Evaluation of biodegradation kinetic testing methods and longterm variability in biokinetics for BTEX metabolism

PII: S0043-1354(98)00257-7

Wat. Res. Vol. 33, No. 3, pp. 733±740, 1999 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98/$ - see front matter

EVALUATION OF BIODEGRADATION KINETIC TESTING METHODS AND LONGTERM VARIABILITY IN BIOKINETICS FOR BTEX METABOLISM M A. R. BIELEFELDT1* and H. D. STENSEL2*

University of Colorado, Department of Civil, Environment, and Arch Engineering, Campus Box 428, Boulder, CO 80309, U.S.A. and 2University of Washington, Department of Civil Engineering, Box 352700, Seattle, WA 98195, U.S.A.

1

(First received August 1997; accepted in revised form May 1998) AbstractÐTwo methods were investigated for measuring the substrate utilization Monod biodegradation kinetics of benzene, toluene, ethylbenzene, o-xylene, and p-xylene (BTEX) by mixed bacterial cultures grown under three di€erent conditions. The mixed cultures were grown in completely mixed reactors that were batch fed every two hours with either BTEX or benzene and operated at a 5-day solids retention time (SRT); a BTEX-fed culture was also grown at a 20-day SRT. Only maximum speci®c substrate degradation rates (k) could be determined in batch tests where BTEX concentrations were measured at discrete time points, since not enough data could be acquired at low substrate concentrations near the half-saturation concentration (Ks) of the compounds. Both k and Ks could be determined in batch tests using an indirect method where dissolved oxygen depletion was continuously measured. The measured biokinetics of each of the cultures varied over time (coecient of variation of the k and Ks values of 27±44% and 43±100%, respectively), despite constant growth conditions. This biokinetic variability should be incorporated into design uncertainty to develop safety factors for reliable bioreactor performance. Results also showed that the operating solids retention time of the reactor and the combination of growth substrates a€ected the biokinetic coecient values of the individual BTEX compounds. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐbiodegradation kinetics, Monod, benzene, toluene, ethylbenzene, o-xylene, BTEX, oxygen respirometry

INTRODUCTION

Accurate determination of biodegradation kinetics is important in order to design cost e€ective and reliable ex-situ biological reactors used to treat contaminated liquids or gases related to such problems as contaminated groundwater, contaminated soil, or industrial wastewaters. A group of common contaminants are the BTEX (benzene, toluene, ethylbenzene, and xylene) compounds. BTEX are volatile, monoaromatic compounds that are common constituents in petroleum products, and are known to be readily degraded by aerobic bacteria (Smith, 1990; Zylstra, 1994). Ecient designs of aerobic bioreactors to treat BTEX contamination requires information on biodegradation kinetics. Biodegradation kinetics are commonly obtained by observing substrate depletion with time in batch tests and ®tting the data with the Monod substrate utilization model (Williamson and McCarty, 1975; Simpkins and Alexander, 1984). Two primary *Author to whom all correspondence should be addressed. [Tel.: +1-303-4928433; Fax: +1-303-4927317; E-mail: [email protected]]. 733

characteristics of biokinetic determination methods are important: the mathematical ability to accurately determine the kinetics from the available data, and the e€ect of the test method on the physiological state of the bacteria and consequently their kinetic response. Using biokinetic Monod models, non-linear parameter ®tting methods have been shown to yield mathematically superior kinetic coecient estimates compared to linear transformation methods such as the Lineweaver±Burke method (Holmberg, 1982; Robinson, 1985; Prats and Rodriguez, 1992; Ritchie and Prvan, 1996). In addition, researchers have shown that batch tests need to be designed with appropriate biomass and substrate concentrations in order to yield mathematically independent parameter estimates. Simpkins and Alexander (1984) discussed the ranges of biomass concentration (X) and initial substrate concentration (So) over which various kinetic models should be used. At high initial biomass to substrate concentrations (X:So), biomass growth is minimal so the no-growth Monod model should be used which provides a maximum speci®c substrate utilization rate (k) rather than the maximum growth rate (mmax) in

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A. R. Bielefeldt and H. D. Stensel

the Monod equation. The recommended range to use the no-growth Monod model was for So of 1±20 mg/L, cell concentrations greater than 106 to 107 per mL (assuming 1 pg of substrate to form 1 cell), and half-saturation coecient (Ks) of <1 mg/L. Ellis et al. (1996a) reported that for So to Ks ratios greater than 1, the Monod maximum speci®c growth rate (mm) and Ks values ®t to experimental data were mathematically unique. In addition, less measurement noise, more frequent sampling (or a greater number of total sample points), and the presence of measured concentrations both above and below the Ks will also increase the reliability of kinetic parameters ®t to experimental data (Holmberg, 1982; Ritchie and Prvan, 1996). In addition to mathematical constraints on design of batch experiments, researchers have shown that the physiological state of the bacteria can be a€ected by the biokinetic measurement procedure, which in turn impacts the biokinetics of the cultures. Grady et al. (1996) states that ``intrinsic'' parameter measurements representing the true maximum kinetic capability of the organisms are obtained in tests conducted at high substrate to biomass concentration (So/X>20 g substrate COD/g cell COD). Conversely, at low substrate to biomass concentrations (So/X less than 0.025) the measured biokinetic parameters are believed to represent the culture at its low growth state common in most treatment or laboratory growth reactors, also called the ``extant'' kinetics. Since the measured kinetics will be used to design treatment reactors, the biokinetics should be measured with biomass at a similar growth condition as will occur in the treatment system, in order to accurately represent the activity during treatment. Ellis et al. (1996a) reported on a batch dissolved oxygen respirometric method which accurately measures extant kinetics. In their work, data on the oxygen depletion over time during substrate degradation was ®t to the Monod kinetic model based on a fourth-order Runge±Kutta ®t, providing estimates of Ks, mm, X, and cell yield (Y). This method has the advantage of a low So which does change the physiologic state of the bacteria, and the collection of numerous data points at these low substrate concentrations. An alternative batch method frequently used to determine Ks is to measure initial substrate depletion rates at a range of initial substrate concentrations, and then ®t k and Ks in a no-growth Monod model to a curve consisting of these initial rates (Bailey and Ollis, 1986). The disadvantage of this method is that higher So/X ratios are often tested, and the So/X range may span both intrinsic and extant kinetics to yield unreliable kinetic values. The range of substrate concentrations tested should be somewhat indicative of the levels which will be encountered in a treatment reactor. For completely stirred tank reactors (CSTRs) there is typically a

low substrate concentration in the reactor. In contrast, sequencing batch reactors have high substrate concentrations at the beginning of the reaction cycle. Another important issue related to biodegradation kinetics is the potential variability of these kinetics in engineered treatment systems. When Ellis et al. (1996b) measured the kinetics of phenol degradation by continuous cultures over time, signi®cant variability in both mm and Ks was found. Determining if such variation is common to other cultures and compounds is important, since variations in biokinetic coecients should result in variations in treatment reactor performance. To further evaluate methods for measuring biodegradation kinetics for other cultures and substrates, and to determine the extent of variability that could occur in a treatment reactor, a series of batch experiments were conducted with mixed cultures degrading benzene, toluene, ethylbenzene, and the xylenes (BTEX). The biodegradation kinetics for ®ve individual BTEX compounds by the cultures were measured by two di€erent methods on multiple testing dates (the e€ects of compound mixtures on biokinetics are reported in the following companion paper). METHODS

Mixed culture growth conditions and plating characterization Three di€erent mixed cultures were started from contaminated vadose zone soil samples from a manufactured gas plant site. The growth reactors were closed, modi®ed 4-L erlenmeyer ¯asks containing approximately 2 L liquid and 2 L headspace. The reactors contained a KOH trap to remove respired CO2, which created a pressure drop to draw oxygen from an attached manometer into the reactor (Strand et al., 1990). All reactors were maintained at 208C in a constant temperature chamber. Continuous mixing of the reactor contents using a magnetic stir bar created a vortex to ensure good gas±liquid mass transfer. Pure BTEX-compounds were spike-fed at 2 hour intervals to the reactors using a syringe pump, giving an average total speci®c BTEX loading of 0.33 g total BTEX/g VSS-day. Nutrient solution was added and cells wasted from the reactors 4 times daily, to maintain an equal hydraulic and solids retention time (SRT) in the reactor. Two cultures were fed a mixture of B, T, E, oX, and pX (25%, 25%, 25%, 12.5%, and 12.5%, respectively) and grown at an SRT of 20 or 5-days; termed the BTEX20 and BTEX5 cultures, respectively. Another culture was fed only benzene and operated at a 5-day SRT, the B5 culture. The cultures were grown for a minimum of three SRTs prior to biokinetic tests. Wall growth in the reactors was minimized by manual removal using a stream of high pressure air. The nutrient solution used both for the growth reactor and dilution of biomass in batch biokinetic tests contained the following compounds at the given concentrations in mg/L: KH2PO4, 700; K2HPO4, 1000; NH4Cl, 400; CaCl2, 50; MgSO4, 30; NaHCO3, 200; NaCl, 10; CuCl2±H2O, 0.055; ZnCl2, 0.148; NiCl2-6 H2O, 0.022; FeSO4-7 H2O, 0.880; Al2(SO4)3-18 H2O, 0.135; MnCl2-4 H2O, 0.282; CoCl2-6 H2O, 0.056; Na2MoO4-2 H2O, 0.032; H3BO3, 0.049.

BTEX biokinetic test methods and variability Replica plating experiments were conducted to determine the relative portions of the BTEX5 and B5 cultures that were able to grow on each individual BTEX compound. Three replicates of 10ÿ5 and 10ÿ6 dilutions of the cultures were plated onto typticase soy broth (TSB) agar plates. After incubating these plates for 36 h in the dark at 208C, the number of colonies on each plate were counted. Plates containing 10±200 colonies were replica plated onto 6 minimal media plates (4.5 g agar to 300 mL nutrient solution) and 1 new TSB plate. To the minimal media plates, either single BTEX compounds or the mixed BTEX-feed was added into the lids of the inverted plates (about 5 drops each of the pure compound) and then sealed with para®lm. In addition, one plate was left with no substrate added to serve as a control. After 5 days of incubation, colonies on each plate were counted. The relative number of colonies on the plates spiked with individual compounds vs the BTEX mixture-spiked plate represented the fraction of BTEX-degraders able to degrade the individual compounds. In addition, the number of colonies on the BTEX-fed plate vs the TSB plate indicated the percentage of the bacteria in the total mixed culture that were BTEXdegraders. Direct method to determine biokinetic coecients in batch tests A direct method was used to measure the maximum speci®c degradation rate (k) of the individual BTEX compounds. First, biomass was removed from the growth reactors just prior to a substrate-feeding cycle and air sparged for approximately 30 min to remove any residual BTEX compounds. A volume of the culture and some amount of fresh nutrient solution to make up 80 mL of total liquid volume were added to 160-mL serum bottles sealed with Mininert sampling valves. After the spike addition of the test compound (4±14 mg/L initial liquid concentration), the batch bottles were incubated on a constant temperature rotary shaker (200 rpm) at 208C. Liquid samples were taken over time (approximately every 30 min), extracted into pentane, and analyzed on a Perkin±Elmer Autosystem gas chromatograph (GC) with a ¯ame ionization detector (Restek DB-5 megabore column, 10 mL/min He carrier gas ¯owrate; oven 508C for 2 minutes then ramp at 258C/min to 908C and hold for 2.4 minutes). Compound concentrations were determined by external standard curves, and corrected for internal standard (ethylene dibromide, EDB) response. The detection limit was approximately 0.2 mg/L for BTEX compounds. The So:biomass ratios used should cause little biomass growth during the test, and were in the acceptable range for ®tting to the no growth Monod model as proposed by Simpkins and Alexander (1984). Biodegradation rates described by the no growth Monod model were determined from the liquid concentration depletion rates by correcting for the BTEX compound partitioned in the headspace using the dimensionless Henry's coecients at 208C of 0.20, 0.24, 0.29, 0.26, and 0.18 L liquid/L gas for B, T, E, pX, and oX, respectively (Howe et al., 1987). R ˆ Robs …1 ‡ HVg =Vl † ˆ kSX=…Ks ‡ S †

…1†

where R = degradation rate, mg/L d; Robs=observed compound depletion rate in the liquid, mg/L d; H = Henry's coecient, mg/L gas per mg/L liquid; Vg=batch bottle headspace volume, mL; Vl=batch bottle liquid volume, mL; k = maximum speci®c degradation rate, g substrate/g VSS-d; S = substrate concentration, mg/L; X = biomass concentration, mg VSS/L; Ks=halfsaturation concentration, mg/L. Killed controls containing BTEX compounds, biomass, and 4 mL of 40% formaldehyde were run with each bottle set, and showed no decrease in BTEX concentrations over

735

time. Biomass was measured by volatile suspended solids (VSS) (APHA, 1992). The VSS of the pre-dilution culture seeded into the bottles and the biomass concentration in the bottle at the end of the batch test were both measured, and typically ranged from 80 to 200 mg/L; the change in VSS concentration during the tests was less than 5%, demonstrating very little biomass growth during the course of the test due to the high biomass concentrations initially present. The ®nal VSS concentration was used to calculate the speci®c degradation rate of the BTEX compounds (g compound/g VSS-d): k = R/VSS. The biokinetics of single compounds were measured on multiple testing dates over a period of 2, 4, and 9 months for the B5, BTEX20, and BTEX5 cultures, respectively. Indirect method to measure biokinetic coecients The Ks of the BTEX compounds could not be determined by the direct method, which indicated that the values were near the detection limit of the GC analysis. This necessitated the use of an alternative indirect oxygen uptake method based on that of Ellis et al. (1996a). In this method, instead of direct BTEX compound measurement, oxygen consumption was continuously measured during substrate degradation, and used to determine k and Ks values. A 250-mL ¯ask was modi®ed to allow a gastight ®t of a Yellow Springs Instruments dissolved oxygen (DO) probe (0.01 mg/L precision), and had a mininert valve port added to allow injection and/or withdrawal of samples. Culture was removed from the growth reactor and air sparged for approximately 30 min to saturate with oxygen and remove residual BTEX compounds. Additional nutrient solution was added when desired to dilute the biomass concentration prior to the test. The culture was added to the test ¯ask to exclude headspace, the DO probe was inserted, and the ¯ask sealed. Contents of the ¯ask was stirred via a magnetic stirrer on a Thermolyne stir plate at speed setting 3. The compound to be tested (typically 2±3 mg/L) was spiked into the system via the mininert sampling port. DO concentration changes were monitored over time and recorded at 10-second to 1minute intervals. Initially, a control ¯ask of culture without the spiked test compound was run in parallel with the test compound culture to correct for the endogenous oxygen uptake rate (RDO e). Subsequently, the test method was modi®ed so that RDO e was determined from the initial oxygen uptake rate during a 5±10 min period prior to spiking the test compound, and a ®nal ``endogenous'' period after the test compound was degraded and the DO depletion rate fell to the pre-spiked rate. The oxygen uptake pro®les were corrected for RDO e and converted into substrate concentrations vs time. Then the substrate depletion curves were ®t to the no growth Monod equation to yield a best ®t of R, Ks, and initial substrate concentration (So) values using the Marquardt±Levenberg algorithm in SigmaPlot (Jandel Scienti®c Software, 1995). The biomass concentration (VSS) measured at the end of the test was used to calculate k (k = R/VSS). Test VSS concentrations ranged from 100 to 640 mg/L, and therefore biomass growth during degradation of the added substrate caused a negligible change in the total biomass concentration. The temperature during the indirect method tests was typically 20± 258C, and measured k values were corrected for temperature e€ects to yield approximate 208C rates using y of 1.08 (Metcalf and Eddy, 1991). RESULTS AND DISCUSSION

Comparison of kinetic measurement methods Figure 1 shows data representative of substrate concentration changes with time for direct method

736

A. R. Bielefeldt and H. D. Stensel

Fig. 1. Linear biodegradation rates of individual BTEX compounds to low substrate concentrations by the BTEX5 culture in a direct method test (VSS = 117 mg/L, 208C); kill controls also shown.

batch tests with single BTEX compounds. Zeroorder substrate removal occurred down to very low substrate concentrations (less than 0.5 mg/L), indicating low Ks values near the method detection limit. There was insucient data at the low substrate concentrations to determine Ks, however linear regression ®ts to the data were used to measure the k of each compound. On average, there were 7 measured concentrations (all above the approximate Ks) from which k was evaluated. An example of the data obtained from the indirect oxygen uptake method is shown; Fig. 2A shows the measured dissolved oxygen (DO) consumption over time; Fig. 2B shows the oxygen uptake due to only benzene degradation after correcting the oxygen uptake data for RDO e; and Fig. 2C shows the calculated benzene concentration depletion. The benzene concentration remaining at any time t is proportional to the initial benzene concentration and the fraction of unconsumed oxygen. The statistical ®t to the Fig. 2C data provided the best-®t and standard deviation of Ks, R (k = R/ VSS), and So. On average, there were 86 total DO measurements made for each test, of which 47 were used in the non-linear ®tting (the rest of the measured values representing the endogenous period before and after substrate addition). On average, there were 7 measured values below the best ®t Ks concentration. For all of the ®t Ks values the average uncertainty (standard deviation divided by best ®t value) was 13%, and for all of the k values the average uncertainty was 4.6%, re¯ecting the good ®t achieved between the data and the no growth Monod model. In addition, Ellis et al. (1996a) reported that indirect method data could yield unique kinetic parameters from the ®tting procedure only for So/Ks>1; in our tests So/Ks was

typically greater than 2, which satis®es this condition. The reproducibility of the measured kinetic values was evaluated by testing replicates under the same conditions on the same day. For both methods, the standard deviation of the k from replicates (data not shown) was less than 0.08 g/g-d, with the coecient of variation (C.V.) ranging from 0 to 14%. The di€erence in k values determined by the indirect vs direct methods when measured on the same date ranged from 2 to 6%. The standard deviation in replicate Ks values for the indirect method ranged from 0.00 to 0.06 mg/L, which is a small concentration di€erence, but yields a high coecient of variance (0±61%) due to the low Ks values. The method error in the biokinetics determined by both methods appears reasonably small. Statistical t-tests were conducted to compare the average k values measured on all test dates by the direct method vs the indirect method (SigmaPlot, Jandel Scienti®c Software, 1995); results are summarized in Table 1. These tests showed that none of the di€erences between the methods were signi®cant at the 95% level, with the exception of toluene with the BTEX5 culture. This di€erence could be due to the fact that many of the biokinetic tests were not conducted on the same date by both methods. The typical So/X ratio (on a chemical oxygen demand, COD, basis) was 0.1 to 0.2 in the direct method vs 0.01 to 0.02 for the indirect method tests. The lack of a notable di€erence between the k values measured by the two methods could be due to the growth reactor batch feeding method in which initial substrate concentrations were approximately 5 to 7 mg/L BTEX, representing So/X ratios of 0.02± 0.07. However, the So/X ratios in the direct method do exceed the growth reactor ratio. From work with CSTRs, Grady et al. (1996) recommended So/ X values less than 0.025 to measure extant kinetics, but these reactors were continuously fed and always contained low substrate concentrations. Since no signi®cant di€erence was noted between the k measured by the two methods, both kinetic test methods are likely representative of the extant kinetics of the cultures from the batch-fed growth reactors used for this research. The kinetic testing results for BTEX compounds illustrate the inadequacies of the direct method and advantages of the indirect method for obtaining biokinetic coecients for compounds with low Ks values. A major advantage for the indirect method is the ability to obtain a large number of data points at low substrate concentrations during the kinetic testing. Besides limitations on the number of data points that can be collected, analytical detection limits may also restrict the ability to determine Ks by the direct method. A similar caution against the use of direct betch-fed experiments with high organism concentrations to accurately measure Ks was given by Williamson and McCarty (1975). The

BTEX biokinetic test methods and variability

737

Variability in kinetic coecients over time

Fig. 2. Representative data from an indirect method test to measure the k and Ks for benzene degradation by the BTEX5 culture (VSS = 183 mg/L, 228C).

primary disadvantage of the indirect method is the fact that biodegradation kinetics for multiple substrates cannot be measured, since it would be impossible to apportion the oxygen depletion between the compounds. Therefore, mixed substrate tests must be conducted using a direct method to measure individual compound concentrations over time.

For all of the cultures, kinetics measured on di€erent dates throughout the growth period showed signi®cant variability over time. Figure 3 shows all the kinetic values measured over the 9 month testing period for toluene by the BTEX5 culture from the direct and indirect methods, with the k values shown in the upper plot, and Ks values in the lower plot. The k values vary randomly over time, and this variance (36%) is greater than would be predicted based on the method uncertainty. Similar results were obtained for the other compounds (data not shown) (Bielefeldt, 1996), with the standard deviation of all of the measured k values across the 9 month period for each compound ranging from 0.23±0.46 g/g d (C.V. 27±44%). The k variations appeared random with no clear trends with time. These changes could be due to variations in the culture population and/or kinetic responses over time, in spite of the constant growth conditions. Similar variability in the measured Ks values over time was also evident (Fig. 3b). The variability in the Ks values for toluene over the 9 month period was 70%, and the range for the other compounds was 43±100% variation (as indicated in Table 1). Biodegradation kinetic values reported in the literature are commonly presented as singular constant values for respective bacteria enrichments. This work showed that kinetic coecients can vary over time for bacteria enrichments degrading BTEX. Grady (Daigger and Grady, 1982; Templeton and Grady, 1988; Ellis et al., 1996b; Grady et al., 1996) has published the most comprehensive work on the variability over time of measured biodegradation kinetics of continuous cultures. For example, one study was conducted on the variability of phenol biodegradation kinetics over a 1 year period by a pure culture grown at a 6-d SRT (Ellis et al., 1996b). The kinetics were measured in batch tests with low initial substrate to

Table 1. Comparison of average and standard deviation of biokinetics measured by the indirect and direct methods, and p value from statistical test for signi®cant di€erence in k values from the two methods Culture

BTEX20

BTEX5

B5

Cmpd

B T E oX pX B T E oX pX B T E oX

Indirect method

Direct method

# test dates

Ks, mg/L

k, g/g-d

# test dates

k, g/g-d

6 6 6 5 5 12 12 10 9 1 3 3 3 4

0.132 0.07 0.182 0.13 0.322 0.24 0.262 0.20 0.302 0.27 0.082 0.03 0.202 0.04 0.212 0.13 0.182 0.18 0.29 0.102 0.11 0.222 0.16 0.292 0.30 0.492 0.15

0.60 20.16 0.77 20.20 0.75 20.22 0.71 20.21 0.32 20.09 0.88 20.24 1.32 20.43 1.05 20.31 1.16 20.44 0.39 0.63 20.22 0.99 20.41 0.49 20.21 0.64 20.14

3 3 3 3 3 5 15 5 6 3 3 3 3 3

0.482 0.12 0.582 0.15 0.522 0.11 0.622 0.14 0.222 0.07 0.782 0.12 0.912 0.23 0.782 0.12 0.812 0.42 0.18 2 0.02 1.212 0.29 1.312 0.39 0.752 0.29 0.822 0.09

p value from di€erence test of k value from D vs ID method

0.30 0.20 0.13 0.56 0.15 0.33 0.01 0.10 0.15 NA 0.05 0.41 0.27 0.10

738

A. R. Bielefeldt and H. D. Stensel

can have a direct impact on uncertainty in the expected treatment eciency of a bioreactor. Reactor designs should incorporate this uncertainty in biodegradation rates into the design safety factors. Comparison of BTEX degradation by di€erent cultures

Fig. 3. Variability in toluene k and Ks values with time which were measured by both the direct and indirect method for the BTEX5 culture, with the overall average (solid line) and standard deviation (dashed lines) shown.

biomass concentration ratios (less than 0.025 on a COD basis). Over the one year period of culture growth, kinetic values were determined on 6 di€erent dates. The measured k values had a coecient of variation (C.V.) of 29%. This variability over time was signi®cantly greater than the average C.V. of 14% among replicate tests on the same date. Similarly, the measured Ks values had a C.V. between replicates of 33 to 100%, and C.V. over time of 80%. The degree of variability of phenol degradation kinetics by the pure culture is comparable to the variability in BTEX kinetics found in this work. Chang et al. (1993) reported the standard deviation of the measured biokinetics for benzene, toluene, and p-xylene with two pure cultures. Three to six experiments were run for each compound, but it is unclear if these biokinetics were measured for replicates on the same day or on di€erent dates. The reported variation for the k and Ks values ranged from 13 to 39% and 8 to 67%, respectively. Variation in the biokinetics of a culture over time appear likely and can result in variation in reactor performance over time. For example, if all other kinetic parameters are constant, the e‚uent concentration from a CSTR is directly proportional to the Ks of the bacteria: S1/S2 = Ks1/Ks2. Therefore, a 50% variation in the Ks value will result in a 50% change in the e‚uent concentration. Depending on the design, this has the potential for exceeding e‚uent concentration limits. Similarly, a change in k of approximately 35% will result in a 30±60% change in the substrate e‚uent concentration (for Y 0.7, endogenous cellular decay rate 0.1±0.2 dayÿ1, and SRT 5±20 d). Clearly, the biokinetic uncertainty

The average and standard deviation of the measured biokinetics (k and Ks) for each compound for each of the cultures are shown in Fig. 4 and 5, respectively. Both the indirect and direct method results are included in the average rates shown in Fig. 4. To determine the e€ect of operating SRT on the resulting biokinetics, the average kinetics of the BTEX20 culture vs the BTEX5 culture were evaluated using t-tests in SigmaPlot (Jandel Scienti®c Software, 1995). The k values for B, T, and E were signi®cantly higher for the BTEX5 vs BTEX20 culture at a 95% con®dence level, and signi®cantly higher for oX at a 93% con®dence level. Figure 5 shows that the cultures had similar Ks values for each individual BTEX compound, with the exception of a signi®cantly higher Ks for B with the BTEX20 culture compared to the BTEX5 culture at a 95% con®dence level. Templeton and Grady (1988) reported that for a pure culture grown on phenol, the shorter SRTs resulted in higher k values and lower Ks values. This e€ect was also observed for the BTEX k values, but only for the benzene Ks values. The similarity in the Ks values of the 5-d and 20-d SRT cultures observed in this work may have been due to the substrate feeding method. Since BTEX compounds were fed to the cultures every two hours, constant liquid concentrations of substrate were not maintained in the reactors as is typical for continually-fed CSTRs. In this case, the BTEX concentration immediately after dosing the BTEX20 and BTEX5 reactors were similar, rather than having higher substrate concentrations in the shorter SRT reactor. Therefore, the levels of induced enzymes in the bacteria in both reactors may have been similar, accounting for the similarity of the observed halfsaturation coecients. There were some e€ects of feeding the culture benzene only (B5) vs the BTEX mixture (BTEX5) on the resulting biokinetics. Despite growth on only benzene, the B5 culture did not have a signi®cantly higher k value for B, or a lower value for T. However, the average k of the BTEX5 culture for E and oX were signi®cantly higher than the B5 culture at a 95% and 88% con®dence level, respectively. Figure 5 shows that the B5 and BTEX5 cultures had similar Ks concentrations for each individual BTEX compound, with the exception of a signi®cantly higher Ks of oX at a 95% con®dence level for the B5 vs BTEX5 culture. The kinetic di€erences for oX seem to agree with the replica

BTEX biokinetic test methods and variability

Fig. 4. Comparison of the average k measured by the direct and indirect methods for each BTEX compound by the three cultures (one standard deviation indicated by the I on the bars).

plating results, which showed that the relative percentage of o-xylene degraders was higher in the BTEX5 culture than the B5 culture. In the B5 culture, the fraction of colonies that grew on the individual compounds vs the BTEX mixture were similar for B, T, and E, and about half for oX. By comparison, for the BTEX5 culture a similar fraction (83±100%) of the BTEX-degrading bacteria could use B, T, E, or oX individually as growth substrates. Therefore, there is some evidence that feeding multiple substrates may have resulted in greater population diversity within the resulting mixed culture than feeding benzene alone. The cultures grown on a mixture of BTEX compounds have a greater likelihood to be more diverse, containing bacteria which utilize di€erent enzyme pathways to degrade the various compounds. However, it is evident that the culture grown on only benzene still possessed a fairly broad capability for BTEX degradation, which is likely attributable to the fact that most of the known enzyme pathways are able to degrade multiple BTEX compounds (Smith, 1990; Zylstra, 1994). SUMMARY

This paper compared two methods which can be used to measure biodegradation kinetics that would be used to design ex-situ biotreatment reactors. For situations when the Ks value is low, the indirect method is best since it is easier to collect numerous data points at low substrate concentrations so that both k and Ks can be accurately determined. However, for higher Ks values (above approximately 0.5 mg/L for BTEX compounds), the advantage for the indirect method will be lost and the direct method should be preferred. In addition, the indirect method cannot be used to determine biodegradation rates of compound mixtures, since the oxygen depletion could not be attributed to the various substrates present. Biokinetic testing during long-term growth of mixed cultures under constant conditions showed that biokinetics can vary signi®cantly over time (by 27±44% for k and 43±100%

739

Fig. 5. Comparison of the average BTEX Ks values measured by the indirect method for the three cultures (one standard deviation indicated by the I on the bars).

for Ks). This level of variability can have signi®cant e€ects on reactor performance predictions, and should be incorporated into design safety factors to reliably meet maximum e‚uent concentration limits. Batch kinetic testing methods should consider the initial substrate concentrations used during testing and they should be representative of conditions in the treatment reactor. In this work, cultures were grown at So/X ratios (on a COD basis) of 0.02± 0.07, and the biokinetics were measured in batch tests using So/X concentrations of 0.01±0.02 in the indirect method and 0.1±0.2 in the direct method. Since the k values from both kinetic measurement methods were similar, it appears that test So/X ratios up to 3 times the growth conditions did not signi®cantly change the biokinetics and can be used to represent extant kinetics of the culture in the growth reactor. Di€erent growth conditions did result in mixed cultures with di€erent BTEX-degradation kinetics, despite the same initial seed source for the bacteria. Growth at a 5-d SRT resulted in faster maximum speci®c degradation rates than growth at a 20-d SRT. Feeding a BTEoXpX mixture to the cultures provided a culture able to transform all of these compounds at similar rates, with the exception of pxylene which was degraded slower. Despite feeding only benzene to a 5-d SRT reactor, the culture maintained the ability to degrade toluene, ethylbenzene, and o-xylene (p-xylene was not tested); however, signi®cantly slower degradation of ethylbenzene and o-xylene was noted in comparison to the BTEX-fed culture. AcknowledgementsÐFunding for this work was provided by the Baltimore Gas and Electric Company and the Mercury Seven Foundation. Thanks also for assistance in the laboratory work by Karl Huntzicker, Erica Yang, Calvin Bell, and Trina Sauer. REFERENCES

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