Environ. Sci. Technol. 2000, 34, 1977-1984
Bioavailability of Soil-Sorbed Biphenyl to Bacteria Y U C H E N G F E N G , ‡,† J E O N G - H U N P A R K , § THOMAS C. VOICE,§ AND S T E P H E N A . B O Y D * ,‡ Department of Crop and Soil Sciences and Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan 48824
Limited bioavailability of organic pollutants in soil may be a detriment to the successful application of bioremediation. The availability of soil-sorbed biphenyl to two biphenyldegrading bacteria, Pseudomonas putida P106 and Rhodococcus erythropolis NY05, was assessed using a kinetic mineralization assay. Biphenyl was aged in four soils of different organic carbon (OC) contents (0.4-7.8%) for up to 274 days. With a biphenyl-soil contact time of 24 h, the initial mineralization rates (IMRs) ranged from 2.6 to 3.5 µg‚L-1‚min-1 for strain P106 and from 3.8 to 0.92 µg‚L-1‚min-1 for strain NY05. These IMRs were higher than those of soil-free controls and those predicted by a coupled desorption/biodegradation model, suggesting both strains of bacteria could access soil-sorbed biphenyl. For strain P106, biphenyl mineralization curves in slurries of four different soils were nearly coincident with those in soil-free systems containing the same total mass of biphenyl. This strain appeared to have immediate and complete access to the pool of sorbed biphenyl. The extent of bioavailability of soil-sorbed biphenyl decreased with increased aging. The decrease in availability was most pronounced within the first 80 days. The effect of soil organic matter content on bioavailability showed different trends for the two organisms.
Introduction The effectiveness of bioremediation of contaminated soils and sediments may be influenced by the accessibility of contaminants to microorganisms. The accessibility of contaminants is related in turn to the extent and mechanism of sorption. Nonionic organic compounds (NOCs) are sorbed by soils and sediments primarily through partitioning into organic matter (1). In general, lower NOC-water solubilities and higher organic matter contents in soil manifest increased sorption. Sorbed NOCs are thought to be more resistant to biodegradation than soluble NOCs (2, 3) as it is generally considered that sorbed chemicals are unavailable to microorganisms unless desorption occurs first (4, 5). However, bacteria thought to be capable of degrading soil-sorbed naphthalene (6) and sediment-bound phenanthrene (7) have been reported. Recently, Laor et al. (8) showed that phenanthrene mineralization was enhanced by sorption to synthetic mineral-humic acid complexes. * Corresponding author phone: (517)353-3993; fax: (517)355-0270; e-mail:
[email protected]. † Present address: Department of Agronomy and Soils, Auburn University, Auburn, AL 36849. ‡ Department of Crop and Soil Sciences. § Department of Civil and Environmental Engineering. 10.1021/es991165e CCC: $19.00 Published on Web 04/05/2000
2000 American Chemical Society
An additional factor influencing the bioavailability of NOCs is the contact time between the contaminants and soils, a parameter that is commonly referred to as aging. There is evidence that the availability of certain organic chemicals decreases with time. For example, ethylene dibromide, a soil fumigant with relatively high water solubility, volatility, and biodegradability, was reported to persist up to 19 years after its last application (9). Laboratory experiments showed that field-aged EDB (9) and simazine (10) were persistent, whereas freshly added 14C-EDB and 14Csimazine were quickly degraded by microbial populations. The diminishing bioavailability of soil-aged chemicals has also been reported for naphthalene (11), phenanthrene and 4-nitrophenol (12), and atrazine (13, 14). The time-dependency of bioavailability is a complex issue due to the involvement of multiple processes including sorption/ desorption, diffusion, chemical transformation, and microbial degradation. These processes, the environmental factors affecting them and the interactions between them, are not fully understood. In particular, questions remain as to the influence of soil organic matter content, soil aggregate size, diffusion within soil aggregates, bacterial species differences, chemical-soil contact times, and desorption/degradation interactions. Biphenyl and its derivatives have been used in industry as a chemical feedstock and in agriculture as a fungicide. Biphenyl-degrading bacteria are ubiquitous in the environment (15). Biphenyl is extensively sorbed by soil organic matter (16, 17). Studies on microbial degradation of biphenyl sorbed to polyacrylic beads suggested that bacteria attached to the solid phase might act on sorbed biphenyl without the initial desorption step (18). In the present study, we examined whether, and to what extent biphenyl-mineralizing bacteria can access sorbed biphenyl, and how the contact time between biphenyl and four soils with different organic matter contents affected biphenyl availability to two biphenyldegrading bacteria.
Materials and Methods Organisms and Growth Conditions. Two bacteria capable of using biphenyl as the sole source of carbon and energy were used in the experiments. Pseudomonas putida P106, isolated from an industrial sewage waste canal in Panama City (15), was obtained from Dennis D. Focht’s laboratory at University of California, Riverside. Rhodococcus erythropolis NY05, isolated from PCB-contaminated soil in Glenn Falls, NY (19), was provided by James M. Tiedje’s laboratory at Michigan State University. Both biphenyl-degrading bacteria were isolated from enrichment cultures obtained by adding soil inocula to mineral salts media containing biphenyl. Pseudomonas putida P106 was grown in mineral salts medium (20) supplemented with 0.2 g of biphenyl/L, and Rhodococcus erythropolis NY05 was grown in the same medium supplemented with 0.48 g of biphenyl/L and 1 mL of vitamin solution (21) per liter. Solid media contained 1.5% (w/v) Noble agar (Difco Laboratories, Detroit, MI); biphenyl was provided as crystals in the Petri dish lids. Nutrient agar plates were made up of 4 g/L (half-strength) nutrient broth and 1.5% agar. Liquid cultures were incubated at room temperature with shaking at 200 rpm on a rotary shaker, and growth was monitored by measuring the absorbance at 600 nm. Cells in the early stationary phase were harvested by centrifugation, washed with 20-mM sterile phosphate buffer (pH 7), and resuspended in the same buffer solutions prior to inoculation. VOL. 34, NO. 10, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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1977
TABLE 1. Properties of the Soils Used in This Study soil Colwood A Capac A Schoolcraft A Capac B
% OC % sand % silt % clay 7.80 3.28 1.44 0.40
64.2 54.6 58.6 60.0
20.7 24.0 22.0 20.0
15.1 21.4 19.4 20.0
pH
CEC [cmol(+)/kg]
6.04 6.85 6.14 6.63
43.0 24.4 11.2 9.60
Soils and Soil Extracts. Three surface soils and a subsurface soil from Michigan were used in the studies. Soils were air-dried, ground, and passed through a 2-mm sieve. Cation exchange capacity and soil pH were determined according to methods described by Page et al. (22). Soil organic carbon (OC) contents and particle size distribution were determined by the Soil and Plant Nutrient Laboratory at Michigan State University. Table 1 summarizes the properties of the soils. Soil samples were sterilized by γ-irradiation (5 Mrad from a 60Co source) in 500-mL polypropylene bottles and were sterilized again by autoclaving after being transferred to serum bottles. To prepare soil extracts from all four soils, 100 g of each soil was suspended in 1 L of phosphate buffer (20 mM) in 2-L Erlenmeyer flasks. The flasks were autoclaved for 30 min and shaken on a rotary shaker for 24 h. Soil suspensions were then frozen overnight and thawed to help destabilize dispersed soil colloids. The supernatants obtained after centrifugation were filtered through two layers of Whatman No.1 filter paper. The soil-extract solutions were autoclaved again and stored until use. Sorption of Biphenyl by Soils. Batch sorption isotherms for all four soils were obtained by equilibrating [U-14C]-labeled (radiochemical purity: >98%, specific activity, 7.6 mCi/mmol, Sigma, St. Louis, MO) and nonradioactive biphenyl with soils at a soil-to-solution ratio of 1:10. Variable volumes of sterile phosphate buffer (20 mM) and nonradioactive biphenyl stock solution were added to 20-mL crimp-top glass vial containing 1.5 g of sterile soil to obtain the appropriate concentrations (50-400 µg/L). 14C-Biphenyl (0.02 µCi) was added last to each tube to bring the final volume to 15 mL. The vials were capped with Teflon-coated butyl stoppers and crimp-sealed with aluminum caps. All sample vials were prepared in duplicate and were incubated on a reciprocating shaker for 24 h at room temperature (22 ( 1 °C). At the end of the incubation period, vials were centrifuged, and 1 mL of supernatant was sampled for determination of the radioactivity. The amount of biphenyl sorbed was calculated by difference between the initial and final solution-phase biphenyl concentrations. For the long-term sorption experiments, vials were shaken for 24 h and then stored in the dark with periodic shaking until sampling. Soil sterility was checked by plating out the soil suspension on nutrient agar plates. Bioavailability Assays. To evaluate the availability of soilsorbed biphenyl to the two biphenyl-degrading bacteria, a mineralization assay, adapted from the method described by Guerin and Boyd (6), was used. Soil-extract controls and soil slurries were set up in 160-mL serum bottles sealed with Teflon-coated butyl stoppers. Sterile soil (7.5 g) was placed into each bottle, and variable volumes of sterile phosphate buffer (20 mM) and nonradioactive biphenyl stock solution were added to obtain the appropriate concentrations. Radioactivity in each serum bottle was about 0.1 µCi. Soil slurries set up for strain P106 contained a biphenyl concentration of ∼400 µg/L, and those for strain NY05 contained ∼140 µg/L. For the aging experiments, soil slurries were shaken for 24 h and then stored in the dark with periodic shaking. Soil-free controls were prepared in soil-extract solutions. Biphenyl concentrations in the controls ranged approximately from 100 µg/L to 400 µg/L for strain P106 and 1978
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25 µg/L to 140 µg/L for strain NY05. The amounts of radioactivity in the soil-free controls were similar to those in the soil slurries. Sterility of the soil slurries was checked as described above. Sterile phosphate buffer controls were set up together with soil slurries to monitor if there were biphenyl losses during aging periods. Soil slurries and soil-free controls were inoculated with 0.75 mL of cell suspension harvested during the early stationary phase. Cell density in serum bottles was ∼107 CFU/ mL for strain P106 and ∼106 CFU/mL for strain NY05. At predetermined time intervals, 1 mL of suspension and 1 mL of headspace were withdrawn from each bottle and injected into a sealed tube containing 1 mL of 2 N HCl. Radioactive CO2 evolved was trapped on NaOH (2 M) saturated filter paper (1 × 8 cm) in a center well hanging from a sleeve stopper. After overnight equilibration, the filter paper strip was transferred to a scintillation vial containing 10 mL of scintillation fluid; 2 mL of 50% (v/v) ethanol used to wash the center well was combined with scintillation fluid. Samples were left in the dark for at least 24 h before liquid scintillation counting. The trapping efficiency, determined using NaH14CO3, was ∼100%. Data Analysis. When the bacterial cell density is high relative to the substrate concentration, little or no cell growth occurs. Under these conditions, biodegradation can be adequately described by the Michaelis-Menten equation. When initial chemical concentration is below Km, first-order kinetic approximations apply. The Km values were determined by measuring mineralization rates over a range of initial biphenyl concentrations and fitting the data to the equation by nonlinear regression analysis. In a first-order rate reaction, the reaction rate is proportional to the first-order rate constant (k) and concentration of the reactant (C):
rate ) -
dC ) k‚C dt
(1)
The integrated form of the first-order product formation equation is
P ) Pmax‚(1 - e-k‚t)
(2)
where P is the percentage of initial radioactivity mineralized, Pmax is the maximal percent mineralized, k is the first-order rate constant, and t is time (4, 23). Mineralization data were fit to the integrated form of the first-order kinetic equation to obtain the estimates for Pmax and k. The initial mineralization rate (IMR, µg‚L-1‚ min-1) is the product of Pmax, k (min-1), and initial biphenyl concentration (µg L-1) and can be visualized as the initial slope of the CO2 production curve. If sorbed biphenyl is totally unavailable to bacteria, only biphenyl in the aqueous phase can be degraded in soil slurries. IMR values of soil slurries would then be expected to equal the soil-free control rate at the same aqueous phase concentration. If the IMRs of soil slurries are above the corresponding control, this indicates that bacteria either have access to sorbed biphenyl or that desorption is rapid relative to degradation. Coupled Desorption/Biodegradation Model. Employing a mass balance on biphenyl in the soil slurries, the initial biphenyl concentration that is potentially available for biodegradation (Co, ug L-1) is expressed as
Co )
Ci 1 + m‚Kd/V1
(3)
where Ci (ug L-1) is initial liquid-phase concentration before adding soil to the batch, m (kg) is amount of soil added, Vl
(L) is liquid volume, and Kd (L kg-1) is the distribution coefficient, which is defined by the ratio of sorbed biphenyl amount in unit soil mass to dissolved biphenyl amount in unit liquid volume in soil slurry. Kd was obtained from the sorption isotherm. The mass balance on contaminant in the biodegradation slurry system is
(
dS dC + m‚ ) V1‚k‚C dt dt
)
- V1‚
(4)
assuming only dissolved biphenyl is available for biodegradation and the biodegradation rate is first order. S (ug kg-1) is solid-phase concentration. First-order mineralization was verified from the experimental data for CO2 evolution. Assuming instantaneous desorption, the desorption rate of biphenyl from the solid phase can be expressed as
dS dC ) K d‚ dt dt
(5)
Rearranging and integrating eq 4, the liquid-phase concentration can be expressed as
C ) Co‚e-Bf‚k‚t
(6)
where
Bf )
1 1 + Kd‚m/V1
(7)
This bioavailability factor (Bf, dimensionless) was used by Zhang et al. (24) to simulate the contaminant biotransformation rate in a batch system. The mass balance for production of CO2 is
dPco2 dC dS ‚V1 ) Yco2‚ ‚V - ‚m dt dt 1 dt
(
)
(8)
where Pco2 (ug L-1) is the production of CO2 and Yco2 (ugCO2 ug-substrate-1) is the yield of CO2 from the contaminant degraded. Substituting eq 5 into eq 8 and rearranging produces
dPco2 1 dC ) Yco2‚ ‚ dt Bf dt
(
)
(9)
Integrating eq 9 gives
Pd ) Pmax‚(1 - e-Bf‚k‚t)
(10)
for soil slurry with instantaneous desorption, and
Pnd ) Bf‚Pmax‚(1 - e-k‚t)
(11)
for soil slurry without desorption, where Pd and Pnd (%) are the percent of CO2 production in a soil slurry with instantaneous desorption and no desorption, respectively. The assumptions implicit in eq 10 (Pd) are that bacteria can utilize only dissolved contaminant in the liquid phase and that desorption occurs instantaneously maintaining equilibrium conditions between the solid and liquid phases during biodegradation. In practice, instantaneous desorption is unlikely to occur in soil assays because most soils have multiple sites with different kinetic behaviors (25). Sharer (26) reported that the reversible portion of sorbed biphenyl was 70% in Capac A soil and 67% in Capac B soil. Furthermore, he noted that 80% of the reversible sites exhibited rate-limited desorption. Therefore, the expression with instantaneous desorption (Pd) may overestimate actual CO2 production in these soil slurries
and should be viewed as a limiting condition. Another limiting case is given by the expression for Pnd, which assumes that sorbed biphenyl does not dissolve into the liquid phase during biodegradation. Therefore, Pnd most likely underestimates CO2 production in these soil slurries for the opposite reasons. It should be noted, however, that both production expressions assume degradation of only liquid-phase biphenyl. Mineralization data were plotted together with the theoretical lines, Pd (eq 10) and Pnd (eq 11), to evaluate sorbed biphenyl bioavailability. If sorbed biphenyl is unavailable to bacteria and only biphenyl in the aqueous phase can be degraded in soil slurries, the amount of initial CO2 production should be equal or less than Pd, which accounts for instantaneous desorption, and should be above Pnd, which assumes no desorption. However, if the initial CO2 production levels in soil slurries are above Pd values, this indicates that mineralization rates are faster than would be expected based on liquid-phase concentrations, possibly indicating that bacteria have access to sorbed biphenyl. If the CO2 production rate is below Pnd values, this indicates slower rates and may suggest that the slurry system inhibits mineralization activity. Chemical Analysis. In the sorption experiments, biphenyl was analyzed by reverse-phase high-pressure liquid chromatography (HPLC) using UV (254 nm) and liquid-scintillation detection. The mobile phase was 80% methanol and 20% water (v/v) at a flow rate of 1 mL/min. Radioactivity was determined by liquid scintillation counting. Samples were prepared in Safety-Solve high flash point cocktail (Research Products International Corp., Mt. Prospect, IL).
Results Sorption of Biphenyl. Organic carbon contents of the soils used in the experiments varied from 0.4% for Capac B to 7.8% for Colwood A. At room temperature, sorption isotherms of biphenyl (50-400 µg/L, 24-h equilibration time) for all four soils were linear with the correlation coefficients ranging from 0.9986 to 0.9999 (11 degrees of freedom, dfn-1), and sorption coefficients increased from 5.78 L/kg to 294 with increasing soil organic carbon contents (Table 2). Sorption of biphenyl to Capac B soil increased significantly over time; however, changes were less dramatic for Schoolcraft A and Capac A, and did not change significantly for Colwood A (Table 2). HPLC analysis indicated that biphenyl was not transformed during the aging periods. Bioavailability of Soil-Sorbed Biphenyl. Both Pseudomonas putida P106 and Rhodococcus erythropolis NY05 used biphenyl as the sole source of carbon and energy and mineralized biphenyl into carbon dioxide. According to Michaelis-Menten kinetics, degradation rates are linearly proportional to substrate concentration when nongrowing cells are used, and substrate concentrations are below the Michaelis-Menten constant (Km). Nonlinear regression estimates of Km were 3.9 mg/L for strain P106 and 0.32 mg/L for strain NY05. Bioavailability assays for strain P106 were carried out using all four soils. Soil slurries and soil-free controls were equilibrated with biphenyl for 24 h before inoculation (2.13 ( 0.28 × 107 CFU/mL). The extents of biphenyl mineralization by strain P106 ranged from 29.1% to 39.2% in all four soils and from 32.7% to 33.7% in the soil-free controls. IMR values were calculated from mineralization data and plotted as a function of the equilibrium aqueousphase biphenyl concentration (Figure 1). To assess whether bacteria might have access to the pool of sorbed biphenyl, IMR values in soil slurries were compared to IMR values in soil-free controls having the same aqueous-phase biphenyl concentrations as those in the soil slurries before inoculation. In the experiments with strain P106, sorption caused a decrease in the aqueous-phase biphenyl concentration from 400 µg/L initially to
