Environ. Sci. Technol. 2008, 42, 6131–6140
In Situ Bioremediation in Heterogeneous Porous Media: Dispersion-Limited Scenario X I N S O N G * ,† A N D E R I C A . S E A G R E N Department of Civil and Environmental Engineering, University of Maryland, College Park, College Park, Maryland 20742
Received June 4, 2007. Revised manuscript received May 5, 2008. Accepted May 7, 2008.
A quantitative framework based on a set of dimensionless numbers was developed to capture the effects of competing interfacial and biokinetic processes and define limits on the application of in situ bioremediation. An integrated numerical modeling and experimental approach was utilized to evaluate the quantitative framework. Experiments were conducted to examine the transport and biodegradation of naphthalene in a saturated, heterogeneous intermediate-scale flow cell with two layers of contrasting hydraulic conductivities. The experiments were carried out in two phases: Phase I, simulating intrinsic biodegradation; and Phase II, simulating an engineered in situ bioremediation. In Phase I, dispersion was identified as the overall rate-limiting process based on the proposed quantitative framework. Two engineered perturbations to the system were selected in Phase II to examine their abilities to enhance in situ biodegradation. In the first perturbation, nitrogen and phosphorus were spiked into the influent solution in excess of the required stoichiometric amounts. This perturbation did not have a significant impact because dispersion, not biokinetics, was the overall rate-limiting process. However, in the second perturbation, advection was increased, resulting in increased longitudinal and vertical transverse dispersion, thereby alleviating the rate-limiting process, and enhancing the overall biotransformation rate.
Introduction The success of in situ bioremediation is made technologically challenging by the inherently complex and heterogeneous nature of the subsurface environment (1). These physical and chemical heterogeneities of the subsurface occur at several scales. Importantly, they influence interfacial masstransfer processes and affect in situ bioremediation by controlling the availability of nutrients and substrates that drive the microbiological processes. If the kinetics of such interfacial physicochemical mass-transfer processes are slower than the potential biodegradation rate, these processes will negatively affect the overall bioremediation rate and the system is mass-transfer limited (2). Therefore, it is important to identify the rate-limiting process in the design and operation of in situ bioremediation systems so that the systems can be appropriately engineered, if necessary, to enhance the overall contaminant degradation rate. Not * Corresponding author phone: +1 415 374 2744; fax: +1 415374-2745; e-mail:
[email protected]. † Present address: ARCADIS U.S., Inc., 155 Montgomery Street, Suite 1500, San Francisco, CA 94104. 10.1021/es0713227 CCC: $40.75
Published on Web 07/09/2008
2008 American Chemical Society
understanding or accounting for the interactions between these scale-dependent physical/chemical heterogeneities and microbiological processes may reduce the effectiveness of field-scale in situ bioremediation (3, 4). In this study, we used a quantitative framework to identify the overall rate-limiting process and the appropriate engineering action that results in an enhanced in situ biodegradation rate. The quantitative framework was based on dimensionless numbers that capture the effects of competing interfacial and biokinetic parameters controlling in situ bioremediation. This research was accomplished using an integrated mathematical modeling and experimental analysis of an intermediate-scale flow cell (ISFC) system. Importantly, compared to other studies using dimensionless numbers to evaluate the impact of coupled processes on bioreactive substrates (e.g., refs 5–9,), an ISFC can incorporate relatively large-scale physical and chemical heterogeneities, while allowing isolation of complex processes under controlled laboratory conditions (10). The ISFC used in this study contained two porous media layers of contrasting hydraulic conductivities, K, and simulated a field scenario characterized by relatively fast biokinetics and low sorption. Due to the heterogeneous hydraulic conductivities of this system, macroscale longitudinal and transverse vertical dispersion significantly impacted in situ bioremediation by controlling the hydraulic mixing of microbial substrates at the interface of the dissolved contaminant plume and the “clean” groundwater, a key macro-scale interfacial process (11–17). We demonstrated that to be effective under these conditions, engineered bioremediation approaches must alleviate this rate-limiting process. The quantitative framework applied in this study can be used to improve our fundamental understanding of the processes controlling contaminant fate and transport in the subsurface and has important implications for the practical implementation of bioremediation. Most engineered bioremediation strategies focus on stimulating biodegradation processes in contaminant plumes. However, as demonstrated in this study, contaminant removal will not be enhanced by efforts to stimulate biodegradation kinetics when they are not limiting. This is significant because numerous studies show that a large fraction of environmental pollutants are unavailable for microbial degradation (18). Thus, at many sites, it would be beneficial to adopt the quantitative framework demonstrated in this study to identify the process limiting biodegradation and design an engineered bioremediation approach to effectively overcome this limitation.
Theory The governing advection and dispersion equation describing transport of an electron donor solute influenced by nonequilibrium sorption and double-Monod biodegradation kinetics, for a two-dimensional domain with horizontal steady-state flow, can be written as,
( )
∂ ∂ S˜ ∂S ∂S ∂ ∂S D + D - Kls S ) - (vxS) + ∂t ∂x ∂x x ∂x ∂z z ∂z Kd ˜ FbM S A qmax φ KS + S KA + A
(
)
(
)
( )( )( )
(1)
with the parameters as defined in the glossary in Supporting Information. The strictly macroscopic multiplicative Monod equation is used to represent dual substrate (electron donor and/or electron acceptor) limited biodegradation (19). Contaminant loss via volatilization is not included, as it was VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Definition of the Dimensionless Numbers
shown to be relatively minor during abiotic control experiments in the ISFC (20). Equation 1 can be converted into the following dimensionless form, ∂S/ 1 ∂2S/ 1 ∂2S/ ∂S/ )- / + + / /2 Pel ∂x Pet ∂z/2 ∂t ∂x
( )(
˜/ St2(S/ - S˜/) - Da2M
S/
A/
Ks/ + S/
KA/ + A/
)
(2)
by introducing x* ) x/L, z* ) z/L, t* ) t/(L/vx), S* ) S/S0, S˜* ˜*)M ˜ /M ˜ 0 and ) S˜/S˜0, A* ) A/A0, KS* ) KS/S0, KA ) KA/A0, M using the dimensionless numbers defined in Table 1, where the characteristic length L is set equal to the aquifer thickness (21). These dimensionless numbers reflect the influences of different mass transfer and biokinetic processes on solute fate in the subsurface, and succinctly capture the complexity of the system. In the full model solution, eq 1 is coupled with mass-balance equations for the electron acceptor and biomass growth and decay, which are not shown here. These equations were solved using Reactive Transport in 3-Dimensions (RT3D), a modular computer code for simulating reactive multispecies transport in 3-dimensional groundwater aquifers (19), as described in more detail in the Supporting Information (Modeling Details). In natural systems, several of the processes described by eq 1 occur simultaneously at different scales of heterogeneities. Nonetheless, for a given combination of environmental conditions, there is one process that will limit the overall in situ bioremediation rate. If that limitation is significant, then the bioremediation is usually not entirely 6132
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successful (3). Indeed, Sturman et al. (3) concluded that the feasibility assessment of an in situ bioremediation project is dominated by the need to correctly identify and estimate the appropriate rate-controlling phenomenon. Therefore, to have successful bioremediation in the field, a systematic method is needed to identify the rate-limiting process and select the appropriate remedial approach for enhancing the in situ biodegradation rate. An integrated quantitative framework that can be used to identify the overall rate-limiting process for a given environmental system is shown in Figure 1. This framework is based on the dimensionless governing equations and a systematic comparison of the dimensionless numbers developed and presented in Table 1 (2, 22). The proposed framework can be used to integrate and compare the key processes that occur in contaminated natural systems and predict the rate-limiting process for a given environmental system. The first two steps in Figure 1 are used to identify the slowest mass-transfer process among advection, dispersion, and sorption/desorption, and the third step compares the slowest mass transfer process with the biokinetics to determine the overall rate-limiting process. This approach is viable as long as the dimensionless numbers are significantly smaller or larger than unity (e.g., 5) (2). If the rate-limiting process can be properly identified, then an appropriate remedial approach can be chosen to alleviate that limitation.
Materials and Methods Model Contaminant and Aqueous Phase. The model compound was naphthalene (Fisher Scientific Co., 99.5%, Hampton, NH), a representative of polycyclic aromatic
FIGURE 1. Quantitative framework for predicting the ratelimiting phenomenon. Application of the flowchart to this work is highlighted by encircling the experimental conditions. This is an expansion of the framework presented in ref 22, which was adapted from ref 2. hydrocarbons, which have critical bioavailability issues at numerous contaminated sites (4). A dilute mineral salt nutrient solution (MSNS) was used in the ISFC experiments to simulate dilute groundwater chemistry (10). The MSNS was prepared in deionized water, using a 10 mM PIPES (Sigma-Aldrich, Inc., 99%, St. Louis, MO) buffer to maintain the pH within a range of 6.8-7.0. During the bioremediation simulation experiments, naphthalene was added to the MSNS at a nominal concentration of 10 mg/L. In addition, sodium bromide (Br-) (Sigma-Aldrich, Inc., St. Louis, MO) was added to the MSNS as a conservative tracer (120 mg/L). The complete solution was filter-sterilized (membrane filters, 0.25 µm, Schleicher and Schuell, Dassel, Germany) as it was pumped into an autoclaved feed bottle. Microorganism. Before initiating the ISFC experiments, the ISFC was inoculated with Pseudomonas fluorescens Uper1, which is able to utilize naphthalene as a sole source of carbon for growth (23). A stock culture of Uper-1 was maintained by periodic subculture on nutrient agar (Difco, Becton, Dickinson and Company) in the presence of naphthalene vapor and stored at 4 °C. The details regarding the Uper-1 inoculum preparation for the ISFC experiments are provided in the Supporting Information (ISFC Setup and Inoculation). Intermediate-Scale Flow Cell. The ISFC is illustrated schematically in Figure 2. A two-dimensional network of sampling ports was located on the front glass wall, with sampling ports in horizontal rows nominally 5 cm apart (labeled by letters) and vertical columns nominally 10 cm apart (labeled by numbers). The details of the ISFC construction are presented in the Supporting Information (ISFC Construction). To simulate macro-scale heterogeneities, the ISFC was set up with two layers of sand with differing hydraulic conductivities. Specifically, Mystic White II (d50 ) 1.2 mm) from U.S. Silica Company (Berkeley Springs, WV) was selected for the top, high-K layer, and Filtersil sand (d50 ) 0.3 mm) from Unimin Corporation (New Canaan, CT) was chosen for the bottom, low-K layer. Saturated hydraulic conductivities were determined in duplicate by the constant head method using a gradient ratio permeameter (see ref 20 for details). The average K values ((standard deviation) for the Mystic White II sand and Unimin Filtersil sand were 4.0 × 10-3 ((1.0 × 10-4) m/s and 6.5 × 10-4 ((8.0 × 10-5) m/s, respectively, giving a K ratio of approximately 7. Both sands were washed by following the procedures of Murphy et al. (10), after which
the sands were autoclaved. The sands were then wet packed into the ISFC as described by Pearce et al. (24). Transport Parameters Estimation. The porosity in each layer of porous media was estimated using the relationship φ ) 1 - Fb/Fp, where Fp is the particle density as determined by a water displacement test, and Fb is the bulk density of the media as determined by dividing the mass of each type of sand used in wet packing by the corresponding sand volume (25). Nonreactive tracer (sodium fluorescein) studies in the ISFC were performed in triplicate and used to estimate the transverse vertical (Rz) and the longitudinal (Rx) dispersivities within each layer by applying a modification (26) of a continuous point-source method (27). The best fit values of the average pore water velocity, vx, and the horizontal hydrodynamic dispersion coefficient, Dx, were obtained by a Fortran program that minimized the sum of the squares of the absolute residuals between the normalized experimental conservative tracer data and the normalized fluxaveraged concentration calculated using the continuous point source model described by Robbins (27). The unconstrained minimization was achieved by solving the nonlinear leastsquares problem using a modified Levenberg-Marquardt algorithm and a finite difference Jacobian. The vertical hydrodynamic dispersion coefficient, Dz, was calculated by assuming the vertical and horizontal transverse dispersion coefficients were equal and using eq (5) in Robbins (27). Finally, Rx and Rzwere estimated from Rx ) Dx/vx and Rz ) Dz/vx, respectively, because diffusion was negligible for nonreactive tracer study conditions. The interlayer transverse dispersivity, Rz′, specified at the interface between the two layers, was quantified by applying a step input of naphthalene in MSNS across both layers during a control experiment prior to inoculating the IFSC. The resulting naphthalene breakthrough data from a sampling port (D9) near the interface were analyzed using MODFLOW and RT3D, and Rxand Rz values for both layers. Rz′ was obtained by adjusting it via trial and error to fit the data of the initial mass pulse from interlayer mass transfer (11). Biokinetic Parameter Estimation. Batch respirometry experiments (Challenge AER-200, Challenge Environmental Systems, Inc.) were performed in duplicate to estimate the kinetic parameters for aerobic naphthalene biodegradation by Uper-1, using cells aseptically collected from the ISFC effluent during the Phase I experiment as the inoculum. A high ratio of initial naphthalene, S0, to biomass, M0, (i.e., S0/M0 > 20, when both concentrations are expressed as chemical oxygen demand (COD)) was used to obtain the “intrinsic” kinetics of the Uper-1 culture (28). During each batch growth assay, time-series profiles for oxygen uptake were obtained. The oxygen uptake data were then used to calculate the true yield coefficient,YX/S, and obtain initial estimates for the maximum specific growth rate (µmax) and half-saturation constant (KS), as described by Brown et al. (29). The final values for µmax,KS, and the maximum specific substrate utilization rate,qmax ) µmax / YX/S, were estimated by fitting the experimentally measured oxygen uptake data to the Monod kinetic model for batch culture using the Fortran program NVOLMA to perform the nonlinear parameter estimation (30). ISFC Operation. Detailed descriptions of the ISFC setup and inoculation are included in the Supporting Information (ISFC Setup and Inoculation). The initial biomass concentration in the ISFC was estimated based on the average heterotrophic plate count (HPC) concentration of 8 × 108 CFU/L measured at sampling ports (A2 and E2) after inoculation. Assuming an individual cell weight of 9.5 × 10-13 g (31, 32), the initial aqueous microbial concentration in the ISFC, M0, was estimated to be approximately 0.8 mg/L on a pore volume basis, which was equivalent to 0.27 mg dry wt/ VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Schematic of ISFC assembly with influent and effluent appurtenances. kg sand in the high-K layer and 0.15 mg dry wt/kg sand in ˜0 the low-K layer, on a solid phase basis as computed from M ) M0φ/Fb (19, 33). The other key initial condition was the dissolved oxygen (DO). Although the ISFC was covered with a glass lid, it was not airtight. As a result, the initial DO concentration in the ISFC, A0, was ∼3.5 mg/L prior to the bioremediation experiments. Similarly the influent solution was in contact with the atmosphere, resulting in an average ((standard deviation) oxygen concentration of 5.7 ( 0.6 mg/L at the influent sampling port. After inoculation, two sets of experiments were performed in the ISFC: Phase I and Phase II. All of these ISFC experiments were performed at room temperature (24 ( 0.6 °C). Phase I simulated intrinsic biodegradation under natural conditions. The MSNS with naphthalene (∼10 mg/L) and Br- (∼120 mg/ L) was pumped into the ISFC for 48 h at a flow rate of 3 mL/min to simulate a pulse input of contaminant, followed by an input of MSNS alone at the same rate until the end of the experiment. This experiment was performed in triplicate (Phase I(1), Phase I(2), and Phase I(3)). In Phase II, the approach of Phase I was followed, except two perturbations of the ISFC were performed to simulate engineered in situ bioremediation approaches. In Phase IIA, the common remedial practice of adding N and P in excess of the stoichiometric requirements to enhance the biodegradation rate was applied. N (ammonium) and P (phosphate) concentrations in the original MSNS were 60% of the stoichiometric amounts required for the biodegradation of naphthalene with a nominal concentration of 10 mg/L (34). In Phase IIA, the N and P concentrations were 5 times of that in Phase I, or 3 times of the stoichiometric requirements. The Phase IIA experiment was performed once. In Phase IIB, the engineered bioremediation perturbation was selected based on the identification of the rate-limiting process using the system parameters confirmed in Phase I and the quantitative framework in Figure 1. As discussed below, dispersion was found to be the overall rate-limiting process. Therefore, the flow rate in Phase IIB was increased to three times of that in Phase I, to increase the dispersion, especially the interlayer transverse dispersion between the high-K layer and low-K layer. It was expected that the increased transverse dispersion would increase the spreading of the naphthalene in the vertical direction and allow more mixing with the DO, thus increasing the biodegradation rate and overall rate of bioremediation (35). To compare the results between Phase I and Phase IIB, the pulse input time was reduced to 16 h to ensure that the naphthalene mass pumped into the ISFC was the same as in Phase I. This experiment was conducted in duplicate (Phase IIB(1) and Phase IIB(2)). 6134
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Tracer (Br-), naphthalene and DO concentrations were monitored during the Phase 1 and Phase II experiments within the high-K layer (ports A2 and B7), within the low-K layer (ports D1, E2, and D7), at the layer interface (ports C2 and C7), and in the influent and effluent. The aqueous microbial numbers were also quantified within both layers (ports A2 and E2) as well as at the layer interface (port C2) and in the effluent. Additional sampling for a naphthalene contour is described in the Supporting Information. Samples of ∼ 1 mL were withdrawn from sampling ports with a syringe pump (Harvard Apparatus, model no. 55-2226) using gastight glass syringes attached to the ports via Teflon tubing (1/16 inch o.d.). The sampling rates were set at 250 µL/min for the influent, effluent and high-K layer, and 25 µL/min for the low-K layer. These sampling rates were less than 5% of the bulk flow rate and no more than two samples were taken at a time. In addition, a temporal moment analysis was used to quantify the naphthalene loss due to biodegradation (e.g., ref 36,). The zeroth moment is defined as the area under the breakthrough curve, which was calculated by linear trapezoidal integration. The areas under the Br- (ABr) and naphthalene (Anaph) breakthrough curves obtained from different sampling ports represent the mass of each species detected after different horizontal flow distances. For comparison between different experimental conditions, the ratio of Anaph/ABr was used to normalize for the effect of dispersion on the naphthalene concentration. In addition, the average pore water velocity in each layer was determined by dividing the Br- travel distance at selected ports by the estimated average time. Analytical Methods. Aqueous naphthalene concentrations were measured by either a liquid-liquid extraction method coupled with gas chromatography (Hewlett-Packard model 6890) using a flame ionization detector (26), or a spectrofluorophotometric (Shimadzu, RF5301 PC) method adapted from Mulder et al. (37), with excitation and emission wavelengths of 273 and 336 nm, respectively. The latter was used during Phase I and Phase II experiments when expediency was required. Fluorescein tracer was analyzed using a spectrofluorophotometric (Shimadzu, RF5301 PC) method adapted from Seagren et al. (38), with excitation and emission wavelengths of 440 and 514 nm, respectively. Bromide was measured using an Orion 94-35 Bromide Electrode (Thermo Electron Corporation, Beverly, MA), connected to an Orion 520A ion meter. DO was determined utilizing an oxygen microelectrode (Microelectrodes, Inc., Bedford, NH), connected to an OM-4 oxygen meter. Cell numbers were determined by total heterotrophic spread-plate counts using R2A agar and incubation at 30 °C for 2 days (39). The initial and final biomass COD for the respirometry studies were
determined by colorimetric assays (Hach Chemical Co., Loveland, CO).
Results and Discussion Parameter Estimation. Particle density (FP) values for the high-K layer and low-K layer were estimated to be 2655 and 2788 kg/m3, whereas the corresponding bulk density (Fb) values were 1415 and 1813 kg/m3, respectively. The porosities estimated from these values were 0.47 and 0.35 for the high-K and low-K layers, respectively. Based on the triplicate tracer studies, the average values (( standard deviation) for Rx and Rz were 1.5 × 10-3 ((7.0 × 10-4) m and 7.6 × 10-5 ((9.7 × 10-6) m, respectively, in the high-K layer, and 6.0 × 10-4 ((2.0 × 10-4) m and 2.0 × 10-3 ((1.0 × 10-3) m, respectively, in the low-K layer. These values compare well with the corresponding values from flow cell experiments in similar porous media (11, 24). The effective molecular diffusion coefficient,D/, was estimated to be 6.5 × 10-7 m2/hr in the high-K layer and 5.8 × 10-7 m2/hr in the low-K layer. These values were obtained by using D/ ) τDm, where the aqueous diffusion coefficient, Dm, was calculated using the Wilke and Chang equation (40), and τ was estimated as τ ) φ-1/3 (41). An example experimental breakthrough curve from the control experiment for sampling port D9 (in the low-K layer near the interface) is presented in the Supporting Information (Figure S1). As expected (11), the interlayer mass transfer produced two stages of tracer breakthrough: an initial pulse resulting from transverse mass transfer from the high-K layer, followed by a mass pulse from the advective-dispersive movement in the low-K layer. Based on the trial and error calibration of these data, the interlayer dispersivity,Rz′, was estimated to be 0.05 m, which is an order of magnitude larger than the value estimated by Szecsody et al. (11). The biokinetic parameter estimates were qmax ) 0.18 1/hr, KS ) 8 mg/L, and YX/S ) 0.34 mg biomass/mg naphthalene. These values compare favorably with those reported by others in the literature (32, 42). The stoichiometric coefficient, YA/S ) 3 mg oxygen/mg naphthalene, was calculated based on stoichiometry for aerobic naphthalene mineralization, ignoring biomass synthesis. The value of the half-saturation constant for oxygen, KA ) 0.1 mg/L, was taken from the literature (43). ISFC Phase I: Intrinsic Biodegradation. The Phase I experiments provided a baseline for comparison to the Phase II results, and allowed for verification of the independent parameter estimates and their application in the quantitative framework. Representative Phase I breakthrough curves (Figure 3) show that, as expected, naphthalene and Brbreakthrough occurred at an earlier stage of the experiment in the high-K layer, compared to the low-K layer (e.g., compare ports A2 and E2). The plume shape resulting in this trend is demonstrated by the naphthalene contour plot in Figure S2 in the Supporting Information. Further, the naphthalene/ Br- pulse arrived at sampling locations near or at the interface earlier than at sampling locations “deeper” in the low-K layer (e.g., compare ports C2 and E2), due to the interlayer mass transfer, as observed by Szecsody et al. (11). At each sampling port, the normalized naphthalene concentration is lower than that of Br- due to the biodegradation of naphthalene. Correspondingly, as the plume passed a port, the oxygen concentration decreased rapidly, presumably due to the aerobic biodegradation of the naphthalene, reaching a minimum value at the same time that the highest concentration of naphthalene passed the sampling port. Subsequently, the oxygen concentration increased as the naphthalene available for biodegradation decreased. The breakthrough curves also show that, as the naphthalene plume moved through the ISFC, further loss due to biodegradation occurred. For example, the ratio of the normalized naphthalene concentration to the normalized
Br- concentration at C7 is lower compared to that at C2. Under these experimental conditions, the dimensionless concentration of oxygen (A/KA) at ports C2 and C7 is in stoichiometric excess relative to the dimensionless concentration of naphthalene (S/KS), indicating that the electron donor naphthalene was the most limiting substrate (44). Finally, the aqueous heterotrophic plate count data were relatively constant over time at ports A2, C2, E2 and in the effluent (Figure 3 (h)). However, the data showed that the microbial biomass increased slightly when the naphthalene plume passed the port and decreased slowly as the naphthalene traveled further downgradient, e.g., see the port A2 data. Similar trends were observed when the experiment was repeated (20). To quantify the magnitude of naphthalene loss due to biodegradation, the moment analysis was performed for the Phase I replicate data, as presented in Table 2. The analysis was focused on sampling ports C2 and C7, because of the importance of interlayer mass transfer and the resulting increased biodegradation. As part of this analysis, velocities in each layer were computed from the individual breakthrough curves, using port B7 for high-K layer and E2 for low-K layer. Several key observations can be made based on the moment analysis. First, significant removal of naphthalene via biodegradation (43-76%) occurred in Phase I by the time the plume reached sampling port C2, with an average of 60%. The largest removal of naphthalene by C2 occurred in Phase I (1), which was the first experiment performed after initially inoculating the ISFC. Given that the inoculum was pumped from the inlet, it is speculated that there was initially a high biomass concentration between the inlet and C2. Second, the extent of biodegradation increased with increasing distance, as demonstrated by the decrease in value of Anaph/ABr from C2 to C7. For the triplicate experiments, the average naphthalene mass loss between C2 and C7 due to biodegradation (Anaph/ABr(C2)-Anaph/ABr(C7)) was 8%, which provides the baseline for comparison with the impact of the engineered biodegradation perturbations in Phase II. Although this value is relatively small, it was reproducible. Finally, following the movement of the naphthalene plume further downgradient, almost all of the naphthalene was removed in the low-K layer via biodegradation by the time the plume exited the flow cell based on the fact that no naphthalene was detected at port D7 (data not shown), while the naphthalene removal in both layers due to biodegradation, coupled with the dilution by the low-K layer flow, resulted in a total removal across the tank of about 80% (1 - Anaph/ABr(effluent)). In general, the simulated naphthalene and Br- results match the experimental study values well in the high-K and low-K layers, which verifies the utilization of the independent transport and biodegradation parameter estimates. However, for ports C2 and C7 located at the interface between the high- and low-K layers, there are some discrepancies between the observed and simulated data, in particular for Br-. As shown at C2 (Figure 3 (e)) and C7 (Figure 3 (f)), although the maximum measured and predicted Br- breakthrough concentrations are similar, the experimental Br- data spread out wider than the RT3D model simulation Br- data. This is a result of interlayer mass transport from the plume passing by in the lower-K layer (e.g., compare the timing of the Brin Figure 3(g) for port E2 and the spread of the Brbreakthrough at port C2). This phenomenon is more pronounced in Phase IIB, and is discussed further below. Interestingly, the naphthalene breakthrough curve is sharper and less asymmetric than the Br- data. Correspondingly the model predictions are much closer to the experimental naphthalene data at ports C2 and C7 than for Br-. As discussed further below, and predicted by Oya and Valocchi (21), this VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Phase I (2) experimental data for the (a) influent naphthalene and Br- data, and (b) effluent naphthalene, Br- and DO, along with experimental breakthrough curves and RT3D model predictions for sampling ports (c) A2, (d), B7, (e) C2, (f) C7, and (g) E2. Also shown are the (h) HPC data. Each data point represents a single analysis. can be explained by the fact that the naphthalene biokinetics were much faster than the dispersion rate, even at the interface; therefore, the naphthalene concentration was dominated by the biodegradation rate, not the dispersion rate. Importantly, as observed by Szecsody et al. (11), the spreading of the plume and early arrival of naphthalene near the interface due to the interlayer mass transfer resulted in a greater period of mixing of the two substrates; thus biodegradation of naphthalene occurred for a longer time than within the layers, e.g., compare C2 with A2 and E2, and C7 with B7. To verify that dispersion was the overall rate-limiting process for the system, the quantitative framework in Figure 1 was applied using the independent parameter estimates summarized in Supporting Information Table S1, and the average pore water velocity values in Table 2. In addition, L 6136
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was set equal to the vertical thickness of the model aquifer and S0 was set equal to the average input naphthalene concentration (9.3 mg/L). The sorption mass transfer rate was assumed to be infinite compared to the dispersion rate because of the minimal sorption of naphthalene to the sands (20). The resulting dimensionless numbers are summarized and quantitatively evaluated in Table 3. As shown in Table 3, both the Pet/l (Peclet no.) and Sh2′ (modified Sherwood no. 2) were much greater than 1, indicating that dispersion was the slowest mass-transfer process. Dispersion was then compared with biodegradation rate via the Da6 (Damko¨hler no. 6). The Da6 values were also greater than 1 in both layers, confirming that dispersion was the overall rate-limiting process, according to Figure 1. This is probably a common situation for many readily biodegradable contaminants under natural conditions (45). Therefore, after confirming that
TABLE 2. Summary of Moment Analysis for the IFSC Experiments high-K layer vx, (m/d) (upper box) low-K layer, vx, (m/d) (lower box)
Anaph/ABr (C2)(1)
Anaph/ABr (C7)
Anaph/ABr(C2) Anaph/ABr(C7)
Anaph/ABr (Effluent)
Phase I (1)(2)
0.30 0.035
0.24
0.18
0.06
0.16
Phase I (2)
0.30 0.035
0.40
0.32
0.09
0.21
Phase I (3)(3)
0.28 0.030
0.57
0.48
0.09
0.20
Phase II A
0.26 0.029
0.33
0.23
0.10
0.20
Phase II B (1)
0.75 0.17
0.58
0.39
0.19
0.17
Phase II B (2)
0.75 0.17
0.78
0.54
0.24
0.26
1 Anaph/ABr represents the naphthalene concentration normalized to the Br concentration at the sampling port location given in parentheses. 2 Note that Br- data were not measured in the phase I (1) experiment. Therefore, the Br- data from the duplicate experiment phase I (2) were used for moment analysis. 3 Phase I (3) was conducted after the ISFC had been reinoculated.
TABLE 3. Application of Quantitative Framework to Identify the Rate-Limiting Process symbol Pet (transverse Peclet no.) Sh2′ (modified Sherwood no. 2) Da6 (Damko¨hler no. 6)
high-K layer
low-K layer
Pet ) 2325 .1: transverse dispersion limits
Pel ) 413.1: longitudinal dispersion limits
Sh2′)∞.1(a): dispersion limits Da6 ) 875 .1: transverse dispersion limits
Da6 ) 1367.1: longitudinal dispersion limits
a Note that the rate comparison using the modified sherwood no. 2, Sh2′, could not be quantitatively made because there was minimal sorption of naphthalene to the sands. Therefore, the sorption mass transfer rate was assumed to be infinite compared to the transverse dispersion rate.
mechanical dispersion was dominate compared to molecular diffusion (35), it was a priori predicted that the best approach to stimulate biodegradation under these conditions would be to increase the mechanical dispersion, thereby improving mechanical mixing of the limiting naphthalene plume with oxygenated water. This was accomplished by increasing advection, i.e., flushing. ISFC Phase IIA: Nutrient Enhanced Biodegradation. Although flushing was expected to be the optimal bioremediation approach, current practices focus on stimulating biodegradation kinetics, e.g., by adding excess N and P. Therefore, this treatment scenario was tested first. The breakthrough curve data are shown in Figure S3 of the Supporting Information. As in Phase I, the naphthalene concentration was reduced relative to Br-, and the DO concentration decreased when the naphthalene broke through. In addition, the naphthalene concentrations in Phase IIA were very similar to those in Phase I. Correspondingly, the amount of biodegradation during transport between C2 and C7 in Phase IIA (10%) was also very similar to that observed in Phase I (average 8%) (Table 2). In comparison, batch measurements of the intrinsic biokinetics of Uper-1 for naphthalene degradation in the presence of the added
nutrients resulted in an average qmax that was significantly greater than the one measured in this study. These data clearly demonstrate that, efforts to stimulate the biodegradation kinetics, although successful in the laboratory, will not be successful in situ if biodegradation is not the overall ratelimiting process, consistent with the quantitative framework analysis above. ISFC Phase IIB: Flushing. As shown in Figure 4, when flushing was implemented to enhance biodegradation in Phase IIB, the Br- and naphthalene brokethrough faster than in Phase I and IIA (as expected), but the effects of increased advection on interlayer dispersion are clearly evident. For example, two breakthroughs occurred at ports C2 and C7 at the interface of the two hydraulic conductivity layers: the initial mass pulse of naphthalene and Br- was due to the interlayer dispersion and advection from the high-K layer, and the second mass peak resulted from the interlayer dispersion and advection in the low-K layer (Figure 4 (c) and (d)). This effect can be detected to a lesser degree at sampling port D7 (Figure 4 (f)), in which case the initial peak is due to dispersion from the high-K layer, and the second peak is due to advection and dispersion in the low-K layer. This phenomenon was also observed by Szecsody et al. (11). The observation of twin Br- and naphthalene peaks in Phase IIB rather than the asymmetric and tailing peaks seen in Phase I may be due to the change in time scale (46). Similar to Phase I, the microbial biomass increased slightly when the naphthalene plume passed a given port and decreased slowly as the naphthalene traveled further downgradient (data not shown). Most importantly, the increase in advection resulted in greater biodegradation. The moment analysis in Table 2 shows that the naphthalene loss due to biodegradation between C2 and C7 was approximately 2.8 times greater in Phase IIB compared to Phase I. The significant increase in biodegradation is supported by the DO data, which show that the minimum DO concentration due to the naphthalene plume passing ports C2 and C7 was lower during Phase IIB compared to Phase I. It is speculated that the enhancement in biodegradation was a result of increased spreading of the limiting electron donor naphthalene plume into the oxygenated groundwater (35), due to greater mixing and dilution (47, 48). Dilution, i.e., the distribution of the plume over an increasing volume of the aqueous phase, depends on the VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Phase IIB (2) experimental breakthrough curves for sampling ports (a) A2, (b) B7, (c) C2, (d) C7, (e) E2, and (f) D7. In each panel, the naphthalene data, Br- tracer data, and DO data are presented, along with naphthalene data from the Phase I (2) for comparison. Each data point represents a single analysis. local dispersion, and the plume shape. Local dispersion should have been increased in Phase IIB compared to Phase I due to the increase in advection and, in turn, mechanical dispersion. Plume shape in the stratified porous media of the ISFC should have also been impacted by the increase in advection, resulting in greater mixing via stretching and deformation of the plume. This distortion of the plume increases the surface area of the plume boundary over which dispersive transport can occur, and increases local concentration gradients, thereby increasing the dilution rate. That an increase in dilution did occur is indicated by the maximum Br- concentration. For example, the maximum normalized Br- concentration at port C7 decreased from approximately 0.7 in Phase I (2) to about 0.4 in Phase IIB (2). Previous laboratory (11) and modeling (12, 13, 33, 49) studies with two layered systems and dual substrate limitation have also demonstrated increased microbial activity and, thus, enhanced biodegradation near the two-layer interface where hydraulic mixing between waters carrying different substrates occurs due to vertical transverse dispersion (see Figure S2 in the Supporting Information). The total removal of naphthalene and DO across the tank was similar in Phase I and Phase IIB, but the removal in Phase IIB occurred over a shorter time period. However, the increase in the naphthalene plume degradation rate resulting from flushing is associated with more rapid plume migration. In this study, as shown in Figure 4 (e), it took 25 h for naphthalene to break through to port E2 in Phase IIB, compared to 90 h in Phase I. Thus, if plume migration is the 6138
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major concern at a contaminated site, flushing may not be a viable alternative, even if dispersion is the overall ratelimiting process (35). In this study, dispersion was correctly identified as the overall rate-limiting process using the dimensionless number analysis and quantitative framework. Consequently, the addition of N and P above stoichiometric requirements in Phase IIA had little or no effect on the bioremediation removal rate. Instead, the increased dispersion resulting from increased advection in Phase IIB was an appropriate engineered bioremediation approach. This manipulation probably resulted in a greater mixing of the limiting electron donor (naphthalene) and the electron acceptor (oxygen) across the interface and, thus, an enhanced amount and rate of biodegradation. The quantitative framework used in this study could be applied to predict the rate-limiting process for in situ bioremediation in contaminated subsurface environments and aid in the selection of appropriate engineered approaches to enhance the biodegradation rate and improve the effectiveness of bioremediation at sites with a broad range of characteristics.
Acknowledgments This research was supported by the National Science Foundation (NSF) under CAREER Grant No. 0093857. We thank Dr. D.R. Lueking from Michigan Technological University for providing the Pseudomonas fluorescens Uper-1 culture. We also thank Eunyoung Hong for working on the
batch measurements of the intrinsic biokinetics of Uper-1 in the presence of excess of N and P.
Supporting Information Available Details regarding the mathematical modeling, ISFC construction, setup and inoculation, and naphthalene plume contour sampling locations and scheduling, an independent parameter estimate summary in Table S1, a breakthrough curve for naphthalene at sampling port D9 during the control experiment in Figure S1, a “snap shot” of naphthalene plume contour plot in Figure S2, Phase IIA experimental breakthrough curves in Figure S3 and Glossary. This material is available free of charge via the Internet at http://pubs.acs.org.
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