Environ. Sci. Technol. 1994, 28, 746-752
Reductive Dechlorination of Tetrachloroethylene by a Chlorobenzoate-Enriched Biofilm Reactor Babu 2. Fathepure'gt and James M. Tledje*
Department of Civil and Environmental Engineering, and Center for Microbial Ecology and Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824 This study evaluates the potential and technical feasibility of treating chloroaliphatics, common groundwater contaminants, using a specialized microbial consortium under fixed-film conditions. The reactor was developed using 3-chlorobenzoate (3-CB) as a substrate under anaerobic conditions and the enrichment that harbored an unusual dechlorinator, Desulfomonile tiedjei DCB-1. The dechlorination rate of both PCE and 3-CB increased with increasing flow rates up to 50 mL/h. The maximum observed dechlorination rates of PCE and 3-CB fed at 6.0 and 1000 pM were 2.0 and 414 pmol L h-l, respectively. This corresponds to a PCE consumption rate of 3.7 nmol h-l (mg of protein)-' i88.9 pmol (g of protein)-l day1]. The rate of PCE dechlorination increased from 2.0 to 10.3 pmol L-l h-' when the influent PCE was increased from 6.0 to 120 pM, respectively; however, concentrations of 60 pM and above damaged reactor performance. PCE was mainly converted to TCE and cis- and trans-DCE at all the tested flow rates. Vinyl chloride (VC) was never detected, thus suggesting dechlorination of PCE to nonchlorinated products. Although the PCE dechlorination activity of the biofilm was dependent on 3-CB,the activity could be sustained for 4-5 days on cheaper substrates such as acetate or benzoate after a 1-day pulse feeding of 3-CB. In addition to PCE dechlorination, the biofilm also dechlorinated other compounds such as chloroform (CF) and 1,1,2-trichloroethane(1,1,2-TCA). The present study is important, since D. tiedjei was previousely shown to dechlorinate many other chloroaromatic and aliphatic compounds. Introduction In recent years bioremediation has received considerable attention as a cost-effective technology for treating contaminated groundwater, sludge, soil, and sediment. Highly chlorinated aliphatic compounds such as PCE, TCE, CF, and other chlorinated compounds with 1-2 carbon atoms are among the most pervasive groundwater contaminants. Most of these chemicals have been extensively used in dry-cleaning and degreasing operations. Chlorinated chemicals have entered soil and groundwater through accidents and leaks at chemical dump sites. These compounds have relatively high water solubility (e.g., 1100 mg/L for TCE and 9300 mg/L for CF at 25 "C) and are highly mobile in aquifer and soil material. Many simple aliphatic halides are well known hepatotoxic and mutagenic agents. Haloolefins, such as VC, have been implicated in causing angiosarcoma of the liver in
* Address correspondence to this author at his present address: Research and Engineering Department, Environmental Services Division, Conoco, Inc., Ponca City, OK 74603. t Department of Civil and Environmental Engineering. 1 Center for Microbial Ecology and Department of Crop and Soil Sciences. 748
Environ. Sci. Technol., Vol. 28, No. 4, 1994
occupationally exposed humans. Also, higher incidences of cancer of the pancreas, lung, and brain were noted in individuals exposed in the work place (1). Other commonly detected groundwater contaminants such as 1,l-dichloroethylene (l,l-DCE),TCE, PCE, CF, and 1,1,2-TCAhave been shown to cause cancer in experimental animals (2, 3). Halogenated chlorinated hydrocarbons such as PCE, TCE, CF, and 1,1,2-TCA alone do not support microbial growth in aerobic environments. However, many of these chemicals are anaerobically transformed to lesser chlorinated compounds by fermentative, sulfate-reducing, and methanogenic bacteria (4-14). The rate of dechlorination declines markedly as the number of chlorines decreases, which results in the accumulation of lesser chlorinated compounds. Thus, reductive dehalogenation by anaerobic bacteria, although seemingly useful may yield VC and CF, which are more dangerous than the parent compounds. Recent research by Freedman and Gossett (10) and de Bruin et al. (14) has shown that PCE can be completely dechlorinated to ethene and ethane, harmless and environmentally acceptable products. In addition, Vogel and McCarty (9) have shown evidence for the formation of l4CO2from V4C1PCE. While these results are encouraging, more research is needed to establish the types of microorganisms and conditions that produce nontoxic and environmentally safe products under anaerobic conditions. Even though compounds such as TCE, DCEs, or methylene chloride (MC) can be metabolized by methanotrophs, the efficacy, economics, and success in achieving complete degradation under in-situ conditions is site specific and not assured. Therefore, research is warranted to explore other microbial systems that may be more effective in treating chlorinated compounds in the field. In the present study, we have developed a continuouslyfed, up-flow biofilm reactor that was inoculated with an enrichment that was previously shown to mineralize 3-CB to chloride and gaseous end products (15, 16). This enrichment harbored a unique anaerobic organism, Desulfomonile tiedjei DCB- 1, which catalyzes reductive dechlorination of 3-CB, pentachlorophenol, and PCE (17). The objectives of the study were to evaluate a fixed-film reactor colonized with a selected dechlorinating community, to enhance the rate of reductive dechlorination of PCE, and to explore the cosubstrate requirements for this process. Conditions that increase rates of reductive dechlorination are particularly important since previous studies show this process to be relatively slow, hence a bottleneck when coupling to aerobic processes for complete mineralization. Experimental Procedure
Chemicals. All the chlorinated chemicals (>98 % pure) were purchased from Aldrich Chemical Co., Milwaukee, WI. Vinyl chloride (0.2 mg/L in methanol) was obtained 0013-936X/94/0928-0746$04.50/0
0 1994 American Chemical Soclety
Flgure 1. System used to feed prlmary substrate, PCE. and reductant to anaerobic biofilmreactor and to collect influentand effluentfor analysis.
from Supelm, Inc., Bellafonte, PA. 3-Chlorobenzoateand benzoate were purchased from Sigma Chemical Co., St. Louis, MO. All the other chemicals used were of reagent grade. Establishment of Biofilm Reactor. A continuouslyfed, up-flow, anaerobic biofilmreador was developedusing a 3-CB-mineralizingenrichment that contained D. tiedjei DCB-I. The biofilm was developed employing a glass column of 50 cm long X 2.5 cm i.d. (Beckman Instrument Inc.) filled with 0.3 cm diameter glass beads as bacterial support. The column was equipped with a water jacket for maintaining the biofilm at 35 "C (Figure 1). Prior to seeding the reactor, the enrichment was grown in batch on a modified RAMM medium ( I S ) supplemented with 0.91.0 mM 3-CB as the primary carbon source. The culture was then pumped into theglass column and allowed to colonize the beads for approximately 1week a t 35 "C. The column had been flushed with N Pprior to inoculation. After 1week of incubation, the reactor was continuously fed with sterile RAMM containing 3-CB as the primary carbon source a t a rate of 2.0 mL/h. Feeding was by a 50-mL gas-tight, glass syringe (Hamilton Co., Reno, NV) mounted on a syringe pump (syringe pump-22, Harvard Apparatus, South Natick, MA). Under these feed conditions and a t 35 'C, the biofilm required approximately 4 months to attain apparent steady state. Apparent steady state is defined as the time when daily measurements of optical density and 3-CB dechlorination activity of the reactor effluent were stable. The measurement of optical density wasdoneat 600nm usinga UVspectropbotometer
(Shimadzu UV-160; Shimadzu Corp., Kyoto, Japan). All the tubings, fittings, and connectors were made of either glass or Teflon. The reactor was covered with aluminum foil to prevent the growth of photosynthetic organisms. The feed to the reactor was prepared as follows: Amounts of 5OmLof RAMMwere dispensed anaerobically into several 100-mL capacity serum bottles, which were sterilized by autoclaving for 20 min, and stored at 4 "C until used. Justprior tofeedingthecohnnn,an appropriate amountof3-CB (0.1 mL = -1mM)fromasterileaqueous stock was added into each serum bottle. The entire contents were then withdrawn into a 50-mL gas-tight glass syringe and delivered to the reactor by a syringe pump. After the biofilm had attained apparent steady state, the RAMM was amended with a known amount of a chlorinated target compound obtained from a freshly prepared aqueous stock. A working aqueous stock solution was prepared by dissolving neat PCE, CF, or 1,1,2-TCA in organic free, deionized water in a serum bottle with no headspace. The bottle was closed with a Teflon-lined rubber septum and aluminum cap. The stock solution was prepared fresh each week. Effluent samples were collected on a daily basis in a flow-through bottle with no headspace. The bottles were closed using Teflon-lined septa and aluminum caps. Influentsamples (5 mL) were collected witha 10-mLglass syringe via a port located near the entrance of the reactor as shown in Figure 1. Dechlorination Studies. Initial studiesdealt with the dechlorination of PCE, CF, and 1,1,2-TCA, fed at low Envlrm. Scl. Technol.. Val. 28. NO. 4. 1994 l b l
concentrations (16 pM or 1 mg/L). The biofilm was acclimated to each of the tested chlorinated compounds separately for 3-4 weeks prior to testing its ability to catalyze dechlorination activity. These studies were conducted at 2 mL/h flow rate (column retention time of 37.5 h) using a 50-mL glass syringe and syringe pump. The dechlorination of PCE at varied flow rates was accomplished by filling several 50/60-mL capacity plastic syringes (Becton Dickinson and Co., Rutherford, NJ) with 50 mL of RAMM containing 3-CB and attaching them to a mechanical vacuum extractor (MVE; Centurion, International Inc., Lincoln, NE) as shown in Figure 1. Additional syringes were added to achieve higher flow rates. The speed of the extractor was calibrated such that about 5 mL of medium was constantly delivered into the biofilm reactor per hour by each syringe. The medium from all syringes was delivered collectively into the biofilm reactor via a 10-mL capacity serum bottle, where an appropriate amount of PCE-saturated deionized water was continuously injected from a gas-tight glass syringe. Also, low concentrations of sodium sulfide (-125 mg/L) as a reducing agent was added to the same bottle to maintain anaerobic conditions in the substrate feed and column. The biofilm was continuously fed in this manner for more than 2-3 weeks for each tested flow rate to obtain apparent steady-state growth. Both influent and effluent samples were collected on a daily basis for analyses. Alternate Electron Donor. A batch study was conducted in which 50 mL of RAMM was supplemented with 6 pM PCE plus acetate (3 mM), methanol (3mM), glucose (1 mM), benzoate (1 mM), or 3-CB (1 mM) as the sole source of carbon and electrons. The bottles (160-mL capacity) were inoculated with 1.0 mL of the reactor effluent that was collected from a periodic sloughing of biomass. Microscopic observation revealed the presence of the distinct long rod typical of D.tiedjei-like organisms (16). Bottles were closed with Teflon-lined rubber stoppers and aluminum caps. All the bottles were incubated static in the dark, at 35 "C for 40 days. Inoculated bottles amended with no added substrate served as the control. At the end of the experiment, all the bottles were analyzed for PCE dechlorination. Pulse Feeding Experiment. Instead of 3-CB in the column feed, benzoate (1 mM) or acetate (3 mM) was substituted as the sole source of carbon and electrons. An appropriate amount of PCE-saturated water was mixed in-line with RAMM. The final concentration of PCE in the reactor feed was maintained at 6-12 pM. Effluent was collected and analyzed for PCE on a daily basis until the reactor's ability for dechlorination dropped markedly. At this time, the feed was respiked with 1 mM 3-CB in place of benzoate or acetate and continuously fed for the next 24 h. At the end of 24 h, the 3-CB was replaced by 1 mM benzoate or 3 mM acetate. Analytical Techniques. Identification and quantification of chlorinated ethylenes were accomplished by using a 5890-A Hewlett Packard gas chromatograph (Hewlett-Packard, Co., Palo Alto, CA) and workstation. The GC was equipped with a HP capillary column (30 m long X 0.53 mm i.d. X 2.6 pm film thickness) and a flameionization detector. An aqueous 5-mL sample was introduced on to the GC column with the help of a Tekmar purge-and-trap unit (Model 4000) equipped with Tenax TA adsorbent. The sample was purged with helium for 16 min at 30 mL/ min and desorbed at 180 "C for 4 min. 748
Environ. Scl. Technol., Vol. 28, No. 4, 1994
Table 1. Biofilm Reactor Characteristics Determined before and after 2.5 Years of Continuous Reactor Operation Initial Conditions working reactor volume (V,), cm3 235 void volume ( VO),cm3 90 porosity (e = Vo/ V,) 0.38 av volume of bead (vb), cm3 0.014 volume occupied by glass beands 145 (Vbt = Vt - VO),cm3 primary carbon source, at 0.9 mM 3-CB reactor temperature, "C 35 feed flow rate (Q), cm3/h 2-70 Conditions at Steady State" biofilm volume ( X v ) ,cm3 15.0 biomass (Xg), mg of protein 0.54 (1.08 g of dry weight) 75.0 void volume (V,), cm3 porosity (c = Vg/Vt) 0.32 superficial velocity (&/A),cm/h 14.3 effective velocity (Vetf = (Q/e)A),cm/h 44.6 kinematic viscosity (v), cm2/h 36.0 Reynolds no. (Re = Vef&)/v 0.32 Determined after the reactor flowrate was 70 mL/h and at steadystate growth.
Chlorinated chemicals were separated using helium as a carrier gas at 6 mL/min. The temperature program was 35 "C for 8 min, increased to 200 "C at 4 "C/min, and followed by an isothermal period of 5 min at the end. The injector and detector temperatures were 220 and 300 "C, respectively. Chlorinated compounds were identified and quantified using freshly prepared external standards treated like samples. 3-Chlorobenzoateand benzoate were determined on a high-pressure liquid chromatograph equipped with a Hiber LiChrosorb 10 pm RP-18 column. The column was coupled to a Hitachi Model 100-40 spectrophotometer with an Altex 100-55 flow cell. The mobile phase consisted of water:acetonitrile:phosphoric acid in the proportion of 66:33:0.1. Peaks were estimated by UV absorption at 230 nm at a flow rate of 1.5 mL/min. Determination of Reactor Biomass. After the biofilm had attained apparent steady state, biomass was determined by breakthrough of a NaBr (50mg/L) conservative tracer and by total protein estimation. The data from the tracer study were used to determine the porosity of the reactor and, hence, the accumulated biovolume. Bromide was measured using a Dionex 2000i ion chromatograph equipped with a Dionex Ionpak HPIC AS4A separation column. Aliquots of 4.5 mL (3 mL/h) of column effluent were continuously collected for 40 h by a fraction collector. Protein Analysis. At the end of 2.5 years of continuous reactor operation under steady state, the reactor was opened, and all the glass beads and column fittings were washed vigorously with l x RAMM salts to recover the biomass. The biomass was concentrated by centrifugation and analyzed for total protein by a modification of the method of Lowry (19). Results
Biofilm Development. Biofilm development was extremely slow. Approximately 2 months of continuous feeding of the reactor with RAMM containing 3-CB was needed before visible growth was seen, and more than 4 months of continuous feeding was necessary to obtain apparent steady-state growth. The column characteristics before and after the growth of the biofilm are shown in Table 1. Due to bacterial growth, the reactor porosity decreased from 0.38 to 0.32, which corresponds to a void
Table 3. Biodegradation of Chlorinated Aliphatics in DCB-1 Biofilm Reactora
Table 2. Biodegradation of PCE in DCB-1 Biofilm Reactor. influent (pM) effluent (pM) % target compd PCE PCE TCE c-DCE t-DCE VCc disappeared* 1.57 2.60 6.33 3-chlorobenzoate
1000
0.06 ND 0.02 0.14 0.10 0.03 ND 0.07 0.15 0.10 0.05 ND 0.21 0.62 0.52 3-chlorobenzoate benzoate 13.0
ND
79.6 86.5 77.9 % substrate removed
98.7
chemicalb PCE CF 1,1,2-TCA
influent (pM)
effluent (pM)
% disappeared
major products
6.33
0.21 2.35 6.75
96.7 68.8 37.0
TCE, DCEs CH2Clz 1,S-DCA
7.54
10.7
The reactor was operated at a flow rate of 2 mL/h (HRT = 37.5 h). Data are the means of samples analyzed daily for 2-3 weeks. b The reactor was fed with one chlorinated target compound for 2-3 weeks before the influent and effluent sampleswere taken for analysis. a
0 The reactor was operated at a flow rate of 2 mL/h (HRT = 37.5 h). Data are the average values of samples analyzed daily for 3-4 weeks. b % target compounddisappeared represents the unaccounted portion of the added PCE. Detection limits for VC = 0.01 mg/L, and ND = not detected or below detection limit.
Y4/ f - 5
volume of 75 cm3 and a biofilm volume of 15 cm3. At 70 mL/h feed rate, the calculated flow rate of medium over the cross-sectional surface of the column (u)was 14.3 cm/h and average linear velocity (Veff)over the void area was 44.6 cm/h. The calculated Reynolds number of 0.32 indicates the laminar flow of the medium (20). Biomass estimated by the tracer study indicated a relatively high biofilmvolume (15 cm3)in comparison with biomass determined by the protein assay (540 mgheactor). This amount of protein corresponds to 1.08 g dry wt and 2 X 1012 microorganisms, based on conversion factors by Balkwill et al. (21) and Nester et al. (22). The higher biofilm volume (15 cm3) determined by the tracer study could be due to the presence of a copious amount of microbial extracellular polysaccharides, glycocalyx, and other inorganic precipitates, consistent with other studies (23-25). Dechlorination Experiments. After the biofilm had attained steady state, the reactor was tested for its ability to dechlorinate PCE at low concentrations, 1.57-6.0 pM (0.26-1.0 mg/L). Approximately, 80% of the added PCE was removed within 37.5 h hydraulic retention time (HRT; Table 2). On a molar basis, only 20% of the PCE was recovered as TCE, DCEs, and residual PCE. We analyzed for VC, but none was found. Our detection limit was 0.01 mg of VC/L. Therefore, it appears that PCE underwent reductive transformation to products beyond VC, yielding unknown nonchlorinated products. Such products could not be measured by the analytical system used in this study. More than 98% of the influent 3-CB was removed during the same retention time (Table 2). No accumulation of benzoate occurred, suggesting complete mineralization of the primary carbon source (3-CB). The 3-CBenriched biofilm was also able to reductively dechlorinate other chlorinated aliphatics such as CF and 1,1,2-TCA. Methylene chloride and 1,2-dichloroethane (1,2-DCA)were the major dechlorinated products of CF and 1,1,2-TCA, respectively (Table 3). Experiments were conducted to evaluate PCE removal at increasing liquid flow rates. The biofilm was continuously fed from 2 to 70 mL of medium per hour with RAMM containing 3-CB (-0.9mM) and PCE (-6.0 pM) at 35 OC. At each of the tested flow rates, the biofilm was continuousely-fed with 3-CB and PCE for approximately 2-3 weeks to obtain apparent steady state prior to collecting samples for analysis. PCE was transformed to TCE and cis- and trans-DCE at all the tested flow rates. The rate of PCE dechlorination increased from 160 to
P
e
Y
C
l
P =
!4
s *-I
/
-
Effl. cis DCE
kG+=--
0 0
10
Flow Rate (ml/h)
Flgure2. Concentrationsof influent PCE and effluent PCEdechkrlnated products as a function of column flow rate.
2000 nmol L-1 h-1 when the reactor flow was increased in a step-wise fashion from 2 to 50 mL/h (Table 4). On a biomass basis, the dechlorination rates correspond to 0.3 and 3.7 nmol h-l (mg of protein)-l at 2 and 50 mL/h flow rates, respectively. Further increases in the flow rate (5070 mL/h) did not result in an enhanced rate of dechlorination. Although, PCE dechlorination rate increased with increasing flow rates, the percent removal (efficiency) fell at flow rates above 25 mL/h. Gas-chromatographic (GC) analysis of effluents showed the formation of TCE and cis- and trans-DCE at all the tested flow rates (Figure 2). At flow rates from 25 to 70 mL/h, a complete mass balance between influent PCE and effluent dechlorinated products was achieved. Potential formation of VC was carefully monitored. No accumulation of VC was detected at all the tested flow rates. Losses due to volatilization were not expected because the effluents were collected using a continuous flow-through vial system with no head space (Figure 1). In addition, effluents were immediately analyzed for VC. The reactor samples were also monitored simultaneously for removal of the primary carbon source, 3-CB. Similar to PCE, the rate of 3-CB dechlorination increased from 26 to 414pmolL-*h-l when the biofilm was fed at increasing flow rates from 2 to 50 mL/h, respectively. These rates correspond to 0.049 and 0.77 pmol h-l (mg protein)-', respectively. Further increase in flow rate from 50 to 70 mL/h resulted in reduced 3-CB dechlorination activity (Table 4). Effect of High Concentrationof PCE. Experiments were conducted to study the effects of high concentrations of PCE on the reactor performance. The amount of PCE dechlorinated increased with increased influent PCE concentration (Table 5). At 5.0 and 33 pM influent Envlron. Scl. Technol.. Vol. 28,
No. 4. 1994 749
Table 4. Effect of Volumetric Loading Rate on PCE and 3-CB Dechlorination Rate and Efficiency8
3-CBdechlorination
PCE dechlorination flow rate* (Q/h)
HRT
2
37.5 18.8 12.5 9.38 7.50 6.25 4.69 3.75 3.00 2.50 1.50 1.07
(e)
dechlorination (Co/Ce) iuM 6.12 5.77 5.39 6.30 6.06 6.23 4.27 5.07 3.26 3.58 3.10 2.15
% removal 96.7 97.0 95.6 94.7 93.2 97.5 92.2 84.5 53.5 56.4 54.4 41.0
rate (Co - Ce/8) 0.16 0.31 0.43 0.67 0.81 1.00 0.91 1.35 1.09 1.40 2.07 2.00
% removal
dechlorination (CO- CA PM 987 865 863 847 836 891 740 632 615 673 621 210
rate (Co - ce/e,
98.7 100.0 96.8 91.5 91.0 91.5 84.5 74.6 74.1 75.2 69.4 23.5
26.3 46.1 69.0 90.3 111.4 142.5 157.9 168.5 205.0 269.2 414.0 196.3 Data are the means of 10-15 samples. * The reactor was continuously fed at each tested flow rate for more than 2-3 weeks prior to sample collection for analysis. The concentration of PCE was around 6.0 pM (1 mg/L) and 3-CB was 0.9 mM. 4 6 8 10 12 16 20 25 30 50 70
Table 5. Effect of High Concentration of PCE on Reductive Dechlorination Ability of Biofilms influent PCE (Co) WM
effluent PCE (Cd PM
removal rate (Co- C,/e)
dechlorination product(s)
5.25 32.6 65.1 102
3.1 27.1 55.7 91.2
2.00 5.07 8.85 10.3
TCE, DCEs TCE, t-DCE TCE TCE
Data are the means of 7-10 samples. 3-Chlorobenzoate(0.8mM) was used as a primary carbon source. The flow rate was 70 mL/h (HRT = 1.07 h).
concentration of PCE, both TCE and DCEs (cis and trans) were detected in the effluents whereas at 65 and 102 pM PCE, TCE was the only dechlorinated product detected. Although the initial rate of PCE dechlorination increased from 2 to 10pmol L-l h-lf3.7 to 19 pmol h-l (mg protein)-11 with increasing PCE concentrations in the feed, the percent removal fell significantly. Only 14 and 11% of PCE was removed at 65 and 102 pM influent PCE, respectively. Presumably, more PCE could be removed with extended residence time. Prolonged, continuous feeding of the reactor (>3 weeks) at 65 and 102 uM PCE resulted in severe damage to the biofilm’s ability to transform both PCE and 3-CB to less chlorinated compounds. It took more than 2 months of continuous feeding of the reactor with no PCE in the feed to rejuvenate the biofilm’s dechlorination activity to the previous level. Evaluation of Alternate Electron Donors. In order to reduce the cost of this dechlorination process, a cosubstrate less expensive than 3-CB is needed. A batch experiment was conducted with sloughed biofilm where substrates other than 3-CB were tested for their ability to sustain PCE dechlorination. Substrate removal of PCE occurred only in bottles supplemented with 3-CB as the carbon source (data not shown). Very little TCE formation was noted in bottles supplemented with acetate, methanol, glucose, or benzoate. These results confirmthe importance of 3-CB for PCE removal by this dechlorinating community. To reduce dependency on the more expensive 3-CB we investigated pulse feeding the reactor with 3-CB while maintaining carbon and electron flow with other cheaper substrates. Benzoate and acetate were chosen as alternate substrates because both are degradation products of 3-CB 750
Envlron. Sci. Technol., Vol. 28, No.
4, 1994
- 1 0
2
4
6
8
0
2
4
6
8
Time (days) Flgure 3. Effect of primary substrate on PCE dechlorination. The substrate fed and its period of feeding is shown by the horizontalbar.
by DCB-1, and both are less expensive and readily available. When the biofilm was fed with benzoate (1mM) or acetate (3mM) at 50 mL/h as the primary carbon source, PCE dechlorination activity began to decline after 4-5 days. However, the activity resumed immediately after a 24-h feed of 3-CB (Figure 3). With benzoate as the primary carbon source, approximately 55 76 of the influent PCE (14.6 pM) underwent dechlorination at the maximum rate of 4.7 pmol L-’ h-l. This activity persisted for approximately5 days, after which time PCE dechlorination activity declined until the next pulse of 3-CB. Similarly, with acetate as the primary substrate, the maximum dechlorination of PCE was sustained for 4 days. Approximately, 54 % of the influent PCE (8.6pM)underwent dechlorination at the maximum rate of 3 pmol L-’ h-l. This experiment was repeated three times at two different flow rates; twice at 2 mL/h and once at 50 mL/h. The results were similar in the three experiments, except at 2 mL/h flow rate, the added PCE was completely removed. These results show that 3-CB is required to sustain PCE dechlorination, but that the activity can be maintained in the absence of added CB for approximately 5 days on alternate substrates likely in the 3-CB degradation pathway.
Discussion The initial development of 3-CB-enrichment biofilm was extremely slow. The reason for this unusually long
time to obtain steady-state growth is not known. The slow growth rate of the 3-CB enrichment and its lack of propensity for attachment may have contributed. Recently, Mueller et al. (26)have shown that initial cell colonization on surfaces was found to depend on many factors such as hydrodynamics, surface free energy, bulk substrate concentration, and cellular properties. An initial adsorption of macromolecules from the bulk liquid onto an inert surface (conditioning film) seems to play an important role in the formation of biofilms. Some conditioning films may reduce or inhibit bacterial adsorption (26,27).Bacteria also have different adsorptive properties specific for attachment media (28). The attached enrichment retained its original dechlorination activity toward both 3-CB and PCE. The majority of the added PCE was removed with no detectable VC in the effluent. Although it was not shown in this study, it is conceived that perhaps further dechlorination of the accumulated DCEs to products such as ethene and ethane occurred at low flow rates since up to 80% of the original PCE was not found in chlorinated volatile products. This conversion to nonchlorinated products has been welldemonstrated in other anaerobic communities (10, 14). Alternatively, the accumulated DCEs might have been further degraded to unknown compound(s) via yet unknown pathways. This observation is important since VC is a carcinogen and commonly encountered intermediate of PCE metabolism in anaerobic environments (9). In addition to PCE, the biofilm also showed the ability to dechlorinate CF and 1,1,2-TCA. This is important since most landfill leachates, industrial effluents, and contaminated groundwaters contain a variety of chloroaliphatic compounds. Although, Fathepure et al. (11)and Cole et al. (18)have recently shown the ability of D. tiedjei DCB-1 to reductively dechlorinate PCE to less chlorinated compounds, the identity of organism(s) in this column responsible for CF and 1,1,ZTCA dechlorination as well as for PCE dechlorination could not be ascertained. Routine microscopic observation of reactor effluents showed the presence of large rods typical of D. tiedjei DCB-1 (16). Furthermore, 3-CB, the selective substrate for strain DCB-1, was required for PCE dechlorination. While this is suggestive of this strain's involvement in dechlorination, we cannot rule out strains with similar biochemical and perhaps morphological properties. The PCE dechlorination rate was enhanced by increasing the reactor flow up to 50 mL/h (HRT = 1.5 h). This rate was almost 100 times higher than that previously measured for this enrichment in batch culture (26 nmol L-l h-l; ref 11). The rate of dechlorination could be further enhanced by increasing the concentration of PCE in the influent (Table 5). However, PCE concentrations of 65 pM (10 mg/L) or greater damaged the biofilm. At higher flow rates (125 mL/h) the percent PCE dechlorination was decreased, but the removal efficiency could be improved by recycling the reactor effluent or by using different adsorbing media. Recently, Fennel1 et al. (29) have shown that by using granular activated carbon as an attachment media, the biodegradation rate of TCE was significantly enhanced and higher quality effluent could be obtained. While our PCE dechlorination rates are substantially higher than the rates found for other microbial systems (11,12),they appear to be lower than the recently reported rates for microbial communities
enriched with PCE as electron acceptor (4,14,30). Much higher rates of PCE removal are expected in the first 5-10 cm of the biofilm reactor bed (9, 14). The rate of 3-CB dechlorination at 50 mL/h flow rate was also faster 10.77 pmol h-l (mg of protein)-l] than the previously reported values for the same enrichment grown in batch. The dechlorination rate of 3-CB by the original enrichment was around 0.3 pmol h-' (mg of protein)-' (15), and later DeWeerd et al. (31) found the rate to be 0.13 pmol h-1 (mg of protein)-' using a defined DCB-1 consortium. The increased rates found in the present study could be due to better growth conditions offered by the continuously-fed, bioreactor system. Effluent analysis by HPLC showed no significant accumulation of benzoate at all the tested flow rates, suggesting that benzoate served as a reductant and a carbon source. PCE was stoichiometrically transformed into TCE and DCEs at flow rates 1 2 5 mL/h. Similar observations were made by Scholz-Muramatsu et al. (4) using biomass from an anoxic fixed bed reactor that was fed with benzoate and high levels of PCE (>1mM). In their study, benzoate was used as electron donor, and PCE was used as electron acceptor. PCE was dechlorinated mainly to cis-1,2-DCE. In an earlier study, using Rhine River sediment, Bosma et al. (30)showed the formation of cis-1,ZDCE from PCE. Only a small amount of VC was detected. Contrary to the previous belief that VC was a major product of PCE under anaerobic condition, recent research has shown the formation of other harmless products, such as ethene and ethane. Freedman and Gossett (10) were able to show a slow conversion of VC to ethene using a methanogenic enrichment culture. Later, DiStefano et al. (32)were able to demonstrate a high rate of PCE conversion to ethene employing the same enrichment. Recently, De Bruin et al. (14) have provided evidence for complete conversion of PCE to ethene and then to ethane using a continuousflow, fixed-bed column reactor with Rhine River sediment and ground granular sludge. Interestingly, complete conversion of PCE to ethane could be achieved only by a combination of Rhine River sediment plus anaerobic granular sludge. 3-Chlorobenzoate was shown to be essential to PCE dechlorination by this microbial community. 3-CB is selective for growth of DCB-1 (16)and induces the PCE dechlorination activity in this strain (18). This later effect is probably responsible for the success of the 3-CB pulse feeding. The pulse feeding strategy should be particularly useful for inducible co-metabolic processes. The dependency of PCE dechlorination activity on 3-CB also indicates that the dechlorinating population is a specific one and not a general one that might be found in systems fed acetate or other common carbon sources. Since most of the affected groundwaters and industrial effluents invariably contain a mixture of highly chlorinated, less chlorinated, and nonchlorinated compounds, bioremediation of such wastes requires both anaerobic and aerobic processes in sequence for the complete destruction of contaminants (13). For such applications, an anaerobic microbial process capable of dechlorinating a wide range of chlorinated hydrocarbons at a high rate is a prerequisite, since reductive processes are usually slow. The present and previous studies ( I 7) indicate that DCB-1 is capable of transforming a variety of chlorinated hydrocarbons including 3-CB, pentachlorophenol,PCE, CF, and 1,1,2-TCA. Based on both redox potential and Environ. Scl. Technol., Vol. 28, No. 4, 1994 751
experimental evidence (10,33-351, the reductive dechlorination steps beyond DCEs are inherently slow. Using bacterial transition metals, Gantzer and Wackett (34) have shown in vitro that a significant portion of PCE was dechlorinated to TCE and cis-1,2-DCE within 1 day of incubation, vinyl chloride after several weeks, and ethylene after several months. A similar observation was made by Freedman and Gossett (10) and Carter and Jewell (35) using anaerobic bacteria. Therefore, it is likely that a bioreactor system that converts PCE to the level of DCEs only would be more cost-effective if the situation demands both a high-rate anaerobic process and an aerobic process.
Vogel, T. M.; McCarty, P. L. Appl. Environ. Microbiol. 1985,49, 1080.
Freedman, D. L.; Gossett,J. M. Appl. Environ. Microbiol. 1989,55, 2144.
Fathepure, B. Z.; Nengu, J. P.; Boyd, S. A. Appl. Environ. Microbiol. 1987, 53, 2671.
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Conclusion
48, 840.
The PCE dechlorinating activity of the enrichment containing D. tiedjei DCB-1 could be established on a fixed-film up-flow reactor, which dechlorinated PCE at a rate 100 times faster than in batch. The HRT could be managed so that no VC was detected. If the flow rate was 1 2 0mL/h, the mass balance indicated that nonchlorinated products may have been produced. If the flow rate was 1 2 5 mL/h, TCE and primarily cis-1,2-DCE were the detected products. The biofilm also transformed CF and 1,1,2-TCAto lesser chlorinated products. This is important since, like PCE, other chlorinated compounds such as CF and 1,1,2-TCA are also resistant to degradation by aerobic bacteria. The biofilm required 3-CB for PCE dechlorination, but the dechlorinating activity could be maintained for 4-5 days on benzoate or acetate after a pulse feeding of 3-CB for 1 day.
Mohn, W. W.; Tiedje, J. M. Microbiol. Rev. 1992,56,482. Cole, J. R.; Fathepure, B. Z.; Tiedje, J. M. Biodegradation submitted for publication. Hanson, R. S.; Phillips, J. A. In Manual of Methods For General Bacteriology; Gerhardt, P., Ed.; American Society for Microbilogy: Washington, DC, 1981; pp 328-364. Characklis, W. G.; Turakhia, M. H.; Zelver, N. In Biofims; Characklis,W. G.,Marshall, K. C., Eds.;John Wiley & Sons, Inc: New York, 1990; pp 265-340. Balkwill, D. L.; Leach, F. R.; Wilson, J. T.; McNabb. J. F.; White, D. C. Microb. Ecol. 1988, 16, 73. Nester, E. W.; Roberts, C. E.; Lidstrom, M. E.; Pearsall,N. N.; Nester, M. T. In Microbiolgy, 3rd ed.; Saunders College Publishing, New York, 1983. Trulear, M. G. Ph.D. Thesis, Montana State University, Bozeman, MT, 1983. Wilderer,P. A.; Characklis,W. G. In Structure and Function of Biofilm, Characklis,W. G . ,Wilderer,P. A., Eds.; Dahlem Workshop Reports. Life Sciences Research Report 46; Wiley-Interscience: New York, 1989. Arcangeli,J. P.; Arvin. E. Appl. Microbiol. Biotechnob 1992,
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This research was supported in part by the Michigan Biotechnology Institute and in part by the State of Michigan's Research Excellence Fund. We thank Ms. Karen L. Clynes and Ms. Laura McCaw for their excellent help in developing and daily maintenance of the biofilm reactor. Also, special thanks are due to Ms. Gabriela Grigorean and Dr. Abbas Fard for their technical assistance. Dr. Lakshmi Bhatnagar of MBI is acknowledged for his interests and help. The authors also thank Drs. Thomas Voice and Craig Criddle for their interest and support and Dr. James R. Cole for assistance with HPLC analysis and useful discussions.
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Carter, S. R.; Jewell, W. J. Water Res. 1993, 27, 607. Received for review October 4, 1993. Revised manuscript received January 7, 1994. Accepted January 10,1994.' ~~~
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Abstract published in Advance ACS Abstracts, February 15,
1994.
