Evaluation of In-situ Sludge Reduction and Enhanced Nutrient Removal in an Integrated Repeatedly Coupling Aerobic and Anaerobic and Oxic-setting-anaerobic System

，，，，，，

（State Key Laboratory of Urban Water Resource and Environment，Harbin Institute of Technology，Harbin 150090，China）

Evaluation of In-situ Sludge Reduction and Enhanced Nutrient Removal in an Integrated Repeatedly Coupling Aerobic and Anaerobic and Oxic-setting-anaerobic System

Shanshan Yang，Wanqian Guo∗，Qinglian Wu，Haichao Luo，Simai Peng，Heshan Zheng，Xiaochi Feng and Nanqi Ren∗

（State Key Laboratory of Urban Water Resource and Environment，Harbin Institute of Technology，Harbin 150090，China）

Aiming to achieve simultaneous good performances of in-situ sludge reduction and effluent quality，an integrated repeatedly coupling aerobic and anaerobic and oxic-setting-anaerobic system（rCAA+OSA）is developed to reduce sludge production and enhance nutrient removal.Considering the mechanism of in-situ sludge reduction in this rCAA+OSA system，the combined effect of energy uncoupling metabolism and sludge cryptic growth maybe attributed to the higher reduction of biomass.Results show that the maximal sludge reduction in this rCAA+OSA system is obtained when the hydraulic retention time（HRT）is controlled at 6.5 h，which an increase in 16.67%reduction in excess sludge is achieved compared with OSA system（HRT of 6.5 h）.When compared the performances of effluent qualities，the enhanced nutrient removal efficiencies also can be observed in this rCAA+OSA system.Three-dimensional excitation emission matrix（3D-EEM）fluorescence spectroscopy is applied to characterize the effluent organic matters（EfOM）under different HRTs in the OSA and the rCAA+OSA systems.Analyses of 3D-EEM spectra show that more refractory humic-like and fulvic-like components are observed in the effluent of the OSA system.On the basis of these results，simultaneous enhanced in-situ sludge reduction and improved nutrient removal can be obtained in the rCAA+ OSA systems.

in-situ sludge reduction；BNPR；EfOM；EEM；rCAA；OSA

1 Introduction

Widespread application of the conventional activated sludge（CAS）process has been employed to deal with a variety of municipal and industrial sewage. However，the application of the CAS exhibits various disadvantages：high waste activated sludge（WAS）production；low nitrogen and phosphorus removal efficiency，and high capital and operating costs of subsequent treatment plants［1-2］.The generation of WAS is considerably high，and the management and disposal expenses of the WAS are costly.More importantly，the restrictive economic，environmental and legal regulations have already been emphasized on the needs for WAS reduction［3-4］.According to Guo et al.［5］，the sludge reduction processes／technologies were mainly classified into two main groups：sludge reduction trough post treatment；and in-situ activated sludge reduction in the process of sewage treatment.In terms of previous studies，sludge reduction by post treatments needed higher operation expenses and more energy consumption［6-7］.What＇s more，this kind of sludge treatment methods cannot lower the sludge production from the source of sewage treatment process itself.Therefore，the research on new in-situ sludge reduction bioreactors is the urgent need for the sustainable development.

According to previous studies，a system constructed by decoupling the anabolism and catabolism of microorganism in activated sludge can lower the production of biomass.Results demonstrated that this kind of bioreactor succeeds in minimizing the excess sludge production from the source of sewage treatment process［8］.Yu et al.［9］proposed a repeatedly coupling of aerobic and anaerobic process（rCAA）fixed-bed reactor，and enhanced sludge reduction was observed in this system.Feng et al.［10］also found：when the aerobic bacteria was converted into an anaerobic region after the aerobic treatment，the uncoupling of themicrobial catabolism and anabolism would happen. Studies have revealed that uncoupling phenomenon induced in the proposed oxic-setting-anaerobic（OSA）system can achieve good performances of in-situ sludge reduction［11-12］.Nevertheless，the nutrient removal quality in the OSA process is lack of reports and investigations.

In order to achieve good performances of enhanced WAS reduction and improved effluent quality，a combination of rCAA and OSA（rCAA+OSA）system is developed in this study.Three-dimensional excitation emission matrix（3D-EEM）fluorescence spectroscopy is introduced to characterize the effluent organic matters（EfOM）under different hydraulic retention times（HRTs）in this combined rCAA+OSA system. The objective of this paper is to optimize the HRTs in the anaerobic tank to achieve remarkable sludge reduction effect and improve nutrient removal efficiency in this combined system.

2 Material and Methods

2.1 Sludge Cultivation

During sludge cultivation period，the activated sludge，taken from Harbin Wenchang sewage treatment plant was seeded into fouridentical lab-scale CAS systems.The composition of the synthetic wastewater was as follows：glucose concentration，580 mg／L；Urea，8 mg／L；NaHCO3，200 mg／L；MgSO4，66 mg／L；CaCl2，6 mg／L；KH2PO4，27.8 mg／L；（NH4）2SO4，112 mg／L；FeSO4，0.3 mg／L；MnSO4，6 mg／L and microelement solution 1.0 mL／L.

2.2 Experiment Operation

After sludge cultivation period，the characteristics and biophase of the activated sludge in the four labscale CAS were maintained insteady states.As shown in Fig.1（a），the OSA system is modified based on CAS by inserting an anaerobic sludge holding tank in the sludge return line（as the control system）.While the sewage treatment tank of the rCAA+OSA system was modified into a repeatedly coupling of aerobic and anaerobic process，and sequentially by inserting an anaerobic sludge holding tank in the sludge return line（Figs.1（b）and 1（c））.The operation parameters of the OSA and the rCAA+OSA systems are shown in Table 1.

Fig.1 Schematic of lab-scale systems

Table 1 Operation parameters of the OSA and the rCAA+OSA systems

In this study，three tested HRTs（6.5，8 and 10.5 h）in the anaerobic sludge holding tanks of the rCAA+OSA systems were conducted.Each experiment was carried out at least 2 months to examine the effects of sludge reduction and effluent quality.In this study，three rCAA+OSA systems with three tested HRTs of 6.5，8 and 10.5 h in the anaerobic sludge holding tanks were numbered rCAA+OSA1＃，rCAA+OSA2＃and rCAA+OSA3＃，respectively.For the OSA system，HRT in the anaerobic tank was controlled at 6.5 h based on previous study［10］served as the reference system. Effluent samples taken from the OSA and the threerCAA+OSA systems were collected to analyze the effluent characteristics.For analysis purposes，all of these samples were filtered through 0.45 μm filters，and the filtered samples were monitored to detect any changes in the effluents.The formation of intermediates and changes in the composition of EfOM samples were quantified and characterized.All the experiments were conducted at room temperature.

2.3 Response Surface Methodology（RSM）

RSM is a mathematical and statistical technique used to analyze the mutual relationships between the response and the independent variables，and to identify the optimal operating conditions for a system［3］. Moreover，this methodology is expected to describe the entire effects of the selected parameters on the process. The results obtained from the orthogonal experiments are therefore analyzed using RSM.In this study，13 experimental runs designed by a two-factor and fivelevel RSM using central composite design（CCD）are used to optimize the relationship between the oxidationreduction potential（ORP）（X1，mV），hydraulic retention time（HRT）（X2，h）and the response，observed sludge production yield（Yobs）（Y，g／（g·d））. The relationship between the uncoded and coded values is shown in Eq.（1）：

Eq.（2）shows a second-order polynomial model：

whereYrepresents the response variable；b0，b1，b2，b11，b22，b12are the regression coefficients from the obtained data；xiis the coded value of the independent variable；Xiis the uncoded value of the independent variable；is the uncoded value of the independent variable at the center point；and ΔXiis the step change value.

2.4 Analytical Methods

Analyses of chemical oxygen demand（COD），mixed liquor suspended solids（MLSS），total phosphorus（TP），and total nitrogen（TN）were determined in accordance with the standard methods［13］.3-D EEM spectroscopy was performed by using a fluorescence spectrometer（FP-6500，JASCO，Inc.）.The detailed operation performance was performed as the study of Yang et al.［3］.All of the sludge samples were centrifuged at12 000×gfor 5 min and filtrated through 0.45 μm pore-diameter cellulose acetate filters.

3 Results and Discussion

3.1 Optimization of Operating Variables and Their Reciprocal Analysis

The levels of the coded and the actual values for the ORP and HRT from preliminary experiments are shown in Table 2.The design matrix and the results obtained based on the experimental CCD design are shown in Table 3.

Table 2 Experimental ranges and levels of the independent variables

Table 3 Response surface central composite design（CCD）and experiments

Second-order polynomial models for coded and actual responses are established as described in Eqs.（3）and（4），respectively：

wherex1andx2are the coded value of the ORP and HRT；X1，X2，andYare the ORP，HRT，andYobs，respectively.

An analysis of variance for the experimental model equations is conducted to examine the significance and the adequacy of the second-order polynomial equation in Table 4.P＜0.000 1 indicates a high significance of the corresponding variable.This implies that the linear modeland quadratic modelare all significant at the 95%confidence level，demonstrating that the combined pretreatments can play a synergistic role in sludge disintegration.In addition，a highsquared regression coefficient，R2of 0.919 9 indicates a high degree of correlation between the predicted and actual responses，demonstrating that the model can fit the response well.The obtained second-order polynomial model is thus adequate and significant.

Table 4 Analysis of variance（ANOVA）results for the improved response surface quadratic mode

2D and 3D contour curves are plotted to represent the interaction between the independent variables and to determine the optimal levels of each independent variable for achieving the optimal response levels.In Fig.2，a relatively higher sludge reduction effect（lowYobs）is obtained when the HRT ranged from 5.5 to 8 h.However，a relatively lower sludge reduction effect（highYobs）is obtained when the HRT ranged from 5.5 to 3 h.Therefore，a lower HRT leads to a lower sludge reduction effect.

Fig.2 The effect of the ORP and HRT and the mutual interaction of these two key parameters onYobs

From Eq.（4），the optimal actual values ofX1andX2are determined to be ORP of-288 mV and HRT of 6.4 h.The obtained optimal HRT in the batch experiments is chosen as the optimal HRT in the rCAA +OSA system.Further study aims to evaluate the impact of HRT on sludge reduction and nutrient removal in the rCAA+OSA system is systematical and comprehensive discussed in this study.

3.2 Performance on Excess Sludge Reduction

Fig.3 summarizes the cumulative discharged excess sludge yields under different HRTs in the OSA and the three rCAA+OSA systems.For the OSA and the three rCAA+OSA systems（HRT＝6.5，8 and 10.5 h），the cumulative discharged excess sludge yields are respective 1 715.72，1 429.72，1 452.34，and 1 578.36 g MLSS.Compared with OSA system，16.67%，15.35%，and 8.01%reduction in discharged excess sludge can be obtained in these combined rCAA+OSA systems.Results obtained in this study demonstrate that when maintained the HRT of 6.5 h in the anaerobic tank of the rCAA+OSA system，the optimized sludge reduction effect can be observed in this combined rCAA+OSA system.In terms of the previous study［9］，this proposed repeatedly coupling aerobic-anaerobic process would make the sludge to be solubilized into low micro-molecule compounds，and then the solubilized organic compounds would be utilized to synthesize new microbial cell through lysiscryptic growth process.Besides，with the introduction of the anaerobic sludge holding tank in the sludge return line，the alternative fasting／feasting environment（an anaerobic retention without nutrient／repeatedly coupling anaerobic-aerobic process with nutrient）would induce the energy uncoupling metabolism，and consequently lower new biomass synthesis.Therefore，according to the operation performances and the bioreactor structural features in this rCAA+OSA system，in-situ excess sludge reduction will be realized by microorganism cell lysis-cryptic growth and energy uncoupling metabolism.

Fig.3 Changes in cumulative excess sludge yields in the OSA and three rCAA+OSA systems

3.3 Performance on Nutrient Removal Efficiency

3.3.1 Performances of COD removal efficiencies in the OSA and three rCAA+OSA systems

Fig.4 shows the performances of COD removal efficiencies in the four OSA and rCAA+OSA1＃，rCAA+ OSA2＃and rCAA+OSA3＃systems.The effluent COD removal efficiency in OSA system is 83.46%，while the effluent COD removal efficiencies in rCAA+OSA1＃，rCAA+OSA2＃and rCAA+OSA3＃systems are 88.15%，87.01%，and 85.49%，respectively.Referring to OSA system，relative higher COD removal efficiencies in rCAA+OSA systems are observed，which implies that this proposed repeatedly coupling anaerobic-aerobic system would solubilize the activated sludge into low micro-molecule compounds，as a result of increasing the organic matters removal efficiencies.Furthermore，the COD removal efficiencies reduce with the increase in the HRTs in the anaerobic sludge holding tank of the rCAA+OSA3＃system.A relative higher HRT in the anaerobic sludge holding tank may cause more residual organic matters remained in the effluent，while a part of refractory residual organic matters would not be participated in the microorganism anabolism during sewage treatment process.Hence，results demonstrate that the optimization of HRT in the anaerobic sludge holding tank can achieve good performances of simultaneous sludge reduction and effluent quality in this rCAA+OSA system.

Fig.4 COD removal efficiencies in the OSA and three rCAA+OSA systems

3.3.2 Performances of BNPR efficiencies in the OSA and three rCAA+OSA systems

The variations of TN and TP removal efficiencies in the OSA and the three rCAA+OSA systems（HRT＝ 6.5，8 and 10.5 h）are shown in Figs.5 and 6.The average TN removal efficiencies in the OSA and the three rCAA+OSA systems are 60.44%，72.64%，71.01%，and 68.80%，respectively.And TP removal efficiencies are respective 56.89%，76.48%，74.72% and 73.51%in average，as shown in Fig.6.According to microbial denitrogenation mechanism［14］，nitrogen removal by nitrification and denitrification is positively correlated to aeration concentration in the biological wastewater treatment zone.For OSA systems，denitrification process（TN removal efficiencies）maybe inhibited in aeration zones［15］.Whereas for the rCAA+OSA system，the remarkable higher nitrogen and phosphorus removal efficiencies must be attributed to the repeatedly coupling anaerobic／aerobic environment in the sewage treatment tank.During biological sewage treatment process，a differing aerated environment for organic substrate competition between phosphorus accumulating organisms and denitrifying bacteria is required［16-17］.Hence，the application of the repeatedly coupling anaerobic and aerobic conditions in sewage treatment process can effectively improve BNPR efficiencies during biological sewage treatment processes［18］.

Fig.5 Comparisons of effluent TN removal efficiencies in OSA and rCAA+OSA systems

Fig.6 Comparisons of effluent TP removal efficiencies in OSA and rCAA+OSA systems

3.4 3-D EEM Spectroscopy Analysis

Chemical analyses methods provide only limited information on the changes in the composition of EfOM，and this is due to the finite chemical parameters that can be measured in the experiment［19］. In terms of the previous studies［3］，molecules including metabolites，proteins，amino，fulvic，humic acids，and enzymes have a characteristic of fluorescent emission.3D-EEM fluorescence spectroscopy technique，which has the advantages of fewer interactions and fewer overlaps with of the spectra from different fluorophores［20］，is more selective and has a wider range than convention alfluorescence measurement or UV／V is spectrometry.Based on previous studies，3D-EEM spectroscopy has been already proved to provide complete information on these fluorescent substances from complex mixtures by changing excitation wavelength and emission wavelength simultaneously［21］.Therefore，in this study，3-D EEM spectra is applied to comprehensively reveal the component changes of the four EfOM from the OSA and the three rCAA+OSA systems.

Although by inoculating the non-fluorescent synthetic wastewater，the generation of fluorescent molecules such as proteins，metabolites，enzymes and pigments in the effluent samples have been observed and showed the characteristics of fluorescent emission. In this study，3-D EEM spectroscopy is introduced to comprehensively reveal the components changed in EfOM from the OSA and the rCAA+OSA systems（Fig. 7）.For these four EEM fluorescence spectra of EfOM samples，there are four main fluorescence peaks can be identified：Peak A represents polycarboxylate-type humic acid region（λEx／λEm：300-350／400-450）；Peak B represents polyaromatic-type fulvic acid region（λEx／λEm：250-300／400-450）；Peak C represents tyrosine／tryptophan protein region（λEx／λEm：250-300／280-350），soluble microbial byproduct；Peak D represents tyrosine／tryptophan amino acid region（λEx／λEm：220-250／280-350）.As shown in Fig.7，Peak A and Peak B fluorescence spectra are the refractory humic-like and fulvic-like components.Peak C and Peak D，which are associated to compounds derived from proteins，represent the soluble microbial by-products and aromatic protein.Compared with EEM spectra in the OSA and three rCAA+OSA systems，more refractory humic-like，fulvic-like components，soluble microbial by-products and aromatic protein are observed in the effluent of the OSA system.Results demonstrate that the characteristic of EfOM becomes more aromatic and refractory from OSA system than from rCAA+OSA systems.Furthermore，when controlled the HRT of 6.5 h in the rCAA+OSA system，there are only a small amounts of humic-like，fulvic-like components，soluble microbial by-products and aromatic protein remained in the EfOM.These observations by EEM spectra are consistent with the performances of COD removal efficiencies in these four systems.In light of the previous studies，the presence of soluble organic matters from biological sewage treatment process have been found to influence the effluent COD standards［22］.

In this study，a combination of the rCAA system with OSA system is proved achieved simultaneous enhanced in-situ sludge reduction and improved nutrient removal.On the basis of these results，further comprehensive investigations in this combined rCAA+ OSA system should be conducted.It is believed that the development of this combined rCAA+OSA system will be a useful biological treatment systemfor the benefit of future investigation and practical application.

Fig.7 EEM fluorescence spectra of EfOM

4 Conclusions

In this study，comparison experiments on optimizing HRTs in the anaerobic sludge holding tank of the rCAA+OSA systems are conducted.Simultaneous enhanced sludge reduction and improved nutrient removal efficiency are obtained in this rCAA+OSA system.Based on experimental observations，the following conclusions can be drawn：

1）Remarkable in-situ sludge reduction and enhanced nutrient removal performances are observed in this rCAA+OSA system.The maximal sludge reduction in this rCAA+OSA system is obtained when the HRT is controlled at 6.5 h in the anaerobic sludge holding tank.An increase in 16.67%reduction in excess sludge is achieved compared with OSA system.

2）The effluent COD，TN，and TP removal efficiencies in the rCAA+OSA1＃system（HRT of 6.5 h）are respective 88.15%，72.64%，and 76.48%，which are increased by 5.61%，16.80%，and 25.61%when compared with OSA system.

3）3-D EEM spectra indicate that more refractory humic-like，fulvic-like components，soluble microbial by-products and aromatic protein generated from OSA are primarily responsible for EfOM quality.Whereas there are only a small amount of humic-like，fulvic-like components，soluble microbial by-products and aromatic protein remained in the EfOM of the rCAA+ OSA1＃system.Therefore，the operation parameters controlled in the biological sewage treatment systems significantly affect the EfOM quantity and quality.

［1］Tian Y，Zhang J，Zuo W，et al.Nitrogen conversion in relation to NH3and HCN during microwave pyrolysis of sewage sludge.Environmental Science and Technology，2013，47（7）：3498-3505.

［2］Guerrero J，Guisasola A，Baeza J A.The nature of the carbon source rules the competition between PAO and denitrifiers in systems for simultaneous biological nitrogen and phosphorus removal.Water Research，2011，45（16）：4793-4802.

［3］Yang Shanshan，Guo Wanqian，Cao Guangli，et al. Simultaneous waste activated sludge disintegration and biological hydrogen production using an ozone／ultrasoundpretreatment.Bioresource Technology，2012，124：347-354.

［4］Jiang J Q，Wang K，Peng X X，et al.Performance of sewage sludge treatment and electricity generation by different configuration microbial fuel cell.Journal of Harbin Institute of Technolog，2013，20（4）：1-6.

［5］Mahmood T，Elliott A.A review of secondary sludge reduction technologies for the pulp and paper industry. Water Research，2006，40（11）：2093-2112.

［6］Yang Shanshan，Guo Wanqian，Meng Zhaohui，et al. Characterizing the fluorescent products of waste activated sludge in dissolved organic matter following ultrasound assisted ozone pretreatments.Bioresource Technology，2013，131：560-563.

［7］Sui P Z，Nishimura F，Nagare H，et al.Behavior of inorganic elements during sludge ozonation and their effects on sludge solubilization.Water Research，2011，45（5）：2029-2037.

［8］Guo Wanqian，Yang Shanshan，Xiang Wensheng，et al. Minimization of excess sludge production by in-situ activated sludge treatment processes：a comprehensive review.Biotechnology Advances，2013，31（8）：1386-1396.

［9］Yu Anfeng，Feng Quan，Liu Zehua，et al.Biological wastewater treatment by a bioreactor with repeated coupling of aerobes and anaerobes aiming at on-site reduction of excess sludge.Water Science and Technology，2006，53（9）：71-77.

［10］Feng Quan，Yu Anfeng，Chu Libing，et al.Performance study of the reduction of excess sludge and simultaneous removal of organic carbon and nitrogen by a combination of fluidized-and fixed-bed bioreactors with different structured macroporous carriers.Biochemical Engineering Journal，2008，39（2）：344-352.

［11］Saby S，Djafer M，Chen G H.Effect of low ORP in anoxic sludge zone on excess sludge production in oxic-settlinganoxic activated sludge process.Water Research，2003，37（1）：11-20.

［12］Ye Fenxia，Li Ying.Oxic-settling-anoxic（OSA）process combined with 3，3，4，5-tetrachlorosalicylanilide（TCS）to reduce excess sludge production in the activated sludge system.Biochemical Engineering Journal，2010，49（2）：229-234.

［13］American Public Health Association（APHA）.Standard methods for the examination of water and wastewater. Washington，DC，2005.

［14］Ni B J，Ruscalleda M，Pellicer-Nàcher C，et al.Modeling nitrous oxide production during biological nitrogen removal via nitrification and denitrification：extensions to the general ASM models.Environmental Science and Technology，2011，45（18）：7768-7776.

［15］Tian Y，Lu Y B.Simultaneous nitrification and denitrification process in a new tubificidae-reactor for minimizing nutrient release during sludge reduction.Water Research，2010，44（20）：6031-6040.

［16］Danesh S，Oleszkiewicz J A.Use of a new anaerobicaerobic sequencing batch reactor system to enhance biological phosphorus removal.Water Science and Technology，1997，35（1）：137-144.

［17］Wang Y Y，Pan M L，Yan M，et al.Characteristics of anoxic phosphors removal in sequence batch reactor. Journal of Environmental Sciences，2007，19（7）：776-782.

［18］Brown P，Ong S K，Lee Y W.Influence of anoxic and anaerobic hydraulic retention time on biological nitrogen and phosphorus removal in a membrane bioreactor. Desalination，2011，270（1／2／3）：227-232.

［19］Lu L，Xing D F，Liu B F，et al.Enhanced hydrogen production from waste activated sludge by cascade utilization of organic matter in microbial electrolysis cells. Water Research，2012，46：1015-1026.

［20］Li W H，Sheng G P，Liu X W，et al.Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor.Water Research，2008，42（12）：3173-3181.

［21］Sheng G P，Yu H Q.Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy.Water Research，2006，40（6）：1233-1239.

［22］Barker D J，Stuckey D C.A review of soluble microbial products（SMP）in wastewater treatment systems.Water Research，1999，33（14）：3063-3082.

X705

：

：1005-9113（2015）05-0017-08

10.11916／j.issn.1005-9113.2015.05.003

2014-07-14.

Sponsored by the National Natural Science Foundation of China（Grant No.51008105 and 51121062），the State Key Laboratory of Urban Water Resource and Environment（Grant No.2014TS06），the Department of Education Fund for Doctoral Tutor（Grant No.20122302110054）and the Special S＆T Project on Treatment and Control of Water Pollution（Grant No.2013ZX07201007-001）.

∗Corresponding author.E-mail：rnq＠hit.edu.cn；guowanqian＠126.com.