城市污水处理系统进水水质对曝气池微生物种群结构的影响

以芜湖市天门山污水处理厂为例,研究城市污水处理系统进水水质对曝气池活性污泥微生物种群结构的影响。采集多个曝气池活性污泥样品进行DNA提取和聚合酶链式反应(PCR)扩增,对采样期间进水主要水质指标进行测定,运用变性梯度凝胶电泳(DGGE)技术分析各活性污泥样品DNA的PCR扩增产物,采用非加权组平均(UPGMA)聚类法分析活性污泥样品的DGGE图谱,选择基于线性模型的冗余分析(RDA)对影响微生物种群结构的水质理化因子进行排序。分析结果表明,污水处理系统处理效果整体比较稳定,COD的去除率在62.3%～97.0%,NH3-N的去除率在81.6%～96.4%,TP的去除率在95.3%～99.7%,系统出水均能达到设计要求。微生物种群结构不稳定,随着进水水质的变化呈现出动态变化过程。其中,BOD5与微生物种群结构的关联度最高。由此得出,系统功能稳定的污水处理厂污泥中微生物的种群结构仍会存在一定变化。     

不同污泥负荷下SBR处理游泳馆污水的微生物群落的演变

污泥负荷直接影响微生物的生长模式,当污泥负荷发生变化时,短时间内微生物群落结构将发生明显变化。为了研究污泥负荷冲击对SBR系统内活性污泥微生物群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCRDGGE)技术,对不同污泥负荷冲击时,SBR处理游泳馆污水中的活性污泥微生物进行了考查。研究表明,在不同污泥负荷冲击的条件下,以MBR污泥为接种污泥,SBR工艺处理游泳馆污水系统内活性污泥微生物群落结构变化明显,多样性指数随着污泥负荷升高而逐渐增加并趋于稳定,但污泥冲击负荷过高多样性指数反而下降,SBR系统内微生物菌种大部分为未经培养菌种,肠杆菌属、甲苯单胞菌属以及γ-变形菌纲细菌等。微生物通过对不同负荷阶段环境条件的适应及演变,逐渐形成了适应相应污泥负荷的微生物种群。     

间歇好氧硫酸盐废水处理系统微生物区系解析

应用PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)技术和16S rDNA序列测定对间歇好氧硫酸盐废水处理工艺的微生物群落结构进行了研究.采集味精厂好氧池原始污泥以及实验室内间歇好氧工艺驯化后不同条件下的活性污泥样品,通过基因组DNA的提取、PCR扩增和DGGE分离,初步分析了各污泥样品的微生物群落多样性,结果表明,PCR-DGGE方法可以在一定程度上反映工艺以及操作条件对微生物群落结构的影响.通过DGGE反复分离纯化及割胶回收,DGGE检验为单一条带后进行测序并提交到GenBank数据库比对,结果表明,间歇好氧硫酸盐系统中优势菌株大多数为未培养细菌,来源于不同的污染环境,具有重要污染物降解的生态功能,其中包括与硫酸盐还原菌(Desulfobulbus propionicus)在系统发育上非常接近的菌株.     

以游泳馆污水为处理对象的SBR中不同污泥负荷下氨氧化菌群落的演变

为了研究污泥负荷对SBR系统内活性污泥微生物中氨氧化菌群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,对不同污泥负荷条件下SBR处理经投加葡萄糖调节的游泳馆污水的活性污泥中氨氧化菌进行了分析。研究结果表明,氨氧化菌的群落结构在不同污泥负荷条件下变化明显,在有机碳源较低的情况下生长旺盛,随着污泥负荷的提高其DGGE图谱条带数量逐渐减少,亮度逐渐减弱;在高污泥负荷环境下,氨氧化菌受到严重抑制,多样性指数大幅下降,并从系统中消失。SBR系统内氨氧化菌大部分为不可培养的变形菌,最常见的氨氧化菌是β变形菌中的亚硝化螺菌和亚硝化单细胞菌。     

应用于PCR-DGGE分析的活性污泥微生物总DNA提取方法比较

探讨适用于PCR-DGGE分析研究的活性污泥细菌和真菌的DNA提取方法。采用5种方法提取活性污泥微生物基因组DNA,以DNA纯度、含量、片段大小及DGGE条带多样性作为考察指标评价提取方法的优劣,以确定最佳实验方案。紫外吸收法和琼脂糖凝胶电泳结果显示,试剂盒法提取的DNA含量最低,其余4种方法获得的DNA含量无显著差异,就DNA纯度而言,试剂盒法最优;除高温裂解法对真菌细胞壁裂解效果较差外,其他4种方法均能不同程度地裂解细菌和真菌细胞;DGGE结果表明,高温裂解法获得的细菌条带最多,基于SDS的细胞裂解法得到的真菌条带最多。综合分析,高温裂解法更适合于活性污泥中细菌的PCR-DGGE分析,基于SDS的细胞裂解法则更适合于污泥中真菌的PCR-DGGE分析。     

溶解氧对晚期垃圾渗滤液短程硝化及微生物群落结构变化的影响

以上海老港垃圾填埋场配套污水处理设施中的污泥为菌种源,在序批式活性污泥反应器(SBR)中对晚期垃圾渗滤液进行短程硝化处理,调节SBR中溶解氧浓度,考察溶解氧对渗滤液短程硝化的影响,分析不同溶解氧条件下污泥微生物群落结构的变化.结果表明,低溶解氧(0.2～0.5 mg/L)条件下,SBR可以获得较高的短程硝化效率,反应17h后,SBR内亚硝态氮/氨氮(质量比)为1.05,氨氮负荷可达到1.5 kg/(kg·d)(以每千克污泥悬浮固体每天承担的氨氮计),出水可以满足后续厌氧氨氧化处理的要求.从污泥变形梯度凝胶电泳(DGGE)图谱中可以看出,SBR微生物群落结构中主要优势种有uncultured Bacteroidetes bacterium、uncultured bacterium、uncultured Candidatus Amoebophilus sp.等.随着溶解氧含量的升高,SBR内微生物群落结构的多样性有所升高,但溶解氧对微生物群落结构影响有限.     

施用城市污泥堆肥对土壤微生物群落结构变化的影响

在温室条件下进行了15周的盆栽实验，考察了施用城市污泥堆肥后，土壤中养分含量的变化规律，重点研究了施用城市污泥堆肥对土壤微生物群落结构变化的影响。实验发现，污泥堆肥能改善土壤养分，有机质和氮、磷含量得到显著提高。经PCR—DGGE分析，施肥1周后土壤中细菌和真菌的群落结构均发生了较大的变化。随着施肥时间的延长，细菌在富含有机质及氮、磷等养分的土壤环境下大量生长，多样性提高，其优势菌群属于γ变形菌、α变形菌和芽单胞菌；随着有机质的不断消耗，细菌的生长活性受到抑制，最终由于养分的缺乏，细菌种群多样性呈现小幅度的降低，优势菌群变为绿弯菌门、γ变形菌亚纲和厚壁菌门。对于真菌，其多样性指数在堆肥前3周逐渐提升，在第3～12周的监测中呈现相对稳定的变化趋势，优势菌群主要为座囊菌纲和散囊菌纲。     

污泥好氧颗粒化过程中氨氧化菌群结构的演替与分析

为了揭示颗粒污泥形成过程中氨氧化菌(AOB)群落结构的演替规律,利用变性梯度凝胶电泳(DGGE)、克隆测序和实时定量聚合酶链式反应(real-time PCR)等分子生物学技术对AOB群落的演替进行了研究。DGGE结果表明,污泥接种驯化期,AOB群结构的变化较为剧烈,在外加选择压的作用下,种群多样性迅速下降;但随着污泥颗粒化的完成而趋于稳定。测序结果表明,接种污泥中的大多数亚硝化单胞菌属因可快速适应工艺的淘洗过程而被保留在系统内,而亚硝化螺菌属逐渐被淘汰。real-time PCR结果表明,在经历了运行初期的淘洗后,AOB含量随着污泥浓度的提高而逐渐增长;但污泥的氨氧化活性随着污泥浓度的增长而降低。     

厌氧、好氧、厌氧/好氧交替状态对活性污泥性质的影响

实验采用活性污泥处理模拟印染废水,研究厌氧、好氧、厌氧/好氧交替3种条件对活性污泥性质的影响。3种实验条件下,污泥沉降比(SV%)均基本保持在18%~25%之间,污泥容积指数(SVI)保持在62~66 m L/g之间。活性污泥混合液中胞外聚合物(EPS)除EPSB-蛋白质浓度持续升高外,其余形式均呈现积累、达到最大值后下降的趋势,其中厌氧/好氧交替条件下EPS浓度最高而好氧条件下最小。在整个实验期间,活性污泥的脱氢酶活性基本呈上升状态,在厌氧/好氧间歇曝气条件下脱氢酶活性最高、好氧条件次之、厌氧条件最低,最终3种条件下的脱氢酶活性分别为31.27、26.63和24.37 mg/(g·h)。活性污泥中ATP浓度基本呈现先增加后减小、再趋于稳定的变化趋势。实验结果表明,活性污泥表观产率系数顺序是好氧厌氧厌氧/好氧交替运行,厌氧/好氧交替实现了系统内污泥减量,微生物的产率系数和能量状态密切相关。脱氢酶活性对污染物的降解影响明显,而实验条件下微生物能量状态和污泥减量并不对污染物降解产生影响。     

PCR-DGGE技术在水解酸化-缺氧法处理采油废水的微生物研究中的应用

采用PCR-DGGE技术直接从水解酸化和缺氧反应器中的污泥样品提取DNA,测定部分菌种的16S rDNA V3区片段序列,通过NCBI基因库比对,初步确定不同生物反应器内优势菌种,并进行了多样性指数分析.结果表明,水解酸化反应器中的生物膜与缺氧反应器中悬浮污泥微生物种群结构存在较大的差异,显示了在不同环境条件下,微生物群落结构的连续动态变化过程.     

Application of 16S rDNA-PCR amplification and DGGE fingerprinting for detection of shift in microbial community diversity in Cu-, Zn-, and Cd-contaminated paddy soils

Seven soils were sampled from farmland at different distances (0.01-5 km) from a copper and zinc smelter. The total contents of heavy metals in these soils ranged from 46 to 4895 mg Cu kg-1, 96 to 1133 mg Zn kg-1, and 6.9 to 28.8 mg Cd kg-1, respectively. The available fractions were highly correlated with total contents of the metals. In order to assess the impact of combined contamination of heavy metals on soil bacterial communities, denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of 16S rDNA sequence of bacteria in soil was used. Bacterial community structure was affected to some extent by heavy metals. The number of DGGE bands in soils increased with increasing distance from the copper and zinc smelter. Clustering analysis of the DGGE profiles showed that bacteria in the seven soils belonged to three clusters. Bacterial communities in three soils sampled at 0.01-0.60 km from the smelter belonged to one cluster, and those in three soils sampled at 0.8-1.2 km from the smelter belong to another cluster. Bacterial community in soil farthest from the smelter belonged to a single cluster. This study demonstrated that heavy metal contamination decreased both biomass and diversity of bacterial community in the soil.     

A preliminary in vitro assessment of GroBiotic-A, brewer's yeast and fructooligosaccharide as prebiotics for the red drum Sciaenops ocellatus

This study examined the effects of brewers yeast, fructooligosaccharide (FOS), and GroBiotic-A, a mixture of partially autolyzed brewers yeast, dairy components and dried fermentation products, on the intestinal microbial community of red drum, Sciaenops ocellatus. Gastrointestinal (GI) tracts were aseptically removed from three sub-adult red drum previously maintained on a commercial diet and placed in an anaerobic chamber. Intestinal contents were removed, diluted and incubated in vitro in one of four liquid media: normal diet alone, diet + 2% (w/w) GroBiotic-A, diet + 2% brewers yeast, and diet + 2% FOS. After 24 and 48 h of incubation at 25 degrees C, supernatants were removed for volatile fatty acid (VFA) analysis and DNA was extracted for denaturing gradient gel electrophoresis (DGGE) analysis. Polymerase chain reaction (PCR) was performed on a highly conserved region of M 16S rDNA and the amplicons were subjected to DGGE. The microbial community (MC) fingerprint was used to distinguish microbial populations. The intestinal contents incubated with GroBiotic-A had significantly (P<0.05) higher acetate and total VFA concentrations at 48 h compared to the other treatments. DGGE analysis demonstrated that the microbial community was significantly altered by Grobiotic-A and brewers yeast.     

SBR中SRT对总细菌群落结构的影响研究

为了揭示序批式反应器不同污泥停留时间(SRT)下总细菌群落结构的异同及SRT变化对总细菌群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)进行研究。通过克隆测序发现,不同的SRT条件下生物多样性和种群结构会有所差异,既存在各SRT条件下相同的优势菌群(Escherichia coli和Aeromonas sp.),也存在某些SRT下特有的优势菌群(Uncultured Peptostreptococcaceae),SRT为40 d时检测到以降解硫酸盐获得能源的优势微生物。研究还表明,SRT为40 d时多样性指数取得最大值,各SRT条件下微生物的种群相似性差别较大。     

环境微生态研究中的分子生物学新技术

随着分子生物学的发展,可用于环境微生态研究的新方法新手段不断出现.利用这些分子生物学技术,可直接对环境样品的总DNA进行分析,绕开菌株分离和培养瓶颈,可以最大限度地获得相关微生物的遗传信息,全面地分析环境中微生物的多样性.对目前广泛应用于环境微生态研究的分子生物学新技术,包括rDNA基因序列分析、变性梯度凝胶电泳、温度梯度凝胶电泳、单链构象多态性、限制性片段长度多态性、随机扩增多态性DNA、核酸杂交和DNA芯片等技术进行了综述.     

Variations of bacterial community structure in flooded paddy soil contaminated with herbicide quinclorac

The denaturing gradient gel electrophoresis (DGGE) method was applied to determine the relative genetic complexity of microbial communities in flooded paddy soil treated with herbicide quinclorac (3,7-dichloro-8-quinoline-carboylic acid). The results obtained showed a significant effect of quinclorac on the development of bacterial populations in soils contaminated with different concentrations of the herbicide at the early time after application. In general, however, the number of populations of the same soil sample treated with the same concentration of the quinclorac differed obviously with increasing incubation time within the early 8 weeks. The scale of differences in banding patterns-showed that the microbial community structures of the quinclorac-treated and non-quinclorac-treated soils were not significantly different after 21 weeks of incubation. Quantification, as demonstrated in this paper, was studied by establishing dose-response relationships. Significant pattern variations were quantified. Prominent DGGE bands were excised, cloned and sequenced to gain insight into the identities of predominant bacterial populations. The majority of DGGE band sequences were related to bacterial genera Clostridium, Sphingobacterium, Xanthomonas and Rhodococcus.     

有机碳源条件下厌氧氨氧化SBBR中的微生物多样性

研究有机碳源对SBBR厌氧氨氧化菌群等微生物的影响。采用16S rDNA序列与PCR-DGGE分析技术相结合的方法,对稳定运行的反应器内的活性污泥和生物膜样品,进行细菌多样性图谱分析,同时采用巢式PCR-DGGE技术对浮霉状菌属(Planctomycetes)细菌进行分析。结果表明,在有机碳源反应系统细菌条带数和多样性指数均高于无机系统,与活性污泥相比,生物膜表尤为明显。当进水不含有机碳源时,氨氧化细菌(ammonia oxidizing bacteria,AOB),厌氧氨氧化菌(anaerobic ammonia oxidizing bacteria,ANAMMOX)为优势功能菌;当进水含有机碳源时,系统中存在的AOB以亚硝化单胞菌(Nitrosomonas sp.)为优势菌群,同时存在反硝化菌,如索氏菌(Thauera sp.)以及厌氧氨氧化菌,它们共同作用完成N的去除。此外,与无机碳源系统相比,有机碳源的存在,有利于浮霉状菌的积累,但压缩了ANAMMOX的生存空间。本研究可为厌氧氨氧化工艺处理低C/N比有机废水提供了理论依据。     

Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE

A laboratory-scale biofiltration system, the rotatory-switching biofilter (RSB), was operated for 199 days using toluene as a model pollutant. The target gaseous pollutant for the biofiltration experiment was approximately 300 ppmv of toluene. Toluene removal efficiency (RE, %) was initially approximately 20% with a 247-ppmv concentration (0.9 g m(-3)) of toluene during the first 10 days. Although the RE decreased several times whenever nitrogen was consumed, it again reached almost 100% when the nitrogen source was in sufficient supply. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to assess the transformation ofmicroflora during operation of the biofilter The results based on a 16S rRNA gene profile showed that the microbial community structure changed with operation time. Although the microflora changed during the initial period (before day 40), transformation of the bacterial component was hardly observed after day 51. Statistical analyses of the DGGE profiles indicated that the bacterial community was almost unaffected by the environmental factors, such as adding ozone, high-level nitrogen supply, increase of loading toluene, and the shutdown of the RSB. The DGGE profile using tmoA-like genes, which encode proteins belonging to the hydroxylase component mono-oxygenases involved in the initial attack of aerobic benzene, toluene, ethylbenzene, and xylene degradation, confirmed the existence of toluene-degrading bacteria. There were at least four kinds of toluene-degradable bacteria having tmoA-like genes up to day 36, which decreased to two species after day 40. Sequence analysis after DGGE profiling revealed that Burkholderia cepacia, Sphingobacterium multivorum, and Pseudomonas putida were present in the biofilter. Only Alicycliphilus denitrificans was present throughout the whole operation period. In the initial stage of operating the RSB, many types of bacteria may have tried to adapt to the conditions, and subsequently, only selected bacteria were able to grow and to degrade toluene.