Denitrifying phosphorus removal: linking the process performance with the microbial community structure

This study investigated the link between the process performance of two denitrifying phosphorus (P) removal systems and their microbial community structure. Two sequencing batch reactors (SBRs) were operated with either acetate or propionate as the sole carbon source, and were gradually acclimatised from anaerobic-aerobic to anaerobic-anoxic conditions. It was found that the propionate SBR was able to sustain denitrifying P removal after acclimatisation, while the enhanced biological phosphorus removal (EBPR) activity in the acetate reactor collapsed after the aerobic phase was eliminated. The results suggested that the anoxic glycogen production rate in the acetate SBR was insufficient to support the anaerobic glycogen demand for acetate uptake. The chemical transformations in each SBR suggested that different types of polyphosphate-accumulating organisms (PAOs) were present in each system, possessing different affinities for nitrate. Microbial characterisation with fluorescence in situ hybridisation (FISH) revealed that Accumulibacter was the dominant organism in each reactor, although different cell morphotypes were observed. A coccus morphotype was predominant in the acetate SBR while the propionate SBR was enriched in a rod morphotype. It is hypothesised that the coccus morphotype corresponds to an Accumulibacter strain that is unable to use nitrate as electron acceptor but is able to use oxygen, and possibly nitrite. The rod morphotype is proposed to be a PAO able to use nitrate, nitrite and oxygen. This hypothesis is in agreement with literature studies focussed on the identity of denitrifying PAOs (DPAOs), as well as a recent metagenomic study on Accumulibacter.     

Modelling biological nutrient removal activated sludge systems - a review

The external nitrification (EN) biological nutrient removal (BNR) activated sludge (ENBNRAS) system shows considerable promise for full-scale implementation. As an aid for this implementation, a mathematical simulation model would be an invaluable tool. To develop such a model, a study was conducted to select the most suitable simulation model to serve as a starting point for further development. For this, the existing available simulation models for BNRAS systems are compared with one another and evaluated against experimental observations in the literature and on ENBNRAS systems. One process immediately apparent to be crucially important is the anoxic growth of phosphorus accumulating organisms (PAOs), with associated PAO denitrification and anoxic P uptake for polyP formation. These linked processes are lacking in the earlier kinetic simulation models for BNRAS systems, which were based on aerobic PAO growth and P uptake only, but have been incorporated into the more recent kinetic models. This provides a substantive body of information on modelling this aspect. Other processes of significance identified to require consideration are anaerobic slowly biodegradable COD (SBCOD) hydrolysis to readily biodegradable COD (RBCOD), and COD loss. Both processes have significant impact on the predicted BEPR performance. Due to the uncertainties associated with the mechanisms and quantification of these two processes, it is concluded that the most extensively validated kinetic simulation model should be selected for development, and that the omissions in this model should be addressed progressively, using the relevant information drawn from the existing models, the literature and observations on ENBNRAS systems.     

Effect of nitrite on phosphate uptake by phosphate accumulating organisms

In biological nitrogen removal processes, nitrite can be formed and accumulated through both nitrification and denitrification. Despite the fact that, in practice, biological phosphate removal (BPR) is often combined with biological nitrogen removal, there are only a few publications reporting the effect of nitrite on BPR. In this study, phosphate-accumulating organisms (PAO) were cultivated in an anaerobic-anoxic-aerobic sequencing batch reactor (SBR). The effect of nitrite on the enrichment of the sludge with PAO, the phosphate uptake rates and the sludge respiration was investigated. The results indicate that (1) presence of nitrite inhibits both aerobic and anoxic (denitrifying) phosphate uptake, (2) aerobic phosphate uptake was more affected than anoxic phosphate uptake, (3) presence of nitrite could be one of the factors enhancing the presence of glycogen accumulating organisms (GAO)--competitors to PAO for substrate in the anaerobic phase, and (4) it is required to monitor and control nitrite accumulation in a full-scale wastewater treatment plants.     

Simultaneous nitrogen and phosphate removal in aerobic granular sludge reactors operated at different temperatures

The main biological conversions taking place in two lab-scale aerobic granular sludge sequencing batch reactors were evaluated. Reactors were operated at different temperatures (20 and 30 °C) and accomplished simultaneous COD, nitrogen and phosphate removal. Nitrogen and phosphate conversions were linked to the microbial community structure as assessed by fluorescent in situ hybridization (FISH) analysis. Anoxic tests were performed to evaluate the contribution of anoxic phosphate uptake to the overall phosphate removal and to clarify the denitrification pathway. Complete nitrification/denitrification and phosphate removal were achieved in both systems. A considerable fraction of the phosphate removal was coupled to denitrification (denitrifying dephosphatation). From the results obtained in anoxic batch experiments dosing either nitrite or nitrate, denitrification was proposed to proceed mainly via the nitrate pathway. Denitrifying glycogen-accumulating organisms (DGAOs) were observed to be the main organisms responsible for the reduction of nitrate to nitrite. A significant fraction of the nitrite was further reduced to nitrogen gas while being used as electron acceptor by denitrifying polyphosphate-accumulating organisms (PAO clade II) for anoxic phosphate uptake.     

Development of a 2-sludge, 3-stage system for nitrogen and phosphorous removal from nutrient-rich wastewater using granular sludge and biofilms

A novel 2-sludge 3-stage process using a combination of granular sludge and biofilm was developed to achieve biological removal of nitrogen and phosphorus from nutrient-rich wastewater. The system consists of a granular sequencing batch reactor (SBR) working under alternating anaerobic/anoxic conditions supplemented with a short aerobic phase and an aerobic biofilm SBR. The wastewater is first fed to the granular SBR reactor, where easily biodegradable carbon sources are taken up primarily by polyphosphate accumulating organisms (PAOs). The supernatant resulting from quick settling of the granular sludge is then fed to the biofilm SBR for nitrification, which produces oxidized nitrogen that is returned to the granular reactor for simultaneous denitrification and phosphorus removal. While maximizing the utilization of organic substrates and reducing operational costs, as do other 2-sludge processes previously reported in literature, the proposed system solves the bottleneck problem of traditional 2-sludge systems, namely high effluent ammonia concentration, due to its high-volume exchange ratios. An ammonia oxidation rate of 32 mg N/Lh was achieved in the biofilm SBR, which produced nitrite as the final product. This nitrite stream was found to cause major inhibition on the anoxic P uptake and also to result in the accumulation of N(2)O. These problems were solved by feeding the nitrite-containing stream continuously to the granular reactor in the anoxic phase. With a nitrogen and phosphorus removal efficiency of 81% and 94%, respectively, the system produces an effluent that is suitable for land irrigation from a wastewater stream containing 270 mg N/L of total nitrogen and 40 mg P/L of total phosphorus.     

Biological nitrogen removal with enhanced phosphate uptake in a sequencing batch reactor using single sludge system

Simultaneous biological phosphorus and nitrogen removal with enhanced anoxic phosphate uptake was investigated in an anaerobic-aerobic-anoxic-aerobic sequencing batch reactor ((AO)2 SBR). Significant amounts of phosphorus-accumulation organisms (PAOs) capable of denitrification could be accumulated in a single sludge system coexisting with nitrifiers. The ratio of the anoxic phosphate uptake to the aerobic phosphate uptake capacity was increased from 11% to 64% by introducing an anoxic phase in an anaerobic aerobic SBR. The (AO)2 SBR system showed stable phosphorus and nitrogen removal performance. Average removal efficiencies of TOC, total nitrogen, and phosphorus were 92%, 88%, and 100%, respectively. It was found that nitrite (up to 10 mg NO2(-)-N/l) was not detrimental to the anoxic phosphate uptake and could serve as an electron acceptor like nitrate. In fact, the phosphate uptake rate was even faster in the presence of nitrite as an electron acceptor compared to the presence of nitrate. It was found that on-line sensor values of pH, ORP, and DO were somehow related with the dynamic behaviours of nutrient concentrations (NH4+, NO3-, and PO4(3-)) in the SBR. These on-line sensor values were used as real-time control parameters to adjust the duration of each operational phase in the (AO)2 SBR. The real-time controlled SBR exhibited better performance in the removal of phosphorus and nitrogen than the SBR with fixed-time operation.     

短好氧泥龄下A2/O和BAF联合工艺的脱氮除磷特性

采用小试装置,研究了短好氧污泥龄下A2/O和BAF联合工艺处理低C/N和C/P污水时的脱氮除磷特性.结果表明,通过提高A2/O工艺段的厌氧区有机负荷和缺氧区硝酸盐负荷对反硝化聚磷菌（DPAOs）进行选择和强化后,其在聚磷菌（PAOs）中的比例维持在28%左右,工艺具有部分反硝化除磷能力,能够减少脱氮除磷过程中对碳源的总需求量.但在联合工艺中,好氧除磷仍是主要的除磷方式.在A2/O工艺段内,好氧污泥龄在满足好氧PAOs存活的同时,还必须满足抑制硝化细菌生长的要求,且为了保证工艺对磷的整体去除效果,混合液在好氧区的接触时间须大于30 min.此外,以保证缺氧区出水中含有1～4 mg/L的硝态氮为原则来控制BAF出水的回流量,可达到较好的脱氮除磷效果.该联合工艺结合了活性污泥工艺和生物膜工艺的优点,运行稳定,出水水质优良,不仅适合于新建污水处理厂,也特别适合于不能脱氮除磷污水处理厂的技术改造.     

Calibration of denitrifying activity of polyphosphate accumulating organisms in an extended ASM2d model

This paper presents the results of an experimental study for the modelling and calibration of denitrifying activity of polyphosphate accumulating organisms (PAOs) in full-scale WWTPs that incorporate simultaneous nitrogen and phosphorus removal. The convenience of using different yields under aerobic and anoxic conditions for modelling biological phosphorus removal processes with the ASM2d has been demonstrated. Thus, parameter η_PAO in the model is given a physical meaning and represents the fraction of PAOs that are able to follow the DPAO metabolism. Using stoichiometric relationships, which are based on assumed biochemical pathways, the anoxic yields considered in the extended ASM2d can be obtained as a function of their respective aerobic yields. Thus, this modification does not mean an extra calibration effort to obtain the new parameters. In this work, an off-line calibration methodology has been applied to validate the model, where general relationships among stoichiometric parameters are proposed to avoid increasing the number of parameters to calibrate. The results have been validated through a UCT scheme pilot plant that is fed with municipal wastewater. The good concordance obtained between experimental and simulated values validates the use of anoxic yields as well as the calibration methodology. Deterministic modelling approaches, together with off-line calibration methodologies, are proposed to assist in decision-making about further process optimization in biological phosphate removal, since parameter values obtained by off-line calibration give valuable information about the activated sludge process such as the amount of DPAOs in the system.     

Phosphate uptake under anoxic conditions and fixed-film nitrification in nutrient removal activated sludge system

Simultaneous enhanced biological phosphate uptake and biological denitrification under anoxic conditions were investigated in a modified lab-scale nutrient removal activated sludge system. The aim of the experiments was to find whether poly-P bacteria are capable of taking up phosphate under anoxic conditions by utilising nitrate as an electron acceptor. The phosphate uptake in anoxic conditions was compared to that in aerobic environment in batch tests. The results of the long-term operation of continuous-flow lab-scale system as well as the results of batch tests showed that the anoxic phosphate uptake with simultaneous denitrification after preceding anaerobic substrate uptake could significantly reduce the extent of competition for organic substrate between poly-P bacteria and denitrifiers. A side-stream nitrification in fixed-film reactor enabled to reduce the losses of organic carbon by aerobic oxidation and to stabilise the slow-growing population of nitrifiers in the system.     

The activated sludge process—3 single sludge denitrification

The general aerobic bi-substrate active-site death-regeneration activated sludge model including nitrification of Dold et al. (Prog. Wat. Technol.12, 47–77, 1980) is extended to include the kinetic behaviour of the denitrification process in single sludge systems. The extension requires a change in the value of only one of the kinetic constants (K′_mp) in the expression for the particulate substrate utilization rate when the environment becomes anoxic. The extended model simulates very closely the response of the multi-reactor nitrification-denitrification process configurations under both constant and cyclic flow and load conditions. Under constant flow and load conditions, the denitrification response predicted can be reduced to that approximated by a zero order reaction dN/dt = ?KX_a with two rates in the primary and one in the secondary anoxic reactor respectively.     

Control of activated sludge filamentous bulking—VII. Effect of anoxic conditions

The effect of anoxic conditions on the occurrence of filamentous organisms in mixed cultures was studied in laboratory activated sludge systems. It was repeatedly demonstrated that anoxic conditions are able to suppress the growth of some undesirable filamentous organisms, for instance, type 021N and Sphaerotilus natans. It was also found that severely filamentous mixed cultures had maximum rates of denitrification or nitrate respiration one order of magnitude lower than non-filamentous mixed cultures. On the basis of these findings it is concluded that some filamentous organisms cannot use nitrate nitrogen as an electron acceptor.     

Enumeration of acetate-consuming bacteria by microautoradiography under oxygen and nitrate respiring conditions in activated sludge

Microautoradiography was used to enumerate bacteria able to take up radiolabelled acetate in activated sludge using oxygen or nitrate as electron acceptors. In each of three wastewater treatment plants (WWTP) with nitrification and denitrification (N-removal), the number of bacteria consuming acetate under aerobic and anoxic conditions was identical in contrast to the acetate removal rates. The rates were clearly lower under anoxic conditions suggesting that the specific activity of the cells and not the number of active cells was reduced under anoxic conditions. The fraction of bacteria able to consume acetate varied in three WWTPs between 47% and 93% of the total number of bacteria as determined by DAPI. In a WWTP without N-removal only 20% of the bacteria were able to consume acetate under aerobic conditions and very few of these were able to do it under anoxic conditions. The cell specific acetate removal rates in all WWTPs were found to be 3.0-13.2 x 10(-15) mol cell(-1) h(-1) under aerobic conditions and between 1.9 and 9.1 x 10(-15) mol cell(-1) h(-1) under anoxic conditions.     

Advancing post-anoxic denitrification for biological nutrient removal

The objective of this research was to advance a fundamental understanding of a unique post-anoxic denitrification process for achieving biological nutrient removal (BNR), with an emphasis on elucidating the impacts of surface oxygen transfer (SOT), variable process loadings, and bioreactor operational conditions on nitrogen and phosphorus removal. Two sequencing batch reactors (SBRs) were operated in an anaerobic/aerobic/anoxic mode for over 250 days and fed real municipal wastewater. One SBR was operated with a headspace open to the atmosphere, while the other had a covered liquid surface to prevent surface oxygen transfer. Process performance was assessed for mixed volatile fatty acid (VFA) and acetate-dominated substrate, as well as daily/seasonal variance in influent phosphorus and ammonia loadings. Results demonstrated that post-anoxic BNR can achieve near-complete (>99%) inorganic nitrogen and phosphorus removal, with soluble effluent concentrations less than 1.0 mgN L⁻¹ and 0.14 mgP L⁻¹. Observed specific denitrification rates were in excess of typical endogenous values and exhibited a linear dependence on the glycogen concentration in the biomass. Preventing SOT improved nitrogen removal but had little impact on phosphorus removal under normal loading conditions. However, during periods of low influent ammonia, the covered reactor maintained phosphorus removal performance and showed a greater relative abundance of polyphosphate accumulating organisms (PAOs) as evidenced by quantitative real-time PCR (qPCR). While GAOs were detected in both reactors under all operational conditions, BNR performance was not adversely impacted. Finally, secondary phosphorus release during the post-anoxic period was minimal and only occurred if nitrate/nitrite were depleted post-anoxically.     

Simultaneous urea hydrolysis, formaldehyde removal and denitrification in a multifed upflow filter under anoxic and anaerobic conditions

A multifed upflow filter (MUF), working under anoxic or anaerobic conditions, coupled with an aerobic biofilm airlift suspension (BAS) reactor was operated in order to treat a wastewater with high formaldehyde (up to 1.5 g L-1) and urea (up to 0.46 g L-1) concentrations. In the MUF, formaldehyde removal, denitrification and urea hydrolysis took place simultaneously. The MUF was operated at 37 degrees C, at a hydraulic retention time (HRT) ranging from 1 to 0.3 d. An organic loading rate (OLR) of 0.5 kg-formaldehyde m-3 d-1 was efficiently eliminated during anaerobic operation and transformed into methane, while a much higher OLR (up to 2 kg-formaldehyde m-3 d-1) was oxidised under anoxic conditions by the nitrite or nitrate from the nitrifying airlift. However, only 80% of urea was hydrolysed to ammonia in an anoxic environment while complete conversion occurred under anaerobic conditions. Moreover, formaldehyde concentrations higher than 50 mg L-1 provoked a loss of efficiency of urea hydrolysis, decreasing to 10% at formaldehyde concentrations above 300 mg L-1. Methane production rate during the anaerobic stage was adversely affected by accumulations of formaldehyde in the reactor causing lower formaldehyde removal efficiency. However, denitrification proceeded properly even at a formaldehyde concentration of 700 mg L-1 in the reactor, although nitrous oxide appears in the off-gas. The COD/N ratios required for complete nitrite and nitrate denitrification with formaldehyde were estimated at 2.1 and 3.5 kg-COD/kg-N, respectively.     

Storage of substrate mixtures by activated sludges under dynamic conditions in anoxic or aerobic environments

In spite of the fact that in most activated sludge plants substrate complex mixtures are removed under alternating anoxic and aerobic conditions, most studies on the dynamic response of biomass are limited to feeding a single substrate (acetate or glucose) under a single redox condition (aerobic or anoxic). In this study, the dynamic response of biomass in a sequencing batch reactor is described in terms of substrate removal and related storage as internal polymers, as functions of single or simultaneous feed of several substrates (acetate, glucose, glutamic acid and ethanol) and of anoxic vs. aerobic conditions. Under anoxic conditions, the four substrates were simultaneously removed at a significantly greater nitrate removal rate than when single substrates were present, so showing that the simultaneous removal was partially due to independent metabolic activities. On the other hand, the removal of every substrate was affected (positively or negatively) by the presence of the others, demonstrating that the substrates can be also used by the same metabolism. As an exception, acetate removal was not affected by the presence of other substrates. As for the comparison of aerobic and anoxic conditions, the acetate uptake rate almost doubled moving from anoxic to aerobic conditions, whereas other substrates were only slightly affected. This difference was probably due to the additional presence of aerobic denitrification, which was much more important for acetate. This also confirmed that acetate removal was independent from other substrates. In all cases, storage was the main mechanism of solids formation, so confirming the general importance of such phenomenon under dynamic conditions, independently from feed complexity and redox conditions.     

Heterotroph anoxic yield in anoxic aerobic activated sludge systems treating municipal wastewater

As input to the steady state design and kinetic simulation models for the activated sludge system, the correct value for the heterotroph anoxic yield is essential to provide reliable estimates for the system denitrification potential. This paper examines activated sludge anoxic yield values in the literature, and presents experimental data quantifying the value. In the literature, in terms of the structure of ASM1 and similar models, theoretically it has been shown that the anoxic yield should be reduced to approximately 0.79 the value of the aerobic yield. This theoretical value is validated with data from corresponding aerobic OUR and anoxic nitrate time profiles in a batch fed laboratory scale long sludge age activated sludge system treating municipal wastewater. The value also is in close agreement with values in the literature measured with both artificial substrates and municipal wastewater. Thus, it is concluded that, in ASM1 and similar models, for an aerobic yield of 0.67mg COD/mg COD, the anoxic yield should be about 0.53 mg COD/mg COD. Including such a lower anoxic yield in ASM1 and similar models will result in a significant increase in denitrification potential, due to increased denitrification with wastewater RBCOD as substrate. In terms of the structure of ASM3, for the proposed substrate storage yields and the aerobic yield of 0.63 mg COD/mg COD, experimental data indicate that the corresponding anoxic yield should be about 0.42 mg COD/mg COD. This is significantly lower than the proposed value of 0.54 mg COD/mg COD, and requires further investigation.     

西安市污水处理厂改良A~2/O工艺的运行效果分析

介绍了西安市污水处理厂改良A^2／O工艺的特点及运行状况。采用对回流污泥预反硝化及分段进水的方法,减少了硝酸盐对聚磷菌（PAOs）碳源摄取释磷行为的影响,基本满足了反硝化和生物除磷（BPR）对碳源的需求。结果表明,该工艺对COD、NH4^＋-N、总氮、总磷的去除率分别为91．91％、78．70％、64．20％和95．52％。     

Kinetics of denitrification reactions in single sludge systems

This paper reports denitrification studies performed using the anoxic reactor of a laboratory scale anoxic-aerobic plant as a batch reactor of variable volume. This was achieved by adding to the anoxic reactor a supplementary flow of nitrate after the shut down of the recirculation line and the interruption of the hydraulic connection to the aerobic reactor. By operating in this way, in a relatively short time, it is possible to get a number of experimental data sufficient to describe the biological process kinetics. The system is extremely flexible and gives kinetic data in short times for different experimental conditions. In fact, it is possible to operate at different COD/NO₃-N ratios simply by changing the influent wastewater flowrate to the anoxic reactor. Two series of tests were performed: in the first series (use of endogenous carbon) a supplementary flow of nitrate was fed to the anoxic reactor while the wastewater influent flow was interrupted; in the second series (use of internal carbon) the influent wastewater flow was fed during the addition of nitrate. The importance of the carbonaceous substrate nature on the denitrification rate was also verified. Data analysis was performed by utilizing the integral method procedure and a zero order kinetics referring to both the substrates COD and nitrate nitrogen was considered. A satisfactory agreement between predicted and experimental data was found. Values obtained for k_D range from 0.07 mg NO₃-N/mg VSS·d, at which the carbon source is mostly endogenous, to 0.25 mg NO₃-N/mg VSS·d, at which the carbon source consists mainly of readily biodegradable COD. Intermediate values occur when the readily biodegradable COD is limiting and denitrification takes place by utilizing the slowly biodegradable one.     

SBR工艺污泥沉降性能的影响因素研究

分别研究了运行方式、运行时间、硝酸盐浓度、曝气量、污泥负荷及曝气时间对SBR工艺中污泥沉降性能的影响.试验结果表明,以厌氧/好氧方式运行时,厌氧≥1 h且好氧≥3h可获得沉降性良好的污泥;以缺氧/好氧方式运行时,缺氧段硝酸盐浓度过高会导致污泥膨胀,缺氧段时间宜控制在1h左右;当有机负荷较高时,曝气量较高或较低均可能导致污泥膨胀,通过降低有机负荷可有效改善污泥沉降性能;在污泥负荷较低、曝气量适当且氮磷充足的条件下,曝气6h时污泥的沉降性较差,将曝气时间延长至10 h,污泥浓度保持稳定,沉降性较好.