Environ. Sci. Technol. 1994, 28, 2387-2392
High-Rate Bioremediation of Chlorophenol-Contaminated Groundwater at Low Temperatures Kimmo T. Jarvlnen,? Esa S. Melin,t and Jaakko A. Puhakka'*$
Institute of Water and Environmental Engineering, Tampere University of Technology, FIN-33720 Tampere, Finland
Aerobic fluidized-bed treatment was employed for psychrotrophic bioremediation of chlorophenol-contaminated groundwater, Laboratory-scale, continuous-flow reactors were inoculated with nonacclimated activated sludge, the groundwater was amended with inorganic nutrients and a phosphate buffer, and continuous groundwater feed was started at 14-17 "C. Chlorophenol concentrations (in mg/ L) in groundwater were as follows: 7-11 for 2,4,6trichlorophenol, 32-36 for 2,3,4,6-tetrachlorophenol, and 1.8-2.3 for pentachlorophenol. After the startup period, the treatment temperature was gradually decreased to the ambient groundwater temperature (7 "C) and further to 4 "C. Steady-state fluidized-bed remediation at 5-h hydraulic retention time resulted in effluent concentrations of less than 0.003 mg/L of each chlorophenol at all temperatures tested. At 5-7 "C, over 99.9% chlorophenol biodegradation was achieved at a chlorophenol loading rate of 740 mg L-l d-1. Inorganic chloride releases were in conformity with the chlorophenol removals indicating mineralization. In conclusion, this system used higher loading rates than previouslyreported for bioremediation, and the effluent quality was close to drinking water standards. Further, this is the first paper on high-rate bioremediation at ambient groundwater temperatures or lower (4-10 "C).
Introduction Chlorophenols (CPs) and particularly pentachlorophenol (PCP) have been widely used as wood-preservatives. They have polluted surface water and groundwater (GW) at numerous sites. In the environment, CPs may be recalcitrant due to inadequate conditions for biodegradation or due to the absense of CP-degrading organisms. In engineered treatment systems, however, CPs can be degraded both under aerobic (1, 2) and anaerobic (3, 4) conditions. Most CP bioremediation studies have been conducted at room temperature (20-30 "C), although actual GW temperatures are usually much lower. In a few long-term batch incubations, low-temperature CP biodegradation has been observed (5-9). To the best of our knowledge, psychrotrophic, high-rate, continuos-flow bioremediation of chlorophenols or other xenobiotic compounds has not yet been reported. Heating of water prior to biological treatment has been aprerequisite (10, 111,which radically increases the treatment expenses. Since 1988, we have studied aerobic and anoxic biodegradation of CPs (12-14). Toxicity removal, growth characterization, and stoichiometry of CP conversion (15) and kinetics of CP degradation (16) in the fluidized-bed system have also been reported. Bioremediation of CP t Present address: Nordic Envicon Oy, Kanslerinkatu 8, FIN-
33720 Tampere, Finland. 1 Present address: Department of Civil Engineering, University of Washington, Seattle, WA 98195. 0013-936X/94/0928-2387$04.50/0
0 1994 American Chemical Society
constituents of contaminated GW in an aerobic fluidizedbed system has been shown (1, 17). In a preliminary experiment at 10 "C, more than 99% of the total CPs from the GW degraded in continuous fluidized-bed treatment (18). In this paper, we report high-rate chlorophenol mineralization from groundwater in a continuous-flow fluidized-bed system at temperatures as low as 4 "C.
Experimental Section Groundwater Samples. In 1987,a CP-contaminated GW aquifer was found from Karkola, Finland. An obvious source of long term pollution was an adjacent sawmill which had used CPs for wood preservation from the 1940s to 1984. The volume of contaminated aquifer has been estimated to by 50 000-100 000 m3, with no estimations of CPs retained in the soil. Total CP concentration of the GW has remained stable (44-55 mg/L) with 2,3,4,6tetrachlorophenol (TeCP) as the main congener (18). In this study, water samples from the Karkola aquifer were collected on four occasions. The aquifer temperature is 6-8 "C throughout the year. After sampling, the GW was stored at +4 "C in 200-L containers in the dark. Fluidized-Bed Systems. Experiments were carried out in four aerobic, continuous-flow fluidized-bed reactors (Figure 1). The inside diameter of reactors was 4 cm with the height of 51 cm. The total liquid volume including recycle tubing volumes was 655 mL. The fluidized-bed volume was set at 460 mL. Biomass from the reactor walls and tubings was removed on a weekly basis. Hydraulic retention times (HRT) and loading rates were calculated based on the fluidized-bed volume; HRTs baaed on the total reactor volume would be a factor of 1.4 longer. Equal volumes of each carrier material were added for biomass retention into each reactor. Carrier material in reactor 1(Rl) was sand, in reactor 2 (R2) it was volcanite, in reactor 3 (R3) it was silica-based microcarrier (Manville Celite R-633), and in reactor 4 (R4) it was pumice. The detailed characterization of microcarriers and carrier effects on chlorophenol batch degradation kinetics will be reported by Melin et al. (19). The bed expansion was set at 50% by adjusting the upflow velocity as follows: in R2, R3, and R4 16-18 m/h and in R146 m/h. Pure oxygen was used for aeration to maintain dissolved oxygen at 8 mg/L or above. Reactors were held in a controlled-temperature incubator in the dark. Temperatures in the four reactors slightly varied during the experiments due to different recycle flow rates. Sand as the heaviest carrier material required the highest recycle flow, resulting in a slightly increased temperature in R1. Inorganic nutrients and a phosphate buffer addition to the feed GW gave the following final concentrations (mg/ L): KzHPOq3H20, 494; KH2PO4 196; NaHC03, 25; (NH4)2S04,10; MgSOq7H20,20; CaC03,5; ZnS0~7Hz0, 2.5. This amendment from the concentrated stock solutions diluted the GW by 2 % . The reactors were inoculated with activated sludge (2.4 g of VSS/reactor) treating chemi-thermomechanicalpulp Envlron. Sci. Technol., Vol. 28, No. 13, 1994 2387
muents
@ = Pump contaminated groundwater at controlled temperatures. Table 1. Experimental Program for Fluidized-Bed Reacton R1-M. temperature ('C) period
days
10-22 31-36 6&56 71-80 115-123 149-158 183-208
HRT N 5 3 5 5 5 5 b
10 6 5 6 8 8 20
R1 16.3-11.1 16.1-11.1 12.9-13.6 9.9-10.7 8.9-9.5
l.c-8.1
R2
R3
R4
15.0-15.5 14.0-14.4 14.1-14.9 15.0-15.5 13.7-14.4 13.8-14.8 11.3-12.1 10.1-11.0 10.5-11.4 8.6-9.2 1.4-7.8 8.1-8.7 1.4-8.0 5.96.3 6.3-6.9 4.1-6.0 3.5-4.6 4.5-5.6 5.1-1.1
a HRT = hydraulicretentiontime(h);Nenwnberofchlorophenol, inorganicchloride,anddiasolvedorganiccmbonaamples. b Hydraulic retention time wae decreased from 3.4to 0.44 h during experimental period I.
mill effluent. The mill did not use chlorine or chlorine dioxide bleaching, Le., the sludge was not acclimated for chlorophenols. The reactors were filled with nutrientamended GW. Then they were operated on semi-batch mode for 1week as follows: 10% of the reactor liquid was replaceddailybynew GW. Onday 7, continuousfeedwas started; HRT was held at 24 h for 2 days, and on day 9, itwasdecreasedto5h. Thefisteffluentinorganicchloride (IC1) measurements on days 8 and 9 showed an increase of 12-14 and 17-18 mg/L, respectively, indicating approximately 5045% CP dechlorination in each reactor. The first experimental period was started on day 10. The temperature was held at 20 "C for the first two days and after that at between 14 and 17 O C . Experimental Program. The temperature effecta were studied in six experimental periods as presented in Table 1. Between the continuous-flow periods prior to each temperature decrease (after experimental periods 2 4 ) , batch experiments were performed. In the batch experiments, GW chlorophenol degradation kinetics were studied at concentrations of 30-35 mg/L (19). After completion of each batch experiment, the reactor temperature was further decreased and stabilized for the next continuous-flow period. All systems were operated continuously for 5 months, with the exception of five 1-2-day periods used for batch experiments. The effect of CP loading rate on process performance at the actual GW temperature (5-7 "C) was studied in R2 by gradually decreasing the HRT (experimental period 7). Chemical Analyses. Biodegradation was monitored by CP analyses wing Hewlett-Packard 5890 Series I1gas 2388 Envtmn. Scl. Technol.. VoI. 28. NO. 13. 1994
chromatograph (GC) (Waldbronin Division B4, Germany) equipped with a B3Nielectron capture detector and the HP-5 column (25 m, 0.32 mm i.d., 0.52 pm). Carrier gas was helium with a flow rate of 1 mL/min. The oven temperature program was 1min at 58 O C , ramped at 10 W m i n to 112 "C, held 1min at 112 O C , ramped at 2.5 "C min/L to 160 "C, and held 16 min at 160 'C. The injection temperature was 225 "C, and the detector temperature was 300 "C. Samples (5.0 mL) were acetylated with 1mL of acetic anhydride using 1mL of 5.2 M K&Os as buffer. The acetylated derivates were extracted in 5 mL of n-hexane; a l.O-*L extract was used for injection. 2,3,6TCP was used as an internal standard. Five-point standard curves (0.010-2.0 mg/L) were used for quantification of 2,4,6-TCP, 2,3,4,6-TeCP, and PCP. The effluent samples were concentrated four times, resulting in an actual quantification limit of 0.003 mg/L. All chromatograms were monitored for possible chlorinated degradation intermediates. Dissolved organic carbon (DOC) removal was measured from acidified samples (pH 2) using either Unicarbo universal carbon analyzer (Elektrodynamo, Helsinki, Finland) or a Shimatzu TOC-5000 carbon analyzer (Shimatzu Corp., Kyoto, Japan) equippedwith an autosampler (ASI-5000, Shimatdzu Co, Kyoto, Japan). Prior to analyses, GC and DOC samples were filtered using 0.45pm Millipore membranes. Chlorine mass balance was completed hy inorganic chloride (ICI) releasemeasurements. IClwasdeterminated with a chloride electrode (W. Ingold AG, Urdorf, Switzerland, Type 15 213 3000) and an Ingold Ag/AgCl reference electrode (Type 373-90-WTE-ICE-S7)using an Orion pH/mV meter (Orion Research Inc. Kusnecht, Switzerland, Model SA 720). The samples were diluted to 50 mL, and 1.0 mL of 5 M NaN08 was added as an ionic strength adjuster. Measurements were carried out at 20 f 1 "C. Suspended solids (SS) and volatile suspended solid (VSS) were measured according to the Finnish Standards (20), using 300-500-mL sample volumes. Sampleswere filtered using Whatman GF/A membranes. The temperature in the reactors was continuously monitored using temperature sensors. The readings were collected into a personal computer. ReferenceCompounds. TCP andPCP were produced by Fluka Chemie AG, Buchs, Switzerland; TeCP was produced by American Tokyo Kasei Inc., Tokyo, Japan; and 2,3,6-TCP was from Aldrich-Chemie, Steinheim, West Germany.
Results
Startup. Four aerobic fluidized-bed processes for CP degradation were seeded with unacclimated sludge and operated at 14-17 "C (for details see the experimental section). Continuous-flow feed of CP-contaminated GW was started with HRT of 5 h nine days after inoculation, and the first experimental period was started on day 10. The feed CP, DOC, and IC1 concentrations in this and subsequent experimental periods are presented in Table 2. Degradation of TCP and TeCP was partial in the beginningofperiodl. NoTCPwasdetectedinanyeffluent samples from R2, R3, and R4, and not after day 14 from R1. During this period, R3 effluent contained no measurable TeCP. TeCP was detected in R2 and R4 effluents only on day 11and in R1 only until day 14. The onset of
Table 2. 2,4,6-Trichlorophenol (TCP), 2,3,4,6-Tetrachlorophenol (TeCP), Pentachlorophenol (PCP), Inorganic Chloride (ICl), and Dissolved Organic Carbon (DOC) Concentrations in Feed Solutions (Mean f Standard Deviations)
TCP (mgiL)
TeCP (mgiL)
PCP (mgiL)
9.75 f 0.17 10.80 i 0.50 10.61 j, 0.64 6.54 f 1.69 8.98 i 0.67 8.67 f 0.76 9.50 f 0.52
33.29 f 1.38 35.44 f 1.43 34.45 f 1.38 26.58 f 0.66 34.64 f 1.53 31.88 f 0.78 33.07 f 2.49
2.23 f 0.24 1.99 f 0.14 1.82 i 0.34 1.84 i 0.10 2.20 i 0.19 1.99 f 0.08 2.11 f 0.21
period 1
2 3 4 5 6 7
T
IC1
DOC (mgiL)
(mg/L)
35.58 f 1.71 27.42 f 1.98 37.67 f 0.47 30.17 f 2.19 36.40 f 1.02 29.20 f 1.47 32.33 f 1.37 22.00 f 2.52 32.50 f 1.87 26.00 f 2.12 31.63 f 1.11 26.38 f 1.22 33.96 f 0.73 31.35 f 2.45
2-
Y
Feed
-t
R1 f R2
+ R3 t R4
0 , 10
12
14
16
20 22 Time (days)
18
30
32
34
36
Figure 2. Pentachlorophenol (PCP) degradation during the startup of four parallelfluidized-bed reactors treating contaminatedgroundwater. Hydraulic retention time (HRT) was 5 h on days 9-30 and 3 h on days
31-36.
PCP degradationwas slower than with other CPs. PCP was detected in each reactor effluent through period 1 (Figure 2). On day 10, IC1 releases were 9-15 mg/L in R1, R2, and R4 and 28 mg/L in R3. Thereafter, IC1 releases were over 22 mg/L in all reactors. After day 14, the degradation of TCP and TeCP was more than 99.9% in all reactors. At the end of period 1, PCP degradation in R1, R2, and R3 reached 90%, while in R4 it was 95%. The mean effluent chlorophenol concentrations, IC1 releases, and DOC reductions are summarized in Table 3. On day 30 (experimental period 2), the HRT was decreased to 3 h. TCP and TeCP degradation was over 99.9% throughout this period (Table 3). PCP degradation remained partial during this period (Figure 1). At the end of the period, PCP removals reached 85-89 % in R1, 96-98 % in R2,92-95 5% in R3, and more than 99 % in R4. After this period, the HRT was adjusted back to 5 h in all reactors. The startup results with the unacclimated activated sludge inoculum show that TCP and TeCP biodegradation started within 10days and with resulting high CP removals. These results also show that PCP degradation remained partial for the first 1.5 months, except in R4 in which effluent PCP concentrations were less than 0.005 mg/L throughout period 2. These results suggest that TCP and TeCP degrading organisms were either initially more abundant or more readily enriched than PCP degraders from the activated sludge.
Effect of Temperature. The effect of four stepwise temperature decreases on CP degradation was studied (experimentalperiods 3-6). Total CP feed concentrations varied from 35 to 47 mg/L, which at an HRT of 5 h resulted in total CP loading rates of 168-226 g L-l d-l. The process efficiencies at each temperature are presented as effluent concentrations in Table 3. CP concentrations remained below detection limit in the effluent even at the lowest temperatures (4-5 "C),with the exception of one R2 sample during period 5. Over 99.8% PCP degradation was observed in each reactor from the beginning of period 3. To obtain mass balances, IC1releases and DOC removals were measured and compared to those calculated from GC measurements (Figures 3 and 4). The mean IC1 releases varied from 14.9% lower to 9.5% higher than calculated from GC results. The DOC removals were 13.631.6% higher than expected from GC results. This suggests removal of unknown GW contaminants as well. CP removal was accompanied by close to stoichiometric IC1 release suggesting mineralization. Effect of CP Loading Rate on Effluent Quality. The CP loading rate was gradually increased by decreasing the HRT in R2 as shown in Figure 5. The temperature was maintained at 5.1-7.1 "C. This experiment was started 183 days after the original startup. The CP degradation remained at the same level as in previous experiments. As an exception, trace amounts of TeCP were measured in the effluent on days 183 and 184 (0.019 and 0.050 mg/L) and of PCP on day 184 (0.006 mg/L). A partial breakthrough of CPs was observed after decreasing the HRT from 1.4 to 1.1h, withacorresponding increase of CP loading rate from 741 to 1000 mg L-l d-' (Figure 5). At loading rates over 1000 mg L-l d-l, TCP concentrations remained at less than 0.1 mg/L; 0.49-6.34 mg/L for TeCP; and 0.60-1.52 mg/L for PCP. At the highest loading rate of 2130 mg L-l d-l (HRT = 0.44 h), the total CP removal was 80 % The degradation of PCP declined more rapidly as compared to TeCP and TCP. IC1 releases and DOC reductions were similar to those predicted from GC results and, thus, indicated mineralization (Figure 6). These results demostrated that fluidized-bed treatment of CP-contaminated GW produced close to drinking water quality effluent at ambient groundwater temperature and at CP loading rates as high as 740 mg L-l d-l. Fluidizedbed treatment resulted in 80% CP removal at the loading rate of 2130 mg of CP L-l d-l and 0.44 h HRT. Biomass Retention. The effluent SS concentrations during experimental periods from 1to 6 are shown in Table 4. During steady-state operation, the VSS concentrations remained below the detection limit. The SS washout was highest during the startup period. During stable, lowtemperature operation, effluent SS concentrations remained below 10 mg/L. At the temperature below 7 "C, effluent SS concetrations slighty increased but did not affect CP degradation. With decreasing HRT (period 7), the effluent SS concentrations somewhat increased (Figure 5) but remained at below 20 mg/L. The results show good biomass retention and immobilization by the carriers.
.
Discussion The results of this study demonstrate that aerobic fluidized-bedbioremediation of a highly contaminated GW Envlron. Sci. Technol., Vol. 28, No. 13, 1994 2389
Table 3. Effluent 2,4,6-TrichlorophenoI (TCP), 2,3,4,6-Tetnchlorophenol (TeCP), and Pentachlorophenol (PCP) Concentrations, Inorganic Chloride (ICU Releases, and Dissolved Organic Carbon (DOC) Removal. during Expsrimental Periods 14. period
reactor
TCP'
1
R1 R2 R3 €24
