Environ. Sci. Technol. 2000, 34, 1751-1757
Carbon Tetrachloride Degradation: Effect of Microbial Growth Substrate and Vitamin B12 Content SIWEI ZOU, H. DAVID STENSEL, AND JOHN F. FERGUSON* Department of Civil & Environmental Engineering, University of Washington, Seattle, Washington 98195
Microbial degradation kinetics of carbon tetrachloride (CT) under reducing conditions were investigated for different cultures, fed with 1,2-propanediol, dextrose, propionaldehyde, or acetate and nitrate, in the anaerobic step of an anaerobic/aerobic operation sequence. Methanogenesis was inhibited due to the aerobic step. CT biodegradation rates followed first-order kinetics with respect to CT concentration and biomass and were not affected by the presence of growth substrate. CT degradation rates increased linearly with higher intracellular vitamin B12 content. The culture fed 1,2-propanediol had the highest vitamin B12 content, which was 3.8, 4.7, and 16 times that of the propionaldehyde-, dextrose-, and acetate-fed cultures, respectively, and its first-order degradation rate constant was 2.8, 4.5, 6.0 times that for those cultures, respectively. No CT degradation occurred with culture liquid, suggesting that intracellular factors were responsible for CT degradation. The propanediol culture was able to sustain a constant CT degradation rate for a 16-day test period without substrate addition. Compared to a propanediol-fed culture grown only under anaerobic conditions, the propanediol culture grown under the sequential anaerobic/aerobic condition resulted in more biomass growth and a greater CT degradation rate per unit of propanediol fed, although its CT degradation rate per unit of biomass was lower.
Introduction Carbon tetrachloride (CT) is a significant pollutant at hazardous waste sites, and its biodegradation has been reported extensively. While aerobic transformation is not favorable, CT can be degraded under denitrifying conditions (1-4), sulfate reducing conditions (5-7), methanogenic conditions (8-11), and fermentation conditions (6, 12). The transformation products reported include less chlorinated methanes (chloroform (CF), dichloromethane (DCM), and chloromethane (CM)), CO, CO2, and CS2, suggesting both reductive and substitutive pathways for CT transformation (5). Although some bacteria can use such chlorinated methanes as CM and DCM to support growth (13, 14), no growth has been shown with CT, suggesting that microbial CT transformation is a cometabolic process. In many cases, the microbial transformation of CT is considered to be closely related to the presence of microbial cofactors, such as porphinoids (cofactor F430) and corrinoids (vitamin B12) (8, 9, 15). In vitro, abiotic degradation of CT, mediated by these cofactors under reducing conditions, has * Corresponding author phone: (206)543-5176; fax (206)685-9185; e-mail:
[email protected]. 10.1021/es990930m CCC: $19.00 Published on Web 03/17/2000
2000 American Chemical Society
been widely reported. Such cofactors can serve as electron carriers passing electrons from a donor to reduce CT. In the presence of a strong reductant such as titanium citrate, dithiothreitol, or sulfide, cofactor F430 and vitamin B12 can dechlorinate CT to either less chlorinated products (CF, DCM, and CM) or to completely non-chlorinated products as CO, CO2, and formic acid at relatively high rates (16, 17). Hashsham et al. (18) reported a 10-fold increase in the CT degradation rate when 2 µM of vitamin B12 was added to their culture grown anaerobically on DCM. Workman et al. (19) studied CT dechlorination by an iron-reducing microbial culture amended with vitamin B12. They found that the culture reduced cobalt(III) in vitamin B12 to cobalt(II), and that the reduced vitamin B12 carried out CT dechlorination. While vitamin B12 addition can significantly enhance microbial CT degradation under reducing conditions, some microorganisms such as methanogens and some acetogens contain elevated levels of this cofactor and have shown CT degradation capability (5, 8-12). However, the relationship between the cellular vitamin B12 content and CT degradation performance has not been well defined. In addition to methanogens and some acetogens, bacteria capable of 1,2-propanediol fermentation have been reported to produce vitamin B12 (20-22). The transformation of 1,2propanediol (propylene glycol) to propionaldehyde requires vitamin B12. Further fermentation of propionaldehyde to n-propanol and propionate does not require vitamin B12 but yields energy for growth. There are no reports regarding CT degradation by these vitamin B12-yielding bacteria. Although, vitamin B12 is only produced under fermentation conditions, the bacteria can grow aerobically (20, 21). Therefore, an anaerobic/aerobic operating sequence with anaerobic propanediol feeding might be advantageous for CT degradation by selecting for vitamin B12 producing bacteria and then maximizing their biomass production through the oxidation of fermentation products under aerobic conditions. The aerobic step inhibits methanogenic activity so that fermentation is the main reaction in the anaerobic step. We have described a biotreatment system (23) operated in an anaerobic/aerobic sequence to treat CT contaminated gases from soil-vapor extraction. In this system, CT is removed and sorbed to powdered activated carbon (PAC) as the gas is sparged through an aerobic reactor. The PAC and biomass mixture is recycled through an anaerobic reactor where the growth substrate is added and CT degradation occurs. The fermentation products produced in the anaerobic reactor are consumed by bacteria when the mixture is cycled to the aerobic reactor. In this study, different bacteria cultures were developed in sequential anaerobic/aerobic reactor systems by feeding with different substrates (propanediol, dextrose, propionaldehyde, or acetate and nitrate). The research objectives were to evaluate CT degradation kinetics for the different cultures, investigate the relationship between intracellular vitamin B12 content and CT degradation, and determine the effect of the presence of growth substrate on CT degradation. The effect of the aerobic step in the anaerobic/aerobic operating sequence on biomass growth and CT degradation was also evaluated.
Materials and Methods Chemicals. CT, chloroform (CF), and dichloromethane (DCM) (purity > 99.9%) were obtained from Aldrich Chemical Co. 1,2-Propanediol (purity > 99.9%), vitamin B12 (purity > 99.9%), and other chemicals used were obtained from Sigma Chemical Co. VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Summary of CT-Degrading Culture Operating Conditions feed
propanediol
dextrose
acetate/nitratea
propionaldehyde
seed source cycle
digester sludge 24-h anaerobic/ 24-h aerobic 30 min/h in anaerobic 401 ( 20 n-propanol propionate
digester sludge 24-h anaerobic/ 24-h aerobic 30 min/h in anaerobic 510 ( 25 acetate propionate
activated sludge 24-h anoxic/ 24-h aerobic 30 min/h in anaerobic 495 ( 20 unknownc
propanediol culture 24 h anaerobic/ 24 h aerobic 30 min/h in anaerobic 415 ( 20 n-propanol propionate
feed schedule biomass (mg/L) main metaboliteb in anaerobic period
propanediol (anaerobic only) propanediol culture 48-h anaerobic 30 min/h in the first 24 h 65 ( 10 n-propanol propionate
a Acetate was fed along with 793 mg per cycle nitrate as NO . b No methane was detected in any of the reactors. c No analysis was done for 3 organic products; acetate was completely consumed, and only a small amount of nitrate was detected.
Cultures and Growth Conditions. The operating parameters and feeding scheme for the different CT-degrading cultures are summarized in Table 1. Four cultures were fed 1,2-propanediol, propionaldehyde, dextrose or acetate and nitrate in the anaerobic step of an anaerobic/aerobic operating sequence. The fifth culture was fed with 1,2propanediol and was operated with only anaerobic conditions. The anaerobic/aerobic 1,2-propanediol-fed and dextrose-fed cultures were inoculated with anaerobic digester sludge from a Seattle municipal wastewater treatment plant. The acetate-fed denitrifying culture was inoculated with activated sludge from the same plant. The rest of the cultures were inoculated from the 1,2-propanediol-fed culture. The basal medium used for all the cultures was modified from reduced anaerobic mineral medium (RAMM) (24) in that three times more cobalt was added, and 20 mg/L potassium sulfate was added as sulfur source instead of sodium sulfide. No reducing agent was added into the media. The reducing conditions were created by bacteria themselves. Resazurin was used as a redox indicator. All cultures were developed in semicontinuously fed reactor systems at 20 °C. Two-liter Erlenmeyer flasks containing 1.5-L liquid volume were used for the reactors and were mixed with magnetic stirrers. A 48-h cycle time was used for the anaerobic/aerobic sequence with anaerobic and aerobic periods of 24 h. The anaerobic period was initiated at the end of the aerobic period by injecting nitrogen gas into the reactors for 30 min through a diffuser. Substrate was fed for 30 min of every hour during the anaerobic period by a peristaltic pump and timer with 580 mg of COD in 300 mL of medium fed each cycle. Air was sparged through a diffuser into the reactor through out the aerobic period. At the end of each anaerobic/aerobic cycle, mixed culture was removed manually to maintain a 10-day solid retention time (SRT) for each culture. The anaerobic-only culture cycle time was also 48 h, and it was fed propanediol for 30 min every hour for only the first 24 h. Biomass was removed at the end of each cycle. After a period of more than 6 SRTs, steady state was assumed, based on a relatively constant biomass concentration at the values shown in Table 1 and on relatively constant amounts of metabolites. Table 1 shows that the propanediol and propionaldehyde fermentation products were n-propanol and propionate, while dextrose formed acetate and propionate. No methane was produced in any of the cultures. For the nitrate-fed reactor, the acetate was consumed in the anaerobic step. The observed biomass yields were 0.22, 0.31, 0.30, and 0.22 g volatile suspended solids (VSS)/g COD for the propanediol, dextrose, acetate/nitrate, and propionaldehyde cultures operated with the anaerobic/ aerobic sequence, respectively. The observed biomass yield was 0.04 g of VSS/g COD for the propanediol culture operated under only anaerobic conditions. The pH in all reactors ranged from 7.1 to 7.4. CT-Degradation Kinetics Tests. CT batch degradation tests were conducted with each of the five cultures. In these 1752
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tests, the effect of initial CT concentration (50, 100, 300, 500 µg/L), the effect of growth substrate concentration (0, 100, 500 mg/L), and the effect of biomass concentration (approximately 100, 300, 500 mg/L) on CT degradation kinetics were evaluated. For the acetate-fed denitrifying culture, the effect of different initial nitrate concentrations on CT degradation rates was investigated. The effect of adding vitamin B12 (10 mg/L) to live cultures on CT degradation rates was also studied for three cultures (propanediol-, dextrose-, and acetate-fed cultures). CT batch degradation tests were conducted with live culture, autoclaved culture, and culture liquid. The autoclaved culture was tested to determine if CT was degraded abiotically by intracellular and/or extracellular factors. Culture liquid was tested to determine if extracellular cofactors were available for CT degradation. The test bottles were prepared as follows. With live cultures, 100 mL of culture suspension was withdrawn from the reactor immediately before feeding and placed in 160-mL serum bottles. The test bottles were then flushed with nitrogen gas and capped with Teflon-lined, butyl rubber stoppers. Fifty milligrams of COD/L of the appropriate substrate was added to each bottle to create reducing conditions, and the bottles were placed in a rotating shaker table overnight at 20 °C. Reducing conditions were established as indicated by resazurin after the preincubation. Occasional redox potential measurements showed that the redox potential in the culture liquid ranged from -150 to -180 mv after preincubation. Autoclaved culture test bottles were prepared in the same way, but the test bottles were autoclaved at 120 °C for 20 min, following overnight incubation. The redox indicator, resazurin, showed that reducing conditions established by the living cultures were maintained. Culture liquid was also prepared for tests in much the same way as for the living culture. Following overnight incubation in serum bottles, 20 mL of liquid was transferred into capped centrifuge tubes in an anaerobic glovebox. After centrifuging at 5000g for 10 min, 15 mL of liquid was transferred to 20-mL anaerobic test tubes in the anaerobic glovebox filled with anaerobic gases composed of 2% H2, 20% CO2, and 78% N2. Reducing conditions were maintained as indicated with resazurin. The prepared test bottles or tubes were spiked with CT and with substrate in some cases, vortexed, and placed in a 20 °C rotating shaker at 160 rpm, tilted with their top downward. CT, CF, and DCM in the headspace of test bottles and tubes were monitored over the test periods. The aqueous concentration of CT, CF, and DCM and their total masses in the batch test bottles were determined from their headspace concentrations. Duplicates were used for all batch test bottles. Sustaining CT Transformation with Propanediol Culture. The ability to sustain CT degradation was tested by repeatedly spiking CT over a 2-week period. The test bottles
were prepared using the procedure described above for live cultures. CT was spiked into the bottles at two different levels (100 and 500 µg/L) after previously injected CT was completely degraded. In some test bottles, propanediol was added at 50 mg/L at the same time CT spiking was conducted. After each CT spike, CT and its metabolites (CF, DCM) were monitored. Reducing conditions were maintained in the bottles over the 2-week period as indicated by resazurin. Effect of Vitamin B12 on CT Degradation Rates. The intracellular vitamin B12 content (µg/g VSS) was determined for each culture and related to CT degradation, and vitamin B12 was also added to the cultures to assess its effect on CT degradation. The vitamin B12 content in the liquid from the propanediol culture was also determined. After the growth reactors reached steady state, the cultures and the culture liquid were sampled 2-3 times for vitamin B12 analysis. The average vitamin B12 content was used to determine its effect on CT degradation kinetics for each culture. The tests of effects of vitamin B12 addition to the live cultures were conducted by adding vitamin B12 stock solution (1 g/L) to a 10 mg/L vitamin B12 concentration in the test bottles before overnight incubation. Analytical Methods. To determine intracellular vitamin B12, the microorganisms were separated by centrifugation at 6000g and washed several times with HPLC grade water. The bacteria were then extracted for cyanocobalamin by boiling in 0.08 M sodium acetate (pH adjusted at 5.6) containing 0.01% KCN (25). The extracted solution was diluted and analyzed by a medical laboratory (Harborview Hospital, Seattle WA) using an IMxB12 assay (Abbott Laboratories, Abbott Park, IL), based on the microparticle enzyme immunoassay technology. A series of aqueous vitamin B12 samples with known concentrations was used to validate the analytical results. The method detection limit was 0.1 µg/L for the aqueous vitamin B12 and 2 µg/g VSS for vitamin B12 in biomass. CT, CF, and DCM headspace concentrations were determined based on standards prepared with known gas-phase concentrations. A 5-50 µL headspace sample was directly injected into a Perkin-Elmer Autosystem gas chromatograph (GC) with an electron capture detector (ECD) connected to a 30-m RTx-1 capillary column (0.32 mm I.D., 1.0 µm df) by Restek Corporation (Bellefonte, PA). The GC conditions were as follows: oven temperature constant at 100 °C, inlet temperature at 200 °C, ECD temperature at 300 °C, helium as carrier gas, and argon/methane as makeup gas. The total masses of CT, CF, and DCM in the test samples were determined using standard curves of GC response areas versus headspace CT, CF, and DCM concentrations for a series of calibration bottles and tubes. The calibration samples contained the nutrient media with the same headspace and liquid volume as the test bottles and tubes and were injected with known amounts of CT, CF and DCM. The calibration samples were placed in a shaker table at 20 °C for 24 h. Aqueous concentrations were calculated from gas phase concentrations using Henry law constants at 0.042, 0.36, and 0.58 mol/L-atm for CT, CF, and DCM, respectively at 20 °C (26). The detection limits were 0.5, 3.5, and 22 µg per liter liquid volume for CT, CF and DCM, respectively. 1,2-Propanediol, propionaldehyde, and n-propanol were determined by a Perkin-Elmer Autosystem GC with a flame ionization detector (FID) connected to a DB-5 Megabore column (0.548 mm I. D., 1.5 µm df) by J&W Scientific (Folsom, CA). The liquid samples were filtered by passing through HPLC-grade 0.45 µm syringe filters by Gelman Science (Ann Arbor, MI) before direct injection into the GC. The GC conditions were as follows: inlet temperature at 250 °C, FID temperature at 280 °C, oven temperature: at 50 °C for 2 min then ramp at 10 °C per min to 120 °C, helium as carrier gas.
The detection limits for the three compounds were around 12 mg/L. Acetate, propionate, and other volatile fatty acids (VFAs) were analyzed by HPLC (Millipore Corporation, Milford, MA) with an UV detector set at 210 nm. The liquid samples were filtered by using HPLC-grade 0.45 µm syringe filters. The eluent (4 mM sulfuric acid in HPLC-grade water) was pumped into an Aminex HPX-87 H column (BioRad, Co.) and kept at 60 °C at a flow rate of 0.6 mL/min. Ten microliters of sample was injected into the system. The detection limits for the VFAs were 10 mg/L. The redox potentials of the culture solutions were measured by using Orion platinum redox combination electrodes (Orion, MA) with Ag/AgCl as its reference electrode. The reading was converted to millivolts (mv) relative to the standard hydrogen electrode (SHE). Biomass concentration was measured by volatile suspended solids (VSS), using glass fiber filters according to Standard Methods (27).
Results Microbial Degradation of CT by Different Cultures. Figure 1 shows aqueous CT concentration changes over test periods in batch bottle tests with the propanediol, dextrose, and acetate cultures. The CT degradation rate was much faster for the propanediol culture than for the dextrose and acetate/ nitrate cultures. Even though the cultures had not been exposed to CT before the tests, there was no lag in CT degradation. About 6%, 8%, and 8% of the CT appeared as CF which also was removed later; neither DCM nor CM was detected for the three cultures. The aerobic bottles (control) with live cultures present showed no CT removal and no formation of CF or DCM, indicating no CT degradation without reduced conditions and no significant sorption of CT to biomass or other losses. For the propanediol and dextrose cultures, their CT degradation rates were not affected by the presence of growth substrate (Figure 1a,b). For the acetate fed denitrifying culture, the CT degradation rate without nitrate addition was significantly higher than that with nitrate addition (Figure 1c). The reduced cell-free liquid from the propanediol culture did not degrade CT (Figure 1a), which indicates that there was not a significant level of extracellular agents in the fermentative culture that promote CT degradation. The measured vitamin B12 in the liquid was very low at 0.30 µg/L, close to the background level of some natural waters (28). There was significant CT degradation in the autoclaved control bottles with the propanediol culture. (No similar test was done with the other cultures.) However the degradation rate in the autoclaved culture was only about 10% of that in the live propanediol culture. CT Degradation Kinetics. Figure 2 shows the average values of ln(C/C0) (CT concentration/initial CT concentration) from batch tests at different initial CT concentrations plotted against time for the dextrose, acetate and propanediol cultures. There are strong linear relations between of ln(C/ C0) and elapsed time for the three cultures with a linear coefficient R 2 from 0.97 to 0.99, suggesting a first-order relationship between CT degradation rate and CT concentration. The error bars in this figure are the standard deviations for data points from four batch tests at different initial CT concentrations. Three levels of biomass concentration (about 100, 300, 500 mg/L) were tested to determine a relationship between CT degradation rate and biomass concentration for the three cultures. The results showed that the CT degradation rate was directly proportional to biomass concentration (data not shown). VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. CT degradation first-order kinetics: linear relationship between -ln (C/C0) and time: propanediol culture at 415 mg VSS/L, dextrose culture at 510 mg VSS/L, and acetate cultures at 495 mg VSS/L.
FIGURE 3. CT degradation first-order rate constants for different cultures. P0, P500, P100+VB12-propanediol culture with no propanediol addition, 500 mg/L of propanediol, 100 mg/L of propanediol, and 10 mg/L of vitamin B12, respectively; D0, D500, D100+VB12dextrose culture with no dextrose addition, 500 mg/L of dextrose, 100 mg/L of dextrose, and 10 mg/L of vitamin 12, respectively; A500, A500+60%N, A500+120%N, A100+VB12-acetate culture with no nitrate addition and 500 mg/L of HAc, 60% equivalent nitrate and 500 mg/L of HAc, 120% equivalent nitrate and 500 mg/L of HAc, and 100 mg/L of HAc and 10 mg/L of vitamin B12 addition, respectively.
FIGURE 1. Batch CT tests: aqueous concentration vs time. a. propanediol culture, [ live culture without propanediol; 2 live culture with 500 mg/L propanediol; * live culture under aerobic conditions; b autoclaved culture; + culture liquid; b. dextrose culture, [ live culture without dextrose; 2 live culture with 500 mg/L dextrose; * live culture under aerobic conditions; c. acetate culture with 500 mg/L acetate for all conditions, [ live culture without nitrate; 2 live culture with 533 mg/L NO3 (66.6% of stoichiometric equivalent); * live culture with 960 mg/L NO3 (120% of stoichiometric equivalent); 9 live culture under aerobic conditions. Based on kinetic testing results, CT degradation in the test bottles can be described as a first-order model with respect to CT concentration as follows
-
dM ) kXC Vdt
(1)
where M and V are the CT mass (mg) and liquid volume (L) in the test bottles, respectively. C is the aqueous CT concentration (mg/L), X is the biomass concentration (g VSS/ L), and k is the first-order rate constant with respect to CT (L/g VSS-h). Figure 3 compares the k values from batch tests for the three cultures at different initial growth substrate concentra1754
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tions and with vitamin B12 addition. The k value for the propanediol culture (3.7 L/g VSS-h) is three to six times greater than for the other cultures. The dextrose culture has a slightly higher k value than the acetate culture. The results also show that the addition of 10 mg/L vitamin B12 enhances CT degradation rates by about three times for the propanediol culture, six times for the dextrose culture, and 10 times for the acetate culture. Again, the presence of the growth substrate did not affect the CT degradation for the dextrose and propanediol cultures, and the presence of nitrate inhibited CT degradation for the acetate/nitrate culture. Our model (eq 1) does not include an effect of growth substrate concentration because it is not significant, nor does it include the effect of nitrate in the acetate/nitrate culture because the data are not sufficient to determine it. Intracellular Vitamin B12 and CT Degradation for the Different Cultures. For the systems operated in the anaerobic/aerobic sequence, the vitamin B12 content was 75 ( 6 µg/g VSS, 16 ( 3 µg/g VSS, 4.5 ( 2 µg/g VSS, and 22 ( 3 µg/g VSS for the propanediol, dextrose, acetate, and propionaldehyde cultures, respectively. The propanediol culture operated under anaerobic-only conditions had the highest specific vitamin B12 content of 121 ( 10 µg/g VSS. Figure 4 shows a plot of the CT degradation first-order rate constant (k) versus the vitamin B12 content for the five cultures. There is a nearly a linear relationship between the k value and the vitamin B12 content with the higher vitamin
reported for a methanogenic consortium fed with a mixture of organic compounds (11). Van Eekert et al. (8) showed that the addition of substrate only slightly enhanced CT removal for methanogenic granular sludge. In contrast, an acetogen Acetobacterium woodii showed a 3-fold increase in the specific CT degradation rate in the presence of growth substrate (fructose), compared to no growth substrate addition (5).
FIGURE 4. CT first-order degradation rate constant versus intracellular vitamin content for different cultures.
Effect of Nitrate. Nitrate was found to inhibit CT degradation for our denitrifying culture. This is similar to results with a denitrifying consortium developed from soil samples from a contaminated site (4) and Shewanella putrefaciens 20 (29) but not to Pseudomonas sp. strain KC (1). In our test, the nitrate inhibition was proportional to the initial nitrate concentration. Initial degradation rates were different, indicating that the removal of nitrate to a low level is not required before CT degradation. Our testing, however, did not routinely include nitrate removal measurements, and we did not explore the mechanism of interaction between nitrate and the CT reactions. Intracellular and Extracellular Factors in CT Degradation. Extracellular factors in our fermentative cultures did not show significant CT degradation. In contrast, a denitrifier, Pseudomonas sp. strain KC (30) and a methanogen, Methanosarcina thermophila (15), have been shown to produce extracellular agents that can rapidly degrade CT.
FIGURE 5. Sustaining CT degradation with the propanediol culture: 9 CT concentration (µg/L) and [ percentage of the cumulative CT added that remains as CF. No propanediol was added during the test. Biomass in the test bottle: 415 mg VSS/L. For each CT spike, 100 µg/L CT was added. B12 content corresponding to higher k values for the five cultures. The highest vitamin B12 (121 µg/g VSS) in propanediol culture under anaerobic-only conditions showed the highest k value (6.1 L/g VSS-h). For the cultures grown under sequential anaerobic/aerobic conditions, the propanediol culture had the highest vitamin B12 content (75 µg/g VSS) and highest k value (3.7 L/g VSS-h), while the propionaldehyde culture had a much lower vitamin B12 content (22 µg/g VSS) and k value (1.2 L/g VSS-h). The acetate culture with the least amount of vitamin B12 had the lowest CT degradation rate. Sustaining CT Degradation with the Propanediol Culture. Figure 5 shows CT degradation with the propanediol culture over 2-week’s CT spiking without propanediol addition. For each spiking, CT was added at about 100 µg/L and complete removal occurred within 5 h. There was no significant difference in the CT degradation rate between the first and the last spikes. CF accumulated with repeated spiking but also was partially degraded. When CF is plotted as of the cumulative CT added, it can be seen that the fraction of CF slightly decreased (Figure 5) during the test. The CF was 10% of the CT added after first spike, but the fraction gradually decreased to 6% at the end of the test. This change was presumably caused by an increase in the CF transformation rate in the later phase as the CF concentration increased over the test period.
Discussion Effect of Growth Substrate. The results of this study show that different growth substrates can develop cultures with different CT degradation performance. However, the presence of the growth substrate had no significant effect on CT degradation. The same lack of growth substrate effect was
It has also been shown that cell free extracts from a methanogenic culture rapidly degrade CT (11). Thus it is clear that biological molecules, perhaps more than live microorganisms, are required to mediate CT degradation under reduced conditions. The significant CT degradation in the autoclaved control bottles with the propanediol culture demonstrated that heatresistant factors contribute to CT degradation by the propanediol culture. However the degradation rate in the autoclaved culture was only about 10% of that in the live propanediol culture, so the presence of enzymes or other heat-labile molecules is important. These combined results imply that the CT degradation is mediated by microbial products but is not an integral part of cellular metabolism. CT Degradation Rates. The kinetic results of this work can be compared to CT degradation rates in other studies, using our first-order model and a rate coefficient of 3.7 L/g VSS-h for the propanediol cultures. At the highest CT concentration tested in our study (500 mg/L), we observed a specific CT degradation rate of about 350 µmol/g VSS-day, which is among the highest reported. Van Eekert et al. (8) reported that specific CT degradation rates for three methanogenic granular cultures ranged from 1.2 to 1.9 µmol/g VSSday at 30 °C. At the same initial CT concentrations, our culture is predicted to have a rate of 740-1180 µmol/g VSS-day. A methanogenic culture operated at 35 °C had a specific CT degradation rate of 27 µmol/g VSS-day at a CT concentration of 7 µmol/L (11), compared to the model prediction of 621 µmol/g VSS-day at the same CT concentration. An acetatefed denitrifying consortium had a first-order CT degradation rate constant of 0.08 L/g VSS-h (31), which is less than the value for the denitrifying culture in this work and much less than that for the propanediol culture. On the other hand, a denitrifying bacterium, Pseudomonas sp. strain KC, has a first-order rate constant of 188 L/g VSS-h at 20 °C (32) (assuming 0.5 g protein per g VSS in the bacteria) during its exponential growth phase, which is much higher than that for the propanediol culture. Vitamin B12 Production and Its Effect on CT Degradation. The high vitamin B12 content for both of the propanediol cultures can be explained by the fermentation pathway for VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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propanediol (20, 21, 23) as shown below.
The initial step of the fermentation is the dehydration of 1,2-propanediol to propionaldehyde, which strictly requires coenzyme B12 or vitamin B12. Propionaldehyde is then fermented into propionate and n-propanol in equimolar amounts, with electrons internally balanced. The bacteria obtain energy from transformation of propionaldehyde to propionate. No vitamin B12 is required for the fermentation of propionaldehyde and its metabolites. Vitamin B12 synthesis has been linked to anaerobic growth on propanediol, compared to sugars, including glucose and glycerol. Toraya et al. (21) reported that Klebsiella pneumoniae ATCC 8724 synthesized more vitamin B12 in a glycerol and 1,2-propanediol medium (90 µg/g dry cell) than in a glycerol medium (35 µg/g dry cell) or in a glucose medium (7 µg/g dry cell). Furthermore, Hosoi et al. (20) reported that Propionibacterium freudenreichii grown in a 1,2-propanediol medium produced two or four times as much as vitamin B12 in comparison with that in a glycerol or glucose medium. In this study, we have shown that there is a distinct reduction in vitamin B12 production when a mixed culture fed propanediol is switched to propionaldehyde, which leads to a significant reduction in CT degradation rate. The propionaldehyde culture, derived from the propanediol culture, grown under the same operating conditions, fed with the same amount of substrate, and yielding the same metabolites, contained approximately one-quarter as much vitamin B12 and had about one-third the CT first-order degradation rate constant after 3 months of operation. A linear relationship between CT degradation rates and aqueous vitamin B12 concentration has been observed by Chiu et al. (1995) for a vitamin B12 amended microorganismfree system reduced by titanium citrate. In the system, vitamin B12 concentrations were used at the low range (0.15-1.45 µg/L). The CT degradation was found to follow zero-order kinetics with respect to CT concentration. The vitamin B12based specific CT degradation rate can be inferred from the paper at 3.8 mg CT/mg vitamin B12-h with 1.25 mM titanium citrate addition, as compared to 0.86 mg CT/mg vitamin B12-h for our biological system at their test concentration of 31 µg/L. The difference could be mainly caused by much stronger reducing conditions created by titanium citrate in their system. A positive relationship between CT degradation rate and intracellular vitamin B12 content was not observed by Van Eekert et al. in a methanol-grown methanogenic sludge (8). Their study showed that a 1.5-2-fold higher vitamin B12 content did not lead to an increased CT degradation rate compared to sucrose or VFA-fed methanogenic sludge. This observation may have been due to the very high vitamin B12 content in all the methanogenic sludges so that vitamin B12 was not a limiting factor. Their methanogenic cultures had an intracellular vitamin B12 content from 450 to 970 µg/g VSS, compared to 75 and 121 µg/g VSS for our propanediol cultures. Methanogenic cultures also contain significant amount of cofactor F430, which is an effective catalyst for CT transformation (17). Hashsham (33) also suggested a vitamin B12 saturation, although their study worked with cultures amended by extracellular vitamin B12. They found that amending vitamin B12 to 1 µM (1.4 mg/L) greatly improved 1756
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the CT degradation rate, but very little benefit was gained when the vitamin B12 was increased from 5 to 10 µM. CT degradation rates with cultures involving intracellular vitamin B12 may be limited by several factors, including the vitamin B12 content, the electron transport system for vitamin B12 reduction, the accessibility of vitamin B12, and other enzymes that might involved. In our study, the results strongly indicate that vitamin B12 was a limiting factor. However, a methanogenic sludge with higher vitamin B12 content developed by Van Eekert et al. (8) had a lower CT degradation rate than ours, suggesting that factors other than vitamin B12 can be limiting. One possible factor could be the accessibility of CT to intracellular vitamin B12. Effect of Anaerobic/Aerobic Exposure on the Performance of CT Degrading Bacteria. No matter what the limiting factors are, the CT degradation rate is directly proportional to the biomass concentration. For a system fed with a given amount of growth substrate, a cost-effective operating strategy would be to maximize the amount of CT-degrading biomass grown. Vitamin B12 can only be effectively produced under anaerobic conditions (22), generally with very low biomass yield. However, some facultative bacteria such as Propionibacterium freudenreichii (21) and some genera of Enterobacteriaceae (22) can grow aerobically and synthesize vitamin B12 anaerobically. The presumption in our anaerobic/ aerobic sequence operation was that vitamin B12-yielding bacteria could grow under aerobic conditions using the metabolites from fermentation of propanediol. Since more growth is possible, the anaerobic/aerobic sequence operation has the potential for producing a greater CT degradation capability than fermentation alone. The performance of the sequential anaerobic/aerobic and the anaerobic-only propanediol-fed cultures depends on both their specific CT degradation rate and their biomass concentration. The CT degradation rate constant was 6.1 L/g VSS-h for the anaerobic-only culture, nearly twice that (3.7 L/g VSS-h) of the anaerobic/aerobic system. However, for the same amount of propanediol fed, the biomass produced was six times higher at 405 mg/L for the anaerobic/aerobic culture compared to the anaerobic-only culture (65 mg/L). Thus, the anaerobic/aerobic culture has a CT degradation rate (µg/L-h) that is about 3.3 times that for the anaerobiconly culture. The higher biomass yield makes up for the lower specific CT degradation rate for the anaerobic/aerobic culture. Sustaining CT Degradation. CT degradation was sustained for 2 weeks with the propanediol culture without propanediol addition and without a significant decrease in the degradation rate (Figure 5). A few reports have shown sustained CT degradation independent of growth substrates. For a culture grown with DCM as the sole energy and carbon source and enhanced with vitamin B12, repeated spikes with CT over 200 days without DCM showed no obvious change in CT degradation when an electron donor (H2) was available (18). Novak et al. (15) also reported consistent CT degradation rates over a 12-day period in cell-free liquid from a Methanosarcina thermophila culture. The observations on CT degradation without growth substrate addition have significant implications for the operation of a CT-degrading system with propanediol feeding. Since CT degradation is not related to the growth substrate utilization other than through production of CT-degrading biomass, continuous application of the growth substrate is apparently not necessary. Further work is needed to determine the maximum amount of CT that can be degraded by the biomass per unit of growth substrate consumed. This study shows the important role of intracellular vitamin B12 in CT-degrading cultures. The results show that high-performance CT-degrading cultures can be developed by selectively feeding substrate that promotes a metabolic
pathway requiring vitamin B12. 1,2-Propanediol was found to support cultures with higher intracellular vitamin B12 content and greater CT degradation rates than cultures grown with dextrose, acetate/nitrate, and propionaldehyde. The sequential anaerobic/aerobic process benefits from the larger amount of biomass that can be grown in the aerobic step using the fermentation products. This process can easily be applied in above-ground reactors.
Acknowledgments Funding for this study was provided by U.S. Department of Energy under Cooperative Agreement #DE-FCO1-95EW55084 through the University of Washington Consortium for Risk Evaluation and Stakeholder Participation (CRESP). The content of this paper does not necessarily represent the views of the Department of Energy.
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Received for review August 11, 1999. Revised manuscript received January 25, 2000. Accepted January 28, 2000. ES990930M
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