Laboratory Evaluation of a Two-Stage Treatment System for TCE Cometabolism by a Methane-Oxidizing Mixed Culture Laurence H. Smith,1 Perry L. McCarty2 1Department of Process and Environmental Technology, Massey University, Palmerston North, New Zealand; telephone: 64-6-356-9099; fax: 64-6-350-5654; e-mail: [email protected]. 2Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford, California 94305-4020 Received 5 July 1996; accepted 30 December 1996 Abstract: The objective of this research was to evaluate INTRODUCTION several factors affecting the performance of a two-stage treatment system employing methane-oxidizing bacteria Biodegradation of trichloroethylene (TCE) contaminated for trichloroethylene (TCE) biodegradation. The system water has gained increased interest due to the potential for consists of a completely mixed growth reactor and a plug-flow transformation reactor in which the TCE is co- TCE destruction rather than transfer to another medium, as metabolized. Laboratory studies were conducted with in air stripping and activated carbon adsorption. Both aero- continuous growth reactors and batch experiments bic and anaerobic bacteria are capable of transforming TCE. simulating transformation reactor conditions. Perfor- The aerobic bacteria generally employ oxygenases, such as mance was characterized in terms of TCE transformation methane monooxygenase (MMO) in methanotrophic bacte- capacity (TC, g TCE/g cells), transformation yield (TY, g ria, to oxidize TCE to TCE epoxide, which is subsequently TCE/g CH ), and the rate coefficient ratio k /K 4 TCE S,TCE mineralized in mixed cultures (Arciero et al., 1989; Ensign (L/mg-d). The growth reactor variables studied were sol- ids retention time (SRT) and nutrient nitrogen (N) con- et al., 1992; Fogel et al., 1986; Nelson et al., 1988; Tsien et centration. Formate and methane were evaluated as po- al., 1989; Wackett and Gibson, 1988). Some of the highest tential transformation reactor amendments. Comparison rates of TCE oxidation have been obtained in methanotro- of cultures from 2- and 8-day SRT (nitrogen-limited) phic pure and mixed cultures, particularly in the cases of growth reactors indicated that there was no significant methanotrophs expressing the soluble form of MMO (Al- effect of growth reactor SRT or nitrogen availability on TC varez-Cohen and McCarty, 1991a; Oldenhuis et al., 1991; or T , but N-limited conditions yielded higher Y Tsien et al., 1989). The design of aerobic treatment systems kTCE/KS,TCE. The TCE cometabolic activity of the 8-day SRT, N-limited growth reactor culture varied significantly for TCE destruction is complicated because TCE oxidation is a cometabolic process; the bacteria do not gain carbon or during a 7-year period of operation. The TC and TY of the resting cells increased gradually to levels a factor of 2 energy for growth from the reaction. Because of the com- higher than the initial values. The reasons for this in- plexity of the processes involved, it is not yet obvious how crease are unknown. Formate addition to the transforma- to optimize a treatment system for TCE biodegradation. tion reactor gave higher TC and TY for 2-day SRT growth One approach suggested by several investigators employs reactor conditions and significantly lower T ,T,and C Y a two-stage treatment system (Fig. 1) in which the culture kTCE/KS,TCE for 8-day SRT N-limited conditions. Methane addition to the transformation reactor inhibited TCE co- produced in a growth reactor is contacted with the TCE- metabolism at low TCE concentrations and enhanced contaminated water in a separate treatment reactor where TCE cometabolism at high TCE concentrations, indicat- TCE degradation occurs (Alvarez-Cohen and McCarty, ing that the TCE cometabolism in the presence of meth- 1991c; Dobbins et al., 1995; Folsom and Chapman, 1991; ane does not follow simple competitive inhibition kinet- McFarland et al., 1992; Tschantz et al., 1995). This reactor ics. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 650– configuration has several advantages over a single reactor. 659, 1997. Keywords: cometabolism; methanotroph; trichloroethy- Since the growth reactor does not receive the contaminated lene; reactor; aerobic water stream, the growth conditions can be controlled to produce a culture with optimum TCE cometabolic activity and the effect of TCE transformation product toxicity (Al- Correspondence to: L. Smith varez-Cohen and McCarty, 1991a; Fox et al., 1990; Henry Contract grant sponsor: Gas Research Institute; Westinghouse Savannah and Grbic-Galic, 1991a; Oldenhuis et al., 1991) is isolated River Corporation from the growth environment. Competitive inhibition be- © 1997 John Wiley & Sons, Inc. CCC 0006-3592/97/040650-10 Figure 1. Proposed treatment system schematic (Alvarez-Cohen and McCarty, 1991c). tween methane and TCE (Henry and Grbic-Galic, 1991b; rate and extent are sought because it is generally believed Lanzarone and McCarty, 1990; Oldenhuis et al., 1989; Sem- that formate does not present complicating effects (Alvarez- prini et al., 1991) is avoided because the methane utilization Cohen et al., 1991b; Brusseau et al., 1990; Henry et al., and TCE cometabolism reactions are separated. Culture 1991a; Henrysson and McCarty, 1993; Koh et al., 1993; growth in a continuous stirred tank reactor (CSTR) and Oldenhuis et al., 1989; Tschantz et al., 1995; Tsien et al., contaminant degradation in a plug-flow reactor (PFR) pro- 1989) such as competitive inhibition that may occur when vide the ideal kinetic conditions for each of these processes. methane is used (Henry and Grbic-Galic, 1991b; Lanzarone In this study, factors of importance to the design of a and McCarty, 1990; Oldenhuis et al., 1989; Semprini et al., two-stage methanotrophic reactor system were evaluated 1991). While intracellular storage materials such as poly-b- through the use of a mixed culture expressing soluble MMO hydroxybutyrate (PHB) might be utilized by the microor- (Alvarez-Cohen et al., 1992). The variables in growth reac- ganisms to regenerate NADH (Gottschalk 1986), its rate of tor conditions studied included the effects of solids retention mobilization for this purpose is slow relative to TCE come- time (SRT) and nitrogen availability. Transformation reac- tabolism rates in some cases (Henrysson and McCarty, tor variables included residence time and formate and meth- 1993). ane as potential amendments. TCE transformation product toxicity has been observed Important biochemical processes that may limit the rate in studies of purified MMO (Fox et al., 1990), in pure and extent of TCE cometabolism include TCE transforma- cultures (Henry and Grbic-Galic, 1991a; Oldenhuis et al., tion product toxicity and the availability of reducing power 1991), and in mixed cultures (Alvarez-Cohen and McCarty, for regeneration of the electron carrier NADH for MMO 1991b; Henry and Grbic-Galic, 1991a; Stensel et al., 1992). activity (Fig. 2). Supplemental formate is commonly used to It is caused by nonspecific covalent bonding of daughter support regeneration of NADH in studies of cometabolic products of TCE epoxide to MMO, and possibly other bio- reactions of methanotrophic cultures when the maximum molecules in the cell, causing cell inactivation (Fox et al., 1990). The extent of enzyme or cell inactivation is propor- tional to the extent of TCE transformation (Alvarez-Cohen and McCarty, 1991a; Oldenhuis et al., 1991). Product tox- icity significantly reduces TCE transformation rates, espe- cially at high TCE/biomass ratios. Methane can function both as an electron donor for NADH regeneration (Fig. 2) and as a competitive inhibitor of TCE cometabolism. However, the reports on the effect of methane on TCE cometabolism have been varied. In many Figure 2. Biochemical pathway for methane oxidation by methanotro- cases, methane acted as a competitive inhibitor (Broholm et phic bacteria (Anthony, 1986). al., 1992; Fennel et al., 1993; Henry and Grbic-Galic, SMITH AND MCCARTY: AEROBIC TREATMENT OF TCE USING METHANOTROPHS 651 1991b; Lanzarone and McCarty, 1990; Leahy et al., 1989; eral salts medium, TCE-saturated water, and the bacterial Oldenhuis et al., 1989, 1991; Phelps et al., 1990; Semprini culture were added to each bottle to give a final liquid et al., 1991), but it has also been found to be stimulatory at volume of 100 mL at initial biomass and aqueous TCE low concentrations and inhibitory at high concentrations concentrations of 150–400 mg VSS/L and 0.9–17 mg/L, (Brusseau et al., 1990; Chang and Alvarez-Cohen, 1995b) respectively. The initial headspace TCE concentration was and to have no effect on TCE cometabolism (Strand et al., measured at least twice prior to addition of the organisms. 1990; Strandberg et al., 1989). Headspace was periodically analyzed for TCE, methane, and oxygen. At least one control bottle without organisms was carried through each experiment and always had insig- MATERIALS AND METHODS nificant TCE losses over 1 week in comparison to sample variability. TCE cometabolism rate coefficients were deter- Experimental Procedures mined by weighted nonlinear least squares analysis of the integrated Monod equation (Smith et al., manuscript in Reagents preparation). Reagents used were TCE (99+% pure ACS reagent, Aldrich Chemicals, Milwaukee, WI), methane (99.0% pure com- Bioreactor Studies of TCE Cometabolism Rate pressed gas, Liquid Carbonic, Chicago, IL), propylene Some TCE cometabolism rate experiments were conducted (99.0% pure compressed gas, Scott Specialty Gases, Fre- in 2-L computer-controlled bioreactors operated under mont, CA), and sodium formate (99.35% pure ACS reagent, batch conditions to simulate the plug-flow kinetics of the Fisher Scientific; Fair Lawn, NJ). transformation reactor (Fig. 1) as described previously (Smith et al., 1997). Samples of mixed culture from the Growth Reactors 7.3-L growth reactor were diluted to 100–200 mg VSS/L in mineral salts medium (Fogel et al., 1986) supplemented Methane-oxidizing mixed cultures were grown in three with 6.5–8.6 mg/L dissolved TCE and in some cases 20 mM semicontinuous stirred tank reactors. One had a 7.3-L liquid sodium formate or 5.6 mg/L dissolved methane.