Environ. Sci. Technol. 1999, 33, 4086-4091
Biosensing 2,4-Dichlorophenol Toxicity during Biodegradation by Burkholderia sp. RASC c2 in Soil Y V O N N E B E A T O N , * ,†,‡ L I Z J . S H A W , § L. ANNE GLOVER,‡ A N D R E W A . M E H A R G , §,| A N D KEN KILLHAM† Department of Plant & Soil Science, University of Aberdeen, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, U.K., Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, U.K., and Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire PE17 2LS, U.K.
Biodegradation of the model pollutant, 2,4-dichlorophenol (2,4-DCP) by Burkholderia sp. RASC c2, in contaminated soil was assessed by combining chemical analysis with a toxicity test using Escherichia coli HB101 pUCD607. E. coli HB101 pUCD607 was previously marked with luxCDABE genes, encoding bacterial bioluminescence and was used as an alternative to Microtox. Mineralization of 14C-2,4DCP (196.2 µg g-1 dry wt) in soil occurred rapidly after a 24 h lag. Correspondingly, 2,4-DCP concentrations in soil and soil water extracts decreased with time and concentrations in the latter were at background levels (98%; Sigma) supplemented with nonradioactive 2,4DCP (>99%; Aldrich) to achieve a final activity of 249 Bq g-1 dry wt and give final concentrations of 49.3, 98.5, 197.0, 295.5, and 469.1 µg g-1 dry wt. Microcosms were incubated at 25 °C for 1 h. Mid-log phase Burkholderia sp. RASC c2 (14) cells cultured in tenth strength PTYG broth (PTYG/10 (14)) with shaking (no. 6 Luckman R100 shaker at 25 °C) were harvested in 1.35 mL aliquots in the presence of 0.1 M CaCl2 by centrifugation (25 °C, 5 min, 11 600g). Supernatant was discarded, cells were resuspended in 1.35 mL sterile minimal salts medium (MSM (16)), and the suspensions pooled. Aliquots (0.5 mL; approximately 107 cfu g-1 dry wt soil) of cells were added to four replicate microcosms for each concentration treatment. Final soil moisture content of microcosms was 60% of the water holding capacity (WHC; 0.31 mL g-1 dry wt) following addition of 2,4-DCP and cells. An alkaline trap (1 mL of 1M NaOH in a test tube) was added to each soil sample, and microcosms were incubated at 25 °C in darkness. 10.1021/es9903025 CCC: $18.00
1999 American Chemical Society Published on Web 10/02/1999
Replicate control samples consisted of additional microcosms (1) without 14C-2,4-DCP for determination of background 14C and (2) without cells for determination of abiotic degradation and volatilization. Control microcosms were prepared as described above, but equivalent volumes of sterile MSM were added to noninoculated and nonspiked control microcosms to achieve a final moisture content of 60% WHC. At time intervals, NaOH solution was removed from the trap and mixed with 4 mL of Ultima Gold scintillation cocktail (Packard, Pangbourne, Berks, U.K.) in a scintillation vial. Fresh 1 M NaOH was added to each trap, and microcosms were returned to the incubator. Radioactivity associated with each sample was quantified using a Tri-Carb liquid scintillation counter 2500TR (Packard) programmed to count each sample for 180 s. Data were expressed as cumulative percentages of the initial spike for each individual replicate. 2,4-DCP Toxicity in Soil. Microcosms were prepared as described before, except that soils were amended with 2,4DCP to give concentrations of 0, 10.0, 24.9, 50.1, 99.9, and 199.9 µg g-1 dry wt at 60% WHC. Soil was incubated for 1 h (25 °C), and a slurry was prepared by adding 4 mL of sterile distilled water. The soil slurry was mixed by vortexing, and extracts were obtained by horizontal shaking at 25 °C for 30 min (no. 6, Luckman R100 shaker) and centrifugation (268g, 5 min, 25 °C). Supernatant was collected, and aliquots were removed for analysis of 2,4-DCP concentration using highpressure liquid chromatography (HPLC) and toxicity assessment using the lux-modified biosensor, E. coli HB101 pUCD607. HPLC Analysis. The liquid chromatography system consisted of a Spectraphysics SP8810 LC pump operating isocratically at 0.5 mL min-1 with HPLC-grade methanol (Rathburn, Walkerburn, U.K.) and Milli-Q water adjusted to pH 2.5 with concentrated H3PO4 (80:20; v/v) as the mobile phase. Separations were made using an Econosil C18 column (250 × 4.6 mm, 5 µm) following injection of a 20 µL sample. 2,4-DCP was detected using UV absorbance at 229 nm (Varian polychrom 9060 diode array detector) and peaks quantified by comparison of peak areas (Varian 4290 integrator) with external standards. Using these conditions, 2,4-DCP retention time was 11 min, and the detection limit was 0.12 µg mL-1. lux-Based Toxicity Assay. Freeze-dried cultures (stored at -20 °C) of E. coli HB101 pUCD607 were prepared using log phase LBG-broth cultures (12). Freeze-dried cultures were resuscitated by resuspending 10 mL of LBG and by incubating at 25 °C for 2 h with shaking (no. 6, Luckman R100 shaker). Cells (1 mL aliquots) were harvested by centrifugation (25 °C, 11 600g, 1 min), washed, and resuspended in 1 mL 0.1 M KCl. Cells were diluted in 0.1 M KCl, and 100 µL aliquots (final cell concentration in assay ) 1.8 ( 0.4 × 105 cfu mL-1) were added to replicate 900 µL soil water extracts. Triplicate toxicity assays using replicate resuscitated E. coli cultures were carried out for each soil water extract. Samples were mixed by vortexing, and their running order for determining light output was randomized. Luminescence was determined by scintillation counting as described for 2,4-DCP mineralization experiments, except the instrument was programmed to count for 15 s using the single photon count option. Mean percentage decrease in luminescence was calculated against the nonspiked control. Data were log transformed to enable analysis by linear regression. An EC50 value was calculated by solving the regression equation for values of Y ) 50% for each of the triplicate assays. 2,4-DCP Toxicity and Concentration in Spiked Soil During Biodegradation. Replicate soil microcosms were prepared as described above but were spiked with one concentration of 14C-2,4-DCP (final activity ) 305 Bq g-1 dry wt soil: final concentration ) 196.2 µg g-1 dry wt soil). After incubation at 25 °C for 1 h, microcosms were inoculated with mid-log Burkholderia sp. RASC c2 cells as described for
FIGURE 1. Mineralization of 14C-2,4-dichlorophenol at 50 µg g-1, b; 100 µg g-1, 9; 200 µg g-1, 2; 300 µg g-1, O; and 500 µg g-1, 0 by Burkholderia sp. RASC c2 in autoclaved soil microcosms incubated at 25 °C in darkness. Bars represent standard errors of four replicates. the mineralization experiment (final moisture content of 60% WHC and 3.4 ( 0.3 × 107 cfu g-1 dry wt). Alkali traps were added to each soil sample, and microcosms were incubated at 25 °C in darkness. Control microcosms were prepared exactly as described earlier. On day 0 at T ) 3 h, four replicate (day 0) samples were randomly selected from control treatment soil samples. Measurement of mineralization was carried out as described before. Similarly HPLC analysis of 2,4-DCP concentration in soil water extracts and toxicity of soil water extract to E. coli were performed as described earlier. In addition, the number of culturable Burkholderia cells in soil water extracts was determined by spot plating serial MSM dilutions on both nutrient agar (NA) and PTYG/ 10 agar. Triplicate samples were also removed from sterile control microcosms and spot inoculated without dilution onto both NA and PTYG/10 agar. All cultures were incubated at 25 °C until discrete colonies could be counted. Soil pellets were resuspended in 10 mL methanol and shaken at 25 °C for 1 h (no. 6, Luckman R100 shaker). Soil was allowed to settle, and duplicate 1 mL samples were removed for determination of 2,4-DCP concentration by HPLC and scintillation counting as described before, using Ultima Gold in a 1:2 sample-to-scintillant ratio. The remaining methanol layer was decanted, and the volume was recorded for mass balance calculations. Soil-associated methanol was evaporated (25 °C, 48 h), and soils were ovendried (105 °C, 16 h). Residual 14C associated with the soil was determined by chromic acid digestion as described by Dalal (17), except test tubes containing NaOH (4 M, 2 mL) were used to trap 14C-CO2 released by oxidation. 14C in the traps was determined by scintillation counting as before, but Hionic-Fluor (Packard) was used as the scintillant in a 1:12 (v/v) sample-to-scintillant ratio. This procedure enabled 99.8 ( 2.2% recovery of a known 14C-2,4-DCP spike added to soil.
Results Dose Dependent 14C 2,4-DCP Mineralization. Mineralization of 14C 2,4-DCP by Burkholderia was detected after an initial lag, and this lag period increased as the concentration of 2,4-DCP increased (Figure 1). At the lowest concentrations (50 and 100 µg g-1 dry wt), mineralization was rapid and was detected within 24 h of inoculation. For initial 2,4-DCP concentrations of 50-200 µg g-1 dry wt, approximately 70% of the initial radioactivity was recovered in the alkaline traps after 10 d. At higher concentrations of 2,4-DCP (300 and 500 µg g-1 dry wt), 66.4 ( 2.2 and 31.8 ( 15.3%, respectively, of initial 14C was evolved by day 19. Greater variability was associated with mineralization of 2,4-DCP at the two highest concentrations. VOL. 33, NO. 22, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Changes in toxicity to E. coli HB101 pUCD607 following 15 min exposure to soil water extracts from 2,4-DCP spiked soil. The x-axis describes 2,4-DCP concentration in the water extract. Bars represent standard errors. Dose Dependent Toxicity of 2,4-DCP to E. coli HB101 pUCD607. An initial experiment investigating toxicity of water extracts from soil spiked with increasing concentrations of 2,4-DCP was carried out using lux-marked E. coli, and the dose response curve is shown in Figure 2. At a soil water concentration of 4.5 ( 0.3 µg mL-1, 2,4-DCP reduced light output of E. coli by 50% (EC50). This corresponded to a soil spike of 34.2 ( 2.4 µg g-1 dry wt soil. At a soil spike concentration of 2,4-DCP at 199.9 µg g-1 dry wt, which was equivalent to 29.5 µg mL-1 in the soil water extract, the mean percentage luminescence of E. coli was 3.9 ( 0.3%. Toxicity Assessment and Chemical Analysis During Biodegradation. Figure 3a shows mineralization of 2,4-DCP (added at an initial concentration of 196.2 µg g-1 dry wt) in autoclaved soil and in autoclaved soil inoculated with Burkholderia sp. RASC c2. Rapid mineralization proceeded in inoculated, spiked soils after a lag phase of 24 h and 30.9 ( 0.5% of the initial 14C added was released as 14CO2 on day 2. ANOVA showed that there was a corresponding, significant (p e 0.001) decrease in 2,4-DCP concentration (measured by HPLC) in soil water extracts from 25.2 ( 1.7 µg mL-1 (20.5 ( 0.6%) on day 1 to below the limit of detection (0.12 µg mL-1) for three out of four replicates on day 2 (Figure 3b). Simultaneously, mean bioluminescence of E. coli in soil water extract expressed as a percentage of the nonspiked, inoculated control increased from 8.4 ( 0.2% on day 1 to 149.5 ( 3.2% on day 2 (Figure 3c) in spiked, inoculated samples. Mineralization continued in inoculated, spiked samples and by day 7, 63.9 ( 2.3% of the initial 14C added was released as 14CO2 (Figure 3a). In comparison, 2.6 ( 0.3% of the initial 14C added was recovered in the NaOH traps of the noninoculated controls by day 7. HPLC analysis of these NaOH traps revealed that the radioactivity was associated with the parent compound and was therefore present as a result of volatilization losses. Concentration of 2,4-DCP in soil water extracted from inoculated soil remained at or below the detection limit (Figure 3b) after day 2. Incubation time had a significant (p e 0.001; ANOVA) effect on 2,4-DCP concentrations in sterile soil water extracts. Concentrations had decreased from 32.2 ( 0.7 µg mL-1 on day 0 to 26.0 ( 0.8 µg mL-1 on day 7. ANOVA showed no significant (p g 0.05) difference between the concentration of 2,4-DCP and 14C in soil water extracts at each time point for the noninoculated treatment. In contrast, from day 1 onward, there was discrepancy between the two chemical methods employed for analysis of water extracts from inoculated soils and the mean percentage 14C was significantly (p e 0.005) higher than the mean HPLC-determined 2,4-DCP concentration (Figure 3b). 4088
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FIGURE 3. Changes in (a) mineralization of 14C-2,4-DCP in inoculated (0) and sterile (9) soil microcosms; (b) 14C-2,4-DCP recovered in soil water extracts by scintillation counting (14C, squares, dashed line) and HPLC analysis (14C-2,4-DCP, triangles, solid line) from inoculated (open symbols) and sterile (closed symbols) soil microcosms; (c) toxicity to E. coli HB101 pUCD607 of soil water extracts from inoculated (0) and sterile (9) soil microcosms; (d) methanol extractable 14C-2,4-DCP recovered from inoculated (open symbols) and sterile (closed symbols) soil microcosms by scintillation counting (14C, squares, dashed line) and HPLC analysis (14C2,4-DCP, triangles, solid line); (e) chromic acid digestion recovery of 14C-2,4-DCP from soil pellets of inoculated (0) and sterile microcosms (9); (f) mass balance of total 14C-2,4-DCP recovered from soil pellet, soil water, and methanol extracts from inoculated (0) and sterile (9) soil microcosms; (g) Burkholderia sp. RASC c2 cell numbers recovered from 14C-2,4-DCP spiked (2) and nonspiked (9) soil microcosms. All microcosms were incubated at 25 °C in darkness. Bars represent standard errors. Following stimulation on day 2, light output of E. coli decreased, and on day 3 was 110.3 ( 3.2% of control samples (Figure 3c). Values remained at approximately 100% for the duration of the experiment (Figure 3c). One way analysis of variance showed that toxicity of water extracts from spiked, sterile soil decreased significantly (p e 0.001) over time. The percentage of methanol extractable 2,4-DCP from noninoculated control microcosms was relatively constant during the experiment at approximately 85% (Figure 3d). Initially, 80.5 ( 3.2% (equivalent to 172.9 ( 6.0 µg g-1) of 2,4-DCP could be extracted by methanol from Burkholderiainoculated soil, but only 4.3 ( 1.3% (8.5 ( 2.6 µg g-1) could be extracted by day 7 (Figure 3d). The decrease in water and
methanol extractable 14C corresponded to increases in mineralized 14C (Figure 3a) and nonextractable 14C (Figure 3e). In contrast to water extracts, there was no significant (p g 0.05) difference between the percentage of 14C and 2,4DCP recovered by methanol extraction from either soil treatment which implied that physically adsorbed 14C was in the form of 2,4-DCP. From day 2 onward, an additional 17.620.4% of the initial 14C added, which was not extractable by water or methanol, could be recovered from Burkholderia sp. RASC c2-inoculated soils by chromic acid digestion (Figure 3e). In contrast, negligible amounts (
