PCR-DGGE技术分析不同包装条件下鱼肉表面优势菌的菌群变化

采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-deformation gradient gel electrophoresis analysis,PCR-DGGE)技术研究3种包装(空气、真空、气调)结合钴60辐照技术对鱼肉表面优势菌菌群的影响,分别选择6种辐照剂量(0、2、4、6、8、10 kGy)照射4℃贮藏18 d后的鱼肉表面优势腐败菌群进行研究。方法:采集第18天不同包装不同剂量的草鱼肉样品,洗脱鱼肉表面菌;提取DNA,扩增16S rDNA的V3可变区,用PCRDGGE技术鉴定优势菌,割胶回收测序。结果表明:不同包装鱼肉表面优势菌群数量和种类不同,在样品中共同的优势腐败菌为假单胞菌,在空气包装中优势腐败菌是假单胞菌、乳酸菌和沙雷菌,沙雷菌在辐照剂量高于8 kGy时被抑制。在真空包装中优势腐败菌是假单胞菌、沙雷菌、热死环丝菌和耶尔森菌,沙雷菌辐照剂量高于6 kGy时被抑制,而耶尔森菌在2 kGy以上即被抑制,热死环丝菌在6、8 kGy辐照条件下才出现,其具有很高的辐照抗性。在气调包装中优势腐败菌是假单胞菌和乳酸菌,但乳酸菌仅在气调包装2 kGy辐照和气调包装8 kGy两种辐照包装条件下是优势腐败菌。结果表明,PCR-DGGE技术可以很好地鉴别鱼肉表面优势腐败菌。

螺旋粉虱成虫体内细菌群落多样性的PCR-DGGE和16S rRNA文库序列分析

为了解螺旋粉虱Aleurodicus dispersus体内细菌多样性和主要优势菌群结构,用PCR-DGGE和16S rRNA文库对采自于海南省番石榴上螺旋粉虱雌、雄成虫体内的细菌群落进行了分析。用PCR扩增体内细菌16S rRNA基因,构建雌、雄虫克隆文库;再用限制性片段长度多态性(restriction fragment length polymorphism,RFLP)方法从文库中筛选不同16S rRNA基因图谱,根据图谱对克隆子进行分型。从螺旋粉虱雌、雄两个样品中共获得10种分类操作单元(operational taxonomic unit,OTUs)。以16S rRNA基因为基础构建系统发育树,系统发育分析表明,螺旋粉虱雌、雄成虫体内优势菌群主要为发酵菌属Zymobacter,杀雄菌属Arsenophonus,泛菌属Pantoea和假单胞菌属Pseudomonas。Candidatus Portiera aleyrodidarum和Arsenophonus sp.可能为其体内共生菌群,在所有样品中均可稳定地检测到。这些微生物可能对螺旋粉虱生长发育、繁殖和性比调控起到重要的协同作用。

芽孢杆菌在肉鸡肠道内的分布及对肠道菌群、消化酶活性的影响

考察枯草芽孢杆菌的芽孢在AA肉鸡肠道内的分布和随粪便的排出规律,及对肠道内细菌群落结构变化和肠道内消化酶活性的影响.试验选取240羽1日龄AA肉鸡,随机分为2组,每组6个重复,采用相同的无抗生素基础日粮饲养3 d后,对照组和试验组分别一次性灌喂无菌水或枯草芽孢杆菌的芽孢(1.5×108CFU?mL-1)各0.5 mL;在不同时间采样进行计数和酶活性测定,同时通过聚合酶链反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术分析此过程肉鸡粪便中细菌群落的变化.结果表明:灌喂的芽孢在十二指肠内开始萌发,以空肠的营养体数量最高,盲肠内则以芽孢为主;粪便中的芽孢数量在24 h时最高,达到7.9×104CFU?g-1,而后逐渐降低,在72 h后基本无法检出.PCR-DGGE结合主成分分析(PCA)表明:对照组与试验组鸡粪中的细菌群落结构存在较大差异,其中试验组中肠道有益菌——乳杆菌属(Lactobacillussp.)含量较对照组有明显提高;同时,灌喂芽孢12 h后,十二指肠内淀粉酶、胰蛋白酶和总蛋白酶活性,空肠内的脂肪酶活性和盲肠内的总蛋白酶活性均高于对照,差异有统计学意义(P<0.05).试验证实芽孢能在AA肉鸡肠道内萌发并停留超过72 h,并对提高肠道内消化酶活性和改善菌落结构发挥作用.

微生物强化对椒坯发酵群落多样性及性质的影响

描述了应用磷脂脂肪酸(phosphoric acid fatty acids,PLFAs)及聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术研究微生物强化发酵椒坯群落多样性变化的规律。研究表明,高盐发酵使椒坯的细菌生物量显著降低;对于强化的样品,因所使用的菌株及强化方式不同,其群落多样性及优势菌差异显著,假丝酵母菌Candida versatilis强化提高了椒坯总生物量,鲁氏酵母Zygosaacharomyce rouxii则相反;共培养强化提高了椒坯以Tetragenoccus和Zygosaacharomyces为主的优势菌的生物量,同时发现嗜盐四链球菌Tetragenoccus halophilus对Vagococcus具有明显的抑制作用。理化研究表明,不同强化方式使样品的总酸(TA)、还原糖(RS)和氨基态氮(FN)等参数略有差异。不同强化方式对挥发性组分贡献不同,其中,共培养强化使椒坯挥发性组分总含量提高了28.46%。该研究揭示了强化发酵对椒坯微生物群落和品质的影响规律,对微生物强化技术在传统发酵的应用有一定的指导作用。

直流电场对根际土壤微生物群落的影响及其机理

电场作用下根际土壤微生物群落的变化与利用电场强化植物修复效率密切相关。文章利用改进的PCR-DGGE（多聚酶链式反应-变性梯度凝胶电泳）方法，研究了不同电场条件下根际土壤微生物群落多样性和相似性的变化，分析了直流电场对土壤微生物群落的影响机理。结果表明，电场对根际土壤微生物群落的影响与电场条件有关，合适的电场条件有助于增加土壤微生物群落的多样性，但是电场形式、强度和施加方式不当则会使土壤微生物群落的多样性和结构受到明显影响。电场作用下土壤性质的变化、电场对土壤微生物的迁移作用和致死效应．以及微生物对环境压力的生理响应等是电场影响土壤微生物群落的主要机制。为了避免电场对根际土壤微生物的不利影响，利用电场强化植物修复时需要采用合适的电场条件。

变性梯度凝胶电泳分析PAH基因外显子3突变

采用ＰＣＲ、变性梯度凝胶电泳、限制性内切酶分析及ＤＮＡ直接序列分析等方法，对５８例南方地区中国人苯丙酮尿症（ＰＫＵ）的苯丙氨酸羟化酶（ＰＡＨ）基因外显子３进行了分析，发现９例ＰＫＵ患者外显子３存在突变，占ＰＫＵ患者的１５．５２％（９／５８），外显子３突变占ＰＡＨ基因突变的７．７６％（９／１１６），６例患者存在ＡｒｇｌｌｌＴｅｒ错义突变，该突变位点的基因内频率为５．１７％。结果提示中国南方和北方人群ＰＫＵ分子遗传学可能存在一定程度的差异。

水果酵素自然发酵过程中优势菌群与有机酸变化规律分析

分析3种自然发酵的水果酵素优势菌群与有机酸变化规律。高效液相色谱法测定有机酸含量变化,聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)分析菌群变化,并将两部分结果运用冗余分析(redundancy analysis,RDA)方法进行变化规律分析,发现发酵过程中细菌多样性指数呈先减小后增大的趋势。乳酸、柠檬酸、苹果酸和草酸在发酵过程中是水果酵素的主要有机酸。PCR-DGGE共检测出19条特异性条带,包括7株乳酸菌,2株肠杆菌,1株肠球菌,2株假单胞菌,以及3株不可培养微生物等。RDA表明对柠檬酸生成贡献率较大的菌株分别为Lactobacillus plantarum>uncultured Brevundimonas sp.>Enterobacter xiangfangensis>uncultured Lactococcus sp.,对乳酸生成贡献率较大的菌株分别为L.plantarum>Lactococcus lactis subsp.>E.xiangfangensis>L.lactis subsp.>uncultured Lactococcus sp.。

Microbial Function, Enzymatic Activities and Diversity in an Anaerobic-Anoxic-Aerobic Reactor System

Enzymatic activities of beta-gincosidase （ β-GLC ）, （ LAP）, and alkaline phosphatase （ APA）, corresponding to nutrient eliminations, and the microbial community structures were analyzed in an anaerobic-anoxic-aerobic reactor system. Results showed that most activity of β-GLC （64.2 μmoi/（ L · h ）） associated with the largest fraction of small- molecular-weight carbohydrates was found in the aerobic reactor, indicating the existence of coupled hydrolysis-uptake mechanism in the aerobic bacteria. Similar activities of LAP presented in the anoxic and aerobic environments, whose increases accompanied by increments in nitrogen uptake rates greatly accelerated the processes of aerobic nitrification and anoxic denitrification. The highest APA activity displayed in the anaerobic reactor, however, dephosphorization performance was mainly achieved under aerobic condition. Microbial community fingerprints generated by polymerase chain reaction amplification and denaturing gradient gel dectrophoresls （ PCR-DGGE ） revealed that Proteobacterium, Actinobacterium, and Nitrosplra were the predominant classes in the activated sindge and there was no evidence of community variations among each function reactor in the system with biomass recycling.

肇庆星湖湿地放线菌多样性

【目的】了解肇庆星湖湿地放线菌多样性及其与底泥理化性质的关系。【方法】应用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-density gradient gel electrophoresis,PCR-DGGE)技术对星湖湿地10个样点放线菌群落进行研究,根据DGGE图谱及克隆测序结果对其物种多样性进行分析,并结合样点理化性质对其空间分布特征进行分析。【结果】通过PCR-DGGE指纹图谱发现各样品放线菌物种丰富度(S)、多样性指数(H')和均匀度指数(J')均有所不同。对不同样点放线菌群落相似性进行比较,发现它们的相似性系数存在着一定的关系,相邻样点间相似性较高。对DGGE优势条带进行回收、克隆、测序,显示优势类群主要分布于类诺卡氏菌科(Nocardioidaceae)、链霉菌科(Streptomycetaceae)、小单孢菌科(Micromonosporaceae)、微球菌科(Micrococaceae)、纤维素单胞菌科(Cellulomonadaceae)和原小单胞菌科(Promicromonosporaceae)。典型对应分析结果表明,星湖湿地放线菌群落结构变化的主要影响因子为底泥水溶性碳(WSOC)和速效磷(A-P)。【结论】肇庆星湖湿地蕴含着大量的放线菌资源,具有较高的物种多样性,需要进一步挖掘这些新资源。

天津独流老醋醋酸发酵过程中细菌群落多样性及动态变化研究

本论文采用PCR-DGGE方法对天津独流老醋醋酸发酵过程细菌群落组成以及其多样性变化进行了研究,重点比较了独流老醋醋酸发酵阶段上下层醋醅中细菌群落组成之间的差异。独流老醋上下层醋醅中均检测到13种细菌,虽然独流老醋在醋酸发酵阶段只对上层的醋醅进行翻醅操作,由于上、下层醋醅仍处于同一发酵体系,细菌的组成一致,但两个区域细菌群落的多样性存在差异。聚类分析将细菌群落组成分为第一阶段和第二阶段两类,Shannon指数表明独流老醋醋酸发酵第一阶段醋醅中细菌多样性高于第二阶段醋酸发酵,而上层醋醅中细菌多样性略高于下层醋醅。巴氏醋杆菌(Acetobacter pasteurianus)是醋酸发酵阶段将乙醇氧化为醋酸的主要微生物,随着发酵的进行丰度逐渐增加,在醋酸发酵后期丰度达到最高。而部分乳酸菌Lactobacillus plantarum、Lactobacillus acetotolerans、等随着发酵的进行丰度逐渐增加,可能与其较强的耐酸性有关。

Cultivation of aerobic granular sludge in a conventional, continuous flow, completely mixed activated sludge system

Aerobic granules were formed in a conven- tional, continuous flow, completely mixed activated sludge system （CMAS）. The reactor was inoculated with seed sludge containing few filaments and fed with synthetic municipal wastewater. The settling time of the sludge and the average dissolved oxygen （DO） of the reactor were 2 h and 4.2 mg. L 1, respectively. The reactor was agitated by a stirrer, with a speed of 250r·min^-1, to ensure good mixing . The granular sludge had good settleability, and the sludge volume index （SVI） was between 50 and 90 mL ·g ^-1. The laser particle analyzer showed the diameter of the granules to be between 0.18 and 1.25 mm. A scanning electron microscope （SEM） investigation revealed the predominance of sphere-like and rod-like bacteria, and only few filaments grew in the granules. The microbial community structure of the granules was also analyzed by polymerase chain reaction-denaturing gradient gel electro- phoresis （PCR-DGGE）. Sequencing analysis indicated the dominant species were α, β, and γ-Proteobacteria, Bacteroidetes, and Firmicutes. The data from the study suggested that aerobic granules could form, if provided with sufficient number of filaments and high shear force. It was also observed that a high height-to-diameter ratio of the reactor and short settling time were not essential for the formation of aerobic granular sludge.

Bacterial community composition and abundance in leachate of semi-aerobic and anaerobic landfills

The abundance and phylogenetic composition of bacterial community in leachate of semi-aerobic and anaerobic landfill were compared through real-time polymerase chain reaction and denaturing gradient gel electrophoresis. In semi-aerobic landfill scenario, the bacterial 16S rRNA copy numbers in leachate had no significant reduction from initial stage to stable period. In the scenario of anaerobic landfill, the largest bacterial 16S rRNA gene copy number was found in leachate at initial stage, but it reduced significantly at stable period. Moreover, methane-oxidizing bacteria population in stable period was lower than that in initial period in both two landfill processes. However, semi-aerobic landfill leachate had more methanotrophic bacteria populations than that in the anaerobic one. Furthermore, according to the sequences and phylogenetic analysis, obvious difference could be detected in bacterial community composition in different scenarios. Proteobacteria and bacteroidetes took up a dominantly higher proportion in semi-aerobic landfill leachate. To summarize up, different landfill methods and its landfill ages had crucial impacts on bacterial abundance and composition in leachate of semi-aerobic and anaerobic landfills.

Interaction and independence on methane oxidation of landfill cover soil among three impact factors:water,oxygen and ammonium

To understand the influence patterns and interactions of three important environmental factors,i.e.soil water content,oxygen concentration,and ammonium addition,on methane oxidation,the soils from landfill cover layers were incubated under full factorial parameter settings.In addition to the methane oxidation rate,the quantities and community structures of methanotrophs were analyzed to determine the methane oxidation capacity of the soils.Canonical correspondence analysis was utilized to distinguish the important impact factors.Water content was found to be the most important factor influencing the methane oxidation rate and Type II methanotrophs,and the optimum value was 15%(w/w),which induced methane oxidation rates 10-and 6-times greater than those observed at 5%(w/w)and 20%(w/w),respectively.Ambient oxygen conditions were more suitable for methane oxidation than 3%oxygen.The addition of 100 mg-N·kg^(-1) drysoil of ammonium induced different effects on methane oxidation capacity when conducted at low or high water content.With regard to the methanotrophs,Type II was sensitive to the changes of water content,while Type I was influenced by oxygen content.Furthermore,the methanotrophic acidophile,Verrucomicrobia,was detected in soils with a pH of 4.9,which extended their known living environments.

Seasonal microbial community shift in a saline sewage treatment plant

Activated sludge was monthly sampled from a saline sewage treatment plant of Hong Kong(China)during June 2007 to May 2008 to analyze the microbial community shift along with environmental variations using denaturing gradient gel electrophoresis of polymerase chain reaction amplified 16S rDNA fragments.Environmental changes resulted into a seasonal microbial community shift characterized by alterations in species number and abundance in the sewage treatment plant.Correspondence analysis and cluster analysis on community structure profile showed that the 12 monthly samples fell into four groups,which is in accordance with season changing in Hong Kong.Canonical correspondence analysis revealed that PO_(4)^(3-)-P and NH_(4)^(+)-N posed more significant effects on community structure than total phosphorus and total nitrogen,respectively.Compared with sludge retention time,influent total phosphorus had an inverse effect on the community structure shift,and chemical oxygen demand and NH_(4)^(+)-N showed a similar effects.Results of this study may contribute to the development of new knowledge involving the microbial community shift in sewage treatment plants.

Applicability of anoxic-oxic process in treating petrochemical wastewater

To explore the applicability of anoxic-oxic (A/O) activated sludge process for petrochemical wastewater treatment,the relationship between bacterial community structure and pollutants loading/removal efficiencies was investigated by gas chromatograph-mass spectrometry (GC-MS),polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and other conventional techniques.It showed that when the concentrations of the influent chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N) were 420～560mg/L and 64～100mg/L,respectively,the corresponding average effluent concentra-tions were 160mg/L and 55mg/L,which were 1.6 and 2.2times higher than those of the national standards in China,respectively,demonstrating the inefficient performances of A/O process.Analysis of GC-MS indicated that refractory pollutants were mainly removed by sludge adsorption,but not by biodegradation.PCR-DGGE profile analysis suggested that the biological system was species-rich,but there was apparent succession of the bacterial community structure in different locations of the A/O system.Variations of bacterial community structure and pollutant loadings had obvious influences on pollutants removal efficiencies.Thus,A/O process was inapplicable for the treatment of complicated petrochemical wastewater,and strategies such as the reinforcement of pre-treatment and two-stage A/O process were suggested.

Microbial and hydrodynamic properties of aerobic granules in a sequencing batch reactor treating landfill leachate

A sequencing batch reactor （SBR） seeded with activated sludge was established for landfill leachate treatment. Small bio-aggregates began to appear after 40-d operation, and gradually changed to mature aerobic granules, with a mean size of 0.36-0.60 ram. Their sludge volume index at 5 min （SVI5 rain）, mixed liquor volatile suspended solids （MLVSS）, and wet density were around 35 ml/g, 3.4 g/L, and 1.062 g/cm3, respectively. The settling velocities of the granules in distilled water ranged from 0.3 to 1.3 cm/s, which were faster than those in landfill leachate with a salt content of 1.4% （w/v）, and also slightly faster than those predicted by Stokes＇ law for porous but impermeable particles. Microbial community evolution during the granulation process and stages under different nitrogen loading rates （NLRs） were monitored and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE）, cloning, and sequencing of 16S ribosomal RNA （rRNA） fragments. Results revealed that some primary and dominant communities in inoculating activated sludge died out gradually; while a few common bacteria, inhabiting soils, municipal wastewater, or activated sludge systems, dominated in the SBR system throughout. In addition, some other dominant species, associated with the aerobic granulation process, were thought to play a significant role in the formation and growth of aerobic granular sludge. During the stable operation time under low NLR, a few species were present in abundance, and may have been responsible for the high organic removal efficiency at this time.

Seasonal dynamic changes in bacterial compositions in the Inner Mongolia desert steppe

Polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)technique was employed to examine the seasonal dynamic changes in bacterial community composition in the Inner Mongolia desert steppe using specific primers F954 and R1369.Bright and reproducible bands were sequenced,and the phylogenic tree was constructed.The results show that the bacterial community composition changed between different seasons.The specific bands were different between the sampling sites with light and heavy levels of degraded grassland.Three main types of bacteria constituting the microbial community in the Inner Mongolia desert steppe belonged to the α,γ and δ-sub phyla of Proteobacteria,Bacteroidetes and Acidobacteria.The unculturable bacteria accounted for 69%of the whole bacterial community of the Inner Mongolia desert steppe.