~ Pergamon Wal.Sci.re« Vol.34.No. 5-6,pp. 403-410. 1996.CopyrightC 19961AWQ. Publishedby ElsevierScienceLtd

Printed in Great Britain.AU righb reserved.0273-1223/96S1500 +OOO

PH: S0273-1223(96)00672-5

MODELING KINETICS OF CHLOROFORMCOMETABOLISM IN METHANOGENICAND SULFATE-REDUCINGENVIRONMENTS

Munish Gupta, Makram T. Suidan and Gregory D. Sayles

Department a/Civil andEnvironmental Engineering. University a/Cincinnati.Cincinnati. OH45221-0071. USA

ABSTRACT

Thekinetics of chloroform cometabolism in methanogenic andsulfate-reducing environments weremodeled.Chloroform inhibits acetate utilization in methanogenic environments and therefore the rate of chloroformcometabolism was modeled using two terms: cometabolism due to primary substrate utilization andcometabolism dueto microbial cell decay (noprimary substrate utilization). Themodified Haldane equation wasused to model chloroform cometabolism dueto primary substrate utilization andtheMonod equation wasusedfor the cometabolism due to microbial cell decay. A commercial software, SigmaPlot (Jandel ScientificSoftware) was used to determine theoptimum parameters for the model. Themodel agreed very well withtheexperimental results. The model parameters confirmed that thesulfate-reducing culture hasa veryhigh potentialforchloroform cometabolism compared to themethanogenic cultures. Also, the methanogenic culture grownon acetate is verysensitive to chloroform compared to the methanogenic culture grown on methanol.Copyright e 1996IAWQ. Published byElsevier Science Ltd

KEYWORDS

Modeling; Chloroform; Biodegradation; Inhibitory; Haldane; Cometabolism; Methanogenic; Sulfate­Reducing; Acetate; Methanol.

INTRODUCTION

Anaerobic treatment has only been used as a post-secondary step for stabilizing waste sludge (anaerobicdigestion) inthepast butrecently it hasreceived considerable attention forthetreatment of industrial wastewatercontaining recalcitrant compounds such as highly chlorinated aromatics and aliphatics. Most of thesecompounds arenon-degradable under aerobic conditions anddegrade very slowly under anaerobic conditions.However, the degradation of thesecompounds can be enhanced, if an easily degradable substrate (primarysubstrate) is added inorderto stimulate thegrowth of anaerobic bacteria. Therecalcitrant compound is thendegraded bythemicroorganisms asa secondary substrate using the primary substrate as the growth carbon andenergy source. Thisis known as cometabolism.

403

404 M.GUPTA etal.

Some of the chlorinated compounds acting as secondary substrates and/or their metabolites are toxic toanaerobic microorganisms andhencecan inhibit the degradation of the primary substrate as well as theirown.The toxicity of the secondary substrate depends on anaerobic environment as well as the nature of themicroorganisms (Gupta etaL, 1994). Therefore modeling of secondary substrate cometabolism is necessary sothatthekinetics of cometabolism aswell astoxicity canbe predicted. Thesemodels will helpin the design andcontrol of anaerobic reactors degrading suchcompounds. Thispaper models and compares the kinetics ofchJorofonn cometabolism intwo methanogenic cultures using acetateor methanol as the primary substrates andina sulfate-reducing cultureusingacetateas the primary substrate.

MODEL DEVELOPMENT

Although biodegradation is a verycomplex process, therehavebeenseveral attempts to model the process ina meaningful waysothatthemodel parameters can beexplained andcompared. Traditionally, Michaelis-Mentenor Monod-Iike expressions havebeenusedfor describing the rate of degradation of a substrate. TheMonodexpression is expressed as:

Rate - (K * S)/(K. + S). (1)

whereK is the maximum rate ofsubstrate utilization andK.is the Monodhalf-velocity constant. The rate ofdegradation ofa substrate, Rate, is firstorder withrespect to its concentration, S, at lowconcentrations (S«I<.) and at highconcentrations (8)> 1<.), Rate is constant regardless of the substrate concentration (zeroorder).

Some of the substrates are toxic to the microorganisms at higher concentrations and hence the rate ofdegradation decreases at higher concentrations ofsubstrate. This is knownas substrate inhibition. TheMonod­like expressions do nottakecareof thereduction ofthe rate at higher substrate concentrations. Several modelshave beenproposed which describe thereduction in rate due to the inhibitory natureof the substrate (Edwards,V.H., 1970). Thesemodels are:

Rate - [K * S]I[K. + S + (S21KJ],Rate- [K * S * (1 +(S!K..»]I[K. +S + (S21KJ],Rate - [K * S]/{[K. +S + (S21KJ]* [1 +(SIK,,)]),Rate- [K * S * exp(-SIKJ]/(K. + S),Rate - K * [exp(-SIKJ] - exp(-SIK.)].

(2)(3)(4)(5)(6)

Equation (2) is knownas the Haldane equation. I<t is known as the inhibition constantand as it approachesinfinity, the Haldane equation takes the sameshapeas the Monodequation (Eqn. (1». Rozich et 01. (1985)evaluated different models (Eqns2-6) for selecting a rate model for acclimated mixed cultures treatingphenol,aninlubitory substrate. They found thattheHaldane equation gave the best representation of the experimentaldata. Several other researchers havealso found the Haldane equation to provide an adequatefit for describingthe kinetics of degradation ofinlubitory substrates (YangandOkos, 1987; Suidan et 01., 1988; Fox and Suidan,1991,SaezandRittmann, 1993).

Themodified Haldane equation shown belowwasproposed byNeufeld et 01. (1980). It is expressed as:

Rate - [K* S]/{K. + S + [8 * (SIKJ"]). (7)

Chloroform cometabolism 40S

This equation consists of anotherparameter,n, known as the order of inhibition,compared to the Haldane equation. For n - "'.... r.q..Qoa

I, the aboveequation becomes the original ~---IHaldane equation (Eqn. (2». As the orderofinhibition, n, increases, the inhibitionbecomes stronger andtherateof degradationdecreases more rapidly. This is shown in !Figure 1. More recently, some researchershave found the modified haldane equation toprovide a better fit of the kinetics of aninhibitory substrate, sincen and, hence, theintensity of inhibition can be varied. Wu eta/. (1988) found the modified haldane modelto be most appropriate for describing the n..n1. Aca. ..... "' ..M=::........U1.. UaWaMl1•• lIoaanaerobic treatment of a solid waste landfillleachate. Morvai et aL(1992) compared several substrate inhibition models andfoundthat the modified modelprovided the best description of biomethanation of acetate. The model was used for both raw sludge andgranular sludge from anUASB reactor. Similar parameters were obtained for both sludges, exceptfor n whichwas about four timeshigher for raw sludge compared to granular sludge. Sincen indicates the intensity ofinhibition, it indicated that the granular sludge was lesssensitiveto inhibition compared to the raw sludge. Ina recent study (Wen et al., 1994) also used the modified haldane equation for describing the kinetics ofrecirculated upflow anaerobic sludge blanket reactortreating phenolic wastewater.

All the above modelsdiscussed above, can be usedto describe the kinetics of degradation of a primary or asecondary substrate. Theprimary difference being that theprimary substrate provides energy and carbon sourceforthegrowth and maintenance ofthemicrobial cells while a secondary substrate is assumed to be a non-growthsubstrate (Horvath, RS., 1972). Some researchers (Criddle, C.S., 1993) have reported that the rate ofcometabolism is linked to the rate of consumption of the primary substrate. In the absence of a primarysubstrate, the rateofcometabolism isrelated to theconsumption ofthemicrobial cell mass(Criddle, C.S., 1993).The rate of cometabotism due to primary substrate consumption is much higher compared to the rate due tocell decay since considerable energy is obtained for cometabotism by primary substrate consumption ascompared to cell decay. Therefore, the ratedueto cell decay isnormally neglected, in the presence of a primarysubstrate. However, for a toxicsecondary substrate suchas chloroform, which inhibits the degradation of theprimary substrate, both the rates should be takeninto account. Hence, the rate of cometabolism can best berepresented bythe sumof the two terms;rate of cometabotism due to primary substrate consumption andtherate due to microbial cell massconsumption (cell decay),

Rate -{[K • S)/{K. + S + [S • (SIIQ.)} } + [(1<' • S)/(K.+ S)] (8)

Therateofcometabotism dueto primary substrate consumption is represented by the modified haldane equation(lst term) and the rate due to cell decayis represented bythe simple Monodequation (2nd term). Both thetermshavedifferent maximum rate of substrate utilization (I<andK') but havethe samemonod halfvelocityconstant (1<.). Ifthesecondary substrate doesnot inhibit the utilization of primary substrate, the cometabolismdue to celldecaycan be ignored andonlythe 1st term(modified Haldane equation) is used.

ESTIMATION OFPARAMETERS

The modified Haldane equation is of non-linear form andits parameters are moredifficult to estimate thanthe

406 M. GUPTA etal.

haldane equation. Neufeld etal: (1980) proposed a two-step procedure to estimate the parameters. In the firststep, K. and K were estimated for high values of S, assuming that 1/S approaches zerq. K and n weredetermined by visual comparison of the data withthe model plot. Several researchers have, since then,usednon-linear leastsquares regression to fit the data andestimate the parameters. But thisapproach is extremelysensitive to theinitial guesses and, hence, it is possible to obtain values of parameters which havelittlephysicalmeaning. D'Adamo et 01. (1984)suggested that setting boundary conditions for the parameter values of theHaldane equation andusingthe non-linear regression technique, meaningful results can be obtained. But therange of the boundssuggested by D'Adamo et 01. (1984)is too wide and, therefore, trial and error may berequired.

4 5 6 7 8 9 10 11 12

Order or Inhibition, D

Fi&Ure 1. Variation ofnonnaliud squareerror with the order orinhibition.

.. 17 ~-----------,

~ 16

1 15t 141 13

112i. 11

10 tI.LU...Ll.LLl.J.IJ,J...Ll.LL.LLLU...Ll.LL.LLLUw.lJ..u.u.Jw.uJ

3

In this paper, a commercial software, SigmaPlot(Jandel Scientific Company) was used forparameter estimation. TheSigmaPlot curvetitterusesnon-linear regression to fit data to equationsthat are nonlinear functions. It uses theMarquardt-Levenberg algorithm to find theparameters that give the best fit of the data.Bounds of the parameter values were used, assuggested by D'Adamo et 01. (1984), so that thefinal parameter valuesliewithin a range. Sincethere is no higher-level bound for n, the orderofinlubition, the following methodology was used.n was fixed to a particular value(starting from 1)and the optimum parameters corresponding tothis value of n, were obtained by SigmaPlot. nwas thenincreased (by increments of 1)andthe optimum parameters wereagain determined. The normalizedsquared errorwas calculated foreach set of parameter values andthe error was plottedas a function of n. Theerrordecreased, reached a minimum value andthenstartedto increase. Thisis showninfigure 2. Theset ofparameters withthe minimum errorwere chosen to be the optimum parameters.

EXPERIMENTAL TESTS & METIIODS

Three different cultures weregrown inseparate stainless steelchemostats. A methanogenic culturewasgrownonacetate, another methanogenic culture wasgrownon methanol and a sulfate-reducing culturewasgrownonacetate. Batch testswereperformed to evaluate thekinetics ofcometabolism of chloroform by eachcultureandthe effect of chloroform ontheconsumption of the primary substrate (acetate/methanol). The batchtests wereconducted using different initial concentrations of chloroform and sameconcentration of primary substrate andwere used to evaluate the rate of cometabolism at those chloroform concentrations. Thus, the rate ofchloroform cometabolism as a function of its concentration, for eachculture, was determined. The completemethodology is described in moredetail in Guptaet01. (1994).

RESULTS & DISCUSSION

Methanogenie Culture grown on Acetate. The rate of chloroform cometabolism for this cultureincreased,thendecreased and finally became constant as the initial chloroform concentration was increased. To furtherinvestigate the constant rate at higher chloroform concentrations, the rate of acetate utilization was alsodetermined at each of the chloroform concentration. The rate of acetateutilization decreased withan increaseinthe chloroform concentration, indicating inhibition of acetateutilization due to chloroform. For higher

Chlorofonn cometabolism 407

chIorofonn concentrations. noacetate consumption wasobserved. which corresponds to the pointof constantrate of chloroform cometabolism (Guptaet al., 1994). Since, therewas no acetate utilization and hence nomicrobial cell growth, thechlorofonn cometabolism occurred dueto microbial cell decay (Criddle, C.S., 1993).Since there is very little energy produced for chloroform metabolism by cell decay, the chloroformcometabolism was limited bythe energy produced and, hence, didnot change with chloroform concentration(Guptaet al., 1994).

FJa:ure3. EJ:perimentallnd model predlcltonl of nte of chloroform cometabolilmfor the methanollenlc culture (ltetate).

700600500

-0- Mealured

- Model Prediction

400300

Chloroform concentntlon, u&ll

200100

..~ 30II

If 25

1& 20B! 15

02e~ 10..o.!.;

As explained before, the rate ofchloroform metabolism occurs dueto theenergy obtained from acetateconsumption as well as themicrobial cell decay. At lowerchlorofonn concentrations, therateof chloroform cometabolism wasmainly governed by the energyobtained by acetate consumption,while at higher chloroformconcentrations, there was noacetate consumption due tochlorofonn inlubition andhence therate of chlorofonn metabolism wasmainly governed by microbial celldecay. Therefore the modelproposed inEquation (8) wasusedto fit the data for chloroformcometabolism. Figure 3 shows therate curve obtained experimentallyandpredicted bythemodel. Since allthe batch testswereconducted induplicate, two values for the measuredrate are shown inthe figure. Table 1 lists the optimum parameters obtained for thisculture.

Table I. Modified Haldane Equation parameten for chlorofonn metabolism in different cultures.

Culture K K. I<t n K'(}lg/l/hr) (}lg/l) (}lg/l) (-) (}lg/l/hr)

Methanogenic 55 100 160 8 8.5(Acetate)

Sulfate-Reducing 4400 2200 3017 5(Acetate)

Methanogenic 103 370 836 2(Methanol)

408 M.GUPTA et al,

J1&ure4. E:lperm-tal and model predlcltonl or rate of chloroform eometaboUamfor the luJrate-reduclnc culture (acetate).

B

3500

8

3000

8

2500

8

-0- Meuured

- Model Prediction

20001500

Chloroform eoneentratlon, ugll

1000

g

500

t 2000• 1800

1 1600

Jj14001200

8! 1000

1 800

~ 600'; 400~

~ 200OL...o-......................................o-l-..............................---..--O....J.......-...................................o-l-......................1-J

o

S uJr. te-Red uel 0 gCulture grown 00

Acetate. The rate curvefor this culture followedthe same pattern as themethanogenic culturegrown on acetate exceptthat the rate did notbecome constant at higherchloroformconcentrations. This canbeexplained sinceacetateconsumption was notinhibited in this culture.The rate of acetateconsumption wasconstant irrespective ofthe chloroformconcentration (Gupta etal., 1994). Therefore,chloroform metabolismwasgoverned byenergy obtained from acetate consumption. Since thechloroform cometabolism dueto celldecay is verysmall compared to cometabolism due to acetate consumption, it canbe neglected in thiscase.Therefore the second terminthe model (Eqn. (8» is ignored andonly thefirst term(modified haldane model)was used for modeling chloroform metabolism in thisculture. Table I al~ shows the optimum parametersobtained for this culture. Figure 4 shows the rate curve obtained by the batch tests as well as the modelpredictions.

Metbaoogeoic Culturegrown 00 Metbanol: Therate curve for this cultureobtained followed the samepattern as for the sulfate­reducing culture and was notconstant at higher chloroformconcentrations. Themethanoldegradation in thisculture wasnot inhibited for chloroformconcentrations up to 770J.lg/l(Gupta et al; 1994) andhence,as for the sulfate-reducingculture, only the first term(modified haldane model) wasused for modeling chloroformcometabolism. Table I liststhe optimum parameterscalculated andFigure 5 showsa comparison of theexperimental rates and themodel predictions.

~ Meuured

- Model Prediction

Chloroform eoncentratlon, uaJI

Ffaure 5. E:lperlmentai and model predlcltonl of rate of chloroform eometabolbmfor the methanoaenlc culture (methanol).

Chlorofonn cometabolism 409

Comparison: Table 1 summarizes the optimum parameters obtained for the different cultures. As seen inFigures 3, 4 & 5, the model predictions faired veryweltwiththe experimental results. The maximum rate ofchloroform cometabotism, K, was more than an order of magnitude higherfor the sulfate-reducing culturecompared to anyof themethanogenic cultures, Thisindicates thatthe sulfate-reducing culturehas the maximumpotential forchloroform cometabolism. The exactrate of cometabotism wiltalsodepend on other parameters(n, K. & K). Also, the methanogenic culture grown on methanol had a higherK value compared to themethanogenic culture grown on acetate, indicating thatmethanol isa bettermethanogenic substratethan acetate,forchloroform cometabolism. The monodhalf-velocity constant, K.. in the Monodequation (Eqn (1», is thesubstrate concentration at which the rate is halfof the maximum rate (K). This is not true in the modifiedHaldane equation, andthe maximum rate of cometabolism occursat the substrateconcentration (S), closetoK. andK,and is givenby,

(9}

This is the critical substrate concentration at which the rate of cometabotism is highest and above thisconcentration, the rate starts to decrease. The critical concentration was the highest for the sulfate-reducingculture (2190 ~gII) which indicates theability of theculture to sustain higherchloroform concentrations. It wasmuch lowerforthe methanogenic cultures, Furthermore, themethanogenic culturegrown on acetate(120 ~gII)

had a lower valuecompared to the other methanogenic culture(506 ~gII), indicating that the methanogenicculturegrown on acetate is the most sensitive cultureto chloroform. The order of inhibition, n, indicates theintensity of inhibition, as shownin figure 1. Again, n was highest (n - 8) for the methanogenic culturegrownon acetate indicating the highest intensity of chloroform inhibition in this culture. The sulfate-reducing culturealsohada high orderof inhibition (n - 5) and the methanogenic culturegrown on methanol exhibited the leastorder of inhibition (n = 2).

SUMMARY & CONCLUSIONS

Thekinetics ofchloroform cometabolism in methanogenic and sulfate-reducing environments usingacetateormethanol as the primary substrate was modeled. The rate of cometabolism was described by two terms:cometabolism due to primary substrateutilization and the cometabolism due to microbial celt decay. Thecometabotism due to celtdecayis small as compared to the cometabolism due to primary substrateutilizationandisnormally neglected. However, it becomes important when the utilization of primary substrateis inhibited.Themodified Haldane equation was used to model chloroform cometabotism dueto primary substrateutilizationandtheMonod equation was usedfor cometabolism due to celldecay. SigmaPlot, a commonly used scientificsoftware, which uses non-linear regression was used for determining the optimum parameters. The modelpredictions agreedveryweltwiththe experimental results. The model parameters confirmed that the sulfate­reducing culture has a veryhigh potential forchloroform cometabotism compared to the methanogenic cultures.It can sustainmuchhigherchloroform concentrations compared to the methanogenic cultures, withoutbeinginluoited. Themethanogenic culture grown on methanol exhibited the minimum intensity of inhibition while themethanogenic culture grown on acetate exhibited the highest inhibition intensity. Also, the methanogenicaJlturegrownon acetate was much more sensitive to chloroform compared to the methanogenic culturegrownon methanol.

ACKNOWLEDGMENTS

The authorswouldliketo thankMr. ArunT. Vemuri for hisassistance in modeling techniques.

410

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