Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge

AIMS: The study investigated the physiology of Amaricoccus kaplicensis to determine whether it could outcompete polyphosphate accumulating bacteria in activated sludge systems removing phosphorus, by preferentially assimilating substrates in the anaerobic stages of these processes. METHODS AND RESULTS: The storage processes were investigated under anaerobic, anoxic and aerobic conditions in both batch and periodically fed cultures in an aerobic sequencing batch reactor (SBR). Amaricoccus kaplicensis showed a high capacity for storing aerobically large amounts of acetate as poly beta-hydroxybutyrate (PHB) at high rates. However, no acetate assimilation under anaerobic conditions and very slow assimilation under anoxic conditions could be detected. CONCLUSION: Amaricoccus kaplicensis in pure culture does not behave as polyphosphate accumulating bacteria competitor; therefore it is difficult to understand why anaerobic/aerobic systems often contain such large numbers of Amaricoccus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Amaricoccus kaplicensis is probably not responsible for the failure of activated sludge systems removing phosphorus, and other organisms capable of anaerobic substrate assimilation should be sought.     

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transformation of phosphorus and relevant intracellular compounds by a phosphorus-accumulating enrichment culture in the presence of both the electron acceptor and electron donor

This study was conducted to obtain a better insight into the metabolic behavior of denitrifying phosphate-accumulating organisms relative to the transformations of relevant intracellular compounds as well as phosphorus and nitrate for enhanced biological phosphorus removal under different combinations of electron acceptor (oxygen or nitrate) and electron donor (acetate). Under anoxic conditions, the amount of polyhydroxybutyrate (PHB) produced per acetate taken up considerably increased with the increasing amount of nitrate reduced whereas the amounts of nitrate reduced and phosphorus released per acetate taken up remained almost constant. However, glycogen utilization occurred during PHB production and then was again observed in response to the initial supplementation of acetate after glycogen accumulation was transiently observed during anoxic phosphorus uptake using nitrate as an electron acceptor. On the other hand, under subsequent aerobic conditions, the additional supplementation of acetate again caused aerobic phosphorus release and PHB production, which showed that PHB production was associated with polyphosphate cleavage regardless of electron acceptor conditions. In contrast to anoxic conditions, glycogen accumulation was observed during PHB production. Based on these observations, the preliminary model for the metabolic behavior of denitrifying phosphate-accumulating organisms was proposed and could well account for the complex transformations of PHB and glycogen together with phosphorus release in the presence of acetate under different electron acceptors.     

Biotechnology for phosphorus removal during wastewater treatment

Advanced biological wastewater treatment for the removal of phosphorus in excess of the normal metabolic requirements of activated sludge type processes has been developed as an alternative to chemical addition. Current laboratory and pilot plant investigations have confirmed that a preliminary anaerobic zone and plug-flow type configuration are necessary for good enhanced biological phosphorus removal. Nitrate in the anaerobic stage inhibits the process whereas acetate enhances phosphorus uptake. The bacteria probably responsible are of the Acinetobacter genus and the presence of stored polyphosphate within these bacteria has been demonstrated. It has also been shown that pure cultures of Acinetobacter do not necessarily take up soluble substrate as phosphate is released during the anaerobic phase, in contrast to the current proposed mechanism, and that in certain cases natural chemical precipitation could make a significant contribution towards overall phosphorus removal. Several studies of pilot and full-scale plants have been reported.     

Characterizing the biochemical activity of full-scale enhanced biological phosphorus removal systems: A comparison with metabolic models

The metabolism of polyphosphate accumulating organisms (PAOs) has been widely studied through the use of lab-scale enrichments. Various metabolic models have been formulated, based on the results from lab-scale experiments using enriched PAO cultures. A comparison between the anaerobic stoichiometry predicted by metabolic models with that exhibited by full-scale sludge in enhanced biological phosphorus removal (EBPR) wastewater treatment plants (WWTPs) was performed in this study. Batch experiments were carried out with either acetate or propionate as the sole carbon source, using sludges from two different EBPR-WWTPs in Australia that achieved different phosphorus removal performances. The results support the hypothesis that the anaerobic degradation of glycogen is the primary source of reducing equivalents generated by PAOs, however, they also suggested a partial contribution of the tricarboxylic acid (TCA) cycle in some cases. The experimental results obtained when acetate was the carbon source suggest the involvement of the modified succinate-propionate pathway for the generation of poly-beta-hydroxyvalerate (PHV). Overall, the batch test results obtained from full-scale EBPR sludge with both substrates were generally well described by metabolic model predictions for PAOs.     

Purple nonsulfur bacteria diversity in activated sludge and its potential phosphorus-accumulating ability under different cultivation conditions

This study investigates the diversity and the potential phosphorus-accumulating ability among the purple nonsulfur (PNS) bacteria. Traditional methods and molecular biotechniques were applied. Microscopic visualization using 4′,6-diamidino-2-phenylindole staining as well as chemical analysis demonstrated that most of the isolated PNS bacteria presented different levels of phosphorus accumulation. Four of the pure cultures, denoted as Rhodopseudomonas palustris CC1, CC7, G11, and GE1, based on their differences in the PNS’s pufM gene, exhibited higher internal phosphorus content compared to other isolated strains in this study. In addition, substantial polyphosphate accumulation was observed after the bacteria entered their stationary growth phase. Among them, the isolated R. palustris G11 could accumulate internal phosphorus up to 13%–15% of its cell dry weight under anaerobic illuminated incubation conditions. When the incubation status was switched from anaerobic to aerobic, the bacterial phosphorus content had a tendency to decrease slightly or remain about the same throughout the whole aerobic stage. The growth rate and biomass were higher when the PNS bacteria grew under photoheterotrophic conditions rather than the chemoheterotrophic ones. Furthermore, the environmental pH value could affect the contents of internal bacterial phosphate. Results of this study demonstrated that PNS bacteria are a group of the polyphosphate-accumulating organisms, of which this ability had never been properly studied. The conditions that PNS bacteria accumulating polyphosphate presented from this study were unique and showed characteristics that were different from the well-known enhanced biological phosphorus removal model.     

Microbial population analysis of nutrient removal-related organisms in membrane bioreactors

Membrane bioreactors (MBR) are an important and increasingly implemented wastewater treatment technology, which are operated at low food to microorganism ratios (F/M) and retain slow-growing organisms. Enhanced biological phosphorus removal (EBPR)-related organisms grow slower than ordinary heterotrophs, but have never been studied in detail in MBRs. This study presents a comprehensive analysis of the microorganisms involved in EBPR in pilot- and full-scale MBRs, using fluorescence in situ hybridization (FISH), as well as an overall assessment of other relevant microbial groups. The results showed that polyphosphate accumulating organisms (PAOs) were present at similar levels in all studied MBRs (10% ± 6%), even those without a defined anaerobic zone. Glycogen accumulating organisms were also detected, although rarely. The FISH results correlated well with the observed P removal performance of each plant. The results from this study suggest that a defined anaerobic zone is not necessarily required for putative PAO growth in MBRs, since polyphosphate storage may provide a selective advantage in fulfilling cell maintenance requirements in substrate-limited conditions (low F/M).     

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass . h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis.     

Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of "Candidatus Competibacter phosphatis", a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by "Candidatus Accumulibacter phosphatis", a known PAO, and did not contain Competibacter. In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter. Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems.     

聚磷菌和聚糖菌的竞争影响因素研究进展

目前, 强化生物除磷工艺(EBPR)以其经济有效而得到广泛的应用, 该工艺关键在于聚磷菌的富集。然而已经发现, 有一类细菌—聚糖菌(GAOs)能够和聚磷菌(PAOs)竞争, 导致除磷效果恶化。关于PAOs-GAOs的竞争, 研究已经很多, 但是其结论有趋同也有矛盾, 有必要对此进行分析讨论。根据近年来国内外的相关报道, 阐述了聚磷菌与聚糖菌的竞争影响因素, 其中碳磷比、碳源种类、温度、pH值是关键因素, 而污泥龄、溶解氧以及水力停留时间等因素对于PAOs和GAOs的竞争也起一定的作用。此外, 在EBPR系统中, 缺氧条件下, 存在反硝化聚磷菌(DPB)和反硝化聚糖菌(DGAO)也会对聚磷菌富集和系统除磷产生影响。最后对EBPR系统未来的发展方向进行了展望。     

Luxury uptake of phosphorus by microalgae in waste stabilisation ponds: current understanding and future direction

There is a need to improve phosphorus removal for the tens of thousands of small communities around the world that currently rely on waste stabilisation ponds for their wastewater treatment. We now know that microalgae in pond systems are capable of accumulating phosphorus within their cells as polyphosphate in a process known as luxury uptake, but this knowledge has not yet been applied to engineer a new process to improve phosphorus removal. This paper summarises the current understanding of this mechanism, discusses the key factors influencing polyphosphate accumulation and provides future direction for research in this field. There have only been a limited number of studies that have focussed on luxury uptake of polyphosphate by microalgae in high phosphorus concentration environments such as those found in waste stabilisation pond systems. However, from this review it has been shown that the environmental factors which influence polyphosphate storage are the phosphate concentration, light intensity and temperature. Currently we are limited to a black box understanding of the bulk population and as a result future research needs to focus on a systematic evaluation of different microalgal genera under a wide range of environmental, biological and process variables in order to reveal the conditions needed to reliably trigger this phenomenon. This will then provide the basis for developing a new algal biotechnology optimised for luxury uptake of polyphosphate. While there are still several key questions that need to be answered there is potential for this to lead to a process which could be as significant to pond systems as the development of enhanced biological phosphorus removal was for the activated sludge process.     

反硝化聚磷菌及其脱氮除磷机理研究进展

水体富营养化是当前水环境保护工作的重点关注问题,微生物修复富营养化水体具有高效、低耗且不产生二次污染等特点,已经成为富营养化水体生态修复的一种重要方式。近年来,对反硝化聚磷菌的研究及其在污水处理工艺中的应用越来越广泛。不同于传统的反硝化细菌联合聚磷菌去除氮磷工艺,反硝化聚磷菌在交替厌氧、缺氧/好氧条件下能同时进行脱氮除磷而被广泛关注与研究。值得注意的是,近几年报道的部分微生物仅在好氧条件下就可进行同时脱氮除磷,但是其脱氮除磷机理仍未理清。基于此,文中总结了目前发现的反硝化聚磷菌和同时硝化反硝化聚磷微生物的种类及特点,并对其脱氮与除磷的关系及其机理进行了系统性分析,对目前反硝化除磷存在的问题进行了梳理,最后对今后的研究方向进行了展望,以期为完善反硝化聚磷菌的脱氮除磷机理及工艺改进提供参考。     

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.
     

Challenges for simultaneous nitrification, denitrification, and phosphorus removal in microbial aggregates: mass transfer limitation and nitrous oxide production

The microbial community composition and activity was investigated in aggregates from a lab-scale bioreactor, in which nitrification, denitrification and phosphorus removal occurred simultaneously. The biomass was highly enriched for polyphosphate accumulating organisms facilitating complete removal of phosphorus from the bulk liquid; however, some inorganic nitrogen still remained at the end of the reactor cycle. This was ascribed to incomplete coupling of nitrification and denitrification causing NO(3)(-) accumulation. After 2 h of aeration, denitrification was dependent on the activity of nitrifying bacteria facilitating the formation of anoxic zones in the aggregates; hence, denitrification could not occur without simultaneous nitrification towards the end of the reactor cycle. Nitrous oxide was identified as a product of denitrification, when based on stored PHA as carbon source. This observation is of critical importance to the outlook of applying PHA-driven denitrification in activated sludge processes.     

A metabolic model of the biological phosphorus removal process: I. Effect of the sludge retention time

The biological phosphorus removal process is a process which depends basically on three internal storage compounds. Poly-beta-hydroxybutyrate (PHB) produced during the anaerobic phase is used as substrate for biomass, polyphosphate, and glycogen formation. The reaction rates of the aerobic processes are primarily determined by the PHB content of the cells. This PHB content is highly dynamic due to the conversions during the anaerobic and aerobic phase of the cycle and the ratio between substrate addition and biomass present in the reactor. The amount of biomass present in the reactor is determined by the sludge retention time and growth rate. A metabolic model of the biological phosphorus removal process was developed and verified over a wide range of growth rates. The effect of different growth rates on the internal fractions of stored components was determined and described mathematically. One set of kinetic parameters was capable of describing the measured conversions of all components observed in the reactor as a function of the sludge retention time. (c) 1995 John Wiley & Sons, Inc.     

Improved understanding of the interactions and complexities of biological nitrogen and phosphorus removal processes

Nitrogen and phosphorus removal from wastewater is now considered essential for the protection of our waterways. Biological nutrient removal processes are generally the most efficient and cost-effective solution to achieve this. While the principles of these processes are well known, intriguing and useful details are being discovered with the recent advances in bio-process engineering and microbial sciences. Phosphorus accumulating organisms have only been identified in recent years, and there are now competing glycogen accumulating organisms being found in biological phosphorus removal systems. These can possibly explain the reasons for the variable phosphorus removal performance of certain systems, and their control can help in the development of more stable and better performing processes. Detailed investigations of the traditional nitrification-denitrification systems, but also of novel developments for nitrogen removal, reveal a more complex and diverse range of processes involved in these transformations. Increasingly, linked phosphorus and nitrogen removal processes are being developed, creating further opportunities to optimise the technologies. However, this might also bring certain risks such as the potential to produce the greenhouse-gas nitrous oxide (N₂O) rather than nitrogen gas as the final denitrification product. A range of recent developments in these areas is covered in this paper.     

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.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: Stoichiometry, kinetics, and the effect of pH

A metabolic model for the stoichiometry of acetate uptake under anaerobic conditions by an enriched culture of glycogen accumulating organisms (GAOs) was developed and tested by experimental studies. Glycogen served as the source of both reducing power and energy to drive the process of acetate uptake. The amount of glycogen consumed and poly-beta-hydroxyvalerate (PHV) accumulated in the cells increased with increasing pH, indicating that the energy requirements for acetate uptake increased with pH. The composition of the accumulated poly-beta-hydroxyalkanoates (PHAs) was adequately predicted using the assumption that acetyl-CoA and propionyl-CoA condense randomly to produce PHA. In addition, the rate of acetate uptake was strongly affected by the pH. The rate decreased with increasing pH and this dependence could be described with a saturation type of expression. A comparison of the rate of acetate uptake at low pH with the rates observed in enriched cultures of phosphorus accumulating organisms (PAOs) indicated that GAOs are able to compete effectively with PAOs in nutrient removal systems under certain conditions.