DU Yong－li(杜勇立)，HUANG Man－h(huán)ong(黃滿紅)，ZHENG Yu(鄭 宇)

1 School of Civil and Architectural Engineering，Central South University，Changsha 410075，China

2 Hunan Provincial Communications Planning，Survey＆Design Institute，Changsha 410008，China

3 School of Environmental Science and Engineering，Donghua University，Shanghai 201620，China

Effect of Sludge Retention Time on the Fate of Proteins and Polysaccharides in AAO Process

DU Yong－li(杜勇立)1，2，HUANG Man－h(huán)ong(黃滿紅)3*，ZHENG Yu(鄭 宇)3

1 School of Civil and Architectural Engineering，Central South University，Changsha 410075，China

2 Hunan Provincial Communications Planning，Survey＆Design Institute，Changsha 410008，China

3 School of Environmental Science and Engineering，Donghua University，Shanghai 201620，China

Effect of sludge retention time(SRT)on the removal of potentialand polysaccharides in an anaerobic/anoxic/aerobic (AAO)process was investigated．The Lowry method and anthrone method were used to detect proteins and polysaccharides．Total removal efficiency of proteins at SRT of 10 to 25 d in the AAO system was higher than 90%．Polysaccharides removal efficiencies were above 80%when SRT was increased from 15 to 25 d，whereas only 81%of polysaccharides was removed at SRT of 10 d．The biodegradation part of proteins and polysaccharides increased from 87．40%to 93%and from 74．22%to 86．94%with increasing SRTs．The ratios of polysaccharides and proteins in extracellular polymer substances(EPSs)were around 1．5－3 in different SRTs．As SRT increasing，polysaccharides and proteins discharged with residual sludge decreased gradually．The amount of EPSs decreased with increasing SRTs．

protein;polysaccharide;sludge retention time(SRT); anaerobic/anoxic/aerobic (AAO) system; extracellular polymer substances(EPSs)

Introduction

Organic matters (OMs) in wastewater are highly heterogeneous，containing substances of various origins［1］．Some studies had reported that major chemical fractions in municipal wastewater were proteins and polysaccharides．For instance，the fractions of total proteins and polysaccharides of total chemical oxygen demand(COD)in several catchments areas of Denmark were，on average，28% and 18%［2］．Narkis et al．identified soluble organics in a physical－chemical wastewater treatment plant in Israel，revealing that 30%of the total COD was proteins，10% of the total COD was polysaccharides in the influent［3］．Though proteins and polysaccharides can comprise a significant portion of organic carbon in wastewater，limited information is available on how these compounds are removed in biological wastewater treatment systems［4－5］．

Anaerobic/anoxic/aerobic(AAO)process for nitrogen and phosphorus removal is widely implemented． Operational parameters，such as sludge retention time(SRT)and internal recycle ratio，have been optimized［6－10］．But it is not clear how these operational parameters affect on the fate of proteins and polysaccharides in the wastewater treatment system．Previous studies found thatseveralremovalwaysofproteinsand polysaccharides in wastewater treatment systems:biodegradation or metabolism and adsorption to sludge［11－12］．Adsorption is one of the sources of extracellular polymer substances(EPSs)，while proteins and polysaccharides in EPSs are postulated to play an important role in flocculation and dewatering［13－15］．Thus it is important to quantify the proteins and polysaccharides in the EPS in order to understand the origins of EPSs and the adsorptions of proteins and polysaccharides．

In this study，the removal characteristics of proteins and polysaccharides in an AAO lab－scale process at different SRTs are presented．Then，the amount of proteins and polysaccharides in the EPS was determined to investigate the fate of proteins and polysaccharides in wastewater．Finally，fate of proteins and polysaccharides in the wastewatertreatmentprocesswere evaluated by mass balance．

1 Materials and Methods

1．1 Experimental equipments

The lab－scale AAO system used in this study consisted of an anaerobic tank(7 L)，an anoxic tank(7 L)，an aerobic tank (21 L) and a clarifier．These tanks were all made of polyethylene glass．The activated sludge was obtained from a full－scale anoxic－aerobic activated sludge plant treating the local domestic and industrial wastewater in Shanghai，China．The electric stirrers were employed in anaerobic and anoxic tanks．The peristaltic pumps were used to feed the system automatically，recycle the sludge from the bottom of clarifier to the anaerobic tank，and transfer the mixed liquor from the aerobic tank to the anoxic tank．The whole system had been operated for more than two months continuously in a laboratory with the temperature controlled at 25℃．During the experiment，dissolved oxygen (DO)in anaerobic，anoxic and aerobic reactors was in the range of 0－0．3，0－0．5 and 3．3－5．7 mg·L－1，respectively．pH was about 6．5－7．5．

According to the recommending value of AAO operational parameters［16］，the hydraulic retention time(HRT)of the above three reactors was set to 1．6，1．6 and 4．8 h，respectively．The sludge was returned from the bottom of the settling reactor to the anaerobic reactor．The mixed liquor was recycled from the aerobic reactor to the anoxic reactor．The circulation ratio for both the sludge and mixed liquor was 1．The whole system was placed in a temperature－controlled room at 25℃．Low nitrogen removal efficiencies have been found in wastewater treatment plants operating SRTs lower than 10 d［17－18］．So in this paper the SRT was changed from 10 d to 25 d，aiming to assess the effect ofSRT variations on the performance ofproteins and polysaccharides removal in AAO system．

1．2 Wastewater characteristics

Real domestic wastewater was taken from the wastewater collection station of a large residential area in Shanghai，China．During the experimental period，the fresh wastewater was introduced into feed tank every 4 h，and the total COD，soluble COD(through 0．45 μm filter membrane)，total organic carbon (TOC)and total nitrogen(TN)of the wastewater are 204－440，108－372，40－138 and 50－70 mg·L－1，respectively．

1．3 Analyses of proteins and polysaccharides

The Lowry method was applied for protein determination．Albumin bovine was used as the standard reagent．The method is described by Raunkj?r et al［19］．The absorbance was detected at 750 nm．

The anthrone method was applied for polysaccharides determination．A modification of the method described by Raunkj?r et al．［19］was used and glucose was used as the standard reagent．Samples were placed in a water bath at 100℃for 10 min and then cooled in a water bath at 4℃ for 5 min．Absorbance at 625 nm was measured．

1．4 Extraction and amount of EPS

The system was operated in steady state by acquiring a steady COD removal efficiency．EPSs are the compounds excreted from bacterial metabolism or lysis，or the compounds adsorbed from the wastewater．Two parts were included in EPS: loosely bound biopolymers (LBB) and tightly bound biopolymer(TBB)［20］．

The extraction procedures of LBB are listed as follows．Sludge sample(900 mL)were centrifuged at 6000 r/min at 4℃ for 15 min．Then，the supernatant was discarded and the pellets were transferred to 15 mL 0．85%(m/v)NaCl solution．Several beadings were added to the solution．Then it was stirred in the ultrasonic instrument for 2 min and the suspension was stirred for 10 min at 150 r·min－1．At the end of the stirring period，the suspension was stirred in the ultrasonic instrument for 2 min，and then centrifuged at 8000 r/min at 4℃ for 10 min．The supernatant was collected for analysis．

After the above pre－treating procedures，the supernatant was discarded and the pellets were transferred to an extraction beaker for determining TBB．A buffer solution at pH 7．0(2．0 mmol·L－1NaH2PO4，1．26 mmol·L－1Na2HPO4，2．5 mmol·L－1NaCl，and 0．5 mmol·L－1KCl)was added to give a final volume of 300 mL 70 g·g－1volatile suspended solids(VSS)．Dowex sodium form cation exchange resin was added to the extraction beaker［21］．The suspension was stirred at 150 r·min－1for 5 h．At the end of the stirring period，the suspension was centrifuged at 5000 r·min－1at 4℃ for 15 min．The supernatant was collected for analyzing．

1．5 Other analytical methods

TOC wasdetermined using a Shimadzu TOC－Vcpn analyzer．COD，total suspended solid(TSS)，VSS，mixed liquor suspended solid(MLSS)，TN，and total phosphorus (TP)measurements were carried out following APHA standard methods［22］．

1．6 Calculations

The mass balance calculation was based on Fig．1．Proteins and polysaccharides concentrations in anaerobic，anoxic，and aerobic reactors were calculated according to Eqs．(1)－(3):

The overall removal efficiency(ORE)of proteins and polysaccharides for the AAO system was calculated according to Eq．(4)．

The totalpercentage ofproteinsand polysaccharides removed from the influent was obtained by summing the percentage of proteins and polysaccharides removed by the anaerobic， anoxic， aerobic and settling reactors． These percentages were calculated using Eq．(5)－(8)，respectively:

In the above equations，ARis the anaerobic removal;AnRis the anoxic removal;AeRis the aerobic removal;SR is the settling removal;Ciis the proteins and polysaccharides concentration in influent，mg·L－1;C1，C2，C3，and Ceare the proteins and polysaccharides concentrations in the effluents from anaerobic，anoxic，aerobic，settling reactors，respectively，mg·L－1;Ce1is the proteins and polysaccharides concentration in the return sludge，mg·L－1;Ci1，Ci2，and Ci3are the proteins and polysaccharides concentrations in mixed liquors in the anaerobic，anoxic，aerobic reactors，respectively，mg·L－1;Q and Qware the influent flow rate(60 mg·d－1) and the waste sludge flow rate，respectively;R and r are the recirculation ratios for activated sludge and mixed liquor，respectively．

Fig．1 Flow chart of the AAO activated sludge system for mass balance calculation

2 Results and Discussion

2．1 Effect of SRT on the removal and fate of the proteins and polysaccharide

In the conditions of differentSRTs， proteins and polysaccharides removal efficiency in each stage of AAO system was shown in Fig．2．

From Fig．2，it could be concluded that proteins removal efficiency in AAO system was around 80%－90%，only little proteins discharged with the final effluent．It was particularly evident that the proteins removal efficiency had been improved only a little with SRT increasing from 10 d to 25 d．Polysaccharides removal efficiency in AAO system was exceeds 80%，and the change of SRT had some influence on the total remove efficiency．When SRT was equal to 10 d，thepolysaccharides removal efficiency was 81．85%，which was the lowest in all SRT conditions．

Fig．2 Removal percentages of the whole process in different SRTs

Fig．3 Removal percentages of each tank account for the total removal quantity

In the condition of different SRTs，the amount of proteins removed in anaerobic tank was larger than that of aerobic tank and anoxic tank(as shown in Fig．3)．The quantity of proteins removed in anaerobic tank exceeded 40．0%of total removed quantity in AAO system，and it got larger with the longer SRT．Maybe it was due to that proteins could be hydrolyzed to volatile fatty acids in anaerobic condition．

In the condition ofdifferentSRT，the quantity of polysaccharides removed in anaerobic tank was nearly equal to that of aerobic tank，accounting for about 40．0% of total removed quantity．The removal ratios hadn't varied much with SRT changing．

2．2 Composition and changes of proteins and polysaccharides in EPS of each tank of AAO system

The average organic carbon concentrations of EPS in the different conditions，which were the sum of TB and LB，were shown in Fig．4．

Fig．4 EPS concentration of each tank of AAO system

Data presented in Fig．4 showed that，in the activated sludge of each reactor，the weight ratio of organic carbon in EPS to volatile suspended solids(VSS)was between 38 and 71 mg·g－1VSS，which agreed with the finding of Benetti［23］．The increase in SRT also affected the quantity of EPS．From Fig．3，the amount of EPS in the period of SRT at 10 d was higher than other periods．It appeared that the change of SRT led to the variety of bacterial metabolism or lysis in the system，which was excreted compound to form part of EPS．

Table 1 Concentrations of proteins and polysaccharides in LB and TB

Table 1 presented the concentrations of proteins and polysaccharides in LB and TB．Data from Table 1 indicated that amount of polysaccharides and proteins in EPS of anaerobic sludge was higher than that of the aerobic sludge or the anoxic sludge．Protein was the major EPS compound，constituting approximately 50% ofthe organic fraction．Meanwhile，polysaccharides constituted only approximately 10% of the whole EPS．From Table 1，it could also be concluded that LB concentrations decreased as the SRT was increasing and the amount of polysaccharides and proteins in EPS at SRT of 10 d was higher than other periods．

Fig．5 Proteins/polysaccharide of different SRTs

Figure 5 gave the ratios of proteins and polysaccharides under different SRTs． The mass ratio of protein to polysaccharide in EPS was around 1．5－3 and it was similar to those obtained by other studies［24－25］．From this figure，it could be concluded that the ratios of proteins and polysaccharides tended to increase when the SRT was increased．

2．3 Mass balance of proteins and polysaccharides in AAO system

As proteins and polysaccharides are not volatile compounds，it is assumed that their removals are due to biodegradation and sorption in the activated sludge process．Therefore，the possible fates of proteins and polysaccharides in the AAO system include discharging with final effluent and the waste sludge，accumulating in the system and being degraded by microorganisms in the activated sludge．The fates of proteins and polysaccharides were calculated based on the following mass balance:

where Mi，Mo，and Mware the proteins and polysaccharidesmasses in the inlet sewage，final effluent，and waste sludge．Mbis the proteins and polysaccharides mass degraded by the AAO activated sludge，and Mais the proteins and polysaccharides mass accumulating in the system．They are calculated according to Eq．(10)－(14):

where V1=3．5 L，V2=3．5 L，V3=10．5 L，and V4=3．5 L are volumes of mixed liquor in the anaerobic，anoxic，aerobic and settling reactor，respectively．

The amounts of proteins and polysaccharides in different removal ways and their contribution to total removal quantity in this research were listed in Fig．6．

Fig．6 Fates of proteins and polysaccharides in AAO system

As shown in Fig．6，when SRT changed from 10 d to 25 d，biodegradation was the main way for removal．The percentage of proteins removed in this way ranged from 87．4%to 93．0%．The percentage of polysaccharides in this way changed from 74．2%to 86．9%．After entering AAO system，the possible removalways of proteins and polysaccharides included discharging with finaleffluent，discharging with residual sludge，accumulating in system，and biodegradation．As SRT increasing，the contribution of the way discharging with residual sludge decreased gradually．Biodegradation at SRT of 10 d was the least of the four SRTs while discharging with final effluent at SRT of 10 d was the highest．

3 Conclusions

Theexperimentalstudy was made by using actual municipal wastewater as the inflow raw water to evaluate the performanceofAAO processin removing proteinsand polysaccharides．The conclusions can be summarized as follows．

(1)Proteins removal efficiency in AAO system was around 80%－90%，while that of polysaccharides exceeded 90% in the conditions of different SRTs．

(2)The quantity of proteins and polysaccharides removed in anaerobic tank were more than 40%of total removed quantity in AAO system．So anaerobic tank played an important role in removing proteins and polysaccharides．

(3)The amount of EPS in the condition of SRT at 10 d washigher than otherconditions． The mass ratios of polysaccharides to proteins in EPSs were around 1．5－3．

(4) Biodegradation was the main way for removing polysaccharides and proteins in AAO wastewater treatment system at SRT of 10 to 25 d．

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X703．1

A

1672－5220(2015)03－0401－05

date:2013－11－01

s:National Natural Science Foundations of China(Nos．21477018，21007010);Hunan Province Ministry of Transportation Scientific Research Projects，China(Nos．200908，201105);Ministry of Transport Science and Technology Program，China(No．2010353343290)

*Correspondence should be addressed to HUANG Man－h(huán)ong，E－mail:egghmh@163．com