强化生物除磷系统的微生物种群及其表征技术*

详细介绍了强化生物除磷系统(enhancedbiologicalphosphateremoval,简称EBPR)中的微生物种群及其表征技术,提出了研究EBPR中微生物种群及其表征技术的发展方向。     

强化生物除磷系统主要微生物及其代谢机理研究进展

强化生物除磷(enhanced biological phosphorus removal,EBPR)工艺在废水除磷处理中应用广泛.主要功能微生物及其代谢机理的研究是有效调控EBPR工艺稳定运行与效能提升的基础.本文选取EBPR系统中最主要的两类微生物(聚磷菌和聚糖菌),从底物吸收机制、糖酵解途径、TCA途径的贡献以及聚磷菌和聚糖菌的代谢相似性等方面对这些微生物的代谢机理进行综述,评价了分子生物学技术在研究EBPR系统微生物学及其代谢机理方面的应用现状,在此基础上对EBPR系统今后的研究方向进行了展望.
     

微生物生态学研究方法进展

微生物培养及显微技术作为鉴定微生物种群的手段有很大的局限性,因为环境中大多数微生物处于“存活但不能培养”的状态。因此．不依赖于微生物培养的生物化学以及分子生物学方法正被广泛地用于微生物生态学研究。主要介绍了荧光技术。基于PCR的分析技术和PLFA等技术在表征微生物多样性研究中的某些进展。     

FISH技术在强化生物除磷中的应用

荧光原位杂交(FISH)是一种微生物生态学研究技术,在强化生物除磷(EBPR)过程中,用来鉴定系统中的优势微生物种群,直接观察其在污泥系统中的形态结构、分布状态,跟踪监测其各个阶段的动态变化,并将其量化。与DGGE/TGGE、SSCP、RFLP、DAPI染色等研究方法,可突破FISHj技术不能提供活性污泥内部种群多样性、检测"未知"微生物的研究局限,增加研究的准确性。发展探针检测水平、发现更有效的染色鉴定方法、与生理生化研究相结合将成为FISH技术在强化生物除磷过程中的发展方向。     

强化生物除磷系统的功能微生物研究进展

人类活动过程中排放的磷是导致水体富营养化的重要原因之一,因此,采取强化生物除磷(Enhanced biological phosphorus removal,EBPR)技术去除污水中磷,减轻对环境不利影响。由于具有经济、可持续的优点,EBPR系统在污水除磷中得到广泛应用,而体系中微生物群落组成合理、功能完整是EBPR系统高效稳定运行的关键所在。为了深入了解EBPR系统除磷机理和实现高效稳定运行,对系统中微生物群落结构和主要功能微生物进行了大量研究。EBPR系统中除了具有聚磷能力的聚磷菌(Polyphosphate-accumulating organisms,PAOs)外,还包括没有聚磷能力的非聚磷菌(non-PAOs),主要为聚糖菌(Glycogen-accumulating organisms,GAOs)和一些辅助细菌等。目前,发现与聚磷相关的功能微生物种类越来越多,研究最多的PAOs和GAOs分别为Accumulibacter和Defluviicoccus。PAOs和GAOs在不同的环境条件下存在竞争或合作关系,但是PAOs在特定条件下是否能够表现出GAOs的代谢特性这一问题还存在争论。除传统碳源、p H和温度等因素影响生物除磷外,外源污染物(如抗生素和重金属)对EBPR系统中功能微生物也产生影响。为了获得高效PAOs,传统分离方法、蓝白斑筛选法和人工构建工程菌的方法先后得到应用。现代分子生物学技术的发展为EBPR系统中功能微生物研究提供了先进可靠的技术手段,通过高效聚磷菌的构建实现高效除磷是未来提高实际污水中生物除磷效率的一个重要发展方向。     

多环芳烃(PAHs)微生物降解的原位表征方法

多环芳烃(polycyclic aromatic hydrocarbons,PAHs)是一类在环境中广泛存在的持久性有机污染物,微生物降解是去除环境中多环芳烃污染的主要途径。传统的有关PAHs微生物降解的研究主要依靠分离培养技术,难以准确认识PAHs微生物降解的原位过程及机制。近年来发展起来的原位表征方法可以在基因及单细胞水平研究PAHs在复杂环境中的微生物降解过程,能够原位表征具有PAHs降解功能的微生物及其功能基因和代谢活性,是阐明PAHs原位降解过程及分子机制的强有力的手段。该文综述了宏基因组技术(meta-genomics)、稳定同位素探针技术(stable isotope probe,SIP)、荧光原位杂交技术(fluorescence in situ hybridization,FISH)、拉曼光谱技术(Raman spectra)以及二次离子质谱技术(secondary ion mass spectrometry,SIMS)等原位表征技术在PAHs微生物降解研究领域的应用及其存在的问题和发展趋势等。PAHs微生物降解过程及机制的原位表征将为缓解与修复PAHs污染提供科学基础。     

生物浸矿反应器中的微生物种群结构及其中可培养微生物的特征

【目的】本文旨在了解生物浸矿反应器中的微生物种群结构及其中可培养微生物的特征。【方法】通过构建微生物冶金反应器中矿浆原样的16S rRNA基因文库,测定16S rRNA基因序列,分析矿浆中种群结构。同时在不同培养条件下,对样品进行富集培养,分离获得纯菌株;并对各个菌株的16S rRNA基因序列,生理生化特征及对不同矿物的氧化能力进行了分析。【结果】研究中所选生物浸矿反应器中主要的微生物物种有细菌:Leptospirillum sp.,Sulfobacillus sp.,Acidithiobacillus sp.,Spingomonas sp.及古菌Sulfolobus sp.,Ferroplasma sp.等菌属。同时分离出5株纯菌株,这些菌分别与Acidithiobacillus thiooxidans,Acidithiobacillus caldus,Acidithiobacillus ferrooxidans,Leptospirillum ferriphilum,Sulfobacillus thermosul fidooxidans相似。分离获得的菌株具有氧化硫或二价铁和不同硫化矿的能力。【结论】生物浸矿反应器是个微生物种类相对简单的生境,利用非培养和培养技术全面地了解生物浸矿体系中的微生物群落及其生理、浸矿特性,有利于洞察生物浸矿过程中微生物种群结构,强化控制种群组成及浸矿活性,从而提高生物湿法冶金的效率。     

DGGE和T-RFLP在堆肥微生物群落结构研究中的应用

对堆肥微生物种群分布及其动态变化的研究进行了分析,论述了分子生物技术中的变性梯度凝胶电泳和末端标记限制性片段长度多态性的原理和特点,以及用于研究堆肥微生物的群落结构演变规律,为分析和筛选堆肥中的微生物提供更加有效、快速的信息,促进堆肥技术的发展。     

rRNA技术及其在分子微生物生态上的应用

传统的基于微生物培养与纯种分离的技术所具有的局限性,以及分子生物学及其有关技术的长足进展,使微生物生态学的研究进入了分子的阶段.其中rRNA技术的建立、发展及其成功应用,为分子微生物生态和微生物系统分类学的研究掀开了崭新的一页.对rRNA分子技术的研究进展、以之为基础的主要方法及其在环境微生物研究中的应用,以及应用过程中所存在的一些潜在问题及其解决办法等作了详细综述.     

有关微生物固定CO2技术及其应用探索

本文首先介绍了固定CO2的意义及方法,然后分析了微生物固定CO2技术流程,接下来阐述了微生物种群及生物反应器类型,最后重点讨论了微生物固定CO2的应用(以微型藻类在CO2吸收与资源化中的应用为例),以期为同行提供有益借鉴与参考。     

Effect of nitrite from nitritation on biological phosphorus removal in a sequencing batch reactor treating domestic wastewater

Although nitrite effect on enhanced biological phosphorus removal (EBPR) has been previously studied, very limited research has been undertaken about the effect of nitrite accumulation caused by nitritation on EBPR. This paper focused on nitrite effect from nitritation on EBPR in a sequencing batch reactor treating domestic wastewater. Results showed that nitrite of below 10 mg/L did not inhibit P-uptake and release; whereas EBPR deterioration was observed when nitrite accumulation reached 20 mg/L. Due to P-uptake prior to nitritation, nitrite of 20 mg/L has no effect on aerobic P-uptake. The main reason leading to EBPR deterioration was the competition of carbon source. Batch tests were conducted to investigate nitrite effect on anaerobic P-release. Under sufficient carbon source, nitrite of 30 mg/L had no impact on poly-β-hydroxyalkanoate (PHA) storage; contrarily, under insufficient carbon source, denitrifiers competing for carbon source with phosphorus accumulating organisms resulted in decrease of PHA synthesis and P-release.     

An innovative membrane bioreactor (MBR) system for simultaneous nitrogen and phosphorus removal

Membrane filtration was integrated with a post-denitrification process to form an innovative membrane bioreactor (MBR) system for effective organic degradation and nutrient (N and P) removal. The system comprised of an aerobic tank, an anoxic tank, an intermediate sedimentation tank, and a membrane filtration tank. The sedimentation tank functioned not only as a rough settler for sludge–water separation before membrane filtration but also as an anaerobic chamber for P release. While half of the influent flowed into the aerobic tank, the other half was fed into the anoxic tank to favor the proliferation of phosphorus accumulating organisms (PAOs). The experiment was conducted continuously for about 430 days. With a short overall treatment time of less than 10 h for municipal wastewater, the MBR-based process could achieve the total organic carbon, total nitrogen, and total phosphorus removals of around 94%, 85%, and 87%, respectively. The growth and activity of PAOs in the MBR system were evidenced by the significant P release in the anaerobic chamber followed by the luxury P uptake in the membrane tank. With the DAPI and PAO_mix probe staining, the increases of PAOs and polyhydroxybutyrate (PHB) in sludge during the experiment were well observed under the fluorescent microscope.     

Assessment of a bioaugmentation strategy with polyphosphate accumulating organisms in a nitrification/denitrification sequencing batch reactor

Different alternative configurations and strategies for the simultaneous biological removal of organic matter and nutrients (N and P) in wastewater have been proposed in the literature. This work demonstrates a new successful strategy to bring in enhanced biological phosphorus removal (EBPR) to a conventional nitrification/denitrification system by means of bioaugmentation with an enriched culture of phosphorus accumulating organisms (PAO). This strategy was tested in a sequencing batch reactor (SBR), where an 8 h configuration with 3 h anoxic, 4.5 h aerobic and 25 min of settling confirmed that nitrification, denitrification and PAO activity could be maintained for a minimum of 60 days of operation after the bioaugmentation step. The successful bioaugmentation strategy opens new possibilities for retrofitting full-scale WWTP originally designed for only nitrification/denitrification. These systems could remove P simultaneously to COD and N if they were bioaugmented with waste purge of an anaerobic/aerobic SBR operated in parallel treating part of the influent wastewater.     

Effects of acetate and nitrite addition on fraction of denitrifying phosphate-accumulating organisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process

The effects of acetate and nitrite on the performance of sequencing batch reactors (SBRs) employing an anaerobic/aerobic/anoxic (AOA) process were investigated. Three types of SBR operations were used: sodium acetate addition at the start of anoxic condition for heterotrophic denitrification (Type 1); sodium acetate addition at the start of aerobic condition for anoxic phosphate removal by denitrifying phosphate-accumulating organisms (DNPAOs) (Type 2: conventional AOA process); and nitrite addition at the start of aerobic condition for inhibition of phosphate-accumulating organisms (PAOs) (Type 3). A track experiment shows that Type 2 led to the best performance of SBRs among the three types. An analysis by fluorescence in situ hybridization (FISH) revealed that nitrite addition decreased the ratio of PAOs with a decrease in phosphorus removal efficiency. The fraction of DNPAOs in Type 2 was the highest at 13%, indicating that Type 2 is suitable for the simultaneous nitrogen and phosphorus removal in the AOA process.     

Modeling and experimental study on the anaerobic/aerobic/anoxic process for simultaneous nitrogen and phosphorus removal: The effect of acetate addition

A mathematical model based on the simulation software AQUASIM was developed to validate an anaerobic/aerobic/anoxic (AOA) process that enables simultaneous nitrogen and phosphorus removal in a single reactor by adding external organic carbon to preclude excess aerobic phosphate uptake by polyphosphate-accumulating organisms (PAOs) and provide phosphate for denitrifying PAOs (DNPAOs). Aerobic batch tests after anaerobic phosphate release with different chemical oxygen demand (COD) concentrations indicated that the effect of COD concentration on the phosphate uptake preclusion could be expressed by a simple formula. The reduction factor reflecting the formula, which retards the aerobic phosphate uptake in the presence of COD, was added to the process rates of aerobic polyphosphate storage and PAOs growth in the model. The improved model, which included the reduction factor, reasonably matched the experimental result regarding aerobic phosphate uptake behavior whereas the model without it did not; thus, the former precisely predicts the AOA process behavior.     

Aerobic phosphorus release linked to acetate uptake in bio-P sludge: process modeling using oxygen uptake rate

The main processes involved in enhanced biological phosphorus removal (EBPR) under anaerobic and subsequently aerobic conditions are widely described in the literature. Polyphosphate accumulating organisms (PAO) are the organisms responsible for this process. However, the mechanisms of PAO are not fully established yet under conditions that differ from the classical anaerobic/aerobic conditions. In this work, we made a comparison between the behavior of PAO under classical EBPR conditions and its behavior when consuming substrate under only aerobic conditions. In addition, oxygen uptake rate (OUR) was measured in the set of experiments under aerobic conditions to improve the characterization of the process. A kinetic and stoichiometric model based on Activated Sludge Model No.2 (ASM2) and including glycogen economy (AnOx model), calibrated for classical anaerobic/aerobic conditions, was not able to describe the experimental data since it underestimated the acetate consumption, the PHB storage, and the OUR. Two different hypotheses for describing the experimental measurements were proposed and modeled. Both hypotheses considered that PAO, under aerobic conditions, uptake acetate coupled to PHB storage, glycogen degradation, and phosphorus release as in anaerobic conditions. Moreover, the first hypothesis (PAO-hypothesis) considered that PAO were able to store acetate as PHB linked to oxygen consumption and the second one (OHO hypothesis) considered that this storage was due to ordinary heterotrophic organisms (OHO). Both hypotheses were evaluated by simulation extending the AnOx model with additional equations. The main differences observed were the predictions for PHB degradation during the famine phase and the OUR profile during both feast and famine phases. The OHO hypothesis described the experimental profiles more accurately than the PAO hypothesis.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

Effect of pH on biological phosphorus uptake

An anaerobic aerobic laboratory scale sequencing batch reactor (SBR) was operated to study the effect of pH on enhanced biological phosphorus removal. Seven steady states were achieved under different operating conditions. In all of them, a slight variation in the pH value was observed during anaerobic phase. However, pH rose significantly during aerobic phase. The increase observed was due to phosphorus uptake and carbon dioxide stripping. When pH was higher than 8.2-8.25 the phosphorus uptake rate clearly decreased. The capability of Activated Sludge Model No. 2d (ASM2d) and Biological Nutrient Removal Model No. 1 (BNRM1) to simulate experimental results was evaluated. Both models successfully characterized the enhanced biological phosphorus removal performance of the SBR. Furthermore, BNRM1 also reproduced the pH variations observed and the decrease in the phosphorus uptake rate. This model includes a switch function in the kinetic expressions to represent the pH inhibition in biological processes. The pH inhibition constants related to polyphosphate storage process were obtained by adjusting model predictions to measured phosphorus concentrations. On the other hand, pH inhibition should be included in ASM2d to accurately simulate experimental phosphorus evolution observed in an A/O SBR.     

Short-term temperature effects on the anaerobic metabolism of glycogen accumulating organisms

Proliferation of glycogen accumulating organisms (GAO) has been identified as a potential cause of enhanced biological phosphorus removal (EBPR) failure in wastewater treatment plants (WWTP). GAO compete for substrate with polyphosphate accumulating organisms (PAO) that are the microorganisms responsible for the phosphorus removal process. In the present article, the effects of temperature on the anaerobic metabolism of GAO were studied in a broad temperature range (from 10 to 40 degrees C). Additionally, maximum acetate uptake rate of PAO, between 20 and 40 degrees C, was also evaluated. It was found that GAO had clear advantages over PAO for substrate uptake at temperatures higher than 20 degrees C. Below 20 degrees C, maximum acetate uptake rates of both microorganisms were similar. However, lower maintenance requirements at temperature lower than 30 degrees C give PAO metabolic advantages in the PAO-GAO competition. Consequently, PAO could be considered to be psychrophilic microorganisms while GAO appear to be mesophilic. These findings contribute to understand the observed stability of the EBPR process in WWTP operated under cold weather conditions. They may also explain the proliferation of GAO in WWTP and thus, EBPR instability, observed in hot climate regions or when treating warm industrial effluents. It is suggested to take into account the observed temperature dependencies of PAO and GAO in order to extend the applicability of current activated sludge models to a wider temperature range.     

Changes in respiratory quinone profiles of enhanced biological phosphorus removal activated sludge under different influent phosphorus/carbon ratio conditions

Changes in the microbial community of an enhanced biological phosphorus removal (EBPR) activated sludge system under different influent phosphorus/carbon (P/C) ratio conditions were investigated through evaluation of population respiratory quinone profiles. A total of 13 types of respiratory quinone homologs consisting of 3 types of ubiquinones (UQ) and 10 types of menaquinones (MK) were identified in this study. The dominant quinones were UQ-8 and MK-7 throughout the operational period. A higher P/C ratio (0.1) in the influent stimulated an increase in the mole fractions of UQ-8, MK-7, MK-8(H₄), MK-9(H₄) and MK-8(H₈), suggesting that actinobacterial polyphosphate-accumulating organisms (PAO) containing partially hydrogenated MK, mainly MK-8(H₄), were contributing to EBPR. However, when the P/C ratio gradually decreased from 0.1 to 0.01, the mole fractions of UQ-8 increased from 0.46 to 0.58, while MK-7, MK-8(H₂), MK-8(H₄), MK-9(H₄), MK-8(H₈) and MK-9(H₆) markedly decreased. These changes in the respiratory quinone profiles suggest that glycogen-accumulating organisms corresponding to some Gammaproteobacteria had become dominant populations with a decrease in actinobacterial PAO. On the other hand, increasing abruptly the P/C ratio to 0.1 further caused an increase in the mole fraction of UQ-8, indicating that Rhodocyclus-related organisms were important PAO.