基于T-RFLP和因子分析的香蒲根际细菌群落研究

以北京市永定河王平湿地为研究对象,应用末端限制性片段长度多态性(T-RFLP)对王平湿地再生水补水口及其远离补水口的香蒲根际细菌和氨氧化细菌(AOB)的群落结构及多样性变化进行分析,在此基础上结合MiCA3比对、典范对应分析(CCA)和多元统计中因子分析(FA),解析湿地不同空间细菌群落功能特性的变化.结果显示再生水补水口细菌群落各多样性指数均明显低于远离补水口细菌群落,而氨氧化细菌群落则呈现相反趋势.基于传统的T-RFLP片段MiCA3比对、CCA和FA相结合的分析表明:对细菌群落,占再生水补水口细菌群落总丰度55.6%的菌群与150bp代表的鞘氨醇单胞菌(Sphingomonas sp.)具相似净化功能,并与总有机碳生物化学循环过程具密切关系,其次占再生水补水口上游细菌群落总丰度75.5%的菌群与81bp为代表的假单胞菌属(Pseudomonas sp.)和Geitlerinema sp.具相似净化功能,并与氮的生物化学循环过程具密切关系.占再生水补水口下游细菌群落总丰度68.7%的菌群与138bp代表的小单孢菌属(Micromonospora sp.)具相似净化功能,同时与重金属Cu、V、和Ti生物化学循环具密切关系.对氨氧化细菌,266bp受铵态氮影响较大,占再生水补水口氨氧化细菌群落总丰度65.5%的菌群与266bp代表的Nitrosomonas sp.具相似的生境特征,适合在高氨环境中生长;与58bp所代表的Nitrosospira sp.具相似功能特性菌群丰度在远离补水口样点中较前者呈现显著增加趋势,表征该类菌群适合在相对低氨环境中生长.     

污水处理工艺对氨氧化菌及细菌群落的影响

采用针对氨氧化菌(AOB)功能基因氨单加氧酶(amoA)的末端限制性片段长度多态性技术(T-RFLP)、克隆测序等方法,研究了北京市2个污水处理厂的4个污水处理系统中AOB的群落结构,同时采用针对16S rRNA基因的T-RFLP技术分析了总细菌的群落结构.T-RFLP指纹图谱分析表明,4个污水处理系统中AOB的优势限制性片段(T-RF)均为291bp和354bp,细菌的优势T-RF为115,117,166,455,465, 468,471,482,800,893bp等.说明污水处理工艺对系统中AOB及细菌的群落结构影响很小.对功能基因amoA的系统发育分析表明,4个污水处理系统中优势AOB均属于Nitrosomonas europaea cluster和Nitrosomonas oligotropha culster.     

点源炼油废水处理系统微生物多样性及代谢功能研究

为了探明点源炼油废水处理系统（简称"系统"）沿程微生物多样性及其活动规律,采用高通量测序技术开展废水微生物群落结构及代谢功能研究.结果表明:①各单元出水Shannon-Wiener指数和Ace指数基本呈上升趋势,均表现为进水 < 涡凹气浮池 < 斜板气浮池 < 调节池 < A/O池.②微生物群落组成显示,各单元出水微生物群落结构差异显著（Bray-Curtis ANOSIM=0.388 1,P=0.008）;进水中变形菌门（Proteobacteria）相对丰度最高,为90.55%.沿程各单元出水优势菌组成发生变化,其中,调节池、涡凹气浮池、斜板气浮池出水中Proteobacteria相对丰度分别降至54.90%、62.59%、52.89%,Epsilonbacteraeota相对丰度分别增至17.03%、29.60%、41.53%,厚壁菌门（Firmicutes）相对丰度分别增至26.13%、6.02%、2.33%;A/O池出水中Proteobacteria相对丰度降至39.69%,髌骨细菌门（Patescibacteria）和拟杆菌门（Bacteroidetes）相对丰度分别增至23.95%、15.65%.③db-RDA分析表明,化学需氧量（COD,P < 0.05）、氨氮（NH₃-N,P < 0.05）显著影响微生物群落结构,其中,NH₃-N去除率与Bacteroidetes（r=0.588,P < 0.05）呈显著正相关,COD去除率与Patescibacteria（r=0.530,P < 0.05）、Bacteroidetes（r=0.706,P < 0.01）均呈显著正相关.④KEGG基因数据库分析可知,各单元出水代谢机制相关基因相对丰度最高,均高于60%,其中,能量代谢相关基因相对丰度大小表现为进水>涡凹气浮池>调节池>斜板气浮池>A/O池;外源化合物生物降解相关基因在一级处理过程中相对丰度波动较小,均在3.00%以下,而生物处理后相对丰度迅速增至15.38%.研究显示:系统沿程微生物多样性基本呈上升趋势,且影响废水中微生物群落变化的最显著因素为COD和NH₃-N;生物处理后外源化合物生物降解相关基因占比迅速增加,表明生物处理单元存在潜在冲击风险.      

涠洲采油废水处理系统运行效果及微生物群落分析

涠洲终端处理厂选用ABR+SBR联合工艺处理采油废水,该系统运行稳定,处理效果好。进水ρ(COD)、ρ(石油类)分别为215~731,9~52 mg/L,系统处理后出水浓度为30~87,2~8 mg/L,去除率为83%~94%,78%~92%。废水中其余指标如S2-、SS和NH3-N去除率分别为99%、94%和70%~90%。利用分子分析法对微生物群落结构研究,表明进水较出水有更高的细菌丰度,出水较进水有更高的真菌丰度。推测活性污泥中Marinobacterium、Marinobacter和Thiomicrospira是系统中采油废水主要降解细菌。此外,真菌群落分布均匀主要为子囊菌门、担子菌门、接合菌门,兼性菌如Aspergillus、Alternaria、Fusarium、Blastobotrys及Meyerozyma在活性污泥中较为丰富,Aspergillus、Alternaria可能是潜在的降解真菌。     

废铁屑缓解硫化物抑制厌氧氨氧化脱氮性能的研究

在上流式厌氧污泥床反应器(UASB)中研究硫化物对厌氧氨氧化(Anammox)系统的长期抑制以及投加废铁屑后对系统性能的影响.结果表明:低浓度(<10mg/L)硫化物对系统无显著影响,当其浓度升至20,30,40mg/L时,使脱氮负荷(NRR)分别降低了13%,42%和51%;40mg/L硫化物使比厌氧氨氧化活性(SAA)降低了63%;Anammox细菌对硫化物抑制具有适应性;长期硫化物造成污泥颗粒结构恶化、上浮结块.投加废铁屑可以显著缓解硫化物抑制,使污泥沉降性能和系统脱氮效能快速恢复, 19d后NRR比无硫阶段提高20%(达4.43kg/(m³·d));SAA仅降低了16.8%.微生物群落表明,硫化物使Candidatus Kuenenia相对丰度从18.81%下降到7.31%,适应后丰度恢复,反应器底层丰度比顶层高出15倍;同时产生了反硝化菌(Arenimonas和Thiobacill).废铁屑投加后Candidatus Kuenenia丰度略有下降,顶层丰度高于底层;系统Thiobacillus丰度提高3.36倍,底层反硝化细菌的相对丰度上升.     

不同种植方式对亚热带红壤微生物多样性的影响

土壤微生物在推动土壤碳循环过程方面发挥着重要作用,然而种植方式的改变对土壤微生物多样性的影响机制还不十分清楚.本研究采集湖南省盘塘县长期定位试验站红壤稻田(PR)、旱地(UC)及水旱轮作(PR)这3种不同种植方式的土壤样品,采用末端限制性酶切片段长度多态性分析(T-RFLP)技术和实时荧光定量(RT-PCR)技术分析了土壤细菌16S rRNA基因的多样性和丰度,研究种植方式改变对土壤微生物数量、群落结构及其多样性的影响.结果表明,3种种植方式的土壤细菌16S rRNA基因数量(以干土计)为2.5×109~1.5×1010拷贝·g-1,与PR相比,UP和UC处理16S rRNA基因丰度显著下降(P0.05).同时,3种种植方式下土壤细菌的优势类群为变形菌(76、90和327 bp;相对丰度47%~53%)和绿弯菌(65 bp;相对丰度10%~12%).冗余分析表明种植方式改变了土壤理化性质,导致土壤细菌群落结构特征的显著变化,而土壤理化性质中有机碳和全氮含量是影响土壤细菌群落结构的主要因子.多样性指数分析(香农指数和均匀度指数)显示种植水稻的土壤细菌多样性最高,显著高于水旱轮作和旱地土壤.可见,种植方式的改变对土壤群落组成和数量造成了深刻的影响,而水稻种植是亚热带红壤可持续利用的一种有效方式,其更有利于土壤有机质的累积,土壤肥力及微生物多样性均较高.     

多壁碳纳米管长期作用对活性污泥系统的影响

为研究MWCNTs(multi-walled carbon nanotubes,多壁碳纳米管)对活性污泥长期作用的影响,建立3组(每组包括3个平行反应器)序批式活性污泥反应器,研究了2种质量浓度(1、20 mg/L)的MWCNTs长期(120 d)作用对活性污泥系统废水处理效果的影响,并采用Illumina MiSeq高通量测序技术研究MWCNTs长期作用后活性污泥中细菌群落结构的变化. 结果表明:20 mg/L的MWCNTs对生物脱氮效果存在明显的抑制作用,TN平均去除率由83.4%降至71.7%;1 mg/L的MWCNTs长期作用后TN平均去除率由83.7%降至79.7%. 1和20 mg/L的MWCNTs对磷的去除均有抑制作用,TP平均去除率分别由98.0%和98.2%降至52.0%和34.0%. Illumina MiSeq高通量测序结果表明,不同质量浓度的MWCNTs长期作用后降低了活性污泥中细菌群落的多样性. 同时,活性污泥中细菌群落结构也发生了变化,其中α-Proteobacteria和γ-Proteobacteria的丰度升高,Bacteroidetes的丰度降低. 1 mg/L的MWCNTs长期作用可致以Defluviicoccus和Micropruina为代表的一类聚糖菌(glycogen-accumulating organisms, GAOs)丰度显著升高,20 mg/L的MWCNTs长期作用可致16个核心属的丰度明显降低.      

转cry1Ab和epsps基因玉米C0030.3.5对土壤固氮细菌丰度和群落结构的影响

为评估转基因玉米种植对土壤氮周转功能微生物的潜在风险,以转cry1Ab和epsps基因玉米C0030.3.5(TM)及其亲本玉米DBN318(PM)为研究对象,于2015年拔节期、抽雄期、乳熟期、完熟期采集根际土和非根际土进行试验,并采用荧光定量PCR(quantitative real-time PCR,q PCR)和末端限制性片段长度多态性(terminal restriction fragment length polymorphism,TRFLP)技术分析土壤固氮微生物nif H基因丰度和多样性.结果表明,TM和PM根际土和非根际土固氮细菌nif H基因丰度随生长时期整体呈现先升高后降低的变化趋势,无论是根际土还是非根际土,同一生长时期2种玉米nif H基因丰度间差异均不显著.相关分析显示,土壤固氮细菌nif H基因丰度与有机质含量呈极显著正相关.T-RFLP结果表明,所获得的14种TRFs中,43 bp和155 bp片段所代表的固氮细菌为共有优势种群,无论是根际土还是非根际土,同一生长时期各T-RFs的相对丰度在TM和PM间差异同样不显著.土壤固氮细菌的Shannon指数和Evenness指数随生长期整体呈现出先升高后降低的变化趋势,无论是根际土还是非根际土,同一生长时期TM和PM的Shannon指数间及Evenness指数间均无显著差异.主成分分析(principal component analysis,PCA)表明TM和PM土壤固氮细菌群落结构组成无显著差异.冗余分析(redundancy analysis,RDA)显示土壤铵态氮和p H对固氮细菌群落结构组成影响显著.     

滇池浮游细菌群落组成的空间分布特征及其与环境因子的关系

为探讨滇池湖体浮游细菌群落组成的空间分布特征,并找出影响浮游细菌群落组成分布的主要影响因子,通过16S rRNA末端限制性片段长度多态性(T-RFLP)分析手段研究了滇池湖体夏季由北向南9个采样点浮游细菌的群落结构.结果表明,9个采样点共得到52个不同的片段(T-RFs),且每种片段至少代表一种细菌.各采样点浮游细菌群...     

DNA和cDNA水平对比研究施肥对稻田土壤细菌多样性的影响

依托长沙农业环境观测研究站不同施肥方式长期定位试验,直接从土壤中抽提总DNA和RNA,应用T-RFLP和RTPCR技术在DNA和c DNA水平对比研究施肥对亚热带稻田土壤细菌丰度和群落结构的影响.结果表明,施肥和秸秆还田显著改变了土壤细菌群落组成(DNA水平)和转录组成(c DNA水平)结构,且不同处理中DNA水平的T-RFLP图谱与cDNA水平相差很大.施肥和秸秆还田降低了土壤中存在的细菌多样性,对土壤中转录的细菌多样性没有影响.土壤16S rRNA基因丰度(DNA水平)平均是其转录丰度(c DNA水平)的337倍,与不施氮肥处理(N0)相比,平衡施肥(NPK)没有改变土壤细菌的数量,在平衡施肥的基础上进行秸秆还田增加了土壤细菌的数量,但高量与低量秸秆还田没有显著差异.RDA分析表明,稻田土壤中铵态氮含量是调控各处理土壤中存在及转录的细菌群落的关键因子.总体而言,不管在DNA还是c DNA水平,研究施肥对土壤细菌丰度的影响均是可行并可信的;但只有在c DNA水平开展研究,才能有效发现细菌群落对环境的适应性.     

Community dynamics of ammonia oxidizing bacteria in a full-scale wastewater treatment system with nitrification stability

To determine whether the functional stability of nitrification was correlated to a stable community structure of ammonia oxidizing bacteria (AOB) in a full-scale wastewater treatment plant, the AOB community dynamics in a wastewater treatment system was monitored over one year. The community dynamics were investigated using specific PCR followed by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amoA gene. The T-RFLP results indicated that during the period of nitrification stability, the AOB community structure in the full-scale wastewater treatment system was relatively stable, and the average change rate every 15 d of the system was 6.6%±5.8%. The phylogenetic analysis of the cloned amoA gene showed clearly that the dominant AOB in the system was Nitrosomonas spp. The results of this study indicated that throughout the study period, the AOB community structure was relatively stable in the full-scale wastewater treatment system with functional stability of nitrification.     

基于纯膜MBBR的CANON工艺稳定运行及抗冲击性分析

应用CANON-MBBR系统处理实际污泥厌氧消化脱水液,考察了系统的稳定运行控制策略。结果表明:稳定期间,系统出水ρ（NH₄+-N）稳定低于25 mg/L,去除率>96%;出水ρ（TIN）<70 mg/L,去除率>87%。系统维持纯生物膜运行,无须进行污泥回流与菌种的补投,实现了稳定的自养脱氮过程。运行期间,考察了水量、DO及温度对系统稳定性的影响,结果表明:在进水流量为80 m3/d（为平稳期1.33倍）、ρ（DO）高达2~4 mg/L和24 ℃低温条件下系统依然保持较高的脱氮性能。高通量测序结果表明,系统优势氨氧化菌（AOB）和厌氧氨氧化菌（AnAOB）分别为Nitrosomonas和Candidatus Kuenenia,其相对丰度均值分别为6.5%和30.2%,亚硝酸盐氧化菌（NOB）的相对丰度始终低于0.1%,NOB被成功抑制。悬浮载体生物膜实现了AOB和AnAOB的高效富集,系统较低的AOB丰度限制了系统脱氮能力的进一步提升。     

有机碳驱动的人工基质表面菌群结构组成及其动态变化

本实验采用平板计数和末端限制性片段长度多态性技术(T-RFLP),分析了有机碳源添加对凡纳滨对虾养殖系统中人工基质(筛绢网)表面生物膜菌群结构组成及其动态变化的影响。研究表明,当人工基质表面菌膜成熟时,实验组人工基质表面异养菌数量高于对照组,但没有显著性差异;弧菌数及弧菌占总异养菌的比例分别极显著低于对照组91.85%和96.98%(p0.01)。T-RFLP图谱分析表明,两处理组中126、432、434、436、485、487和488 bp等T-RF相对丰度变化显著;其中,29 d时实验组菌群种类丰富度指数和Shannon-Weiner指数分别高于对照组100%和33.9%。移动窗口分析表明,对照组菌群结构变化率△t为23.2%26.38%,高于实验组的12.4%0.3%。综上可得,有机碳驱动不仅能够促进异养菌的生长,竞争性抑制弧菌的增殖,而且可以提高养殖池塘中人工基质表面菌群结构的稳定性。     

Fe2+激活过硫酸盐耦合活性炭深度处理焦化废水

采用Fe²⁺激活过硫酸盐（PS）耦合活性炭处理焦化废水生化出水.在原水TOC为86.4mg/L,色度338倍的条件下,研究PS和Fe²⁺投加量,初始pH值等因素对处理效果的影响.结果表明：PS和Fe²⁺投加量分别为1.5和4mmol/L,不调节pH值（8.0）,反应60min,色度和TOC去除率可达87.17%和68.16%.经Fe²⁺/PS体系处理的废水采用A,B两种活性炭进行吸附处理,结果表明：B炭的吸附效果较好,且可去除Fe²⁺/PS体系残留的PS.B炭15g/L,反应120min时,出水色度为14倍,TOC 11.86mg/L.Fe²⁺激活PS氧化法耦合活性炭吸附深度处理焦化废水时,总色度去除率95.86%,总TOC去除率86.27%.对生化出水,Fe²⁺/PS体系出水和活性炭吸附出水进行三维荧光光谱扫描分析,结果表明：Fe²⁺/PS体系能氧化分解废水中部分类腐植酸物质,而活性炭吸附则可进一步去除了废水中残留的类腐植酸物质.     

Community analysis of ammonia-oxidizing bacteria in activated sludge of eight wastewater treatment systems

We investigated the communities of ammonia-oxidizing bacteria(AOB) in activated sludge collected from eight wastewater treatment systems using polymerase chain reaction(PCR) followed by terminal restriction fragment length polymorphism(T-RFLP),cloning,and sequencing of the α-subunit of the ammonia monooxygenase gene(amoA).The T-RFLP fingerprint analyses showed that different wastewater treatment systems harbored distinct AOB communities.However,there was no remarkable difference among the AOB TRFLP profiles from different parts of the same system.The T-RFLP fingerprints showed that a full-scale wastewater treatment plant(WWTP) contained a larger number of dominant AOB species than a pilot-scale reactor.The source of influent affected the AOB community,and the WWTPs treating domestic wastewater contained a higher AOB diversity than those receiving mixed domestic and industrial wastewater.However,the AOB community structure was little affected by the treatment process in this study.Phylogenetic analysis of the cloned amoA genes clearly indicated that all the dominant AOB in the systems was closely related to Nitrosomonas spp.not to Nitrosospira spp.Members of the Nitrosomonas oligotropha and Nitrosomonas communis clusters were found in all samples,while members of Nitrosomonas europaea cluster occurred in some systems.     

Flow characteristic and wastewater treatment performance of a pilot-scale airlift oxidation ditch

A pilot-scale airlift oxidation ditch using bubble diffuser and baffle as aerator was operated in a wastewater treatment plant (WWTP) to investigate its flow characteristic and wastewater treatment performance. Compared with the conventional oxidation ditch process, effective depth and oxygen utilization efficiency of this new process was improved by underwater aeration. Furthermore, it had a reversed velocity distribution, which decreased from the bottom to the top on vertical section. Velocity measurement showed that a velocity over 0.2 m/s at the bottom was sufficient to prevent sludge settlement during long term operation. Application of these concepts would save land area and energy consumption by about 25%–50% and 55%, respectively. In this new system, organic biodegradation and nitrification could be well achieved. Denitrification could occur steadily in the straight part by adjusting the airflow rate. An average TN removal rate of 63% was achieved with dissolved oxygen (DO) concentrations between 0.6 mg/L and 1.5 mg/L. The main pollutants in the effluent could meet the strictest discharge standard (COD<50 mg/L, NH₄ ⁺−N < 5 mg/L, and TN < 15 mg/L) in China now.     

吸附-全程自养脱氮(A-CANON)工艺处理实际市政污水

以上海某污水处理厂市政污水为研究对象,考察市政污水在常温（25℃）条件下先经吸附段（A段）去除回收部分碳源后利用全程自养脱氮（CANON）工艺处理的脱氮效果.A-CANON工艺稳定运行175个周期,A段水力停留时间（HRT）=20min,DO=2mg/L,COD去除率始终高于60%,出水C/N降至1.5左右;CANON段出水中COD及TN浓度分别低于50mg/L和15mg/L,NH₄⁺-N浓度一般低于5mg/L,基本满足城市污水厂一级A排放标准.16S rDNA高通量测序结果显示,采用A-CANON工艺后,CANON系统中的优势厌氧氨氧化菌属为Candidatus_Jettenia和Candidatus_Brocadia.其中,Candidatus_Jettenia的丰度由第85周期的1.79%增加到第175周期的13.51%,较同期丰度降低1.4%的Candidatus_Brocadia表现出更强的环境适应能力.研究结果表明采用A-CANON工艺可有效处理市政污水并回收其中碳源.为该工艺在市政污水处理中的应用提供了理论依据和技术支持,有望实现市政污水碳、氮处理的可持续性.     

Bioaugmentation and quorum sensing disruption as solutions to increase nitrate removal in sequencing batch reactors treating nitrate-rich wastewater

Bioaugmentation of denitrifying bacteria can serve as a promising technique to improve nutrient removal during wastewater treatment. While denitrification inhibition by bacterial quorum sensing (QS) in Pseudomonas aeruginosa has been indicated, the application of bacterial QS disruption to improve nitrate removal from wastewater has not been investigated. In this study, the effect of bioaugmentation of P. aeruginosa SD-1 on nitrate removal in sequencing batch reactors that treat nitrate rich wastewater was assessed. Additionally, the potential of a quorum sensing inhibitor (QSI) to improve denitrification following bacterial bioaugmentation was evaluated. Curcumin, a natural plant extract, was used as a QSI. The chemical oxygen demand (COD) and initial nitrate concentration of the influent were 700±20 mg/L and 200±10 mg/L respectively, and their respective concentrations in the effluent were 56.9±3.2 mg/L and 9.0±3.2 mg/L. Thus, the results revealed that bioaugmentation of P. aeruginosa SD-1 resulted in an increased nitrate removal to 82%±1%. Further, nitrate was almost completely removed following the addition of the QSI, and activities of nitrate reductase and nitrite reductase increased by 88%±2% and 74%±2% respectively. The nitrogen mass balance indicated that aerobic denitrification was employed as the main pathway for nitrogen removal in the reactors. The results imply that bioaugmentation and modulation of QS in denitrifying bacteria, through the use of a QSI, can enhance nitrate removal during wastewater treatment.     

高浓度食品添加剂废水中试研究

根据食品添加剂废水水质变化大,成分复杂特点,提出了"水解酸化—接触氧化—臭氧催化氧化—曝气生物滤池(BAF)"的组合工艺。废水COD从进水2000～7000mg/L降到100mg/L以下,最低为33mg/L,排放水质达到国家排放标准。水解酸化系统使废水平均COD从5290mg/L降到2323mg/L,并使大颗粒难降解分子部分转化为小颗粒可降解分子,为后续的接触氧化系统处理提供良好的条件,接触氧化出水平均COD为268mg/L。接触氧化出水含较多难生物降解有机物,经O3氧化预处理后在COD下降45%的情况下其BOD5/COD由0.3升为0.44,更易于生化降解。废水经曝气生物滤池平均出水COD为66mg/L。中试研究表明,水解酸化系统和臭氧催化氧化(负载MnO2的陶粒为催化剂)-曝气生物滤池深度处理系统是该工艺处理高浓度废水稳定达标的关键。