啤酒废水处理系统中活性污泥微型生物群落结构分析

为了分析啤酒厂废水处理系统活性污泥运行状态,选取了啤酒厂污水处理系统中不同阶段的微型生物群落结构作为研究对象。本文利用16S rDNA及18S rDNA特异性引物作为分子标记,通过PCR扩增污水处理系统中的环境生物群落DNA,以变性梯度凝胶电泳(DGGE)技术分离检测PCR产物获得微生物群落的DNA指纹图谱。研究结果显示,经过活性污泥处理的啤酒厂废水中细菌群落和真核生物群落结构发生了明显的改变。其中在细菌群落DGGE图谱中检测到21条特异性条带,而在真核生物DGGE图谱检测到10条。UPGMA聚类分析显示,进水与活性污泥中的细菌及真核群落结构十分相似(相似性>0.67),而与出水的差异较大(相似性<0.41),这表明了进水对活性污泥生物群落结构的重要影响。     

传统浓香型白酒大曲发酵过程中细菌和真菌动态变化研究

研究采用聚合酶链反应-变性梯度凝胶电泳(PCR-DGGE)技术对浓香型白酒大曲生产过程中细菌和真菌群落的多样性进行了研究。结果表明,16S r DNA的条带数、优势度、多样性和相似性在DGGE模式中明显不同,反映了不同阶段存在的细菌群落的广泛多样性。真核微生物群落的条带丰度和优势度在不同时期有所不同。     

PCR-DGGE指纹技术研究复合保鲜剂对冷藏带鱼贮藏期间微生物变化的影响

为探讨复合保鲜剂的作用机理和延长水产品货架期,应用聚合酶链式反应(PCR)-变性梯度凝胶电泳(DGGE)指纹技术研究复合保鲜剂对冷藏带鱼贮藏期间微生物种群多样性演替规律的影响。采用SDS(sodium dodecyl sulfate)化学裂解法,分别从对照组与复合保鲜剂处理组的冷藏带鱼中提取总细菌基因组DNA,同时进行16S rDNA的V3可变区PCR扩增,再通过DGGE得到动态变化的指纹图谱。DGGE图谱表明,产品在贮藏初期具有丰富的微生物群落,说明污染微生物的多样性,但经过一段时间后,只有少数种类细菌存活并最终成为主导菌群;通过对冷藏带鱼DGGE图谱上的主要条带进行测序分析,获得12种细菌。对照组与复合保鲜剂处理组在腐败后期的特定腐败微生物种类具有高度的相似性。嗜冷杆菌为带鱼贮藏初期的优势菌,随着贮藏时间的延长,希瓦氏菌与假单胞菌的比例逐渐增加,在贮藏过程中成为主要优势菌,荧光假单胞菌与弧菌在贮藏期间占有较大比例。同时,冷藏带鱼经复合保鲜剂处理后,其中的希瓦氏菌与假单胞菌受到明显抑制。     

宏基因组测序技术在传统酿造食品微生物群落分析中应用研究进展

我国传统酿造食品发展历史悠久,种类丰富,以其独特的魅力深受消费者喜爱。传统酿造食品中微生物群落的多样性是影响产品风味、品质与安全的重要因素,因此微生物群落结构特征、演替变化和功能基因挖掘等成为近年来的研究热点。高通量测序技术以其通量高和结果准确等优势,成为传统酿造食品微生物群落研究中的重要工具。该文以基于高通量测序技术的宏基因组测序技术出发,综述了宏基因组测序技术在传统酿造食品微生物群落分析研究中的进展,并分析讨论其面临的主要问题和发展趋势,为酿造食品的科学研究和工业生产提供相关理论基础。     

基于PCR-DGGE技术对库尔勒香梨“黑头病”病变组织生物群落的初步研究

为了解库尔勒香梨"黑头病"病变组织中真菌群落结构特征,应用PCR-DGGE技术对库尔勒地区4个不同保鲜库中12个样品真菌群落结构进行了研究。根据DGGE图谱对生物多样性进行了分析。结果表明,所有样品群落结构存在一定的差异,造成差异的原因可能与库尔勒香梨的种植环境有关;不同样品的9号条带在整个病变过程中优势度最高,其代表的菌株,可被认为是导致库尔勒香梨"黑头病"的主要致病菌;对DGGE的优势条带序列分析,同源性最高的微生物属于链格孢属(Alternaria)。该研究初步揭示了库尔勒香梨病变组织中微生物群落的变化规律,为库尔勒香梨"黑头病"的研究提供理论依据。     

液熏罗非鱼片加工过程中微生物群落的PCR-DGGE 分析

为弄清液熏罗非鱼片加工过程中微生物污染的源头,以进一步防控产品的微生物污染提供依据。应用基于细菌16S rDNA 的PCR-DGGE(PCR-denaturing gradient gel electrophoresis,PCR- 变性梯度凝胶电泳)技术分析液熏罗非鱼片主要加工关键环节的微生物群落结构,提取样品中的细菌总DNA,对细菌的16S rDNA 的V6～V8 区段进行PCR 扩增后,进行变性梯度凝胶电泳(DGGE),对DGGE 图谱进行微生物多样性分析,对主要条带进行序列分析并构建系统发生树。结果表明：10 个条带所代表的优势种很可能来源于以下几个属：巨型球菌属(Macrococus)、微球菌属(Micrococus)、肠道细菌属(Enterobacter)、假单胞菌属(Pseudomonas)、弧菌属(Vibrio)、突柄杆菌属(Prosthecobacter)、布特菌属(Buttiauxella),其中肠道细菌属、微球菌属、假单胞菌属和弧菌属细菌都具有使产品腐败的潜能。本研究表明：液熏罗非鱼片中呈现微生物多样性,PCR-DGGE 技术可用于研究液熏罗非鱼片加工过程中的微生物群落结构及变化,且具有一定的优越性。     

传统分离培养结合DGGE技术研究新疆传统发酵酸驼乳中乳酸菌的多样性

目的:分析新疆传统发酵酸驼乳中乳酸菌多样性,为我国传统乳制品微生物资源的利用提供基础数据。方法:采用传统分离培养和基于16S r DNA的PCR-DGGE方法,对12份酸驼乳样品中分离出的87株可培养乳酸菌进行形态、生理生化结合16S r DNA分子法鉴定菌株;对DGGE图谱上的15条16S r DNA目标条带切胶回收测序。结果:传统培养法显示样品中乳酸菌总数在106~108CFU/m L之间,其中植物乳杆菌为优势菌群,次优势菌群为乳酸片球菌;通过DGGE目标条带序列分析与相似性比对,植物乳杆菌、瑞士乳杆菌丰度最高,是优势菌群。此外,鉴定出干酪乳杆菌、屎肠球菌、乳明串珠菌、芽孢杆菌。DGGE图谱显示:样品混合菌群中有5条条带无对应的纯菌株,而纯菌株在混合菌群的图谱上都有相应的条带。结论:传统分离培养与PCR-DGGE在分析优势细菌种群上结果一致,均为植物乳杆菌;用传统分离培养未鉴定到屎肠球菌和芽孢杆菌。DGGE图谱能更全面地反映样品中的弱势菌群,体现样品细菌多样性的真实水平。     

高通量测序技术及其在黄酒微生物多样性研究中的应用

黄酒是我国传统的发酵酒精饮料，口感醇厚、风味独特。由于传统微生物培养方法具有一定局限性，无法全面反映黄酒酿造微生物多样性，而高通量测序技术具备方法简单、通量更高、成本较低和速度较快等特点，将其应用于黄酒微生物多样性的研究中，为认识黄酒酿造微生物多样性提供了有利手段。该文综述了高通量测序技术的发展阶段、原理、方法、操作流程、数据分析方法及其在黄酒微生物多样性研究中的应用，并对黄酒微生物多样性的研究方向进行了展望，以期为全面解析传统黄酒酿造微生物多样性与群落结构提供参考。     

应用PCR-DGGE技术分析不同性状窖泥的细菌群落结构

基于聚合酶链式反应-变性梯度凝胶（PCR-DGGE）技术分析了不同性状窖泥的细菌群落DGGE指纹图谱,结果表明,通过DGGE指纹图谱能够区分新窖泥、窖壁泥和窖底泥,其中以窖壁泥退化前后图谱变化最明显。PCR-DGGE图谱优势条带测序结果显示,窖泥中细菌微生物包括拟杆菌门、放线菌门、厚壁菌门、变形菌门4类,其中新窖泥微生物种类最丰富,涵盖以上门类,特有优势微生物为Enterobacterale科、双歧杆菌属（Bifidobacterium）、假单胞菌属（Pseudomonas）及里氏杆菌属（Riemerella）。紫单孢菌科（Porphyromonadaceae）仅存在于窖底泥中,Tumebacillus属微生物只存在于窑壁泥中。通过窖泥的DGGE图谱对比分析,能够对窖泥类型做出初步判定,并能及时发现窖泥退化现象,为工厂窖泥培养、检测和窖泥优化提供了理论支持。     

黄酒酿造过程细菌群落结构变化初步研究

应用变性梯度凝胶电泳(DGGE)技术对黄酒酿造过程细菌群落的结构变化进行分析研究.以细菌通用引物扩增16S rDNA基因V3高变异区,扩增产物进行DGGE,获得表征细菌群落结构的指纹图谱.分析结果表明,浸米水、麦曲和酒母的细菌群落结构各不相同,其中麦曲的细菌群落多样性较为丰富;前酵和后酵阶段细菌群落结构存在差异,同一阶段细菌群落结构组成类似.测序结果显示,存在于黄酒酿造过程中的细菌主要有乳杆菌(Lactobacillus)、葡萄球菌(Staphylococcus)、糖多孢菌(Saccharopolyspora)、肠杆菌(Enterobacteriaceae)、布丘氏菌(Buttiauxella)等种类,同时,黄酒酿造过程中还存在着不可培养(Uncultured bacterium)的优势细菌.PCR-DGGE技术是研究黄酒酿造过程细菌群落结构变化的一种有效手段.     

Succession of a microbial community during stable operation of a semi-continuous garbage-decomposing system

The microbial community in a garbage-decomposing system was analyzed using denaturing gradient gel electrophoresis (DGGE) on the basis of 16S rDNA. The system treated 1 kg of garbage everyday for two months at ambient temperature with almost constant decomposition efficiency, although a transient pH increase occurred. Succession of the banding pattern of the DGGE profile suggested that the bacterial community was not directly affected by the continuous addition of non-sterilized garbage into the open system, but changed with the fluctuation of pH. These resistance and resilience characteristics of the community structure may be effective to keep the decomposition efficiency stable. The analyses of the DNA sequences from the DGGE bands suggested the existence of uncultured or novel bacteria as well as Lactobacillus sp., Corynebacterium spp., Enterococcus spp., and Staphylococcus sp. A specific PCR detection was performed to evaluate the existence of Escherichia coli within the community. E. coli 16S rDNAs were not detected from the decomposing system.     

Assessing the impact of nanomaterials on anaerobic microbial communities

As the technological benefits of nanotechnology begin to rapidly move from laboratory to large-scale industrial application, release of nanomaterials to the environment is inevitable. Little is known about the fate and effects of nanomaterials in nature. Major environmental receptors of nanomaterials will be soil, sediment, and biosolids from wastewater treatment. Analysis of anaerobic microbial activity and communities provides needed information about the effects of nanoparticles in certain environments. In this study, biosolids from anaerobic wastewater treatment sludge were exposed to fullerene (C60) in order to model an environmentally relevant discharge scenario. Activity was assessed by monitoring production of CO2 and CH4. Changes in community structure were monitored by denaturing gradient gel electrophoresis (DGGE), using primer sets targeting the small subunit rRNA genes of Bacteria, Archaea, and Eukarya. Findings suggest that C60 fullerenes have no significant effect on the anaerobic community over an exposure period of a few months. This conclusion is based on the absence of toxicity indicated by no change in methanogenesis relative to untreated reference samples. DGGE results show no evidence of substantial community shifts due to treatment with C60, in any subset of the microbial community.     

酱油发酵过程中细菌群落结构的动态变化

本文采用16S r DNA PCR-DGGE(变性梯度凝胶电泳)指纹图谱和系统发育分析方法,以高盐稀醪发酵工艺生产的广东酱油为研究对象,揭示了酱油生产发酵过程中细菌群落结构的多样性及动态变化。从样品中提取总细菌DNA,用降落PCR扩增16S r DNA V3片段序列,再通过分析DGGE图谱选择特异性条带,进行割胶回收、测序及Blast分析。DGGE图谱表明,发酵初期样品具有丰富的微生物群落,但之后只有少数种类细菌存活,整个酱油发酵过程微生物群落结构的演变规律是由复杂到简单,这也说明酱油发酵环境具有抑制微生物生长的作用。测序结果表明,代表最相似菌为魏斯菌属(Weissella cibaria)和非培养的肠杆菌属(Uncultured Enterobacter sp.),其次是嗜盐四联球菌(Tetragenococcus halophilus)、类肠膜魏斯氏菌(Weissella paramesenteroides)、弗氏柠檬酸杆菌(Citrobacter freundii)、产气肠杆菌(Enterobacter aerogenes)和肠杆菌属(Enterobacter sp.BF1-8)。     

Microbial community analysis in the denitrification process of saline-wastewater by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA and the cultivation method

The metallurgic wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of nitrogen compounds and salts. Biological nitrogen removal from this wastewater was attempted using a circulating bioreactor system equipped with an anaerobic packed bed or an anaerobic fluidized bed. The denitrification capability of the system with the anaerobic packed bed was more stable than that of the system with the anaerobic fluidized bed. The NOx removal rate of the anaerobic packed bed was as high as 97%. Microbial community analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments and the cultivation method revealed that the community diversity varied in accordance with wastewater composition such as the level of salinity and so on. Phylogenetic analysis suggested that the taxonomic affiliation of the dominant species in the anaerobic reactors was to the gamma-Proteobacteria including Halomonadaceae species. The PCR-DGGE method as a non-cultivation method was found to be a powerful tool for analysis of the microbial community, because the cultivation method could detect only a fraction of the microbial species present in these systems. The genetic diversity of the isolated bacteria belonging to the gamma-Proteobacteria which reduced both nitrate and nitrite in the anaerobic packed bed was higher than that of the bacteria in the anaerobic fluidized bed. This suggested that a genetically diverse microbial community stabilized the denitrifying performance in the anaerobic packed bed.     

PCR-DGGE分析资中冬尖发酵过程中细菌多样性

目的:采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denatured gradient gel electrophoresis,PCR-DGGE)技术分析资中冬尖发酵过程中微生物群落结构及其动态变化。方法:从资中采集4份不同发酵年份冬尖样品,提取样品总DNA,采用巢式PCR法扩增细菌16S r DNA V3区,扩增产物采用DGGE分离,对细菌主要优势条带进行克隆、测序、构建系统发育树。结果:冬尖在发酵过程中细菌多样性较丰富,且群落结构发生了较大变化。不同发酵时间样品间,细菌群落结构相似性为9%~67%,其中第2年与第3年样品相似性最高,达67%。资中冬尖发酵过程中,所涉及的细菌主要分为3类,分别为厚壁菌门(Firmicutes)、变形杆菌门(Proteobacteria)和非培养菌,其中厚壁菌门为优势菌群,占53%;变形杆菌门占37%;非培养菌仅占10%。结论:采用PCR-DGGE技术能更全面、更真实地反映资中冬尖在发酵过程中微生物群落的多样性及其动态变化,为冬尖的生产工艺和风味物质的形成提供理论支撑。     

Microflora assessments using PCR-denaturing gradient gel electrophoresis of ozone-treated and modified-atmosphere-packaged farmed cod fillets

Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rDNA sequence was used to characterize changes in the microbial flora caused by ozone (O3) treatment of farmed cod (Gadus morhua). Portions of cod were produced under controlled conditions, bathed in fresh water supplemented with 2 ppm of O3 for 30 min, and packaged in modified atmosphere (MA: 60% CO2 and 40% N2) before 4 degrees C storage. Control samples were packaged in MA or air, without prior O3 treatment. Samples were analyzed by PCR-DGGE to determine the predominant bacterial flora and to examine possible differences in the microbial community due to O3 treatment. The DGGE analysis during the storage period showed that the O3 treatment produced no significant difference in the microbial flora compared with the controls. Sequencing of 16S rDNA detected the specific spoilage bacteria Photobacterium phosphoreum, Pseudomonas spp., Shewanella baltica, and Shewanella putrefaciens as the predominant bacteria in all samples. PCR-DGGE results were supported by culture and sensory analyses used in predicting product shelf life. Aerobic plate count, H2S-producing bacteria, and psychrotrophic bacterial counts demonstrated no significant extension of the shelf life of MA-packaged, O3-treated cod fillets.     

Phylogenetic diversity and metabolic potential of activated sludge microbial communities in full-scale wastewater treatment plants

The activated sludge process is an essential process for treating domestic and industrial wastewaters in most wastewater treatment plants (WWTPs). This process consists of a mixture of general and special microorganisms in a form of a complex enrichment population. Thus, the exploration of activated sludge microbial communities is crucial to improve the performance of activated sludge process. In this study, we investigated the phylogenetic diversity and metabolic potential of activated sludge microbial communities in full-scale WWTPs. Four 16S rRNA gene clone libraries were constructed from activated sludge samples. In all samples, Proteobacteria was the most abundant phylogenetic group, followed by Bacteroidetes and Firmicutes. The dominance of Proteobacteria was further demonstrated by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP). Some specific genera, e.g., Nitrosomonas, Thauera, and Dechloromonas, which significantly correlate with the functions and performance of wastewater treatment, were abundant in all samples. A large number of unclassified sequences were found in the library, suggesting that a wide variety of novel species may inhabit complex activated sludge communities. The structures of the bacterial community did not differ significantly among samples. All samples utilized the vast majority of 31 carbon sources of an EcoPlate (Biolog), suggesting that activated sludge microbial communities possess high metabolic potential and equivalent functions required for wastewater treatment.     

水井坊窖底泥微生物群落结构的DGGE分析

为了补充和验证研究浓香型白酒不同窖泥的微生物群落特征分析,该研究采用DGGE技术对水井坊酒厂窖龄分别为50及150年相同窖池不同部位窖底泥（三点取样法）进行细菌、古菌和真菌微生物群落特征分析。研究结果显示：50年和150年窖龄窖泥的微生物群落的演替特征和分布特征具有一定相似性,进一步验证了50年可能是窖池微生态发生较大变化的一个分界线这一推测的准确性;不同部位的窖底泥的微生物群落结构具有一定的差异性,因此在今后对窖底泥的实验研究中,采用多点取样法进行分析。     

PCR‐DGGE analysis on microbial communities in pit mud of cellars used for different periods of time

Polymerase chain reaction denaturing gradient gel electrophoresis (PCR‐DGGE) was used to analyse microbial community evolution in the pit mud of cellars used for different periods of time in production of Chinese Luzhou‐flavour liquor. The pit mud was collected from the cellars and the microbial DNA was extracted from the microbes in the pit mud. The Bf 968 primer was used for PCR‐DGGE to analyse the variable region 6 (V6) to variable region 8 (V8) of the microbial 16S rDNA. It was found that the band number, dominance, diversity and similarity of the 16S rDNA were clearly different in the DGGE patterns, because of the great diversity expressed by the different microbial communities in the different‐aged cellars. It is concluded that mutual collaboration and constraint exist between the different microbial communities in the different‐aged cellars, and this relationship leads to an evolutional change in the structures and in the numbers of the microbial communities in the pit mud of the cellars. Changes become more obvious with increasing age of the liquor cellars. Copyright © 2012 The Institute of Brewing & Distilling