Bioresource Technology 177 (2015) 194–203

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Economical evaluation of sludge reduction and characterization of effluent organic matter in an alternating aeration activated sludge system combining ozone/ultrasound pretreatment Shan-Shan Yang, Wan-Qian Guo ⇑, Yi-Di Chen, Qing-Lian Wu, Hai-Chao Luo, Si-Mai Peng, He-Shan Zheng, Xiao-Chi Feng, Xu Zhou, Nan-Qi Ren State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, PR China

h i g h l i g h t s  AAMA + O3/US2# system was economically feasible that can give a 14.04% saving of costs.  55.08% sludge reduction was achieved in AAMA + O3/US2# system compared with AAMA1#.  Less humic substance and soluble microbial products were generated from AAMA + O3/US2#. +

 NH4 -N removal and TTC-ETS activities showed significant positive correlations.  Appropriate sludge lyses recycling gave rise to the improvement in microbe activity.

a r t i c l e

i n f o

Article history: Received 26 August 2014 Received in revised form 2 November 2014 Accepted 4 November 2014 Available online 11 November 2014 Keywords: Combined ozone/ultrasound pretreatment Sludge reduction Nutrient removal Microbial activity Economic benefits

a b s t r a c t An ozone/ultrasound lysis–cryptic growth technology combining a continuous flow anaerobic–anoxic– microaerobic–aerobic (AAMA + O3/US) system was investigated. Techno-economic evaluation and sludge lyses return ratio (r) optimization of this AAMA + O3/US system were systematically and comprehensively discussed. Economic assessment demonstrated that this AAMA + O3/US system with r of 30% (AAMA + O3/US2# system) was more economically feasible that can give a 14.04% saving of costs. In addition to economic benefits, a 55.08% reduction in sludge production, and respective 21.17% and 5.45% increases in TN and TP removal efficiencies were observed in this AAMA + O3/US2# system. Considering the process performances and economic benefits, r of 30% in AAMA + O3/US2# system was recommended. Excitation–emission matrix and Fourier transform infrared spectra analyses also proved that less refractory soluble microbial products were generated from AAMA + O3/US2# system. Improvement in 2,3, 5-triphenyltetrazolium chloride electron transport system (TTC-ETS) activity in AAMA + O3/US2# further indicated that a lower sludge lyses return ratio stimulated the microbial activity. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Conventional activated sludge (CAS) process, which involves the transformation of dissolved and suspended organic contaminants into biomass during sewage treatment process (Mohammadi et al., 2011), is widely used for the municipal and industrial wastewater treatment, domestically and internationally. However, one significant disadvantage to CAS process is the high generation of waste activated sludge (WAS) during sewage treatment process. The management and disposal costs of the WAS account for up to 60% of the whole operation expenses (Lin et al., ⇑ Corresponding author. Tel./fax: +86 451 86283008. E-mail address: [email protected] (W.-Q. Guo). http://dx.doi.org/10.1016/j.biortech.2014.11.009 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

2012), and the restrictive economic, environmental and legal regulations have imposed restrictions on the conventional sludge treatment methods, e.g. land application, incineration and land filling (Yang et al., 2011). In view of the environmental burden and the expensive costs, the solution of this increasing WAS problem has become one of the most stringent challenges in sewage treatment field (Li et al., 2013). Therefore, a profound research effort on exploiting and developing new methods for WAS minimization is urgently needed. The mechanisms for in-situ activated sludge reduction technologies are commonly classified into four groups (Guo et al., 2013): chemical or/and physical lysis–cryptic methods combining with the activated sludge processes; uncoupling metabolism; worms’ predation; and improved/novel developed processes. In recent

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Nomenclature AAMA + O3/US system anaerobic–anoxic–microaerobic–aerobic combining ozone/ultrasound system BNPR biological nitrogen and phosphorus removal CAS conventional activated sludge COD chemical oxygen demand DO dissolved oxygen DPAO denitrifying phosphate-accumulating organisms EEM excitation–emission matrix EfOM effluent organic matters FTIR Fourier transform infrared spectroscopy MLSS mixed liquor suspended solids NH+4-N ammonia nitrogen O3 ozone

years, sludge reduction by cell lysis–cryptic growth has aroused much public concern and interest (Lan et al., 2013; Lin et al., 2012; Zuriaga-Agustí et al., 2012). According to the previous investigations, this technique can be realized by means of various treatment methods including thermal, microwave, alkaline, ultrasonic, and ozone oxidation pretreatments, which these pretreatments combining the existing bio-reactors (e.g. CAS, membrane bio-reactor, sequencing batch reactor (SBR) and etc.) used so far at lab, pilot and real scales have already achieved many positive results (Ma et al., 2012; Yang et al., 2013). Zuriaga-Agustí et al. (2012) reported that dosing 2.5 mg chlorine dioxide/g TS in a SBR achieved 43.4% reduction in excess sludge production, while results demonstrated that this technology severely deteriorated effluent quality. Lan et al. (2013) achieved a 42.4% sludge reduction in a SBR under 70 MPa disruption pressure of high-pressure-homogenization, and this lysis–cryptic growth system was found imposed negligible changes in the sludge activity. Dytczak et al. (2007) combined an ozonation stage with a SBR, results found that 20% returned ozonated sludge had no negative impact on the effluent quality of the SBR process. Ma et al. (2012) proposed a continuous operated lysis– cryptic growth system combining ultrasonic and alkaline technologies. Results indicated that 56.5% reduction in excess sludge could be achieved in this pilot-scale lysis–cryptic growth system. Lin et al. (2012) reported a combination of ultrasonic and chlorine dioxide processes. When combined the ultrasonic + chlorine dioxide technology with a SBR, 55% reduction in excess sludge was observed by recycling 70% ultrasonic + chlorine dioxide disrupted sludge. However, obvious disadvantages induced by the ultrasonic + chlorine dioxide technology were the increases in effluent phosphorus and nitrogen concentrations. Based on the previous investigations, although most studies have already demonstrated the ability of sludge reduction by using different sludge lysis–cryptic growth technologies combining bio-reactors, few literatures have paid attention to the worsening of the effluent quality (Yan et al., 2009; Lin et al., 2012). As is well-known, substandard discharging high levels of nitrogen and phosphorous are widely recognized as potential causes of the eutrophication. The breakthrough of water turbidity, oxygen depletion and algae blooms caused by eutrophication would permanently harm human health and severely damage the environment (Amini et al., 2013). Thus, the development of sludge lysis– cryptic growth technologies combining bio-reactors aiming at achieving simultaneous excess sludge reduction and superior biological nitrogen and phosphorus removal (BNPR) efficiency will become the focal points in the future study. Focusing on the application of sludge lysis–cryptic growth technologies, ultrasound (US) and ozonation (O3) are potentially regarded as the wonderful tools (Lin et al., 2012; Yang et al., 2013). US pretreatment, recognized as an effective and promising

PAO r SBR SMP TC TN TP TS TTC-ETS US WAS

phosphate-accumulating organisms sludge lyses return ratio sequencing batch reactor soluble microbial products total cost total nitrogen total phosphorus total solids 2,3,5-triphenyltetrazolium chloride electron transport system ultrasound waste activated sludge

pretreatment, has been proved little negative impact on environment (Guo et al., 2011). Whereas when the US pretreatment is devoted to the WAS, a large proportion of US energy will be absorbed by the liquid (Xu et al., 2010), thus the application of ultrasound sludge lysis–cryptic growth technology might be limited by coupling the lysis step in the bio-reactors. To solve this problem, a combined ozonation and US pretreatment has been proved to lower the US energy consumption and enhance the WAS disruption (Yang et al., 2013). However, to our knowledge, there has been few literature study on this O3/US pretreatment coupling with bio-reactors. In order to provide a comprehensive basis for practical application, comprehensive researches on the application of the combined O3/US pretreatment for process performances are necessary. In this study, an O3/US lysis–cryptic growth technology combining an alternating anaerobic–anoxic–microaerobic–aerobic system (AAMA + O3/US system) was investigated. The objectives of this study are (1) to evaluate the economic assessment of the combined O3/US pretreatment time in this AAMA + O3/US system, (2) to optimize the impacts of different O3/US sludge lyses return ratios on the performances of sludge reduction and BNPR, (3) to analyze the effects of effluent organic matters (EfOM) composition by excitation–emission matrix (EEM) and fourier transform infrared spectra (FTIR) spectra, and (4) to investigate the relationship between the activities of microorganisms and the performances of nutrient removal during sewage treatment process. It is expected that the results obtained in this study can provide a comprehensive basis for the future investigation of an O3/US pretreatment-based wastewater treatment and sludge reduction system. 2. Methods 2.1. Waste activated sludge The waste activated sludge taken from the secondary setting tank of Harbin Wenchang sewage treatment plant was used in this study. Before the experiments started, the seeded sewage activated sludge was screened through a sieve to separate large debris from the activated sludge, and then, the waste activated sludge was settled and washed two or three times to remove any residuals in the supernatant. 2.2. Pretreatment experiments To prepare the O3/US sludge lyses, sludge samples discharged from the corresponding continuous flow systems were pretreated by a combined O3/US apparatus. For the combined O3/US apparatus

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applied in this study, ozone was generated from pure oxygen using an ozone generator (DHX-SS-1G, Jiujiu ozone, Harbin, China), and a low-range gaseous flow meter was installed to adjust the ozone flow rate. The operation parameters of the ozone apparatus were 300 W of the maximum rated power and 12 g(O3)/h of the ozone output. A laboratory-scale ultrasound instrument with a diameter of 8 mm titanium probe transducer, was applied (Shanghai Sonxi Ultrasonic Instrument, Shanghai, China). The optimized parameters applied in this study were performed as 0.154 g(O3)/g(TS)h ozone dose and 1.445 W/mL ultrasound energy density in terms of the previous study (Yang et al., 2013). 2.3. Experiments on the combined O3/US pretreatment time optimization For achieving simultaneous in-situ excess sludge reduction and enhanced effluent quality, an alternating anaerobic–anoxic–

microaerobic–aerobic system combining two-point O3/US sludge lyses recycling lines was performed in this study. Two nitrate recycling flows were returned into the anoxic and microaerobic zones from the subsequent aerobic zone, as shown in Fig. 1b. The detail operation performances of the AAMA + O3/US system were described in Section 2.4. The operation parameters of the AAMA + O3/US system were shown in Table S1 (Supporting Information, SI). To evaluate the economic efficiency of the combined O3/US pretreatment time in this AAMA + O3/US system, six comparison experiments on investigating the relationship between the combined O3/US pretreatment time and the economic assessment were conducted before starting the subsequent process optimization experiments. In these six comparison systems (the C1#, C2#, C3#, C4#, C5#, and C6# systems), the optimization of the combined O3/US pretreatment time was conducted at respective 10, 20, 30, 40, 50, and 60 min. The corresponding 30% daily discharged excess

Fig. 1. Schematic of the control (a) and the AAMA + O3/US systems (b).

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sludge which was pretreated by the combined O3/US sludge technology was concurrently sonicated and ozonized, then returned back to the six alternating aeration systems. The control system was performed without O3/US sludge lyses recycling flows (Fig. 1a). The formula of the economic analyses for the control and the AAMA + O3/US systems was showed in SI Section S3. 2.4. Experiments on the process optimization After the combined O3/US pretreatment time optimization experiments, four combined AAMA + O3/US systems numbered 1#, 2#, 3#, and 4# were maintained in stable states. AAMA1# was performed as the control system without O3/US sludge lyses recycling flows. The three AAMA + O3/US2#, AAMA + O3/US3#, and AAMA + O3/US4# systems were performed with different proportion O3/US sludge lyses return ratios (30%, 60%, and 90% of the produced excess sludge) recycled into the anoxic and microaerobic zones by controlled the sludge return ratio of 1:1, and two liquid flow meters were installed to adjust the O3/US sludge lyses return ratios.  For AAMA + O3/US2#, respective 15% of the pretreated O3/US sludge lyses were returned back to the anoxic and microaerobic reactors.  For AAMA + O3/US3#, respective 30% of the pretreated O3/US sludge lyses were returned back to the anoxic and microaerobic reactors.  For AAMA + O3/US4#, respective 45% of the pretreated O3/US sludge lyses were returned back to the anoxic and microaerobic reactors. Air supply was sprinkled at the bottom of the microaerobic and aerobic zones by using a flow controller (Fig. 1). Excess sludge withdrawal started when the mixed liquor suspended solids (MLSS) concentration exceeded 4000 mg/L. The components of the influent synthetic wastewater applied in this study were shown in Table S2. 2.5. Analytical methods 2.5.1. Chemical analytical methods Chemical oxygen demand (COD), total solids (TS), MLSS, total phosphorus (TP), total nitrogen (TN), and ammonia nitrogen (NH+4-N) were monitored according to the standard method (APHA, 2005). Dissolved oxygen (DO) value was measured by a DO probe (Germany WTW Company pH/Oxi 340i main engine, pH meter, Germany). During experiment operation period, the measurements of COD, MLSS, and TS were performed daily, and analyses of TP, TN, and NH+4-N were conducted three or four times per week. The removal efficiencies of COD, TP, TN, and NH+4-N were calculated by subtracting effluent concentrations from influent concentrations and dividing the results by influent concentrations, then displaying the results in percentage format. The specific operation steps of 2,3,5-triphenyltetrazolium chloride electron transport system (TTC-ETS) activity were performed in SI. In this study, analyses of sludge and liquor samples were conducted by three replicates at room temperature. 2.5.2. EEM and FTIR spectra EEM spectra were measured by using a fluorescence spectrometer (FP-6500, JASCO, Inc.). EEM spectra were operated by scanning emission wavelength from 220 to 600 nm at 1 nm increments and by varying excitation wavelength from 220 to 450 nm at 5 nm increments. Excitation and emission slits widths were set at 3 nm and 5 nm, and the scanning speed was set at 2000 nm/min. The major functional groups of organic matters in effluent organic matters (EfOM) were analyzed by using FTIR spectra (Spectrum

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One B, PerkinElmer, USA). In this study, the analyses of all the soluble components were filtered through 0.45 lm filtration. 3. Results and discussion 3.1. Economic assessment on the combined O3/US pretreatment time in the AAMA + O3/US system In order to evaluate the economic assessment for different combined O3/US pretreatment time, the average daily produced excess sludge from the control and the six comparison systems was shown in Table 1. For the control system, the average daily produced excess sludge was 30.19 ± 1.51 g(TS)/d. While with corresponding 30% produced excess sludge returned back to the six comparison systems, respective 55.08%, 59.99%, 62.70%, 66.11%, 67.61%, and 69.79% reduction in average daily produced excess sludge could be achieved compared with the control. Although good results of in-situ sludge reduction could be achieved in the six combined AAMA + O3/US systems with higher combined O3/US pretreatment time, an economic evaluation is needed to establish the feasibility of implementing this combined O3/US sludge pretreatment technology. According to the study of Elbeshbishy et al. (2010), the costs of dewatering, transportation and landfill were estimated at $250/ton TS, while the costs of electricity was estimated at $0.07/kW h. Take C1# test as an example, the daily produced excess sludge from the control and the C1# systems were respective 30.19 ± 1.51 g(TS)/d and 13.56 ± 0.68 g(TS)/d. For the C1# system, 30% excess sludge (4.068 ± 0.20 g(TS)/d) generated from the AAMA + O3/US system was pretreated by a combined O3/US technique. Thus, the actual daily discharged excess sludge from the C1# system was 9.492 ± 0.47 g(TS)/d by subtracting the amount of the pretreated returned sludge. According to the formula of the economic analyses in SI (Section S3), the economic analyses results for the control and the C1# systems were calculated as follows: U Ozone dose input cost of electricity (TC O3 ) is: TC O3 = (0.154 g(O3)/g(TS)h  4.068 g(TS)/d  300 W  103 kW/ W)/12 g(O3)/h  $0.07/kW h  1/6 h = $0.18  103/per day. U Ultrasound energy input cost of electricity (TC US ) is: TC US = 83 W/g(TS)  103 kW/W  4.068 g(TS)/d  $0.07/ kW h  1/6 h = $3.94  103/per day. U Cost of discharged excess sludge yield (TC DES ) is: TC DES = $250/g(TS)  106  9.492 g(TS)/d = $2.37  103/per day. . For the control system, the total cost (TC1) is: TC1 = $250/g(TS)  106  30.19 g(TS)/d = $7.55  103/per day. . For C1# system, the total cost (TC2) is: TC2 = TC O3 + TCUS + TCDES = $6.49  103/per day. . Net saving compared to the control ($) = TC1  TC2 = $1.06  103/per day. Table 1 shows the summary economic assessment results for the control and the six sludge reduction systems. When compared to the control system, the economic assessment results demonstrated that this combined system with 10 min combined O3/US pretreatment time was more economically feasible that can give 14.04% reduction in costs compared with the control system per day. In addition to the economic benefit observed in C1# system, a 55.08% saving of excess sludge production could be observed. Although the increase in the combined O3/US pretreatment time

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Table 1 Economic assessment for different combined O3/US pretreatment time compared to the control.

a

Batch testsa

Daily produced excess sludge (g(TS)/d)

The costs of sludge treatment ($/ per day) (103)

The costs of combined pretreatments ($/per day)

The total cost ($/per day) (103)

Net saving compared to the control ($/per day)

Control C1# test C2# test C3# test C4# test C5# test C6# test

30.19 ± 1.51 13.56 ± 0.68 12.08 ± 0.60 11.26 ± 0.56 10.23 ± 0.51 9.78 ± 0.49 9.12 ± 0.46

7.55 2.37 2.11 1.97 1.79 1.71 1.59

– 4.12  103 7.18  103 9.96  103 12.03  103 14.34  103 16.02  103

7.55 6.49 9.30 11.94 13.82 16.05 17.62

– 1.06  103 1.75  103 4.39  103 6.27  103 8.5  103 10.07  103

C1#, C2#, C3#, C4#, C5#, and C6# represented the six comparison tests of combined O3/US pretreatment time conducted at 10, 20, 30, 40, 50, and 60 min.

would improve the effect of sludge reduction in this combined AAMA + O3/US system, the electricity costs of the combined ozone dose and ultrasound energy inputs would be going to increase in multiples (Table 1). Thus, from economics point of view, the optimized combined O3/US pretreatment time of 10 min was selected. 3.2. Process performances 3.2.1. Excess sludge reduction Fig. 2a shows the yields of the cumulative produced excess sludge from the control and the three combined AAMA + O3/US systems. During 60 d stable operation, the cumulative produced excess sludge yield in AAMA + O3/US4# system (r of 90%) was 657.68 ± 32.88 g, which obtained 63.69% reduction compared with the control system (1811.40 ± 90.57 g). For AAMA + O3/US2# and AAMA + O3/US3#, the produced excess sludge yields were respective 813.60 ± 40.67 g and 729.67 ± 36.48 g, corresponding to 55.08% and 59.72% reduction in sludge production were observed compared with the control AAMA1#. Therefore, it can be found that this AAMA + O3/US system can effectively reduce excess sludge during sewage treatment process. In this AAMA + O3/US system, when the activated sludge is lysed by the O3/US pretreatment, the contents and nutrients contained in the microbial cell are released into the sludge supernatant. These released organic nutrients from the cell lysis will participate in the metabolism cycle of other microorganisms and a portion of the carbonaceous materials is reused for vital activities of other microorganisms (Mohammadi et al., 2011). By this way, the production of excess sludge will be reduced by microorganism cell lysis–cryptic growth in the AAMA + O3/US systems. On the other hand, according to the energy uncoupling theory, when the microorganism stayed in a fasting (insufficient food under an anoxic condition)/feasting (sufficient food under an oxic condition) condition (Westgarth et al. 1964; Chen et al., 2001), the dissipation of adenosine triphosphate will limit the microorganism anabolism, as a result of decreasing the amount of energy available for new biomass synthesis (Aragón et al., 2009). For this AAMA + O3/US system, with the recycle of O3/US sludge lyses as the extra carbon source returned back into the anoxic and microaerobic zones, it may form a coupling alternative fasting/feasting environment during sewage treatment process. Under the alternative fasting/feasting environment in the AAMA + O3/US system, the catabolism was no longer coupled to the anabolism, as a consequence, the new microbial biomass could not be synthesized (Coma et al., 2013). Hence, based on the advantages of the system operation, enhanced excess sludge reduction in this AAMA + O3/US system might be induced by the coupling mechanisms of cell lysis–cryptic growth and energy uncoupling metabolism. 3.2.2. COD removal efficiencies Fig. 2b shows the performances of COD removal efficiencies in the control and the three combined AAMA + O3/US systems. The effluent COD removal efficiency in AAMA1# was 92.69 ± 2.16%, in

comparison to 93.07 ± 2.12%, 91.36 ± 2.11%, and 90.58 ± 2.09% in the three AAMA + O3/US systems, respectively. Referring to Fig. 2b, COD removal efficiencies in the combined AAMA + O3/US system were not negatively affected by the return of sludge lyses. Based on the study of Lin et al. (2012), the disintegrated excess sludge returned back to the combined AAMA + O3/US systems undergoes three pathways: (1) a portion of sludge was biodegraded into CO2, H2O, and other byproducts; (2) a portion of sludge was biologically assimilated to become new activated sludge; and (3) the rest of the sludge was not assimilated and remained as inactive sludge. So higher sludge lyses return ratios controlled in the AAMA + O3/US system might cause more residual sludge lyses which were not participated the microorganism anabolism, remained in the effluent. Hence, suitable sludge lyses return ratio aiming at achieving good performances of simultaneous sludge reduction and effluent quality is obviously preferable.

3.2.3. Biological nitrogen and phosphorus removal efficiencies Fig. 3 shows the corresponding profiles of TN, NH+4-N and TP for the control and the three combined AAMA + O3/US systems. In Fig. 3a, the TN removal efficiency for AAMA1# was 67.36 ± 1.54%, while the effluent TN removal efficiencies for the three AAMA + O3/US systems were respective 81.52 ± 1.94%, 78.05 ± 1.78%, and 76.96 ± 1.79%. Compared with AAMA1#, better TN removal efficiencies could be observed in these AAMA + O3/US systems, and eminent TN removal efficiency in AAMA + O3/US2# system was also obtained. Fig. 3b showed the effluent NH+4-N removal efficiencies in the control and the three combined AAMA + O3/US systems. With an average influent NH+4-N concentration of 23.96 mg/L, the effluent NH+4-N removal efficiencies in the control and the three AAMA + O3/US systems were 83.64 ± 1.97%, 87.19 ± 1.90%, 85.14 ± 1.86%, 84.27 ± 1.89%, respectively. The effluent NH+4-N removal efficiencies for the control and the three combined AAMA + O3/US systems exhibited the same regularity as the TN removal performances. In the AAMA + O3/US systems, denitrification process occurred in the anoxic and microaerobic zones principally depended on the supply of O3/US pretreated sludge lyses as the external carbon sources. According to the advantages of the O3/US sludge lyses dosing phases in this study, the returned O3/US sludge lyses in the anoxic and microaerobic phases would provide a certain amount of external carbon sources, which could be utilized by denitrifying bacteria, and enhanced the TN removal efficiency (Tong and Chen, 2009). Otherwise, the simultaneous nitrification and denitrification occurred at relatively low DO concentration (usually controlled the DO of 0.5 mg/L), would consequently improve the nitrogen removal efficiency (Yoo et al., 1999). However, based on the study of Wu et al. (2014), the shortage of easily degradable carbon source in the denitrification process limited the biological nitrogen removal efficiency. Accompanied by the existence of more non-degradable organics (i.e. humus or humic acid), the alternate anoxic/aerobic mode did not contribute to nitrogen removal (Wang et al., 2013a). Referring to Fig. 3a, relative lower TN removal efficiencies can be observed in

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the AAMA + O3/US3# and AAMA + O3/US4# systems, implying that more sludge lyses had increased the amount of non-degradable organics and hence impacted the TN removal efficiencies. Therefore, it is further demonstrated that suitable sludge lyses return ratio controlled in the AAMA + O3/US system would achieve good performances of biological nitrogen removal efficiency. Comparable results of TP removal efficiencies in the control and the three AAMA + O3/US systems are depicted in Fig. 3c. For AAMA1#, the TP removal efficiency of 85.17 ± 1.96% was observed. While the TP removal efficiencies for the three AAMA + O3/US2#, AAMA + O3/US3#, and AAMA + O3/US4# were respective 89.81 ± 2.13%, 84.79 ± 1.93%, and 80.64 ± 1.88%. Considering the different sludge lyses return ratios controlled in the three AAMA + O3/US systems, the differences in TP removal efficiencies must be due to the different quantities of returned sludge lyses back into the AAMA + O3/US systems. Higher effluent TP concentrations in AAMA + O3/US3# and AAMA + O3/US4# must be due to the high quantities returned sludge lyses which contained more phosphorus in activated sludge. Although there were no obvious improvements in the four systems when comparing the high or low values of TP and NH+4-N, results of this study demonstrated that an appropriate sludge lyses return ratio maintained in the AAMA + O3/US system had no negative impact on the effluent quality. For the AAMA + O3/US systems, phosphorus removal process was completed by both phosphate-accumulating organisms (PAO) and

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denitrifying phosphate-accumulating organisms (DPAO). Through sequential alternating aeration conditions, PAO and DPAO stored phosphorus, then an amount of phosphorus was taken up when an oxygen electron acceptor is supplied under aerobic condition (Du et al., 2012). Additionally, DPAO utilized nitrate or O2 as an electron acceptor, and simultaneously enhanced biological phosphorus removal efficiency under anoxic and microaerobic and aerobic phases. Hence according to the phosphorus removal mechanism of microbial metabolism, the phosphorus removal efficiency could be improved in this AAMA + O3/US system. 3.3. EfOM variation by EEM and FTIR spectra analysis The application of EEM fluorescence spectroscopy is an instrumental sensitivity technique, which is more selective and has a wider range (Lu et al., 2012). Generally, the fluorescence peak intensity variations between different EEM spectra are the indication for increment or decrease of fluorescing materials (Feng et al., 2014). As shown in Fig. 4, there were four main fluorescence peaks could be identified from the four EfOM samples of the combined AAMA + O3/US systems. Peak A (polycarboxylate-type humic acid region, Ex/Em 300–400/400–500 nm) and Peak B (polyaromatictype humic acid region, Ex/Em 250–300/400–500 nm) were the main peaks of fluorescence spectra contained in the EfOM samples. Peak C and Peak D were associated to components derived from

Fig. 2. The cumulative excess sludge production yields (a) and COD removal efficiencies (b) in the control AAMA1# and the three AAMA + O3/US systems.

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proteins in which the fluorescence implies the presence of aromatic amino acids such as tyrosine and tryptophan. See Fig. 4 and Table 2, the effluent fluorescence intensities of Peak A (246.72) and Peak B (173.58) in AAMA + O3/US4# with higher sludge lyses return ratio (r = 90%) were higher than the control and the other two combined AAMA + O3/US systems. In addition, the intensity of Peak D in AAMA + O3/US3# and AAMA + O3/US4# showing increments in EfOM spectra suggested that tyrosine amino acid also accumulated in the effluent with the increase in sludge lyses return ratio. Whereas, the effluent fluorescence intensities indicated that there were less fluorescing materials,

including humic acid, fulvic acid, tyrosine/tryptophan amino and protein components observed in the effluent of the AAMA + O3/US2# system (Fig. 4 and Table 2). According to the study of Esparza-Soto et al. (2011) (glucose is the sole carbon source), the increment in an accumulation of refractory humic-like compounds in the EfOM demonstrated that EfOM was composed mainly by soluble microbial products (SMP). SMP, which comprised a majority of soluble organic material, have been proved to influence the effluent COD performances (Ni et al., 2011). In this study, different ratios of O3/US sludge lyses recycled back to the AAMA + O3/US systems had different effects

Fig. 3. The TN removal efficiencies (a), NH+4-N removal efficiencies (b), TP removal efficiencies (c) in the control AAMA1# and the three AAMA + O3/US systems.

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on SMP production. Therefore, by spectra analyses (Fig. 4 and Table 2), it could be concluded that the combined AAMA + O3/US systems with lower sludge lyses return ratio (e.g. r = 30% in this study) would induce less refractory humic and protein-like substances concentrated in the effluent. FTIR spectroscopy has also been widely utilized for the systematic characterization of the EfOM in recent years. In terms of previous investigations, FTIR spectroscopy analysis could provide plentiful available and valuable information on structural properties and functional groups of organic matters (Zhu et al., 2012). For the four EfOM samples from the control and the three combined AAMA + O3/US systems (Fig. 1S), the FTIR spectra were quite similar in the profile and expressed the similar characteristics in comparison, but different in the adsorption intensities. The C@O peaks (1730–1700 cm1), representing the redundant quantities of aromatic and carboxylic structures, indicated a typical characteristic of humic and fulvic acids in EfOM (Tian et al., 2011). So EfOM from AAMA + O3/US4# contained more humic and fulvic substances than other three combined AAMA + O3/US systems. The strong peaks at 1200–1000 cm1 in EfOM proved the presences of polysaccharides, aromatics, and carbohydrates (Tian et al., 2011). As shown in the FTIR spectra, the polysaccharides and carbohydrates were the main products from the EfOM samples in the four combined AAMA + O3/US systems during the phase of substrate utilization. Evidenced by the peak at 1200–1000 cm1

in AAMA + O3/US2#, less polysaccharides and carbohydrates in the EfOM samples were observed in the AAMA + O3/US2# with lower sludge lyses return ratio (r = 30%) than in AAMA + O3/US3# and AAMA + O3/US4#. As well as the analyses of EEM and FTIR spectra, relative higher COD, TN, NH+4-N and TP removal efficiencies were all observed in AAMA + O3/US2# (r = 30%). For a better-developed sludge reduction system, consideration must be given to both the excess sludge production and the effluent quality. Thus, O3/US sludge lyses return ratio of 30% in this AAMA + O3/US system was recommended. 3.4. Effects of returned O3/US pretreated sludge lyses on TTC-ETS activity TTC-ETS which is applied effectively as a proxy for the potential respiratory capacity of sludge organisms, has already been used as an artificial electron acceptor to detect dehydrogenase activity, and in turn, metabolically active bacteria in activated sludge (Yin et al., 2005). Fig. 2S showed the activities of TTC-ETS of sludge microorganisms in the control and the three combined AAMA + O3/US systems. As shown in Fig. 2S, different O3/US sludge lyses return ratios in the combined AAMA + O3/US systems exhibited different TTCETS activities. With the average TTC-ETS values of 70.45 ± 1.60, 88.73 ± 2.05, 76.48 ± 1.88, and 71.62 ± 1.71 lg/mg h for AAMA1#, AAMA + O3/US2#, AAMA + O3/US3#, and AAMA + O3/US4#, the

Fig. 4. EEM fluorescence spectra of EfOM: (a) the control AAMA1#, (b) AAMA + O3/US2#, (c) AAMA + O3/US3#, and (d) AAMA + O3/US4#.

Table 2 The fluorescence spectral identification of EfOM in the control AAMA1# and the three AAMA + O3/US systems. Systems

AAMA1# system AAMA2# + O3/US system AAMA3# + O3/US system AAMA4# + O3/US system Int.: intensity.

Peak A

Peak B

Peak C

Peak D

Ex/EM

Int.

Ex/EM

Int.

Ex/EM

Int.

Ex/EM

Int.

300–400/400–500 300–400/400–500 300–400/400–500 300–400/400–500

214.97 107.68 213.49 246.72

250–300/400–500 250–300/400–500 250–300/400–500 250–300/400–500

115.62 97.52 124.09 173.58

250–300/300–350 250–300/300–350 250–300/300–350 250–300/300–350

563.86 83.66 674.61 556.36

220–250/300–350 220–250/300–350 220–250/300–350 220–250/300–350

186.49 75.32 274.65 233.15

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Table 3 Correlations between NH+4-N removal efficiencies and electron transport system (ETS) activities for the control AAMA1# and the three AAMA + O3/US systems. TTC-ETSa NH+4-Na NH+4-Nb NH+4-Nc NH+4-Nd

TTC-ETSb

TTC-ETSc

TTC-ETSd

0.955e 0.946e 0.923e 0.921e

a The removal efficiency of NH+4-N (%) and concentration of TTC-ETS (lg/mg h) in AAMA1# system, percent. b The removal efficiency of NH+4-N (%) and concentration of TTC-ETS (lg/mg h) in AAMA2# + O3/US system, percent. c The removal efficiency of NH+4-N (%) and concentration of TTC-ETS (lg/mg h) in AAMA3# + O3/US system, percent. d The removal efficiency of NH+4-N (%) and concentration of TTC-ETS (lg/mg h) in AAMA4# + O3/US system, percent. e Correlation is significant at 0.01 level (2-tailed).

comparison of the total biological activities in the four continuous flow systems was: AAMA + O3/US2# > AAMA + O3/US3# > AAMA + O3/US4# > AAMA1#. According to the results obtained in this study, improvements in TTC-ETS activities could be observed in AAMA + O3/US2#, indicating that the microorganisms acclimatized with a relative low sludge lyses return would stimulate the activity of microbe in activated sludge. In terms of the previous studies, it was accepted that the performances of the NH+4-N removal depended mainly on the biomass and the activities of the microorganisms contained in the activated sludge (Cheng et al., 2011). Consequently, in this study, relationships between the NH+4-N removal efficiencies and the TTC-ETS activities in the control and the three AAMA + O3/US systems were investigated by statistical correlation analysis (Table 3). As observed from Table 3, significant positive correlations (0.955, 0.946, 0.923, and 0.921 for the control and the three AAMA + O3/US systems) between the NH+4-N removal efficiencies and TTC-ETS activities variation were observed. These significant positive correlations make it possible to predict NH+4-N removal efficiencies by measuring the TTC-ETS activities of sludge, indicating that the NH+4-N removal performances depended mostly on the metabolism of the sludge microorganisms in activated sludge systems. This observation was consistent with the study of Wang et al. (2013b), who reported a linear relationship between the NH+4-N removal efficiencies and the TTC-ETS activities of the sludge. In terms of the previous studies, sludge pretreatments could bring many additional benefits, including enhanced excess sludge minimization, volatile sulfur compound reduction, and improved sludge dewaterability (Dhar et al., 2012). Comprehensive investigations on the performances of sludge reduction and nutrient removal in the AAMA + O3/US systems demonstrated that this combined system could obtained enhanced in-situ sludge reduction and better effluent quality. Moreover, based on the economic analyses calculation formula (SI, Section S3), the total costs for the control AAMA1# and the three AAMA + O3/US2#, AAMA + O3/US3#, and AAMA + O3/US4# systems were respective $7.55  103/per day, $6.49  103/per day, $8.60  103/per day, and $10.26  103/per day. When compared the economic analyses of these four systems, r of 30% was recommended in this AAMA + O3/US system for more economic benefit than the control AAMA1#, AAMA + O3/US3# and AAMA + O3/US4# systems. Therefore, in this study, O3/US lysis–cryptic growth technology combining bio-reactors was found to have wider development prospects for future design and management. We believe that this combination technology will be an economic feasible wastewater treatment system for in-situ excess sludge reduction and nutrient removal.

4. Conclusions Economic assessment demonstrated that the AAMA + O3/US2# system was more economically feasible that can give a 14.04% saving of costs compared with AAMA1#. In addition to the economic benefits, 55.08% reduction in excess sludge could be observed in AAMA + O3/US2# system, and average TN and TP removal efficiencies in AAMA + O3/US2# increased by 21.17% and 5.45% compared with AAMA1#. Results also demonstrated that a small percentage of sludge lyses recycling gave rise to the improvement in microbe activity. It is believed that this AAMA + O3/US system will be an economic feasible wastewater system. Acknowledgements This research was supported by the National Nature Science Foundation of China (Grant Nos. 51008105 and 51121062). The authors also gratefully acknowledge the financial support by the State Key Laboratory of Urban Water Resource and Environment (Grant No. 2014TS06), the Harbin Institute of Technology Fund for young top-notch talent teachers (AUGA5710052514), Academician Workstation Construction in Guangdong Province (2012B090500018), and Shanghai Tongji Gao Tingyao Environmental Science & Development Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 11.009. References Amini, M., Younesi, H., Lorestani, A.A.Z., Najafpour, G., 2013. Determination of optimum conditions for dairy wastewater treatment in UAASB reactor for removal of nutrients. Bioresour. Technol. 145, 71–79. APHA, 2005. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. Aragón, C., Quiroga, J.M., Coello, M.D., 2009. Comparison of four chemical uncouplers for excess sludge reduction. Environ. Technol. 30, 707–714. Chen, G.H., Yip, W.K., Mo, H.K., Liu, Y., 2001. Effect of sludge fasting/feasting on growth of activated sludge cultures. Water Res. 35, 1029–1037. Cheng, L., Li, X.C., Jiang, R.X., Wang, C., Yin, H.B., 2011. Effects of Cr(VI) on the performance and kinetics of the activated sludge process. Bioresour. Technol. 102 (2), 797–804. Coma, M., Rovira, S., Canals, J., Colprim, J., 2013. Minimization of sludge production by a side-stream reactor under anoxic conditions in a pilot plant. Bioresour. Technol. 129, 229–235. Dhar, B.R., Nakhla, G., Ray, M.B., 2012. Techno-economic evaluation of ultrasound and thermal pretreatments for enhanced anaerobic digestion of municipal waste activated sludge. Waste Manage. 32 (3), 542–549. Dytczak, M.A., Londry, K.L., Siegrist, H., Oleszkiewicz, J.A., 2007. Ozonation reduces sludge production and improves denitrification. Water Res. 41, 543–550. Du, H.W., Yang, L.Y., Wu, J., Xiao, L., Wang, X.L., Jiang, L.J., 2012. Simultaneous removal of phosphorus and nitrogen in a sequencing batch biofilm reactor with transgenic bacteria expressing polyphosphate kinase. Appl. Microbiol. Biotechnol. 96, 265–272. Elbeshbishy, E., Nakevski, A., Hafez, H., Ray, M., Nakhla, G., 2010. Simulation of the impact of SRT on anaerobic digestibility of ultrasonicated hog manure. Energies 3 (5), 974–988. Esparza-Soto, M., Núñez-Hernández, S., Fall, C., 2011. Spectrometric characterization of effluent organic matter of a sequencing batch reactor operated at three sludge retention times. Water Res. 45, 6555–6563. Feng, X.C., Guo, W.Q., Yang, S.S., Zheng, H.S., Du, J.S., Wu, Q.L., Ren, N.Q., 2014. Possible causes of excess sludge reduction adding metabolic uncoupler, 3,30 ,40 ,5-tetrachlorosalicylanilide (TCS), in sequence batch reactors. Bioresour. Technol. 173, 96–103. Guo, W.Q., Ding, J., Cao, G.L., Ren, N.Q., Cui, F.Y., 2011. Treatability study of using low frequency ultrasonic pretreatment to augment continuous biohydrogen production. Int. J. Hydrogen Energy 36 (21), 14180–14185. Guo, W.Q., Yang, S.S., Xiang, W.S., Wang, X.J., Ren, N.Q., 2013. Minimization of excess sludge production by in-situ activated sludge treatment processes – a comprehensive review. Biotechnol. Adv. 31 (8), 1386–1396. Lan, W.C., Li, Y.Y., Bi, Q., Hu, Y.Y., 2013. Reduction of excess sludge production in sequencing batch reactor (SBR) by lysis–cryptic growth using homogenization disruption. Bioresour. Technol. 134, 43–50.

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