Environ. Sci. Technol. 1991. 25, 1068- 1074
Effects of Electron Acceptors on Halogenated Organic Compound Biotransformations in a Biofilm Column Gordon D. Cobbt and Edward J. Bouwer"
Geography and Environmental Engineering, The Johns Hopkins University, Baltimore, Maryland 2 1218 The transformability of trihalomethanes, carbon tetrachloride, l,l,l-trichloroethane, 1,2-dibromomethane, tetrachloroethylene, dibromochloropropane, and chlorinated benzenes was evaluated by a biofilm utilizing a mixture of primary electron acceptors (oxygen, nitrate, and sulfate). These compounds at concentrations commonly found in groundwater were continuously administered for 4 years to a biofilm column reactor that resembled polluted groundwater environments. Acetate was the primary substrate to support microbial growth. Sequential biofilm zones of aerobic respiration, denitrification, and sulfate reduction developed within the column. Transformation of the halogenated aliphatic compounds coincided with the onset of sulfate reduction in the column. The temporary absence of nitrate and sulfate in the column feed decreased the steady-state removals for several of the halogenated aliphatic compounds. These results suggest that sulfate was an important primary electron acceptor. Aerobic transformations of the chlorinated benzenes were incomplete due to the rapid depletion of oxygen and limited aerobic zone at the column inlet.
Introduction Synthetic organic compounds are difficult to control in the environment; their widespread usage, uncontrolled disposal, and chemical/physical properties make them common groundwater contaminants. Biotransformation can be a significant process affecting the fate of organic contaminants in the subsurface. Diverse and metabolically active microorganisms have been found in both shallow and deep aquifers, and they have been observed to transform some commonly recognized contaminants (I,2). Increased knowledge of the factors that affect the biotransformation of potentially hazardous organic micropollutants is needed. An important environmental factor influencing biotransformations is the electron acceptor utilized by microorganisms for deriving energy from an electron donor. Microorganisms preferentially utilize electron acceptors that provide the maximum free energy during respiration (3). Of the common electron acceptors used by microorganisms, oxygen typically provides the most free energy to microorganisms during electron transfer. Use of nitrate, Mn(IV),Fe(III), sulfate, and carbon dioxide typically yields decreasing amounts of free energy during electron transfer according to the order listed. There is limited capacity for mixing in the subsurface, and rates of oxygen replenishment from the atmosphere are extremely slow following consumption by aerobic reactions. The coupling of mass transport and microbial reaction in the subsurface results in temporal and spatial gradients of electron acceptor concentrations. These temporal and spatial changes in oxidation-reduction conditions can play a critical role in determining the stability, speciation, mobility, and persistence of a large number of organic and inorganic chemicals in the subsurface (4). This study was undertaken to Present address: ENVIRON Corp., 4350 North Fairfax Drive, Arlington, VA 22203. 1088 Envlron. Scl. Technol., Vol. 25, No. 6, 1991
identify the importance of electron acceptor availability to biotransformation of certain trace organic contaminants. Experimental studies with biofilms using a single electron acceptor showed that halogenated aliphatic compounds, such as trichloroethylene and chloroform, could be transformed under methanogenic and sulfate-reducing conditions; however, these compounds tended to persist under aerobic conditions (5). Several chlorinated benzenes could be biotransformed under aerobic conditions, but were stable in the absence of molecular oxygen (6). Basic laboratory studies that simulated polluted groundwater were conducted to evaluate biotransformation of trace levels of halogenated aliphatic and aromatic compounds by an acetate-supported biofilm column in the presence of a mixture of primary electron acceptors. The transformation patterns observed suggest that sulfate was an important primary electron acceptor.
Experimental Section Continuous-Flow Biofilm Column Studies. A continuous-flow, laboratory-scale biofilm column reactor (Figure 1)with a mixture of oxygen, nitrate, and sulfate as primary electron acceptors was operated for more than 4 years (February 1985 to October 1989) to evaluate biotransformation of some halogenated organic compounds commonly found in groundwater. Bacteria were provided acetate as primary substrate to evaluate the biotransformation of trace levels of halogenated organic compounds as secondary substrates. The glass column was 100 cm long X 1.1cm i.d. and filled with 3-mm glass beads. A defined sterile mineral salts solution containing 60 mg/L acetate as primary organic substrate, 10 mg/L ammonia, 8 mg/L oxygen (approximate air saturation concentration), 10 mg/L nitrate, and 10 mg/L sulfate as primary electron acceptors, and a mixture of halogenated organic compounds was continuously applied to the column in an upflow mode. The column feed composition is shown in Table I. The acronyms that are subsequently used to refer to the halogenated organic compounds in the text are given in Table I. Glass beads were used as the biofilm support media in order to minimize sorptive effects while creating porous media flow conditions to simulate the subsurface. Primary settled sewage obtained from the Back River Wastewater Treatment Plant (Baltimore, MD) was used to seed the column. This sample was diluted 1:lOOO with the phosphate buffer solution (Table I) and introduced into the column for a contact period of 48 h. A concentrated aqueous solution of acetate and halogenated organic compounds was then delivered to the column from a 20mL gas-tight syringe driven by a syringe pump at a rate of approximately 1.0 mL/130 mL of mineral salts solution to initiate biofih growth. This yielded feed concentrations between 20 and 120 pg/L for the halogenated organic compounds listed in Table I. A gas-tight syringe was utilized to reduce volatilization losses, since many of the halogenated organic compounds are highly volatile in water. A peristaltic pump maintained a mineral salts feed rate of 0.45 mL/min to the column, resulting in a packed-bed detention time of 1.5 h. All tubing, fittings,
0013-936X/91/0925-1068$02.50/0
0 1991 American Chemical Society
Biofilm Column Reactor
Perisleitic Pump
8' Influent Sampling Vial
Influent Feed Reservoir
Flgure 1. Schematic diagram of mixed electron acceptor biofilm column reactor.
Table I. Biofilm Column Reactor Feed Composition compound
concn, mg/L
Primary Substrate acetate
60
Electron Acceptors oxygen nitrate (as NO3-) sulfate (as SO4,-)
8a 10 10
Secondary Substrates bromodichloromethane (BDCM) bromoform (BF) carbon tetrachloride (CT) chloroform (CF) dibromochloromethane (DBCM) dibromochloropropane (DBCP) 1,2-dichlorobenzene (12DCB) 1,3-dichlorobenzene (13DCB) 1,4-dichlorobenzene (14DCB) ethylene dibromide (EDB) tetrachloroethylene (TeCE) 1,2,4-trichlorobenzene (124TCB) l,l,l-trichloroethane (TCA)
b b b b b b b b b b b b b
Inorganic Nutrients NH4Cl MgS04.7HzO NalSOd NaNOj CaCl, NaHC03 FeC13.6H20 FeC1,.4H20 MnC12.4H20 Na2Mo04.2Hz0 cuc1, Na,Se03 H3BO3 ZnC1, AlCl3 CoC12.6H20 Ni(N03)2
29.8 22.5 1.85 13.8 27.5 20 0.25 1.0 0.125 0.025 0.0075 0.005 0.0125 0.0125 0.0125 0.0125 0.0125
Buffer pH 7.1 KHOPO,
K2HPO; NazHP04.7H20
8.5 21.8 33.4
"This value represents the saturated dissolved 0, concentration. *All influent secondary substrate concentrations were between 20 and 120 pg/L.
syringes, and vials were made of either Teflon or glass in order to minimize sorption. The column was operated in an environmental chamber a t 22.5 "C in the dark to prevent growth of photosynthetic microorganisms.
Influent and effluent samples were collected frequently in continuous-flow-through bottle reservoirs without headspace in order to prevent volatilization losses of the halogenated organic compounds. The sampling reservoirs and tubing were autoclaved weekly to prohibit microbial growth external to the column. Concentration profiles with column depth were determined from samples collected at several of the sampling ports located along the length of the column (Figure 1). Samples were taken from the sampling ports with a B-D Yale, 2-mL glass syringe (Becton-Dickinson, Rutherford, NJ) starting at the port closest to the column effluent and progressing to the port closest to the column influent. The samples were withdrawn at a volumetric rate closely matched to the feed flow rate to minimize disturbance of the concentration profiles. The samples were analyzed for halogenated organic compounds, acetate, oxygen, nitrate, and sulfate. Effect of Nitrate and Sulfate on Biotransformations. The addition of both nitrate and sulfate as primary electron acceptors to the biofilm column was discontinued after 881 days of operation (July 1, 1987) to observe the response to halogenated organic compound biotransformations. Nitrate addition to the feed solution was resumed after 1005 days of operation (November 2, 1987). Sulfate addition to the feed solution was resumed after 1040 days of operation (December 7,1987), thus restoring the normal feed solution. Application of the normal feed solution continued throughout the remainder of the column operation. Stop-Flow Experiments. The feed flow to the biofilm column was occasionally discontinued for up to 50 h in order to investigate the effect of longer detention time on the halogenated organic compound biotransformations. The short-term semibatch conditions within the biofilm column allowed additional contact time between the biofilm and halogenated organic compounds. Because of the limited pore volume within the reactor, it was not possible to collect large enough samples from the reactor ports a t the end of the stop-flow contact time without significantly disturbing the concentration profiles. Therefore, an indirect method was used to obtain the halogenated organic concentrations within the column at the end of each stop-flow period. A bromide tracer was added to the feed after the feed flow to the column was resumed, and the column effluent was continuously monitored for 4.5 h to determine exiting porewater concentrations. One effluent sample was taken every 6 min and contained approximately 2.5 mL of liquid volume, 2 mL of which was diluted 1 : l O and used for pentane extraction gas chromatography to assay for halogenated compounds, and then for ion chromatography analysis for bromide. This first effluent sample analyzed after flow was reinitiated reflected the concentrations in approximately the last 2.5 cm3 of liquid volume a t the effluent end of the column. Each subsequent sample was used to reflect the concentrations progressively closer to the influent end of the column. A pore volume of 2.5 cm3 corresponded to a column segment of approximately 7.14 cm in length. Following the stop-flow period, the breakthrough behavior of bromide was used to estimate the contribution of reinstated feed levels of the halogenated organic compounds to the measured effluent concentrations. Consequently, the concentrations of the halogenated organic compounds in the biofilm column prior to reinitiating feed flow were determined by subtracting the concentration due to the influent feed (bromide tracer contribution) from the measured effluent concentration. The concentrations calculated for each 7.14-cm column segment were assigned Environ. Sci. Technol., Vol. 25, No. 6, 1991
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to the midpoint of each segment. Details of this analysis are presented elsewhere (7). Analytical Techniques. The halogenated organic compounds were assayed by pentane extraction gas chromatography with electron capture detection (ECD) with a detection limit of 0.1 pg/L in water, according to the method of Henderson et al. (8). Column influent and effluent samples were taken in 20- and 60-mL serum bottles (Pierce Chemical Co., Rockford, IL), respectively. Samples from porta along the column (2 mL) were diluted with Milli-Q water (1:lO) in a 20-mL serum bottle. All serum bottles were sealed without headspace with Teflon-faced silicone septa (Wheaton Products, Millville, NJ) and capped with aluminum seals (Wheaton). Samples were extracted by replacing 1 mL of sample with 1 mL of pentane and were placed on a shaker table for 30 min to provide adequate contact time for the organic compounds to partition into the pentane phase. 1,1,2-Trichloroethane was utilized as the internal standard for quantification. A 1-pL sample of the pentane extract was injected onto a fused-silica capillary column (Hewlett-Packard 5890A gas chromatograph with a J&W Scientific DB-530 m long X 0.32 mm id., 0.25-pm film thickness chromatographic column). Helium was used as the carrier gas, while a 95% argon/5% methane mixture was used as the ECD make-up gas. Injected pentane extracts were run at an initial temperature of 35 "C for 2 min, after which the temperature was raised to 57 "C at 2.5 "C/min and then at 200 "C at 8.0 "C/min. The signal from the electron capture detector was recorded by a reporting integrator (Hewlett-Packard 3393A). Anionic inorganic compounds (Cl-, Br-, SO4*, and NO3-) and acetate were assayed by ion chromatography with a detection limit of approximately 0.1-0.5 mg/L (Dionex Corp. 2010i ion chromatograph module). Samples (1mL) collected either from the column influent, effluent, or ports along the reactor were passed through 0.45-pm filters (Gelman Science Acrodiscs) prior to injection onto the anion ion chromatography column. The eluant consisted of a mixture of 0.0028 M NaHC03 and 0.0022 M Na2C03. Anion detection was accomplished by using an electrical conductivity detector in conjunction with anion suppression. The external standard method was employed for quantitation where sample peak areas were compared with areas of known concentration for each compound. Dissolved oxygen content and pH were analyzed by procedures given in ref 9.
Results Within 1day after the mixed solute feed to the column was started, the effluent concentrations of dissolved oxygen and nitrate decreased to below detection limits. Therefore, aerobic heterotrophs and denitrifiers immediately colonized the glass beads in the column. About 2 months was required for the aerobic and denitrifying biofilm growth to attain an apparent, visual steady-state thickness. The relatively high acetate loading allowed the microorganisms to rapidly consume the dissolved oxygen. Dissolved oxygen was completely depleted in the bulk liquid solution prior to the first sampling port, located at 2.5-cm depth from the influent end of the column. Significant sulfate utilization did not occur in the biofilm column until after approximately 90 days of operation (Figure 2). A t this time, black biomass accumulated downstream of the denitrifying biofilm (initially located roughly 30-40 cm from the inlet). This black growth is often associated with biological sulfide production, giving visual confirmation that sulfate respiration occurred in the biofilm column. 1070 Envlron. Scl. Technol., Vol. 25, No. 0, 1991
0
20
60
40
80
100
120
140
160
Time of Operation (days)
Flgure 2. Effluent concentrationversus time for sulfate, CT, BDCM, and BF (average influent concentrations 11.0 f 1.0 mg/L, 46 f 16 pg/L, 72 f 13 pglL, and 74 f 24 wg/L, respectively). i
"
"
"
"
'
-
-
i
u 0
0
a,
U
20
m
E 0
cs
0
0
20
40
60
80
100
Column Length (cm)
Flgure 3. Acetate, nitrate, and sulfate concentration profiles within biofilm column after 875 days of column operation.
Over the first several months of operation, nearly complete breakthrough was observed for the halogenated organic compounds in the acetate-supported biofilm column with oxygen and nitrate as predominant electron acceptors. About the same time that sulfate started to be utilized as an electron acceptor in the biofilm column (Figure 2), significant transformations of CT, BDCM, and BF were observed (Figure 2). These results suggest that sulfate respiration was a favorable redox condition for biotransformation of the halogenated aliphatic compounds. Transformation of DBCP was observed after a ---month lag period, suggesting the involvement of a biological mechanism following an acclimation period. Since the mixed electron acceptor biofilm column was continuously operated for over 4 years, long-term and steady-state utilization data were obtained for the halogenated organic compounds. A comparison of the steady-state removal efficiencies at various times throughout the study period is provided in Table 11. Although average influent concentrations varied due to different preparations of organic stock solutions, percent removals remained relatively constant with time. Several of the halogenated compounds were transformed to near or below the detection limit (BF, CT, BDCM, DBCM, and DBCP). Removal efficiencies for the other halogenated aliphatic compounds studied, CF, TCA, EDB, and TeCE, ranged between 39 and 64%. The chlorinated benzenes studied (13DCB, 12DCB, 14DCB, and 124TCB) were also partially transformed with removal efficiencies ranging between 40 and 55%. Concentrations of acetate, nitrate, and sulfate measured in the bulk liquid within the biofilm column after 875 days of operation are given in Figure 3. Oxygen concentrations are not shown because oxygen could not be detected in the
200
-
Effluent
-I
c
I
200
C
.-
L
150 0 C
s
100
r
V
.
750
850
950
1050
"
1150
I
750
950
850
Days of Column Operation
1050
1150
Days of Column Operation
Figure 4. Influent and effluent concentrations versus time for BF in biofilm column in the presence and absence of nitrate and sulfate.
Figure 6. Influent and effluent concentrations versus time for EDB in biofilm column in the presence and absence of nitrate and sulfate.
-
z I 1)
.-
-D-
100
0
. d
gE
80
Steady-State (833-890 days) No NO3 or SO4 (912-998 days) Restore NO3 + SO4 (1084-1112 d)
0
5
60
u
40 0 u-
e
20
In 04 750
850
950
1050
1150
0
c
0
20
Days of Column Operation
Figure 5. Influent and effluent concentrationsversus time for DBCP in biofilm column in the presence and absence of nitrate and sulfate.
bulk liquid a t a sampling point 2.5 cm downstream from the column inlet. The primary electron acceptor profiles indicate that aerobic respiration was limited to the first few millimeters of the column, a zone of denitrification extended to a depth of roughly 5 cm into the column, and the remainder of the biofilm in the column was dominated by sulfate-reducing microorganisms. Most of the acetate utilization occurred within the first 20 cm of the column and was coupled to the utilization of the electron acceptors (Figure 3). Although excess acetate was available, no evidence for methanogenic activity was observed within the biofilm column throughout the study period. The effect of the primary electron acceptor concentrations on steady-state secondary utilization of the halogenated organic compounds was examined by removing both nitrate and sulfate from the normal column feed solution for several months starting a t day 881. Within 2 weeks after nitrate and sulfate were no longer available as primary electron acceptors, the effluent concentrations of BF (Figure 4) and DBCP (Figure 5) increased. Similar concentration increases in the effluent also occurred for CT, BDCM, DBCM. All of these halogented aliphatic compounds were normally transformed to near or below the detection limit in the presence of nitrate and sulfate. In contrast, the effluent concentrations for compounds that showed only partial removal with the normal feed, such as TCA and EDB (Figure 61, remained unaffected during the absence of nitrate and sulfate. The concentration fluctuations observed for the organic compounds in the influent samples were partially attributable to a "pulselike" syringe pump delivery system. Periodic preparation of new organic stock feed solutions also contributed to changes in influent concentrations. The biofilm activity damped out most of the observed influent concentration variations in effluent samples.
40
60
80
100
Column Length (cm)
Figure 7. Effect of nitrate and sulfate absence on BF concentratlon profiles in biofilm column.
20/
.....m... ...&..
-*-
W
Steady-S;(ydays) , No NO3 or SO4 (912-998 days) Restore NO3 + SO4 (1084-1112 d) New Steady-State (1153-1295 d)
,
0 0
20
40
80
80
100
Column Length (cm)
Figure 8. Effect of nltrate and sulfate absence on ED6 concentratlon profiles in biofilm column.
After nitrate addition to the feed solution was resumed at 1005 days of operation, no apparent changes in removals were detected for any of the halogenated organic compounds over the next 1-month period (Figures 4-6). After readdition of sulfate as a primary electron acceptor on day 1040, an approximately 1-3-month lag period occurred prior to a detectable increase in percent removals for BF (Figure 4), CT, BDCM, and DBCM. DBCP required the longest time, approximately 6 months, to achieve its prior steady-state effluent concentration (Figure 5). The effect of the temporary absence of nitrate and sulfate on the BF concentration profiles within the column is shown in Figure 7. In comparison to the normal steady-state profile, BF utilization decreased over the last 90 cm of the column without nitrate and sulfate available to the biofilm. This region of the column was dominated by the sulfate-reducing biofilm. The BF utilization profile gradually approached the previous steady-state profile after the normal feed with nitrate and sulfate was resumed Environ. Scl. Technol., Vol. 25, No. 6, 1991
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Table 11. Steady-State Removal in Multiple Electron Acceptor Biofilm Column over Time of Operation compound
390-490 days out' % rem
in"
CT BDCM DBCM BF DBCP EDB TCA TeCE CF l2DCB 13DCB 14DCB 124TCB
in'
500-615 days out' % rem
in"
76 f 29 135 50 111 f 41 148 f 56 136 f 50 96 f 34 79 f 41 38f 15 116 f 43
*
BDLb BDLb BDLb BDLb 12 f 5 43 f 9 36 f 9 21 f 5 70 f 12
>99 >99 >99 >99 91 f 5 56 f 18 55 f 26 46 f 25 39 f 25
72 f 27 138 f 40 116 f 34 160 f 44 145 f 42 100 f 28 86 f 28 40 f 14 117 f 32
Halogenated Aliphatics BDLb >99 80 f 22 BDLb >99 168 f 36 BDLb >99 127 f 30 BDLb >99 177 f 43 13 f 3 91 f 3 169 f 36 42 f 6 59 f 13 120 f 23 34 f 4 60 f 14 102 f 23 22 f 6 44 f 25 45 f 13 57 f 4 51 f 14 160 f 28
BDLb BDLb BDLb BDLb 15 f 5 43 f 8 38 f 4 21 f 6 66 f 13
>99 >99 >99 >99 91 f 3 64 f 10 63 f 13 52 f 19 59 f 11
44 f 14 46 f 16 61 f 22 31 f 10
23 f 6 25 f 6 34 f 8 15 f 4
48 f 21 45 f 23 44 f 25 51 f 20
50 f 14 48 f 14 66 f 18 33 f 9
Chlorinated Benzenes 26 f 7 48 f 21 54 f 13 27 f 6 44 f 21 51 f 11 38 f 9 43 f 20 71 f 14 17 f 4 47 f 20 31 f 9
24 f 5 24 f 5 34 f 7 15 f 4
55 f 53 f 52 f 50 f
" Values, in pg/L, represent average concentrations and their standard deviations. from the column feed durine this Deriod. l 80
"
"
"
"
'
--O-
-+--A-
--D-
10 hours 20 hours 30hours 40 hours 50 hours Steady-State
20
40
14 14 14 19
in'
1360-1390 days out' % rem
58 f 8 121 f 20 129 f 18 132 f 19 156 f 31 156 f 34 78 f 11 59 f 10
BDLb BDLb BDLb BDLb 19 f 4 79 f 22 41 f 4 35 f 4 2 f .4c 27 f 4
83 f 79 f 89 f 67 f
16 15 17 15
47 f 7 46 i 15 53 f 8 35 f 5
>99 >99 >99 >99 88 f 3 50 f 18 47 f 9 40 f 12 -1400 43 f 14 41 f 15 4 0 f 15 47 f 14
Below detection limit. This compound was removed
steady-state profiie for up to 50 h. This behavior of 13DCB again supports the hypothesis that sorption was not responsible for the removals and suggests that a biological mechanism was responsible for the loss of EDB and TCA with increasing contact time.
t
$
0
725-810 days out' % rem
60
80
100
Column Length (cm)
Flgure 9. EDB concentratlon profiles measured in biofilm cdumn during stop-flow experiments.
(days 1084-1112). The data averaged over the period from day 1153 to 1295 indicated that the new steady-state profile had become similar to the previous steady-state response. The absence of nitrate and sulfate in the column feed did not significantly alter the utilization of EDB within the biofilm column (Figure 8). The measured decrease in EDB concentration occurred prior to the first sampling port, suggesting transformation under aerobic conditions. The concentration profiles for 13DCB remained constant along the length of the column. Since most of the halogenated compounds studied have a smaller octanol/water partition coefficient than 13DCB (IO), the negligible loss of 13DCB rules out sorption onto biomass as a likely loss mechanism for the removals of the other halogenated organic compounds. As the packed-bed detention time was increased from 1.5 to 50 h in the stop-flow experiments, the extent of removal for EDB (Figure 9) increased. For contact times of 20 h and longer, the active biofilm in the initial 50 cm of the column reduced the EDB concentration below detectable levels. A 50-h reaction time resulted in marginal improvement in the biotransformations in comparison to the 40-h data. The acetate and electron acceptor concentration profiles within the column (Figure 3) suggest limited microbial reactions in the last 20 cm of the column. This was substantiated by the persistence of EDB in the last 20 cm of the column during the stop-flow experiments (Figure 9). Furthermore, this region of the column lacked a visible biofilm. The stop-flow response for TCA was essentially identical with that for EDB. However, the stop-flow profiles for 13DCB did not change relative to the 1072 Environ. Sci. Technol., Vol. 25, No. 6, 1991
Discussion A biofilm column was operated with acetate (primary substrate) and a mixture of terminal electron acceptors, which resulted in the development of three distinct biofilm zones: aerobic, denitrifying, and sulfate-reducing regions. Several of the halogenated organic compounds were transformed in this biofilm column. The nearly complete transformation of CT, BDCM, DBCM, BF, and DBCP coincided with the onset of sulfate reduction in the column. The temporary absence of nitrate and sulfate as primary electron acceptors caused the effluent concentrations to significantly increase for these five halogenated compounds. The reduced removals occurred mainly in the portion of the column that was predominantly characterized by sulfate reduction during the normal column feed. The effluent concentrations once again decreased only after the normal feed with sulfate was resumed, indicating the importance of sulfate reduction to the transformations. Several months of operation were required for effluent concentrations to decrease to prior steady-state values. These lag periods after the normal feed with nitrate and sulfate was resumed on day 1040 were consistent with the delay in biotransformation observed when the column was initially started (Figure 2). Therefore, the loss of sulfate-reducing biomass was most likely the cause of the reduced removals when sulfate was absent. A significant portion of BF was removed during the absence of nitrate and sulfate (Figure 4) in contrast to DBCP (Figure 5) and EDB (Figure 6), which indicates complete breakthrough during this time period. The reason for this difference was not confirmed, but perhaps BF was being selectively used as a terminal electron acceptor in the absence of sulfate. Bae et al. (11, 12) demonstrated that decreasing the availability of a primary electron acceptor can enhance the removals of halogenated aliphatic compounds if reductive dehalogenation is the initial metabolic reaction. In their studies of CT reductive dechlorination in a denitrifying biofilm, the CT behaved as an electron acceptor that competed with the nitrate for available electrons inside the bacterial cells. CT removal
increased when nitrate (primary electron acceptor) was removed from the column for a period of 8 days, presumably because the bacteria used more of the CT as an electron acceptor. Clearly, more information is needed on the chemical requirements for biotransformation of halogenated organic compounds. Removal efficiencies for the other halogenated aliphatic compounds, CF, TCA, EDB, and TeCE, were less and ranged from 39 to 64% with the normal packed-bed detention time of 1.5 h. The environmental conditions required for transformation of the halogenated aliphatic compounds in the mixed electron acceptor biofilm column were consistent with those previously reported for biofilm columns with single electron acceptors (13,14). The highly reducing environments of sulfate respiration and methanogenesis favored the transformation of the trace halogenated aliphatic compounds. Chlorinated benzenes were demonstrated to be biotransformed in past work with an entirely aerobic biofilm column (6). The partial removals of chlorinated benzenes in the mixed electron acceptor column are attributed to biotransformation kinetics that were too slow to yield complete removal in the limited aerobic zone that developed. Furthermore, the excess acetate available at the column inlet provided little selective pressure for aerobic heterotrophic microorganisms to utilize the chlorinated benzenes at their low concentrations. The energy that was available from metabolism of the chlorinated benzenes was negligible compared to the energy provided by the acetate. The temporary lack of nitrate and sulfate in the column feed did not significantly influence the removal efficiencies for EDB and TCA, suggesting the involvement of a fermentative microorganism. A Clostridium sp. was recently isolated from the effluent of an anaerobic suspendedgrowth reactor that was able to biotransform TCA, CF, and CT by reductive dehalogenation (15). This fermentative bacterium wtm obligately anaerobic and was unable to carry out dissimilatory sulfate or nitrate reduction. On the basis of these characteristics, this microorganism may have been responsible for some of the biotransformations observed in the mixed electron acceptor biofilm column. Significant removal of BF and EDB occurred near the column inlet, which contained predominantly aerobic and denitrifying biofilms (Figures 7 and 8). Furthermore, the fraction removed in the inlet segment of the column did not change in the absence of nitrate and sulfate as electron acceptors. EDB was mineralized in soils under aerobic conditions (16, 17). Consequently, aerobic biotransformation is a possible pathway for some of the losses observed. Additional work needs to be conducted to clarify the role of specific microorganisms and redox conditions for the removals. The influence of electron acceptor conditions on the change in concentration of specific halogenated organic compounds was the primary focus of this research. Therefore, no conclusions can be drawn about the extent of mineralization resulting from the disappearance of the compounds. Of the possible physical, chemical, and biological mechanisms for the removals, a biological mechanism was the most likely. The acclimation times required and larger extent of removal in the stop-flow experiments implicate a biological mechanism. Sorption onto biomass was an unlikely loss mechanism, as evidenced by the negligible loss of 13DCB within the column during normal operation (1.5-h detention time) and during the stop-flow studies (up to 50-h detention time). A possible chemical loss mechanism is nucleophilic substitution between HSand the halogenated aliphatic compounds (18). Stoichio-
metric conversion of 10 mg/L sulfate in the feed would produce approximately 0.10 mM sulfide under sulfatereducing conditions in the biofilm column. However, the half-life of EDB at 25 "C in the presence of 0.67 mM HSwas found to be 40 days (19). Consequently, the nucleophilic substitution reaction rate is too slow with the 0.10 mM sulfide produced by the biofilm to explain the removal of EDB within the 1.5-h detention time. Hydrolysis rates for the compounds are also too slow for this chemical reaction to be important in the biofilm column (20,21). A combination of biological and chemical mechanisms cannot, with certainty, be ruled out; further work is needed to elucidate the relative importance of chemical and biological factors. Limited information was obtained on reaction intermediates and products. When CF was omitted from the feed, significant CF was detected in the effluent (Table II), suggesting its formation within the biofilm column. Reductive dechlorination of CT to CF is the most likely explanation for this formation of CF (13). The production of a volatile hydrocarbon in EDB radiotracer studies is additional evidence for reductive dehalogenation [data not shown (7)]. The removals observed in the mixed electron acceptor biofilm column provide valuable insight into relative rates of biotransformation for the halogenated compounds. Biotransformation rates were the highest for CT, BDCM, DBCM, BF, and DBCP. These compounds were nearly completely transformed during normal operation of the biofilm column with 1.5-h packed-bed detention time. Slower biotransformation rates were observed for CF, TCA, EDB, and TeCE as these compounds were only partially transformed within the 1.5-h contact time. The longer detention time of between 20 and 50 h achieved during stop-flow operation of the biofilm column was sufficient for nearly complete transformation of EDB and TCA. A biofilm model, presented elsewhere, was used to evaluate the biotransformation rate parameters (22). The relative biotransformation rates for the halogenated aliphatic compounds in the mixed electron acceptor biofilm column were comparable to the kinetic results for a single acetate-supported sulfate-reducing biofilm column (14). The biotransformation rates for the chlorinated benzenes were markedly slower in this study compared to those from a single aerobic column (6). Oxygen limitation and excess primary substrate acetate were probably responsible for the reduced apparent biotransformation rates for the trace concentrations of the chlorinated benzenes.
Conclusions This study has helped to clarify the biotransformation of halogenated organic compounds in the presence of a mixture of primary electron acceptors. Sequential use of oxygen, nitrate, and sulfate as primary electron acceptors occurred within the continuously fed biofilm column. Oxygen and nitrate were completely utilized by the biofilm within 1day of initiating the feed to the column, whereas, a 3-month lag period occurred before sulfate was utilized as electron acceptor. Nearly complete transformation of the one-carbon halogenated aliphatic compounds was observed following the onset of sulfate reduction within the 1.5-h liquid detention time. Removals were less for the two- and three-carbon halogenated compounds. The temporary absence of nitrate and sulfate in the column feed decreased the removals for several of the halogenated aliphatic compounds. Readdition of sulfate was required for effluent concentrations to once again decrease to below detection. Therefore, sulfate reduction was a favorable redox condition for many of the biotransformations, and Environ. Sci. Technot., Vot. 25, No. 6, 1991
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sulfate was needed to maintain the biotransformations over the long term. Shorbterm increases in the liquid detention time for up to 50 h allowed nearly complete utilization of the two- and three-carbon halogenated compounds. On the basis of this behavior, removals were attributed to biotransformation, rather than sorption or chemical reactions. Aerobic biotransformations of the chlorinated benzenes were incomplete due to slow kinetics in the limited aerobic zone at the column inlet. These results demonstrated the importance of electron acceptor availability and detention time on the biotransformation of subsurface organic contaminants. Registry No. BDCM, 75-27-4; BF, 75-25-2; CT, 56-23-5; CF, 67-66-3; DBCM, 124-48-1; DBCP, 96-12-8; 12DCB, 95-50-1; 13DCB, 541-73-1; 14DCB, 106-46-7; EDB, 106-93-4; TeCE, 12718-4; 124TCB, 120-82-1; TCA, 71-55-6; 02, 7782-44-7.
Literature Cited Ghiorse, W. C.; Wilson, J. T. Adv. Appl. Microbiol. 1988, 33, 107-172. Thomas, J. M.; Ward, C. H. Enuiron. Sci. Technol. 1989, 23, 760-766. Stumm, W.; Morgan, J. J. Aquatic Chemistry, 2nd ed.; Wiley: New York, 1981. Barcelona, M. J.; Holm, T. R.; Schock, M. R.; George, G. K. Water Resour. Res. 1989, 25, 991-1003. Bouwer, E. J.; McCarty, P. L. Appl. Environ. Microbiol. 1983, 45, 1286-1294. Bouwer, E. J.; McCarty, P. L. Environ. Sci. Technol. 1982, 16,836-843. Cobb, G. D. Modeling and Experimental Simulations of Organic Contaminant Biotransformations in Subsurface Environments. Ph.D. Dissertation, T h e John Hopkins University, Baltimore, MD, 1989. Henderson, J. E.; Peyton, G. R.; Glaze, W. H. In Zdentification and Analysis of Organic Pollutants i n Water;
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Keith, L. H., Ed.; Ann Arbor Science Publishers: Ann Arbor, MI, 1976; pp 105-112. (9) Standard Methods for the Examination of Water and Wastewater, 17th ed.; American Public Health Association: Washington, DC, 1989. (10) Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and Biology; Wiley: New York, 1979. (11) Bae, W.; Odencrantz, J. E.; Rittmann, B. E.; Valocchi, A. J. J . Contam. Hydrol. 1990, 6 , 53-68. (12) Bae, W.; Rittmann, B. E. Effects of Electron Acceptor and Electron Donor on Biodegradation of CCl, by Biofilms. Proceedings of the 1990 Environmental Engineering Conference, ASCE, 1990; pp 390-397. (13) Bouwer, E. J.; McCarty, P. L. Appl. Environ. Microbiol. 1983,45, 1295-1299. (14) Bouwer, E. J.; Wright, J. P. J . Contam. Hydrol. 1988,2, 155-169. (15) Galli, R.; McCarty, P. L. Appl. Environ. Microbiol. 1989, 55,837-844. (16) Pignatello, J. J. Appl. Environ. Microbiol. 1986,51,58&592. (17) Pignatello, J. J. J.Environ. Qual. 1987, 16, 307-312. (18) Morrison, R. T.; Boyd, R. N. Organic Chemistry;Allyn and Bacon: Boston, MA, 1974; pp 452-491. (19) Barbash, J. E.; Reinhard, M. Environ. Sci. Technol. 1989, 23, 1349-1358. (20) Mabey, W.; Mill, T. J . Phys. Chem. Ref. Data 1978, 7, 383-415. (21) Jeffers, P. M.; Ward, L. M.; Woytowitch, L. M.; Wolfe, N. L. Environ. Sci. Technol. 1989, 23, 965-969. (22) Cobb, G. D.; Bouwer, E. J. Model Verification of Organic Contaminant Biotransformation by Biofilms in Porous Media under Steady-State Mixed Electron Acceptor Conditions. Submitted for publication in Water Res.
Received for review August 23,1990. Revised manuscript received November 26,1990. Accepted January 23,1991. This research was supported by the National Science Foundation through Grant ECE-8451060 (Presidential Young Investigator Award).
