Bioresource Technology 154 (2014) 44–50

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Bioresource Technology

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Characteristics of self-alkalization in high-rate denitrifying automaticcirculation (DAC) reactor fed with methanol and sodium acetate

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.11.097

⇑ Corresponding author. Tel./fax: +86 571 88982819.E-mail address: pzheng@zju.edu.cn (P. Zheng).

Wei Li a, Ping Zheng a,⇑, Jun Guo b, Junyuan Ji c, Meng Zhang a, Zonghe Zhang a, Enchao Zhan a,Ghulam Abbas a,d

a Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, Chinab College of Environmental Science and Engineering, Tongji University, Shanghai 200000, Chinac College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, Chinad Department of Chemical Engineering, University of Gujrat, Gujrat, Pakistan

h i g h l i g h t s

� Self-alkalization of high-rate denitrifying reactor was remarkable.� Self-alkalization with acetate as carbon source was stronger than that of methanol.� Excellent self-adaptation to alkalinity was found in high-rate denitrifying reactor.� The self-adaptation mechanism of high-rate denitrifying reactor was revealed.

a r t i c l e i n f o

Article history:Received 23 September 2013Received in revised form 25 November 2013Accepted 30 November 2013Available online 16 December 2013

Keywords:Denitrifying reactorHigh-rateSelf-alkalizationAdaptation

a b s t r a c t

Denitrification is a self-alkalization process. In this experiment, the characteristics of self-alkalization inhigh-rate heterotrophic denitrifying automatic circulation (DAC) reactor fed with methanol and sodiumacetate were investigated, respectively. The results showed that, (1) The self-alkalization of high-ratedenitrifying reactors was remarkably strong both with methanol and sodium acetate as carbon sources,while the effluent pH was much lower than the stoichiometric values and the malfunction fromself-alkalization of denitrification was far less serious than expected. (2) The self-adaptation of thereactors was attributed to the neutralization of carbon dioxide (oxidization product of organic matter)and the self-adaptation of denitrifying sludge. The formation and discharge of calcium carbonateprecipitates gave rise to lower effluent pH and alkalinity than the stoichiometric values.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Simultaneous elimination of nitrogen and organic matters bybiological denitrification has become increasingly important inwastewater treatment (Kapoor and Viraraghavan, 1997;Nancharaiah and Venugopalan, 2011; Hu et al., 2012). Thedevelopment of denitrifying reactor can improve denitrificationtechnology (Rabah and Dahab, 2004). The denitrifying granularsludge reactor was reported in 1975 for the first time (Miyaji andKato, 1975) and it has been a focus in the field of denitrification tech-nology due to its excellent performance and low cost. The maximumnitrogen removal rate (NRR) and COD removal rate (ORR) reportedso far in literature are 25 kg N m�3 d�1 (Bode et al., 1987) and67.5 kg COD m�3 d�1 (Franco et al., 2006), respectively.

Denitrifying bacteria played a key role in denitrifying reactorand the reactor performance was largely dependent on theirgrowth and metabolism (Lew et al., 2012; Volcke et al., 2012). Likeother species, denitrifying bacteria have their own pH or alkalinityrange for growth. Drastic variations in environmental pH can harmmicroorganisms by inhibiting the activity of enzymes andmembrane transport proteins. It has been reported that thesuitable pH range for denitrifying bacteria growth is 7.5–9.0 (Glassand Silverstein, 1998; Lee and Rittmann, 2003). However, thedenitrification is a self-alkalization process and it often leads to apH of reaction solution over 9.7 at high substrate concentrations(Jin et al., 2012). Methanol is the most popular carbon source fordenitrification both in experiments and engineering applicationsEq. (1). If the alkalinity from denitrificaiton with methanol ascarbon source was taken as a standard, the carbon sources canbe divided into 3 types: weak alkalizer, moderate alkalizer andstrong alkalizer. Methanol and sodium acetate Eq. (2) belong to

W. Li et al. / Bioresource Technology 154 (2014) 44–50 45

moderate alkalizer (normal alkalizer because of its popularity) andstrong alkalizer, respectively.

NO�3 þ 1:08CH3OH ! 0:056C5H7NO2 þ 0:47N2 þ 0:52HCO�3þ 0:24CO2�

3 þ 1:68H2O ð1Þ

NO�3 þ 1:06CH3COO� ! 0:15C5H7NO2 þ 0:42N2 þ 0:66HCO�3þ 0:70CO2�

3 þ 0:73H2O ð2Þ

½Hþ� ¼ Ka2½HCO�3 �½CO2�

3 �() pH ¼ pKa2 � lg

½HCO�3 �½CO2�

3 �ð3Þ

where Ka2, ½HCO�3 �, ½CO2�3 � and [H+] were secondary ionization

constant of carbonate (10�10.30, 30 �C), HCO�3 concentration, CO2�3

concentration and H+ concentration, respectively.Surprisingly, the effluent pH of high-rate denitrification reactor

was much lower than the stoichiometric value and the malfunctionfrom self-alkalization of denitrification was far less serious thanexpected. Here the self-adaptation to alkalinity of heterotrophicdenitrification in a high-rate reactor and its mechanism wereinvestigated to promote the development and application ofhigh-rate denitrifying reactor.

2. Methods

2.1. Synthetic wastewater

In one reactor, NaNO3 and CH3OH (carbon source) were addedto get concentrations of 1 g NO�3 –N L�1 and 5 g COD L�1, respec-tively. In the other reactor only CH3OH was replaced by CH3COONa(carbon source) with the same COD concentration. The other con-stituents of the mineral medium were (g L�1): KH2PO3 0.05, CaCl2

0.4, MgSO4�7H2O 0.1 and 1 ml L�1 of trace elements solution. Thetrace elements solution contained (g L�1): 5 EDTA, 5 MnCl2�4H2O,3 FeSO4�7H2O, 0.05 CoCl�6H2O, 0.04 NiCl2�6H2O, 0.02 H3BO3, 0.02(NH4)6Mo7O2�4H2O, 0.01 CuSO4�5H2O and 0.003 ZnSO4. The pH ofsynthetic wastewater was in the range of 7.1 ± 0.2.

2.2. Reactor operation

The experimental work was carried out in two parallelplexiglass-made denitrifying automatic circulation (DAC) reactors(one fed with CH3OH, the other fed with CH3COONa) with inner

Fig. 1. DAC reactor system.

diameter of 0.06 m and height of 0.45 m (Fig. 1). The effective vol-ume was 1.25 L (Li et al., 2013). 1 L denitrifying granular sludge ob-tained from other lab-scale reactor was used as seeding sludge. TheVSS/SS and settling velocity (Vs) were 0.55 and79.41 ± 13.77 m h�1 (Li et al., 2013), respectively. The reactor wasstarted up at high NRR of 25 kg m�3 d�1 (Bode et al., 1987) witha fixed effluent recycling ratio (recycling flow to inflow ratio) of2.0. Both the synthetic wastewater and the recycling liquid weremixed in the manifold at the bottom of reactor. The loading ratewas increased by shortening hydraulic retention time (HRT) (Tanget al., 2011). Experimental temperature was set at 30 ± 1 �C.

2.3. Analytical methods

The influent and effluent samples were taken using a syringeand filtrated with disposable Millipore filter units (0.45 lm poresize) for analyses of pH, alkalinity, calcium, nitrate, nitrite, ammo-nium and chemical oxygen demand (COD). The pH, alkalinity,calcium, nitrate, nitrite, ammonium, COD, suspended solids (SS)and volatile suspended solids (VSS) were determined accordingto the Standard Methods (APHA, 2005).

For analysis of total calcium in granules, samples of 20 ml weredirectly transferred into a digestion tube and 2.5 ml 65% HNO3,7.5 ml 37% HCl and 10 ml distilled water were added. Hereafter,the mixture was heated for two hours at 80 �C, subsequentlycooled, and diluted with demineralized water in a volumetric flaskof 100 ml. Then, calcium was analyzed according to the StandardMethods (APHA, 2005).

For scanning electron microscopy (SEM) analysis, granules werefixed for 2 h in 2.5% glutaraldehyde. After rinsing twice with so-dium cacodylate buffer, the granules were fixed for 1.5 h in 1% os-mium tetroxide. After rinsing with demineralized water, theaggregates were dehydrated in an ethanol series (10%, 30%, 50%,70%, 90% and 100%, 20 min per step) and subsequently dried atcritical point with CO2. After gold/palladium sputter coating, theaggregates were examined by SEM. SEM was performed accordingto Zhang et al. (2009). Energy-dispersive X-ray spectroscopy (EDS)was used for element content analysis of the precipitates ingranules. The samples tested were the same as in SEM analysis.

2.4. Specific denitrification activity (SDA) assays

The denitrification batch assays were performed in serum bot-tles with a volume of 120 ml. A synthetic wastewater with NaNO3

concentration of 200 mg L�1 was prepared. The NO�3 –N/COD ratiowas fixed at 5. The pH were adjusted as needed (glycine-NaOH buf-fer was used for pH of 9.3 ± 0.1 and pH of 9.7 ± 0.1, Na2HPO4–NaH2-

PO4 buffer was used for pH of 7.8 ± 0.1). The biomass concentrationwas about 1 g VSS L�1. The temperature was 30 ± 1 �C. Gas and li-quid phases were purged with 95% Ar-5% CO2 for 20 min. The ser-um bottles were sealed tightly with butyl rubber caps. The NO�3 –Nconcentration was monitored during the incubation. SDA was esti-mated from a decrease of substrate concentration and biomassconcentration.

3. Results and discussion

3.1. Performance of the DAC reactor

3.1.1. Performance of the DAC reactor fed with methanolWith the initial NLR of 25 kg m�3 d�1, the DAC reactor fed with

methanol was operated by shortening HRT at fixed influent sub-strate concentration. The performance of DAC reactor is depictedin Fig. 2. During the whole operation (0–70 day), the NLR increasedfrom 24.58 to 56.40 kg m�3 d�1, with an increase of NRR from

Fig. 2. Volumetric capacity (A, B) and removal efficiency (C, D) of the high-rate DAC reactor fed with methanol.

Fig. 3. Volumetric capacity (A, B) and removal efficiency (C, D) of high-rate DAC reactor fed with sodium acetate.

46 W. Li et al. / Bioresource Technology 154 (2014) 44–50

24.41 to 56.35 kg m�3 d�1. On the 65th day, the NLR reached up to56.40 kg m�3 d�1 and the NRR increased to 56.35 kg m�3 d�1

(Fig. 2A). At the same time, the COD loading rate (OLR) reachedup to 288.69 kg m�3 d�1, and the COD removal rate (ORR)

increased to 180.18 kg m�3 d�1 (Fig. 2B). The nitrogen removalratio was above 99%, and the effluent nitrogen concentration wasbelow 6.86 mg L�1 with an average of 0.78 mg L�1 (Fig. 2C). TheCOD removal ratio was higher than 62%, and the effluent COD

W. Li et al. / Bioresource Technology 154 (2014) 44–50 47

concentration was lower than 1944.04 mg L�1 with an average of1394.70 mg L�1 (Fig. 2D).

3.1.2. Performance of the DAC reactor fed with sodium acetateThe DAC reactor fed with sodium acetate was also operated by

shortening HRT at fixed influent substrate concentration. The per-formance of DAC reactor is depicted in Fig. 3. During the wholeoperation (0–70 day), the NLR increased from 25.08 to57.24 kg m�3 d�1, with an increase of NRR from 25.02 to57.13 kg m�3 d�1 (Fig. 3A). On the 67th day, the NLR reached upto 57.24 kg m�3 d�1 and the NRR increased to 57.13 kg m�3 d�1

(Fig. 3A). At the same time, the OLR reached up to 277.07 kg m�3 -d�1, and the ORR increased to 232.57 kg m�3 d�1 (Fig. 3B). Thenitrogen removal ratio was above 99%, and the effluent nitrogenconcentration was below 3.56 mg L�1 with an average of1.82 mg L�1(Fig. 3C). The COD removal ratio was higher than 70%,and the effluent COD concentration was lower than 1500 mg L�1

with an average of 1139.34 mg L�1 (Fig. 3D).These results demonstrated that the performance of high-rate

DAC reactor was good when methanol or sodium acetate was usedas carbon source. Both the NRR and ORR observed for these two

Fig. 4. Profile of effluent pH, effluent alkalinity and effluent HCO�3 =CO2�3 ratio of d

reactors were surpassed the top levels (25 kg N m�3 d�1 and67.5 kg COD m�3 d�1) reported so far in the literatures (Bodeet al., 1987; Franco et al., 2006). The effluent nitrogen concentra-tions for both reactors were below 10 mg L�1 which was the USdrinking water standard (USEPA, 1987).

3.2. Self-alkalization in the DAC reactor

During the whole operation, profiles of effluent pH and effluentalkalinity of denitrifying reactor fed with methanol and sodiumacetate were shown in Fig. 4. The NRR increased from 25 to55 kg N m�3 d�1 and effluent pH increased from 9.10 to 9.45 whenmethanol served as carbon source (Fig. 4A). Surprisingly, the efflu-ent alkalinity decreased from 3625.82 to 3121.32 mg CaCO3 L�1

(Fig. 4C). The NRR increased from 25 to 55 kg N m�3 d�1 and efflu-ent pH increased from 9.60 to 9.88 (Fig. 4B) when sodium acetateserved as carbon source. While the effluent alkalinity decreasedfrom 6938.13 to 5929.12 mg CaCO3 L�1 (Fig. 4D).

At the NLR of 45 kg N m�3 d�1, acid–base titration was con-ducted at 25 �C with 0.2 M HCl to determine the influent and efflu-ent alkalinity. The results were shown in Fig. 5. The effluent

enitrifying reactor fed with methanol (A, C, E) and sodium acetate (B, D, F).

Fig. 5. Acid–base titration curves by 0.2 M HCl for influent and effluent liquid (25 ml) at the NLR of 45 kg N m�3 d�1. (A) was for methanol and (B) was for sodium acetate.

48 W. Li et al. / Bioresource Technology 154 (2014) 44–50

alkalinities were notably higher than the influent counterpart val-ues for both the DAC reactors (methanol and sodium acetate). Andthe alkalinity increment of 5465.46 ± 159.17 mg CaCO3 L�1 in thereactor fed with sodium acetate was notably higher than the coun-terpart value of 3531.53 ± 42.04 mg CaCO3 L�1 in the reactor fedwith methanol. In addition, there were two abrupt ranges on eacheffluent acid titration curve (Fig. 5A and B). The midpoint of thefirst abrupt range was located near the pH of 8.3, and the midpointof the second abrupt range was located near the pH of 3.9. Thesetwo points were well fitted with the first stoichiometric point(pHsp1 = 8.32) and the second stoichiometric point (pHsp2 = 3.89)of carbonate-bicarbonate buffering solution. In other words, thecarbonate-bicarbonate buffering system originated from hetero-trophic denitrification was the main source of alkalinity in effluentsolution. Moreover, in the same pH range of 3.9–8.3, the acid con-sumption of denitrifying effluent with sodium acetate as carbonsource was 2.76 mM, significantly higher than the counterpart va-lue of 1.54 mM when methanol served as carbon source. In otherwords, buffering capacity of the former was much higher than thatof the latter.

According to the ionization equilibrium, the effluent pH is clo-sely related to the HCO�3 =CO2�

3 ratio Eq. (3). With methanol as acarbon source, the measured effluent HCO�3 =CO2�

3 ratio was 9.00–4.92 (Fig. 4E) and the pH was 9.10–9.45 which showed a goodagreement with the theoretical value of 9.33–9.59 calculated fromEq. (3). With sodium acetate as carbon source, the measuredeffluent HCO�3 =CO2�

3 ratio was 2.38–1.67 (Fig. 4F) and the pH was9.60–9.88 which was also near the theoretical value of 9.90–10.06 calculated from Eq. (3).

Methanol is the most popular carbon source for heterotrophicdenitrification. With methanol as carbon source, 3.57 g CaCO3

alkalinity can be produced per g NO�3 –N by denitrification accord-ing to the Eq. (1) (McCarty et al., 1969). If the alkalinity fromdenitrification with methanol as carbon source was taken as astandard, the carbon source can be divided into 3 types: weak alka-lizer, moderate alkalizer and strong alkalizer. With sodium acetateas carbon source, 7.36 g CaCO3 alkalinity is produced per gNO�3 –Nby denitrification according to Eq. (2) (Elefsiniotis et al., 2004). So,sodium acetate belongs to strong alkalizer.

Table 1Ca distribution in DAC reactors and their effects on alkalinity loss at high NLR.

Carbon resource NLR(kg N m�3 d�1)

Influent Ca(mmol d�1)

Effluent Ca(mmol d�1)

Sludge(g VSS

Methanol 45 202.50 ± 10.70 19.69 ± 1.13 15.76 ±Sodium acetate 45 200.80 ± 9.40 21.94 ± 0.56 17.22 ±

3.3. Self-adaptation to alkalization in the DAC reactor

3.3.1. Neutralization of carbon dioxideNitrate reduction is a process of self-alkalization because of the

hydrogen ions consumption (Jetten et al., 2009). In heterotrophicdenitrification, nitrate reduction is coupled with the oxidation oforganic matter (Wu et al., 2012) which can produce and releasecarbon dioxide. The saturation concentration of CO2 in water isabout 0.033 mol L�1 at normal temperature and pressure. Andthe carbonic acid (H2CO3), generated by dissolving CO2 in liquid,can neutralize OH� (Formula 4) and produce HCO�3 and CO2�

3 underalkaline conditions. Thus, in this experiment, the carbon dioxideproduced by denitrificaiton played a certain role of neutralizationin the effluent liquid.

ð4Þ

3.3.2. Formation and discharge of carbonate precipitationThe stoichiometric alkalinity of reactor effluent was

3570 mg CaCO3 L�1 (methanol) and 7360 mg CaCO3 L�1 (sodiumacetate), according to Eqs. (1) and (2), respectively. And the corre-sponding stoichiometric pH was 9.99 and 10.36, respectively(Fig. 4). While the measured effluent alkalinity was 3625.82–3121.32 mg CaCO3 L�1 (methanol) and 6938.13–5929.12 mgCaCO3 L�1 (sodium acetate), respectively. And the correspondingmeasured pH was only 9.10–9.45 and 9.60–9.88, respectively. Eq.(3) showed that the pH decrease was related to the increase ofHCO�3 =CO2�

3 ratio. But why were the HCO�3 =CO2�3 ratios higher than

the stoichiometric values in both effluent liquids? Why was the to-tal effluent alkalinity lower than the stoichiometric values?

Calcium (Ca) is the key element in the formation of granularsludge and it precipitates easily in alkaline conditions (Van Lange-rak et al., 1998). The dose in previous researches was generally100–600 mg L�1 (Yu et al., 2001). In this experiment, the influent

discharged�1)

Ca in sludge(mmol g VSS�1)

Ca discharge bysludge (mmol d�1)

Maximum alkalinitylose (mg CaCO3 L�1)

3.94 9.75 ± 0.70 156.42 ± 49.45 278.08 ± 87.912.72 22.47 ± 0.47 388.21 ± 69.21 690.15 ± 123.04

Table 2Specific denitrifying activity of granular sludge at different NLR and pH.

Substrate NLR (kg N m�3 d�1) pH SDA (mg g VSS�1 h�1) pH SDA (mg g VSS�1 h�1)

Methanol 45 9.3 ± 0.1 38.92 ± 4.46 7.8 ± 0.1 25.21 ± 2.245 9.3 ± 0.1 18.68 ± 5.02 7.8 ± 0.1 17.58 ± 4.05

Sodium acetate 45 9.7 ± 0.1 40.00 ± 3.66 7.8 ± 0.1 33.83 ± 3.855 9.7 ± 0.1 22.69 ± 3.72 7.8 ± 0.1 24.23 ± 2.15

W. Li et al. / Bioresource Technology 154 (2014) 44–50 49

calcium concentration was 3.60 ± 0.19 mmol L�1, and the effluentcalcium concentration was 0.35 ± 0.02 mmol L�1 (Methanol) and0.39 ± 0.01 mmol L�1 (acetate sodium) (Table 1), respectively. Thatmeans 99% of the Ca was retained in the reactor. Morphologicalcharacteristics of the granules were observed using scanning elec-tron microscope (SEM) to characterize the formation of carbonateprecipitation. The results were shown in (Fig. S1). In both reactors(methanol and sodium acetate), lots of agglomeration of crystals ingranular sludge could be easily found. The result was in accordwith the report by Jin et al. (2012). The average contents of calciumin precipitates were 37.55% (methanol) and 34.71% (sodium ace-tate) from the granular sludge (Fig. S1). The solubility of CaCO3

was only 12 mg L�1 at normal temperature and pressure, whichwas far lower than the counterpart value of Ca(HCO3)2. Therefore,the formation of CaCO3 caused by Ca2+ and CO2�

3 accounted for thereduction of effluent alkalinity.

According to the conservation of Ca in the reactor, the differ-ence between influent Ca and effluent Ca was almost equal tothe discharged quantity of Ca by discharging excessive sludgeevery day (Table 1). Moreover, assuming all the Ca in excess sludgewas converted into carbonate precipitate (CaCO3), the alkalinitylosses in the two reactors were 278.08 ± 87.91 mg CaCO3 L�1

(methanol) and 690.15 ± 123.04 mg CaCO3 L�1 (sodium acetate)(Table 1). These values were almost equal to the difference be-tween the stoichiometric alkalinity and the detected alkalinity inthe effluent (Fig. 4C and D).

3.3.3. Adaptation of denitrifying sludgeDenitrifying sludge is the fundamental part of denitrification

reactor. In this experiment, sludge samples were taken from thereactors with the NLR of 5 kg N m�3 d�1 (reactor operated by Liet al. (2013)) and 45 kg N m�3 d�1 (this experiment). To investigatethe adaptation of denitrifying sludge, the determinations of SDAwere carried out at normal denitrification pH of 7.8 (Hiscocket al., 1991; Mateju et al., 1992; Lee and Rittmann, 2003) and efflu-ent pH, respectively. The results were listed in Table 2. At the high-load of 45 kg N m�3 d�1, the SDA of denitrifying sludge with meth-anol as carbon source was 38.92 ± 4.46 mg g VSS�1 h�1 with the pHof 9.3 ± 0.1, which was much higher than the counterpart value of25.21 ± 2.24 mg g VSS�1 h�1 with the pH of 7.8 ± 0.1. At the normalNLR of 5 kg N m�3 d�1, the SDA of denitrifying sludge with metha-nol as carbon source was 18.68 ± 5.02 mg g VSS�1 h�1 with the pHof 9.3 ± 0.1, which was approximately equal to the counterpart va-lue of 17.58 ± 4.05 mg g VSS�1 h�1 with the pH of 7.8 ± 0.1. Besides,similar results were obtained in the SDA test of denitrifying sludgewith sodium acetate as carbon source. These results suggested thatthe denitrifying sludge in high-rate denitrification reactor couldwell adapt to high pH after the long-term operation at high NLRs.

4. Conclusions

The self-alkalization of high-rate DAC reactors were remarkablystrong both with methanol and sodium acetate as carbon sources,while the DAC reactor could well self-adapted to the high-pH and

high-alkalinity. The NRR and ORR were more than 55 kg N m�3 d�1

and 180 kg COD m�3 d�1, respectively.The self-adaptation to high-pH and high-alkalinity was attrib-

uted to the neutralization of carbon dioxide (oxidization productof organic matter) and the self-adaptation of denitrifying sludge.The formation and discharge of calcium carbonate precipitatesgave rise to lower effluent pH and alkalinity than the stoichiome-tric values.

Acknowledgements

This research was supported by the Natural Science Foundationof China (31070110), the Natural Science Foundation of China(51278457), the Specialized Research Fund for the DoctoralProgram of Higher Education of China (20110101110078) andthe Natural Science Foundation of Zhejiang Province (Z5110094).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.11.097.

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