Environ. Sci. Technol. 1998, 32, 2450-2460
Modeling the Production of and Competition for Hydrogen in a Dechlorinating Culture DONNA E. FENNELL AND JAMES M. GOSSETT* School of Civil and Environmental Engineering, 223 Hollister Hall, Cornell University, Ithaca, New York 14853
A comprehensive biokinetic model employing Michaelis-Menten-type kinetics, H2 thresholds, and thermodynamic limitations on donor fermentation was used to describe both fermentation of electron donors and competition for the evolved H2 between hydrogenotrophic tetrachloroethene dechlorinators and methanogens. Model simulations compared favorably to experimental data where delivery of H2 to a tetrachloroethene dechlorinator was accomplished using the donors butyric acid, ethanol, lactic acid, and propionic acid. Fermentations of the different donors were characterized by different dynamic patterns of H2 generation that were captured successfully by the model. Experimental data and model simulations show that the ability to use H2 at appreciable rates at low levels provides a competitive advantage to dechlorinators over methanogens. Slowly fermented substrates producing lower H2 levels;kinetically accessible to dechlorinators, but too low for significant use by methanogenic competitors;were more effective and persistent “selective” stimulators of dechlorination than rapidly fermented substrates producing higher H2 levels;accessible to both dechlorinators and methanogens. Model simulations suggest that adding excessive levels of rapidly fermented, high H2-level-generating donors in an attempt to overcome competition, instead results in a dominant methanogen population and an eventual failure of dechlorination. When stimulating dechlorination, the quality of the donor as well as the quantity added must be considered.
Introduction Many of the recently identified tetrachloroethene (PCE) dechlorinators have been reported to use molecular hydrogen (H2) as an electron donor (1-4), and of these, some only use H2 (3, 4). H2 is therefore an important electron donor to consider for in situ stimulation of reductive dechlorination of PCE. H2 is produced by fermentative organisms, and in natural environments, it is consumed by (among others) methanogens, sulfate-reducing bacteria, and ferric ironreducing bacteria. Investigators have used H2 level as one of a number of parameters to characterize zones in contaminated plumes as methanogenic, sulfate reducing, or iron reducing (5). Each hydrogenotroph has a different threshold or minimum level of H2 at which it can operate. Under H2limiting conditions, H2 is poised at a level that corresponds to the dominant microbial activity. At sites contaminated * Corresponding author. Phone: (607) 255-4170; fax: (607) 2559004; e-mail:
[email protected]. 2450
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 16, 1998
with chlorinated solvents, hydrogenotrophic dechlorinators must also compete for this limited donor. Two recent reports suggest that dechlorinators possess a higher affinity for H2 than do methanogens. Smatlak et al. (6) reported a 10-fold lower half-velocity coefficient (100 nM) for H2 use by dechlorinators than for hydrogenotrophic methanogens (960 nM) in a mixed culture. Ballapragada et al. (7) reported half-velocity coefficients of 9-21 nM for H2 use by dechlorination in a mixed culture. Thermodynamic considerations suggest that this high affinity for H2 may be generally true of hydrogenotrophic dechlorinators. Since PCE dechlorination is energy yielding down to extremely low H2 levels (8), it is likely that dechlorinators would have evolved with the ability to use H2 at low, but still energetically favorable, concentrations. Many different organic substrates become H2 sources when fermented under anaerobic conditions (Table 1). However, the levels of H2 resulting from their fermentation can differ by orders of magnitude, depending on the intrinsic thermodynamics of the particular fermentation reaction. We previously investigated four different organic H2 sourcess butyric acid, ethanol, lactic acid, and propionic acidswith widely different H2-production ceilings (i.e., maximum levels of H2 that could be thermodynamically achieved via fermentation). These studies demonstrated that substrates fermented only under low H2 partial pressures (e.g., butyric and propionic acids) are superior donors for stimulating dechlorination while minimizing competing methanogenesis (9). If anaerobic reductive dechlorination is to become a widely used bioremediation technology, it is important that we understand and be able to model and predict the fate of applied donor and the extent to which it is channeled to desirable dechlorination or is wasted on stimulating the growth of competing organisms. Many of the existing subsurface pollutant fate-andtransport models that are advocated for modeling dechlorinating systems were developed for predicting petroleum hydrocarbon transport and degradation. However, halogenated solvents serve as electron acceptors and are used only by a limited number of microorganisms with a restricted suite of electron donorssdonors often in limited supply. In contrast, petroleum hydrocarbons serve as donors and the organisms that oxidize them are nearly ubiquitous; consequently, modeling dechlorination using petroleum-hydrocarbon-derived models is generally inappropriate. Furthermore, many of the available fate-and-transport models use half-life coefficients computed from historical data and firstorder kinetics for predicting dechlorination. Such a simplistic approach is unwise because the presence, availability, and kinetics of the crucially important electron donors are not even considered, and it is unrealistic to assume that the same donor, redox, and microbial population conditions that were present in the past will continue into the indefinite future. Wiedemeier et al. (10) provided an overview of some of the many analytical and numerical fate and transport models currently available for evaluating contaminant transport and degradation. Most were developed for fuel hydrocarbons. All but a few use first-order decay as the kinetic model for contaminant degradation (some also have zero- or multipleorder options). RT3D (11), which includes a kinetic package for reductive dechlorination, BIOPLUME III (12), and UTCHEM (13) incorporate more elaborate biodegradation schemes including Monod kinetics. One model includes both chloroethene and electron-donor degradation kinetics, equations for the conversion of an applied donor to endproducts thought to be used by dechlorinators, and competitive S0013-936X(98)00136-9 CCC: $15.00
1998 American Chemical Society Published on Web 07/03/1998
TABLE 1. Fermentation Reactions for Hydrogen Donors Examined during This Study (25)a
a
Fermentation to Acetate and H2 butyrate-+ 2H2O f 2acetate- + H+ + 2H2 ethanol + H2O f acetate- + H+ + 2H2 lactate- + 2H2O f acetate- + HCO3- + H+ + 2H2 propionate- + 3H2O f acetate- + HCO3- + H+ + 3H2
∆G 35°C ° (kJ/mol) 123.16 84.85 71.01 166.9
Fermentation to Propionate and Acetate ethanol + 2/3HCO3- f 2/3propionate- + 1/3acetate- + 1/3H+ + H2O lactate- f 1/3acetate- + 2/3propionate- + 1/3HCO3- + 1/3H+
-26.41 -40.26
All species as aqueous.
inhibition between PCE and trichloroethene (TCE) (14). None of these models, however, incorporates biokinetic expressions necessary to model closely coupled syntrophic associations of hydrogen-producing and hydrogen-using microorganisms, with all their attendant, competitive considerations. These expressions must be included for an accurate description of donor degradation and its resulting stimulation of either reductive dechlorination or competing processes. In microbial applications apart from dechlorination, several models have been used to describe processes dependent upon syntrophic associations of hydrogenic fermenters and hydrogenotrophic methanogens and their finely regulated interactions involving interspecies H2 transfer (15-21). These models incorporate mathematical expressions for limitation on the rate of donor degradation when product formation begins to thermodynamically limit the fermentation. We now report the development of a comprehensive biokinetic model to provide a more highly developed description of the microbial interactions that occur in mixed microbial communities including dehalorespirers. The model developed during this study encompasses the kinetics of dechlorination, thermodynamically controlled donor fermentation, methanogenic use of H2 and acetate, and the growth of all involved microbial populations. Such a complex descriptionscurrently lacking in fate-and-transport models that are used for analyzing bioremediation schemes and data from naturally attenuated sitesswill enable more accurate modeling of reductive dechlorination stimulated by H2.
Experimental Methods The experimental data used here for comparison with the model and most of the experimental methods were reported previously (9). A methanol-enriched, PCE-dechlorinating culture that was operated at 35 °C was used as inoculum for the cultures that were developed in these studies. The data shown come from semicontinuously operated reactors (160 mL serum bottles with 100 mL culture volumes), which were enriched with PCE and an electron donorseither butyric acid, ethanol, lactic acid, or propionic acid. A vitamin solution and fermented yeast extract (FYE) were routinely added as nutritional supplements during long-term operation (9). The serum bottles were incubated at 35 °C with rotaryplatform agitation. Data were obtained from long-term operation, as well as from time-intensive studies in which reactants, intermediates, and products were monitored after batch additions of PCE and an electron donor. Particulate Organic Matter. Biomass content of cultures was estimated from particulate organic nitrogen (PON). A 100 mL volume of enrichment culture sample or a basalmedium blank was filtered through a SUPOR-200, 0.2 µm filter (Gelman Sciences). Total Kjeldahl nitrogen (TKN) analysis was performed on the prepared filters according to Standard Methods (method 421) (22). The biomass content of the culture (mg of VSS/L) was estimated using the difference between the TKN of the sample and the TKN of
the blank and assuming a microbial cell composition of C5H7O2N (23).
Model Development Model Implementation. The model was constructed and implemented in STELLA Research 4.02 (High Performance Systems), and it mimicked semicontinuous operation of 100 mL enrichment cultures in 160 mL serum bottles (9). Pulse inputs of donor, PCE, and FYE were simulated, as were wasting and purging events. Chloroethenes were modeled with an aqueous phaseson which the biokinetics were basedsand a gaseous phase. Mass transfer between the aqueous and gaseous phases was incorporated in the model for the chloroethenes and ethene (ETH) using constants determined elsewhere (6, 24). H2 and CH4 were assumed to be in phase equilibrium. [Modeling interphase transport of H2, in particular, required an extremely small time step (dt), ca. 0.0005 h, to avoid numerical instabilities; given the extremely small dt values necessary to capture dynamics of gas/liquid exchange, we felt justified in making an equilibrium assumption. We validated this assumption by performing a few simulations that included a mass-transfer module for H2.] A more complete model description appears elsewhere (25). Kinetic Model for Donor Fermentation. The model describing the degradation of the H2 donors used MichaelisMenten kinetics, but with the inclusion of the thermodynamic influence of product (e.g., acetate and H2) formation on the rate of fermentation.
dMtdonor -kdonorXdonor(S - S*) ) dt KS(donor) + S
(1)
where Mtdonor is the total amount of donor in the bottle (µmol); kdonor is the maximum specific rate of donor degradation (µmol/mg of VSS h); Xdonor is the donor-fermenting biomass in the bottle (mg of VSS); KS(donor) is the half-velocity coefficient for the donor (µmol/L); S is the donor concentration (µmol/ L); S* is the hypothetical donor concentration that, under the instantaneous culture conditions, would result in ∆Grxn ) ∆Gcritical, given the concentrations of all the other reactants and products at that instant; and t is the time (h). S* is the “equilibrium” concentration of S, and it is related to ∆Gcriticalssome marginally negative free energy that the organisms must have available to live and grow (26-31):
(
)
[products] S*[other reactants]
∆Gcritical ) ∆G°35°C + RT ln
(2)
S is the actual substrate concentration at a particular time and is related to ∆Grxnsthe free energy available from the fermentation at that point in time:
(
∆Grxn ) ∆G°35°C + RT ln
)
[products] S[other reactants]
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Subtracting and simplifying,
[
)]
(
∆Grxn - ∆Gcritical S - S* ) S 1 - exp RT
TABLE 2. Kinetic Parameters (35 °C) Utilized in the Model ) SΦ
(4)
where,
(
Φ ) 1 - exp
)
∆Grxn - ∆Gcritical RT
(5)
butyrate- f acetate- + H2 ethanol f acetate- + H2 lactate- f acetate- + H2 propionate- f acetate- + H2 ethanol f propionate- + acetatelactate- f propionate- + acetate-
Note that 1 g Φ g 0. Φ ) 1 when ∆Grxn
