Jian－Hui Wang，Jun Yin，Hai Lu，Ying－Zi Lin

(1.School of Municipal and Environmental Engineering，Harbin Institute of Technology，Harbin 150090，China;?2.Key Laboratory of Songliao Aquatic Environment，Ministry of Education，Jilin Jianzhu University，Changchun 130118，China)

1 Introduction

In wastewater treatment，biodegradation has been used for the removal of organic matter.The effect of this method depends upon the activity of activated sludge.Now，all the methods used to estimate the biological activity of activated sludge remains a lot of problems.Mass measurement of suspended solids or volatile suspended solids are most widely used［1］.Such method can not distinguish between living cells and debris of either organic orinorganic origin.Moreover，this method does not detect the true rate of treatment system accurately.Deoxyribonucleic acid (DNA)and RibonucleicAcid (RNA)measurementsdonot distinguish between active and inactive bacterial cells.Also，DNA/RNA methods depend upon the advanced instruments.Lopez’s group［2］consulted that adenosine triphosphate(ATP)reflected the activity of activity sludge，because they found the linear relationships between ATP content of activated sludge and their respective cell residence times，loading rates，substrate removal and oxygen utilization rate.However，ATP measurement is relative complexity of its quantitative estimation.In spite of Active Bacteria Number(ABN) is considered the most sensitive parameter of estimating the activity of activated sludge，the choice of culture medium and the separation of sludge flocs during the determination process raised controversy.

An traditionally used method is to measure the activity of some enzymes involved in oxidative substrate removal［3］.Dehydrogenase(DH)activityofthe electron transport system(ETS)has been found to correlate with oxygen uptake rate(OUR)［4］or sludge oxygen uptake rate(SOUR)［5］.In addition aerobic phase，OUR/SOUR do not characterize the activity of activated sludge during anaerobic phase.DH is responsible for taking hydrogen from a substrate and passing it along a set of carriers to free oxygen or to synthetic acceptors further converted to colored products.DH activity of the ETS can be conveniently measured by the use of artificial electron acceptors like some tetrazolium salts， predominantly 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride(INT)［6］.Several authors have utilized this method to estimate the biological activity of plant tissue［7］.The method was also used in medicine to evaluate the activity of brain cells.

In the present study，the activity of cultivated activated sludge was measured in aerobic and anaerobic phase by utilizing INT as the electron acceptor of DH present in the metabolically active biomass.Bacteria converted INT to a colored water-soluble formazan product，estimated by photometry［8－12］.This feature obviates the need for formazan crystal solubilization prior to absorbance measurements，as required when using other tertazolium salts such as 2，3，5-Triphenyltetrazolium chloride(TTC)and 3-(4，5-Dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide (MTT)［13－14］.Comparingwith themethodscited above，this method is quicker and easier to perform.Moreover，the analysis requires neither complicated nor expensive instruments.

In this paper，the active sludge cultured was firstly investigated in SBR in order to validate the method for aerobic and anaerobic phase.Then，this method was further applied to the bioactivity estimation in anaerobic phase.

2 Materials and Methods

2.1 Experimental Setup

A sequencing batch reactor(SBR)with a working volume of 40 L(as shown in Fig.1).Temperature was maintained at25 ℃ and the aeration flux was 1.0 m3/h.The operating time was 10 h，including aeration time 6 h，COD concentrations 150 mg/L，stirring time 2 h and sedimentation period 2 h.

Fig.1 Schematic diagram of SBR

2.2 Synthetic Wastewater Composition

The synthetic wastewater contained domestic sewage as the organic substrates at concentrations of 300 mg/L COD.The complete composition of the feed was(mg/L):KH2PO4，15(6 as P);(NH4)2SO4，145(45 as N);NaHCO3，375 and pH was 7.2.

2.3 Activated Sludge

Activated sludge of SBR was domesticated from sewage treatment plant aeration tank. Sludge concentration was about 5000 mg/L with particle size of 1.5 mm.

2.4 Sampling and Analytical Procedures

2.4.1 INT－ETS activity

The sample(0.5 mL)was pipetted into separate 10－mL centrifuge tube in 0.1 mL of 0.2%INT.The tube was positioned at 37°C in a stable temperature horizontal shaking bath in darkness for 30 min to produce Iodonitrotetrazolium Formazan(INTF).The sludge was prepared for quantitative analysis of INTF through a refinementofINT-ETS activity.The multiphase mixture was mixed with 1 mL formaldehyde (37 wt.%)and centrifuged(4500 r/min)for 5 min.The sludge after centrifugal separation was vortexed with 5 mL of methanol and extracted for 10 min.The extract liquor was then measured at 485 nm for INTF quantification. The residues were centrifuged (4500 r/min)for 5 min to separate the sludge.The sludge discs was dried at 105 ℃ for 1 h and weighed.The activity of INT-ETS was calculated by Eq.(1)［15－16］:

where UTis the activity of INT-ETS［mg INTF/(gTSS·h)］; D485is absorbance;V is volume of extractants(mL); KTis gradient of the standard curve slope;W is the amount of sludge(g);t is the cultivate time(h).

2.4.2 Other conventional parameters

CODCrwas determined using a COD fast detector model 5B-1(DR1010，Hach，America);NO3－-N，and NH4+-N were measured by thymol spectrophotometry， N-(1-naphthyl)-1， 2-diaminoethane dihydrochioride spectrophotometry and Nessler＇s reagent spectrophotometry，respectively.

3 Results and Discussion

3.1 Activity of INT-ETS During One SBR Cycle

The changes in the activity of INT-ETS，COD and the concentration of nitrogen species during one SBR cycle are determined(as shown in Fig.2).

As seen，the activity of INT-ETS decreases with the prolonging of reacting time during the aerobic phase (as shown in Fig.2).It should be noted an inflexion at approximately 30 min，and the corresponding activity of INT-ETS is 190.65 mg/(g·h).Base on the results of COD determination，it indicates that most of the COD is removed during the aerobic phase and the nitration of ammonia nitrogen is about to begin.That is to say，before the inflexion point，the activity of INT-ETS is attributed to aerobic heterotrophic bacteria; after the inflexion，the activity of INT-ETS is attributed to autotrophic nitrifying bacteria.The experiment results demonstrate that INT-ETS activity of sludge can effectively reflect the variation of the organic degradation procedure in SBR system.At the beginning of nitrification，there is an INT-ETS activity jump when the aeration time is 40 min.Meanwhile，it is observed that the increase of nitrate concentration is far less than decrease of ammonia nitrogen.This can be attributed to simultaneous nitrification and denitrification.INT-ETS activity rises at initial stage，then decreases when the nitrification starts，finally reaches to be stable at the end of nitrification(300 min aeration later).

Fig.2 Profiles of INT-ETS activity of activated sludge and COD，ammonia，nitrate and nitrite in SBR process

During the anaerobic phase，the activity of INTETS is increased slowly，which is associated with denitrification of nitric acid and nitrous acid.INT-ETS begins to decline from 420 min to 450 min and then becomes stable.The corresponding nitrate also experiences a sharp decline at 420 min and almost decreases to 0 after 30 min.It indicates that the denitrification reaction has ended and can explain that INT-ETS activity can show the changes of denitrification process timely.

3.2 Effect of NH4+-N Concentration of Influent Water on the Activity of INT-ETS

When the influent COD is approximately 300 mg/L，influent ammonia nitrogen is 14.5 mg/L and 42.0 mg/L，the activity of INT-ETS，COD，ammonia nitrogen，nitrite nitrogen and nitrate nitrogen during one SBR cycle are determined，as shown in Figs.3(a)and 3(b).

It can be seen that the main difference of Figs.3(a) and 3(b)is the appearing time of plateau phase.This can be attributed to the reaction time of nitrification of ammonia nitrogen.During denitrification phase，the difference of reaction time is due to the difference of the concentration ofnitrate nitrogen.Also，the appearing time of inflexion point is different.INT-ETS activity variation rule of the COD removal phase in two kinds of experimental conditions are almost the same with previous experiment;however，the appearance time ofthe stable stage ofINT-ETS activity is influenced due to different influent ammonia nitrogen concentration resulted in different nitrification duration.Denitrification terminates at 90 min and 240 min when influent ammonia nitrogen concentration is 14.5 mg/L and 42.0 mg/L，respectively，and INT-ETS activity stable stage appears at that time too.The appearance time of the turning point is changed since different nitrate production leadstodifferentdenitrification duration.

Large amount of ammonia nitrogen consumed by microbial assimilation made only 3.5 mg/L nitrate produce at the end of nitrification when influent ammonia concentration is14.5 mg/L，and then convert to nitrogen in denitrification within 20 min.Meanwhile，INT-ETS activity only rises for a while，and then tends to be stable.Correspondingly，33.0 mg/L nitrate produces at the end of nitrification when influent ammonia concentration is 42.0 mg/L，which prolongs the denitrification process to 450 min and extends INT-ETS activity to be stable as well.

3.3 Effect of Organic Contaminants Concentration of Influent Water on the Activity of INT-ETS

When the influent ammonia nitrogen is approximately 14.0 mg/L，COD is 293 mg/L and 685 mg/L，the activity of INT-ETS，COD，ammonia nitrogen，nitrite nitrogen and nitrate nitrogen during one SBR cycle are determined respectively，as shown in Figs.4(a)and 4(b).

Fig.3 The influence of influent ammonia concentration on the SBR process

It can be seen that the behavior of activity of INTETS is correlated to a variation in organic contaminants concentration.No significant changes in COD removal phase is found，comparing with the results mentioned above.When the influent COD is 293 mg/L，most of the COD is removed after 30 min of operation.At the same time，the curve of the activity of INT-ETS is seen.

In Figs.4(a)and 4(b)during the nitrification phase，the change laws of the activity of INT-ETS fits the resultsmentioned above.However，the main difference is the appearing time of the key point of the beginning of nitrification and the reaction time.This is because more organic and nitrogen assimilation the influent water contains，the shorter the nitrification lasts.When the influent COD is 293 mg/L，the key point appears at 30 min and 120 min，which signs the beginning and end of nitrification.The reaction time of nitrification is 90 min.When the influent COD is 685 mg/L，the reaction time of nitrification is 60 min and the key point is 120 min，which signed the end of nitrification.

Fig.4 The influence of influent COD concentration on the SBR process

During the denitrification phase，the activity of INT-ETS has different change laws under different two kinds of experiment conditions.It can be seen that this is because the concentration ofnitratenitrogen is different，when the aeration is ended compare with the transform of nitrogen.At the end of the oxic phase，approximately 5.0 mg/L of nitratenitrogen is produced，when the COD ofinfluentwater is 293 mg/L. The nitratenitrogen converts to hydronitrogen in the first 20 min.At the same time，the activity of INT-ETS increases and then decreases to a stable value.However，when the COD of influent water is 685 mg/L，only 0.5 mg/L of nitratenitrogen is produced.The nitratenitrogen begins to convert rapidly to hydronitrogen completely.Meanwhile，the activity of INT-ETS is basically constant without obviously rising phenomenon，and it is almost the same.

4 Conclusions

Based on the results of this study，it can be concluded that INT-ETS activity can effectively and duly reflect bio-reaction procedure of SBR system and it showsthatthe measurementsofXTT sodium reduction can be a useful technique for estimating activated sludge activity in steady-state and transient regime cultures，and can be a good tool for monitoring and controlling activated sludge water systems.

［1］Blenkinsopp S A，Lock M A.The measurement of electron transport system activity in river biofilms.Water Research，1990，24(4):441－445.

［2］Lopez J M，Koopman B，Bitton G.INT-dehydrogenase test for activated sludge process control. Biotechnology Bioengineering，1986，28(7):1080－1085.

［3］Awong J，Bitton G，Koopman B.Oxygen uptake rate and INT-dehydrogenase activity of acitinomycete foams.Water Research，1985，19(7):917－921.

［4］Christine M C，Pernelle J J，Morin L，et al.Relevance of the INT test response as an indicator of ETS activity in monitoring heterotrophic aerobic bacterial populations in activated sludges.Water Research，1998，32(4):1213－1221.

［5］Weddle C L，Jenkins D.The viability and activity of activated sludge.Water Research，1971，5(8):621－640.

［6］Fonseca A C，Summers R S，Hernandez M T.Comparative measurements of microbial activity in drinking water biofilters.Water Research，2001，35(16):3817－3824.

［7］Kim C W，Koopman B，Bitton G.INT-dehydrogenase activity test for assessing chlorine and hydrogen peroxide inhibition of filamentous pure cultures and activated sludge.Water Research，1994，28(5):1117－1121.

［8］Yin Jun，Tan Xuejun，Ren Nanqi.Evaluation of TTC and INT-electron transport system activity tests for heavy metal inhibition of activated sludge.Environmental Science，2005，26(1):58－62.

［9］Bielefeldt A R，Cort T L.Dual substrate biodegradation of a nonionic surfactant and pentachlorophenol by Sphingomonas chlorophenolica RA2.Biotechnol Bioeng，2005，89(6): 680－689.

［10］Cort T L.Effects of Surfactants on Pentachlorophenol Biodegradation bySphingomonasChlorophenolicum sp.Strain RA2.Colorado:University of Colorado，2000.

［11］Cort T L，Bielefeldt A R.A kinetic model for surfactant inhibition of pentachlorophenol biodegradation.Biotechnol Bioeng，2002，78(6):606－616.

［12］Cort T L，Song M S，Bielefeldt A R.Nonionic surfactant effects on pentachlorophenol biodegradation. Water Research，2002，36(5):1253－1261.

［13］Shen T，Wang J Y.Biochemistry.Beijing:Higher Education Press，1990.256－262.

［14］Wang Feifei，Ding Yuanhong，Ge Lei，et al.Effect of high-strength ammonia nitrogen acclimation on sludge activity in sequencing batch reactor. Journal of Environmental Sciences，2010，22(11):1683－1688.

［15］Shi Hanchang，Ke Xiyong，Zhang Wei，et al.Study on the biologicalactivity ofactivated sludge using a rapid biological activity tester.Environmental Science，2004，25 (1):67－71.

［16］Mathew M，Obbard J P.Optimization of the dehydrogenase assay for measurement of indigenous microbial activity in beach sediments contaminated with petroleum.Biotechnol lett，2001，23(3):227－230.