附录毕业设计(论文)外文资料翻译学院(系)：资源与环境工程学院专业：环境工程姓名：学号：外文出处：http://protein.bio.msu.ru/biokhimiya/ontents/v65/full/65030405.html附件： 1.外文资料翻译译文；2.外文原文。
微生物中多聚磷酸盐细菌加强生物废水中清除磷的能力摘要活性污泥处理工艺在厌氧和有氧（厌氧—好氧法）环境交替进行方法可以提高的废水中磷的去除效果（EBPR）。
据了解，聚磷菌（PAB）在厌氧—好氧法中发挥重要作用。
本文对微生物的新陈代谢和群落结构描述有限，主要突出在EBPR过程中的选择作用。
微生物在厌氧—好氧法中，碳源丰富的厌氧环境和碳源缺乏的好氧环境交替进行，促进了聚磷菌重要的新陈代谢特征。
其中包括有机质的吸收，以及把它们转化为细胞内聚磷菌自身储存的PHA和水解产物，并在厌氧条件下释放能量。
假设细胞内神经的功能是作为调节器，调节细胞的氧化还原平衡。
能另储存有助与聚磷菌在厌氧环境中维持氧化还原平衡，吸收各种类型的有机质，增强微生物的选择功能。
聚磷菌不能由其他物质组成，各种各样的细菌除外。
要确定EBPR工艺中微生物群落的结构，需要通过分子技术细心观察在各种EBPR中，每一种聚磷菌的活动情况，因为许多聚磷菌都是不可用的培养基。
关键词：活性污泥厌氧—好氧法生态学生物加强清除磷酸盐微生物群落聚磷菌废水处理工艺当过量的含磷废水排入不外流的水体，湖泊或内陆海水时会造成水体富营养化。
（海藻过量生长繁殖）要在污水排入水体之前去处水中的磷。
厌氧、好氧条件交替控制活性污泥法已经成功的用于提高水体中磷的去处效果。
这种厌氧好氧交替运行的工艺已经得到普遍运用，在厌氧段、好氧段池体的空间布局以及利用设备的污泥回流系统等方面有显著效果。
例如这种被称为EBPR的厌氧—好氧或厌氧—缺氧过程。
据研究显示，聚磷菌在EBPR厌氧好氧法中具有重要作用。
EBPR要实现高而稳定的性能，必须保持聚磷菌在系统中的活性。
基本的厌氧—好氧法的图表可以说明其中的问题。
这一过程的特点是结构上存在一种厌氧阶段，保持绝对厌氧条件,没有氧气,也没有no2-/no3-为活性污泥细菌提供电子受体。
有机质的供应一部分来自进入厌氧段的污水，一部分是反应器中回流污泥补充碳源。
在EBPR过程中，加快厌氧段有机质的吸收率是细菌得到微生物的关键。
这种PAB繁殖机制可以如下表述。
通常，在厌氧阶段活性污泥向污水中释磷，同时吸收有机质。
在后期的好氧段，吸收的磷，远大于在厌氧段前期释放的磷。
污水中的磷被去处了，它作为一种物质积累到细胞里。
多聚磷酸盐是一种高能化合物，它水解能为细胞多种生化反应提供足够的能量。
在厌氧阶段，多聚磷化物的水解使PAB获得足够的能量以满足它们吸收有机质。
没有电子受体（氧，NO2-/NO3-）好氧细菌和反硝化细菌没有足够的能量利用有机质，也不能完成PAB的利用。
因此采用厌氧段使PAB具有优势，更好的处理污泥中的磷。
处理系统中的过量污泥并收集含高浓度磷的污泥，这样可以提高除磷效率。
数量极少的纯培养基在EBPR中扮演重要角色。
EBPR 中新陈代谢方面的研究主要是基于对浓集的混合培养基的研究而不是纯培养基。
这方面的不足就是缺乏准确的有关EBPR的微生物学和生物化学方面的资料。
因此，EBPR中PAB的微生物学变的不容易理解。
EBPR工艺中聚磷菌的碳代谢虽然厌氧—好氧法对于EBPR从工程角度来说已经是成熟的工艺方法，但它还不能清楚的解释一些微生物方面的定义在微生物的新陈代谢过程中，厌氧段通过废水中细菌的酶化作用完成了碳化合物的吸收。
由于污泥在厌氧条件下完成了和碳化合物的充分接触，生物体能更有效的利用碳质，在厌氧环境中占据优势。
因此，在厌氧条件下，PAB能实现对碳质的高速吸收的原因是我们一直关注的重要课题。
据了解，短链脂肪酸醋酸有利于EBPR中碳的来源，并且在EBPR中新陈代谢已经作为碳质的模型正在进行研究。
在这项研究上有一个决定性问题，就是事实上没有一个细菌可以从EBPR工艺中孤立起来，来显示EBPR污泥的主要特征。
任何孤立的纯文化每一个细菌在掩样杨。
这就是EBPR中的微生物被研究原因。
这种高浓度PAB培养基通常从模拟实验获得，模拟厌氧—好氧法处理废水。
在一组醋酸作为碳源的厌氧实验中，含高浓度PAB的活性污泥利用短链迅速吸收醋酸，在细胞内累计PHAs释放磷。
吸收的醋酸作为PHs转化和积累。
据发现在高浓度PAB中PHAs的积累由4部分组成3HB, 3HV, 3H2MB,和3H2MV。
分析这些PHAs的化学成分并证明是由上述四个单位组成。
至于碳水化合物，有人证明了它存在于厌氧—好氧活性污泥中，当醋酸作为碳源被吸收时，高浓度PHAs在厌氧段形成。
醋酸转化为PHAs需要减少电能，因为PHAs比醋酸不易合成。
为了解释在没有电子受体情况下减少电能这个过程，Mino 和Arun提出一个假设模型。
该模型中，在假设降低PHAs能量情况下，厌氧环境中存储的乙酰部分氧化为二氧化碳。
这种模式现在被称为Mino模型,其相关的一些研究者已证实，理论化学计量学根据模型依照显示能定量地解释通过PAB 污泥将醋酸盐和糖朊转换成PHA ，成功地采用了类似的概念来解释在EBPR中厌氧吸收率问题。
EBPR中厌氧碳新陈代谢模型另一个假说是由Matsuo、Comeau和 Wentze提出来的。
根据这种假说，TCA循环假设在厌氧条件下进行，把一部分醋酸氧化成二氧化碳并减少能量。
这种模式通常只在厌氧或好氧环境下进行循环。
对于这一矛盾的热力学理论，人们已经在厌氧或好氧环境中发现完整的TCA循环。
这些微生物利用硫元素和电子受体通过氧化醋酸完全转化二氧化碳。
据认为,这种情况的产生主要是要求减少能量生成代谢，就像Mino模型；而不是TCA循环那种预言。
原因如下：（1）这种理论能很好地解释实验观察到醋酸厌氧吸收率的现象，通过高浓度PAO，PHA的形成、乙二醇的应用、二氧化碳的生成。
（2）13C示踪实验器材的使用指出：醋酸通过厌氧污泥吸收的不是二氧化碳,因此不会通过循环进行代谢。
（3）实验用13C-器材,显示乙二醇转化为厌氧代谢的淤泥。
另一方面,有证据表明有可能介入的局部TCA循环发电,减少电能是在EBPR 厌氧阶段。
即13C的碳被转化高浓度PAB，醋酸-污泥浓缩被认为是绝对厌氧条件下释放二氧化碳。
迄今为止,这是唯一可能的实验结果显示了运行周期迈进的阶段厌氧Ebpr的过程. 循环的功能迈进的碳排放源的厌氧吸收率以及对微生物的筛选过程Ebpr有待进一步调查.EBPR的过程中,受到其他微生物碳厌氧环境和丰富的碳有氧环境恶劣. 这一交替的、综合和退化三种形式临时医院引起循环和新陈代谢,是通过这些微生物完成的。
这种微生物循环是能量的消耗，而不是微生物的能源利用效率。
然而，这种微生物循环使PAB在厌氧—好氧环境中进行选择。
如何解释这一规定在细胞循环代谢是由Pramanik发现的。
这一模式包含了一整套涉及细胞代谢途径和能源需求及高分子合成代谢物如何运输并跨越细胞膜.模型不仅支持假设，还提供了生物代谢途径,以及能源供应,而且还表明,在代谢途径中规则成立。
强化社会结构生物学微生物磷清除过程不动杆菌首次作为PAB被提出来，很少有研究人员质疑不动杆菌是否仅仅是EBPR中的一种细菌。
它有可能被认为高磷EBPR淤泥清除能力是一组由微生物,试图找出几个不动杆菌以外生物体。
现在,新的强有力的工具的运用,对微生物体结构的分析,了开发和利用EBPR淤泥。
其中化学分析方法与分子分析与方法,如荧光在原地交错(渔)、图书馆克隆方法、热梯度电泳(DGGE)、终端限制碎片长度白细胞(T生物)等。
高EBPR浓度污泥的微生物多样性已成功利用这种新技术。
分子分析适用于活性污泥结构的特点分析，醌生物样品的种类数量可确定,应当明确反映研究样本形态组成。
有人建议由几个不同EBPR污泥组织，醌最丰富、Q-8,仅占总数约PAB污泥的31%（磷含量1.94、60mG悬浮固体);第二个最丰富的人,Q-10,占8.5%; 第三、MK-8(H4)、6.5%。
换句话说,有几个不同污泥微生物群体,已确认的其他研究人员也用它，T-样品的分离,PCR-16S更直接表明不同的人口，数量约19至24年各主要见于高度PAB污泥浓缩。
（磷含量、悬浮固体12%）。
Dgge 的技巧也显示分离,扩大碎片rDNA和EBPR淤泥中的一些主要的DNA序列不同的碎片,暗示研究Ebpr结构多样性。
这些成果有力地表明,没有一个是PAB或基因型数量有限,但也会涉及各类细菌。
Bond应用PCR克隆启动两种活性污泥,高磷清除绩效果以及典型的新陈代谢,PAB等。
他们发现这个组织数量相当惊人，高磷污泥比低磷污泥大幅度提高了。
这一结果显示,有特定集团作用. 然而,只有14%的被占领，基因总数在少数的高磷污泥。
现在还不能确定这能否为观察到高磷清除绩效。
讨论之前,有报道EBPR结构中有一种压倒优势(细菌总数81%)。
就目前而言,这是唯一的一个案例,主要是细菌的主要表现是EBPR负责。
用DAPI进行双重染色与rRNA的探针，针对不同对象确定细菌组繁殖在原地。
因此,在检验污泥时这两个群体被认为在累积磷。
报告说,阳性菌G+C高含量DNA扮演重要角色,因为较高EBPR发生这种细菌组发现了一个克隆EBPR的过程。
大多数基因阳性菌具有很强的DNAG+C 含量，依据实验样品的rDNA碎片从高浓度-污泥浓缩(磷含量,12%的悬浮固体)、污泥很低磷酸盐含量(2%悬浮固体)。
认为阳性菌具有很强的DNAG+C的结构不只是PAB的重要组成部分。
醌分析使用方法,该市污水处理厂污泥运作模式相类似,不论对方采取何种过程污泥。
从淤泥中EBPR程序和常规程序分子形态十分相似。
比较不同启动模式醌淤泥建议采用的厌氧阶段进入，全面启动常规污泥过程不会导致大量细胞变化。
上述这些结果又会导致下述结论:细胞拥有独特的新陈代谢特点,把生物和微生物群体分开。
最可能的阿尔法-、试用、伽玛射线的类别和阳性菌具有很强的DNAG+C的内特性。
展望未来这次审查显示,PAB不是由少数受限制物质组成,但也会转化成各类细菌。
在EBPR中细菌的种类不同,负责功能不同。
在EBPR过程中，明确界定微生物Ebpr社会结构和过程的机制来描述PAB生态选择，在研究加强和行为发生个别种类对EBPR的需要。
因为许多PAB似乎是不可能的结构,只有分子方法能实现这些目的。
这可能意味着,新陈代谢的关键基因的EBPR常见细菌不同。
最有趣最重要的是确定这种基因并且找出它是怎样的规则。
Microbial Selection of Polyphosphate-Accumulating Bacteria in Activated Sludge Wastewater Treatment Processes for EnhancedBiological Phosphate RemovalAbstract:Activated sludge processes with alternating anaerobic and aerobic conditions (the anaerobic-aerobic process) have been successfully used for enhanced biological phosphate removal (EBPR) from wastewater. It is known that polyphosphate-accumulating bacteria (PAB) play an essential role for EBPR in the anaerobic-aerobic process. The present paper reviews limited information available on the metabolism and the microbial community structure of EBPR, highlighting the microbial ecological selection of PAB in EBPR processes. Exposure of microorganisms to alternate carbon-rich anaerobic environments and carbon-poor aerobic environments in the anaerobic-aerobic process induces the key metabolic characteristics of PAB, which include organic substrate uptake followed by its conversion to stored polyhydroxyalkanoate (PHA) and hydrolysis of intracellular polyphosphate accompanied by subsequent Pi release under anaerobic conditions. Intracellular glycogen is assumed to function as a regulator of the redox balance in the cell. Storage of glycogen is a key strategy for PAB to maintain the redox balance in the anaerobic uptake of various organic substrates, and hence to win in the microbial selection. Acinetobacter spp., Microlunatus phosphovorus, Lampropedia spp., and the Rhodocyclus group have been reported as candidates of PAB. PAB may not be composed of a few limited genospecies, involve phylogenetically and taxonomically diverse groups of bacteria. To define microbial community structure of EBPR processes, it is needed to look more closely into the occurrence and behavior of each species of PAB in various EBPR processes mainly by molecular methods because many of PAB seem to be impossible to culture.KEY WORDS:activated sludge, anaerobic-aerobic process, ecological selection, enhanced biological phosphate removal (EBPR), Lampropedia, microbial community, (PHAs), polyphosphate-accumulating bacteria, wastewater treatment Phosphate can cause eutrophication (extraordinary growth of algae) when it is excessively discharged into closed natural water bodies like lakes and inland seas. To control eutrophication, phosphate removal from wastewater is often required before wastewater is discharged to the receiving water bodies. Activated sludge processes with alternating anaerobic and aerobic conditions have been successfully used for enhanced biological phosphate removal (EBPR) from wastewater. This anaerobic-aerobic alternation can be achieved either by spatial configuration of anaerobic and aerobic zones in series in continuous flow systems with sludge recycle or by temporal arrangement of anaerobic and aerobic periods in sequence batch reactors. Such EBPR processes are referred to as the anaerobic-aerobic oranaerobic-oxic process. It has been shown in previous studies that polyphosphate-accumulating bacteria (PAB) play an essential role for EBPR in the anaerobic-aerobic process. To achieve high and stable EBPR performance, it is essential to maintain PAB in the system.A basic configuration of the anaerobic-aerobic process is schematically shown in Fig. a. This process is structurally characterized by the presence of an anaerobic stage in which absolute anaerobic conditions are kept with neither oxygen nor NO2-/NO3- available as electron acceptor for activated sludge bacteria. Organic substrates are supplied from influent wastewater into the anaerobic stage and the return sludge comes into contact with the carbon source only in the anaerobic stage. Faster uptake of organic substrates in the anaerobic stage is the key for bacteria to win in the microbial selection in the EBPR process. The mechanism of proliferation of PAB can be described as follows. It is typically observed in the anaerobic stage that the activated sludge releases Pi to the bulk solution with concomitant uptake of organic substrates. In the subsequent aerobic stage, it takes up more Pi than has been released in the previous anaerobic stage. The Pi removed from the wastewater is accumulated in the cell as polyP. Polyphosphate is a high-energy compound and its hydrolysis can supply energy to various biochemical reactions in the cell. In the anaerobic stage, the hydrolysis of intracellular polyP enables PAB to obtain the energy they need to take up organic substrates. Without electron acceptors (oxygen, NO2-/NO3-), aerobic bacteria and denitrifying bacteria are unable to obtain the energy required for the utilization of organic substrates, and they are thus unable to compete with PAB. Therefore, the introduction of the anaerobic stage leads to the precedence of PAB and to a rise in phosphorus content of the sludge. By withdrawing the phosphorus-rich sludge from the system as excess sludge, high phosphate removal efficiency can be achieved.Fig. 1. a) Basic concept of anaerobic-aerobic process for EBPR. b) Behavior of PO4-P, orthophosphate present in the bulk solution; glycogen, glycogen stored in the cells; PHA, polyhydroxyalkanoates stored in the cells.Although the anaerobic-aerobic process for EBPR is an established process from an engineering point of view, it has not been clearly defined in microbiological terms. For example, the phylogenetic or taxonomic groups responsible for EBPR have not been identified, and general structures of the EBPR microbial community have not been successfully described yet. Very few pure cultures have been isolated as candidates of PAB playing a key role in EBPR processes. Studies on metabolic aspects of EBPR have been mainly done based on enriched mixed cultures but not on pure cultures. This has resulted in lack of definitive and conclusive informationabout the microbiology and biochemistry of EBPR. Thus, the mechanism of microbial ecological selection of PAB in EBPR processes has been understood very poorly. The present paper reviews limited information available on the metabolism and the microbial community structure of EBPR, highlighting the selection of PAB in EBPR processes.CARBON METABOLISM ADOPTED BY POLYPHOSPHATE- ACCUMULATING BACTERIA IN EBPR PROCESSES In terms of microbial metabolism, the anaerobic stage involves the uptake of organic substrates from wastewater by bacteria. Since the sludge comes into contact with organic substrates under anaerobic conditions, organisms that can utilize organic substrates more rapidly in an anaerobic environment gain precedence. Therefore, the reason why PAB can achieve a very high rate of organic substrate uptake under anaerobic conditions has been a major subject of concern. It has been well known that short chain fatty acids like acetate are favorable carbon sources for EBPR, and acetate metabolism has been intensively studied as a model carbon metabolism substrate in EBPR. A critical problem in such studies lays in the fact that none of the bacteria isolated from EBPR processes have exhibited all the key characteristics of the EBPR sludge and that any isolated pure cultures had never been verified to be primarily responsible for EBPR in an anaerobic-aerobic system until recently . This is the reason that metabolic aspects of EBPR have been studied using mixed cultures enriched with PAB. Such PAB-enriched cultures have usually been obtained from lab-scale activated sludge reactors simulating the anaerobic-aerobic process fed with synthetic wastewater.In anaerobic batch experiments with acetate as the carbon source, the activated sludge enriched with PAB typically take up acetate rapidly, accumulate PHAs in the cell, consume previously stored intracellular carbohydrate, and release Pi as a result of utilization of stored polyP. These typical behaviors of key substances involved in EBPR are graphically shown in Fig.. The acetate taken up is converted to and accumulated as PHAs. Satoh et al. [found that the PHAs accumulated in the PAB-enriched sludge are composed of four monomeric units: 3HB, 3HV, 3H2MB, and 3H2MV. Inoue et al. analyzed the chemical structure of these PHAs by NMR and verified that they are co-polymers composed of the above four monomeric units. As for carbohydrate, Liu et al. proved enzymologically that the carbohydrate stored in the anaerobic-aerobic sludge is a polymer of glycosyl units with the alpha-1,4- and the alpha-1,6-linkages, or glycogen. When acetate is fed as the carbon source, 3HB-rich PHAs are formed in the anaerobic stag. The conversion of acetate to PHA requires reducing power, because PHA is a more reduced compound than acetate. To explain the source of the reducing power under the conditions without electronacceptors, a hypothetical model was proposed by Mino et al. and Arun et al.. In that model, anaerobic degradation of stored glycogen to acetyl-CoA as well as its partial oxidation to CO2 is assumed to account for the generation of the reducing power for PHA synthesis. This model is now called the Mino model, and its relevance has been confirmed by several researchers. The outlines of the model are shown in Fig. The theoretical stoichiometry based on the model could quantitatively explain the observed conversions of acetate and glycogen to PHA by PAB-enriched sludges, as shown in the table. Satoh et al. [successfully applied a similar concept to explain the anaerobic uptake of propionate in EBPR processes (see the table).Fig. 3. A conceptual model for anaerobic carbon metabolism in an EBPR process (after references).Another hypothesis was postulated by Matsuo et al., Comeau et al. [, and Wentzel et al. [to account for the source of the reducing power in anaerobic acetate metabolism. According to this hypothsis, the TCA cycle is assumed to operate under anaerobic conditions in order to oxidize a part of acetate to CO2 and to generate reducing power in the form of NADH. This model is referred to as the Comeau-Wentzel model. Usually the TCA cycle operates only under aerobic or anoxic conditions. The oxidation of succinate to fumarate in the TCA cycle requires a terminal electron acceptor with a redox potential (E0´) more positive than that of fumarate/succinate couple (+32 mV). Only O2 (O2/H2O, E0´= +818 mV), NO3- (NO3-/NO2-, E0´ = + 433 mV), and NO2- (NO2-/N2-, E0´ = +970 mV) appear to meet these conditions. Contradictory to this thermodynamic theory, a complete TCA cycle has been found to operate in some anaerobic eubacteria or archae. These microorganisms can oxidize acetate completely to CO2 via the TCA cycle by utilizing elemental sulfur, thiosulfate, or sulfate as electron acceptor. It is believed, however, that major part of the required reducing power should be generated through the glycogen metabolism as described in the Mino model rather than through the TCA cycle as predicted by the Comeau-Wentzel model. The reasons are as follows: 1) the theoretical stoichiometry for the glycogen metabolism can explain very well the experimentally observed anaerobic acetate uptake, PHA formation, glycogen utilization, and CO2 production by PAO-enriched sludges ; 2) a 13C tracer experiment using NMR indicated that the acetate taken up by the sludge anaerobically was not oxidized to CO2 and thus not metabolized through the TCA cycle, and 3) experiments using 13C-NMR [demonstrated that glycogen is involved in the anaerobic metabolism of EBPR sludges.On the other hand, there is evidence that indicates the possibility of partial involvement of the TCA cycle in the generation of reducing power by PAB in the anaerobic stage of the EBPR process. Namely, 13C-labeled carbon in the acetate fedto a PAB-enriched sludge was found to be released as CO2 under absolute anaerobic conditions. So far, this is the only experimental result indicating the possible functioning of the TCA cycle in the anaerobic phase of the EBPR process. The function of the TCA cycle in the anaerobic uptake of carbon sources by PAB as well as its contribution to the microbial selection in the EBPR process remains to be further investigated.In the EBPR process, microorganisms are exposed to alternate carbon-rich anaerobic environments and carbon-poor aerobic environments. By this alternation, synthesis and degradation of three kinds of biopolymers (polyP, PHA, and glycogen) are induced and metabolic cycling through these biopolymers is established in microorganisms. Such metabolic cycling is energy consuming and not favorable for microorganisms in terms of energy utilization efficiency. Ecologically, however, this metabolic cycling enables PAB to win in the microbial selection in the anaerobic-aerobic process. To explain how this metabolic cycling is regulated in the cell, a metabolic flux model was developed by Pramanik et al.. This model contains a complete set of metabolic pathways involved in biosynthesis and energy production and accounts for energy requirements for macromolecule synthesis and metabolite transport across the cell membrane. The model not only supports the hypothesis that the biopolymer metabolism provides a means for PAB to balance intracellular energy supplies, but also suggest pathways at which metabolic regulation should occur.MICROBIAL COMMUNITY STRUCTURE OF ENHANCED BIOLOGICALPHOSPHATE REMOV AL PROCESSWhen Acinetobacter was first proposed as PAB, there were very few researchers who raised the question of whether Acinetobacter is the only bacterium responsible for EBPR. It may have been somehow assumed that EBPR sludges with high phosphate removal capability were dominated by a single group of microorganisms, and few attempts were made to find candidates of PAB other than Acinetobacter. Now, new and powerful tools for the analysis of microbial community structures have been developed and used to analyze EBPR sludges. They include chemotaxonomic methods like quinone profiling and molecular methods like the fluorescent in situ hybridization (FISH), the clone library approach, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphisms (T-RFLP), etc.High microbial diversity of the EBPR sludge has been demonstrated by using these new techniques. Quinone profiling was applied to characterize activated sludge community structures. The type of quinone in biological samples can be quantitatively determined, and the quinone patterns should explicitly reflect thechemotaxonomic composition of the examined samples. It was suggested that EBPR sludges consist of several different chemotaxonomic groups. The most abundant quinone, Q-8, accounts for only about 31% of total quinone in a PAB-enriched sludge (phosphorus content, 1.94 mmoles or 60 mg/g of suspended solids); the second most abundant one, Q-10, accounts for 8.5%; the third, MK-8(H4), 6.5% (calculated from Hiraishi et al.). The FISH technique with group-specific oligonucleotide probes targeting rRNA showed that an EBPR sludge contained the alpha-, beta-, and gamma-subclasses of proteobacteria, the cytophage group, and Gram-positive bacteria with high G+C DNA contents (13, 25-33, 10-12, 1, and 17-27% of the total cell count, respectively). In other words, the EBPR sludge phylogenetically consisted of several different microbial groups, which has been confirmed using FISH by other researchers as well. The T-RFLP of PCR-amplified 16S rDNA more directly showed the high population diversity; about 19-24 different numerically dominant ribotypes were observed in a highly PAB-enriched sludge (phosphorus content, 12% of suspended solids). The DGGE technique also showed that PCR-amplified 16S rDNA fragments of EBPR sludges contained a number of dominant DNA fragments with different sequences, implying that the examined EBPR communities had high genotypical diversity. All these results strongly suggest that PAB are not a single genotype or a few limited genospecies, but involve phylogenetically and taxonomically diverse groups of bacteria.Bond et al. applied PCR cloning to two activated sludges, one with high phosphate removal performance as well as the typical metabolism of PAB and the other without. They found that the Rhodocyclus group belonging to the beta-subclass of proteobacteria was present in significantly higher numbers in the high-phosphate sludge than in the low-phosphate sludge. This result suggests that the Rhodocyclus group may have a specific role in EBPR. However, the Rhodocyclus group occupied only 14% of the total isolated clones in the high-phosphate sludge. It is not certain that this can account for the observed high phosphate removal performance. As discussed before, an EBPR community was reported in which a type of Rhodocyclus was overwhelmingly dominating (81% of total bacteria) bacterium was shown to be essentially responsible for EBPR. Kawaharasaki et al. used dual staining with DAPI for polyP and rRNA-targeted oligonucleotide probes specific to different bacterial groups to identify PAB in situ. Many of the Gram-positive bacteria with high G+C DNA content and the alpha-subclass of proteobacteria gave the fluorescent DAPI signal of polyP. Therefore, these two groups were considered to accumulate polyP in the EBPR sludge examined. Christensson et al. reported that the Gram-positive bacteria with high G+C DNA content was suspected to play an important role in EBPR becauserelatively high occurrence of this bacterial group was observed in a clone library from an EBPR process. A significant portion of the clones of Gram-positive bacteria with high G+C DNA content was phylogenetically close to Terrabacter tumescens. Based on comparison of the electrophoregrams of T-RFLP of 16S rDNA fragments from a highly PAB-enriched sludge (phosphorus content, 12% of suspended solids) and a sludge with very low phosphate content (2% of suspended solids), Liu et al. concluded that the Gram-positive bacteria with high G+C DNA contents are not the only major component of PAB. Hiraishi et al. showed using the quinone profiling approach that the quinone patterns of activated sludges treating municipal sewage were similar to each other irrespective of the type of activated sludge process; sludges from EBPR processes and conventional processes had very similar quinone patterns. The comparison of quinone patterns from different activated sludges suggested that introduction of the anaerobic stage into the fully aerobic conventional activated sludge process does not result in significant population shift. These findings described above may again lead to the conclusion that PAB, which physiologically possess unique metabolic characteristics, should include different phylogenetic and taxonomic bacterial groups: most probably the alpha-, beta-, and gamma-subclasses of proteobacteria and the Gram-positive bacteria with high G+C DNA contents are the candidates.FUTURE PERSPECTIVESThe present review shows that PAB are not composed of a few limited genospecies, but involve phylogenetically and taxonomically diverse groups of bacteria. The type of bacteria responsible for EBPR may vary among different situations. To clearly define the microbial community structure of EBPR processes and to describe mechanism of ecological selection for PAB in EBPR processes, a closer look into occurrence and behavior of individual species of PAB in various EBPR processes will be needed. Since many of PAB seem to be impossible to culture, molecular methods are surely powerful tools for this purpose. 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