左右的片断。依据获得的18S rDNA特异性序列,结合已知真菌的18S rDNA部分序列,构建了不同宿主的YLS分子系统树。结果显示, 褐飞虱3种不同致害性种群体内的YLS同属子囊菌亚门（Ascomycotina）的核菌纲（Pyrenomycetes）,并与此纲中的Hypomyces chrysospermus亲缘关系相对最近。     

一株产虾青素酵母菌株的鉴定

从市售海水鱼内脏中分离得到一株产虾青素的酵母,编号为NZ- 01.采用传统形态学鉴定方法及rDNA序列分析法分别对从NZ- 01进行鉴定.形态学鉴定结果表明该菌为胶红酵母(Rhodotorula mucilaginosa),分别用特异性引物对rDNA 序列的18S rDNA D1/D2区、26S rDNA D1/D2区和ITS区进行扩增,PCR产物测序,将测序结果登录GenBank进行BLAST分析,结果均与形态学观察结果一致,NZ- 01为胶红酵母.     

一株耐热脂肪酶产生菌的鉴定及其脂肪酶基因的克隆

目的:从北方堆肥土样中分离、筛选获得产耐热脂肪酶的嗜热菌株 FS32b,确定该菌株的分类学地位及其所产耐热脂肪酶基因.方法:通过研究该菌株 16S rDNA 基因序列的系统进化树,进行菌种分类鉴定并利用PCR技术获得其脂肪酶基因.结果:FS32b 与报道过的 Bacillus subtilis X60646 有紧密的亲缘关系,二者的 16S rDNA 序列相似性为 99%,其脂肪酶基因经序列测定分析表明,该菌株含长度为 639bp 的耐热脂肪酶基因的完整开放阅读框架(ORF).此片断编码有 213 个氨基酸的酶.结论:FS32b初步鉴定为Bacllus subtilis,其脂肪酶基因的克隆为以后高效基因工程菌的构建奠定了基础.     

水浮莲中-株西瓜枯萎病拮抗菌的分离、筛选及鉴定

从水浮莲(Pistia stratiotes L.)的叶中分离出1株对西瓜枯萎病有明显拮抗作用的内生细菌XJPL-YB-26,其发酵液上清在280 nm处有最大紫外吸收峰.利用软件Primer 6.0设计16S rDNA引物并对其基因组DNA进行扩增并测序得到XJPL-YB-26的部分16S rDNA序列.GenBank接收号为EU251191.经 Blastn调出与菌株16S rDNA同源的序列,并用软件MEGA 3.1按Neighbor-Joining方法构建16S rDNA系统发育树.菌株XJPL-YB-26与AB271744处于同一分支,相似性为99%,最终鉴定为枯草芽孢杆菌Bacillus subtilis.     

高通量测序技术分析猪粪堆肥过程中微生物群落结构变化

为了解猪粪堆肥过程中微生物群落结构组成及多样性的变化,采集猪粪堆肥过程的三个代表性样品—新鲜猪粪、高温堆肥、腐熟堆肥,利用Illumina Miseq高通量测序技术对16S rRNA V4～V5可变区序列进行测序,分别获得37 009、42 470、36 713条有效序列及328、280、160个操作分类单元(OTU)。Alpha多样性分析表明,在堆肥过程中微生物群落丰富度呈现降低趋势,而多样性呈现先上升后下降趋势。随着堆肥的进行,在门水平上,厚壁菌门、拟杆菌门和软壁菌门相对丰度降低,而变形菌门和放线菌门相对丰度升高;在属水平上,Turicibacter、Terrisporobacter、Parabacteroides、Clostridium sensu stricto、Corynebacterium等来自动物肠道的微生物相对丰度明显下降,Thermopolyspora、Thermomonospora、Thermobifida、Halocella等耐热耐盐微生物成为最主要优势菌。堆肥过程不同菌群优势度的变化是微生物与堆肥中各理化因子相互作用的结果。     

不对称还原苯乙酮的酵母菌株筛选与分子鉴定

利用苯乙酮作为模式底物,对145株菌株进行初筛和复筛,获得一株具有高效立体选择性的酵母菌株YS6-2,能够不对称还原苯乙酮生成(S)-1-苯基乙醇.在苯乙酮浓度为70mmol/L时,底物的初始转化率达26.8%,产物(S)-1-苯基乙醇的对映体过量值为98.8%.基于形态学、生理生化特征、18S rDNA和26S rDNA D1/D2区域的分析表明,YS6-2为胶红酵母(Rhodotorula muci-laginosa).     

Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

一株以葡萄糖为底物产2,3-丁二醇微生物菌株的筛选及鉴定

从自然界中筛选出一批以葡萄糖为底物发酵产2,3-丁二醇的菌株,经初步发酵测定发酵液中2,3-丁二醇含量,其中菌株6-7的2,3-丁二醇产量最高达49.6g/L。对其进行常规生理生化鉴定实验,并结合16SrDNA序列分析,比对结果表明,菌株6-7与Bacillus subtilis strain BIHB332相似性达99%。在细菌分类学上属于枯草芽孢杆菌属,将其命名为Bacillussubtilis6-7。其特点是属于环境友好和食品安全型菌株,因此,利用Bacillus subtilis6-7生产2,3-丁二醇具有良好的工业应用价值。     

生物垃圾堆肥的过程研究及腐熟度评价

目的：研究静态强制通风发酵工艺对合肥市农贸市场的生物废弃物进行好氧堆肥处理过程的物质变化和腐熟效果。方法：分析发酵过程中温度、含水率、挥发分含量、纤维素、半纤维素和木质素含量等物理指标和化学指标。结果：一次发酵中堆肥的温度在55℃以上的有3d,经过一次发酵和二次发酵,堆肥中挥发分含量由85.4%降低到51.2%。纤维素的含量由20.90%减少到15.48%,半纤维素的含量由7.20%减少到1.36%,,纤维素、半纤维素在二次发酵中降解率比一次发酵的小,木质素虽在一次发酵中基本没有降解。但在二次发酵中得到有效降解,木质素含量由8.30%减少到5.38%。堆肥后物料在70d堆肥后,基本上达到腐熟程度。结论：温度、含水率、挥发性物质、淀粉实验、生物可降解度实验等指标检测可以评价静态强制通风发酵工艺对合肥市农贸市场的生物废弃物进行好氧堆肥处理过程的物质变化和腐熟效果。     

The microbial signature of aerosols produced during the thermophilic phase of composting

Aims:  The microbial diversity of bioaerosols released during operational activities at composting plants is poorly understood. Identification of bacteria and fungi present in such aerosols is the prerequisite for the definition of microbial indicators that could be used in dispersal and exposure studies.
Methods and Results:  A culture-independent analysis of composting bioaerosols collected at five different industrial open sites during the turning of composting piles in fermentation was performed by building 16S rDNA and 18S rDNA libraries. More than 800 sequences were analysed. Although differences in the phylotypes distribution were observed from one composting site to another, similarities in the structure of microbial diversity were remarkable. The same phyla dominated in the five bioaerosols: Ascomycota among fungi, Firmicutes and Actinobacteria among bacteria. For each phylum, some dominant phylotypes were common to at least four bioaerosols. These common phylotypes belonged to Thermomyces , Aspergillus , Penicillium , Geobacillus, Planifilum , Thermoactinomyces , Saccharopolyspora , Thermobifida and Saccharomonospora .
Conclusions:  The microbial signature of aerosols produced during the thermophilic phase of composting was determined. The similarities observed may be explained by the selection of thermophilic and sporulating species.
Significance and Impact of the Study:  Several bacteria and fungi identified in this study may represent potential indicators of composting bioaerosols in air.     

Isolation and characterization of rat ribosomal DNA clones

Four EcoRI fragments, which contain the transcribed portion of the rat rDNA repeat, have been isolated from a rat genome library cloned in lambda Charon 4A vector. Three of the fragments, 9.6, 6.7, and 4.5 kb, from clones lambda ChR-B4, lambda Nr-42, and lambda ChR-C4B9, contained part of the 5'-NTS, the 5'-ETS, 18S rDNA, ITS-1, 5.8S rDNA, 28S rDNA and approximately 3.5 kb of the 3'-NTS. Two EcoRI fragments, from clones lambda ChR-B4 and lambda ChR-B7E12, which coded for the 5'-NTS, the ETS, and most of the 18S rDNA, differed by 1 kb near the EcoRI site upstream of the 5' terminus of 18S rRNA. Restriction maps of the cloned DNA fragments were constructed by cleavage of the fragments with various restriction endonucleases and Southern hybridization with 18S, 5.8S, and 28S rRNA. These maps were confirmed and extended by subcloning several regions of the repeat in pBR322.     

Effects of Continuous Thermophilic Composting (CTC) on Bacterial Community in the Active Composting Process

The method of continuous thermophilic composting (CTC) remarkably shortened the active composting cycle and enhanced the compost stability. Effects of CTC on the quantities of bacteria, with a comparison to the traditional composting (TC) method, were explored by plate count with incubation at 30, 40 and 50°C, respectively, and by quantitative PCR targeting the universal bacterial 16S rRNA genes and the Bacillus 16S rRNA genes. The comparison of cultivatable or uncultivatable bacterial numbers indicated that CTC might have increased the biomass of bacteria, especially Bacillus spp., during the composting. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to investigate the effects of CTC on bacterial diversity, and a community dominated by fewer species was detected in a typical CTC run. The analysis of sequence and phylogeny based on DGGE indicated that the continuously high temperature had changed the structure of bacterial community and strengthened the mainstay role of the thermophilic and spore-forming Bacillus spp. in CTC run.     

一株产蓝色素链霉菌18-A-5的鉴定与系统发育分析

从原始热带雨林土壤中,分离到一株产蓝色色素菌株18-A-5,对其进行了系统分类学研究。形态学特征观察表明,在高氏合成一号培养基上初产蓝绿色色素,日久为深蓝色,基内菌丝蓝色,气生菌丝灰白色,产灰色孢子,孢子丝直或柔曲,形成长孢子链,孢子圆柱形。其DNA的G＋C摩尔分数为62.4%,16S rDNA序列分析结果(GenBank登陆号为EU054353),18-A-5与生靛链霉菌Streptomyces indigoferus ATCC23924^T 、草绿色链霉菌Streptomyces herbaricolor ATCC23924^T具有极高的同源性,达100％,聚类分析表明,18-A-5与生靛链霉菌Streptomyces indigoferus、草绿色链霉菌Streptomyces herbaricolor两株菌聚类在一起,分支置信度为74%。结合生理生化特性、细胞壁化学组成分析、脂肪酸分析等将菌株18-A-5定名为草绿色链霉菌Streptomyces herbaricolor。并对该蓝绿色可溶性色素性质进行了耐酸碱性、热稳定性、抗菌谱等初步分析。     

复合微生物菌剂在剩余污泥堆肥中的作用研究

应用复合微生物菌剂对剩余污泥进行堆肥试验,较系统地研究了复合微生物菌剂在剩余污泥堆肥系统中的作用。结果表明:接种复合微生物菌剂进行剩余污泥堆肥,与对照组相比,不但能够提高堆肥温度,而且高温持续时间长,堆肥反应速率加快,腐熟时间缩短,当接种量为7%(体积比)时,腐熟时间比对照组提前了12 d。