Modification on the conventional procedure to measure AOC in drinking water

Journal of EnvirorunentalVal. 16, No.6,pp.996- 0002004CNI1-2629/17Article ID: 10-074206-09969050CLC number: X830.2; R123.6Documnent code: AModification on the conventional procedure to measure AOC in drinking waterL Fu-zhi' ，SANG Jun-qiang'" , ZHANG Xi-hui' ，W ANG Zhan-sheng'(1. Research Center for Enviroonmental Engineering & Management ,Shenzhen Graduate School ，Tsinghua University, Shenzhen 518055. China. E-mail: sangjunqiang @ tsinghua . edu. en; 2. Department of Environmental Science and Engineering, Tsinghua University . Beijing 100084. China)Abstract: Additional phosphorus will be introduced to water sample if the conventional procedure is used to measure assimilable organiccarbon(AOC) in drinking water. It has been shown that there are the cases that phosphorus is the limiting nutrient for microbial growth indninking water. The measured value of AOC would not be able to indicate appropriately the regrowth potential of bactria in this case. Theconventional procedure used to measure AOC was modified to avoid the introduction of additional phosphorus to water sample in thisstudy. It was shown that it was feasible to measure AOC in water using the modified procedure. Furthermore, the measured value of AOCdetermined by the moditied procedure could indicate appropriately the regrowth potential of bacteria in drinking water despite either organicsor phosphorus was the limiting nutrient for bacterial regrowth.Keywords: drinking water; assimilable organic carbon; bacterial regrowth; phosphorus: organicsAOC can indicate appropriately the regrowth potential ofIntroductionheteotrophic bacteria in drinking water when either organicsA0C( assimilable organic carbon) has been used as oneor phosphorus is the limiting nutrient for bacterial regrowth.of the main parameter to indicate the regrowth potential ofMaterials and methodsheterotrophic bacteria in drinking water for a long time. Themeasurement of AOC is based on the conventional hypothesis1.1 Water samplesthat biodegradable organic carbon is the only limiting nutrientThree different water samples were prepared by addingfor the regrowth of heterotrophic bacteria in drinking watersome nutrients to deionized water. The concentration ofdistribution systems ( Bruce，1984; Peter. 1990; Mark ,organic carbon added to all the waler samples was 200 μg/L1996; Liu, 2000; Van. 1982). The prineiple to determine( CH, COONa, measured as acetate-C) . The concentration ofAOC is thal the reproduction of Pseudomonads FluorescentPO: -P( NaH2 PO, ) appended to the three water samples wasstrain P17 and Spillum strain NOX is in direct proporion to0, 1 and 20 μg/L, respectively.the concentration of assimilable organic carbon in drinkingIn order to meet the demand of microbes for otherwater( Van， 1982; Miettinen， 1996). At present， thenutrients，a mixture of inorganic nutrienls includingprocedure advanced by Dr. Liu is widely used in China 10NH, NOg. MgSO, ●7H2O, CaCl, ●2H20, KCl and NaCl wasmeasure A0C in drinking waler( Liu, 2000).added to all the water samples. The concentration of eachRecently，the cases that phosphorus is the limitinginorganic nutient added to the water samples were 250 pg/Lnutrient for bacterial regrowth in drinking water have beenof N( including NH' -N and NO; -N)，10 ug/L of Mg’”, 27found( Miettinen. 1996; 1997a; Sathasivan. 1999; Minnaμg/L of Ca, 53 ug/L of K' and 40 ug/L of Na"2002; Sang，2003). This changes the conventional thinkingrespectively .that biologically available organie carbon is the sole limiting1.2 Analysisnutrient for bacterial regrowth. It has been shown that whenAOC of all water samples was determined using both thephosphorus is the limiting nutrient for bacterial regrowth, theconventional andmodified procedure. BRP ( Bacterialreproduction of P17 will be depressed and in direct proportionRegrowth Potential) of the two water samples with 1 ug/L and1o the concentration of phosphorus rather than the20 μg/L P0:5 -P was measured according to a methodconcentration of biological available organic carbon inmodified based on the method proposed by Sathasivandrinking water( Markku, 1999). Then the feasibility 1to use( Sathasivan, 1999) .the measured value of AOC deternined by the conventionalThree basic steps were followed when BRP waprocedure advanced by Liu to indicate the regrowth potentialdetermined: (1)_ Preparation of inoculum: Raw water wasof heteotrophic bacteria in drinking water should betreate中国煤化工process fllwed bychallenged if phosphorus is the limiting nutrient for bacterialcoagu: fltration. About 200 mlregrowth .of the cahucu mas ULIOICU, luuwdtedYHCNM H Gled tn Iie dankd a 200The purpose of this study is to modify the conventionalfor 5 d then fitered with a 2 μm polycarbonate membraneprocedure advanced by Liu so that the measured value offilter. The filtrate was used as inoculum for BRP analysis.No.6Modification on the conventional procedure to measure AOC in drinking water(2) lnoculation of water sample: Water sample to befrom inoculum to water samples during the inoculation of P17analyzed was poured into prepared 50 ml vials andand NOX if the conventional procedure is used to determinepasteurized in 65C water bath for 30 min. After cooling, theAOC.water sample was inoculated with 0.5 ml of the inoculum.According to the conventional procedure, the inoculumAferwards, the water sample was incubated 7 d at 20C inof P17(or NOX) to be inoculated to water samples isthe dark. (3) Enumeration of bacteria: Microbial counts inprepared by inoculating 100 μl of the originally preparedthe waler sample after incubation were determined by spreadinoculum of PI7(or N0X) to 50 ml of culture medium whichplaling on R2A-agar plates ( Standard Methods for thecontains 2000 μg/L of acetale-C ( CH, COONa is used )Examination of Water and W astewater，1995). R2A-agar( Wang, 1999). The detailed cormposition of the conventionalplates were incubated 7 d at 22- 25C before the CFUculture medium used to inecubate P17 and NOX is listed in( colony forming unit) were counted.Table 1.It can be seen from Table 1 that the concentration of2 Results and discussionK: HPO。in the conventional culture medium is 7000 pg/L and .2.1 Limitation of the conventional procedurethe concentration of KH2PO。 is 3000 μg/L. Then theConsiderable amount of phosphorus will be introducedphosphorus concentration in the medium is 1931 ug/L.Table 1 Detailed composition of the conventional culture mediurm used to incubate P17 and NOXCompoundAcetate-CK,HPO。KH,PO4MgSO, .7H20(NH,):SO2NaClFeSO,Conentration. pg/L20007000030001001000000In addition，because the concentration of P17( or NOX)properly the potential of bacterial regrowth in the water .in the prepared inoculum is very high. the volume of the2.2 Modification on the conventional procedureinoculum inorulated to water samples is usually very small.In order to make the measured value of AOC could beIn order to avoid operating eror as possible duringused as a feasible and proper parameter indicating theinoculation, the inoculum needs to be diluted before it isregrowth potential of bacterial in drinking water even ifinoculated. According to the practice, the concentration ofphosphorus was the limiting nutrient for bacterial regrowth,P17(or NOX) in the inoculum should be diluted to 1/2- - 1/3two modifications were applied to the conventional procedureof its initial concentration and the actual volume of dilutedin this study.inoculum inoculated to water samples is usually about 80 μl.One of the modifications was to reduce the phosphorusThe ultra-pure water amended with several inorganic nutrientsconcentration in the conventional culture medium used to(Table 2) is used to dilute the inoculum according to theincubate PI7 and NOX. The concentration of K; HPO4 in theconventional procedure. It can be seen from Table 2 that themodified culture medium was reduced to 200 μg/L and theconcentration of P0O;- -P in the diluting water is very high andconcentration of KH,P0。 was reduced to 100 1ug/L.the actual concenlration is 3047 pug/L. .concentrations of all the other nulrients remained he same toTable2 Concentration of inorganic nutrients in the water used to dilutethe conventional culture medium. Then the phosphorusinoculumconcentration in the modified culture medium was 59 1g/Land the ratio of organic carbon to phosphorus was 100: 3,K2 HPO,KNOtNH。Clwhich could meet the demand of P17 and NOX. The otherConcentration, ng/L17.114476.4modification was that K,HPO。 was not added to the dilutingBecause the actual volume of diluted inoculumwaler used to dilute the inoculum . Since this diluting waterinoculated to water samples is about 1/500 ( the actual ratiowas only used to dilute the inoculum during the inoculation oflies on the concentration of P17 or NOX in the inoculum) ofPI7 and NOX, this modification could not adversely affect onthe volume of the water samples， the concentration ofthe growth of P17 and NOX.phosphorus in the water samples will increase by about 4- 5It could be calculaled according to the two modifcationsμg/L due to the introduction of phosphorus from the dilutedthat phosphorus introduced to water samples duringinoculum. Compared with organics, microorganisms requireinoculation of PI7(or NOX) would be less than 0.1 ug/L,much less phosphorus .Therefore,' if phosplhorusthereby the remarkable increase of phosphorus concentrationconcentration in the water sample to be analyzed is extremelyin water中国煤化工low and phosphorus is limiting nutrient for bacterial regrowth,ndCNMH(to incubate P17 andthe measured value of AOC will be greatly afected due to theNOX using Tue mouea meuium, r'17 and NOX wereintroduction of additional phosphorus from inoculum if theincubated in both the modified mediurm and the conventionalconventional procedure is used . Then the effect of phosphorusmedium，respectively. The final concentration of P17 andconcentration on the measured value of AOC might not beNOX in the modified medium was compared with the finalseen and 防数雅red value of AOC can not indicateconcentration of P17 and NOX in conventional medium after998u Fu-zhi et al. .Vol,16incubation. The results are shown in Fig.1 and Fig. 2( threethe limiting nutrient for bacterial regrowth in this waterindependent results were shown in each figure) .sample and the increase of phosphorus could not changeobviously the measured value of AOCp7 and AOCxox .By the conventional procedure and modified procedure ,AOC of this water sample was measured using P17 and NOX,0.8respectively. Three independent results are shown in Fig.3s0.6tand Fig. 4, respectively .客0.4L复0.2-25020Analysis sequence2 1500 Conventional medium 2 Modified mediumFig.1 Final concentration of P17 in the mdied and conventona! medium5(2.40D[ I5 2.0口Conventional procedure 2 Modified procedureFig.3 Meusured value of AOCpr in the waler sanpe with 20 pug/l. P0:- P员0.8It could be seen from Fig.3 that the measured value of昌0.AOCp) determined by the modified procedure was similar tothe measured value determined by the conventional procedureand was around 200 μg/L. The similar results were gotten口Conventional mediuma Modifed mediumfrom the measurement of AOCNox in this water sample( Fig.Fig.2 Final conrentration of NOX in the molified and conventioual medium4). This indicated that if organics was the limiting nutrientIt can be seen from Fig.l that there was litle differencefor bacterial regrowth in drinking water， the modifiedbetween the concentration of P17 in the inoculum prepared byprocedure was ftting for the measurement of A0C and thethe modified medium and the concentration of PI7 in themeasured value was the same as the value determnined by theinoculum prepared by the conventional medium. The similarconventional procedure .result was gotten from the incubation of NOX in the lwomedia(Fig.2). This was to say that this modification onculture medium had litle efet on the growth of P17 and，200NOX. This result indicated that it was feasible to incubate。150PI7 and NOX using the modified medium.8 1002.3 Comparison between the two procedures for AOC0一|measurementoLIn order to investigate whether the modified procedurewas feasible to determine AOC in water and whether theAnalysis squencemeasured value of A0C could indicate the regrowth potentialFig.4 Measured value of AOCwun m the waler samples with 20 1u/1. P0O?- .Pof bacteria, both the modified procedure and the conventionalprocedure were used to measure AOC in waler samples and2.3.2 Measured value of AOC in water sample withoutthe results were compared .PO;: .P2.3.1 Measured value of AOC in water sample with 20Since the concentration of organie carbon in this waterμg/L PO: .Psample was 200 ug/L and no phosphorus was added, it couldBecause CH,COONa is the reference substance forbe inferred that phosphorus was the limiting nutrient forAOCp17 and AOCw)x measurement and it can be assimilatedbacterial regrowth in this water sample and the increase ofconplelely by both PI7 and NOX. only one of P17 and NOXphosphorus could obviously change the measured value olis needed to determine the overall AOC in all the walerAOC中国煤化工。PO:- -P adition wassamples used in this experiment and the measured values oAOCpT and AOCNox should be around 200 μg/L in theory imeasMYHCNMHG using P17 and Nox,organic carbon is the limiting nutrient for bacterial regrowthrespectively, by the conventional procedure and the modifed(Liu, 2000). Since the concentration of organic carbon inprocedure. The results are shown in Fig.5.the water sample was 200 ug/L and the concentration ofIt could be seen frorm Fig.5 that the measured value ofphosphenugs :20 1p/L, it could be sure tha organies was AOC in the waler without phophons adition delermined byNo.6Modification on the conventional procedure to measure AOC in drinking water999for baclerial regrowth. ht could be seen from Fig. 6 that the240BRP in the water sample with 0.5 ug/L phosphorus was muchless than the BRP in the water sample with 20 pg/Lphosphorus . This indicaled that although the concentration of120 Horganic carbon in the two water samples was the same, th30 Hability of substrates in the two water samples to support the0-growth of bacteria was different. The regrowth polential ofMeasured using PI7 Measured using NOxbacteria in the water sample with 20 μg/L phosphorus was口Coanventional procedure 2 Modified procedurehigher than in the water sample with 0.5 rg/ L phospborus.Fig.s Measurd value of AOCrr und AOCwov in the water sumple withoutIt was shown that the two measured values of AOCp，PO/2-P aditiondetermined by conventional procedure in the two watersamples had lttle difference. That was to say, the two valuesthe modified procedure was much lower than the measuredcould not indicate the actual difference between the regrowthvalue of AOC determined by the conventional procedure.potential of bacteria in the Iwo water samples. On the otherBecause considerable amount of phosphorus was introduced tohand, it could be seen from Fig .6 that the measured value ofthe water sample during the inoculation of PI7 and NOXA0Cp7 in the water sample with 0.5 μg/L PO:" -Pwhen the conventional procedure was used to determine AOC,determined by the modified procedure was much less than thethe introduced phosphorus could meet the demand of P17 andmeasured value of AOCpr in the water sample with 20 ug/LNOX for reproduction, which resulted in the measured valuePO,~ -P determined by the modifed procedure. This resultof AOCpr and AOCNox being about 200 μg/L. However,when the modified procedure was used, lttle phosphoruswas corresponding to the BRP of the lwo water samples andcould be introduced to the water sample, the growth of P17illuminated that the measured value of AOC determined bythe modified procedure could indicate properly the regrowthand NOX would be depressed remarkably because of thedeficiency of phosphorus. This made the measured value ofpotential of bacteria in drinking water despite phosphorus orAOCm7 and AOCrox be much less than the measured valueorganics was the limiting nutrient for bacterial regrowth.In addition， it could be seen from Fig. 6 that thedetermined by the conventional procedure .measured value of AOCp; in the water sample with 20 μg/L2.3.3Relation between the measured value of AOCphosphorus determined by modified procedure was similar toand BRPBRP indicales the growth of an assemblage of indigenousthe measured value of AOCp17 determined by conventionalheterotrophie bacteria in drinking water. Therefore， BRPprocedure. This indicated that the measured value of AOCshows exartly the regrowth potential of bacteria in drinkingdetermined by the two procedures could indicale properly thewater with limited substrales to support their growth. In thisregrowth potential of bacteria in drinking waler if organics wasexperiment，BRP in water samples with 0.5 pug/L and 20the limiting nutrient for bacterial regrowth.ug/L P0,- -P was analyzed and AOCm> was determined by2.4 DiscussionIt could be seen from this study that if phosphorus wasboth the conventional procedure and the modified procedure .the limiting nutrient for bacterial regrowth in drinking waterThe results are shown in Fig.6.and the conventional procedure advanced by Liu was used to2040-determine AOC, the measured value of AOC could nol show00 -00 texactly the regrowth potential of bacteria in the water due tothe introduction of phosphorus from the inoculum of PI7 and手12060NOX. If the modified procedure advanced in this study was父8used，this limitation could be avoided and the measured40value of AOC could indicate exactly the regrowth potential ofPhosphorus limitation Organics limiationbacteria in waler.口AOCer mcasured by modifed proedureIt should be noted that the term AOC has been advanced口AOCr measured by conventional procedare日BRPover ten years before the cases that phosphorus may becomethe limitith in drinking waterFig.6 Measured vabue of AOCp> und BRP in waer smples wih 0.5 and 20are four中国煤化工Irs to the portion of1ng/LPO% PBDOCMYHC NMH Ge carbon) that isSince organie carbon in al the waler samples was 200converted to baclerial biomass( Van，1982; Liu. 2000).pg/1. if the phosphonus was 0.5 ug/L. phosphorus would beTherefore, the actual AOC in drinking water is not related tothe limiting nutrient for bacterial regrowth. If the phosphorusthe phosphorus concentration from this point of view.were 20 1ug/L, organice carbon would be the limiting nutrientHowever. AOC is used as a parameter to index the regrowth.1000U Fu-zhi et al .Vol. 16potential of bacteria in drinking water in practice. ItsIt was shown that if organics was the limiting nutrient forimportance for drinking water industry would lose if AOCbacterial regrowth in water， the measured value of AOCp7could nol be used as a parameter to index the regrowthnd AOCNoX determined by the modified procedure wapotential of bacteria. In addition, as far as the technique tosimilar to the corresponding measured value determined bymeasure AOC is concerned, the measurement of AOC is athe conventional procedure. This indicated that it wabioassay, so the measured value of AOC is related to thefeasible to measure AOC in drinking waler using the modifiedgrowth of PI7 and NOX. If phosphorus is the limitingprocedure . If phosphorus was the limiting nutrient for bacterialnutrient for bacterial regrowth in the drinking water to beregrowth in drinking water， the measured value of A0Canalyzed，the growth of P17 and NOX should also bdetermined by the modified procedure was corresponding toaffected. Then the feasibilit to use the measured value ofthe BRP of the drinking water and was less than the measuredAOC determined by conventional procedure to index thevalue determined by conventional procedure . This ilumninatedregrowth potential of bacteria in drinking water should bethat the measured value of AOC determined by the modifiedchallenged .procedure could indicate properly the regrowth potential ofIt could be seen from this study that if organics was thebacteria in drinking water if phosphorus was the limitinglimiting nutrient for bacterial regrowth in drinking water, it isnutrient for bacterial regrowth .feasible to use the measured value of AOC determined byThe results indicated that the measured value of A0Ceither the conventional procedure or the modified proceduredetermined by the modified procedure could indicaleas the index of the regrowth polential of bacteria because theappropriately the regrowth potential of bacteria in drinkingtwo values are the same. The two measured values alsowater despite organics or phosphorus was the limiting nutrientrepresent the conventional AOC that is converted to bacterialfor bacterial regrowth .biomass from BDOC. However, if phosphorus was theReferences :not feasible to use the measured value of AOC determined byBruce E R, Vermon L s.1984. Arhieving bilgically stable drinking water[J].」AWWA. 76(10): 106- - 114.the conventional procedure as the index of the regrowthGibbs R A. Scutt J E. Croll B T，1993. Asimilalble organie cartbonpotential of bacteria. ht has been showm that the regrowthconcentrations and bacterial numbers in a waler distibution system[Jj. " aterSei Technol, 27: 156- 166.potential of bacteria in the drinking water of some area is notrelated to the measured value of AOC ( Gibbs，1993 ;liuW」，Wang Y J. /hang 1. P n al.. 200 Delerminaion onf asimilbleMiettinen，1997b). The main reason is that phosphorus isMark w 1.. NaneyJW. Darrell BS. 1996. Flloswale studies of fartors related twocolifurm regrowth in drinking water[J]. Appl Environ Microbiol. 62(7):the limiting nutrient for baclerial regrowth in this waler2201-2211.( Mettinen, 1996; 1997a).MarkkuJ 1. Mietinen I T. Vartiainen T时al..9999 A new sensitive bicarsays shown in Fig. 6，if phosphorus is the limitingfor determimation of microbally arailable phosphorus in water[J]. ApplEnviron Microbiol. 65(5); 2032 - - 2034.nutrient for bacterial regrowth， the measured value of AOCMetinen I T，Vartiainen T. Martikaiuen P J.1996. Contarmnination of drinkingdelermined by the modified procedure was less than thewater[J]. Narur, 381: 654- 655 .Mietinen I T. Vartiainen T, Marikainen P J.1997a. Phwosphorus and bacterialmeasured value determined by the conventional procedure ，growth in drinking water[J]. Appl Environ Mecrdial. 63(8): 3242- -3245.but the measured value determined by the modifed procedureMetinenI T,Vartiainen T, Martikainen P J. 1997b. Microbial prowth andasumnlable organir carbon in Finnish drinking waters[ J. w uter Sri Terhnol,showed exactly the regrowth potential of bacteria in drinking35: 301- 306.waler.Therefore，despite organics or phosphorus was theMinna M K. leena K K. Markku 」I. et al.. 2002. The mirobial eonumunitylimiting nutrient for bacterial regrowth in drinking water, thstrurture of drinking water biofGluns can br ffleted by phusplors avilbility[J]. Appl Emiron Microbiol. 68(1): 434- -439.measured value of AOC determined by the modified procedurePplter M H. 1990. Measurment of bidegradable organic natter and buecterialcan be used as the proper parameter Lo index the regrowthgrouth potential in drinking water- a review[J]. J AWWA. 82(7): 78-potential of bacteria in drinking water although the measuredSahasivan A. Ohgaki s. 1999. Application of new laternal regrowth potentialvalue of AOC can not represent all the portion of BD0C thatnethud for water dirtributon system- a edlear eridence of puspborusis converted to bacterial biomass if phosphorus is the limitingSangJQ, YuC z，ZhangX H er al.. 2003. Phophurus linitaion on bat tenalnutrient for bacterial growth.regrowth in drinking wuter[J]. Journal of Envrour-rntal Seicnces, 15(6):3 ConclusionsSluandard mxthods for the exanination of water and wastewater[ M].1995. 19thrd. Wwashingtun DC: Amrican Publie Heulth Asoriation .In this study, the conventional procedure advanced byVan [-ining the concentration of easilyLiu used lo measure A0C was modified to avoid the中国煤化Ihwa.24 10) s40)introduction of additional phosphorus to water samples to beiang:YHCNMHGhly plluludl aw wuler M].analyzed so that the measured value of AOC can indicateBejung: Constructron Industry Press of China.properly the regrowth potential of bacteria despite organics or(Reeived for reviev November 24.2003. Accepled March 24. 2004)phosphorus was the limiting nutrient for bacterial regrowth indrinking water.