Envlron. Sci. Technol. 1993, 27, 691-698
Strategy Using Bioreactors and Specially Selected Microorganisms for Bioremediation of Groundwater Contaminated with Creosote and Pentachlorophenol+ James G. Mueiier,’i$ Suzanne E. Lank,* Derek Ross,§ Richard J. Coivin,§ Douglas P. Middaugh,ll and Parmely H. Pritchardil SBP Technologies, Inc., Gulf Breeze, Florida, The ERM Group, Exton, Pennsylvania, and U S . EPA, ERL, Gulf Breeze, Florida 32561
A two-stage, sequential inoculation bioreactor strategy for the bioremediation of groundwater contaminated with creosote and pentachlorophenol (PCP) was evaluated at bench scale (1.2 L) and pilot scale (454 L). Bioreactor performance using specially selected microorganisms was assessed according to chemical analyses of system influent, effluent, and bioreactor residues, a chemical mass balance evaluation, and comparative biological toxicity and teratogenicity measurements. During pilot-scale operations, the concentration of creosote constituents was reduced from ca. 1000 ppm in the groundwater feed (flow rate 114 L/day) to 99% 1. Notably, the cumulative concentration of carcinogenic polycyclic aromatic hydrocarbons was reduced from 368 ppm in the feed to 5.2 ppm in the system effluent. Moreover, the toxicity and teratogenicity of the bioreactor effluent were significantly reduced. In general, field data correlated well with those obtained from bench-scale studies.
Introduction A wide variety of bioreactor technologies have been developed for the treatment of solid, liquid, and gaseous matrices contaminated with myriad organic chemicals ( I ) . Because the physicochemical variables (e.g., pH, nutrient concentrations, biomass, oxygen-transfer rate, contaminant loading rate, etc.) of the bioreactor can be precisely controlled, conditions can be optimized for the desired microbial activities and performance can be maximized. Moreover,effectivemixing may help alleviate certain masstransfer problems, and surfactants, detergents, or solubilizing agents can be added to increase the aqueous solubility of hydrophobic contaminants, thereby enhancing the bioavailability of target chemicals. Herein lie the advantages of bioreactors versus other bioremediation approaches (e.g., composting, land farming, and in situ treatment). For these same reasons, bioreactor inoculation is often a viable technique to rapidly establish active biomass and enhance the desired biological activity. Together, these factors act to maximize the kinetics of biodegradation, hence enhancing the biomediation processes. Depending on the characteristics of the pollutant chemicals and the nature of the contaminated matrix, alternative biological treatment strategies such as solidphase (land farming), in situ bioremediation or composting are often more applicable. Hence, bioreactor operations t Research contribution no. 822 of the Gulf Breeze Environmental Research Laboratory. t SBP Technologies, Inc. 5 The ERM Group.
11 U.S. EPA. 0013-936X/93/0927-0691$04.00/0
0 1993 Amerlcan Chemlcal Society
do not always represent the remedial technology of choice, especially when relatively easily biodegradable organic chemicals are the contaminants of concern ( 2 , 3 ) . When bioreactor or other above-ground technologies are employed, disadvantages associated with their use include the following: (1) the need to add the material to the bioreactor via excavation or other form of physical removal (e.g., groundwater pumping and vacuum extraction), (2) numerous factors intrinsic to the contaminant removal process (e.g., efficacy of aquifer pumping and soil washing), and (3) additional expense (e.g., operating costs and initial capital for start-up). Despite these limitations, many examples can be cited in which bioreactor strategies have been applied successfully for the biodegradation of organic contaminants. For instance, biofiltration has been used extensively to remove volatile organiccompounds from air emissions (4,5),vaporphase bioreactors have treated chlorinated aliphatics in the gaseous state (6, 7),and fixed film bioreactors have been used for the treatment of kraft bleaching effluent containing a variety of chlorinated aromatic chemicals (8) and for petroleum refinery effluent (9). With varying success,attempts have been made to apply reactor technologies to the treatment of soil and water contaminated with the chemicals found in organic wood preservatives such as creosote (e.g., polycyclic aromatic hydrocarbons) or pentachlorophenol (PCP) (10-15). However, one challenge frequently encountered is the limited ability to efficiently biodegrade high molecular weight (chemicals containing four or more fused rings) polycyclic aromatic hydrocarbons (HMW PAHs) present in contaminated soils and waters. This limit is due to structural aspects of these chemicals and their strong tendency to partition to biomass, sludge, and bioreactor residues (1618). Previously, we described the isolation and characterization of microorganismscapable of utilizing HMW PAHs and other persistent creosote constituents as sole sources of carbon and energy for growth (19, 20). Additionally, an axenic culture of Pseudomonas sp. strain SR3 was shown to mineralize PCP when supplied as a sole carbon source in liquid medium (21). The objective of these studies was to evaluate, at the bench- and pilot-scale levels, the ability of a sequential inoculation process employing “specially selected” microorganisms to enhance the effectiveness of bioremediation technologies for the treatment of groundwater contaminated with creosote and PCP. The performance of the bioreactors was judged on the basis of mass balance evaluations of chemical analyses of bioreactor influent, effluent, and residues (off-gases,settled biomass, reactor sludges) and comparative biological toxicity and teratogenicity measurements. Envlron. Scl. Technol., Vol. 27, No. 4, 1993 891
Experimental Section
American Creosote Works Site Description. The American Creosote Works (ACW) site at Pensacola, FL, is an abandoned wood-preserving facility that was in operation from 1902 until closure in 1981 (12, 22, 23). Because of high-volume usage and improper disposal of creosote and PCP over this 79-year period, soil and groundwater at this site have been extensively contaminated. Recent analyses of the surface soil, the subsurface soil, and the shallow aquifer (1 m below land surface) verified the presence of creosote constituents, PCP, chlorinated dioxins, and chlorinated furans in all tested materials (24, 25). Groundwater Extraction. For the laboratory and bench-scale studies described herein, groundwater was recovered from a depth of 6-7 m through an on-site monitoring well (ACW 3201, installed by the US. Geological Survey, immediately adjacent to and directly downgradient from the most contaminated materials at the ACW site (23-25). Groundwater was removed using Teflon-coated Bev-a-line tubing (15-mm i.d.) and an electric pump. Groundwater was delivered directly into clean, sterile, 1.0-L Wheatsn bottles fitted with Teflonlined screw caps and stored on ice for transport to the laboratory. Upon arrival at the laboratory, samples were stored in darkness a t 4 OC until used. Larger volumes of groundwater were obtained from monitoring well SBP MW-1, installed directly adjacent to well ACW 320 at a depth of 13.3 m to a thin clay aquitard (24,251. The stainless steel well (10-cm casing diameter) was screened (slot size 0.05 cm) from a depth of 4.3 to 11.0 m. Water was removed from the well at a rate of 30.3 L/min to minimize draw-down and reduce the uptake of nonaqueous-phase liquids shown to be located throughout the aquifer (24, 25). Inoculum Preparation and Viability Tests. Previously, we described the isolation of seven bacterial strains, designated CRE1-7 (originallydescribed as strains FAE1-'I), from creosote-contaminated soil at the ACW Superfund site, Pensacola, FL, (19). Another member of this community, P s e u d o m o n a s paucimobilis strain EPA505, was later described for its ability to mineralize fluoranthene as a solecarbon source (20). Similarly,strains CRE8-13 were isolated from ACW soil enrichments with fractions of specification creosote 450 containing acidic (e.g., phenolic) or basic (e.g., heterocyclic) creosote constituents. P s e u d o m o n a s sp. strain SR3 was isolated from a PCP-contaminated soil from northwest Florida for its ability to utilize PCP as a sole carbon source (21). These 15isolates represent the specially selected bacterial strains used in these studies. To generate inocula for bench- and pilot-scale studies, small-scale fermentations were performed with each of the 15individual bacterial cultures in Luria-Bertani broth. Once cells reached the late log phase (ODsoonmca. 1.01, they were concentrated (10 000 rpm for 10 min at room temperature), washed once in sterile phosphate buffer (pH 7.0), and then suspended in phosphate buffer plus 5% DMSO or 20% glycerol. For PCP-degrading strain SR3, cells were grown with glucose and yeast extract using a two-stage PCP induction process as described by Resnick and Chapman (26). On average, 10 L of cells was concentrated to a final volume of 50 mL-resulting in a final cell density between 5 X loll and 1X lo1*cells/mL. Cell concentrates were stored at -70 OC until used. 692
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Prior to use in laboratory and field studies, the percent recovery of viable cells of each strain from inoculant preparations was determined by direct plate counting on nutrient agar (Difco). Additionally, the activity of the individual strains and the 13-member community (reconstituted from inoculant preparations) toward creosote and PCP present in groundwater recovered from the ACW site was evaluated. These studies were performed in two 1.2-L Biostat M bioreactors (see followingsection) operated in the batch mode. Each bioreactor was filled with 1.0 L of mineral salts (MSII) medium (19) containing 1000 r L of a filter-sterilized creosote stock solution (7.0 mL of site creosote-PCP oil plus 3.0 mL of DMSO) yielding an ACW groundwater medium containing an initial concentration of 700 mg of creosote-PCP oil/L plus 300 mg of DMSO/L. The use of solubilizing agents or surfactants to enhance the aqueous solubility of hydrophobic chemicals has been well documented (17, 18,27,28). Initially, both bioreactors were inoculated with strains CRE1-13 at a density of 1 X lo6 cells/mL for each strain. Both bioreactors were operated in the batch mode for 6 days. On days 4,5, and 6, strains SR3 and EPA505 were added to only one of the bioreactors at a concentration of 1X lo6cells/mL. Duplicate 5.0-mL samples of bioreactor contents were removed daily for chemical analyses (see Analytical Methods). Previous studies showed limited biodegradation of HMW PAHs (ca. C50%) and PCP (C5%) without the inoculation of ACW groundwater under shake flask (13) and identical bioreactor conditions (data not shown). Sequential Inoculation, Continuous-Flow Bioreactor: Bench-Scale Studies. The first (BR1) of two 1.2-L Biostat M bioreactors (B. Braun Biotech, Allentown, PA), connected in series, was filled with 1.0 L of fresh MSII medium plus 0.03% Triton X-100 (Sigma Chemical Co.) and 0.5 mL of creosote-PCP oil (recovered from the ACW site aquifer as a dense nonaqueous-phase oil). This yielded an average total concentration of 149.4 mg/L of the 40 monitored creosote constituents plus 9.9 mg of PCP/L (the difference in the amount of creosote added and that detected by gas chromatography was attributed to the presence of water and fine particulates in the oil recovered from the ACW site aquifer). For initial inoculation, concentrated cells (1.0 mL containing ca. 5 X loll cells) of strains CRE1-13 were removed from storage at -70 "C, thawed at room temperature, and then added to BR1 for an initial inoculum density of ca. 1X lo7cells of each strain/mL. The reactor was operated for 24 h in the batch mode to allow for acclimation of added biomass. Operational parameters were controlled electronically [pH 7.2, dissolved oxygen (DO) 90% of saturation, 28.5 "C, 350 rpml and monitored daily as previously described (12). After this acclimation period the bioreactor contents were again spiked with 0.5 mL of the ACW creosote-PCP oil and the system was converted to a flow-through mode of operation. Feed solution was introduced into BR1 at arate of 0.7 mL/min (24-hhydraulic retention time). Feed solutions were prepared daily by adding 1.0 mL of creosotePCP oil and 0.3 mL of Triton X-100 to 1.0 L of MSII medium in a Teflon bottle, resulting in an average total concentration of 273.0 mg/L for 40 monitored creosote constituents plus 16.9mg of PCP/L. These solutions were mixed constantly with a magnetic stir bar, and feed was moved through Teflon and Viton tubing with a peristaltic
BIOREACTOR 1
BIOREACTOR 2 (2a. b ana c )
Ground Water
Concentrate
Discharge
-
EPA505 and SR3
Effluent Polishing
Fbm 1. Schematic of the twwtage. continuous-How. sequential Inoculathm bioreactw system at the pilot scale: (A)sampling points.
pump. A level control probe was used to maintain the volume of BR1 at 1.0 L. This probe was connected to a peristalticpump which, once activated, intermittently (5-s pulsed pumping) transferred material from BR1 to the second bioreactor (BR2)through Teflon and Viton tubing. As effluent from BR1 was transferred, a cell suspension (ca. 1X lo7cells/mL) of EPA505 and SR3 (see inoculum preparation section) was added to BR2 at a rate of 0.3 mL/min. The volume of reactor contents in BR2 was maintained at 1.0 L by a second-level control probe connectedto another peristalticpump. Emuent from BR2 was transferred into a 15-L holding vessel. Continuous-flow operations with daily additions of EPA505 and SR3 to BR2 were performed for 32 days. Duplicate 5.0-mL samples were taken daily from both bioreadors and analyzed as described below (see Analytical Methods). Feed solutions were also analyzed daily. At the conclusion of the study, activated carbon traps were extracted and analyzed to monitor for losses via volatilization. A t the same time, each reactor was drained and undissolved sludge and oily creosotic material adhering to the internal surfaces of the bioreactors and flow lines were collected by washing with methanol and then methylene chloride. Sequential Inoculation, Continuous-Flow Bioreactor: Pilot-Scale Studies. System Design and Operation. The two-stage, sequential inoculation, continuous-flowbioremediation process (Figure 1)was fieldtested at ACW with freshly recovered groundwater highly contaminated by creosote and PCP. The pilot-scale system used a 454-L completely stirred tank reactor (CSTR) (EIMCO, Denver, CO) designated FBRl (field bioreador 1)and three 227-L stirred tank batch reactors (designated bioreadors FBR2a-c). System operations were initiated by filliig FBRl with a mixture of city water and/or contaminatedgroundwater (feed)plus inocula The inoculum consisted of 50 mL (ca. 5.0 X 1OI2 cells) of frozen concentrate of strains CRE1-13. FBRl was then operated in the batch mode for a period of 3 4 days to allow for acclimation of the initial inoculum. Operational parameters and reaction conditions were controlled and maintainedasfollows: >2.5mgofDO/L,23-25"C,pH7.0-7.5, TCODNP ratio of 400:51. Microbial activity and performance of FBRl was monitored daily (see Analytical Methods. Once biomass and activity measurements stabilized, continuous-flow operations were initiated by introducing
equilibrated groundwater feed to FBRl at an average rate of 114 L/day (4-day hydraulic retention time). Continuouslymixedfeedsolutionwas amendedwithTritonX-100 (0.01-0.03%), urea, diammonium phosphate, and magnesium sulfate to obtain desired levels and contained ca. 1250 ppm total monitored chemicals (see Analytical Methods). Daily additions ofPseudomonospaucimobilis strain EPA505 and Pseudomonas sp. strain SR3 (ca. 5 X lo6cellslml) were made to each filled chamber of FBRZ. Feeding continued for two hydraulic retention times (8 days). Effluent from FBRl was concurrentlydelivered to FBR2a-c. OnceachamberofFBRZwasfilledwithefiluent from FBR1, effluent flow was diverted to another compartment. Material in the filled chamber of FBR2 waa then held in the batch mode for 4 days. As each batch of FBR2 completed treatment, polymer was added to aggregate solids (biomass) and the contents were clarified by settling for 18 h under static conditions. Following clarification, duplicate samples of clarified effluent (10 mL) and settled biomass (5.0 mL) were collected for extraction and analysis of residual creosote and PCP. On-site analyses of the operational parameters and reaction conditions were performed daily with samples from FBR1, each utilized chamber of FBR2, and groundwater feed. Additionally, daily measurements of creosote and PCP concentrations were obtained with individual or, whenever possible, duplicate samples of bioreactor contents and groundwater feed. Samples (5.0 mL) from the reactors were placed in clean (solvent-rinsed), glass tubes fitted with Teflon-lined screw caps, preserved by adjusting the pH to >12.5 with NaOH, and transported on ice to the laboratory for processing ( 4 2 - h holding time a t 4 "C). Intermittent assays to monitor bacterial populations were also performed (see Analytical Methods). Off-gasesfrom FBRl were passed through an activated carbon trap (100 g of carbon) at a rate of 3.40 m3/h for three 30-min periods at the beginning, middle, and end of the bioreactor run (total air flow through FBRl was regulated at 10.90 m3/h). At the end of each bioreactor run,bioreactor sludges and line residues were removed with a pressurized steam cleaner and wash water was collected. Triplicate 5.0-mL samples of composited wash water were used to determine the amount of creosote and PCP removed from the system through adsorption. Analytical Methods. Physical and Chemical Assays. On-line pH measurement of FBRl was performed using Envlm. SCl. Tschnol.. VOI. 27.
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a 3-ft submersible electrode (Cole-Parmer, Chicago, IL). A pH of 7.0-7.4 was maintained through the automated addition of 1.0 M NaOH or 0.76 ?4 HCl solutions. The pH of FBR2 chambers was monitored with a Corning pH/ conductivity probe. Laboratory measurements of DO used a calibrated 0 2 probe (Corning, NY). Oxygen uptake rates (mg of 0 2 L-l h-l) were calculated as changes in DO measured over time (10 min) with a YSI oxygen meter, Model 51B (29). Total suspended solids (TSS) were determined gravimetrically after heating at 105 OC for 120min (29). Volatile suspended solids (VSS) values were determined after heating at 550 OC for 20 min (29). Total chemical oxygen demand (TCOD) was measured by the HACH reactor digestion method (30). Soluble chemical oxygen demand (SCOD)values were obtained with filtrate (0.7-pm glass fiber filters) of bioreactor contents and effluent (30). Reactive phosphorus concentrations were determined according to the PhosVer 3 (ascorbic acid) method (30). Ammonia nitrogen and nitrate nitrogen were measured by the salicylate and cadmium reduction methods, respectively (30). Extraction of PCP and creosote constituents present in liquid and activated carbon samples was performed as previously described (12, 13, 32), and samples were analyzed by gas chromatography (12, 13). Viable heterotrophs and phenanthrene-degrading microbes present in reactor samples were enumerated by direct plate counts (12, 13). T o x i c i t y Assays. Toxicity and teratogenicity analyses using embryonic fish, M e n i d i a beryllina, were performed with 1, 10, and 100% sample solutions as previously described (13, 32-34). In brief, single, blastula-stage embryos were exposed to respective test solutions for 7-9 days. Thirty test embryos were used for each exposure concentration and for a control group maintained in clean dilution water. Observations were made daily for survival, normal development, and teratogenic responses in each embryo or larva. Four end points were used to define responses: (1)embryotoxic, (2) teratogenic, (3) hatched with terata, and (4) hatched normal (32-34). Microtox assays with bioreactor samples were performed according to manufacturer’s specifications (Microbics Corp., Carlsbad, CA) with a modification of running the basic test in duplicate. Conventional toxicity assays for static, nonrenewal 48-h LC50 Mysidopsis bahia tests and 96-h LC50 Ceriodaphnia dubia tests were performed according to EPA guidelines (35). For all toxicity and teratogenicity tests, samples were prepared in serial dilutions to test a range of decreasing concentrations. Prior to biological assays, samples were filtered through Whatman No. 1 paper to remove suspended solids, which were shown to inhibit these tests (Middaugh and Heard, unpublished data). No differences were observed in the chemical composition of materials after filtering (data not shown). Treatment effectiveness was quantified by demonstrating changes in LC50/EC50 and no effect concentrations. R e s u l t s a n d Discussion
Analytical Chemistry Data. The concentrations of 21 PAHs (three groups), 9 heterocyclic and 11 phenolic compounds, and PCP found in ACW groundwater are shown in Table I. The proportion of each of these chemicals was, in general, similar to that found in specification creosote no. 450 (a standard creosote used 694
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Table I. List, Grouping, and Distribution of 40 Individual Chemicals Monitored by Chemical Analyses
chemical/grouping
av concn, mg/La site specif groundwater creosote 450
group 1PAHs 1-methylnaphthalene 13.33 (0.9) 17256 (3.8) 2-methylnaphthalene 2.86 (0.2) 46895 (10.2) 2,3-dimethylnaphthalene 8.81 (0.6) 2312 (0.5) 2,6-dimethylnaphthalene 19.42 (1.4) 7120 (1.6) biphenyl 2.27 (0.2) 8067 (1.8) naphthalene 1.11(0.1) 55701 (12.1) subtotal 47.8 (3.4) 137351 (30.0) group 2 PAHs 2-methylanthracene 74.33 (5.2) 11435 (2.5) acenaphthylene 14.77 (1.0) 1076 (0.2) acenaphthene 139.76 (9.9) 44872 (9.8) anthracene 60.85 (4.3) 16269 (3.6) anthraquinone 19.42 (1.4) 9892 (2.2) fluorene 141.33 (10.0) 35778 (7.8) phenanthrene 356.76 (25.1) 101323 (22.1) subtotal 807.2 (56.9) 220645 (48.2) group 3 PAHs benz [ a ]anthracene 38.95 (2.7) 7993 (1.7) benzo[a]pyrene 37.58 (2.7) 3408 (0.7) benzo [bl fluoranthenei 11.91 (0.8) 2774 (0.6) benzo [kl fluoranthene benzo[ blfluorene 35.99 (2.5) 7707 (1.7) chrysene 37.58 (2.7) 8211 (1.8) fluoranthene 230.86 (16.3) 40567 (8.8) 171.13 (12.1) 30168 (6.6) pyrene subtotal 564.0 (39.8) 100828 (21.9) total PAHs 1419.0 (100) 458824 (100) heterocyclic compds acridine 4.11 (2.3) 346 (0.5) carbazole 30.42 (17.1) 7886 (11.7) 84.42 (47.6) dibenzofuran 28711 (42.8) dibenzothiophene 55.98 (31.5) 7146 (10.7) isoquinoline 0.10 (
