Environ. Sci. Technol. 2008, 42, 3011–3017
Electrolytic MethanogenicMethanotrophic Coupling for Tetrachloroethylene Bioremediation: Proof of Concept SERGE R. GUIOT,* RUXANDRA CIMPOIA, RAMONA KUHN, AND AUDE ALAPLANTIVE National Research Council, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, QC, H4P 2R2 Canada
Received August 29, 2007. Revised manuscript received December 20, 2007. Accepted January 16, 2008.
Coupling of methanogenic and methanotrophic catabolisms was performed in a single-stage technology equipped with a water electrolysis cell placed in the effluent recirculation loop. The electrolysis-generated hydrogen served as an electron donor for both bicarbonate reduction into CH4 and reductive dechlorination, while the O2 and CH4, supported the cometabolic oxidation of chlorinated intermediates left over by the tetrachloroethylene (PCE) transformation. The electrolytical methanogenic/methanotrophic coupled (eMaMoC) process was tested in a laboratory-scale setup at PCE loads ranging from 5 to 50 µmol/Lrx · d (inlet concentrations from 4 to 11 mg/L), and at various hydraulic residence times (HRT). Degradation followed essentially a reductive dechlorination pathway from PCE to cis-1,2-dichloroethene (DCE), and an oxidative pathway from DCE to CO2. PCE reductive dechlorination to DCE was consistently over 98% while a maximum oxidative DCE mineralization of 89% was obtained at a load of 4.3 µmol PCE/ Lrx · d and an HRT of 6 days. Controlling dissolved oxygen concentrations within a relatively low range (2–3 mg/L) seemed instrumental to sustain the overall degradation capacity. Degradation kinetics were further evaluated: the apparent halfsaturation constant (KS) had to be set relatively high (29 µM) for the simulated data to best fit the experimental ones. In spite of such kinetic limitations, the eMaMoC system, while fueled by water electrolysis, was effective in building and sustaining a functional methanogenic/methanotrophic consortium capable of significant PCE mineralization in a single-stage process. Hence, degradation standards are within reach so long as the methanotrophic DCE-oxidizing potential, including substrate affinity, are optimized and HRT accordingly adjusted.
Introduction Due to its widespread use as dry cleaning solvents and degreasing agents for military and industrial applications, chlorinated ethenes such as tetrachloroethene (PCE) and trichloroethene (TCE) are prominent and priority groundwater pollutants. PCE, which appreciably can be transformed under anaerobic conditions, is refractory under conventional aerobic conditions (1, 2). Conversely, anaerobic PCE degradation is often incomplete, with pathways stalling at * Corresponding author phone: +1 (514) 496-6181; fax: +1 (514) 496-6265; e-mail:
[email protected]. 10.1021/es702121u CCC: $40.75
Published on Web 03/18/2008
Published 2008 by the American Chemical Society
partially degraded intermediates, unless Dehalococcoides species are actively present in which case complete dechlorination from PCE to ethene is possible (3, 4). However, Dehalococcoides species may be inhibited by some chloroorganics such as the chloroform and 1,1,1-trichloroethane (5), outcompeted by other dehalorespiring microorganisms for chlorinated ethenes and/or electron donors (6), highly sensitive to oxygen (3), and not competent for all chemicals present when as often the contamination is mixed. Accordingly the combination of anaerobic and aerobic conditions may remain a cost-effective alternative for complete PCE biodegradation. Sequential anaerobic and aerobic biodegradation is usually carried out in two systems, zones or phases, at a proportionally increased cost. An integrated anaerobic/ aerobic multispecies biosystem was recently developed for the bioremediation of groundwater. The technology used anaerobic granules from industrial upflow anaerobic sludge bed (UASB) plants as precursors for coupling anaerobic/ aerobic populations within a single biofilm (7, 8). When oxygenation is controlled in the presence of organic substrate, O2 penetration into the biofilm is limited; hence, aerobic activity can develop at the periphery of the granule without affecting the activity of strict anaerobes near the core. This concept has been applied to the biodegradation of various chlorinated solvents, namely PCE (7) and TCE (8, 9). Of particular interest in such a process is the indigenous production of methane by methanogens in the innermost core, which migrates outward, countercurrent to the O2 diffusion flux. The concomitant presence of O2 and CH4 is expected to promote the growth of methanotrophic bacteria (10, 11) which can then cometabolically oxidize the intermediates left over, thanks to soluble or/and particulate methane monooxygenases (sMMO, pMMO). However it has been shown that methanotrophic growth was, in fact, limited as a result of competition by heterotrophic bacteria for O2 (12). To decrease the potential competition for O2 between heterotrophs and methanotrophs, organic carbon can be replaced by an electron donor such as molecular hydrogen. Hence water electrolysis could be incorporated in the biosystem as an integrated source of both oxygen and hydrogen. It is not new to combine electrolysis with a biological system, but to our knowledge, up to now the utilization of both gas species of electrolysis has not been exploited. Processes involving oxygenation by electrolysis have been reported where O2 alone was used (13, 14). In another case, H2 generated by electrolysis has been used as electron donor for denitrifying bacteria, but the oxygen was discarded and at a cost (15). Novel in this process is the exploitation of both gas species. Hydrogen serves as electron donor to both anaerobes for the reductive dechlorination of PCE into intermediates, and methanogens for reducing water–carbonates into CH4; whereas oxygen is used as electron acceptor by methanotrophs to oxidize indigenous CH4 while cometabolically degrading the chlorinated intermediates. The objective of the present study will be to test the concept of electrolytic methanogenic-methanotrophic coupling (eMaMoC) for the biotreatment of recalcitrant chlorinated aliphatics such as PCE, in a single-stage laboratoryscale reactor, and in so doing obtain a better understanding of the degradation pathways associated with such a complex consortium, identify operational factors which are key to its effectiveness, and evaluate the limits of the technology. VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3011
Materials and Methods Reactor Setup. A 5 L upflow reactor was equipped with an electrolytic cell placed on the liquid recirculation loop for uninterrupted reactor supply in both H2 and O2 (Figure S1, Supporting Information). Dilution water (buffer), nutrient solution, trace metal solution, and PCE solution were combined in a single influent stream and entered the reactor via the effluent recirculation line. The dilution water consisted of (mg/L): NaHCO3, 775; KHCO3, 990; Ca(NO3)2 · 4H2O, 27; K2HPO4, 3540. The nutrient solution contained (mg/L): KH2PO4, 808; K2HPO4, 1032; NH4HCO3, 12 460. The chloridefree trace metal solution contained (mg/L): FeSO4 · 7H2O, 856; H3BO3, 5; ZnSO4 · 7H2O, 13; MnSO4 · H2O, 60; Co(NO3)2 · 6H2O, 33; NiSO4 · 6H2O, 9; (NH4)6Mo7O24 · 4H2O, 273; AlK(SO4)2 · 12H2O, 2; Na2-EDTA, 33; MgSO4 · 7H2O 1630; Na2SeO4, 6; Na2WO4 · 2H2O, 6; cystein, 320. The PCE solution was prepared in an SKC Quality sample bags (Tedlar, SKC Inc., EightyFour, PA) at a concentration of 20–50 mg/L of PCE. Ethanol, which facilitated the dissolution of PCE, was adjusted so as to have the desired chemical oxygen demand (COD) load. The dilution water:trace metal solution:nutrient solution ratio in the feed was between 410 and 430:1:1 (vol/vol/vol). Inoculum. For the first run, the reactor was inoculated with nonadapted anaerobic sludge: 84% (dry weight) were anaerobic granules, originating from an industrial UASB reactor, treating fruit-juice wastewater (Rougemont, QC, Canada), and the balance (16%, dry weight), anaerobic digester sludge from a municipal wastewater treatment plant (Repentigny, QC, Canada). Following inoculation the initial reactor biomass content was 20.7 g VSS (volatile suspended solids)/liter of reactor (Lrx). The second reactor (II) was inoculated with nonadapted anaerobic granules of the same origin as reactor I, with an initial biomass content of 41.5 g VSS/Lrx. Analytical Methods. The COD and VSS were determined according to Standard Methods (16). Analysis of gas phase PCE and metabolites was determined by gas chromatography (GC) (Agilent Technologies 6890N Network GC System, Hewlett-Packard, Wilmington, DE) with a flame ionization detector (FID) and two 1.8 m Carbopack B/1% SP-1000 columns (Supelco, Bellefonte, PA). Analysis of PCE and chlorinated metabolites in the liquid were assessed from gas measurements as described above, in the headspace of 20 mL glass vials containing 10 mL of liquid sample, and sealed and heated for 60 min in an 80 °C water bath as detailed previously (9). The reactor gas composition (O2, H2, CH4, CO2, N2) was measured by GC and FID as described earlier (17). Dissolved methane measurements were determined from gas headspace GC analysis of liquid samples at 20 °C, and the total methane content of the sample was calculated using Henry’s law (23.6 mg CH4/L · atm at 20 °C). Dissolved O2 (DO) was measured in the reactor liquid using a polarographic probe (model no. 01972–00, Cole Palmer Instruments, Chicago, IL). Reactor Balances. The in-reactor rate of CH4 consumption was deduced from the balance between the CH4 production and its off-gas release. As shown in Table S1 (Supporting Information), the anaerobic functions for both hydrogen and ethanol degradation were highly competitive to the aerobic ones. Hence the H2 consumption as well as the ethanol consumption rates were used to stoichiometrically estimate a fair CH4 production rate (¼ mol CH4/mol H2, and 1.5 mol CH4/mol ethanol). The H2 consumption rate was obtained by subtracting off-gas H2 from the H2 produced, which was obtained by doubling the O2 volume produced. Oxygen production rate was estimated by summing the balance between DO in the liquid recirculation at the reactor inlet and liquid effluent multiplied by the recirculation flow rate and the off-gas O2 release flow rate. 3012
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The PCE dechlorination was estimated from the molar balance between the PCE input and PCE ouputs (liquid effluent and off-gas). PCE mineralization was calculated from the molar balance between inlet PCE and all organochlorines at both off-gas and liquid outlets. Specific Activity Tests. Specific activities were presented on a mixed culture basis. Anaerobic activities for ethanol, acetate, and hydrogen were determined in serum bottles by measuring the rate of substrate depletion, individually and under nonlimiting conditions, as described previously (18). Methanotrophic activity was evaluated as described earlier (9). The specific activity was obtained by reporting the rate of substrate depletion to the VSS content in the test bottle. The specific reductive dechlorination activity was based on the PCE degradation rate under strict anaerobic conditions. A biomass sample was inoculated in 120 mL serum bottles containing anaerobic phosphate buffer to obtain ca. 2 g VSS/L in 20 mL of liquid. The bottles were flushed with H2/CO2 (80:20%, vol./vol.) and capped with a Mininert valve to avoid absorption of chemicals. PCE (Aldrich, Milwaukee, WI; HPLC grade, 99.9+%) diluted in ethanol was added to obtain an initial content of ca. 2 mg PCE/bottle, along with 100 mg ethanol-COD/L. The control bottle contained 0.1 g/L NaN3 for inhibiting biological activity. All tests were performed in triplicates. The bottles were incubated in the dark on a rotary shaker (New Brunswick Scientific Co., Edison, NJ) at 20 °C and 250 rpm. Initial PCE concentration was measured after four hours once equilibrium was reached, then measurements were repeated once daily, using the headspace method as described above. Total chloroethene concentrations were estimated using Henry’s constants (19). At the end of the test the VSS were measured in each bottle, and specific activity was calculated as described above. Mineralization was assayed in 120 mL serum bottles equipped with a 1 M KOH trap and spiked with 14C-uniformly labeled cis-1,2-DCE or 14C-uniformly labeled TCE (America Radiolabeled Chemicals Inc., St. Louis, MO), to obtain 100 000 dpm/L, and nonradioactive cis-1,2-DCE or TCE (HPLC grade, 99.9+%, from Supelco, Bellefonte, PA), to obtain an initial concentration of 2 mg L-1 as described previously (9). Initial biomass content was ca. 2 g VSS/L.
Results and Discussion Reactor Performance at Low HRT. Immediately following reactor inoculation, specific hydrogenoclastic, acetoclastic, and methanotrophic activities of the mixed inoculum were assessed, as 96 ( 15 mg H2/g VSS · day, 125 ( 28 mg acetate/g VSS · day and 17 ( 3 mg CH4/g VSS · day, respectively. Methanotrophic functions were already present in the inoculum since in industrial reactors, methane is concomitant to traces of oxygen continuously supplied through the influent. An initial adaptation period of PCE feeding under anaerobic conditions was carried out, so that the reductive dechlorination rate will not be limiting at the onset of anaerobic/aerobic coupled conditions. Ethanol, the carbonsource was fed at a rate of 0.7 to 1.2 g COD/Lrx · day. The hydraulic retention time (HRT) was initially set at 3.5 days, and the effluent recirculation flow rate, at 3.9 L/h so as to maintain an upflow liquid velocity (vUP) of 0.5 m/h. Then, HRT was decreased to values ranging between 1 and 1.24 days, the effluent recirculation flow rate, increased at 15 L/h and vUP at ca. 2 m/h. The PCE load fluctuated between 15 and 25 µmol/Lrx · day (2.5–4.1 mg /Lrx · day), for an inlet PCE concentration between 24 and 52 µM (4–8.6 mg/L). Adaptation of the biomass to PCE was evident after one month of operation, once the results demonstrated an increase in the PCE removal efficiency from 84 to over 95%. Furthermore, upon increasing the PCE load to 25 µmol/Lrx · day, the PCE dechlorination efficiency was still maintained at over 95%
FIGURE 1. PCE removal (1) and mineralization (2) efficiency, in parallel to the inlet dissolved oxygen concentration (--o--) as a function of the reactor operational time (Reactor I). with cis-1,2-DCE as the only end product of the reductive dechlorination process (only traces of vinyl chloride or ethene were detected at some occasions). At day 83, water electrolysis was turned on. The applied electrical power varied from 0.8 to 1.1 W, thereby generating oxygen at a rate between 240 and 440 mg O2/Lrx · day, 20–70% of which were estimated to be transferred into the aqueous phase. The reactor was operated steadily for 2 months under coupled anaerobic/aerobic conditions at an HRT of ca. 1 day, a vUP of ca. 1 m/h, a temperature of 25 °C, and an ethanol feeding rate of ca. 250 mg COD/Lrx · day. The PCE load fluctuated between 15 and 50 µmol/Lrx · day (inlet PCE concentration ranging from 20 and 52 µM, or 3.3–8.6 mg/L). Figure 1 shows the time-profile of PCE mineralization alongside the DO and the PCE dechlorination efficiency. Over the course of reactor operation, a PCE dechlorination efficiency of over 98% was achieved for the most part, suggesting a well established reductive dechlorinating consortium capable of thriving under coupled conditions, as electrochemical reductive dechlorination at the cathode could not support more than 5% of PCE removal, while chemical organochlorine oxidation could be excluded, at the voltage range applied (details are in the Supporting Information). However, after DO had peaked to 10 mg/L, the PCE removal capacity was compromised as was reflected by a slight but noteworthy (below 90%) decrease in the PCE dechlorination efficiency. Also observed was that PCE mineralization consistently followed a pattern similar to that of DO within the reactor (Figure 1). However, after DO had peaked to 10 mg/L, from day 121 and onward, the PCE mineralization efficiency started to decrease although DO was maintained around 5–6 mg/L. Several reasons might explain the observed decline in mineralization efficiency. The increasing CH4 production and consumption activity (Table 1) may have increased the competition with DCE for MMO enzymes, hence, decreased the mineralization rate and efficiency (20, 21). Additionally type II methanotrophs, which favor conditions of low O2 and high CH4 (10), probably predominated in the inoculum and at the initial stages of the reactor operation, may have become inhibited (sMMO damage or repression) during the later stages of operation by elevated O2 concentrations (22). Fast-
growing type I methanotrophs, possessing only pMMO, may then have proliferated over type II methanotrophs when oxygenation conditions increased (23). If so, the DCE oxidation rate could have decreased, as the rate of pMMO chloroethene degradation might be much less than that of sMMO (24). It is also possible that the ever increasing reactor DO slowly disrupted the integrity of the biofilm periphery as shown before in ref 11. Methanotrophs are known to produce exopolymeric substances that may serve to protect them against toxic metabolites (25). Thus a disturbance of the biofilm may have occurred rendering it susceptible to substances such as chlorinated ethenes (22), chloral hydrates (26), or epoxides (27). At this stage of the study, one can conclude at least that single stage anaerobic/aerobic coupling is working. It is clear that anaerobic activities were maintained, both reductive dechlorination (conversion of PCE into DCE over 97%) and strict anaerobic activity (the methanogenic activities at the end were measured as 104 ( 4 mg H2/g VSS · day and 45 ( 9 mg acetate/g VSS · day; the acetogenic activity was 193 ( 6 mg ethanol/g VSS · day). Methanotrophic activities of granules increased as well (24 ( 2 and 29 ( 0 mg CH4/g VSS · day, at days 132 and 140, respectively, as compared to 15 ( 4 mg CH4/g VSS · day at the end of the anaerobic phase). Chlorinated intermediates could then be oxidized, even though their mineralization was limited due to either operational (e.g., a HRT of 1 day is relatively short), or intrinsic (e.g., kinetics) restrictions. In order to clarify this, as well as to validate the reproducibility of the concept, a second reactor was started up with fresh inoculum, and modifications to operational conditions, namely the HRT. Reactor Performance at High HRT. The second reactor (II) was operated under anaerobic conditions for one week in a batch mode with 1 mg/Lrx of PCE pulsed daily. Immediately following, water electrolysis was turned on. The applied electrical power fluctuated around 0.5 ( 0.3 W, with oxygen generated at a flow rate of 28-80 mg O2/Lrx · day, 73-100% of which was estimated to be transferred into the aqueous phase. The reactor was operated in a continuous mode with a PCE loading rate of ca. 11 ( 3 µmol/Lrx · day (PCE inlet concentration of 40–60 µM), a much lower ethanol loading rate of 40 mg COD/Lrx · day, a temperature of 22 ( 0.5 °C, a pH of 7.2 ( 0.1 and a constant vUP of 0.43m/h (effluent recirculation flow rate of 3.3 L/h). Here, the HRT was increased to ca. 5 day. A pumping malfunction occurred with the dilution water stream (days 27 through 32), resulting in a higher HRT range (7–16 day), with alterations to PCE loading (7 ( 3.8 µmol/Lrx · day) and the PCE inlet concentration (67 ( 0.6 µM). Accordingly, data of this first operational phase were segregated into three periods, as displayed in Table 2. A second phase was then carried out to test the effect of an increased O2 transfer rate with same O2 supply (i.e., similar power applied to the electrolysis cell). To achieve this, at day 56, the effluent recirculation rate was increased, so as to reach a vUP of 0.75 m/h (effluent recirculation flow rate of 5.9 L/h). Consequently, the DO concentration at the base of the reactor, increased from 1.5 to 2.3 mg/L, on average. At midphase II, an operational problem led to the loss of some biomass (biomass content decreased to 31.1 gVSS/Lrx as measured at day 123). To compensate for the loss of biomass, PCE load was slightly diminished to ca. 9 µmol/Lrx · day (inlet PCE concentration between 38 and 49 µM). As a result of parameter changes, data were segregated accordingly. At the end of the reactor run, the PCE load was even more reduced (around 6 µmol/Lrx · day), in an attempt to elevate the PCE mineralization efficacy. At reactor closure, the biomass content was quantitatively measured at 39 g VSS/Lrx, as compared to the 41.5 g VSS/Lrx at inoculation. This means that biomass content remained steady over the six months of reactor operation, except for the short episode as mentioned VOL. 42, NO. 8, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Reactor I Performance Results over Time As a Function of PCE Load and Influent DOa period
DO in solid washout CH4 production CH4 released
#
days
0
40–83
na
1
84–97
2
98–108
5.70 (0.99 4.66 (0.49 1.91 (0.74 8.48 (1.89 5.29 (0.70 na
3 109–116 4 117–120 5 121–133 6 140–142
mg/L
mgVSS/Lrx · day
mmol/Lrx · day
mmol/Lrx · day
49 (42 135 (225 124 (27 218 (66 225 (51 249 (28 nd
3.81 (1.19 4.97 (2.13 7.37 (1.34 8.30 (1.9 9.91 (2.13 7.81 (2.03 1.3
3.66 (2.53 2.10 (0.97 2.69 (0.49 4.12 (0.52 2.51 (1.94 1.78 (0.43 1.36
PCE in mg/L
µM
4.05 24.4 (2.19 (13.2 4.52 27.2 (0.71 (4.3 5.51 33.2 (0.50 (3.0 7.66 46.1 (1.12 (6.7 4.37 26.3 (0.18 (1.1 5.12 30.8 (1.06 (6.4 3.41 20.6 (0.19 (1.2
PCE loading PCE outb DCE outb
PCE mineralizationc
µmol/Lrx · day
µM
µM
% µmol/g VSS · day
21.12 (11.61 27.1 (4.7 33.6 (3.4 46.0 (6.5 24.2 (5.3 30.1 (11.3 14.16 (5.1
1.32 (2.52 0.39 (0.31 0.30 (0.21 1.49 (1.89 0.89 (0.19 0.68 (0.17 0.3 (0.9
18.4 (10.0 13.3 (3.4 15.1 (3.6 33.0 (1.3 11.3 (0.5 24.6 (5.4 20.3 (0.8
a na: non applicable. nd: not determined. b Includes off gas loss: PCE
