interaction of denitrification and p removal in anoxic p removal systems

Desalination 201 (2006) 82–99

*Corresponding author.

Interaction of denitrification and P removal in anoxic P removalsystems

Jignesh Patel, George Nakhla*Department of Chemical and Biochemical Engineering, The University of Western Ontario,

London, Ontario, N6A 5B9, CanadaTel. +1 (519) 661-2111 ext. 85470; Fax +1 (519) 850-2921; email: [email protected]

Received 30 April 2004; accepted 11 March 2006

Abstract

This paper compares municipal wastewater (MWW) as a carbon source with other volatile fatty acids (VFAs)such as acetic acid, propionic acid and butyric acid in biological nutrient removal (BNR) processes. Biomassspecific denitrification rates (SDNR) using acetic acid, butyric acid, propionic acid, MWW and primary effluentfrom batch studies were 107.2, 77.1, 37.9, 31.1 and 30.0 mg NO3–N/g VSS·d respectively. SDNR for acetic acidderived from batch experiments and respirometric studies were identical while for the MWW the discrepancybetween the two methods was about 31%. Phosphorus (P) release was observed only after nitrate concentrationdropped to <1 mg/L. The extent of P release and subsequent uptake was highest with acetic acid and lowest withpropionic acid. P release correlated negatively with nitrate uptake under anaerobic conditions.

Keywords: Volatile fatty acids; Denitrification; Phosphorus removal; Respirometer; Municipal wastewater

1. Introduction

One of the major focus areas of current re-search in biological nutrient removal (BNR) is tomake the best use of available chemical oxygendemand (COD) for nitrogen (N) and phosphorus(P) removal. Effective P removal selectivelyrequires the existence of short-chain fatty acidsand hence the nature of the organic substanceplays a key role in P removal. A study performedon a sequential batch reactor (SBR) [1] with

glucose proved that the presence of volatile fattyacids (VFAs) was critical in the selection of abacterial population with a high capacity forenhanced biological phosphorus removal (EBPR).Carlsson et al. [2] have demonstrated that it isprimarily VFAs in the influent wastewater thataffect the release of phosphorus. Experimentalwork done by Abu-ghararah et al. [3] showed thatpropionic acid was less effective for EBPR thanacetic acid in short-term experiments with MWW.Summarizing data from four full-scale systemswith prefermenters (in Canada and Australia), Von

0011-9164/06/$– See front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.desal.2006.03.522

J. Patel, G. Nakhla / Desalination 201 (2006) 82–99 83

Muench [4] showed acetic acid ranged from 49to 71% and propionic acid ranged from 24 to 33%of total influent VFAs (by weight). Moreover,acetate and propionate are the two most commonVFAs present in domestic wastewaters [5].

Combining simultaneous nitrification denitri-fication (SND), via nitrites, with anaerobic–anoxicEBPR has the capacity of achieving simultaneousN and P removal with a minimal requirement forCOD [6]. In systems with SND, higher denitri-fication rate and a lower biomass yield have alsobeen reported during aerobic growth comparedto conventional nitrification–denitrification sys-tems [7,8]. Moreover, it has been reported thatdenitrification can be accomplished by denitrify-ing phosphate accumulating organisms (DPAOs)using the same COD, in anaerobic–anoxic EBPRsystems, allowing simultaneous nitrate/nitrite re-duction and P uptake [9,10]. Furthermore, DPAOsare 40% less efficient in generating energy, andthus have a 20–30% lower cell yield compared toaerobic polyphosphate accumulating organisms(PAOs) [11,12]. The limitation of C: N: P for BNRprocess has been primarily addressed for aerobicP uptake processes in the past [13–16] and hencethe lack of these ratios for anoxic P uptake isapparent.

The delicate balance between organic carbon,N and P levels has a major impact on the degreeof P removal that can be achieved in BNR systems.EBPR from domestic sewage with low/mediumorganic content is not sufficient when denitrifica-tion preferentially competes for available carbonsource [17]. In the anoxic P uptake, the competi-tion for carbon between denitrifiers and PAOs isless pronounced because DPAOs can sequentiallyor simultaneously uptake nitrate and P utilizingthe same available readily biodegradable COD(RBCOD). Other advantages of the anoxic P up-take are separate optimization for P and N removal,smaller reactor volumes, lower energy require-ments, reduction of oxygen requirements, minimalutilization of oxygen for P removal and minimalloss of COD by aerobic oxidation [18]. Having

mentioned this, the nature and magnitude ofRBCODs play an imperative role in anoxic P uptake.

While most of the work done on nature of car-bon sources largely focused on synthetic sub-strates i.e. acetic acid, propionic acid and butyricacid [1,5,19–23], interaction of mixed substratesin a complex matrix such as in MWW has notbeen thoroughly investigated. Furthermore, sincefull-scale EBPR plants have been predicated onaerobic P uptake, research on the impact of carbonand C:N:P ratios for anoxic P uptake has beenscant. No quantitative correlation between P re-lease and nitrate uptake has been delineated. Themethods utilized for these studies primarily track-ed the fate of soluble species in water. Literaturestudies [24,25], on domestic wastewater supple-mented with acetate/methanol, have shown that Prelease occurs only when denitrification is com-pleted, i.e. when nitrate concentration dropped be-low the detection limit, along with a simultaneousdrop in redox potential. A few other researchers[26,27] have reported the release of P in the pre-sence of nitrate together with a synthetic substratesuch as meat extract or acetic acid. However, Prelease decreased with increasing concentrationof nitrates. Gerber et al. [28,29] have studied therelease of P in the presence of nitrate upon additionof three of the twelve substrates investigated, i.e.formate, acetate and propionate while P releasewas not induced until the nitrate was consumedin all other substrates studied.

The overall objective of this study is to shedmore light into the intricate correlation of nitrateuptake and P release (as discussed above), evaluatethe impact of alternative carbon sources and theirnature on denitrification and P removal, and toexamine the fate of NO3–N and P simultaneouslyduring the anoxic phase. This paper mainly de-scribes the impact of different substrates on anoxicP removal and denitrification. Also the depend-ency of P removal on nitrates has been studied.Additionally the paper demonstrates the feasibilityof using anoxic respirometry to determine denit-rification rates.

84 J. Patel, G. Nakhla / Desalination 201 (2006) 82–99

2. Materials and methods

2.1. System description

Fig. 1 shows the detailed experimental set upfor biological nutrient removal using a modifiedmembrane bioreactor (MBR) system. The systemis novel and innovative in terms of removing twobasic units, fixed-film nitrification and final settler,from the DEPHANOX process [30] hence con-serving much of the installation, operational andmaintenance costs. Canadian and US patentapplications for the system have been recentlyfiled [31].

The system consists of anaerobic, anoxic andaerobic acrylic reactors of 5, 5 and 10 L respec-tively and a clarifier made of clear PVC of 15 Lvolume (working volume 10 L). ZW-1 (ZENON

Fig. 1. Schematic of MBR process for biological nutrient removal.

Environmental Systems Inc., Oakville, ON) mem-branes with a pore size of 0.04 µm and nominalmembrane surface area of 0.047 m2 were immers-ed in the aeration tank and were used as a finalseparation unit. In the anaerobic reactor, VFAsare sequestered by the poly-P bacterial cells(PAOs) as poly-β-hydroxyalkanotes (PHA) whilephosphate is released and most of the organicmatter is biologically adsorbed onto the sludgeflocs. After the anaerobic reactor, the NH4-richsupernatant is separated from organic substrate-rich sludge in the clarifier. The sludge is taken tothe anoxic reactor for denitrification and P uptakewhile supernatant is sent to aerobic reactor by-passing the anoxic stage for external nitrificationand P uptake. Mixed liquor is recycled fromanoxic to anaerobic and from aerobic to anoxic

ANAEROBIC BIO-REACTOR

P release & COD removal

P uptake & Denitrification

Sludge (Organic-rich)230 .4 L/d

Supernatant (NH4 and PO4 rich)51.2 L/d

Effluent

Recycle 2240 L/d

Recycle 1201.6 L/d

AIR

WastewaterStorage Tank

Valve

Pump

P Vacuum Gauge

MEMBRANE BIO-REACTOR

P uptake & Nitrification

P

Submerged Membrane

(ZW - 1)

CLARIFIER

ANOXIC BIO-REACTOR

Drain

Overflow(Sludge Wastage)

Effluent WaterTank

Influent80 L/d

Sludge Wastage


reactor in order to keep the biomass concentrationconstant in the system. Air (2–4 L/min), intro-duced at the bottom of the membrane modules,served two purposes: supplying oxygen for thebioreactor and creating turbulent flow around themembrane surface to minimize membrane fouling.Peristaltic pumps (Masterflex, Model 77521-40,77200-12, 77200-60, IL, USA) were used to trans-fer wastewater from one unit to another and avacuum pump was used to collect effluent fromthe membranes and to transfer it to the effluenttank. All three reactors were continuously mixedusing overhead laboratory mixers (Stir-Pak,Model 4554-14, 3–250/50–5000 rpm, IL, USA).Returned activated sludge from a local wastewatertreatment plant was used for startup. The influentsynthetic and municipal wastewater was fed at aflow rate of 80 L/d to the system at a hydraulicretention time (HRT) of 6 h and sludge retentiontime (SRT) of 10 d to monitor the biologicalnutrient removal process.

The synthetic wastewater (SWW) used in the

Table 1Influent characteristics of synthetic and municipal wastewater

*numbers within parenthesis are number of samples

SWW MWW Parameter (mg/L) Range Average ± SD (n) Range Average ± SD (n)

TSS 0–8 2±3 (11) 82–190 160±35 (11) VSS 0–8 2±3 (11) 72–176 128±29 (11) COD 269–377 300±19 (19) 230–399 329±56 (11) SCOD 254–324 293±22 (19) 91–165 123±28 (11) BOD 186–288 246.8±36.5 (10) 110–192 159±28 (10) SBOD 156–276 237.2±34.7 (10) 60–108 82±17 (10) TKN 22–35.8 27±4.4 (9) 12.8–28.9 22.1±5.8 (10) STKN 22–35.8 27±4.4 (9) 12–25.5 17.5±4.1 (10) NH4

+–N 21.9–29.5 25±2 (19) 9.7–19.9 14.7±4 (13) NO3–N 0.4–1.2 0.75±0.2 (19) 0–0.7 0.3±0.4 (13) NO2–N 0.006–0.084 0.032±0.026 (19) 0–0.590 0.136±0.204 (13) Total P 4.4–6.6 4.8±0.5 (19) 3.7–5.6 4.4±0.7 (13) SP (PO4–P) 4.3–6.6 4.8±0.6 (19) 1.5–4.2 2.2±0.7 (13) COD:TKN — 10.6 — 14.9 COD:TP — 57.4 — 74.8 COD:N:P — 100:9.4:1.7 — 100:6.7:1.3 Alkalinity as CaCO3 240–265 250 190–262 228.4

above system contained trace metals, salts, N, Pand carbon sources necessary for biomass growth.Sodium acetate and glycerol contributed to thetotal influent COD of about 300 mg/L. Ammon-ium sulfate and potassium phosphate monobasic/dibasic were added as a source of N (~25 mg/L)and P (~5 mg/L). Other trace metals included inthe waste were magnesium, calcium, potassium,iron, copper, molybdenum, manganese, zinc andcobalt. Sodium bicarbonate was used to maintainthe alkalinity. To test the system with real wasteat the later stage, the grit chamber effluent fromthe Adelaide Pollution Control Plant (London,ON) was fed as a MWW. The typical charac-teristics of the SWW and MWW used in the abovesystem are shown in Table 1.

2.2. Respirometric tests

Respirometric tests were conducted using anAER-208 respirometer (Challenge EnvironmentalSystem Inc., Fayetteville, Arkansas, USA). The


respirometer tests consisted of eight 250 mL reac-tion bottles, flow measuring cells, interface mod-ule and a computer for data processing and stor-age. The reaction bottles were maintained at a con-stant temperature of 23°C using a water bath andwere mixed continuously (90 rpm) using magneticstirrers. When operating in the anoxic mode, gasesproduced by biological reactions in bottles flowthrough each cell under the influence of a slightpressure buildup inside the bottles. A KOH trapconsisting of KOH pellets was used to removecarbon dioxide present in the gases produced.

The respirometer was used to study the effectof different carbon sources on specific denitrifica-tion rates (SDNRs) under anoxic conditions. Car-bon sources tested were acetic acid, MWW, andcarbon adsorbed onto sludges. Duplicate reactionbottles were prepared for each of these carbonsources for accuracy measurements. The seed usedin the study was taken from the anoxic reactor ofthe aforementioned modified MBR system. In thecase of acetic acid, 250 mL of seed was used with0.1 mL of concentrated acetic acid while forMWW, 200 mL of seed was added to 50 mL ofMWW. For carbon adsorbed onto sludges, 125 mLof anaerobic sludge was taken along with 125 mLof seed. All the bottles were finally purged withnitrogen (N2) gas. NO3–N (20 mg/L) was addedin the form of KNO3 (potassium nitrate) to eachreaction bottles and the initial samples were col-lected soon after. A control was prepared withoutany carbon source and sodium azide of 1g/L toinactivate microorganisms.

2.3. Batch experiments

The batch reactor (1 L) was set up to test theeffect of different carbon sources on P removaland observe the effect of denitrification on P re-lease under sequencing anoxic–aerobic condi-tions. The different carbon sources used in thestudy were acetic acid, butyric acid, propionicacid, MWW and primary effluent. Effluent of thegrit chamber of the ‘Adelaide Pollution Control’

plant, London, ON was used as MWW and pri-mary effluent was collected from the same plantafter passing through primary clarification. Theseed used in the study was taken from the anoxicreactor of the aforementioned modified MBRsystem. The concentrated form of acetic acid(0.3 mL), butyric acid (0.19 mL) and propionicacid (0.3 mL) was mixed with one liter of seed toprepare different batches to result in initial CODsin the range of 300–400 mg/L to reflect the waste-water strength. In the case of MWW and primaryeffluent, 500 mL of each were mixed with 500 mLof seed. NO3–N was added in the form of KNO3(potassium nitrate) while P was added in the formof KH2PO4 (potassium phosphate monobasic) toeach batch test.

2.4. Analytical methods

The samples from the batch reactor and res-pirometer were analyzed for total suspended solids(TSS) and volatile suspended solids (VSS) accord-ing to standard methods for the examination ofwater and wastewater (methods 2540 D and 2540 E)[32]. The soluble fraction of the mixed liquor ob-tained by centrifuging (3000 rpm for 10 min,Beckman Coulter, Allegra 6 series, California,USA) and then filtering through a 0.45 µm glassfiber filter paper (Whatman, 47 mm 1822 047)was used for analysis of soluble components, i.e.soluble COD (SCOD), soluble phosphorus (P) andNO3–N. HACH equipment (HACH Odyssey DR/2500 spectrophotometer and COD heating reactor)were used to measure SCOD (method 8000),NO3–N (method 10020) and soluble phosphorus(method 8114). For all parameters, individual stan-dards were run during each analysis. Calibrationverification standards were prepared and analyzedfor spectrophotometer calibration. All samples andstandards were stored in an air tight container andrefrigerated. Test results were accepted only if theminimum accuracy of measuring the standardswas 95%. Also occasionally samples were givento a certified local environmental laboratory foraccuracy checks.


3. Results and discussion

In the comparative study of BNR by modifiedMBR system [33] with the SWW and MWW, itwas observed that P release in the anaerobicreactor was lower when treating MWW thanacetate-based SWW. For the SWW run the systemwas operated for 4 turnovers of the mean SRTwhile for the MWW run it was operated for 6 turn-overs of the mean SRT to ensure attainment ofsteady state. The measured steady-state averageP content (%P), by weight based on VSS, of thesludges was 6.2% during the SWW run and 4.1%during the MWW run. As depicted in Fig. 2, Prelease was in the range of 2.7–7.9 g/d(corresponding to 9.5–28 mg/L based on theanaerobic flow of 281.6 L/d including recycle)for the SWW run vs. 0–0.8 g/d (corres-pondingto 0–2.8 mg/L) for the MWW run. The same trendof lower P uptake in the MWW run vis-à-vis theSWW run was observed with respect to the Puptake in the anoxic reactor (Fig. 3) with P uptakeof 1.2–6.1 g/d for the SWW run and 0–0.8 g/d forthe MWW run.

In both the SWW and MWW runs, 96% of Premoval (corresponding to 4.2 mg/L) was ob-

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90 100Time (days)

Solu

ble

Phos

phor

us (g

/d)

AN infAN eff

SWW MWW

Fig. 2. Phosphorus release in anaerobic reactor.

served in the system. Effluent P of ~0.1 mg/L wasalso observed in both cases. Despite the sameoverall P removal, it is interesting to contrast theP release and P uptake patterns for both waste-waters. The lack of high P release during theMWW run may be attributable to the very lowconcentrations of RBCOD in the raw wastewater.The acetic acid and propionic acid in the MWWwere ~40% and 33% of the total VFA concen-tration (based on COD) respectively and the con-centration of the total VFAs was about 125 mg/Las COD. To compare, Von Muench [4] has observ-ed that acetic acid ranged from 49 to 71% andpropionic acid ranged from 24 to 33% of totalinfluent VFAs (by weight). The acetic acid con-tributed only 13% of total COD of the MWW inthis study. However, that the system was capableof removing P for 6 turnovers of the mean SRTwithout a significant P release. A mass balanceperformed on the solids and P shows that the totalcumulative MLVSS wasted during the MWW runwas 422 g while the observed P removal acrossthe system was about 20.8 g. Accordingly, P re-moval due to EBPR corresponded to about 11.1 gafter accounting for the 9.7 g of P wasted with thebiomass based on the typical P content of 2.3%.


0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90 100Time (days)

Solu

ble

Phos

phor

us (g

/d)

ANO infANO Eff

SWW MWW

Fig. 3. Phosphorus uptake in anoxic reactor.

Fig. 4 shows the correlation between P releaseand NO3–N uptake in the anaerobic zone of themodified MBR system. It clearly shows a goodcorrelation for the SWW run indicating that Prelease under anaerobic conditions was inverselyproportional to nitrate uptake. Consequently, therewas low P release in the range of 9.5–18.8 mg/Lwhen nitrate uptake was more than 2 mg/L. Con-versely, P release was in the range of 20.7–28.0 mg/Lwhen nitrate uptake was lower than 2 mg/L. Thereason for poor correlation in the case of MWWrun is the very low P release, thus increasing theimpact of experimental error. These observationsemphasize that in order for P release to occur,nitrate concentrations must be below 2 mg/L andalso confirm that denitrification precedes P releaseeven for DPAOs in the same fashion as reportedfor conventional EBPR systems. Thus whileDPAOs have the ability to consume P and nitratessimultaneously and sequentially [9,10], they pre-ferentially uptake nitrates until the oxidation–reduction potential (ORP) is sufficiently low tomaintain true anaerobic conditions, after whichthey switch to P release. This implies that forDPAOs the separation of P uptake, release and

denitrification is strictly a function of the nitratesand consequently the ORP. Thus, the presence ofnitrate determines the prevailing metabolism in areactor/zone, i.e. anoxic conditions (denitrifica-tion) when it is present and anaerobic conditions(P release) when it is absent.

3.1. Respirometry studies

Respirometry tests were performed to deter-mine the effect of different carbon sources ondenitrification. The conditions for conducting therespirometry test are presented in Table 2. It shouldbe noted that the lower initial SCOD concentration(Table 2) in the case of MWW is only due to the 1in 5 dilution while conducting the respirometrytests. Although the total COD was not measuredin this particular case, it should be noted that thetotal COD of the MWW was ~320 mg/L. Fig. 5shows the temporal N2 produced. The reliabilityof the test as reflected by the close agreementbetween duplicates (not shown in Fig. 5) is noted.It is apparent that acetic acid was the most readilybiodegradable followed by the anaerobic sludgefrom the intermediate clarifier (Fig. 1), and the


MWW. The aforementioned observation wasconfirmed by the total gas production, the rate(slope of the graph), and the time lag. For aceticacid, gas production increased immediately to atotal of 5 mL, corresponding to the theoretical nit-rate removal of 23 mg NO3–N/L at 23°C, closelymatching the initial nitrate concentration of

y = -4.45x + 25.60R2 = 0.82

y = -0.28x + 1.06R2 = 0.05

0

5

10

15

20

25

30

0 1 2 3 4 5

NO3-N Uptake (mg/L)

Solu

ble

Phos

phor

us R

elea

se (m

g/L)

SWWMWW

Fig. 4. Correlation between phosphorus release and nitrate uptake.

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7Time (hr)

N2 P

rodu

ctio

n (m

L)

Acetic Acid SludgeMWW Control

Fig. 5. Cumulative N2 production in respirometer.

21.2 mg/L. For the organic–rich sludge from theintermediate clarifier of the process depicted inFig. 1, following a lag phase of 1.67 h, nitrogenproduction totaled 2.14 mL, corresponding to thetheoretical nitrate removal of 9.8 mg NO3–N/L,which is 46% of the initial nitrate concentrationof 21.3 mg/L. Similarly for MWW a lag phase of


Table 2Substrate effect on denitrification (respirometer)

*represents initial values#Sludge collected from the intermediate system clarifier(refer to Fig. 1)

Substrate Acetic acid Sludge# MWW SCOD, mg/L* 515.0 39.0 38.0 NO3–N, mg/L* 21.2 21.3 19.7 VSS, gm/L* 5.6 5.7 5.4 S0/X0, mg/gm* 92.0 6.8 7.0 Denitrification rate, mL N2/L.h

12.6 4.8 2.6

SDNRmax, mg/g VSS.d

62.2 23.3 13.5

2.67 h was observed and only 0.7 mL of gas wasproduced i.e. 3.23 mg NO3–N/L were denitrified.It is conspicuous that the RBCOD of this MWWwas limiting denitrification. The results confirmthat the thickened anaerobic sludge is a better car-bon source than MWW. This is potentially attri-butable to the adsorbed COD on the sludges duringthe anaerobic phase [18] as well as potential fer-mentation in the clarifier even though there wasno direct indication of this by pH measurements.Several researchers have observed the pheno-

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7Time (hr)

N2 P

rodu

ctio

n R

ate

(mL/

L.hr

)

Acetic Acid SludgeMWW Control

Fig. 6. N2 production rate in respirometer.

menon of sorption of SCOD onto sludges in theirstudies with domestic sewage [34–37]. It is usuallyconsidered that biosorption is caused by electro-static or hydrophobic interactions according to thenature of the organic matter [36].

Fig. 6 shows the denitrification rates for thedifferent carbon sources used. Maximum specificdenitrification rates (SDNRmax) were calculatedusing maximum denitrification rates during ex-ponential periods, initial VSS in reaction bottlesand accounting for 24.3 L/mole of N2 at 1 atmand 23°C. As presented in Table 2, SDNRmax of62.2, 23.3 and 13.5 mg NO3–N/g VSS·d werecalculated for acetic acid, sludge and MWW res-pectively, thus emphasizing that acetic acid is thebest substrate of all three for denitrification.SDNRmax with the sludge in respirometric studiesof 23.3 mg NO3–N/g VSS·d, was comparable tothat achieved for anoxic denitrification understeady state conditions of 24.1 mg NO3–N/g VSS·din the modified MBR system. Upon comparingthe SDNRmax for MWW and system sludges (fromthe intermediate clarifier) in Table 2, it is evidentthat the system sludges affected a 73% higherdenitrification rate than the MWW. The lower rateof denitrification for the MWW, compared to theother two, clearly highlights the lack of readily


biodegradable substrate. The carbon limitation inthe MWW alluded to above is likely to influencethe extent of denitrification more than the maxim-um denitrification rate, considering that the initialSCOD was similar for both MWW and sludge. Itis indeed intriguing that the sludge exhibited high-er SDNRmax than the MWW, since sorbed carbonhas to desorb to affect denitrification. It is there-fore postulated that the increased SDNRmax in thesludge (from the intermediate clarifier) is a resultof the change in nature of soluble organic matteri.e. more RBCODs than in the MWW. This maybe the result of fermentation in the system clarifier.

It is interesting to note the two significant peaks(Figs. 5 and 6) for acetic acid compared to othersubstrates. Since the seed used in the study wasfrom a BNR system using MBR treating MWW,the likely reason is the adsorbed carbon on thesludges contributing to the second peak. The acet-ate, as a readily biodegradable, contributed to theinitial N2 generation of 2.35 mL during 1.33 hand reached steady state thereafter. The N2 pro-duction rate again picked up at 3.33 h, 2 h later,accumulating another 2.65 mL N2 to the total pro-duction of 5 mL N2. This closely compares withthe 2.14 mL N2 generated after a lag phase of1.67 h for sludges. The maximum denitrificationrate of 5.2 mL N2/L·h in the second phase withacetic acid also narrowly balances with 4.8 mLN2/L·h measured for sludges.

It may be argued that the second peak observedin the case of acetic acid (Fig. 6) is due to thehydrolysis or fermentation of organics associatedwith the seed (taken from the anoxic reactor ofthe MBR system shown in Fig. 1). However, itshould be noted that the contact time for pre-fermentation of sludges generally ranges fromabout 1–2 d [38–41] unlike ~2 h in the presentrespirometry study. Moreover, neither the MWWnor the sludge (from the intermediate clarifier)exhibited the same phenomenon as acetic acid.Hence, it is more likely that the observed secondpeak in the case of acetic acid was due to the sorp-tion of carbon onto sludge rather than hydrolysisor fermentation of the sludge.

3.2. Batch studies

The summary of substrate effects on P releaseand denitrification observed during the batchstudies is reported in Table 3. Figs. 7–11 showthe effect of acetic acid, butyric acid, propionicacid, MWW and primary effluent on P release anddenitrification. All the batch experiments weredesigned to ensure sufficient carbon availability.For the tests with butyric and propionic acid, therationale for using the initial COD of ~400 mg/Lwas their relatively slower biodegradability. Itshould be noted that the lower initial SCOD con-centration (Table 3) in the case of the MWW isonly due to the 1 in 2 dilution while conducting abatch experiment. It is noted that the 1:5 dilutionof MWW in respirometry and 1:2 in the batchstudies resulted in comparable initial SCODs asapparent from Table 2 and 3. This is primarilydue to the wide variability in the MWW charac-teristics as reflected in Table 1. Also, the initialSCOD concentration in the case of the primaryeffluent is different from that of the MWW(Table 3), as both the primary effluent and MWWsamples were taken on different days from a localwastewater treatment plant. It should be noted thatall the batch tests were conducted at different timesduring the MWW run of the system shown inFig. 1, however the initial P content of the sludgewas relatively the same at 4.5–4.9% (by weightof VSS). The incongruity of the high initial P con-centration of 19 mg/L during acetic acid experi-ment compared to the other substrates was due tothe sludge being left overnight, thus developinganaerobic conditions and releasing P.

Scrutiny of the P release and denitrificationpatterns depicted in Figs. 7–11 clearly emphasizesthat all the substrates, except acetic acid, exhibitedthe same trend of sequential denitrification and Prelease i.e. nitrate must be reduced to low levelsto stimulate P release. Acetic acid demonstratedsimultaneous P release and denitrification. Thismay have occurred due to the readily biodegrad-able nature of acetic acid resulting in a rapid deple-tion of NO3–N (Fig. 7). The overnight P release


Table 3Substrate effect on P release and denitrification in batch tests

a represent the values at the end of anoxic and anaerobic phaseb duration of denitrification is the time to reach NO3–N concentration below 0.8 mg/Lc duration of P release is the time when P release was initiated to the time it reached maximum Pd estimated COD consumption is based on 7 mg COD/mg NO3–N and 7.5 mg COD/mg P released while the measuredCOD consumption is the consumption during the anoxic–anaerobic phas

Substrate Acetic acid Butyric acid Propionic acid MWW Primary effluent

SCOD, mg/L Initial Anaerobica Final

297 121 27

397 297 131

425 361 189

39 37 32

65 32 30

VSS, gm/L 4.97 2.24 2.66 2.78 4.32 S0/X0, mg/mg 0.06 0.177 0.16 0.014 0.015 NO3–N

Initial, mg/L Anoxic, mg/La Duration of denitrification, minb SDNRmax, mg/g VSS.d

5.9 0.3 15 107.2

9.5 0.5 80 77.1

5.1 0.5 60 37.9

4.7 0.8 60 31.1

7.5 0.4 80 30.0

Phosphorus (P) Initial, mg/L P released, mg/L Final, mg/L ∆P, mg/L Duration of P release, minc P release rate, mg P/g VSS.d P uptake rate, mg P/g VSS.d

19.2 18.3 0.4 18.8 90 58.9 135.9

2.9 4.3 1.9 1.0 100 27.6 32.1

4.3 2.2 2.0 2.3 70 17.0 31.1

4.7 1.8 1.7 3.0 80 11.7 19.1

4.6 1.3 1.6 3.0 100 4.3 17.9

COD consumptiond Measured Estimated

176 176.5

100 95.3

64 48.7

2 40.8

33 59.5

in the case of acetic acid renders the unique con-clusion of simultaneous denitrification and P re-lease less certain, but some literature studies [21,28,42] do support this phenomenon and hence itis likely that acetic acid has this property. Forpropionic acid, butyric acid, MWW and primaryeffluent, P release did not occur until nitrate con-centration was below 1 mg/L as confirmed fromFigs. 8–11. A similar phenomenon was also ob-served by Malnou et al. [25] in the study of theeffect of nitrate on P uptake. For MWW, it can beconfirmed that a longer time was needed to start

the P release, even though the NO3–N concen-tration was below 1 mg/L, compared to the othersubstrates studied. The postulated reason is thelonger time needed to ferment particulate CODpresent in the MWW to RBCOD. For MWW andprimary effluent, the low COD drop during the Prelease shows a striking example of RBCODlimitation. Interestingly for both MWW and pri-mary effluent, there was no major change in theSCOD concentration, which remained at 37 mg/Lat the end of anoxic conditions.

SDNRmax followed descending order from


acetic acid, butyric acid, propionic acid, MWWand primary effluent. SDNRmax for acetic acid wasfound to be 107.2 mg NO3–N/g VSS·d comparedto 58.1 mg NO3–N/g MLSS·d or 83.0 mg NO3–N/g MLVSS·d (SDNR, based on 70% MLVSS)achieved by Takai et al. [20] with an initial NO3–N of 50 mg/L. Chuang et al. [42] have observed

0

5

10

15

20

25

30

35

40

0 25 50 75 100 125 150 175 200 225Time (min)

P, N

O3-

N (m

g/L)

0

50

100

150

200

250

300

CO

D (m

g/L)

PNO3 - NCOD

Anoxic Aerobic

Fig. 7. Effect of acetic acid on soluble P removal and denitrification.

Anoxic Aerobic

Fig. 8. Effect of propionic acid on soluble P removal and denitrification.

SDNR of 4 mg NO3–N/g MLSS·h (137.1 mgNO3–N/g MLVSS·d based on 70% MLVSS) withacetic acid as a substrate. Takai et al. [20] in theirstudy of the effect of volatile fatty acids on denit-rifying activity have emphasized the relative inef-fectiveness of denitrification with propionic acid,although they reported SDNRmax of 77 mg NO3–

y = -0.07x + 4.98R2 = 0.98

0

1

2

3

4

5

6

7

0 25 50 75 100 125 150 175 200 225 250 275Time (min)

P, N

O3-

N (

mg/

L)

0

50

100

150

200

250

300

350

400

450

500

CO

D (m

g/L)

PNO3 - NCOD

Anoxic Aerobic


y = -0.12x + 9.06R2 = 0.95

0

1

2

3

4

5

6

7

8

9

10

0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375Time (min)

P, N

O3-

N (

mg/

L)

0

50

100

150

200

250

300

350

400

450

500

CO

D (m

g/L)

PNO3 - NCOD

Anoxic Aerobic

Fig. 9. Effect of butyric acid on soluble P removal and denitrification.

y = -0.06x + 3.78R2 = 0.78

0

2

4

6

8

10

12

14

16

18

0 25 50 75 100 125 150 175 200 225 250 275 300 325 350Time (min)

P, N

O3-

N (m

g/L)

0

5

10

15

20

25

30

35

40

45

50

CO

D (m

g/L)

PNO3 - NCOD Anoxic Aerobic

Fig. 10. Effect of municipal wastewater on soluble P removal and denitrification.

N/g MLSS·d for propionic acid which was muchhigher than that observed in this study. The studyperformed by Elefsiniotis et al. [43] showed thatacetic acid was preferred by denitrifiers, followedby butyric, propionic and valeric acids; similar tothe sequence observed in the present study. Forcomparison with the findings of this study, Gerber

et al. [29] observed denitrification rates of 60 mgN/g MLSS·d (or 85.7 mg NO3–N/g MLVSS·d)for acetate, 50.4 mg N/g MLSS·d (or 72.0 mgNO3–N/g MLVSS·d) for butyrate and 40.8 mg N/g MLSS·d (or 58.3 mg NO3–N/g MLVSS·d) forpropionate at COD concentrations of 200 mg/L.SDNRmax for MWW was about 30% lower than


that of acetic acid (Table 3). Henze et al. [44] intheir study of denitrification rates using severalorganic components have shown that the denitri-fication rate with domestic wastewater was aboutone third of the value obtained with acetic acid ormethanol. For primary effluent, SCOD reductionof only 11 mg/L was observed during the denitrifi-cation process. Since the requirement to denitrify7.1 mg NO3–N/L (7.5 minus 0.4 mg NO3–N/L) isabout 49.7 mg/L of COD, it is apparent that theCOD remaining was probably produced by hyd-rolysis or fermentation of particulate COD. Bycomparing SDNRmax values for both primary efflu-ent and MWW, it is apparent that both substrateswere the same.

The release of 18.3 mg P/L even at a high initialP concentration of 19.2 mg/L was noticeable inthe case of acetic acid. Comparing, Jun et al. [19]has also reported P release of 31.8 mg/L in theirbatch study with acetate. With the lowest final Pconcentration achieved with the maximum Prelease, as expected, acetic acid was observed tobe the best substrate for denitrification and P re-moval compared to other substrates. Propionicacid exhibited slightly higher overall P removal(2.3 mg/L) than butyric acid (1 mg/L) though their

y = -0.09x + 6.72R2 = 0.94

0

1

2

3

4

5

6

7

8

9

10

0 25 50 75 100 125 150 175 200 225 250 275Time (min)

P, N

O3-

N (

mg/

L)

0

10

20

30

40

50

60

70

CO

D (m

g/L)

PNO3-NCOD

Anoxic Aerobic

Fig. 11. Effect of primary effluent on soluble P removal and denitrification.

final effluent P concentrations were the same(Table 3). In the study of the effects of VFAs inan anaerobic process [45], propionate degradationwas the slowest and most sensitive process of allthe VFAs degradation. Lemos et al. [46] and Ran-dall et al. [47] have demonstrated that one of thereasons for low P release for propionic acid com-pared to acetic acid may be attributed to the form-ation of mainly (90%) poly-hydroxyvalerate(PHV) as PHA in the case of propionate comparedto poly-hydroxy butyrate (PHB) in the case ofacetate. Pijuan et al. [5] have reported in theirstudy with propionate that PHAs comprised 36%PHB and 64% PHV as compared to 86% PHBand 14% PHV with acetate as a sole carbon source.It can be noted that butyric acid had the lowest Puptake (relative to the initial P concentration) de-spite higher P release than the propionic acid,MWW and primary effluent (Table 3). It can beargued that this was due to the lowest initial Pamong all other aforementioned substrates. How-ever, the final effluent P concentration of 1.9 mg/Lwith butyric acid clearly indicates that the lowinitial P concentration did not limit the EBPR.Moreover, EBPR mainly depends on the P contentof the sludge rather than the initial P concentration.


Although the higher P release in the case of butyricacid relative to propionic acid seems to contradictthe findings of Pijuan et al. [48], it must be assertedthat the sludge used in their study was acclimat-ized to propionate. Some literature studies [3,47]have reported that higher P removal was observedfor acetic and isovaleric acid among all other 1–5carbon VFAs, because of the predominance ofPHB. It was difficult to ascertain the exact causefor this behavior of butyric acid as no measure-ments of PHAs were done in this study.

Although, MWW has exhibited lower P releasethan acetic, propionic and butyric acid, the finaleffluent P concentration was comparable to thatachieved for propionic and butyric acids. Jun etal. [19] has shown that the P uptake and overall Premoval were well developed even though Prelease was not comparatively high with milk andstarch in their study of five kinds of organic sub-strate. It is interesting to note that the final effluentSCOD (32 mg/L), in the case of MWW and pri-mary effluent, at the end of the anoxic periodremained essentially constant during the subse-quent aerobic phase i.e. was non-biodegradable.This clearly indicates that the SCOD was generat-ed by fermentation during the anoxic phase andconsumed simultaneously for P release and de-nitrification. Thus, the rate of denitrification andP release is limited by hydrolysis of particulateorganics. P uptake rate followed a pattern similarto the P release rate (Table 3) in the order of aceticacid, butyric acid, propionic acid, MWW and pri-mary effluent.

It is interesting to note that in the literature, Premoval has been undoubtedly correlated with Prelease [19,49]. The results of the batch studieswith propionic acid, MWW and primary effluentshow that P release as low as 1.3–2.2 mg/L canstill result in P removal. The results of the con-tinuous flow system with the MWW [33] alsocorroborate this since P release of <2.2 mg/L stilldid not hamper the overall P removal. Gerber etal. [28,29] have shown that the rate of P release isgoverned by a number of variables such as the

maximum amount of phosphate available forrelease, amount of short chain fatty acids availableand nitrate present.

Typical ratios of COD consumed to P release(COD/P) are 6.7 g VFA/g P [50] and 7.5 g/g [19].Assuming 7 mg COD/mg NO3–N and 7.5 mg COD/mg P, the theoretical COD requirement would be176.5, 95.3 and 48.7 mg/L for acetic, butyric, pro-pionic acids respectively, if denitrification and Prelease occur sequentially or simultaneously. Thisclosely approximated the observed COD reduc-tions of 176, 100, 64 mg/L at the end of anaerobicphase measured experimentally. For the MWW,the theoretical COD requirement comes out to be40.8 mg/L based on nitrates consumed and P re-leased. This is much higher than the COD reduc-tion observed during the process, re-emphasizingthe point of fermentation and hydrolysis of par-ticulate COD discussed above. By comparing theCOD consumption of the heterotrophs in thisstudy for denitrification, it is apparent that appro-ximately 7 mg COD/mg NO3–N is required. Thisis about 20% lower than typical value of 8.7 mgCOD/mg NO3–N (based on a yield of 0.67 mgCODbiomass per mg COD) for ordinary hetero-trophic denitrifiers [50].

From a kinetic point of view, it appears thatthere is a relationship between SDNRmax and Prelease rate (Table 3). The highest denitrificationrate corresponds to the highest P release rate (inthe case of acetic acid) and the lowest denitrifica-tion rate corresponds to the lowest P release rate(in the case of MWW). However, the trend maynot be linear. Given the close agreement betweenSDNRmax, extent of ∆P and P released, it appearsthat the propionic acid is the predominant VFA inthe MWW.

It is interesting to contrast the denitrificationrates derived from respirometric studies (Table 2)and those derived from batch studies (Table 3). Itmust be emphasized that the denitrification ratesreported in Table 2 have been calculated by con-verting the gas (mainly N2 since the CO2 wasabsorbed in the KOH trap) production rate to an


equivalent N2 mass rate and normalized it tobiomass. This calculation for SDNR would be trueif all the denitrified nitrogen ended up in N2 gas,however, some of the nitrogen is indeed utilizedin biomass synthesis. Neglecting the later nitrogenwill underestimate the SDNR values. Since theestimated COD removal based on the theoreticalrequirement (7 mg COD/mg NO3–N) for denitrifi-cation and P uptake closely matched the experi-mental data (as discussed above), the theoreticalrequirement of 7 mg COD/mg NO3–N was usedto estimate the approximate quantity of nitrogenutilized in cell synthesis. Using the typical yieldof denitrifiers of 0.63 mg CODbiomass/mg COD(0.44 mg VSS/mg COD) and the 12% N contentof biomass (C5H7O2N), the N incorporated in bio-mass growth is 0.37 mg N per mg NO3–N denit-rified i.e. the amount of N produced as N2 gas is0.63 mg N (1 minus 0.37) per mg NO3–N denitri-fied. Accordingly, the respirometry-based denitri-fication rates for acetate, sludge and MWW(Table 2) would translate to actual SDNR rates of98.7, 37 and 21.4 mg NO3–N/g VSS·d. The SDNRrates for acetate using batch and respirometry datawere identical, while the rates for MWW differby about 31%. This discrepancy is primarily attri-buted to the difficulty of precisely delineating theanoxic yield for MWW to incorporate hydrolysisof particulate matter.

4. Conclusions

The following conclusions can be drawn basedon the findings of this research:• Nitrates play a very important role in phos-

phorus release. P release did not occur untilthe NO3–N concentration was below 0.8 mg/Lin all substrates studied except for acetic acidwhich, unlike other substrates, supported sim-ultaneous denitrification and P release.

• SDNRmax followed descending order fromacetic acid, butyric acid, propionic acid, MWWand primary effluent.

• Denitrification rates for acetate using respiro-

metry and batch tests were identical, while forthe MWW respirometry denitrification rateswere 31% lower than from batch studies.

• With the study on different carbon sources itwas found that P release and P uptake rateswere higher in the case of acetic acid and prog-ressively decreased in the order of butyric acid,propionic acid, MWW and primary effluent.It should be noted that these results wereobtained in batch studies and different resultshave been reported for long term cultivationwith these substrates, most notably acetate/propionate mixtures resulting in superior re-moval to acetate dominated wastewater [49].

• P removal can occur even with the low P re-lease. The same final P concentrations wereobserved in the case of propionic acid, MWWand primary effluent even with lower P releasethan butyric acid. It is notable that other re-searchers have reported high net P removalseven in control reactors receiving no VFA sub-strate, and thus with low P releases and up-takes, in batch experiments using EBPR bio-mass for a single-treatment cycle [22]. How-ever, the significance and reason for these ob-servations are uncertain and further researchis required to ascertain exactly what is occur-ring.

Acknowledgment

We are thankful to Natural Sciences and Engi-neering Research Council (NSERC) of Canadafor funding this project, ZENON EnvironmentalSystems Inc. for donating membranes andAdelaide Pollution Control plant staff for pro-viding municipal wastewater. The authors wouldalso like to acknowledge the Canada Foundationof Innovation for the infrastructure support.

References[1] A.A. Randall, L.D. Benefield, W.E. Hill, J.P. Nicol,

G.K. Boman and S.R. Jing, The effect of volatile


fatty acids on enhanced biological phosphorus re-moval and population structure in anaerobic/aerobicsequencing batch reactors, Wat. Sci. Technol., 35(1)(1997) 153–160.

[2] H. Carlsson, H. Aspegren and A. Hilmer, Interactionsbetween wastewater quality and phosphorus releasein the anaerobic reactor of the EBPR process, Wat.Res., 30(6) (1996) 1517–1527.

[3] Z.H. Abu-ghararah and C.W. Randall, The effect oforganic compounds on biological phosphorus re-moval process, Wat. Sci. Technol., 23(46) (1991)585–594.

[4] E. Von Muench, DSP prefermenter technology book,Brisbane, Old., Australia: Science Traveller Inter-national CRC WMPC Ltd, 1998.

[5] M. Pijuan, A.M. Saunders, A. Guisasola, J.A. Baeza,C. Casas and L.L. Blackall, Enhanced biologicalphosphorus removal in a sequencing batch reactorusing propionate as the sole carbon source, Bio-technol. Bioeng., 85(1) (2004) 56–67.

[6] R.J. Zeng, R. Lemaire, Z. Yuan and J. Keller, Simul-taneous nitrification and denitrification, and P re-moval in a lab-scale sequencing batch reactor, Bio-technol. Bioeng., 84(2) (2003) 170–178.

[7] O. Turk and D. Mavinic, Preliminary assessment ofa shortcut in nitrogen removal from wastewater, Can.J. Civil Eng., 13 (1986) 600–605.

[8] O. Turk and D. Mavinic, Maintaining nitrite buildupin a system acclimated to free ammonia, Wat. Res.,23 (1989) 1385–1388.

[9] T. Kuba, G.J.F. Smolders, M.C.M. van Loosdrechtand J.J. Heijnen, Biological phosphorus removalfrom wastewater by anaerobic and anoxic sequenc-ing batch reactor, Wat. Sci. Technol., 27 (1993) 241–252.

[10] J.P. Kerrn-Jespersen, M. Henze and R. Strube,Biological phosphorus release and uptake underalternating anaerobic and anoxic conditions in afixed-film reactor, Wat. Res., 28 (1994) 1253–1255.

[11] E. Murnleitner, T. Kuba, M.C.M. van Loosdrechtand J.J. Heijnen, An integrated metabolic model forthe aerobic and denitrifying biological phosphorusremoval, Biotechnol. Bioeng., 54 (1997) 434–450.

[12] T. Kuba, A. Wachtmeister, M.C.M. van Loosdrechtand J.J. Heijnen, Effect of nitrate on phosphorus re-lease in biological phosphorus removal systems,Wat. Sci. Technol., 30 (1994) 263.

[13] J.L. Barnard, Design and Retrofit of WastewaterTreatment Plants for Biological Nutrient Removal.Technomic Publishing, Pennsylvania, 1992.

[14] M.C. Goronszy, In: Course notes on IntermittentlyOperated Activated Sludge Plants. Dept. of Chem.Eng., University of Queensland, Australia, 1992.

[15] S.H. Isaacs and M. Henze, Controlled carbon sourceaddition to an alternating nitrification–denitrificationwastewater treatment process including biologicalP removal, Wat. Res., 29 (1994) 77–89.

[16] I. Yeom, Y. Nah and K. Ahn, Treatment of householdwastewater using an intermittently aerated mem-brane bioreactor, Desalination, 124 (1999) 193–204.

[17] R. Tasli, D. Orhon and N. Artan, The effects of sub-strate composition on the nutrient removal potentialof sequencing batch reactors, Wat. S.A., 25(3) (1999)337–344.

[18] R. Sorm, J. Wanner, R. Saltarelli, G. Bortone and A.Tilche, Verification of anoxic phosphate uptake asthe main biochemical mechanism of the Dephanoxprocess, Wat. Sci. Technol., 35(10) (1997) 87–94.

[19] H.B. Jun and H.S. Shin, Substrate transformationin a biological excess phosphorus removal system,Wat. Res., 31(4) (1997) 893–899.

[20] T. Takai, A. Hirata, K. Yamauchi and Y. Inamori,Effects of temperature and volatile fatty acids onnitrification–denitrification activity in small-scaleanaeobic–aerobic recirculation biofilm process, Wat.Sci. Technol., 35(6) (1997) 101–108.

[21] N. Artan, R. Tasli, Ö. Nevin and D. Orhon, The fateof phosphate under anoxic conditions in biologicalnutrient removal activated sludge systems, Biotech-nol. Lett., 20(11) (1998) 1085–1090.

[22] C.R. Hood and A.A. Randall, A biochemical hypo-thesis explaining the response of enhanced biologicalphosphorus removal biomass to organic substrates,Wat. Res., 35(11) (2001) 2758–2766.

[23] R. Tasli, N. Artan and D. Orhon, The influence ofdifferent substrates on enhanced biological phos-phorus removal in a sequencing batch reactor, Wat.Sci. Technol., 35(1) (1997) 75–80.

[24] D.W. Osborn and H.A. Nicholls, Optimization ofthe activated sludge process for the biological re-moval of phosphorus, Prog. Wat. Technol., 10 (1978)261–277.

[25] D. Malnou, M. Meganck, G.M. Faup and M. duRostu, Biological phosphorus removal: study of themain parameters, Wat. Sci. Technol., 16(10/11)(1984) 173–185.

[26] M.C. Hascoet and M. Florentz, Influence of nitrateson biological phosphorus removal from wastewater,Wat. S.A., 11 (1985) 1–8.

[27] A. Iwema and A. Meunier, Influence of nitrate on


acetic acid induced biological phosphate removal,Wat. Sci. Technol., 17(11) (1985) 289–294.

[28] A. Gerber, E.S. Mostert, C.T. Winter and R.H. deVilliers, The effect of acetate and other short-chaincarbon compounds on the kinetics of biologicalnutrient removal, Wat. S.A., 12 (1986) 7–12.

[29] A. Gerber, R.H. de Villiers, E.S. Mostert and C.J.van Riet, The phenomenon of simultaneous phos-phate uptake and release, and its importance in bio-logical nutrient removal. In Biological PhosphateRemoval from Wastewaters. R. Ramadori, ed.,Pergamon Press, Oxford, 1987.

[30] G. Bortone, R. Saltarelli, V. Alonso, R. Sorm, J.Wanner and A. Tilche, Biological anoxic phosphorusremoval — The Dephanox process, Wat. Sci.Technol., 34(1–2) (1996) 119–128.

[31] G. Nakhla and J. Patel, Treatment of wastewatercontaining phosphorus and nitrogen, US PatentApplication No. 11/119929, and Canadian PatentApplication No. 2506038 (filed: May 2005).

[32] American Public Health Association (APHA),American Water Works Association (AWWA),Water Environment Federation (WEF), StandardMethods for the Examination of Water andWastewater, 20th ed., Washington, D.C., 1998.

[33] J. Patel and G. Nakhla, Simultaneous nitrification–denitrification with anoxic P uptake in MBR system,Wat. Environ. Res., in press.

[34] S. Hough and B. Narayanan, Waste activated sludgeanaerobic contact waste stream treatment process,US patent application publication, Appl. No.: 10/265,264, Pub No.: US 2004/0094475 A1, 2004.

[35] K. Fujie, H. Hu, B. Lim and H. Xia, Effect of bio-sorption on the damping of influent fluctuation inactivated sludge aeration tanks, Wat. Sci. Technol.,35(7) (1997) 79–87.

[36] A. Guellil, F. Thomas, J.C. Block, J.L. Bersillon andP. Ginestet, Transfer of organic matter betweenwastewater and activated sludge flocs, Wat. Res.,35(1) (2001) 143–150.

[37] L. Novák, L. Larrea and J. Wanner, Mathematicalmodel for soluble carbonaceous substrate biosorp-tion, Wat. Sci. Technol., 31(2) (1995) 67–77.

[38] G.J. Hatziconstantinou, P. Yannakopoulos and A.Andreadakis, Primary sludge hydrolysis for biolo-gical nutrient removal, Wat. Sci. Technol., 34(1–2)(1996) 417–423.

[39] I. Karlsson and G. Smith, Pre-precipitation facilitatesnitrogen removal without tank expansion, Wat. Sci.Technol., 22(7/8) (1990) 85–92.

[40] P.P. Brinch, K. Rindel and K. Kalb, Upgrading tonutrient removal by means of internal carbon fromsludge hydrolysis, Wat. Sci. Technol., 29(12) (1994)31–40.

[41] G.H. Kristensen, P.E. Jorgensen, R. Strube and M.Henze, Combined pre-precipitation, biologicalsludge hydrolysis and nitrogen reduction — A pilotdemonstration at integrated nutrient removal, Wat.Sci. Technol., 26(5–6) (1992) 1057–1066.

[42] S.H. Chuang, C.F. Ouyang and Y.B. Wang, Kineticcompetition between phosphorus release and denitri-fication on sludge under anoxic condition, Wat. Res.,30(12) (1996) 2961–2968.

[43] P. Elefsiniotis, D.G. Wareham and M.O. Smith, Useof volatile fatty acids from an acid-phase digesterfor denitrification, J. Biotechnol., 114(3) (2004)289–297.

[44] M. Henze, Carbon sources for denitrification ofwastewater. Seminar on Nutrients Removal fromMunicipal Wastewater, 4–6 September 1989, Tam-pere University, Tampere, Finland, 1989.

[45] P.F. Pind, I. Angelidaki and B.K. Ahring, Dynamicsof the anaerobic process: Effects of volatile fattyacids, Biotechnol. Bioeng., 82(7) (2003) 791–801.

[46] P.C. Lemos, C. Viana, E.N. Salgueiro, A.M. Ramos,J.P.S.G. Crespo and M.A.M. Reis, Effect of carbonsource on the formation of polyhydroxyalkanoates(PHA) by a phosphate-accumulating mixed culture,Enz. Microb. Technol., 22 (1998) 662–671.

[47] A.A. Randall and Y. Liu, Polyhydroxyalkanoatesform potentially a key aspect of aerobic phosphorusuptake in enhanced biological phosphorus removal,Wat. Res., 36 (2002) 3473–3478.

[48] M. Pijuan, J.A. Baeza, C. Casas and J. Lafuente,Response of an EBPR population developed in anSBR with propionate to different carbon sources,Wat. Sci. Technol., 50(10) (2004) 131–138.

[49] Y. Chen, A. Randall and T. McCue, The efficiencyof enhanced biological phosphorus removal fromreal wastewater affected by different ratios of aceticto propionic acid, Wat. Res., 38 (2004) 27–36.

[50] Metcalf and Eddy, Wastewater Engineering:Treatment and Reuse, 4th ed., McGraw-Hill, NewYork, 2003.

interaction of denitrification and p removal in anoxic p removal systems

Documents