Environ. Sci. Technol. 1990, 24, 1042-1049
Continuous Coculture Degradation of Selected Polychlorinated Biphenyl Congeners by Aclnetobacter Spp. in an Aerobic Reactor Systemt Peter Adriaens and Dennis D. Focht
Department of Soil and Environmental Sciences, University of California, Riverside, California 9252 1 ~~~~
~
A coculture of two Acinetobacter spp. was applied to degrade polychlorinated biphenyls during a 42-day incubation study in a continuous aerobic fixed-bed reactor system, filled with polyurethane foam boards as support for bacterial biofilm development. The reactor was supplied with mineral medium containing 500 ppm sodium benzoate as a growth (primary) substrate, while the incoming airstream was saturated with biphenyl vapors to induce for PCB cometabolism in Acinetobacter sp. strain P6. The chlorobenzoates thus generated from 4,4‘-dichlorobiphenyl(4,4’-DCBP), 3,4-dichlorobiphenyl(3,4-DCBP), and 3,3’,4,4’-tetrachlorobiphenylwere further metabolized by Acinetobacter sp. strain 4-CB1. The chlorobenzoate metabolites, as well as ring-fusion product (A, = 442 nm) from the PCB congeners, accounted for the degradation of 63% (2.8 mM) of the 4,4’-DCBP, 100% (0.5 mM) of the 3,4-DCBP, and 32% (0.12 mM) of the 3,3’,4,4’-TCBP added. Respectively, 6.5% of 4,4’-DCBP and 11% of 3,4-DCBP were mineralized by the coculture. Upon application of a shock load of 4,4’-DCBP and 3,3’,4,4’-TCBP, the biofilm responded with a concurrent higher release of chlorobenzoates and chloride through cosubstrate utilization. Cosubstrate degradation kinetics of the PCBs and chlorobenzoates at concentrations below the affinity constant value (KM)showed that chlorobenzoate utilization rates were 6 times lower and chlorobiphenyl utilization rates 15 times lower than benzoate metabolization rates by both Acinetobacter spp. Continuous degradation by this stable coculture may thus provide a working model for biodegradation of other polychlorinated biphenyls. Introduction
Polychlorinated biphenyls (PCBs) have been in the forefront of public and scientific concern for over 20 years, because of their persistance in nature. Moreover, it has become apparent that no single naturally occurring microorganisms are yet able to mineralize chlorinated PCBs higher than monochlorobiphenyls (I). Huang and Focht (2) used the multiple chemostat method (3) to create a 3-chlorobiphenyl-degrading strain through enhanced genetic recombination of a 3-chlorobenzoate utilizing Pseudomonad and Acinetobacter sp. strain P6 isolated by Furukawa et al. (4). Aside from these studies, only initial cometabolic transformation of higher chlorinated PCBs, mainly to their respective chlorinated benzoic acids, has been reported (4-7). Therefore, it appears that degradation of PCBs has to be achieved either through a stable consortium of successively operating bacteria or by genetically engineered recombinant strains, containing the gene pool for the consecutive steps involved in PCB degradation (8). The first approach has shown to be successful in closed systems for selective PCB congeners (9) and Arochlor mixtures (10). Abbreviations: 4-CBP, 4-chlorobiphenyl;4,4’-DCBP, 4,4’-dichlorobiphenyl; 3,4-DCBP, 3,4-dichlorobiphenyl; 3,3’,4,4’-TCBP, 3,3’,4,4’-tetrachlorobiphenyl; 4-CB, 4-chlorobenzoate;3,4-DCB, 3,4dichlorobenzoate;3-C-4-OHB,3-chloro-4-hydroxybenzoate. 1042 Environ. Sci. Technol., Vol. 24, No. 7, 1990
In terms of reclamation technology, however, it is important to investigate whether the applied bacteria are able to maintain their successive degrading potential in a continuous system. A widely used approach is the model microbial film fermentor, which has mainly been used to predict kinetic properties of a biofilm by using easily biodegradable (11-15) or more complex xenobiotic substrates (16-18) as primary (growth) substrates or as the sole source of nitrogen. Whereas a lot is known about primary metabolism in microbial film fermentors, few reports exist on the nature and kinetics of cosubstrate (no growth) utilization by microbial biofilms in general (19, 20) and their importance in biofilm degradation of xenobiotics in particular (21-23). Since biofilm degradation kinetics are the result of simultaneously occurring transport (described by Fick’s law of diffusion) and microbial reaction rates (described by Monod kinetics), several investigators have shown the influence of microbial films on the diffusional characteristics of the reactive species involved (24-27). A stagnant layer of water at the film surface provides one barrier to solute transfer, while a thickening biofilm provides increasing resistance to solute diffusion to the deeper regions of the film. Because of its low solubility and importance as an electron acceptor, oxygen is most likely to be one of these limiting substrates (14,28). Moreover, PCBs pose additional engineering problems because of their extremely low water solubility (29)and their slow rate of biodegradation. In their study on the degradation kinetics of 14C-labeled PCBs in soils, Focht and Brunner (30) concluded that the rate-limiting step in the cometabolic-commensal metabolism of PCBs had to be the initial oxidation, since the rate of 14C02production was directly related to the population density of biphenyl (BP) oxidizers. In the present study, we describe a continuous-flow, packed-bed bioreactor, using polyurethane (PU) ether foam as the bacterial attachment surface. PU foam is an effective trap for concentrating PCBs from dilute air and water samples (31, 32) and has proved to be the most effective substratum in a comparative study on microbial colonization velocity in methane fermentors (33). Hence, the following study was undertaken (i) to demonstrate the applicability of a stable coculture of Acinetobacter spp. to degrade selected PCB congeners, which included a tetrachlorobiphenyl, in a continuous system, (ii) to determine the extent of PCB transformation by a bacterial biofilm, and (iii) to describe the degradation (transformation) kinetics of the cosubstrates, separately for PCB oxidation by Acinetobacter sp. strain P6 and chlorobenzoate metabolism by Acinetobacter sp. strain 4-CB1. Materials and Methods 1. Microbial Film. The biological fixed film was a coculture of two Acinetobacter spp. Acinetobacter sp. strain P6, as has been described by Furukawa et al. (4) and Kohler et al. (7), grows on benzoate, biphenyl, and 4chlorobiphenyl as sole carbon and energy sources. Acinetobacter sp. strain 4-CB1 was isolated on 4-chlorobenzoate as sole source of carbon and energy and is described by Adriaens et al. (9). Although it does not grow
0013-936X/90/0924-1042$02.50/0
0 1990 American Chemical Society
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Figure 1. Schematic diagram of the continuous-flow fixed-bed reactor system (AC, activated carbon; BP, biphenyl; BA, benzoate; PCB, polychlorinated biphenyl congeners: Rec., recycling flow; SP,sampling ports; XAD, Amberlite resin).
on 3,4-dichlorobenzoate, it dehalogenates this substrate to 4-carboxy-1,2-benzoquinone. 2. Media. The minimal salts medium (MM) (9),consisting of 40 mM phosphate buffer (KH2P04,Na2HP04, pH 7.3) was supplemented with 500 mgL-’ sodium benzoate as the primary growth substrate for both Acinetobacter spp. Cosubstrates (PCBs), coated on a Chromosorb column and inserted in the medium supply line, were provided at a daily supplement of 13.7 pmol of 4,4’-DCBP, 5.6 pmol of 3,4-DCBP, and 1 pmol of 3,3’,4,4’-TCBP. Solid media contained 1.5% Bactoagar (Difco, Detroit, MI). A few crystals of biphenyl and 4,4’-DCBP were placed in the upper lid of the agar plate, which was incubated upside down. 3. Chemicals. Biphenyl (BP), benzoate, 4-chlorobenzoate, 3-chloro-4-hydroxybenzoate,3,4-dichlorobenzoate, and sodium hydroxide were obtained from Aldrich Chemical Co., Milwaukee, WI. 4-Chlorobiphenyl (4-CBP) and 4,4’-DCBP were obtained from Pfaltz and Bauer Inc., Waterbury, CT. 3,CDichlorobiphenyl and 3,3’,4,4’-TCBPwere obtained from Analabs, Norwalk, CT. Hexane, methanol, ethyl acetate, and acetone were obtained from Fisher Scientific, Fair Lawn, NJ. 4. Bioreactor. The experimental bioreactor was designed to facilitate continuous measurement of the biofilm reaction rate following a perturbation from steady state. The main components of this system are shown in Figure 1: a fixed-bed reactor with the biofilm and the medium supply tank. The bacteria were continuously supplied with benzoate medium (500 ppm) as a primary growth substrate at a rate of 1.06 mL/min. The medium was pumped through a PCB-coated Chromosorb (GAW, 45-60 mesh, 10 g) column, solubilizing (on a daily basis) 13.7 pmol of 4,4’-DCBP, 5.6 pmol of 3,4-DCBP, and 1 pmol of 3,3’,4,4’-TCBP. A biphenyl-saturated upflowing airstream (350 ml-min-’) provided for enzyme induction in Acinetobacter sp. strain P6. A liquid recycle loop carried medium between top and bottom of the fixed bed at the same pumping rate as the fresh medium. The high recycle flow rate (100%) ensured that the entire liquid contents of the system were well mixed. The fixed bed was 770 mL in volume, water displacement by the airstream was 120 mL, and the total internal volume of the reactor was 1770 mL. Therefore, the actual volume of liquid in the reactor to-
taled 880 mL. The bed, consisting of reticulated (no cells) polyurethane ether foam boards (30 pores/in.), was a generous gift from Recticel (Geochem N. V., Wetteren, Belgium). This bioreactor confirmation provides for a gradient in degree of colonization and differential exposure of the f i to the PCBs supplied. Sampling ports were installed at different heights in the reactor for removing liquid samples by Syringe. Finally, on the basis of the medium supply rate required for the steady-state biofilm, the hydraulic retention time was calculated at 27.6 h. This was thought to provide for sufficient exposure of the film to the PCBs and their more water-soluble key intermediates, as indicated by batch culture studies (9). 5. Extraction Procedures. Effluent samples (100 mL) were taken daily, acidified (pH 2) with 10 N H2S04,and extracted three times with 20 mL of ethyl acetate. The pooled extracts were dried over Na2S04,evaporated to dryness, and redissolved in 2 mL of methanol for gas chromatography (GC) and high-performance liquid chromatography (HPLC) analyses. Foam samples, taken after 42 days, were subjected to a 4-h Soxhlet extraction in a 1:l mixture of hexane and acetone (70 mL each). The yellow extract was dried over Na2S04,evaporated to dryness, and redissolved in 5 mL of methanol before GC, HPLC, and UV-vis (ultravioletvisible light range) analyses. The recovery efficiency for chlorobiphenyls (4-CBP and 4,4’-DCBP were tested) was 93 f 270, vs 75% for biphenyl due to volatilization of this compound. 6. Analytical Procedures. Oxygen uptake was measured polarographically with an oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH), which was mounted to a reaction vessel (2 mL) held at a constant temperature (28 “C). The assay mixture contained 40 mM phosphate buffer (pH 7.2) and 50-100 mL (0.2-0.4 mg of protein) of the washed cell suspension (0.2 g wet weight mL-9. The reactions were started by adding concentrated methanol solutions. The final concentration in the reaction mixture varied with the substrate assayed. Inorganic chloride was determined turbidimetrically at 330 nm by the AgNO, precipitation method (34). Biphenyl, 4-CBP, 4,4’-DCBP, 3,4-DCBP, and 3,3’,4,4’TCBP were analyzed on an HP 5890 A gas chromatograph equipped with a flame ionization detector (FID) (Hewlett-Packard, Palo Alto, CA) according to previous methods (9). Benzoate, 4-CB, 4-carboxy-1,2-benzoquinone, and 3,4DCB were analyzed on a Beckman Programmable Solvent Module (System Gold 126, Beckman Instruments Inc., San Ramon, CA), according to the methods described previously (9). 3-Chloro-4-hydroxybenzoatewas identified by mass spectrometry according to previous methods (9) and quantified by HPLC as described above. Ring-fission product in effluent samples and PU foam extracts were analyzed on a spectrophotometer (Uvicon 860, Kontron Instruments, Everett, MA), and concentrations were calculated by using log EU2 = 4.5 (35). Protein contents of cells were determined by the biuret method with bovine serum albumin as a standard (36). 7. Scanning Electron Microscopy (SEM). Polyurethane foam specimens from the top, middle, and bottom sections of the reactor were prepared for SEM according to Weber et al. (37), except for the following modifications. Samples were stored in an absolute ethanol solution (200 proof, Goldshield Chemical Co., Hayward, CA) for critical point drying (Tousimis Research Corp., Environ. Sci. Technol., Vol. 24, No. 7, 1990
1043
Table I. Physical Characteristics of PCB Congeners and Degradation Products parameter
units
benzoate
biphenyl
4,4'-DCBP
3,4-DCBP
3,3'4,4'-TCBP
4-CB
3,4-DCB
Mr aq sol vol rate
g/mol mg/L mmHg mg of octanol/mg of H 2 0 mg of HzO/mg of C mL/mol cm2/day cm2/day
126 >lo00 NA 1.87 1.18 68.0 0.642 0.514
154 7.1 f 0.3 9.5 x 10-3 4.1 2.6 124.7 0.447 0.358
222 0.07 f 0.01 1.9 x 10-5 5.28 3.3 145.2 0.408 0.326
222 0.009 f 0.001 1.8 x 10-3 5 3.2 141.5 0.414 0.331
290 0.002 k 0.0003 2.3 x 104 6.52 4.1 161.2 0.383 0.306
156 >300 NA 2.68 1.7 78.4 0.590 0.47
192 >lo0 NA 3 1.9 88.6 0.550 0.44
kOW
Kw" mol volb diff D, diff D f
oConversion of K,
to K, according to K, = 0.63K0, (47). *Values for the PCB isomers were taken from Opperhuizen et al. (29).
Rockville, MD). The foam samples were then mounted on aluminum stubs and coated with 30 A of gold in a Sputter Coater (SC 500 A, Emscope, Kent, England) to minimize charging and increase the conductivity of the biological material. A scanning electron microscope (JOEL JSM-35C, JOEL Ltd., Tokyo, Japan) was operated at an accelerating voltage of 15-20 keV for observing the specimen. 8. Determination of Biofilm Kinetic Parameters. The yield coefficients, Y, were evaluated in batch reactors, containing minimal medium, supplied with 500 mgL-l of benzoate. The yield coefficients were calculated from Y = -AX/AS (1) where AX is the increase in biomass concentration and AS the change in substrate concentration in time intervals of 1 2 h. The decay coefficient b was evaluated with resting cells of Acinetobacter sp. strain 4-CB1 and Acinetobacter sp. strain P6, harvested on 500 mgL-' benzoate supplied minimal medium. The washed cells (0.2 mg wet weight mL-l) were resuspended in 50 mL of 40 mM buffer solution and incubated at 28 "C in 125-mL serum bottles on a rotary platform shaker (150 rpm). Samples (10 mL) were taken every 2 days and filtered through a 0.45-pm filter (MilliporeCorp., Bedford, MA) in a 25 mm i.d. filter holder (Gelman Sciences Inc., Ann Arbor, MI). The filters were weighed prior to and after filtration to determine the volatile suspended solids (VSS) concentration. The cell decay coefficient was calculated by using b = In ( X b / X ' J / t (2) where Xb is the initial concentration of resting cell suspension and X't the concentration of cells at time t. The specific substrate utilization rate k of both Acinetobacter spp. for benzoate was calculated from k = p-/ Y (3), where pmaris the maximum cell growth rate (l/h). The half-velocity constant (or substrate affinity coefficient) KMfor all substrates was based on oxygen uptake studies. Resting cells were prepared according to Adriaens et al. (9). Cells were grown in 1 L of minimal medium (MM) or MM+ supplemented with the respective growth substrates (benzoate, BP, 4-chlorobenzoate, and 4-CBP). The reaction in the assay was initiated by addition of 40-400 nmol of both BP and 4-CBP and 2-200 nmol of both benzoate and 4-chlorobenzoate. The oxygen uptake rates (V) were plotted vs substrate concentration, and the KM value was calculated from the Lineweaver-Burk plot. The standard procedure for calculating the KMvalues, as exemplified for 4-chlorobenzoate, is represented in Figure 2.
9. Determination of Physical Parameters. Biomass organic carbon of the top, middle, and bottom sections of the foam (X,) was quantified on a dry weight of biofilm basis, as described by Worden and Donaldson (39,except for the following modifications. Oven-dried foam samples 1044
Environ. Sci. Technol., Vol. 24, No. 7, 1990
0
25
50
15
100
125
150
175
200
S IW
Figure 2. Standard procedure used for calculation of the K, values shown in Table 111, as demonstrated for 4chlorobenzoate (4CB) by the Michaelis-Menten and Lineweaver-Burk plots of oxygen uptake rates ( V ) obtained on increasing concentrations (S) of 4-CB, by Acinetobacter sp. strain 4-CB1 cells grown on 4-CB.
(24 cm3.g-l foam) were treated twice with 100 mL of a 1 M NaOH solution, the first time for 15 min and then overnight, with occasional vigorous swirling to remove the biofilm. The foam was flushed five times with distilled water to get rid of biomass remnants before drying. A 1:lO dilution of the NaOH extracts with 1N H3P04was sparged with O2 for 10 min to remove inorganic carbon as C02. Sample analysis was performed on a 200-pL injection volume in a total organic carbon analyzer (Dohrman DC 80, Xertex Corp., Santa Clara, CA), using citric acid (400 and 2000 ppm carbon) as a standard. The biofilm thickness (dehydrated) was calculated as Lf = weight of dry PU foam/(weight of evaporated H 2 0 X surface area of PU foam X pH20,where the evaporated water weight was 9.5 and 9.1 g for samples from top and bottom, and middle section, respectively. The surface area of the foam samples taken was 1600 cm2. Dissolved oxygen in the stirring tank and the reactor was continuously monitored by an oxygen probe immersed in the medium. The oxygen concentration values remained constant when the biofilm reached stability. Calculation of the molecular diffusivity, D,, was based on the Wilke and Chang equation (39),using the molecular volume for benzoate from Table I. 10. Experimental Setup. The biofilm was grown on benzoate (500 ppm) MM at a pumping rate of 1.06 mL. m i d . After 14 days, the PCB-coated Chromosorb column was inserted in the medium supply line. This column contained 100 mg of 4,4'-DCBP, 50 mg of 3,4-DCBP, and 5 mg of 3,3',4,4'-TCBP. On day 9, a dry run (no liquid medium in the reactor) was imposed on the biofilm. On day 17, a shock load of 150 mg of 4,4'-DCBP and 15 mg of 3,3',4,4'-TCBP was injected in the bioreactor as a hexane solution. On day 32, the growth substrate was switched from benzoate to succinate (500 mgL-I). The continuous reactor was terminated after 45 days. Samples were taken at regular time intervals (every day or every 2 days) and
Table 11. Kinetic Parameters of Both Acinetobacter Spp. in Batch Culture
Acinetobacter sp. parameter
units
strain P6
mg of C/mg of substr way M mg of substr/mg of C-day h l/h
benzoate 0.31 0.35 42.X lo4 21.7 2.5 0.28
subst
2KM k td pmax
strain 4-CB1 benzoate 0.39 0.45 75 x lo" 11.1 3.8 0.18
Table 111. Affinity Constant (K,) Values (pM) of Both Acinetobacter Spp. for All Growth Substrates Involved" assay substrate BA 4-CB BP 4-CBP
strain 4-CB1 onb BA 4-CB 7.5 39.5 NA NA
8.5 15.8 NA NA
BA 4.2 NA 12.2 97.0
strain P6 onb BP 4-CBP 4.3 NA 3.2 ND
The values are based on O2 uptake measurements. determined: NA, not amlicable. a
ND NA ND 45
Table IV. Kinetic Parameters of the Benzoate-Grown Biofilm on 30 Pores/Inch PU foam boards parameter
symbol
unit
substr supply rate O2 concn-influent O2 concn-effluent bacterial density in film yield of bacterial mass
r, DOi DO, Xf
mgday-' mgL-l mgL-' mgof C/cm3 mgofC/mg of substr l/day cm2/day cm2/day cm h
sp biomass decay coeff mol diff in H20a mol diff in biofilm" biofilm thicknessb hydraulic residence timeC
Y b
D, Df Lf HRT
value 1526 8.4 6.7 40.0 f 4.2 0.35 f 0.02 0.4 0.642 0.514 (58 f 1) X lo4 27.6
" Molecular diffusivities D, are calculated from the Wilke and Chang equation (39), while a Df/D, ratio of 0.8 was applied for diffusivity in the biofilm (40). bThe biofilm thickness (dehydrated) was calculated as Lf = weight of evaporated H 2 0 X PU surface area. 'A 100% recirculation of the medium is taken into account.
--.
a PI
t
ND, not
monitored for degradation products. The physical characteristics of the PCB congeners and degradation products are given in Table I.
Results Kinetics of a Benzoate-GrownBiofilm of Acineto bacter Spp. Basic microbial kinetic parameters of Acinetobacter sp. strain 4-CB1 and Acinetobacter sp. strain P6, were determined in separate batch culture experiments, using benzoate as the growth substrate. Because the growth rate (pmax)of Acinetobacter sp. strain P6 was higher than that of Acinetobacter sp. strain 4-CB1 (Table 11),the former should be more competitive for growth on benzoate and, therefore, better able to colonize on the PU foam. A comparison of saturation constant values (KIM) with both Acinetobacter spp. is given in Table 111. The results reflected the importance of induction and/or acclimatization of both Acinetobacter spp. to the chlorinated substrates. The KM (based on oxygen consumption) for 4-chlorobenzoate metabolism was more than twice as great for cells of Acinetobacter sp. strain 4-CB1 grown on benzoate than on 4-chlorobenzoate. When Acinetobacter sp. strain P6 was grown on benzoate, the KIMfor BP and 4CBP was 4 and 2 times greater than when cells were grown on the respective substrates. In order to describe the dynamics of the biofilm, and degradation of the PCBs, kinetic and physical parameters were calculated (Table IV). While the kinetic parameters ( Y and b) were based on studies in batch reactor systems (eqs 1and 2), the physical parameters of the stable biofilm were calculated as mentioned previously. The biofilm was considered stable when the effluent concentration of benzoate reached a constant value of 45 k 7 ppm, which meant that the benzoate used by the biofilm was in equilibrium with oxygen consumption. Stability was reached after 14 days. Diffusion of benzoate in the biofilm, calculated as a flux (cm2-day-l)was therefore assumed not limiting. The overall difference in oxygen concentration between influent and effluent was measured to be 1.7 mgL-'. The bacterial density in the film ( X , ) was lower in the middle section of the reactor than in the top and bottom sections; 32 mg of C ~ c m - and ~ , 44 mg of C~cm-~, respectively. Accordingly, the biofilm thickness was 59 x
Figure 3. Development of the biofilm of Acinetobacter spp. on 30 poredin. PU foam support material (a, 20 kV, X9400; b, 15 kV, X260).
lo4 and 57 x lo4 cm for the top and bottom sections, and the middle section, respectively. Table IV thus represents the average values of these parameters. The physiological development of the biofilm and the importance of extracellular polymers in the makeup of the film matrix is shown in Figure 3. Continuous Degradation of PCBs. In addition to 500 mg-L-' benzoate as primary substrate, the following water-soluble amounts of PCBs were supplied: 13.7 pmobday-l of 4,4'-DCBP, 5.6 pmoloday-' of 3,4-DCBP, and 1 pmol-day-' of 3,3',4,4'-TCBP. No PCB was detected in effluent samples, indicating that everything was trapped Environ. Sci. Technol., Vol. 24, No. 7, 1990
1045
Table V. Degradation of PCB Isomers by a Coculture of Two Acinetobacter Spp. during a 45-Day Period”
congener (mM added) 4,4’-DCBP (4.400)
metabolite
4,4’-DCBP ring-fission product 4-CB inorganic chloride 3,4-DCBP (0.468) 3,4-DCBP ring-fission product 3,4-DCB 3-C-4-OHB quinone inorganic chloride 3,3’4,4’-TCBP (0.376) 3,3’4,4’-TCBP ring-fission product 3,4-DCB 3-C-4-0H B quinone inorganic chloride
chloride recovery, mM aqueous solid phase phaseb
