Positive role of nitrite as electron acceptor on anoxic denitrifying phosphorus removal process

Chinese Science Bulletin◎2007SCIENCE IN CHINA PRESSSpringerPositive role of nitrite as electron acceptor on anoxicdenitrifying phosphorus removal processHUANG RongXin', LI Dong', LI XiangKun', BAO LinLin', JIANG AnXi1' & ZHANG Jiel'.2f1 School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China;2 School of Architecture and Civil Engineering, Being Univerity of Technology, Beijing 10022, ChinaLiteratures revealed that the electron acceptor-nitrite could be inhibitory or toxic in the denitriftyingphosphorus removal process. Batch test experiments were used to investigate the inhibitory effectduring the anoxic condition. The inoculated activated sludge was taken from a continuous double-sludge denitrifying phosphorus and nitrogen removal system. Nitrite was added at the anoxic stage.One time injection and sequencing batch injection were carried on in the denitriftying dephosphorusprocedure. The results indicated that the nitrite concentration higher than 30 mg/L would inhibit theanoxic phosphate uptake severely, and the threshold inhibitory concentration was dependent on thecharacteristics of the activated sludge and the operating conditions; instead, lower than the inhibitoryconcentration would not be detrimental to anoxic phosphorus uptake, and it could act as good electronacceptor for the anoxic phosphate accumulated. Positive effects performed during the denitrifyingbiological dephosphorus all the time. The uility of nitrite as good electron acceptor would provide anew feasible way in the denitrifying phosphorus process.denitrifying phosphorus removal bacteria, nitrite, electron acceptor, inhibitory concentrationFrom the beginning of 1960s, the eutrophication in to anoxic phosphate uptake and can serve as electronclosed water is becoming more and more severe over the acceptor for anoxic phosphate uptake. Exposure toglobel!. Biological nutrient removal (BNR) technology higher concentration levels (roughly 8 mg NO2-N/L oris developing and the application focuses on the waste-greater) inhibits anoxic phosphate uptake completely,water treatment domain4. In the early studies of bio- and toxic aerobic phosphate uptake severelyI4.51.logical nutrient removal processes, it was assumed thatThe validation of the positive role of nitrite in phos-phosphorus- accumulating organisms (PAOs) could notphorus removal may allow one to exploit phosphorususe nitrate or nitrite as electron acceptor, sometimes theremoval even when nitrite accumulation is taking placeintermediate products of biological nitrogen removal in an anoxic process. In this study, several types of batchprocess, nitrate and nitrite, could become inhibitory ortests were conducted under different concentrations oftoxic substances in BNR process3- 51. However, severalnitrite and different operation modes by using DPBrecent publications have reported that phosphorus re-sludge. The purpose of this research is to investigate themoval occurs in the presence of nitrate in activatedthreshold inhibitory concentration of nitrite, validate thesludge system'o 10. Using a chemically bond oxygen in role of nitrite in phosphorus removal process, and ex-NO,-N as electron acceptor, which was called denitri-plore a feasible way to know how the DPB work utilizesfying phosphorus removal bacteria (DPB) technology,Received October 24, 2006; accepted March 27. 2007had gained acceptance in the BNR processes. Thedoi: 10.1007/511434-007-0315-9experiments"- 5 show that nitrite at low concentration'Corresponding author (email: huangrongxinhit@ 163.com)Supported by the Science and Technology Project of Heilongiang Province (Grantlevels (up to about 4- 5 mgNO2-N/L) is not detrimental No. GA0IC201-03)www.scichina.com www.springerlink.comChinese Science B中国煤化工o.16 21792183"TYHCNMHGnitrite as electron acceptor in the denitrifying phospho-Table 1 Composition of the nutrient solutionrus removal system.ChemicalsConcentrationKI0.031 Materials and methodsNaMoO4. 2H2O0.061.1 Materials and equipmentMnCl2. 4H2O0.12CuSO4.5 H2OThe lab-scale continuous double-sludge denitrifyingFeCl3.6H2O1.5phosphorus and nitrogen removal system used for ex-CoCl2.6H2O0.15periments is shown in Figures 1 and 2. Raw municipalZnSO4.7 H2Owastewater is pumped into anaerobic tank (1), wherephosphate is released and most organic substrates areTable 2 The influent characteristic of two different batch tests (unit:absorbed and predominantly stored as PHB (poly-B-hydroxybutyrate) in bacterial cells. The first settler (2)TermCOD TP NHCl T(C) pHseparates DPB (denitrifying phosphorus removal bacte-Max/min300/200 32/0 45/34 25 7.6/6.8P-release batch tests25ria) activated sludge from ammonia and phosphorus-richP-uptake batch tests≤30 30. 3825 7.4supernatant. The supernatant then goes into contact oxi-dation tank (3) where nitrification occurs. Nitrification1.2 Experimental proceduresludge in the second settler (4) is pumped back into re-actor (3). The organic-substrate-accumulating sludge inThe reactor had a working volume of 4 L. Each batch ofsettler (2) bypass the nitrification tank and is mixed withoperating cycle lasted for 8 h, which included a 120-minthe nitrified effluent from (3) together in anoxic stageanaerobic phase and a 300-min anoxic phase with each(5). Here nitrate or nitrite is removed mainly by DPBbeing followed by a 30-min settling phase. Syntheticsludge as electron acceptor for anoxic P-uptake. Post-wastewater was pumped into the reactor during the firstaeration step (6) allows air stripping from the sludge5 min of the anaerobic phase. At the end of the settingbefore settler (7).stage, 2 L wastewater was discharged. In the anoxicSludge obtained from the lab-scale continuous douphase, nitrite solution (5 mg/mL) was pumped into theble-sludge denitrifying phosphorus and nitrogen removalreactor with intermittent feeding during the first 240 min.system (Figure 1)[2] was used. A cylindrical vessel withSludge retention time (SRT) was maintained for about4 L working volume was used as a reactor. Synthetic10 d by wasting sludge mixed liquor at the end of aero-wastewater was used for the operation consisting of so-bic or anoxic stage. During the anoxic phase, nitrogendium acetate (used as carbon source), KH2PO4 (phos-gas was purged above the liquid surface to secure strictphorus source), NH4CI. Other trace components in syn-anoxic condition and the liquor was mixed at the samethetic wastewater are shown in Table 1. The water char-time. The pH was also strictly controlled at 7.0- 8.0 byacteristics of influent at the two different batch tests are addition of 0.5 mol/L HCI or NaOH to avoid phosphatelisted in Table 2.precipitation throughout the experiment.3InfluentEfluentAiPeristalticpumpNitrification sludge recycleSludge bypassSludge recycleFigure 1 The layout of two-sludge denitrifying BNR process. 1, Anaerobic stage; 2, 4, 7, settler; 3, contact oxidation stage; 5, anoxic stage; 6, post-中国煤化工2180HUANG RongXin et al. Chinese Science Bulletin | August 2007 IvMYHCNMH G4supplied to all the fermentors throughout the batch teststo maintain anoxic condition. Phosphorus, nitrite andMLSS were monitored in the batch tests to study at whatconcentrations the nitrite works.1.3 Analysis item and methodsThe water samples were filtered through 0.45 um fibermembrane before analysis. Nitrite nitrogen (NO2-N) andorthophosphate (PO4-P) were analyzed by UV-2550 UI-traviolet Spectrophotometer (Shimadzu Company, Ja-pan). COD and MLSS were measured according to Na-tional Standard Methods (APHA, 1985).Figure 2 The real experimental equipment of the two-sludge BNR2 Results and discussion1.2.1 Phosphorus release batch tests. The batch tests2.1 Qualification of nitrite inhibiting concentrationwere conducted under strictly anaerobic state. Thin the denitrifying biological phosphorus removalsludge was taken from a lab-scale denitrifying phospho-Nitrite solutions of different concentrations were addedrus and nitrogen removal system at the end of anoxicrespectively at the end of anaerobic period when theP-uptake phase. 2000 mL of sludge (MI .SS betweenphosphorus-release was finished. Then the momentary3000 and 3500 mg/L) was put into five 4L laboratoryconcentrations of nitrite-nitrogen, and total-phosphorus infermentors and sufficient carbon source was added (thethe beaker were measured. At the same time, the capabilityamount of carbon source was determined by previousand ratio of phosphorus uptake were determined andbatch tests). COD, phosphorus and MLSS were moni-compared. The detailed results are reported in Figure 3.tored in the batch tests. N2 gas was fed continuously intothe fermentors to prevent the invasion of air. Each batch40test lasted for 2 h.351.2.2 Phosphorus uptake batch tests. In this stage,synthetic wastewater provided enough phosphorus望25sources (the concentration of total phosphorus was about2030 mg/L) and carbon sources as lttle as possible. Toomany carbon sources would result in the denitrifying10一-Input35mLnitrogen removal other than the phosphorus removal in-+Input30mLwork. The sludge was loaded with sufficient nitrite at5t -✧Input 25 mL- Input 20 mLanoxic conditions. The nutrient solution was also pu40 8012016200 240into the mixed liquor at a flow of 1 mL per 5 L waste-Time (min)water along with the experiments. N2 gas was also usedFigure 3 The TP change under different nitrite concentrations with oneto prevent the air invasion.time injection.1.2.3 Nitrite inhibition batch test. A series of batchtests were conducted to assess whether nitrite wouldFigure 3 shows that nitrite can be used as electronindeed inhibit phosphorous uptake. After 2-h anaerobicacceptor without affecting the denitrifying phosphorusstage, 2-L synthetic wastewater (about 30 mg/L-P anduptake basically when lower than 30 mL of the nitritethe MLSS between 3000 and 3500 mg/L, few COD re-solution was injected. It could be seen that phosphorusquirements) was put into anoxic beakers before theremoval ratios of the 25 and 20 mL one-time injectioncommencement of the tests. Different amounts of nitritewere different, the latter was better than the former ob-(with initial concentrations of 20, 25, 30, 35, 40 mg/L)viously. There was no phosphorus-releasing found. Bywere added into the five fermentors at the beginning ofcontrast, no phosphorus uptake brings forth when 30, 35the study. The batch test lasted for 5 h. The N2 gas wasand 40 mL of nitrite solution were added at the begin-中国煤化工HUANG RongXin et al. Chinese Science Bulletin | August 2007 | \2181YHCNMH Gning of the stage. Phosphorus uptake was severely in-NO-N solution: 10 mhibited, and the phosphorus released into the mixed liq-NO-N solution: 15 mL| NO3-N solution: 5 mLuor by means of the bacteriolysis. Because the concen-20tration of nitrite solution was too high to exceed the en-→TP18durance concentration scope of denitrifying phosphorus十Nitrite-nitrogen |16bacteria, the inhibitory function happened. The DPB21A14died or dissolved into the water. The phosphorus uptake|1211appeared again after the inhibitory phenomenon, but8lasted for a short time. As seen clearly from Figure 3, theshortest inhibitory time was 90 min with 30 mL injec-tion; the longest inhibitory duration appeared with the40 mL injection, and lasted for the whole 240 min dur-0 30 60 90 90120150 180210 240 240 270300ing the anoxic period.Time (min)There was a platform in the total phosphorus changecurve when different concentrations of high strengthFigure4 The TP and NO2 -N change curves with nitrite solutions underinterval addition.nitrite solution were added at volumes of 30, 35 and 40mL respectively. The so-called “inhibitory platform”4.0occurs 30, 90 and 120 min later before the commence-- - Maximum P-uptake ratioment of anoxic phase. The values of nitrite-nitrogen con-3.0centration were 30.23, 34.87 and 38.75 mg/L at theseplatforms. Anoxic phosphate uptake was inhibited se2.0 tverely when DPB exposed to high concentration of morethan 30 mg NO2 -N/L, which led to the halt of phospho-.0上rus uptake.2.2 Positive role of phosphorus uptake at the anoxic750100507200250300phase with intermittent input of nitriteThe experiments on high concentration of one-time .Figure 5 The maximum phosphorus uptake rate under each additioninjection showed that the inhibitory function was done phase.when the concentration of nitrite was higher than 30mg/L. The concentration of nitrite is hard to reach 25Figure 4 shows that it is possible to use nitrite asmg/L usually except in the shortcut nitrification reac-electron acceptor to achieve the P-uptake during the an-tor.oxic phase so long as its concentration does not exceedConsidering that there exists a limited concentration an inhibition level.of 30 mg/L of nitrite, the chemicals were input for threeAs shown in Figures 4 and 5, the mixed liquor waspassels, one was 5 mL nitrite solution injection (theabundant in nitrite at the beginning of first stage, so theconcentration of about 5 mg/mL) at the commencementmaximum P-uptake ratio came out during this phase.of the experiment; another injection was in 90 min afterThe value was 3.99 mgTP/(gMLSS .h), and the nitritethe first passel of nitrite injection, which was based on a decreased to near zero in 90 min. Then the second addi-lot of previous prophase experiments, at that time thetion of another 10 mL nitrite solution commenced. Thenitrite in the liquor had reacted away; the third 5 mL maximum P-uptake ratio decreased to 2.16 mgTP/injection happened 240 min later. Al1 the experiments(gMLSS .h). Shortage of nitrite was caused by "secon-ended in 5 h. The nitrite concentration was close to zero dary phosphorus-release" in the liquor at the end of thisat that time. The commencement concentrations of totalphase. When less amount of 5 mL nitrite solution wasphosphorus and MLSS were about 27.35 and 3000 mg/L，added, nitrite was used as electron acceptor. The DPBthe concentration change curves of total phosphorus and began to take up more phosphorus from the liquor oncenitrite are shown in Figure 4 and the correspondingagain. But during this period, the phosphorus absorptionmaximum P-uptake ratios are shown in Figure 5.rate is only 1.63 mgTP/(gMLSS .h).中国煤化工2182 .HUANG RongXin et al. Chinese Science Bulletin | August 2007 |vMYHCNMH GHowever, from basic microbiological viewpoint, it is(1) Nitrite was not always an inhibitor to BNR proc-not clear why nitrate or nitrite could not be used as aness, and it was possible to use nitrite as electron acceptorelectron acceptor for phosphorus removal. Using a so long as its concentration did not exceed an inhibitionchemically bond oxygen as electron acceptor has ad-level (which was dependent on the type of sludge andvantages such as COD and energy (aeration) saving, andoperating condition). Instead, it is an effective alterna-a lower sludge production in the overall phosphorus andtive electron acceptor to oxygen or nitrate.nitrogen removal process 16,17]. Although nitrite may not(2) Nitrite concentrations (below 25 mgNO2-N/L) didbe as good as nitrate in replacing oxygen as electronnot adversely affect anoxic phosphate uptake in acti-acceptor, it is possible to use nitrite as electron acceptorvated sludge. Below this concentration range, phosphateso long as its concentration does not exceed an inhibi-uptake ratio increased with the increasing of nitritetion level, which is dependent on the characeristics ofconcentration.(3) Exposure to higher nitrite concentrations (highersludge and operating conditions.than 30 mg NO2-N/L), anoxic phosphate uptake was3 Conclusionstotally inhibited. The inhibition lasted for several hoursafter the nitrite exposure. The critical nitrite concentra-In this study, the maximum inhibitory concentration of tion, in which severe nitrite inhibition of phosphate up-nitrite-nitrogen was examined, after that the positive roletake occurs, has been found varying from experiments toof nitrite in the denitrifying phosphorus removal processexperiments and, hence, the performance efficiency ap-was investigated by means of intermittent injection. 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Water Res,lease and uptake under alternating anaerobic and anoxic conditions in2004, 38: 3760- 3768a fixed-film reactor. Water Res, 1994, 28: 1253- 12554 Jnkins D, Tandoi V. The applied microbiology of enhanced biological12 Kerm-Jespersen J P, Henze M. Biological phosphorus uptake underphosphate removal accomplishments and needs. Water Res, 1991, 25:anoxic and anaerobic conditions. Water Res, 1993, 27: 617- - 6241471- 14783 WeonS Y, Lee C W, Lee S I, et al. Nitrite inhibition of aerobic growth5 Van Starkenburg W, Rensink J H, Rijs G B J. Biological P removal:of acinetobacter sp. Water Res, 2002, 36: 4471- 4476State of the art in the Netherlands. Water Sci Tech, 1993, 27: 14 Jens M, Eva A, Steven I. Effect of nitrite on anoxic phosphate uptake317- -328in biological phosphorus removal activated sludge. Water Res, 1999,6 Kuba T, Smolders G J F, van Loosdrencht M C M, et al. 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