Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis

Samples of Coffea arabica were collected during the different stages of the fermentation from two production sites in Tanzania. The yeasts community was identified by genotyping using ITS-PCR and sequence analysis of the D1/D2 domain of the 26S rRNA gene. For confirmation, denaturating gradient gel electrophoresis (DGGE) of PCR-amplified 26S rRNA gene was performed to detect yeast directly from coffee samples without cultivation. Yeast counts were in the range 4.0 x 10(4) - 5.0 x 10(7) CFU/g with an increase during fermentation. Three yeasts species were dominant. The predominant yeast found during fermentation and drying was Pichia kluyveri. Pichia anomala was found in high numbers during drying of coffee beans. Hanseniaspora uvarum was the predominant yeast during fermentation but decreased during drying. Kluyveromyces marxianus, Candida pseudointermedia, Issatchenkia orientalis, Pichia ohmeri and Torulaspora delbrueckii occurred in concentrations of 10(3) CFU/g or below in coffee samples. Saccharomyces cerevisiae and Candida xestobii were not isolated by cultivation, but by the DGGE technique. A good agreement was found between the sequence analysis of the D1/D2 domain of the 26S rRNA gene and sequencing of the DGGE bands.     

不同屠宰工艺(剥皮和烫毛)对猪胴体表面微生物的多样性影响及关键点的控制研究

应用变性梯度凝胶电泳(Denaturinggradientgelelectrophoresis(DGGE))和经典微生物培养相结合的方法研究了剥皮和烫毛工艺中猪胴体表面污染的微生物数量和细菌多样性的变化以及3.5%乳酸处理后细菌总数和大肠菌群的变化。结果显示:不同猪胴体屠宰工艺中的微生物种类并不完全一致,微生物的污染多是由于前期的屠宰工艺引入;与剥皮工艺相比,烫毛后污染的微生物种类多,初始污染程度也较为严重;乳酸处理显著降低了剥皮工艺中的细菌总数,使出库猪胴体表面细菌总数降低到2.95logCFU/cm2,完全达到HACCP微生物的控制要求,但是没有降低烫毛工艺出库时的细菌总数,因此对不同的屠宰工艺应采取不同的关键点控制措施。     

应用DGGE技术研究玉米储藏过程中的霉菌群落时空分布特征

研究储粮过程中霉菌污染的发生规律,旨在减少霉菌及真菌毒素的污染,提高我国粮食安全。利用变性梯度凝胶电泳研究粮库中不同储藏时间和空间的玉米样品中霉菌群落的特点。结果表明,本研究中储藏玉米的霉菌污染以青霉、曲霉和毛霉为主,其霉菌群落的变化与储藏时间具有较强的相关性,而与其在粮库中的空间位置相关性较小。在储藏时间方面,储藏1a和3a的样品中霉菌群落具有较大的差异,而储藏2a样品的霉菌群落处于过渡期状态。本实验探究储藏玉米中霉菌群落的时空分布特征,可以为建立准确可靠的霉菌群落模型提供理论支持和数据支持。     

基于DGGE方法分析低氯化钠浓度对发酵白菜原核微生物群落结构的影响

为了探究氯化钠浓度对发酵白菜体系中微生物群落结构的影响。以4%、6%和8%氯化钠浓度发酵白菜,通过DGGE(denaturing gradient gel electrophoresis)方法及Quantity One软件分析原核微生物群落结构;通过回收DGGE电泳中荧光强度强、不同时间差异的电泳带,经克隆后测定碱基序列、与Gen Bank库序列对比鉴定。结果表明6%氯化钠浓度发酵白菜微生物多样性指数H(Shannon)、丰富度指数R(Species Richness)在发酵全过程中低于4%、8%组,均匀度指数E(Species Evenness)较4%、8%组的稳定。说明氯化钠浓度6%的发酵泡菜在整个发酵期微生物群落结构相对稳定且微生物种类小于氯化钠浓度4%、8%组。从DGGE电泳带荧光强度和存在的时间可知:在氯化钠浓度6%的发酵白菜体系中,Leuconostoc citreum(柠檬明串珠菌)、Uncultured bacterium(非培养细菌)、Leuconostoc mesenteroides(肠系膜明串珠菌)是白菜发酵中期、后期的优势微生物,Leuconostoc citreum(柠檬明串珠菌)、Leuconostoc mesenteroides(肠系膜明串珠菌)是乳酸菌。氯化钠浓度4%和8%发酵白菜中乳酸菌未成为优势微生物。说明氯化钠浓度6%较4%和8%发酵白菜原核微生物群落的变化接近于自然发酵蔬菜微生物变化规律,研究结果表明6%氯化钠浓度有利于白菜的发酵。      

Monitoring of population shifts in an enriched nitrifying system under gradually increased cadmium loading

The changes in nitrifying bacterial population under cadmium loading were monitored and evaluated in a laboratory scale continuous-flow enriched nitrification system. For this purpose, the following molecular microbiological methods were used: slot–blot hybridization, denaturing gradient gel electrophoresis (DGGE), real-time PCR followed by melting curve analysis, cloning and sequence analysis. The initial cadmium concentration was incrementally increased from 1 to 10 mg/l which led to a drop in ammonia removal efficiency from 99 to 10%. Inhibition was recovered when cadmium loading was stopped. During the second application of cadmium, nitrifying population became more tolerant. Even at 15 mg/l Cd, only a minor inhibition was observed. To investigate the variations in ammonia and nitrite oxidizing bacteria populations in a period of 483 days, ammonia monooxygenase (amoA) and 16S rRNA genes-based molecular techniques were used. An obvious shift was experienced in the diversity of ammonia oxidizers after the first application of 10 mg/l Cd. Metal-tolerant ammonia oxidizing species became dominant and the microbial diversity sharply shifted from Nitrosomonas and Nitrosococcus sp. to Nitrosospira sp. which were observed to tolerate higher cadmium loadings. This result indicated that the extent of nitrification inhibition was not only related to the metal concentration and quantity of microorganisms but also depended on the type of species.     

Identification of microorganisms involved in reductive dehalogenation of chlorinated ethenes in an anaerobic microbial community

In this study, we report on phylogenetic and physiological characterization of an anaerobic culture capable of reductive dehalogenation of tetrachloroethene (PCE) obtained from a PCE-contaminated site. The culture was enriched using different combinations of electron donors (hydrogen and acetate) and electron acceptors (PCE, cis-1,2-dichloroethene (cDCE) and controls without chlorinated ethenes). The resulting subcultures were analyzed using three different approaches: chemical analysis to document conversion of chlorinated ethenes; polymerase chain reaction (PCR) of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE) to compare community compositions; fluorescence in situ hybridization (FISH) to quantify specific groups of microorganisms using oligonucleotide probes previously designed or newly designed based on the sequences retrieved from sequence analysis of specific DGGE bands. Members of two genera which contain bacteria capable of reductive dehalogenation were detected in the culture: Dehalococcoides and Desulfitobacterium. The combined analyses suggested that Dehalococcoides-like bacteria are associated with complete dehalogenation of chlorinated ethenes to ethene with hydrogen as electron donor; and Desulfitobacterium-like bacteria, in contrast, are associated with incomplete PCE dehalogenation to cDCE and appear to be able to use acetate as electron donor. In addition, Sporomusa-like bacteria were identified, which most likely act as homoacetogens. The results demonstrated that combination of culture enrichment with different substrates, DGGE, and FISH allowed a detailed qualitative and quantitative characterization of the dominant microorganisms associated with reductive dehalogenation.     

Effects of selected pharmaceutically active compounds on treatment performance in sequencing batch reactors mimicking wastewater treatment plants operations

The impact of four pharmaceutically active compounds (PhACs) introduced both individually and in mixtures was ascertained on the performance of laboratory-scale wastewater treatment sequencing batch reactors (SBRs). When introduced individually at concentrations of 0.1, 1 and 10 μM, no significant differences were observed with respect to chemical oxygen demand (COD) and ammonia removal. Microbial community analyses reveal that although similarity index values generally decreased over time with an increase in PhAC concentrations as compared to the controls, no major microbial community shifts were observed for total bacteria and ammonia-oxidizing bacteria (AOB) communities. However, when some PhACs were introduced in mixtures, they were found to both inhibit nitrification and alter AOB community structure. Ammonia removal decreased by up to 45% in the presence of 0.25 μM gemfibrozil and 0.75 μM naproxen. PhAC mixtures did not however affect COD removal performance suggesting that heterotrophic bacteria are more robust to PhACs than AOB. These results highlight that the joint action of PhACs in mixtures may have significantly different effects on nitrification than the individual PhACs. This phenomenon should be further investigated with a wider range of PhACs so that toxicity effects can more accurately be predicted.     

Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors

Methanogenic community structure and population dynamics were investigated in two anaerobic reactors treating a dairy wastewater, an Inverted Fluidized Bed (IFB) and Expanded Granular Sludge Bed (EGSB). A combination of real-time PCR, denaturing gradient gel electrophoresis and statistical techniques was employed. Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h during the 200-day trial. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on absolute and relative abundance quantification data, demonstrated that the methanogenic communities developed in a different manner in the IFB, compared to the EGSB reactor. In our study, relative abundance-based quantification by NMS and moving-window analysis appeared to be a valuable molecular approach that was more applicable to reflect the changes in the anaerobic digestion process than approaches based either on qualitative analysis, or solely on absolute quantification of the various methanogenic groups. The overall results and findings provided a comparative, quantitative and qualitative insight into anaerobic digestion processes, which could be helpful for better future reactor design and process control.     

耐除草剂转基因大豆对根际土壤固氮微生物nifH基因多样性的影响

通过大田试验,以两种耐除草剂转基因大豆（PAT、ALS）及相应非转基因亲本大豆（PAT1、ALS1）和当地主栽大豆中黄13为材料,采用聚合酶链式反应-变性梯度凝胶电泳（PCR-DGGE）技术及扩增产物序列分析方法,探讨耐除草剂转基因大豆种植对根际土壤固氮微生物nifH基因多样性的影响。结果表明, PAT、ALS与相应亲本大豆PAT1、ALS1相比,根际土壤固氮微生物群落组成相似度均在60%左右。PAT、ALS根际土壤固氮微生物nifH基因多样性指数（H）和均匀度指数（EH）与相应亲本大豆相比差异均不显著（p〉0.05）。测序结果表明,PAT、ALS与相应亲本大豆根际土壤中固氮微生物主要隶属于蓝藻门（Cyanobacteria）、变形菌门（Proteobacteria）和厚壁菌门（Firmicutes）。不同处理下nifH基因与理化因子的典范对应分析结果表明,速效磷（AP）、硝态氮（NO3--N）对固氮微生物区系的影响达到显著水平（p〈0.05）。以上结论表明,与PAT1、ALS1相比,两种耐除草剂转基因大豆对根际土壤固氮微生物nifH基因多样性的影响不显著。     

芝麻香型白酒高温大曲微生物总DNA提取的改良方法

从常温研磨、蛋白酶K和R nase的加入对传统CTAB法进行改良,并结合试剂盒法,提出了一种高效提取高温大曲微生物总DNA的方法。该方法获得的总基因片段大小约21Kb;A260/A280=1.878;A260/A230=1.706;PCR反应抑制物少,可直接用于16S rDNA的扩增;并通过构建克隆文库技术和变性梯度凝胶电泳(DGGE)分析后发现高温大曲中细菌多样性丰富,能一定程度上反映微生物群落结构和组成。研究结果表明该方法提取的DNA适用于芝麻香高温大曲中微生物的分子生态学研究。