Effects of metabolic uncouplers on excess sludge reduction and microbial products of activated sludge

Bioresource Technology 185 (2015) 1–6

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Effects of metabolic uncouplers on excess sludge reduction and microbial products of activated sludge Fang Fang a,b, Hai-Lan Hu b, Min-Min Qin b, Zhao-Xia Xue a,b, Jia-Shun Cao a,b,⇑, Zhi-Rong Hu c a

Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098, China College of Environment, Hohai University, Nanjing 210098, China c EnviroSim Associates Ltd, McMaster Innovation Park, 175 Longwood Road South, Suite 114A, Hamilton, Ontario L8P 0A1, Canada b

h i g h l i g h t s  Three uncouplers all resulted in significant sludge reduction.  The EPS content and composition were changed with three uncouplers.  The PHB production was stimulated by three uncouplers.

a r t i c l e

i n f o

Article history: Received 9 January 2015 Received in revised form 11 February 2015 Accepted 12 February 2015 Available online 19 February 2015 Keywords: Activated sludge Metabolic uncouplers Sludge reduction Extracellular polymeric substances (EPS) Polyhydroxybutyrate (PHB)

a b s t r a c t The present study investigated the influences of three metabolic uncouplers (pCP, oCP and oNP) on excess activated sludge reduction and microbial products of extracellular polymeric substances (EPS) and intracellular storage product (polyhydroxybutyrate, PHB) in short-term tests. Results showed sludge was reduced 58.2%, 59.8% and 80.8%, respectively, at pCP, oCP and oNP concentrations of 20 mg/L. The dosage of three uncouplers had no obviously influences on COD removal and sludge settleability, but had significant inhibition effect on ammonia removal, especially for oNP. Low concentration of pCP and oNP (5 mg/L) dosing resulted in protein and polysaccharide content increased in EPS, however, they were decreased at high pCP and oNP concentrations (>5 mg/L). To oCP, the protein content in EPS was increased linearly with oCP concentration. Furthermore, metabolic uncouplers addition stimulated the production of PHB. Among three uncouplers, oCP could be an alternative uncoupler for sludge reduction in activated sludge process. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Conventional activated sludge process which is widely used in municipal and industry sewage treatment plants (WWTPs), will produce a large quantity of excess sludge. These excess sludge should be treated before discharge and the treatment costs of the excess sludge were high, which may be accounted for 50–60% of the total operation costs of wastewater treatment plants (Horan, 1999; Campos et al., 2009; Feng et al., 2013). Therefore, effective strategies should be developed to reduce sludge production through either sludge post treatment or sludge in-situ treatment. Due to the high operational complexity and expenses of the post treatment methods, in-situ excess sludge reduction processes have ⇑ Corresponding author at: Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098, China. Fax: +86 25 83786701 (J.-S. Cao). E-mail addresses: [email protected] (F. Fang), [email protected] (J.-S. Cao). http://dx.doi.org/10.1016/j.biortech.2015.02.054 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

drawn great attentions (Guo et al., 2013). Different approaches of excess sludge in-situ reduction have been applied and the mechanism of these methods can be categorized as cell lysis–cryptic growth, maintenance metabolism, predation on bacteria, and uncoupling metabolism (Liu, 2003; Wei et al., 2003; Chen et al., 2004; Ye and Li, 2005; Tian et al., 2013). Because of the advantages of convenience, high-efficiency and easy operation, uncoupling metabolism achieved by adding metabolic uncouplers has been proven to be a highly useful approach (Li et al., 2012; Qiao et al., 2012; Feng et al., 2013). The mechanism of metabolic uncoupling reduction is to dissociate the energy coupling between catabolism and anabolism. Thus, a part of energy extracted from substrates is wasted through futile cycles and energy supply for anabolism is limited, leading to a decrease in biomass production (Chen et al., 2000; Tian et al., 2013). Different metabolic uncouplers, such as 2,4-dinitrophenol (dNP), para-nitrophenol (pNP), 2,4-dichlorophenol (dCP) and 3,30 ,40 ,5-tetrachlorosalicylanilide (TCS) addition led to excess

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sludge production reduced (Aragon et al., 2009; Chen et al., 2006; Ye and Li, 2005; Tian et al., 2013). Chen et al. (2002) reported by adding 0.8–1.0 mg/L TCS, 40.0% of the excess sludge yield was reduced. Tian et al. (2013) and Zhang et al. (2013) had proved the effectiveness of 2,6-DCP on biomass growth in long-term sludge culture and found the sludge yield was decreased about 40%. In these studies, the sludge production was decreased with metabolic uncouplers addition. However, what the other part substrate is used for and where the other energy goes are still unknown yet. In recent years, some studies investigated the link between microbial products and sludge reduction with metabolic uncoupler addition. Extracellular polymeric substances (EPS), an extracellular microbial product, consisted of proteins, polysaccharides, humic acid, DNA and lipids, were demonstrated to provide a protective barrier for the bacteria inside activated sludge (Frølund et al., 1996; Henriques and Love, 2007; Sheng et al., 2010). It is reported adding 2,6-DCP in activated sludge system resulted in the significant increase in EPS content (Tian et al., 2013; Zhang et al., 2013). When dosing TCS or TCP to sequencing batch reactors, the contents of tryptophan, tyrosine protein-like substances and tryptophan, tyrosine amino-like substances were increased (Feng et al., 2013). Furthermore, other microbial product, such as intracellular storage product of polyhydroxybutyrate (PHB) which is important in nutrient removal is usually found in activated sludge systems (Majone et al., 1999). However, the investigations of the influences of the metabolic uncouplers on intracellular storage product of PHB were still scarce. Therefore, in this study the influences of different metabolic uncouplers on excess sludge reduction and nutrient removal were investigated. In addition, the effects of metabolic uncouplers on microbial products of EPS and PHB production were also evaluated. Hopefully, the results would provide a better understanding of metabolic uncouplers on activated sludge in the biological wastewater treatment systems. 2. Methods 2.1. Activated sludge, reactor and wastewater The activated sludge used for experiments was collected from Jiangning municipal wastewater treatment plant in Nanjing, China. Before experiments, the sludge was cultivated in a sequencing batch reactor (SBR) with the working volume of 3.6 L at room temperatures. The reactor was operated sequentially within a 6-h cycle, including 7 min of influent filling, 328 min of aeration, 10 min of settling, and 15 min of effluent discharging. The hydraulic retention time was 12 h and the sludge retention time was maintained at 15 d. The dissolved oxygen (DO) concentration was controlled above 4 mg/L and the mixed liquid suspended solid (MLSS) level was kept at about 2500 mg/L by withdrawing the excess sludge. The cultivation continued for 2 months without the addition of metabolic uncouplers. A synthetic wastewater was used for cultivation, which composed of sodium acetate (770 mg/L) as a sole carbon source for bacterial growth, NH4Cl (153 mg/L), KH2PO4 (43.8 mg/L), MgSO4 (20 mg/L), and CaCl2 (20 mg/L). In addition, other microelement solution of 1.0 ml/L was added, which contained (unit in mg/L): EDTA 50; ZnSO47H2O 22; MnCl24H2O 5.1; FeSO44H2O 5.0; (NH4)6Mo7O244H2O 1.1; CuSO45H2O 1.8; and CoCl26H2O 1.6. HCl or NaOH was applied to adjust pH around 7.0. 2.2. Batch experiments Batch experiments were performed in triplicate to investigate the effect of different metabolic uncouplers on sludge growth

and microbial products. In this study, o-chlorophenol (oCP), pchlorophenol (pCP) and o-nitrophenol (oNP) were chosen as metabolic uncouplers, and their concentrations were 5, 10, 15 and 20 mg/L, respectively. Meanwhile, control tests without the addition of uncouplers were also conducted in parallel. For all batch experiments, initial biomass and influent substrate concentrations were fixed at about 2500 mg MLSS/L and 300 mg COD/L, respectively. All batch tests were carried out for 6 h, and the beakers were aerated with air pumps to maintain a DO concentration above 4 mg/L. 2.3. EPS extraction The EPS of activated sludge were extracted using the cation exchange resin (CER) technique according to Frølund et al. (1996). The sludge samples were harvested by centrifugation at 5000 rpm for 15 min, and then the pellets were washed twice with 100 mmol/L NaCl solution. The sludge pellets were re-suspended to its original volume with a phosphate buffer. After that, the sludge suspension was mixed with CER in sodium form (Dowex Marathon C, 20–50 mesh, Sigma–Aldrich) with a dosing of 60 g/g VSS and followed by stirring at 200 rpm for 6 h at 4 °C. The samples were then centrifuged at 10,000 rpm and 4 °C for 30 min and filtered through 0.45-lm cellulose acetate membrane. 2.4. Analytic methods  COD, NH+4-N, NO 2 -N, NO3 -N, MLSS, mixed liquor volatile suspended solids (MLVSS), and sludge volume index (SVI) were measured according to the Standard Methods (APHA et al., 2005). The observed sludge yield (Yobs) was defined as the excess biomass generated per COD consumed. The efficiency for excess sludge reduction is evaluated by the reduction percentage of Yobs compared with the control test (Li et al., 2012). The content of protein was determined using the modified Lowry method using bovine serum albumin as the standard (Frølund et al., 1996), and the content of polysaccharide was measured with the anthrone method utilizing glucose as the standard (Raunkjaer et al., 1994). The intracellular storage product of PHB was measured according to Fang et al. (2009). Three-dimensional excitation–emission-matrix (EEM) fluorescence spectrometry was used to characterize the EPS of activated sludge under metabolic uncouplers dosing. All EEM spectra were measured using a luminescence spectrometry (F7000, Hitachi, Japan). The EEM spectra were collected with subsequent scanning emission spectra from 200 to 450 nm at 0.5 nm increments by varying the excitation wavelength from 200 to 600 nm at 10 nm increments. Excitation and emission slits were both maintained at 5 nm, and the scanning speed was set at 1200 nm/min for all measurements. The spectrum of the deionized water was recorded as the blank (Sheng and Yu, 2006). The software MatLab 7.0 (MathWorks Inc., USA) was employed for handling EEM data.

3. Results and discussion 3.1. Influences of metabolic uncouplers on sludge reduction The influences of pCP, oCP and oNP at different concentrations on sludge yield and sludge reduction are shown in Fig. 1. When pCP concentration was increased from 0 to 20 mg/L, sludge production in terms of Yobs was declined from 0.45 to 0.18 mg VSS/ mg COD. In the presence of 20 mg/L pCP, the sludge reduction reached about 60.0% compared to the control test without uncouplers addition. Increase in oCP concentration also led to a decrease in Yobs. At an oCP concentration of 20 mg/L, the Yobs was 0.19 mg

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Fig. 1. Effects of metabolic uncouplers on sludge yield and sludge reduction: (A) pCP; (B) oCP; and (C) oNP.

VSS/mg COD, which supposed a sludge reduction of approximately 57.8% in comparison with the control (0.45 mg VSS/mg COD). These results were consistent with those obtained by Yang et al. (2003). The sludge production exhibited the same trend as pCP and oCP when oNP concentration was increased from 0 to 20 mg/ L. However, when oNP concentration was 20 mg/L, the value of Yobs reached 0.09 mg VSS/mg COD, which was lower than that reported by Yang et al. (2003). These results suggested that addition of three metabolic uncouplers all can significantly reduce sludge production. With the addition of metabolic uncouplers, the proton gradient across cell membrane which drives oxidative phosphorylation for adenosine triphosphate (ATP) generation was diminished. As a result, the energy generated from the oxidation of organic substrate would be lost as heat rather being captured in ATP (Low and Chase, 1999; Yang et al., 2003). Consequently, growth efficiency of activated sludge is reduced with the dosing of the three metabolic uncouplers. Furthermore, comparison of the three uncouplers, oNP was the strongest metabolic uncoupler in terms of sludge reduction. As previous study, a lower acidity constant (pKa) would diminish the proton motive force for ATP generation, resulting in a high sludge reduction (Yang et al., 2003). Among the three metabolic uncouplers, oNP belongs to nitrophenolic uncoupler and its pKa is lowest, leading to the highest sludge reduction.

3.2. Pollutant removal and sludge characteristics with metabolic uncouplers dosage Fig. 2 illustrated the COD and NH+4-N removal efficiency with the addition of pCP, oCP and oNP at different concentrations. As shown in Fig. 2(A), the COD removal efficiencies were gradually decreased with the increase of the three metabolic uncouplers concentration. At pCP and oCP concentrations of 5 mg/L, the COD removal efficiencies were slightly lower than that of the control. When pCP and oCP concentrations were 20 mg/L, the COD removal efficiencies were 75% and 73%, respectively. The COD removal was not significantly influenced with chlorophenols as metabolic uncouplers. Compared with chlorophenolic uncouplers, nitrophenolic uncoupler of oNP

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had relatively significant influence on COD removal efficiency. When the oNP concentration was 20 mg/L, the COD removal efficiency was 68% and decreased by approximately 25% as compared to the control. Therefore, chlorophenolic uncoupler-induced reduction in COD removal efficiency is much less important compared to lowered sludge production (Chen et al., 2006; Yang et al., 2003; Zheng et al., 2008). The effects of three uncouplers on NH+4-N removal efficiency are shown in Fig. 2(B). The NH+4-N removal efficiency was decreased to 71% at a pCP concentration of 5 mg/L and was maintained about 65% with pCP concentration in the range of 10–20 mg/L. To oCP, NH+4-N removal efficiency was declined to about 77% when the concentration of oCP was ranged from 5 to 20 mg/L. However, oNP dosage in activated sludge had remarkable influence on NH+4-N removal efficiency. It was decreased from 91% to 27% with an increase of oNP concentration from 0 to 20 mg/L. The NH+4-N removal efficiencies were all decreased with the addition of the three metabolic uncouplers. The results were consistent with those reported by Zhang et al. (2013), who also found the oxidation of NH+4-N was significantly interfered with the addition of uncouplers. This inhibition effect was resulted from ammonia-oxidizing bacteria (AOB) activity inhibition by metabolic uncouplers and was proved by ammonia uptake rate (AUR) decrease with metabolic uncouplers addition (Tian et al., 2013). In addition, the SVI values of activated sludge after metabolic uncouplers dosage were investigated. Compared to the control, the SVI values increased nearly by 4%, 25% and 13%, respectively, with the addition of pCP, oCP and oNP concentrations in a range of 5–20 mg/L (Fig. 3). However, the SVI values remained in normal levels, indicating that the three uncouplers had slight influence on the settleability of activated sludge.

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3.3. EPS content in the presence of three uncouplers

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The EPS contents and compositions of activated sludge in the presence of three metabolic uncouplers at different concentrations are illustrated in Fig. 4. The dosage of three metabolic uncouplers all led to an increased production of EPS. At a pCP and oNP concentration of 5 mg/L, EPS content increased to 11.8 and 14.9 mg/g VSS, respectively, compared with 7.2 mg/VSS for the control. When the pCP or oNP concentration increased from 10 to 15 mg/L, EPS content decreased. With the further increasing pCP and oNP dosage to 20 mg/L, EPS content was increased to 14.9 and 11.3 mg/g VSS, respectively. To oCP, EPS content was increased with the increase of oCP concentration from 0 to 20 mg/L. The results were in consistent with those reported by Li et al. (2012), who found that EPS production was promoted in the presence of TCS. Furthermore, the linear regression suggests that EPS production is linearly proportional to the initial oCP concentration with a correlation coefficient of 0.96. Dosage of metabolic uncouplers stimulated the production of EPS, suggesting that the biomass was sensitive to the presence of metabolic uncouplers and produced more EPS act as function through retardation of metabolic uncouplers penetration to protect sludge microorganisms for better survival in the unfavorable environment (Henriques and Love, 2007; Tian et al., 2013; Zhang et al., 2013). The metabolic uncouplers dosage influences not only the quantity but also the composition of EPS. Compared with control test, two main constituents of EPS-protein and polysaccharide were both increased with 5 mg/L pCP and oNP, indicating that low concentration metabolic uncouplers of pCP and oNP could stimulate the protein and polysaccharide production in EPS. But a remarkable reduction in the protein content occurred when pCP and oNP concentration were further increased from 5 to 15 mg/L. The results suggested that certain concentration of pCP and oNP inhibited the production of protein. The phenomenon was similar with that reported by Wang et al. (2014), who found that D-tyrosine dosage would decrease the protein content of activated sludge. Differently, the protein contents increased significantly with the increase of oCP concentration from 0 to 20 mg/L, while the polysaccharide levels changed slightly. This finding was in agreement with previous study by Li et al. (2012), who found that the protein content increased significantly with TCS dosage. EEM spectroscopy was proven to be an appropriate and effective method to characterize the EPS (Sheng and Yu, 2006). The threedimensional EEM spectra of extracted EPS from activated sludge after the dosage of metabolic uncouplers of pCP, oCP and oNP are

oCP 350 15 mg/L 300

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200 200 200 200 200 250 300 350 400 450 250 300 350 400 450 250 300 350 400 450 250 300 350 400 450 250 300 350 400 450 400 400 400 400 400 oNP oNP oNP oNP oNP 350 0 mg/L 350 5mg/L 350 15 mg/L 350 20mg/L 350 10 mg/L 300 300 300 300 300 250

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F. Fang et al. / Bioresource Technology 185 (2015) 1–6

and oNP were investigated in batch experiments. The influence of three uncouplers on sludge yield, nutrient removal efficiency and sludge settleability were evaluated. The EPS contents and intracellular storage product of PHB content were determined with three uncouplers at different concentrations. The results indicated that among three uncouplers, oCP could be an alternative metabolic uncoupler to achieve sludge reduction in activated sludge process.

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Fig. 6. Effect of different uncouplers on the PHB contents at the end of the reaction.

illustrated in Fig. 5. Three main peaks were identified from the EEM fluorescence spectra of the EPS extracts. The first peak (Peak A) is located at the excitation/emission (Ex/Em) wavelengths of 230/ 300 nm, which is described as tyrosine amino acid. The second peak (Peak B) is identified at Ex/Em of 240/350–370 nm, describing as tryptophan amino acid. The third peak (Peak C) is at Ex/Em of 270/ 370–380 nm, which is ascribed to tryptophan protein (Chen et al., 2003; Feng et al., 2013; Yang et al., 2012). As shown in Fig. 5, the three fluorescent features were not influenced by the variations of the kinds and concentrations of the metabolic uncouplers. 3.4. Effect of three uncouplers on PHB production Due to the particularly importance of intracellular storage product of PHB in activated sludge, PHB contents at the end of the experiments in the presence of metabolic uncouplers were investigated. As illustrated in Fig. 6, PHB contents were increased with the increase of three uncouplers concentration from 0 to 10 mg/L. The maximum PHB accumulation was observed as 56.8, 42.9 and 40.4 mg/g VSS, respectively, at pCP, oCP and oNP concentration of 10 mg/L. Then, PHB contents were decreased after further increasing of three uncouplers concentration from 10 to 20 mg/L. In addition, the yield of PHB by pCP addition was higher than those of oCP and oNP in comparison. These results indicated that metabolic uncouplers had significant influences on the production of intracellular PHB, which were in disagreement with that no obvious variation in terms of intracellular substances with TCS, TCP and combined uncouplers addition (Feng et al., 2013). With metabolic uncouplers dosing, the energy coupling between catabolism and anabolism was dissociated, thus, the electron and energy distribution in activated sludge were changed (Feng et al., 2014). A part of electrons and energy from substrates may be used for polymerized products storage and this fraction could be increased at metabolic uncouplers dosing. Thus, intracellular polymerized products of PHB was increased at low concentration of metabolic uncouplers (610 mg/L) (Low and Chase, 1999; Tian et al., 2013). However, when metabolic uncouplers concentrations were high (>10 mg/L), the substrate utilization efficiency was inhibited because of the toxicity of metabolic uncouplers, resulting in the decrease in the electrons and carbon available from substrate. Therefore, the PHB contents were declined. The exact reason is unknown and more investigations are needed in further research. 4. Conclusion The sludge reduction and metabolic products of activated sludge with the dosing of three metabolic uncouplers-pCP, oCP

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