Methanotrophic cometabolism of trichloroethylene (TCE) in a two stage bioreactor system

War. Res. Vol. 26. No. 2. pp. 259-265. 1992 Printed in Great Britmn. All rights reserved

0043-1354/92 $5.00 + 0.00 Copyright ~: 1992 Pergamon Preu pk;

TECHNICAL NOTE METHANOTROPHIC COMETABOLISM OF TRICHLOROETHYLENE (TCE) IN A TWO STAGE BIOREACTOR SYSTEM MICHAELJ. McFARLANDI*•, CATHERINEM. VOGEL/ and JIM C. SPAIN 2 t Utah State University. Civil and Environmental Engineering. Logan. LIT 84322-4110 and 2Engineering and Services Laboratory, Tyndall Air Force Base, FL 32403-6001, U.S.A. (First received Norember 1990; accepted in revisedform July 1991) Abstract--Competitive inhibition of trichloroethylene (TCE) removal by a mixed methanotrophic consortia was minimized by using a two stage bioreactor system supplied with sodium formate as reducing equivalents. A maximum TCE removal rate of 21.1 mg TCE per g volatile solids per day was observed when the influent formate concentration was 20 raM during continuous methane addition. Termination of methane while maintaining the same formate loading resulted in a TCE removal rate of 25.5 mg TCE per g volatile solids per day suggesting that methane competitively inhibits TCE removal. Formate serves as a noncompetitive substrate for methane monooxygenase system which is responsible for TCE removal. Key words--trichloroethylene, methane monooxygenase, reducing equivalents, formate

INTRODUCTION

Many aquifers underlying United States Department of Defense (DOD) facilities are contaminated by chlorinated solvents such as trichlorocthylenc (TCE). Due to its potential hcalth threat, methods to permanently remove TCE from groundwaters remains a priority for DOD. The least cost alternative for permanent removal of TCE is biological degradation. Although there is much activity in in situ bioremediation research, above-ground bioreactors are a preferred approach since they offer better process control together with more effective containment of toxic transformation products. TCE does not serve as a primary growth substrate for microorganisms and is biodegraded under aerobic conditions only through the process known as cometabolism or cooxidation (Folsom et al., 1990; Little et al., 1988; Roberts et al., 1989). Although much is known about the biochemical fundamentals of cometabolic metabolism, little progress has been made regarding the engineering application of cometabolic systems to remediate TCE contaminated groundwaters. One group of microorganisms that has the ability to cometabolize trichlorocthylene are methanotrophic bacteria. These organisms meet their energy requirements through the oxidation of methane under aerobic conditions. The enzyme system responsible for both methane and TCE oxidation has been *Author to w h o m all correspondence should be addressed. 259

identified as methane monooxygenase (MMO) (Little et al., 1988). Although the principal catabolic function of MMO is to catalyze the conversion of methane to methanol, its low substrate specificity enables it to mediate TCE oxidation. Figure I outlines the biological pathway by which methanotrophic metabolism leads to: (1) energy production for growth, (2) precursors for biomass formation and (3) TCE cooxidation. Figure 1 indicates that catalytic activation of methane monooxygenase enzyme requires reducing equivalents in the form of nicotinamide adenine dinucleotide (NADH) for methane oxidation to proceed. This reduction (or energy requirement) of MMO provides a unique engineering approach to regulating MMO activity. Under normal methanotrophic growth conditions, reducing equivalents are generated internally by the oxidation of formaldehyde and formate. For biotreatment purposes, MMO activation may be regulated by appropriate exogenous additions of reducing equivalents. When TCE is the substrate for MMO, the first chemical intermediate is TCE epoxide (Fig. I). This compound is unstable and is rapidly transformed to glyoxylic acid with the removal of chloride. Glyoxylic acid may then be chemically or biologically oxidized to carbon dioxide. Operation of methanotrophic bioreactor for TCE remot'al

Although various studies have reported biodegradation of TCE in actively growing methanotrophic cultures, it has been recently observed that active

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methanotrophic growth may actually reduce TCE removal rates (OIdenhuis et al., 1989; Tsien et al., 1989). This is due to the fact that nonlimiting concentrations of methane can saturate the catalytic site of the MMO enzyme responsible for TCE oxidation (Fig. I). Comparison of the half velocity constant of soluble MMO enzyme from the methanotroph Methylosinus trichosporium OB3b for TCE (K, = 200/~m) to that of methane (K, = 2/~m) illustrates the much stronger affinity of the enzyme for methane viz ~ vi'. trichloroethylene (Oldenhuis et aL, 1989). Conversely, it has been reported that the addition of reducing equivalents in the form of formate can enhance the rate of TCE degradation in pure cultures (Tsien et al., 1989). An advantage of using formate as a source of reducing equivalents is that, unlike methane, it will not compete with TCE for the catalytic site of MMO. It should be noted that formate acts to stimulate the activity of existing MMO and that MMO production is induced by methane during microbial growth conditions. To reduce competitive inhibition of TCE by methane, a two stage system was proposed in which methanotrophic growth and TCE removal processes were physically separated. A schematic diagram of the two stage bioreactor is given in Fig. 2. Details of

reactor design and operation are contained in the Materials and Methods section. RESEARCH GOAL AND OBJECTIVES The goal of the present research was to evaluate the technical feasibility of a two stage methanotrophic biological system to degrade trichloroethylene (TCE). The specific research objectives included: (a) development of a mixed methanotrophic culture obtained from local environment, (b) monitoring of TCE removal in abiotic and biotic reactor systems, and (c) evaluation of the use of reducing equivalents (e.g. formate) to improve methanotrophic TCE removal rates. MATERIALS AND METHODS Experimental approach The experimental approach consisted of three phases. The first phase was an abiotic control study in which TCE laden distilled water was pumped through the plug flow reactor system. Results from this phase provided information regarding; (I) time for breakthrough of TCE and (2) steady state concentrations of TCE obtainable in the absence of microorganisms. In the second phase, methanotrophic bacteria from the biological growth reactor were introduced into the TCE

Technical Note

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removal unit. Comparison of the effluent TCE concentrations in the presence of microorganisms to that obtained in the abiotic control experiments provided an estimate of methanotrophic degradation of TCE under methane limiting conditions. In the third phase, sodium formate was added as reducing equivalents for the MMO enzyme. Evaluation of TCE removal from this experimental phase provided insight into the effectiveness of enzyme activation on TCE transformation. Reactor design and operation The bioreactor system consisted of two units; a methanotrophic growth unit and a TCE biodegradation unit. A glass chemostat (New Brunswick Inc.) having a 1.4 I. working volume was used as the mcthanotrophic growth unit. The chemoslat was equipped with a stainless steel impeller operated at 350 rpm. The hydraulic retention time for this unit was maintained at 24 h while the temperature was held at 28"C during the entire study. Methane. oxygen and nutrients were added continuously to promote growth of methanotrophic bacteria. Methane and air were added at flow rates of 0.1 and 0.41/min, respectively, while the nutrient flow rate was maintained at

I ml/min. The composition of the nutrient salts media fed to the methanotrophic growth unit was described in Cornish et al. (1984). The TCE biodegradation unit consisted of a 400ml stainless steel plug flow reactor that was operated with zero reactor headspace. The plug flow reactor was equipped with stainless steel valves (Supelco, Bellefonte, Pa) for TCE sampling. The contents of the biological growth reactor was pumped into the TCE removal unit at a flow rate of l0 ml/min resulting in an average hydraulic retention time (HRT) of 40 rain for the second unit. The effluent from the plug flow reactor was aerated in the TCE stripping unit to remove any residual TCE before being recycled to the biological growth unit via a gravity feed line. The TCE stripping unit consisted of a 250 ml glass side arm flask in which a glass diffuser was used to supply air for volatilization of untreated TCE in the effluent from the plug flow unit. Pure trichloroethylene (TCE) was introduced to the biotreatment system through a syringe pump (Harvey Appartus Compact Syringe Pump--model 975). Based on the density of TCE (c. 1.4 g/ml) and the diameter of the syringe used, the volume flow rate required to reach the desired steady state TCE concentration (15 mg/1 TCE) was approx. 0.00003 ml/min. A solution of sodium formate was

262

Technical Note

introduced to the reactor using a second identical syringe pump (Fig. 2). The pump flow rate was adjusted to give a steady state concentration of 20 raM. Tygon9 tubing was used for transfer of microbial cells from the bioreactor and for the pumping of the nutrient feed. The remainder of the fluid transfer lines were i" i.d. stainless steel tubing. All stainless steel tubing was connected using stainless steel Swagelock~ fittings.

Methanotrophic inoculum The mixed methanotrophic inoculum was obtained locally from the wastewater treatment lagoon located at Tyndall Air Force Base, Florida. A 10 ml sediment sample was diluted 1:50 with nitrate salts medium and placed in the methanotrophic growth reactor. Methane and air were added at flow rates of 0.1 and 0.4 I/rain, respectively. After approx. 4 days. a dense biomass culture had developed. The culture had an orange-red color and existed mainly as large flocculent particles or dense microbial films associated with the chemostat wall and impeller. Analytical procedures Liquid samples (5ml) to be analyzed for TCE were withdrawn from the plug flow reactor using a Lurelock'~ glass syringe (Becton Dickinson, Rutherford. N.J.). The samples were injected through a stainless steel tube into a l0 ml glass vial (Pierce Co.. Rockford. III.)containing a 5 ml solution of hexane. Samples were injected underneath the hexane solution to minimize TCE volatilization loss. The hexane solution contained tetrachloroethylene (PCE) at a concentration of 0.5 mg/I as an internal standard. After introduction of the sample, the vials were sealed using a Teflons lined silicone septa capped by a crimped aluminum lid. Solvent-solvent extraction of the TCE was accomplished by shaking the sample bottles for approx. 30 s followed by centrifugation for 3 rain at 1200rpm to ensure complete separation of hexane/water phases (IEC Clinical Centrifuge--Damon/lEC divisionl. For quantitation of TCE, a 1 pl sample was withdrawn from the hexan¢ phase and injected on the gas chromatograph. A Hewlett-Packard 5890 gas chromatograph equipped with an electron capture detector (ECD) interphased with a 18652A A/D converter was used for TCE analyses. Separation of TCE and PCE peaks was accomplished using a 10' packed column containing 10% SP-1000 80/100 Supelcoport packing. Reported TCE removal rates were estimated by evaluating the loss of TCE at the reactor inlet sampling port before and after a particular reactor operating condition was imposed. Total TCE concentrations were used in estimation of removal rates and no attempt was made to differentiate between aqueous and sorbed TCE. The first sampling port was located approx. 2" from the base of the plug flow reactor. It was assumed that the sampling port was sufficiently far from the reactor inlet to be characterized by plug flow behavior. Dissolved oxygen was measured in both reactor stages using a Clark type oxygen electrode (Model 5331 Yellow Springs Instrument Co., Yellow Springs, Ohio). Reactor pH was measured by an Orion Research pH meter Model 601A/Digital lonanalyzer. Biomass concentrations were estimated using volatile solids analyses according to the procedure outlined in Standard Methods (APHA et aL, 1989). Reagent grade trichloroethylene (TCE) was purchased from MCI, Cincinnati, Ohio. Reagent grade tetrachloroethylene (PCE) was supplied by J. T. Baker Phillipsburg. N.J. Reagent grade hexane used was purchased from Fisher Scientific (OPTIMA). Methane and nitrogen gases were obtained from stock supplies at Tyndall Air Force Base while air for the biotreatment system and stripping unit was supplied from an in-house compressor located at the Engineeringand Services Center.

RKSULTS Liquid-liquid hexane extraction procedure yielded recovery efficiencies of 90.7% using standard TCE solutions. This recovery efficiency was comparable to TCE recoveries using similar internal standard methods (Oldenhuis et al., 1989). Results from all three phases of the experimental program are contained in Fig. 3. All data reported are averages of duplicate analyses.

Abiotic control The change in TCE concentration with time in the plug flow reactor system is shown in Fig. 3. A steady state TCE concentration of 15 mg/l was reached after approx. 50 bed volumes (i.e. 33 h). Results from the abiotic control study suggested that normal plug flow behavior did not occur in the second stage of the biotreatment system. Under normal conditions, a steady state TCE concentration would have been expected after approx. 2-3 reactor volumes. There were at least two reasons why steady state behavior may have occurred much later in the present system. First, the decision to pump pure TCE into the biotreatment system meant that the syringe pump flow rate had to be maintained at 0.00003 ml/min. With such a small flow rate, a small "dead" space in the syringe needle, such as a small bubble, would have taken a significant amount of time to be displaced. Another possible contribution to the delayed response was that some TCE may have been adsorbed to the Teflon~ coated sampling valves, Effluent from the stripping unit yielded a TCE concentration of 0.07mg/1 which represents a 99% removal efficiency. It was anticipated that this level of residual TCE in the stripper effluent had an insignificant effect on methanotrophic bacterial growth. Since a TCE concentration of 15 mg/I is known to be nontoxic to methanotrophic organisms (Oldenhuis et al., 1989), this concentration was used in the biological degradation study. After 80 bed volumes, methanotrophic bacteria were pumped into the plug flow reactor from the biological growth unit to evaluate biotic removal of TCE.

Biotic remoral o f TCE Immediately after introduction of biomass to the plug flow unit, TCE concentrations were observed to decrease (Fig. 3). Initial indications suggested that the methanotrophic bacteria were removing TCE. However, with time, the TCE concentrations steadily increased. Visual inspection of the samples withdrawn from the bottom and top of plug flow reactor indicated that the biomass had settled and may have become inactive. The volatile solids concentration at the bottom of the reactor varied from 1.2 to 1.6 g VS/I while at the top of the plug flow unit, the volatile

Technical Note

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solids concentration was approx. 0.01 g/I. To increase the active biomass in the system, waste cells from the chemostat were recycled to the biotreatment system. This led to a further reduction in the effluent TCE concentration initially, but, within 24 h, the TCE concentration had increased. The TCE concentration at the reactor influent sampling port eventually reached 30mg/I which was twice the abiotic steady state concentration suggesting that TCE sorption to microbial cells was occurring. The sampling port, which was located approx. 2" above the actual plug flow reactor inlet, was characterized by settled methanotrophic biomass. (It should be noted that no attempt was made to distinguish between TCE sorbed to settled biomass and dissolved TCE. The TCE concentrations reported are for total TCE contained in sample.) It is difficult to discern the exact mechanism responsible for the initial removal of TCE from the reactor system. Previous studies have suggested that microbial sorption of TCE by organisms rich in lipids, such as algae or, in this case, methanotrophic bacteria, may be more than an order of magnitude greater than that found for "typical" bacteria (Smets and Rittmann, 1988). An average linear partition coeffient for TCE of 2.4 (ml H20/mg VS) was reported for algae by Stunts and Rittmann (1988). If it can be assumed that algae and methanotrophic bacteria have similar sorption properties, the relative contributions of sorption versus biodegradation in the present reactor system may be evaluated. For a steady state concentration of 15 mg TCE/I, the sorption density (rag TCE/g VS) for

TCE in the present system can be calculated as follows: sorption density = 2.4 ml H20/mg VS x 15 mg TCE/I H20 (I) = 0.036 mg TCE/g VS. The largest biomass concentration found in the bottom of the reactor was 1.6g/I, thus the total removal of TCE can be calculated as follows: TCE sorbed by cells = 0.036 mg TCE/g VS x 1.6 g VS/I

(2)

= 0.06 mg TCE/I. Using the reported linear partition coefficient suggests that only 0.6% of the initial TCE removed could be accounted for by sorption alone. Although removal of the remainder may be attributed to biodegradation, this may not be necessarily correct since the partition coefficient reported by Smets and Rittmann (1988) was estimated in experiments using TCE concentrations ranging between 5 and 500/~g/I. A TCE concentration of 15,000/~g/i would not necessarily be expected to demonstrate the same sorption behavior. Measurement of reactor operational conditions indicated that the pH of the treatment system remained at 7.0 over the entire study. Dissolved oxygen concentrations in the chemostat varied between 8.0 and 8.2mg/I while the dissolved oxygen level in the plug flow reactor was approx. 1.5 mg/1.

264

Technical Note

Formate enhanced biodegradation o f TCE

To estimate the effect of reducing equivalents addition on TCE removal, formate was added continuously to the biotreatment system at a concentration of 20 raM. Results of formate addition are given in Fig. 3. Formate additions resulted in rapid reduction in TCE concentration in the plug flow reactor. The concentration of TCE at the influent of the plug flow reactor decreased from 29.2 to 1.4 mg/l TCE within 24 h after formate addition. The volatile solids concentration at the influent of plug flow reactor was estimated to be 1.3 g volatile solids per liter. This biomass level results in a maximum rate of TCE removal of 21.1 mg TCE/g VS-day. After 2 days of continuous operation, the formate loading was discontinued. Termination of formate addition led to a rapid increase in TCE concentration suggesting that the presence of reducing equivalents were limiting the TCE removal rate (Fig. 3). Due to the possible inhibition of TCE oxidation by residual methanc entering the plug flow reactor, it was dccidcd to evaluate the cffects of adding formate in thc absence of mcthane addition to the biological growth unit (Fig. 3). Methanc addition was terminated for a period of 7 h. During this timc frame, thc concentration of TCE in the bottom of the reactor decreased from 22.3 to 10.2mg/I TCE which is equivalent to a TCE removal rate of 25.9 mg TCE/g VS-day (volatile solids conccntration--l.6g VS/I).

DISCUSSION

It is clear that a mixed methanotrophic culture is more effective in biodegrading TCE when supplied with suitable reducing equivalents (e.g. formate) under limiting methane concentrations. However, the enzymes responsible for transformation will not retain their biological integrity indefinitely and will eventually be degraded in the absence of methane. A simple solution to this problem is to create a sludge blanket reactor that is to receive periodic additions of fresh methanotrophic cells. The TCE contaminated water and formate could be added continuously to such a system with periodic removal of inactive biomass. In order to eliminate potential competitive inhibition of TCE degradation by methane, methane could be stripped from the influent to the plug flow unit. In addition, periodic oxygen additions to the influent of the plug flow unit would also assist in reducing the presence of residual methane. The concentration of TCE used in the present study was low compared to what may be encountered in actual field situations. Although TCE concentrations much above 25 mg/I have been found to be toxic to growing populations of methanotrophic bacteria (Oldenhuis et al., 1989), no one has reported the toxicity of TCE to nongrowing cultures. It will be important to establish the toxic threshold of TCE on

nongrowing methanotrophic biomass cultures if the two stage bioreactor system is to be used under field conditions. With regard to TCE sorption to methanotrophic biomass, results from the present study suggest that sorption may be nonlinear. This is not surprising since others have reported that the sorptive capacity of microbial biomass rich in lipids increases with increasing TCE concentration (Smets and Rittmann, 1988). This suggests that it is not just the amount of organic matter present that determines the extent of sorption of hydrophobic compounds to microbial cells but also its nature and structure. Further work is required to characterize the concentration effects of TCE sorption to methanotrophic biomass. CONCLUSION Results from this preliminary study suggest that the methanotrophic two stage bioreactor system is effective in removing TCE from contaminated groundwater. However, the mixed methanotrophic culture was only effective in biodegrading TCE when it was supplemented with formate, in the absence of appropriate levels of exogenous reducing equivalents, the removal of TCE occurred mainly by adsorption to biological floes. A maximum TCE removal rate of 21.1 mg TCE/g VS-day was estimated during both formate and methane addition. This may be a very conservative estimate given the fact that 24 h had elapsed before a TCE measurement was made. Actual maximum TCE removal rate may have been much greater. The TCE removal rate estimated in the present study is significantly greater than that reported recently by Phelps et al. (1990) for a mixed methanotrophic culture (i.e. 15 mg TCE/g VS-day). When formate was added a second time with simultaneous termination of methane addition, the TCE removal rate increased to 25.9 mg TCE/g VSday. The increase of TCE removal in the absence of methane is consistent with results reported by others (Oldenhuis et al., 1989; Tsien et al., 1989). Acknowledgements--The authors would like to thank the

Engineering and Services Laboratory and the Air Force Officeof Scientific Research for sponsorship of this research. This research was supported by Contract No. F49620-88-C0053 AFOSR and managed by Universal Energy Systems. The authors would like to acknowledge Ms Ivonne Cardona Harris for preparation of this manuscript. REFERENCES

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