Resting-State Bioaugmentation - American Chemical Society

R. B. KNAPP,* AND R. T. TAYLOR. Lawrence Livermore National Laboratory, L-206, P.O. Box 808,. Livermore, California 94551. A field test has demonstrat...
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Environ. Sci. Technol. 1996, 30, 1982-1989

TCE Remediation Using In Situ, Resting-State Bioaugmentation A. G. DUBA, K. J. JACKSON, M. C. JOVANOVICH, R. B. KNAPP,* AND R. T. TAYLOR Lawrence Livermore National Laboratory, L-206, P.O. Box 808, Livermore, California 94551

A field test has demonstrated that an in situ biofilter using resting-state cells effectively remediated groundwater with about 425 ppb of trichloroethene (TCE) as the sole contaminant species. About 5.4 kg (dry weight equivalent) of a strain of methanotrophic bacteria (Methylosinus trichosporium OB3b) was suspended in 1800 L of groundwater (5.4 × 109 cells/ mL) and injected into an aquifer through a single well at a depth of 27 m, several meters below the water table. The injected groundwater was devoid of TCE and growth substrates but was amended with a phosphate solution (10 mM) to buffer the pH and phenol red (20 µm) to act as a tracer. Approximately 50% of the injected bacteria attached to the sediments, forming an in situ, fixed-bed bioreactor of unknown geometry. Contaminated groundwater was subsequently withdrawn through the biofilter region by extracting at 3.8 L/min for 30 h and then at 2.0 L/min for the remaining 39 days of the field experiment. TCE concentrations in the extracted groundwater decreased from 425 to less than 10 ppb during the first 50 h of withdrawal, which is equivalent to a 98% reduction. TCE concentration extracted through the biofilter gradually increased to background values at 40 days when the experiment was terminated.

Introduction The widespread use of chlorinated ethenes as solvents and their subsequent disposal has resulted in extensive contamination of groundwater. In particular, groundwater contaminated with trichloroethene (TCE) occurs at many government installations and industrial plants. At these sites, natural advective and dispersive transport processes have led to large, relatively dilute contaminant plumes, commonly with contaminant concentrations less than 1 ppm. In the U.S. Department of Energy complex alone, there are more than 20 sites with TCE-contaminated groundwater; estimated remediation costs are in the billions of dollars range and expected cleanup times are on the order of decades (1). High costs and extended cleanup times derive from the high mobility of the solvents, the large volume of affected groundwater, the large variations * Corresponding author telephone: (510)423-3328; fax: (510)4223118; e-mail address: [email protected]




in hydraulic conductivity within the contaminated aquifers, and the application of the 5 ppb drinking water standard (2) as the regulatory limit. Previously Tested Remediation Methods. Pump-andtreat is the industry and regulatory standard for remediating groundwater contaminated with volatile organic compounds (3). However, pump-and-treat is viewed as both ineffective and expensive for many applications because it is maintenance-intensive, operations are on the scale of decades, multiple plume volumes need to be extracted, remediation goals are elusive, and it merely transfers contamination from the aqueous phase to the atmosphere, to a solid medium for later disposal, or to a surface treatment facility (3, 4). In contrast, in situ bioremediation has been proposed as an attractive alternative, largely because it destroys contaminants without bringing groundwater to the surface. It also has the potential to reduce remediation costs (5). One difficulty of in situ bioremediation of chlorinated solvents such as TCE is the cometabolic nature of the degradation reaction; microorganisms derive neither carbon nor energy from the reaction, and so the in situ bacterial population must be externally supported. In situ bioremediation of TCE-contaminated groundwater has not yet received commercial acceptance. The field tests reported to date have focused on biostimulation. Jackson et al. (6) conducted an in-well experiment of an alternative to the typical biostimulation approach. Biostimulation is attractive because of its simplicity and promise of low cost. In biostimulation, a suite of electron acceptorsssuch as oxygensand nutrients is injected into the subsurface to increase the population of indigenous, contaminant-degrading microorganisms. The injected compounds are inexpensive, and the surface operations are straight-forward. The process has three main aspects: (1) selecting an injection package that is appropriate for the indigenous microbial community, the subsurface environment, and the contaminant(s) of interest; (2) increasing population densities over a large area so that the degradation rates are high; and (3) achieving contact between the stimulated community and the contaminated fluid. Extensive biostimulation field tests conducted at Moffet Field, CA successfully demonstrated the first two points (7-10). Achieving contact between contaminant and the stimulated population was not tested because the contaminants were injected along with the nutrient package, thereby forcing contact. With this forced contact, however, 90% TCE degradation of a 1 ppm contaminant stream was demonstrated at the end of a 60-day interval in which phenol-degrading microorganisms were stimulated (7). Much poorer results were obtained for TCE degradation when methanotrophs were stimulated (8-10). In general, injection of growth media displaces contaminated groundwater, thereby separating the soluble contaminant inventory from the stimulated bacterial population. Biostimulation is most applicable in aquifers where a significant fraction of the contaminant mass is sorbed to the solid media; this can cause efficient mixing between contaminant and injected fluid and can result in substantial biodegradation (11). However, biostimulation field studies conducted without a tracer in the injection

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 1996 American Chemical Society

package are ambiguous because of the inability to discriminate between biodegradation and dilution (12, 13). Additional challenges in field engineering of biostimulation include competitive inhibition (10), preferential growth near the injection well (14), and a limited ability to select which bacterial strains are stimulated. In Situ Microbial Filter Concept. We adopted an in situ microbial filter strategy (15) that avoids the undesirable separation of contaminants from bacteria and eliminates competitive inhibition of biodegradation. For this restingstate, bioaugmentation approach, naturally occurring bacteria are grown in surface bioreactors, separated from their growth medium, resuspended in an aqueous solution that is devoid of added growth nutrients, and then injected into the subsurface. During the injection phase, a portion of the bacteria attaches to the aquifer material and forms a fixed-bed biofilter. After the microbial injection is carried out, subsequent ambient or induced flow delivers contaminated groundwater to the biofilter region, resulting in contaminant biodegradation. The degree of biodegradation depends on the flux of contaminants, the attached population density, and the contaminant residence time in the biofilter, just as in any chemical reactor. The emplaced biofilter will eventually expire because the injected, nondividing bacteria have both a finite biotransformation capacity for degrading contaminants (15-17) and a finite timeslongevitysduring which they will remain catabolically active in the resting state (15). As used in this study, biotransformation capacity is the maximum contaminant mass that can be degraded by a given mass of cells in the absence of an extracellular source of carbon, energy, or electron acceptor/donor; this is limited because the degradation of TCE consumes intracellular electron donors (e.g., NADH) and inactivates the key enzyme system that catalyzes the oxidation reaction. Methylosinus trichosporium OB3b, a naturally occurring, nonpathogenic, obligate methanotroph, was used in this and our previous studies (6, 15-20). This methanotroph is a known oxidative, cometabolic degrader of TCE and other chlorinated solvents (21, 22). Additional attributes that make this bacteria attractive include a rapid initial TCE transformation rate, a high resting-cell biotransformation capacity for TCE (∼0.25 mg of TCE per mg of dry cell weight), adequate attachment/detachment properties, ease of large-scale batch cultivation in a bioreactor, and, if properly cultured, an ability to sustain soluble methane monooxygenase (sMMO) TCE-degrading activity in the resting state for extended periods of time (15, 17-20, 2325). Previous work demonstrated that these precise batch cultivation conditions can produce bacteria that maintain significant TCE-degrading activity in the resting state for 6-7 weeks for cells stored in suspension (15) and up to 15 weeks for attached cells (25). This is a significant advance since previous field biostimulation experiments (8-11) report loss of cell activity within several hours to a few days after the substrate supply is terminated. We wish to emphasize that because the attached bacterial population receives neither carbon nor energy from cometabolism and because methane is not present in the subsurface system, the performance of the in situ microbial filter would be expected to diminish with time as longevity or biotransformation capacity is exceeded. Thus, in order to continue in situ bioremediation by this process, regular replenishment of the bacterial population by reinjection is required. The frequency of this reinjection

ultimately controls the economic feasibility of the approach. We report below the results of a recent field study that tested our ability to establish a resting-state biofilter in the subsurface, to degrade substantial amounts of TCE, and to sustain biodegradation over significant periods of time.

Experimental Procedures Suitability of a given site for implementation of an in situ microbial filter approach depends on the hydrology, physical characteristics, and aqueous geochemistry of that site. Site suitability criteria for use of M. trichosporium OB3b include pore size in the aquifer sediments, pH, dissolved O2 content, and contaminant content of the groundwater. In the following section, these characteristics are discussed for the site of the first field test of the in situ microbial filter process. Field Site. The TCE plume at the Chico Municipal airport is about 500 m wide and 2000 m long (Figure 1). TCE is the sole contaminant, and it occurs with a maximum known concentration between 1.0 and 1.5 ppm at the northeast source region. For the most part, dissolved TCE is restricted to a single unit within the Tuscan formation that acts as a partially confined aquifer. The Tuscan formation is highly heterogeneous in grain-size distribution, porosity, and permeability (26). Drilling information and outcrops of this unit suggest a sequence of discontinuous layers, some of which are composed of cobbles and some finer-grained material. Average porosity and permeability were estimated from drawdown tests at well 13 (Figure 1) to be 40% and 3 µm2, respectively. Groundwater velocity in the area is estimated at 30 cm/day, northeast to southwest. Well 13 was selected for bacterial injection, and wells NW and SE were selected as monitoring points (Figures 1 and 2). The water table was measured at a depth of 25.88 m just prior to bacterial injection, but it rose approximately 70 cm during the 39-day experiment due to heavy and frequent rains at the site. Packers were emplaced in each of the three wells; a packer was installed approximately 1 m from the bottom of well 13, and wells NW and SE had dual packers isolating this same depth interval. Downhole pumps were located with intakes at 27.7 ( 0.1 m in each well (Figure 2). A multiple tracer testsbromide, phosphate, and phenol redswas conducted in well 13 2 weeks before bacterial injection to assess aquifer properties in the depth interval where the biofilter was to be emplaced. Approximately 2400 L of amended groundwater was injected into the aquifer at a rate of about 4.7 L/min. This was immediately followed by injecting 600 L of groundwater, with no amendments, at the same rate. Groundwater was then extracted at 3.8 L/min for 22 h. Tracer concentrations were measured in samples collected periodically from wells NW and SE throughout the injection/withdrawal process and at well 13 during the withdrawal process (Figure 3). Site Suitability. Aqueous geochemistry constraints for M. trichosporium OB3b were favorable (Table 1) to completely mineralize TCE at the observed contaminant concentration (425 ppb). The aquifer material also was determined to be capable of accommodating the transport of cells. This requires pore throats greater than about 20 µm, which are probably consistent with a 3-µm2 permeability medium, although its heterogeneous nature leaves this issue with some uncertainty. The rate of TCE degradation in site groundwater by washed M. trichosporium cells was assayed, and parameters




FIGURE 1. Schematic of the TCE plume, hydrostratigraphy, and test wells at the Chico Municipal Airport, Chico, CA. Well 13 was the injection/withdrawal well for our field experiments; NW and SE were observation wells. TCE contours are labeled in ppb (modified from Brown and Caldwell internal reports), and monitoring wells are designated by dots. Well 12 was used as a source of clean groundwater. The hatched lines at the bottom of the wells in the cross-sectional insert denote screened intervals. The upper-most layer is the Red Bluff formation, and the lower three layers are the Tuscan formation.

in the Michaelis-Menten relationship were extracted (15, 17). The apparent Km’s ranged from 70 to 104 µM, and the Vmax’s ranged from 82 to 98 nmol min-1 (mg of cells)-1, similar to values obtained previously (17). Using methods described previously, we also demonstrated the ability of washed cell suspensions, at average volumetric biocatalyst densities expected in well-site sediments, to effect the oxidative biotransformation of low levels of TCEs131 ng to 1.0 µg/mLs(15, 17). A biotransformation capacity of 0.19 mg/mg of dry cell wt was obtained for TCE in well 13 groundwater by a repetitive-transfer protocol (15, 17). The longevity of the resting-state activity for TCE is a critical parameter. Previously, we achieved a half-life of about 5 weeks for M. trichosporium OB3b suspended or attached in the presence of Higgins’ phosphate buffer solution; subsequent TCE degradation was assayed both with and without formate at 21 °C (15, 17, 19). For the Chico site, cells stored as suspensions in well 13 water up to 21 days still maintained ∼70% of their original activity with TCE when assayed either with or without formate. Laboratory measurements of cell attachment behavior to material of similar mineralogy to the injected interval in well 13 gave rates and maximal attached populations (about 9 × 108 ( 3 × 108 cells/g of sediment) similar to those found previously (17, 20, 24).




FIGURE 2. Illustration of the downhole packers in the three wells with a cross-section of a highly idealized biofilter with a radius of 1.1 m. Depth below surface is indicated in scale to the right.

Biomass Production. Kilogram amounts of M. trichosporium OB3b were batch-cultivated at 30 °C and pH 6.8-


Aqueous Geochemistry

FIGURE 3. Comparison of measured bromide concentrations at the NW and SE observation wells during the tracer test with those predicted from a computer simulation assuming a homogeneous aquifer.

7.2 in a 1500-L fermentor (IF-1500, New Brunswick Scientific, Inc., Edison, NJ). The inoculum for the IF-1500 was grown in an 80-L fermentor (MPP-80, New Brunswick), and its inoculum, in turn, was generated in a 5-L bioreactor (Bioflo III, New Brunswick). Except for a few minor changes, such as the timing of the gas flow-rate shifts and the agitation speeds, biomass production in the IF-1500 was a direct scaleup of our past optimized runs in a 5-L Bioflo III using a modified Higgins’ salts minimal medium lacking added copper (15, 18, 19, 23). IF-1500 cells were harvested at about 80 h by continuous centrifugation (CEPA Model 261 centrifuge). The resulting wet cell paste was then packed into large (4-L) wide-mouth polypropylene bottles, closed with a screw-cap, and then stored in a refrigerator at 2-4 °C. Care was taken to completely fill each bottle with cell paste to assure a minimal head space. The forgoing cultivation, cell-harvest procedure yielded an average of ∼2.7 kg of dry cell wt/IF-1500 run; two runs each with ∼1000 L of liquid medium were carried out to create the M. trichosporium OB3b biomass for the field test at well 13. The mean wholecell TCE-degrading activity of the freshly harvested bacteria from these two runs was 63 nmol min-1 mg-1 dry wt with formate and 23 nmol min-1 mg-1 dry wt without formate in the assay incubation mixtures. These rates compare well to past values we have reported for 5-L bioreactor grown cells (15, 17-19, 23). An important practical finding was that cells stored as above as a cold paste retained 90% of their sMMO catalytic activity for TCE (( formate) for at least 1 month. Biofilter Design and Emplacement. The polypropylene bottles containing M. trichosporium OB3b in the form of a paste were transported to the field site packed in crushed ice. The cells were resuspended at a density of about 5.4 × 109 cells/mL in groundwater withdrawn from well 12 (Figure 1). Groundwater from well 12 originated from the same aquifer interval that is contaminated at well 13 but contained no detectable TCE (