Free nitrous acid inhibition on anoxic phosphorus uptake and denitrification by poly-phosphate accumulating organisms

Nitrite has been found in previous research an inhibitor on anoxic phosphorus uptake in enhanced biological phosphorus removal systems (EBPR). However, the inhibiting nitrite concentration reported varied in a large range. This study investigates the nitrite inhibition on anoxic phosphorus uptake by using four different mixed cultures performing EBPR with pH considered an important factor. The results showed that the protonated species of nitrite, HNO(2) (or free nitrous acid, FNA), rather than nitrite, is likely the actual inhibitor on the anoxic phosphorus uptake, as revealed by the much stronger correlation of the phosphorus uptake rate with the FNA than with the nitrite concentration. All the four EBPR sludges showed decreased anoxic phosphorus uptake rates with increased FNA concentrations in the studied range of 0.002-0.02 mg HNO(2)-N/L. The phosphorus uptake by all four cultures was completely inhibited at 0.02 mg HNO(2)-N/L. Granular sludge appeared to be more tolerant to HNO(2) than flocular sludge likely due to its stronger resistance to the transfer of nitrite into the bacterial aggregates. Furthermore, denitrification by the phosphorus-accumulating organisms (PAOs) was also found to be inhibited by HNO(2). The denitrification rate decreased by approximately 40% when the FNA concentration was increased from 0.002 to 0.02 mg HNO(2)-N/L.     

Impact of nitrite on aerobic phosphorus uptake by poly-phosphate accumulating organisms in enhanced biological phosphorus removal sludges

Impact of nitrite on aerobic phosphorus (P) uptake of poly-phosphate accumulating organisms (PAOs) in three different enhanced biological phosphorus removal (EBPR) systems was investigated, i.e., the enriched PAOs culture fed with synthetic wastewater, the two lab-scale sequencing batch reactors (SBRs) treating domestic wastewater for nutrient removal through nitrite-pathway nitritation and nitrate-pathway nitrification, respectively. Fluorescence in situ hybridization results showed that PAOs in the three sludges accounted for 72, 7.6 and 6.5 % of bacteria, respectively. In the enriched PAOs culture, at free nitrous acid (FNA) concentration of 0.47 × 10^?3 mg HNO₂-N/L, aerobic P-uptake and oxidation of intercellular poly-β-hydroxyalkanoates were both inhibited. Denitrifying phosphorus removal under the aerobic conditions was observed, indicating the existence of PAOs using nitrite as electron acceptor in this culture. When the FNA concentration reached 2.25 × 10^?3 mg HNO₂-N/L, denitrifying phosphorus removal was also inhibited. And the inhibition ceased once nitrite was exhausted. Corresponding to both SBRs treating domestic wastewater with nitritation and nitrification pathway, nitrite inhibition on aerobic P-uptake by PAOs did not occur even though FNA concentration reached 3 × 10^?3 and 2.13 × 10^?3 mg HNO₂-N/L, respectively. Therefore, PAOs taken from different EBPR activated sludges had different tolerance to nitrite.     

Denitrifying phosphorus removal and impact of nitrite accumulation on phosphorus removal in a continuous anaerobic-anoxic-aerobic (A2O) process treating domestic wastewater

A lab-scale anaerobic-anoxic-aerobic (A(2)O) process was operated to investigate denitrifying phosphorus removal and nitritation-denitritation from domestic wastewater, especially regarding the impact of nitrite accumulation caused by nitritation on phosphorus removal. The results showed that mean total nitrogen (TN) removal was only about 47% and phosphorus removal was almost zero without the pre-anoxic zone and additional carbon source. Contrastively, with configuration of pre-anoxic zone, TN and phosphorus removal was increased to 75% and 98%, respectively, as well as denitrifying phosphorus removal of 66-91% occurred in the anoxic zone. Nitritation-denitritation was achieved through a combination of short aerobic actual hydraulic retention time and low dissolved oxygen levels (0.3-0.5 mg/L); however, phosphorus removal deteriorated with increase of nitrite accumulation rates. The free nitrous acid (FNA) concentration of 0.002-0.003 mg HNO(2)-N/L in the aerobic zone inhibited phosphorus uptake, which was major cause of phosphorus removal deterioration. Through supplying the carbon sources to enhance denitrification and anaerobic phosphorus release, nitrite and FNA concentrations in the aerobic zone were reduced, and phosphorus removal was improved. Compared with nitrification-denitrification, nitritation-denitritation reduced the carbon requirement by 30% and performed biological nutrients removal well with mean TN and phosphorus removal of 85% and 96%, respectively.     

Nitrite (not free nitrous acid) is the main inhibitor of the anammox process at common pH conditions

Nitrite is a substrate but also an inhibitor of anaerobic ammonium oxidation (anammox).There is currently no consensus on whether ionized nitrite (INi) or free nitrous acid (FNA) is the actual inhibitor of the process. The inhibition by INi and FNA on the anammox process has been analysed using a wide range of INi and FNA concentrations and by altering the pH and total nitrite conditions. The inhibitory impacts of both species were quantified through a rational inhibition equation, considering INi and FNA as substrate inhibitor and non-competitive inhibitor, respectively. Inhibitory constants were calculated with strong statistical support as 561 mg INi-N l^?1 and 0.117 mg FNA-N l^?1. Based on the model, INi is the main inhibiting species of the anammox process at pH > 7.1, which are the most common conditions occurring in field applications of anammox.     

Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia and free nitrous acid

Inhibition of ammonium oxidation and nitrite oxidation by free ammonia (FA) and free nitrous acid (FNA) was studied using three different sludges. An uncompetitive inhibition model fit the experimental data well when the reactions were under FA inhibition, whereas a noncompetitive model fit well under FNA inhibition. The estimates of the inhibition constant (K_I) of nitrite oxidation were 46 μM for FA and 1.7–6.8 μM for FNA, each of which was significantly smaller than that of ammonium oxidation, which were 290–1600 μM for FA and 12 μM for FNA. The much smaller values of K_I for nitrite oxidation reflected the susceptibility of that reaction to inhibition by FA and FNA, which could lead to accumulation of nitrite during nitrification. A kinetic model for simultaneous inhibition by FA and FNA was derived. The model predicted that nitrite oxidation should be affected more seriously than ammonium oxidation by the simultaneous inhibition, which would accelerate the accumulation of nitrite in a strong nitrogenous wastewater treatment. It also indicated that a complete removal of ammonia could be achieved with high accumulation of nitrite in a sequencing batch reactor, which is impossible in a continuous-flow reactor.     

好氧和缺氧条件下游离亚硝酸对氨氧化菌和亚硝酸盐氧化菌的选择性抑制

【背景】稳定短程硝化是实现城市污水厌氧氨氧化技术的瓶颈,目前国内外关于游离亚硝酸(Free nitrous acid,FNA)对硝化菌活性的影响大多是在曝气条件下进行研究,鲜有关于缺氧条件下FNA对硝化菌活性影响的报道。【目的】探究好氧和缺氧下FNA对氨氧化菌(Ammonia oxidizing bacteria,AOB)和亚硝酸盐氧化菌(Nitrite oxidizing bacteria,NOB:Nitrospira和Nitrobacter)活性的抑制影响。【方法】采用序批式反应器(Sequencing batch reactor,SBR),基于混合液悬浮固体浓度(Mixed liquid suspended solids,MLSS)为8 300 mg/L的全程硝化污泥条件,通过批次试验分别考察好氧和缺氧下FNA(初始浓度为1.16 mg/L)处理48 h后,AOB和NOB活性的变化情况。【结果】好氧FNA处理活性污泥48 h后,FNA浓度维持在1.16-1.17 mg/L,游离氨(Free ammonia,FA)浓度小于0.017 mg/L,AOB、Nitrospira、Nitrobacter丰度均未发生明显变化;过曝气至99 h时,与空白组相比,比氨氮氧化速率(r~+_(NH4-N))、比亚硝酸盐氮氧化速率(r_(NO2-N))均出现小幅下降,分别由3.5、4.828 mg N/(g VSS·h)降至3.3、4.668 mg N/(g VSS·h),且亚硝酸盐氮累积率(Nitrite accumulation rate,NAR)始终低于33.2%。缺氧FNA处理活性污泥48 h后,FNA浓度维持在0.64-1.16 mg/L,FA浓度低于0.039 mg/L,AOB丰度变化较小,而Nitrospira、Nitrobacter丰度均明显下降,分别由3.002 9×10~9、4.245×10~8 copies/g VSS降至1.666 5×10~8、5.163 8×10~7 copies/g VSS;过曝气至99 h时,与空白组相比,r~+_(NH4-N)值下降幅度较小,而r_(NO2-N)值明显降低,由4.828 mg N/(g VSS·h)降至0.007 mg N/(g VSS·h),且在过曝气0-292 h内,NAR均大于94%。【结论】好氧FNA处理活性污泥48 h后对AOB和NOB无明显抑制作用,但缺氧FNA处理活性污泥48 h后对AOB具有轻微抑制作用,而对NOB具有强烈的抑制作用,可以实现稳定的短程硝化。     

亚硝酸盐对污水生物除磷影响的研究进展

亚硝酸盐作为生物硝化和反硝化的中间产物, 存在于污水生物脱氮除磷系统中。对于生物强化除磷工艺亚硝酸盐既是电子受体用于反硝化除磷, 同时又是抑制剂影响生物除磷过程。本文综述了聚磷菌在厌氧、好氧和缺氧环境中的代谢机理, 在此基础上分别从好氧除磷和反硝化除磷两方面介绍了亚硝酸盐对污水生物除磷影响的研究, 同时概述了亚硝酸盐对生物除磷的抑制机理, 并对该领域的研究提出了个人见解。     

Impact of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO)

The use of saline water as secondary quality water in urban environments for sanitation is a promising alternative towards mitigating fresh water scarcity. However, this alternative will increase the salinity in the wastewater generated that may affect the biological wastewater treatment processes, such as biological phosphorus removal. In addition to the production of saline wastewater by the direct use of saline water in urban environments, saline wastewater is also generated by some industries. Intrusion of saline water into the sewers is another source of salinity entering the wastewater treatment plant. In this study, the short-term effects of salinity on the anaerobic metabolism of phosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) were investigated to assess the impact of salinity on enhanced biological phosphorus removal. Hereto, PAO and GAO cultures enriched at a relatively low salinity level (0.02 % W/V) were exposed to salinity concentrations of up to 6 % (as NaCl) in anaerobic batch tests. It was demonstrated that both PAO and GAO are affected by higher salinity levels, with PAO being the more sensitive organisms to the increasing salinity. The maximum acetate uptake rate of PAO decreased by 71 % when the salinity increased from 0 to 1 %, while that of GAO decreased by 41 % for the same salinity increase. Regarding the stoichiometry of PAO, a decrease in the P-release/HAc uptake ratio accompanied with an increase in the glycogen consumption/HAc uptake ratio was observed for PAO when the salinity increased from 0 to 2 % salinity, indicating a metabolic shift from a poly-P-dependent to a glycogen-dependent metabolism. The anaerobic maintenance requirements of PAO and GAO increased as the salinity concentrations risen up to 4 % salinity.     

Comparative proteomic analysis reveals insights into anoxic growth of Methyloversatilis universalis FAM5 on methanol and ethanol

Methyloversatilis universalis FAM5 is a facultative methylotrophic bacterium that has been found in a variety of natural and engineered ecosystems. The goal of this study was to investigate M. universalis FAM5 responses to different electron/carbon donors, e.g. methanol or ethanol, during anoxic growth in chemostats with nitrate as the electron acceptor. During steady‐state anoxic growth on either methanol or ethanol, over 90% of the influent nitrate was reduced primarily to nitrite. The cell yield on methanol was lower, possibly due to high energy requirements for C₁ assimilation. Label‐free proteomics further revealed that methanol‐grown cells displayed elevated concentrations of the enzymes involved in C₁ metabolism (H₄MPT/H₄F pathways, formate oxidation and serine cycle). In contrast, C₂ metabolism (glyoxylate shunt and tri‐carboxylic acid cycle) and polyhydroxy‐β‐butyrate (PHB) synthesis related proteins were overrepresented during subsequent growth on ethanol. Notably, the expression of respiratory nitrate reductase was not affected by the carbon sources applied. Furthermore, the changes in the proteome upon switching back to methanol were mostly reversible. Therefore, M. universalis displays wide‐ranging responses to adapt between growth on methanol and ethanol. Such metabolic versatility could be particularly useful in wastewater treatment systems, which need to switch between different electron donors, while still reliably meeting effluent nitrogen discharge goals.     

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi‐species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite‐oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia‐oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients. Biotechnol. Bioeng. 2010;105: 1115–1130. © 2009 Wiley Periodicals, Inc.     

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

A sequencing batch reactor system for high-level biological nitrogen and phosphorus removal from abattoir wastewater

A sequencing batch reactor (SBR) system is demonstrated to biologically remove nitrogen, phosphorus and chemical oxygen demand (COD) to very low levels from abattoir wastewater. Each 6 h cycle contained three anoxic/anaerobic and aerobic sub-cycles with wastewater fed at the beginning of each anoxic/anaerobic period. The step-feed strategy was applied to avoid high-level build-up of nitrate or nitrite during nitrification, and therefore to facilitate the creation of anaerobic conditions required for biological phosphorus removal. A high degree removal of total phosphorus (>98%), total nitrogen (>97%) and total COD (>95%) was consistently and reliably achieved after a 3-month start-up period. The concentrations of total phosphate and inorganic nitrogen in the effluent were consistently lower than 0.2 mg P l⁻¹ and 8 mg N l⁻¹, respectively. Fluorescence in situ hybridization revealed that the sludge was enriched in Accumulibacter spp. (20–40%), a known polyphosphate accumulating organism, whereas the known glycogen accumulating organisms were almost absent. The SBR received two streams of abattoir wastewater, namely the effluent from a full-scale anaerobic pond (75%) and the effluent from a lab-scale high-rate pre-fermentor (25%), both receiving raw abattoir wastewater as feed. The pond effluent contained approximately 250 mg N l⁻¹ total nitrogen and 40 mg P l⁻¹ of total phosphorus, but relatively low levels of soluble COD (around 500 mg l⁻¹). The high-rate lab-scale pre-fermentor, operated at 37°C and with a sludge retention time of 1 day, proved to be a cheap and effective method for providing supplementary volatile fatty acids allowing for high-degree of biological nutrient removal from abattoir wastewater.     

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.     

Effect of influent nutrient ratios and hydraulic retention time (HRT) on simultaneous phosphorus and nitrogen removal in a two-sludge sequencing batch reactor process

A laboratory-scale anaerobic–anoxic/nitrification sequencing batch reactor (A₂N-SBR) fed with domestic wastewater was operated to examine the effect of varying ratios of influent COD/P, COD/TN and TN/P on the nutrient removal. With the increased COD/P, the phosphorus removals exhibited an upward trend. The influent TN/P ratios had a positive linear correlation with the phosphorus removal efficiencies, mainly because nitrates act as electron acceptors for the phosphorus uptake in the A₂N-SBR. Moreover, it was found that lower COD/TN ratio, e.g. 3.5, did not significantly weaken the phosphorus removal, though the nitrogen removal first decreased greatly. The optimal phosphorus and nitrogen removals of 94% and 91%, respectively were achieved with influent COD/P and COD/TN ratios of 19.9 and 9.9, respectively. Additionally, a real-time control strategy for A₂N-SBR can be undertaken based on some characteristic points of pH, redox potential (ORP) and dissolved oxygen (DO) profiles in order to obtain the optimum hydraulic retention time (HRT) and improve the operating reliability.     

1H-NMR study of the metabolome of an exceptionally anoxia tolerant vertebrate, the crucian carp (Carassius carassius)

The crucian carp (Carassius carassius) can tolerate anoxia for days to months, depending on the temperature. In this study, we applied ¹H-NMR-based metabolomics to polar extracts of crucian carp brain, heart, muscle and liver samples obtained from fish exposed to either control normoxic conditions, acute anoxia (24 h), chronic anoxia (1 week) or reoxygenation (for 1 week following chronic anoxia) at 5 °C. Spectra of the examined tissues revealed changes in several energy-related compounds. In particular, anoxic stress resulted in decreased concentrations of phosphocreatine (muscle, liver) and glycogen (liver) and ATP/ADP (liver, heart and muscle) and increased concentrations of lactate (brain, heart, muscle) and beta-hydroxybutyric acid (all tissues). Likewise, increased concentrations of inhibitory compounds (glycine, gamma-amino butyric acid or GABA) and decreased concentrations of excitatory metabolites (glutamate, glutamine) were confirmed in the anoxic brain extracts. Additionally, a decrease of N-acetylaspartate (NAA), an important neuronal marker, was also observed in anoxic brains. The branched-chain amino acids (BCAA) valine/isoleucine/leucine increased in all anoxic tissues. Possibly, this general tissue increase can be due to an inhibited mitochondrial function or due to protein degradation/protein synthesis inhibition. In this study, the potential and strength of the ¹H-NMR is highlighted by the detection of previously unrecognized changes in metabolites. Specifically, myo-inositol substantially decreased in the heart of anoxic crucian carp and anoxic muscle tissue displayed a decreased concentration of taurine, providing novel insights into the anoxia responses of the crucian carp.     

Metabolic Characteristics of a Glycogen-Accumulating Organism in Defluviicoccus Cluster II Revealed by Comparative Genomics

Glycogen-accumulating organisms (GAOs) may compete with phosphate-accumulating organisms (PAOs) for short-chain fatty acids (VFAs) in anaerobic polyhydroxyalkanoates (PHA) synthesis, but no consequently aerobic polyphosphate accumulation in enhanced biological phosphorus removal (EBPR) process, thus deteriorating the EBPR process. They are detected frequently in the deteriorated EBPR process, but their metabolisms are still far from our comprehensions for there is seldom pure culture. In this study, a nearly complete draft genome of a GAOs in Defluviicoccus cluster II, GAO-HK, is recruited from the metagenome of activated sludge in a full-scale industrial anoxic/aerobic wastewater plant. Comparative genomics reveal similar metabolisms of PHA and glycogen in GAOs of GAO-HK, Defluviicoccus tetraformis TFO71 (TFO71) and Competibacter phosphatis clade IIA (CPIIA), and PAOs of Accumulibacter clade IIA UW-1 (UW-1) and Tetrasphaera elongata Lp2 (Lp2). Although there are similar gene cassettes related with polyphosphate metabolism in these GAOs and PAOs, especially for Defluviicoccus-relative bacteria and UW-1, ppk1 in GAOs are diverse from those in the identified PAOs, implying the difference of polyphosphate metabolism in GAOs and PAOs. Additionally, genes related to the dissimilatory denitrification are absent in TFO71 and GAO-HK, implying that additional nitrate or nitrite may favor PAOs over Defluviicoccus-relative GAOs. Therefore, PAOs suffering from competition of Defluviicoccus-relative GAOs might be rescued with the additional nitrate/nitrite, which is important to improve the stability of EBPR processes.     

A comparative study of methanol as a supplementary carbon source for enhancing denitrification in primary and secondary anoxic zones

A comparative study on the use of methanol as a supplementary carbon source to enhance denitrification in primary and secondary anoxic zones is reported. Three lab-scale sequencing batch reactors (SBR) were operated to achieve nitrogen and carbon removal from domestic wastewater. Methanol was added to the primary anoxic period of the first SBR, and to the secondary anoxic period of the second SBR. No methanol was added to the third SBR, which served as a control. The extent of improvement on the denitrification performance was found to be dependent on the reactor configuration. Addition to the secondary anoxic period is more effective when very low effluent nitrate levels are to be achieved and hence requires a relatively large amount of methanol. Adding a small amount of methanol to the secondary anoxic period may cause nitrite accumulation, which does not improve overall nitrogen removal. In the latter case, methanol should be added to the primary anoxic period. The addition of methanol can also improve biological phosphorus removal by creating anaerobic conditions and increasing the availability of organic carbon in wastewater for polyphosphate accumulating organisms. This potentially provides a cost-effective approach to phosphorus removal from wastewater with a low carbon content. New fluorescence in situ hybridisation (FISH) probes targeting methanol-utilising denitrifiers were designed using stable isotope probing. Microbial structure analysis of the sludges using the new and existing FISH probes clearly showed that the addition of methanol stimulated the growth of specific methanol-utilizing denitrifiers, which improved the capability of sludge to use methanol and ethanol for denitrification, but reduced its capability to use wastewater COD for denitrification. Unlike acetate, long-term application of methanol has no negative impact on the settling properties of the sludge.     

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.     

Enrichment of denitrifying glycogen-accumulating organisms in anaerobic/anoxic activated sludge system

Denitrifying glycogen-accumulating organisms (DGAO) were successfully enriched in a lab-scale sequencing batch reactor (SBR) running with anaerobic/anoxic cycles and acetate feeding during the anaerobic period. Acetate was completely taken up anaerobically, which was accompanied by the consumption of glycogen and the production of poly-beta-hydroxy-alkanoates (PHA). In the subsequent anoxic stage, nitrate or nitrite was utilized as electron acceptor for the oxidation of PHA, resulting in glycogen replenishment and cell growth. The above phenotype showed by the enrichment culture demonstrates the existence of DGAO. Further, it was found that the anaerobic behavior of DGAO could be predicted well by the anaerobic GAO model of Filipe et al. (2001) and Zeng et al. (2002a). The final product of denitrification during anoxic stage was mainly nitrous oxide (N(2)O) rather than N(2). The data strongly suggests that N(2)O production may be caused by the inhibition of nitrous oxide reductase by an elevated level of nitrite accumulated during denitrification. The existence of these organisms is a concern in biological nutrient removal systems that typically have an anaerobic/anoxic/aerobic reactor sequence since they are potential competitors to the polyphosphate-accumulating organisms.     

Failure of an enriched nitrite-DPAO population to use nitrate as an electron acceptor

The metabolism of denitrifying polyphosphate accumulating organisms (DPAO) is not completely known. Recent reports suggest the existence of two types of DPAOs: those that can use nitrate and nitrite as electron acceptors (nitrate-DPAO) and those that can only use nitrite (nitrite-DPAO). Then, the survival of nitrite-DPAO in nitrate reducing environments is due to the existence of flanking denitrifying species, which reduce nitrate to nitrite. This works aims at a better understanding of the nitrite-DPAO population. For this aim, a nitrite-DPAO population was previously selected in a SBR using nitrite as electron acceptor. Then, nitrate utilisation by nitrite-DPAO was studied within a short-term period (4 days) and within a long-term period (50 days) with simultaneous nitrite and nitrate additions. The results obtained clearly indicate that nitrite-DPAO fail to use nitrate as electron acceptor even after 50 days of periodic dosing of nitrate and agree with the dual DPAO theory. Moreover, this failure casts doubts on the feasibility of nitrite based EBPR systems (i.e. partial nitrification + nitrite-DPAO) because these systems will not be able to denitrify an occasional nitrate inlet, which will remain in the effluent.