Grantee Research Project Results
2001 Progress Report: Understanding Seasonal Variation of Bioavailability of Residual NAPL in the Vadose Zone
EPA Grant Number: R827133Title: Understanding Seasonal Variation of Bioavailability of Residual NAPL in the Vadose Zone
Investigators: Holden, Patricia , Keller, Arturo A.
Institution: University of California - Santa Barbara
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1998 through September 30, 2001
Project Period Covered by this Report: October 1, 2000 through September 30, 2001
Project Amount: $425,000
RFA: EPA/DOE/NSF/ONR Joint Program on Bioremediation (1998) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management
Objective:
The current regulatory trend towards accepting intrinsic bioremediation as a long-term management scheme for residual organic pollutants in the subsurface for fuel spills is based on statistical evaluations of plume behavior for a limited array of pollutants. The effectiveness of intrinsic bioremediation towards protecting water supplies, and human health is not known for many pollutants. In addition, the patterns of biodegradation that occur over seasonal time scales, and the seasonality of intrinsic bioremediation and its intermediate effectiveness are unknown. Residual nonaqueous phase liquids (NAPLs) in the vadose zone, if left in place, have the potential to mobilize with seasonal moisture fluctuations. These seasonal variations in mass transfer along with the intrinsic microbial biodegradative response to moisture variations will determine bioavailability, which is a measure of the potential effectiveness of intrinsic bioremediation. This research focuses on determining the effect of seasonal fluctuations in moisture content and soil temperature on abiotic and biotic fate and transport of hydrocarbons in the subsurface.
The main objectives of this research project are to:
· Incorporate our mathematical model into a macroscopic numerical model that simulates multiphase flow and mass transfer, such as UTCHEM, to study field sites where mass transfer limitations are important and evaluate the accuracy of our model in predicting the rate of mass transfer;
· Develop and test a genetic reporter system that can be used to understand the expression of genes encoding surface active compound production in porous media; and
· Determine the effect of carbon to nitrogen (C/N) and carbon substrate quality on extracellular polymeric substances (EPS) production in unsaturated systems.
Progress Summary:
Simulation of Seasonal Variations in Natural Attenuation of NAPL in Unsaturated and Saturated Zone. There are several numerical models to analyze and predict the fate and transport of NAPLs in the subsurface environment. These models also can be used to assess the effectiveness of remedial actions at NAPL contaminated sites. For this work, we used UTCHEM, a comprehensive multiphase flow simulator, which can be used to simulate spills of either LNAPLs or DNAPLs (Pope, et al., 1999). In addition, an arbitrary number of injection and pumping wells can be specified so that physicochemical and bioremediation approaches can be modeled. We conducted simulations of spills of pure toluene under four different climate conditions using a modified version of UTCHEM to investigate the effects of seasonal changes on the rate of attenuation of toluene in unsaturated and saturated zone. We also varied the attached biomass, which is denoted as number of attached bacterial cells of biological species per gram of dry soil (cells/gram of solid), to evaluate the resulting biodegradation under different climatic conditions.
The full development of the UTCHEM model, a multiphase, multicomponent, three-dimensional numerical model incorporated with biodegradation capabilities, is described in detail by Delshad, et al. (1996). We added default boundary configurations suitable for studies of fluid flow and transport in the unsaturated and saturated zone. An analytic method for calculating the soil thermal profile also is added. In addition, we incorporated temperature-dependent physicochemical properties, as well as three-phase relative permeabilities based on our previous work. The boundary conditions were designed to allow the movement of gaseous and aqueous phases across the top. Pressure was specified for the lower portion of the right-hand boundary to fix the water table level and allow discharge. All other boundaries were impermeable to fluid flow. A constant head was maintained on the left-hand side of the saturated zone. A 50 m ´ 20 m domain is discretized with 43 grid blocks in the horizontal direction (x) and 45 grid blocks in the vertical direction (z). The soil surface is at z = 0 m. The initial water table is set in -10 m, so the modeled area includes both unsaturated zone and saturated zones. Groundwater flows from left to right. The leaking tank is located 19 m away from the left boundary. The tank is a cylinder with diameter D = 0.3 m, height H = 2.8 m, and the top surface is 1 m below the ground surface. The leak rate is 0.1 m3/day, lasting 10 days, resulting in a total spill volume of 0.1 m3. The total simulation time is 500 days. The time step is adjusted automatically according to changes in the concentration gradient, within the limits of maximum and minimum time step set in the input file. Toluene is the only NAPL in these simulations.
Four different climatic conditions were considered, as shown in Table 1. Case 1 is based on soil surface temperatures and rainfall rates from Madison, WI. Soil temperature at land surface varies over 28°C from summer to winter with an average temperature of 8°C. Average precipitation rate is 1.5 m/yr in Case 1. Cases 2 to 4 are based on soil temperatures averaging 21°C, considering a site in Sacramento, CA, but with different rainfall rates. Case 2 considers a constant rainfall rate of 2.74 x 10-3 m/day (1 m/yr). Case 3 considers no rainfall. Case 4 uses the actual rainfall records from a local weather station in Sacramento (National Climatic Data Center, 2000) with an average of 0.5 m/yr. The precipitation for Case 4 falls mostly between October and May, which creates strong seasonal differences in soil moisture. We present these simulations based on the four weather conditions to illustrate: (1) contrasts between natural attenuation in different temperature and moisture conditions; and (2) biodegradation under different bioavailability corresponding to different weather condition and biomass.
Weather Case | 1 | 2 | 3 | 4 |
Average Temperature (°C) | 8 | 21.3 | 21.3 | 21.3 |
Precipitation Rate (m/yr) | 1.5 (Constant) | 1.0 (Constant) | 0.0 (Constant) | 0.5 (Average) |
Our results for a 500-day simulation are provided in Figure 1. The results
of our simulations showed that toluene migrates farther from the source with
increasing rainfall rate because toluene is dissolved easily into fresh water.
In contrast, gas phase mass transfer of toluene is lower with increasing rainfall
rate because the addition of water filled pore space limits NAPL-gas mass transfer
area. The implications are that under rainfall conditions, NAPL dissolves and
moves more rapidly towards the water table.
The evolution of toluene in three phases (aqueous, gas, and NAPL) plus the total mass of toluene are shown in Figure 2. The mass of remaining toluene decreases over time (see Figure 2), because there are net losses to the atmosphere, losses to the groundwater, and loss due to biodegradation. The influence of periodic rainfall is most evident in Case 4 (see Figure 2) where the initial rainfall of the season (October, in Sacramento) causes an increase in the aqueous phase toluene, a decrease in the gas phase toluene (see Figure 2), and an increase in the NAPL phase toluene (see Figure 2) because the gas phase toluene that is displaced exceeds the aqueous phase solubility and condenses back into the NAPL phase.
The oscillating behavior in toluene mass in the gas phase also is due to lower soil temperature in the winter months (days 300 to 400), which shifts some of the toluene vapors back to the NAPL phase. This temperature-dependent effect is noticeable in all four cases, because we simulate a time-dependent soil temperature profile in all of them. Figure 3 illustrates the relative percentage of toluene in aqueous, gaseous, and NAPL phases for the four cases, after 500 days. It is clear that gas phase diffusion dominates the transport of toluene compared to transfer to the aqueous phase (see Figure 3). In all cases, the aqueous phase can only store a small fraction of the toluene mass, due to the relatively small aqueous solubility of toluene.
UTCHEM assumes biodegradation reactions occur only in the aqueous phase (Pope, 1996). Generally speaking, biodegradation is limited by electron acceptors (e.g., oxygen in this case), dissolved substrate (e.g., aqueous phase toluene), and biomass. Figure 4 explores the role of toluene bioavailability for biodegradation: the toluene biodegraded is directly proportional to the availability of dissolved toluene, which is dependent on the aqueous phase saturation. The y-axis presents the mass of toluene degraded over time. The rate of biodegradation with time presented in Figure 4 corresponds to the behavior of aqueous phase toluene in Figure 2. The much slower biodegradation rate of Case 3 illustrates the importance of making the substrate bioavailable to the microbes in the aqueous phase. With no rainfall, the soil moisture in Case 3 drops significantly, stressing the microbes and reducing the availability of toluene.
Figure 1. Simulation of Toluene Spill in Four Cases. It shows aqueous phase toluene concentration (left) and NAPL saturation (right) after 500 days. Case (1) cold weather (8°C) with constant rain rate (1.5 m/yr) for Madison, Wisconsin; Case (2) warm weather (21°C) with constant rain rate (1.0 m/yr); Case (3) warm weather (21°C) with no precipitation (0 m/yr); Case (4) warm weather (21°C) with actual rainfall records (average 0.5 m/yr) for Sacramento, CA.
Figure 2. Fate and Transport of Toluene Under Four Weather Conditions
Figure 3. Relative Percentage of Toluene Mass in Each Phase After 500 Days
Figure 4. Toluene Biodegraded in Four Cases Within 500 Days
In Figure 5, we show the effect of the attached biomass on the toluene biodegradation rate. Two different initial biomass concentrations are explored, namely 100 and 10,000 cells/g soil. With more initial biomass, more toluene is biodegraded over the period of study, as is shown in Cases 2, 3, and 4. The differences are not very significant, because the rate of biodegradation appears controlled mostly by the bioavailability of the substrate, rather than the biomass concentration. Case 1 presents a slightly different behavior: toluene biodegraded with 100 cells/g soil exceeds that with 10,000 cells/g soil after 250 days. This might be explained by a depletion of oxygen after 250 days.
Figure 5. Comparison of Biodegradation of Toluene With Two Different Initial Biomass Concentrations Under Four Climates
Based on our UTCHEM simulations, we conclude that rainfall is a dominant process affecting partitioning and attenuation of toluene NAPL in soil. Rainfall affects the rate of mass transfer to the aqueous phase, and the gaseous diffusion rate and the bioavailability, causing the biodegradation of toluene. Low rainfall infiltration rates reduce the bioavailability of residual NAPL; this results in a more extended period of natural attenuation. Dry conditions promote gas phase diffusion as a dominant mass transfer process, and, under these conditions, loss is mostly to the atmosphere. Soil temperature, another seasonally varying environmental factor, appears to control the mass of toluene that is in the gas phase because of the temperature dependency of NAPL partitioning. The result is that gaseous phase toluene was predicted to condense back to the residual NAPL pool during cooler temperature regimes. Through these simulations, we have demonstrated the efficacy of our modified UTCHEM model as a tool for understanding the effects of seasonally varying moisture and temperature on residual NAPL biodegradation. These simulations provide insight into the effects of rainfall amount, frequency, and soil temperature on residual NAPL bioavailability and biodegradation.
Biotic Mechanisms in Bioavailability. Three local (pore scale) mechanisms have been proposed to account for bioavailability and subsequent bacterial metabolism of sparingly soluble hydrocarbons: (1) dissolution and diffusion of dissolved hydrocarbons to cells with uptake via active or passive transmembrane transport; (2) invagination of hydrocarbon NAPL into cells with subsequent intracellular metabolism of the hydrocarbon inclusions; and (3) bacterial production of surface-active compounds such as surfactants (biosurfactants) and emulsifiers (bioemulsifiers) that increase the local pseudosolubility of hydrocarbons, and thus improve mass transfer. We studied Pseudomonas aeruginosa in sand and liquid culture as model systems for understanding biosurfactant and bioemulsifier production in soils. While surface-active compounds are known to accumulate in liquid culture when hexadecane is the carbon source and either nitrogen (N), phosphorus (P), or iron (Fe) are limiting, little is known about the accumulation of surface active compounds at the pore scale in unsaturated porous media. As surfactant accumulation affects interfacial tension, which inherently affects NAPL spreading, we must understand the real importance of biosurfactant accumulation during conditions of intrinsic bioremediation.
In our last project period, we reported that four strains (one doubly proficient in producing bioemulsifying protein and biosurfactant, one singly deficient in producing bioemulsifying protein, one single mutant in biosurfactant, and one double mutant) confirmed the accumulation of surface active compounds under N-limitation in liquid culture. We also reported that, in sand culture, the strains biodegraded hexadecane at equivalent rates regardless of the ability to produce surface active compounds in liquid culture. These results suggested that surface-active compounds were not produced in unsaturated porous media; in this case, sand. To investigate this, we produced a molecular reporter system based on the regulatory gene for the PA bioemulsifying protein and on the gene for green fluorescent protein (GFP). We also produced a gene fusion between gfp and a regulatory element for rhlR, the gene involved in the regulation of rhamnolipid synthesis. In that, rhlR is constituitively produced in P. aeruginosa in small amounts, this latter fusion served as a positive control for gfp expression in our culture systems. The GFP-based reporter systems allowed us to know when genes related to surface-active compound production were being expressed.
Using our reporter systems, we discovered that genes that encode surface active compounds are expressed in porous media as well as in "hanging drop" slides where an oil (hexadecane), water and glass (cover slip) interface could be visualized using confocal scanning laser microscopy. In our hanging drop slides, we observed that cells were fluorescent green, indicating expression of genes for surface-active compounds, at the oil water interface, but not in the bulk phase. This finding follows from previous work suggesting that surface-active compounds promote the adhesion of bacteria to hydrophobic interfaces where cells can directly access sparingly soluble hydrocarbons, and in this case, hexadecane.
We also found that in sand culture, the genes in our gene fusions were expressed, suggesting that the products of transcriptional activity (i.e., surface-active compounds), were produced. Interestingly, the transcriptional activity did not coincide with enhanced biodegradation. We offered a comparative analysis of liquid culture versus sand culture where we examine mainly the surface area of NAPL (hexadecane) as the controlling factor in surface-active compound effects. We conclude that the lower surface area in liquid culture, as opposed to the higher surface area of well distributed NAPL in sand, enhances the importance of surface-active compounds to bioavailability and biodegradation. Our report was the first to show directly that genes for surface-active compounds are expressed in unsaturated porous media.
In our work related to understanding the role of extracellular polymeric substances (EPS) in biodegradation, we continued studies to determine the nutritional conditions promoting EPS production. We found that there were limited differences in EPS production by P. aeruginosa growing as an unsaturated biofilm across a range of C/N conditions of 1 to 24, whether glucose or hexadecane was the C source. We did find that higher N (provided through a C/N of 1) promoted higher protein in EPS, but we did not find higher carbohydrate or alginate masses in EPS as a result of increasing C/N to a value of 24. Our work across these carbon sources and C/N condition ranges, in addition to biochemical analysis, included an atomic force microscopy of cells grown as unsaturated biofilms. The purpose of this imaging was to visualize the cell surface properties and to test, to the degree applicable, differences in surface adhesion forces as a result of EPS variations.
Although this imaging work provided little insight into the differences in EPS-mediated surface adhesive properties, it resulted in an analysis of cell size as a function of C source (glucose and hexadecane) and C/N. We report that these gram negative bacteria growing as unsaturated biofilms elongate when they are nutritionally starved, either by providing unfavorable C/N, low bioavailability carbon (hexadecane), or by increasing the diffusional path length for carbon. Our results suggest that bacteria in unsaturated biofilms respond to starvation very differently than cells growing in liquid where the paradigm is that starvation causes cells to become more spherical. Again, we observe that bacteria can adapt to low nutritional conditions-in our prior study by surface-active compound production, and here by morphological changes to cell shape and size.
References:
Pope G, et al. Three-dimensional NAPL fate and transport model. EPA Report 600/R-99/011, U.S. Environmental Protection Agency, Cincinnati, OH, 1999.
Delshad M, Pope GA. UTCHEM biodegradation model description and capabilities. Center for Petroleum and Geosystems Engineering, 1996, pp. 1-17.
National Climatic Data Center. Climatic data set for Sacramento, CA, 2000.
Delshad M, Pope GA, Sepehrnoori K. A compositional simulator for modeling surfactant enhanced aquifer remediation. Journal of Contaminant Hydrology 1996;23(4):303-328.
Future Activities:
Our future research will be directed at determining the effect of C/N and carbon substrate quality on EPS production in unsaturated systems over a higher range of C/N, and improving our ability to visualize bacteria in unsaturated porous media for the purposes of improving our microscale modeling capabilities for biodegradation simulations. During the next reporting period, which is a final phase intended to conclude our research project, we also are completing the publication of our work that was supported by this grant.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 20 publications | 6 publications in selected types | All 6 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Holden PA. Biofilms in unsaturated environments. In: Doyle R, ed. Microbial Growth in Biofilms: Special Environments and Physicochemical Aspects. Methods of Enzymology 2001;337:125-143. |
R827133 (2001) |
not available |
|
Holden PA, Pierce D. New environmental scanning electron microscopic (ESEM) observations of bacteria on simulated soil substrates. Microscopy and Microanalysis, 2001;7(Suppl 2):736-737. |
R827133 (2001) |
not available |
Supplemental Keywords:
sediments, chemical transport, chemicals, toxics, polycyclic aromatic hydrocarbon, PAH, bacteria, terrestrial, cleanup, environmental chemistry, biology, ecology, modeling, bioavailability, ecosystem protection, environmental exposure, waste, water, bioremediation, chemical mixtures, groundwater remediation, hydrology, nonaqueous phase liquid, NAPL, biodegradation, biological attenuation, contaminated sediment, exopolymeric substances, fate and transport, mass transfer, natural bioattenuation, seasonal variation, sediment, vadose zone., RFA, Scientific Discipline, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Hydrology, Contaminated Sediments, exploratory research environmental biology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Chemical Mixtures - Environmental Exposure & Risk, Ecosystem Protection, chemical mixtures, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Bioremediation, Groundwater remediation, Ecological Indicators, ecological effects, ecological exposure, fate and transport, ecology, NAPL, sediment, contaminated sediment, biodegradation, chemical transport, mass transfer, seasonal variation, bioremediation of soils, biological attenuation, vadose zone, exoplymeric substances, sediments, natural bioattenuation, groundwaterProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.