WATER RESEARCH ww ekeviercom/locate/watres Water Research 38 (2004) 3760 3768 Effect of nitrite on phosphate uptake by phosphate accumulating organisms T. Saito“**, D. Brdjanovic?, M.C.M. van Loosdrecht® “Department of Civil Engineering, Collegeof Science and Technology, Nihon University, 1-8 Kanda-Surugadai, Chiyadacku, Tokyo, Japan “Department of Municipal Infrastructure, UNESCO-IHE Institute for Water Education, Westuest, 7, PO Box 3015, 2001 DA Delfi, The Netherlands “Department of Biochemical Engineering, Delft University of Technology, Jullanalaan 67, 2628 BC Delft, The Netherlands Received 11 November 2003; received in revised form 12 May 2004; accepted 28 May 2004 Abstract In biological nitrogen removal processes, nitrite ean 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 ellect of nitrite on BPR. In this study, phosphate accumulating organisms (PAO) were cultivated in an anacrobic-anoxic-aerobie 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) aerobie phosphate uptake was more uffected than anoxic phosphate uplake, (3) presence of nitrite could be one of the factors enhancing the presence of glycogen accumulating organisms (GAQ)—competitors to PAO for substrate in the anaerobic phase, and (4) il is required to monitor and control nitrite accumulation in a full-scale wastewater treatment plants, © 2004 Elsevier Lud. All rights reserved. Keywords: Biological phosphate removal; Phosphate accumulating organisms; Nitrite; Inhibition; Denitrification; Glycogen accumulating organisms 1, Introduction phosphate-accumulating organisms (PAO) are respon- sible Tor this complex process. PAO have as character Biological phosphate removal (BPR) has been intro- duced into municipal wastewater treatment to control eutrophication of surface waters and is presently a well- established process in practice. In BPR processes, *Corresponding author. Department of Civil Engineering College of Science and Technology, Nihon University, 18 Kanda-Surugadai, Chiyoda-ku, Tokyo, Japan, Tel: +8I-+ 3259-0672; fax: +81-3-3293-3318. E-mail address: saito@isilest.hon-u.ae jp (T. Saito) 0043-1354) 4:10. 1016). watres.2004.05.023, istic the potential to use intracellular polyphosphate as ‘energy reserve in the absence of electron aceeptors such as nitrate or oxygen. In an anaerobic compartment, this ccan be used (o accumulate substrate (preferential volatile fatty acids) inside the cell as polyhydroxyalkanoates (PHA) (Sato et al, 1992; Smolders et al., 1994). The consumption of polyphosphate leads to release of Phosphate into the liquid media. Under aerobic or anoxic conditions, PHA is used for growth and phosphate uptake, leading to @ net phosphate removal see front matter (> 2004 Elsevier Lid. All rights reserved.T. Saito et ak. ( Water Research 38 (2004) 3760-3768 3761 from the wastewater. By wasting phosphate-rich sludge, phosphate is removed from the water line and could eventually be further efficiently recovered by chemical coagulation (van Loosdrecht et al., 1997). Nowadays, full BPR or combination of BPR and chemical phosphate removal replace pure chemical provesses ‘more and more, mainly because of the absence of wemicals addition and the lower sludge production. However, performance of BPR can become unstable, especially when it is applied in combination with biological nitrogen removal process. This instability of BPR has been explained by several mechanisms, namely: (@) competition with glycogen accumulating organisms (GAO) (Liu et al, 1997), (b) introduction of nitrate into anaerobic phase (Hascoet and Florent, 1985), (@) excessive aeration (Brdjanovie et al., 1998). So far, there is sufficient evidence to believe that these mechanisms ean be responsible for lowering the phosphate removal capacity at full-scale plants. Still unsolved dilemmas regarding BPR exist, such as the factors favoring presence of GAO over PAO. Revently, the existence of PAO that can utilize nitrate as an electron acceptor instead of oxygen has been ‘confirmed experimentally and practically (Kerrn-Jesper- sen and Henze, 1993; Kuba et al., 1993). The merits of the anoxic phosphate removal is that both the amount of COD and oxygen required for nutrients removal can be significantly reduced, since stored PHA is used simultaneously for denitrification and phosphate uptake (Kuba et al, 1996b), Several processes have been developed making specific use of these organisms (Kuba et al, 1996b; Bortone et al., 1996; van Loosdrecht et al., 1998; Hu et al., 2001), Nitrite is a normal intermediate in the denitrification process and is therefore not a strange electron acceptor for PAO that can denitrily. The possibility of utilizing nitrite as an electron acceptor has been recently examined (Ahn et al., 2001), However, if long term studies with nitrite fed systems have, as yet not been reported in the literature, feasibility of nitrite utilization is still unclear. In fact, high concentration of nitrite in the mixed liquor is reported to inhibit anoxic phosphate uptake (Kuba et al, 1996a; Meinhold et al 1999), While there are a few reports investigating the cllect of nitrite on anoxic phosphate uptake (Meinhold etal, 1999; Ahn etal, 2001), no reports on the eet on. aerobic phosphate uptake is available, In general, nitrite exhibits strong toxicity against bacteria on their growth and respiration process (Rowe et al, 1979; Yarbrough et al, 1980) so that elevated nitrite concentrations are expected t0 negatively affect BPR process. In order to establish more stable performance of BPR process, the llect of nitrite should be adequately evaluated. This research was, therefore, conducted with the aim to evaluate the effect of nitrite on phosphate uptake under aerobic and anoxic conditions. Hereto an enriched culture of PAO bacteria was used 2. Experimental setup 21. Cultivation of shudge enriched by PAO ‘An anaerobic-anoxie-aerobie sequencing batch reae- tor (SBR) with 2L working volume and equipped with dissolved oxygen electrode and temperature (20-25°C) and pH control (7440.1), was used to cultivate sludge enriched with PAO, The cycle was a G-hour cycle consisting of 2h of anaerobic phase, 2.5h of anoxic phase, 0.5h of aerobic phase and 1h of settling and discharging phase. During the initial 3min of the first (anaerobic) phase, IL of influent solution containing acetate and phosphate (the composition is listed below) was fed into the reactor. After 2h of anaerobie phase 50ml of a concentrated nitrate solution (1,875mgN/L) was introduced during the first 90min of the anoxic phase. The influent COD/NOs-N ratio (the ratio of the amount of COD added in the anaerobic phase to the amount of nitrate added in the unoxic phase) and COD/ PO.-P ratio (the ratio of the amount of COD to the amount of phosphate in the anaerobic feed solution) were 4.3 (gCOD/gN) and 26.7 (gCOD/gP), respectively Anaerobic and anoxic conditions were maintained by slowly sparging nitrogen gas through the system. Alter 2.5h of anoxic phase, aeration was provided for 30min, ‘Aeration was suflicient to keep DO concentration above 4.0mgO2/L throughout the aerobic phase. At the end of the G-hour cycle, 1.0SL of supernatant was discharged (resulting in HRT of 12h), Sludge retention time was controlled at average 12.5 days by sludge wasting at the end of aerobic phase. The system was inoculated with fresh activated sludge from a nitrogen and phosphate removing full-scale plant in the Netherlands. The SBR was continuously operated for more than 90 days. In order to study in a controlled way, 8mg/l of allylthiour ca was regularly added to suppress nitrifying activity ‘The influent solution consisted of NaAc (400mgCOD/ 1), KsHPO, (49mg/L), KH3PO, Q8mg/L), NHACl (QO7mgiL), MgSOs-7H.0 (180mg/L), CaCh-21:0 (4mg/L), and trace elements 0.3mL/L, Composition of trace elements: EDTA (10 g/L), FeSO, 7H20 (1.54 1), HBO, (50mg/L), CuSO4-SH;0 GOme/L), MnCi-4H0 (120mg/L), KL (180mg/L), Naz MoO,-2H,0 GOmg/L), ZiSO.-7H0 (120me/L). CoClg- 6130 (150mg/L). 2.2. Batch experiments for inhibition of BPR by nitrite 2.2.1. Inhibition of phosphate uptake Phosphate uptake tests were conducted with various nitrite concentrations under three dillerent electron acceptor conditions (oxygen, nitrite and mixture of nitrite and nitrate) to examine the effect of nitrite on both anoxic and aerobic phosphate uptake. Sludge sample was taken from SBR at the end of anaerobic3762 T. Saito et ak. ( Water Research 38 (2004) 3760-3768 phase and washed by phosphate-free mineral solution Tris-buller solution was used to control pH around 7.0 For aerobic test, aeration was provided throughout the experiments. Initial concentration of phosphate and nitrite were 4SmgP/L and 0-I2mgN/L, respectively For anoxic test, nitrogen gas was provided throughout the experiment. Initial concentrations of phosphate and nitrite were 0.0mgP/L and O-I2mgN/L, respectively For the anoxic test using minture of nitrate and nitrite, initial phosphate, nitrate and nitrite concentration were 9.0mgP/L, 2.6 and 0-I2mgN/L, respectively. 2.2.2. Inhibition of oxygen respiration OfF-ine respiration measurements in a biological ‘oxygen monitor (BOM) were done to examine the effect of nitrite on oxygen respiration. Sludge was taken from SBR at the end of anaerobic phase. Oxygen utilization ale was measured under various nitrite concentrations (0-7meN/L). 2.3. Analytical procedures The performance of SBR system was monitored on # daily basis by sampling at the end of each SBR cycle phase. On several oceasions, a more detailed, so-called, SBR cycle measurements was performed. An extensive sampling program was designed for each of the batch experiments performed. Phosphate, COD (HAC), nitrate and nitrite were analyzed by Dr. Lange measurement kits, MLSS and MLVSS were measured according to Dutch standard method. Fluorescence in situ hybridization (FISH) analysis was conducted using modified method of Amann (1995). Sludge was taken from SBR at the end of the aerobic phase and was fixed by 4% paraformaiehyde solution in phosphate bufler. Hybridization was performed with 35% formamide solution at 46°C for 1.Sh, PAOMIX (Crocetti et al, 2000) labeled by Fluos, GAOMIX (Crocetti et al, 2002) labeled by Cy-3 and EUB338 probe labeled by Cy-5 were used to detect PAO, GAO and eubacteria, respectively 3. Results 3.1. Cycle measurements Typical patterns of soluble compounds are shown in Fig. |. As expected, the characteristic PAO activity was observed. During the anaerobic phase, acetate (COD) was taken up (S0mg/L) and phosphate was released GOmg/L). In anoxic and subsequent aerobic phases, phosphate was taken up, 20 and 10 mgP/ During the anoxic nitrate-feeding ph ‘mulated, although it was completely denitrilied at the tend of the anoxic phase and no nitrite was introduced 200, 100 ol 60 : g z s |é B ol Boo £ 2 E ts z ger eel : 8 é g wl 5 ol 6 Fig. 1. Cycle behaviour of soluble compounds at day 21 in SBR, ‘operation, into the subsequent aerobic phase. During the first 40 days of operation, the observed P/C ratio (the ratio of the phosphate released to the acetate consumed under anaerobic condition) in the anaerobic phase was still low and was not more than 0.2gP/gCOD. Net phosphate removal was only 33% of the influent concentration. After 40 days of operation, nitrite accumulated only in small quantities, No nitrification was observed through- ‘out the operation, because of regular addition of allylthiourea. 3.2. Sludge acclimatization ‘The accumulation of PAO in the system was relatively slow compared lo previous experiments in our labora: tory (Smolders et al, 1995; Kuba et al., 1993). This is illustrated by the development of phosphate concentra tion at the end of anaerobic and aerobic phases as shown in Fig. 2. The phosphate concentration at the end ‘of anaerobie phase gradually increased from 30mg al day 4 to 8OmgP/L at day 80 and the phosphate concentration at the end of cycle decreased from I3meP/L at day 4 to 4mgP/L at day 80. These results strongly indicate that PAO gradually accumulated in the system, However, the gradual and rather slow improve- ment of PAO performance suggests that the activity bad been suppressed for some reason. Hascoet and Florent, (1985) suggested that introduction. of nitrate into anaerobic zone reduces phosphate removal activity Kuba et al. (1994) also reported that in the presence of both nitrate and acetate, lower PIC ratio is observed, because of direct use of acetate through TCA cycle. Since in this study, neither nitrate nor nitrite wasT. Saito et ak. ( Water Research 38 (2004) 3760-3768 3763 4100 60 [Aerobie phosphate uptake = ee 40 = 0 aa $5 E BE 20 & 0} End ot anaerobic 22 6 E ao Ls goon: 28 Z g3° E20 fot aerobic pase -- =O --- £2 40 ODBP.O Ceo, 2 «40 «6080100 A Ee 20f--- perimental period (days) Fig. 2. Course of phosphate concentration at the end of 25 anaerobic and acrobie phase in SBR operation. 2 0 ~ Nivate 544... An a oS A 4 aR bie centering the anaerobic phase, and HAe was not entering the anoxic phase, these possibilities are excluded. One of the reasons could be the competition with GAO. Actually, FISH probes verified the inferred presence of GAO (see below). The low P/HAC ratio also indicates the presence of GAO (Zeng et al, 2003), Nitrite and nitrate concentration at the end of anoxic phase and the amount of phosphate taken up under anoxic and aerobic conditions are shown in Fig. 3. Interesting observation is that during the initial 40 days, nitrite accumulated in the anoxic phase (up to 20mgN/ L), but did not accumulate afterwards. Correspondingly, total phosphate uptake (the sum of anoxic and aerobic phosphate uptake) after day 40 increased much faster in comparison with the preceding period. Furthermore, 2 significant increase of aerobic phosphate uptake was observed (relative to that of anoxic). In addition, 2 two- step progress was observed in terms of phosphate release: a slow increase until 40 days and [aster increase afterwards (as documented by Fig. 2), It strongly looks as if nitrite was limiting the accumulation of PAO in the system. Moreover it seems that the limiting effect of nitrite is mainly exerted during the aerobic period, because the aerobic phosphate uptake was mainly improved. The faster increase in anaerobic phosphate release after day 40 indicates that not only. the phosphate uptake bul most likely also the growth of PAO was negatively affected. The effect of nitrite was underlined by an experimental accident at day 80. Malfunctioning of the acetate-feeding pump caused nitrite accumulation: no avetate was added in the anaerobic phase during 6 eycles and therefore nitrite accumulated. As a result of the nitrite accumulation, the phosphate removal activity drastically decreased (Figs. 2 and 3). The observed pattern of phosphate and nitrite concentration strongly indicates that nitrite could have severe effect on the activity of PAO. Moreover, FISH probing showed a strong decrease in the number of PAO after the incident (see below) o 2 40 6 80 100 Experimental periods (days) Fig. 3. Phosphate uptake under aerobie/anoxie condition in ‘one eyele (upper graph) and nitrate/nitrite concentration at the end of anoxic phase (lower graph) tn nitrate (© without nitrate (mgPigVSSh) Phosphate uptake rate o 2 4 6 & 10 12 Initial nitrite concentration (aN) Fig. 4, The effect of ii Phosphate uptake rate the: Aanoxie phosphate uptake rate nitrite concentration on sults ofthe batch experiments on 3.3. Effect of nitrite on anoxic phosphate uptake Two types of anoxic phosphate uptake tests with different electron acceptors were conducted. In the first test, both nitrate and nitrite were present in the mixed liquor to test the effect on ordinary anoxic phosphate uptake with nitrate. In the second test, nitrate was not present and nitrite was both the sole electron acceptor and the suspected inhibitor for anoxic phosphate uptake simultaneously. The initial phosphate uptake rates at varying initial nitrite concentrations are shown ia Fig. 43764 T. Saito et ak. ( Water Research 38 (2004) 3760-3768 ‘The figure illustrates a typical substrate inhibition curve obtained in the test with nitrite only. Highest phosphate uptake nate was observed around 3mgN/L of initial nitrite concentration. At nitrite concentration in excess of 3mgN/L, phosphate uptake rate gradually decreased. At nitrite concentration below 3mgN/L, phosphate uptake rate decreased because of electron acceptor limitation. In the presence of nitrate, there is always enough electron acceptor. Therefore, at low nitrite concentrations, the maximal phosphate uptake rate is not influenced by nitrite. At higher nitrite concentra- tions, the inhibition effect of nitrite was not influenced (compensated) by the presence of nitrate. Fig. $ shows the eflect of the initial nitrite concentra- tion on the denitrification rate in the form of nitrate equivalents by considering the oxidation state of nitrite relative to nitrate, Results show that denitrification rates in both tests were almost constant at around 8mgN/ EVSSh. The only exception is the test without nitrate at initial nitrite concentration less than 2.SmgN/L, whi results in lower denitrification rate due to electron acceptor limitation. No inhibition ellect was observed for denitrification up to I2mgN/L of initial nitrite concentration, These results suggest that nitrite does not have negative impact on the enzyme system related to denitrification, but rather on the enzyme system related (0 phosphate uptake and potentially polyphosphate formation. From these results, P/N ratio (the ratio of the phosphate taken up to the denitrified nitrate-equiva- lents), which expresses elliciency of denitrification for phosphate uptake, were calculated. Fig. 6 shows the relationship between the observed P/N ratio and phosphate uptake rate. It ean be clearly stated that P/ NN ratio is strongly dependent on the phosphate uptake rate, According to measurements by Kuba et al. (1996c), the maximal P/N ratio would be around 3, if ATP gained in denitrification was only used for phosphate 10 . with arate 23 a5 ~ ae we gge woes ° o 2 4 6 8 10 2 Initial nitrite concentration (mgN/) Fig. S. The effect of initia nitrite concentration on denitritiea- tion rile the results of the aeh experiments on anoxic Phosphate uptake rate 0.20 im with nitrate 0.16|.. & without nitrate -- o12). 0.08 0.04 PIN ratio (molP/mole’) 0.00 0 3 6 9 12 15 Phosphate uptake rate (mgP/gVSS.h) Fig. 6. Relationship between P/N ratio and phosphate uptake rate the results of the butch experiments on anoxic phosphate uptake rate 20 3 86 = 12 E @ ,{- Mie B PY o amon S |.4 2monn & 4}-0 6mgni -- ~ & | 6 12mann 1020 3040 Time (min) Fig. 7. Results of acrobic phosphate uptake tests in the presence of nitrite uptake. The total contribution of phosphate uptake in the energy conversions is clearly limited. It is therefore logical that P/N ratio increases linearly with increasing phosphate uptake rate, It is also clear that there is no real difference between nitrite or nitrate in this case, indicating that the other cell processes are not strongly influenced by nitrite in the studied concentration range, ‘This confirms the observation that denitrification as such is not affected by the presence of nitrite. 34. Effect of nitrite on aerobie phosphate uptake From the SBR experiments, il was already concluded that aerobic phosphate uptake might be most sensitive (o nitrite. Therefore the effect of nitrite on aerobic phosphate uptake was investigated, Batch experiments were conducted in aerobic conditions with various trite concentrations. Parts of the results are shown in ig. 7. 2mgN/L of nitrite causes already a tion of aerobic phosphate uptake (2.4 mgP;T. Saito et ak. ( Water Research 38 (2004) 3760-3768 3765 more than 6mgN/L of nitrite results in almost complete inhibition (less than 0.6meP/zVSSh), whereas the maximal phosphate uptake rate (in absence of nitrite) was 24meP/gVSSh In addition, to assess the effect of nitrite on spiration, oxygen utilization rates were measured at various nitrite levels. Parts of the results are shown in Fig. 8. After addition of nitrite, oxygen utilization rates, suddenly dropped. Observed oxygen utilization rates are 55, 25 and &gO,/m*h in the presence 0, 2 and 6mgN/L. of nitrite, respectively. The loss of oxygen respiration activity is lower than the loss of phosphate uptake rate This again shows a lower tolerability of phosphate uptake than respiration against nitrite exposure. Sensitiveness of metabolic activities (phosphate up- take and respiration) to nitrite exposure was further compared between aerobic and anoxic activities, Re- lative activity was calculated by dividing the observed activity with the potential activity, which was obtained from cach batch test as the highest activity with neither inhibition nor electron acceptor limitation. Fig. 9 illustrates the sensitivity of phosphate uptake and respiration (which is expressed as % activity) to initial nitrite concentration Nitrite up to 4meN/l is not influential for amoxic phosphate uptake, while % activity of aerobic phosphate uptake at TmgN/l_ of nitrite is already only 20%. Even at low concentration nitrite strongly inhibits aerobic activity of PAO. The cllect is clearly stronger than under anoxic conditions, At I2mgN/L, aerobic phosphate uptake was almost completely lost, while anoxic phosphate uptake was still at 65%, Similar effect is noticeable for the respiration activity. Oxygen utilization rates are strongly affected by the presence of nitrite. Approximately 2 and SmgN/L of nitrite in the system results in SO and 20% of respiration activity, respectively, whereas nitrate respiration still maintained normal at these concentrations. Potentially, this is due to the fact that nitrite is metabolized under 10 Nitrite DO (mgO2 /) ae) Nitrite addition o 3 6 8 2 6% 18 Time (min) Fig. 8. Results of BOM measurement with enriched PAO sludge. 100 Denitrification 20 %% Activity ‘Oxygen respiraion Aerobie uptake 0 5 10 15 20 Initial nitrite concentration? (maNl) Fig 9. Effect of nitrite on activity of PAO. (‘Initial nitrite concentrations of oxygen respiration tests are the calculated value from the amount of nitrite added in BOM measurements shown in Fig. 8) ‘anoxic condition. It could mean that the concentrations near the cell are lower than in the bulk liquid. 4. Discussion 4.1. Nitrite inhibition of phosphate uptake From SBR operation, we observed the interesting findings that (I) aerobic phosphate uptake and most likely aerobic growth was severely suppressed even when nitrite was only present during the anoxic phase, and 2) after a nitrite accident, bio-P activity was clearly ‘damaged. These findings suggest that nitrite has toxicity rather than inhibition against PAO, so that specific concentration or dose of nitrite (mgN/gVSS) might be a more appropriate expression than nitrite concentration, 1) to access the nitrite effect on PAO activity. In less than O.4mgN/eVSS and around AmgN/gVSS were observed as threshold for aerobic and anoxic phosphate uptake, respectively. A relatively higher tolerance of PAO's anoxic activity against nitrite probably comes from anoxic metabolism of nitrite ‘giving lower concentration near the cell While several researchers have reported widely distributed concentrations of nitrite as threshold on ‘anoxic phosphate uptake, nitrite effect on PAO activity was not evaluated properly. Meinhold et al. (1999) used sludge from a BIODENIPHO™ pilot plant and reported that at concentration in excess of SmgN/L of nitrite, ‘anoxic phosphate uptake was strongly inhibited. Ala et al, (2001) cultivated sludge containing denitrifying fraction of PAO in an anaerobic-aerobic SBR and reported that even 40mgN/L of nitrite did not show any3766 T. Saito et ak. ( Water Research 38 (2004) 3760-3768 detrimental effect on anoxic phosphate uptake. Both of them used mixed sludge so that the nitrite level on PAO. cannot be well evaluated. Lee et al. (2001) reported that sludge cultivated in an anaerobie-acrobic-anoxic-aero- bie SBR, which was experiencing nitrite on daily basis, was tolerant t0 LOmgN/L of nitrite, They explained widely reported nitrite level as the results of degree of acclimatization. However, considering that they used mixed cultures growing on wastewater, the effect of acclimatization cannot be clearly evaluated. Hu et al (2003) used enriched sludge with PAO cultivated under anaerobic and anoxic conditions. They reported that 35mgN/I of nitrite did not show negative effect on anoxic phosphate uptake. However, in their case, pH was not controlled (70-83). This range of pH could easily have chemical precipitation (Maurer et al., 1999), So far, there is only one published paper (Kuba et al, 1996a) using enriched PAO culture with properly controlled pH around 7. ‘They reported that S-l0mgN/L of nitrite strongly inhibited anoxic phos- phate uptake. However, the value was not obtained from batch test but from SBR operation. This paper is the first report evaluating nitrite effect on PAO activity by the specific batch test using enriched PAO culture and proper pH control. The experiments clearly indicate that especially the phosphate uptake process was vulnerable for nitrite. ‘The exact biochemical ba ground is unknown and deserves attention in future research, 4.2. Effect of nitrite on PAO and GAO competition Inhibition of phosphate transport may not be fatal for PAO, but obviously has severe effeet on their metabo- lism and competitiveness. If phosphate uptake is reduced, phosphate release and PHA storage under subsequent anaerobic condition will decrease. Lower PHA storage will result in less phosphate uptake under subsequent anoxie or aerobic condition. If non-poly-P bacteria, like GAO, exist in the system and if they are less sensitive to nitrite as compared to PAO, the competition for organic carbon could be much more severe for PAO. Kuba et al. (1996a) reported that enrichment culture of denitrifying PAO was affected by nitrite accumulation, P/C ratio dropped from 0.45 10 0.30 (gP/eCOD) and the lower P/C ratio lasted for relatively long period. This observation suggests that inhibition of phosphate uptake gave competitive ad- vantage to GAO in utilizing organie carbon over PAO (Zeng et al., 2003). In our study, low P/C ratios were indeed observed for a long period, supporting the suggestion of GAO in the system. Results of FISH analysis, expressed as relative abundance to total gram- (Table 1), show that not only PAO but JAO accumulated in the system. It is remarkable JAO accumulated in the system much more than also that Table | Proportion of PAO and GAO among general gram-positive bacteria (ay) ° 6s 3s PAOs (%) 20.30 30 40 2 GAOS ("0 5 50 60 o “Aller mitrte accident on day 80. PAO, which must be potentially the result of nitrite inhibition to PAO. ‘The situation worsened further on expense of PAO alier the nitrite accident on day 80, alter which the PAO population decreased to 20% on day 85. These observations clearly indicate that nitrite exposure strongly inhibits PAO activity and enhanees activity of GAO. However, the ellect of nitrite on GAO is not clear yet, and needs further study. From the present study it seems that the hypothesis that BPR can be negatively, influenced by the presence of elevated nitrite needs to be taken seriously in evaluating full-scale failures of BPR. 43. Effect of nitrite on phosphate removal in actual WwTPs Im the combined process of phosphate and nitrogen removal such as A,O process, it is well-known that phosphate removal could become unstable and less ellicient than A/O process. This is for a large part due to the direct competition for substrate between PAO and normal denitrifying heterotrophs, when nitrate is enter- ing the anaerobic compartment (Hascoet and Florent 1985). However, itis also clear that nitrite accumulation in the aerobic phase is highly detrimental for PAO. This can be either due to the low oxygen concentration that cllects the nitrite oxidizers more strongly than the ammonium oxidizers (Garrido et al, 1998). It could also be the result of elevated temperatures because ammonium oxidizers have a comparative higher growth rate al temperatures above 20-25°C than nitrite oxidizer (Hunik, 1993; Hellinga et al, 1998). This would imply that in’ warmer climates the risk for deterioration of BPR due to competition of GAO is higher. It is interesting to note that occurrence of GAO in European plants always seem to be low, whereas from Australia regular reports, significant GAO populations occur. ‘Also when BPR needs to be implemented in (warmer) industrial wastewater treatment systems, this effect should be evaluated, 5. Conelusions ‘The results of this study show that: (1) Nitiite strongly inhibits aerobic phosphate uptake and respiration rate. Therefore, more attentionT. Saito et ak. ( Water Research 38 (2004) 3760-3768 3767 should be paid at fullseale wastewater treatment plants to nitrite accumulation from the perspective Of stability of BPR process, ») Aerobie phosphate uptake is more sensitive to nitrite in comparison to anoxic phosphate uptake. Systems which are characterized by high contribution of anoxic phosphate uptake to total phosphate uptake, could have comparably more stable performance regarding BPR, especially when it is applied at installations in which nitrite easily accumulates in mixed liquor. (3) Nitrite could be an important factor in the competi: tion between PAO and GAO bacteria. References ‘Ahn, J., Daidou, T, Tsuneda, S., Hirata, A., 2001, Metabolic behavior of denitrifying phosphate-accumulating organisms under nitrate and mitrle electron aeveptor eonektions J. Biosci. Bioeng. 92 (5), 442 446. Amann, R.L, 1995. In situ identification of microorganisms by ‘whole cell hybridization with FRNA-targeted nucleic acid probes, In Molecular Microbial Ecology Manual. Kluwer ‘Academic Publications, Dordrecht Bortone, G., Saltareli, R. Alonso, V., Sorm, R., Wanner, J. Tilche, A., 1996. Biological anoxic phosphorus removal the DEPHANOX process. Water Sci, Technol, 34 (1 2), 119 128. 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