A~2/O和SBR系统中微生物群落结构比较分析

利用16sRNA高通量测序手段对A~2/O脱氮除磷工艺和A/O-SBR除磷工艺中微生物群落结构和脱氮、除磷微生物丰度变化进行全面的分析。根据测序结果,共获得20个门、150个属微生物。微生物多样性分析结果显示,在两个系统中主要菌门为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、浮霉菌门(Planctomycetes)3个菌门,这3个门的细菌总数占微生物总数的80%左右。在属级水平上,A~2/O工艺的优势微生物为脱氮单孢菌属(Dechloromonas),丰度为10.00%。在SBR工艺中,陶厄氏菌属(Thauera)为优势菌属,其丰度为7.90%。Candidatus Accumulibacter是SBR好氧除磷系统中的主要除磷微生物,其相对丰度为2.80%。脱氮单孢菌属(Dechloromonas)、陶厄氏菌属(Thauera)为A~2/O工艺中主要脱氮微生物,Candidatus Accumulibacter为代表性的除磷微生物,其丰度分别为10.20%、3.70%和0.50%。     

中试厌氧氨氧化反应器的启动与调控

研究了中试厌氧氨氧化(Anaerobic ammonium oxidation,Anammox)反应器的启动性能。结果表明,以硝化反硝化污泥、短程硝化污泥、厌氧絮体污泥和厌氧颗粒污泥混合接种,经过255d的运行,可在常温下(5oC～27oC)成功启动中试Anammox反应器,反应器的基质氮去除速率可达1.30kg/(m3·d)。厌氧氨氧化是致碱反应,厌氧氨氧化成为反应器内的主导反应后,进水pH宜控制在厌氧氨氧化适宜范围的偏低水平(6.8左右)。亚硝酸盐既是Anammox菌的基质,也是抑制剂,控制进水亚硝酸盐浓度(13～36mg/L)有助于厌氧氨氧化反应。菌种是生物反应器的功能之源,向中试装置投加少量厌氧氨氧化污泥(投加比2%),可大大加速中试Anammox反应器的启动进程。     

利用PCR-DGGE分析有机物对厌氧氨氧化菌群的影响

为了研究有机物对Anammox菌群的影响,以及微生物与脱氮的关系,为工艺改进提供依据。使用SBR厌氧氨氧化反应器,从反应器不同TOC/NH_4~+-N阶段采集活性污泥样品,利用聚合酶链式反应—变性梯度凝胶电泳(PCR-DGGE)技术,分析了样品中微生物种群结构。结果表明反应器中主要微生物包含变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、厚壁菌门(Firmicutes)和绿菌门(Chlorobi);其中变形菌门(β-变形菌和γ-变形菌)为优势菌群。TOC/NH_4~+-N从0逐渐增加至2.0的过程中,反应器中的反硝化菌(变形菌门)不断增长,Anammox菌群在TOC/NH_4~+-N为0.4阶段得到最大程度的富集,此时反应器内部微生物多样性也最高;随着有机物含量增加,Anammox菌生长受到严重抑制,反应器微生物物种多样性也逐渐下降。荧光定量(qPCR)分析表明Anammox菌含量从1.30×10~(11) copies/mL下降至3.18×10~9 copies/mL,而DB含量从1.57×10~9copies/mL增加至3.74×10~(10) copies/mL。说明随着C/N的增加,反应器脱氮能力逐渐从Anammox过渡到反硝化过程。通过测定反应器内壁附着污泥,还发现其微生物丰度和含量均高于同时期反应器内部活性污泥样品,推测厌氧微生物菌群更适宜在静态基质生长。     

有机碳源下废水厌氧氨氧化同步脱氮除碳

为明确有机碳源胁迫下,厌氧氨氧化反应器的同步脱氮除碳规律及功能微生物群落结构的动态变化,采用成功启动的厌氧氨氧化UASB反应器,通过逐步提升进水有机负荷,探究有机碳源下废水厌氧氨氧化同步脱氮除碳。研究表明,当进水化学需氧量(Chemical oxygen demand,COD)浓度从172 mg/L升至620 mg/L,反应器维持较高的脱氮效率,氨氮和总氮去除率均在85%以上,并对COD具有平均56.6%的去除率,高浓度COD未对Anammox菌活性构成显著抑制作用。聚合酶链式反应和变性梯度凝胶电泳(PCR-DGGE)图谱和割胶测序结果表明,变形菌门Proteobacteria、浮霉菌门Planctomycetes、绿曲挠菌门Chloroflexi以及绿菌门Chlorobi等微生物共存于同一反应体系中,推测反应器内存在复杂的脱氮除碳途径。而且,代表厌氧氨氧化的部分浮霉菌门微生物能耐受高浓度有机碳源,在高有机负荷下依旧发挥着高效的脱氮作用,为反应器高效脱氮提供了保障。     

厌氧氨氧化颗粒污泥研究进展

厌氧氨氧化(Anaerobic ammonium oxidation,Anammox)工艺是一种新的生物脱氮技术。一经问世即得到人们青睐,现已成为废水脱氮的升级技术。厌氧氨氧化菌(Anaerobicammoniumoxidation bacteria,AnAOB)是Anammox工艺的功能之源。以颗粒污泥形态存在的AnAOB是Anammox颗粒污泥床脱氮系统的重要支柱。由于AnAOB生长缓慢且对环境条件变化敏感,Anammox脱氮系统不仅启动缓慢,而且运行极易失稳甚至崩溃。值得庆幸的是,AnAOB可自主选择、组合和固定功能菌群落而形成Anammox颗粒污泥,并通过其优良的重力沉降性能和高效的基质转化性能保障Anammox脱氮系统的持续工作。本文综述了AnAOB的种类和特性及Anammox颗粒污泥的组成、结构和功能,以期为Anammox工艺的优化和拓展提供参考。     

利用EGSB反应器富集高纯度厌氧氨氧化菌

以储存60 d的ANAMMOX污泥为种泥,采用EGSB反应器富集高活性、高纯度的ANAMMOX菌富集培养物,考察反应器的脱氮能力,同时采用分子生物学方法鉴定培养前后ANAMMOX富集培养物中的优势菌种及ANAMMOX菌的相对丰度。在反应器运行的185 d中,最高氮负荷达3.0 kg N/(m3·d),氨氮与亚硝酸盐氮的去除效率均大于85%。培养后ANAMMOX富集培养物中的优势菌种由待定荧光布罗卡地菌Candidatus Brocadia fulgid和待定巴西布罗卡地菌Candidatus Brocadia brasiliensis变为待定亚洲杰特氏菌Candidatus Jettenia asiatica。FISH结果表明ANAMMOX菌所占总细菌的相对丰度明显增加,由(57.69±4.79)%提高到(83.32±4.40)%。定量PCR结果证实了培养后的富集培养物内ANAMMOX菌的拷贝数由1.14×1011拷贝/g湿重提高到3.69×1011拷贝/g湿重。     

厌氧氨氧化启动过程Anammox菌富集规律和差异分析

为明确厌氧氨氧化反应器启动过程中Anammox菌的富集规律,采用荧光原位杂交(FISH)和实时荧光定量PCR(q-PCR)分析技术,对未添加填料、添加多面空心球以及添加竹炭的3个UASB反应器厌氧氨氧化启动过程中Anammox菌的增长规律进行分析。研究表明,Anammox菌的相对数量和绝对数量均随着启动时间呈逐渐递增趋势,在稳定运行阶段的第123天,无填料、多面空心球和竹炭反应器中Anammox菌分别占总细菌的23.3%、32.6%和43.7%,单位VSS污泥中Anammox菌16S rRNA基因拷贝数分别为(25.64±2.76)×107、(47.12±2.76)×107和(577.99±27.25)×107拷贝数g–1 VSS。竹炭反应器中Anammox菌最大生长率和最短倍增时间分别为0.064 d?1和10.8 d,最大生长率分别是无填料和多面空心球反应器的1.78倍和1.88倍。因此,填料添加特别是竹炭的添加可极大地促进Anammox菌的选择性生长和繁殖。FISH和q-PCR分析技术均适用于Anammox菌的富集规律研究,但因其检测目标存在差异,造成两者表征结果有所不同。     

污水处理系统中厌氧氨氧化菌分布及影响因素

基于厌氧氨氧化的污水生物脱氮工艺近年来发展迅速,污水处理系统中厌氧氨氧化菌的分布和多样性成为了重要的研究方向。目前,在污水处理系统中曾检测出多种厌氧氨氧化(Anaerobic ammonium oxidation,Anammox)菌,最常被检测出的是待定布罗卡地菌Candidate Brocadia和待定斯图加特库氏菌Candidate Kuenenia的Anammox菌,并且研究发现单一生境下往往只存在一种类型的Anammox菌,但是影响Anammox菌分布和多样性的因素与机制目前仍不明确。系统总结了污水处理系统中,不同工艺形式和运行条件下的Anammox菌分布情况,归纳分析了关键因素对Anammox菌分布的影响,包括底物浓度和微生物比生长速率、污泥性质与微生物生境、多重因素的联合作用和影响等。在此基础上,阐述了Anammox菌分布机制研究的工程意义,并对该领域的研究方向和思路进行了展望。     

生物电化学耦合厌氧氨氧化强化脱氮及其微生物群落特征

为探究生物电化学强化厌氧氨氧化(anaerobic ammonia oxidation,anammox)脱氮作用过程,采用双室微生物电解池(microbial electrolysis cell,MEC)富集电活性微生物,构建耦合厌氧氨氧化阴极的生物电化学系统。具体地,在外加0.2 V电压条件下改变不同总氮进水浓度于30°C进行暗培养批次实验研究,结合循环伏安法、电化学阻抗谱、高通量测序方法等多种表征手段研究了强化脱氮机理。结果表明,在初始总氮浓度分别为200、300和400 mg/L时对应获得了96.9%±0.3%、97.3%±0.4%和99.0%±0.3%的总氮去除率,且阴极电极生物膜表现出良好的电化学活性。高通量测序结果表明外加电压富集了除厌氧氨氧化菌以外的其他脱氮功能菌群:反硝化菌(Denitratisoma)、Limnobacter和氨氧化菌SM1A02和Anaerolineaceae、亚硝化菌(Nitrosomonas europaea)和硝化螺菌属(Nitrospira)等,这些具有电化学活性的微生物构成了体系的氨氧化胞外产电菌(ammonium oxidizing exoelectrogens,AOE)和反硝化电养菌(denitrifying electrotrophs,DNE),它们连同厌氧氨氧化菌Candidatus Brocadia构成了系统的脱氮微生物群落结构。AOE和DNE的种间直接电子传递作用协同厌氧氨氧化是强化系统总氮去除的关键原因。     

厌氧氨氧化膨胀床反应器的运行性能

试验研究了以竹炭为载体的厌氧氨氧化膨胀床反应器的运行性能.接种反硝化污泥,用模拟废水可成功启动该反应器:运行至144 d时,容积总氮去除率达到3.02 kg N/m3/d.这是国内文献报道的最高水平.动力学分析表明.这种反应器的最大容积总氮去除率可迭12.77 kg N/m3/d.具有很大的脱氮潜能.反应器的启动过程可分为菌体自溶、活性延滞和活性提高三个阶段.与此相应,污泥性状也从黄褐色絮状污泥变为棕灰色颗粒污泥和红色颗粒污泥.红色颗粒污泥以杆茵和球菌为主.厌氧氨氧化活性可达0.56mg TN/(mgprotein)/h,它们是反应器厌氧氨氧化功能的主要承载者.     

高通量测序分析多种典型生境中厌氧氨氧化细菌的多样性分布特征

【背景】厌氧氨氧化过程是氮素循环过程的重要途径之一,在氮素循环中发挥重要作用。先前的研究已经证实了厌氧氨氧化细菌存在于多种生境中,但对其多样性分布还没有系统的研究。【目的】对厌氧氨氧化细菌在不同类型生境中的多样性分布规律进行深入分析,充分展示其在不同生境中的群落结构特点,并揭示多样性分布与环境因素之间的关系。【方法】在建立厌氧氨氧化细菌16S rRNA基因序列数据库的基础上,运用高通量测序技术分析其在不同生境中的多样性分布特征。【结果】厌氧氨氧化细菌在红树林、海湾和河口生境中的多样性水平较高,而污泥和红壤的多样性水平明显较低。系统发育分析表明,这些生境中的厌氧氨氧化细菌主要由Candidatus Brocadia、Ca.Scalindua和未明确分类地位的菌属组成;从河流到红树林生态系统,随着盐度的增加,厌氧氨氧化细菌的优势种属由Ca. Brocadia转变到Ca. Scalindua,相关性分析也表明了盐度是导致不同生境中厌氧氨氧化细菌群落结构差异的主要因素。【结论】不同生境中存在不同的厌氧氨氧化细菌种群结构,环境条件的差异影响了厌氧氨氧化细菌的种群分布和系统演化。     

Dispersal and control of anammox granular sludge at high substrate concentrations

This paper reports about the dispersal and control of anammox granular sludge at high substrate concentrations. The results demonstrate that anammox granular sludge would turn into flocculent sludge when the substrate concentrations exceed the inhibitory threshold concentrations, with an apparent drop in the settling velocity of anammox sludge from 73.73 to 16.49 m/h. Moreover, the sludge was washed out of the reactor and a decrease in the nitrogen removal rate from 23.82 to 16.97 kg N/(m³/day) was observed. The dominant anammox bacteria in the granular and flocculent sludge were Candidatus Kuenenia stuttgartiensis; however, the contents of heme c and extracellular polymeric substances in the flocculent sludge were much lower than in the granular sludge. Furthermore, the chemical composition of extracellular polymeric substances was different. The high nitrite concentrations more than the inhibitory threshold concentrations were regarded as the reason for the observed granular sludge dispersal and deterioration in reactor performance. The apparent dispersed granular sludge and malfunction of reactor performance could be recovered by means of washing out the residual substrate from the reactor and then re-running the reactor from low substrate concentrations.     

Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor

Anammox process has attracted considerable attention in the recent years as an alternative to conventional nitrogen removal technologies. In this study, a column type reactor using a novel net type acrylic fiber (Biofix) support material was used for anammox treatment. The Biofix reactor was operated at a temperature of 25°C (peak summer temperature, 31.5°C). During more than 340 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m³/d with TN removal efficiencies reaching 81.3%. When the reactor was used for raw anaerobic sludge digester liquor treatment, an average TN removal efficiency of 72% was obtained with highest removal efficiency of 81.6% at a nitrogen loading rate of 2.2 kg-N/m³/d. Results of extracellular polymeric substances (EPS) quantification revealed that protein was the most abundant component in the granular sludge and was found to be almost twice than that in the sludge attached to the biomass carriers. The anammox granules in the Biofix reactor illustrated a dense morphology substantiated by scanning electron microscopy and EPS results. The results of DNA analyses indicated that the anammox strain KSU-1 might prefer relatively low nutrient levels, while the anammox strain KU2 strain might be better suited at high nutrient concentration. Other types of bacteria were also identified with the potential of consuming dissolved oxygen in the influent and facilitating survival of anammox bacteria under aerobic conditions.     

Nitrogen removal performance of a hybrid anammox reactor

The anaerobic ammonium oxidation (anammox) process has attracted considerable attention in recent years as an alternative to conventional nitrogen removal technologies. In this study, an innovative hybrid reactor combining fluidized and fixed beds for anammox treatment was developed. The fluidized bed was mechanically stirred and the gaseous product could be rapidly released from the anammox sludge to prevent washout of the sludge caused by floatation. The fixed bed comprising a non-woven biomass carrier could efficiently catch sludge to reduce washout. During the operation, nitrogen loading rates to the reactor were increased to 27.3 kg N/m³/d, with total nitrogen removal efficiencies of 75%. The biomass concentration in the fluidized bed reached 26-g VSS/L. Anammox granules were observed in the reactors, with settling velocities and sludge volumetric index of 27.3 ± 6.5 m/h and 23 mL/g, respectively. Quantification of extracellular polymeric substances revealed the anammox granules contained a significant amount of extracellular proteins.     

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Nitrogen removal via simultaneous partial nitrification,anammox and denitrification (SNAD) process under high DO condition

The simultaneous partial nitrification, anammox and denitrification (SNAD) process for treating domestic wastewater was investigated in a sequencing batch reactor (SBR). The SBR was operated with air flow rate of 500 L h^?1 at 30 °C. Domestic wastewater was used as influent and Kaldnes rings were used as biomass carriers. In the beginning, long aeration condition was implemented to cultivate nitrification biofilm. Afterwards, intermittent aerobic condition was conducted during the cycle operation. The influent organic matter loading rate was improved by reducing the aeration and mixing times. Consequently, when the SNAD biofilm reactor was fed with the organic matter loading rate of 0.77 (kg COD m^?3 d^?1), the bio-bubbles appeared in the reactor and the total inorganic nitrogen (TIN) removal efficiency decreased. After the organic matter loading rate decreased to 0.67 (kg COD m^?3 d^?1), the reactor showed excellent nitrogen removal performance. The TIN removal efficiency varied between 80 and 90 %, and the average TIN removal loading rate was 0.22 (kg TIN m^?3 d^?1). Additionally, the scanning electron microscope (SEM) observation confirmed that the anammox bacteria located in the inner part of the carriers. Finally, the microbial community analysis of 16S rRNA gene cloning revealed that the anammox bacteria on the carriers consisted of three main genuses: Candidatus Brocadia sp., Candidatus Brocadia caroliniensis and Candidatus Brocadia fulgida.     

Adaptation of a freshwater anammox population to high salinity wastewater

For the successful application of anaerobic ammonium oxidation (anammox) in wastewater practice it is important to know how to seed new anammox reactors with biomass from existing reactors. In this study, a new high salinity anammox reactor was inoculated with biomass from a freshwater system. The changes in activity and population shifts were monitored. It was shown that freshwater anammox bacteria could adapt to salt concentrations as high as 30 gl(-1) provided the salt concentration was gradually increased. Higher salt concentrations reversibly inhibited anammox bacteria. The nitrogen removal efficiency and maximum anammox activity of the salt adapted sludge was very similar to the reference freshwater sludge. Fluorescence in situ hybridization analysis revealed that the freshwater anammox species Candidatus "Kuenenia stuttgartiensis" was the dominant in both salt adapted sludge and freshwater sludge. These results show that gradual adaptation may be the key to successful seeding of anammox bioreactors.     

Anaerobic ammonium oxidation (Anammox) in immobilized activated sludge biofilms during the treatment of weak wastewater

This work studied the formation of molecular nitrogen by the microbial population of immobilized activated sludge of the domestic wastewater treatment plants (WWTP) that employ the technology developed by ZAO ECOS Company. The technology includes physicochemical water pretreatment and treated water recycling. A hard flexible fibrous brush carrier is used for the immobilization of microorganisms. The presence of both aerobic and anaerobic microorganisms and functioning of the methanogenic microbial community was shown in the biofilms developing on the carrier fibers and in suspended sludge. The high efficiency of nitrogen removal at a low C/N ratio was established to be due to the conjugated nitrification, denitrification, and anammox processes, whose functioning was demonstrated by laboratory cultivation methods and by studying the processes in batch and continuous reactors. Fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH) revealed bacteria belonging to the order Planctomycetales, particularly their anammox group. This work is the first evidence of the important role of the anammox process in the combined system of physicochemical and biological treatment of weak wastewater (BCDEAMOX).     

Reactivation of effluent granular sludge from a high-rate Anammox reactor after storage

In this study, effluent sludge from a high-rate Anammox reactor was used to re-start new Anammox reactors for the reactivation of Anammox granular sludge. Different start-up strategies were evaluated in six upflow anaerobic sludge blanket (UASB) reactors (R₁–R₆) for their effect on nitrogen removal performance. Maximal nitrogen removal rates (NRRs) greater than 20 kg N/m³/day were obtained in reactors R₃–R₅, which were seeded with mixed Anammox sludge previously stored for approximately 6 months and 1 month. A modified Boltzmann model describing the evolution of the NRR fit the experimental data well. An amount of sludge added to the UASB reactor or decreasing the loading rate proved effective in relieving the substrate inhibition and increasing the NRR. The modified Stover–Kincannon model fit the nitrogen removal data in the Anammox reactors well, and the simulation results showed that the Anammox process has great nitrogen removal potential. The observed inhibition in the Anammox reactors may have been caused by high levels of free ammonia. The sludge used to seed the reactors did not settle well; sludge flotation was observed even after the reactors were operated for a long time at a floating upward velocity (F_s) of greater than 100 m/h. The settling sludge, however, exhibited good settling properties. Scanning electron microscopy showed that the Anammox granules consisted mainly of spherical and elliptical bacteria with abundant filaments on their surface. Hollows in the granules were also present, which may have contributed to sludge floatation.     

Accelerating effect of hydroxylamine and hydrazine on nitrogen removal rate in moving bed biofilm reactor

In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N?H? and NH?OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L?1 of each NH?OH and N?H?, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO??. Various combinations of N?H?, NH?OH, NH??, and NO?? were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N?H? concentration (4.38 mg N L?1) present in these batches was 5.43 mg N g?1 TSS h?1, whereas equimolar concentrations of N?H? and NH?OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.