Environ. Sci. Technol. 1996, 30, 1596-1603
Survival and Activity of an Atrazine-Mineralizing Bacterial Consortium in Rhizosphere Soil S. ALVEY AND D. E. CROWLEY* Department of Soil and Environmental Sciences, University of California, Riverside, California 92521
Plant rhizosphere effects on atrazine degradation were examined in soil inoculated with an atrazinemineralizing bacterial consortium. The consortium, consisting of three bacterial species, was isolated from an agricultural soil having previous long-term exposure to the herbicide. Atrazine mineralization and metabolite formation were monitored by measuring 14CO evolution from microcosms amended with 2 radiolabeled atrazine and by HPLC of soil extracts. In noninoculated soil, ca. 11% of 14C-chain-labeled atrazine was N-dealkylated, while only 2.4% of the ringlabeled atrazine was mineralized after 5 weeks. Corn plants had no effect on atrazine mineralization or ethyl-side-chain N-dealkylation in noninoculated soils, but the formation of hydroxyatrazine was significantly enhanced in planted soil. Growth of corn in sterilized soil suggested that hydroxyatrazine formation was caused by plant metabolism of atrazine. Introduction of the atrazine-mineralizing consortium into the soil significantly increased the rate of atrazine mineralization in comparison to noninoculated soil. After 4 weeks, 71% of the atrazine was mineralized in nonplanted soil, whereas 84% of the atrazine was mineralized in soil with corn plants. There was no significant difference in the rate of atrazine mineralization by the consortium in nonplanted and planted soil. However, atrazine-mineralizing populations at the end of the incubation were higher in the planted soil, which contained 8.1 × 104 degraders g-1 of soil versus 2.7 × 103 degraders g-1 in soil without plants. The results demonstrated that bioaugmentation with the atrazine-mineralizing consortium greatly enhanced the rate of atrazine mineralization. Long-term survival of the consortium and degradation of atrazine to hydroxyatrazine were both enhanced in rhizosphere soil, but corn seedlings had no significant effect on the rate of atrazine mineralization, either by the indigenous microflora or in soil inoculated with atrazine-mineralizing bacteria.
* Corresponding author; telephone: 909-787-3785; fax: 909-7873993; e-mail address:
[email protected].
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Introduction Triazine-based compounds are among the most commonly used herbicides in the United States (1) but have been identified as a threat to groundwater quality in many areas where they have been applied for agriculture (2, 3). The half-life for transformation of the most commonly applied triazine herbicide, atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), to nonphytotoxic or less toxic metabolites is generally reported to take from 60 days to more than 1 year, whereas complete mineralization has been estimated at 98%. Nonradioactive atrazine was purchased
from Chemservice (Westchester, PA) and was 99% pure. The atrazine and [14C]atrazine were dissolved in water and sterilized by autoclaving, after which 10-mL aliquots were added to each microcosm to a final concentration of 3 mg of atrazine kg-1 of soil with a specific activity of 0.16 µCi/ mmol. The microcosms were incubated in the greenhouse under ambient conditions during May-July 1995; temperature ranged from 20 to 40 °C, light intensity at midday was ca. 1200 µE m-2 s-1. The corn plants were observed to be healthy for the 5-week growth period but grew at a relatively slow rate as compared to growth under field conditions. After 5 weeks, the corn plants (shoots and roots) were harvested and the microcosms were replanted with wheat (see below). Analyses of atrazine and metabolite accumulation in the soils were conducted after 5 and 8 weeks, immediately following the plant harvests. Wheat Bioassay. A bioassay was performed to determine if atrazine and its metabolites were present at toxic levels in the soil after the initial 5-week incubation. Ten wheat seeds were planted per microcosm in three of the five replicates for each treatment. Percent germination and survival for a 3-week period were compared to control soil that had not been amended with atrazine. Experiment 2. A second experiment examined the influence of corn plants on survival and activity of an atrazine-mineralizing consortium (5) inoculated into the same soil used in experiment 1. Planted microcosms were either nonfertilized or amended with 100 mg kg-1 (NH4)2SO4. Nonplanted soils received the same fertilizer treatment regime and included a poisoned control containing 10 000 mg kg-1 HgCl2. No sterile treatments were included in the experimental design. There were six replicate microcosms in each treatment, half of which were inoculated with the atrazine-mineralizing consortium. The microcosms were incubated in a controlled-environment, plant-growth chamber (Conviron, Model E15, Controlled Environments Inc., Asheville, NC) with a light/dark cycle of 15/9 h and a photon flux density of 370 µE m-2 s-1. Temperature was maintained at 22 °C, and relative humidity was 65%. All microcosms received 0.5 µCi of ring-labeled atrazine. The initial concentration of atrazine in the microcosms was 6 mg of atrazine kg-1 of soil and had a specific activity of 0.08 µCi mmol-1. Inoculum. Inoculum of the atrazine-mineralizing consortium (5) was produced in mineral salts (MS) medium with atrazine (33 ppm) as the sole source of nitrogen and glucose (1000 mg kg-1) as a carbon source. The MS medium consisted of 10 mM K2HPO4, 3 mM NaH2PO4, 1 mM MgSO4, and 10 mL of chloride-free trace element stock solution, which contained the following in mg L-1: CaSO4, 200; FeSO4‚7H2O, 200; MnSO4‚H2O, 20; NaMoO4‚2H2O, 10; CuSO4, 20; CoSO4‚7H2O, 10; H3BO3, 5. Inoculum was harvested after 4 days of growth in liquid medium by centrifugation at 6000 rpm for 10 min. The cells were washed three times in sterile P-saline buffer (8.5 g of NaCl, 0.3 g of KH2PO4, and 0.6 g of Na2HPO4; adjusted to pH 7) and suspended in double-deionized water to an OD600 of 0.1. Inoculated microcosms received 7 mL of the suspension to provide a cell density of 4.5 × 106 g-1 of soil as determined by plating on tryptone-soy agar. Bacterial Identification. When separated on tryptonesoy agar medium, the consortium was shown to contain three bacterial species. Identification was accomplished by fatty acid methyl ester analysis according to standard protocols provided by Microbial ID (MIDI, Newark, DE).
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The three bacterial species were identified as Clavibacter michiganese, Pseudomonas sp., and Cytophaga sp. and had similarity indices of 0.584, 0.229, and 0.170, respectively. Most Probable Number. After the soil had been incubated for 28 days in the microcosms in experiment 2, the entire soil from each microcosm was removed and thoroughly mixed for enumeration of the atrazinemineralizing consortium in each microcosm. A 1-g aliquot of the mixed soil from each microcosm was taken and suspended in 9 mL of sterile, MS medium containing 100 mg kg-1 atrazine in a 20-mL scintillation vial to make the 10-1 dilution. Atrazine is only soluble to 33 ppm in water; thus a significant portion was in a solid phase. However, upon degradation or mineralization of atrazine, it has been our experience that the solid atrazine readily solubilizes. A dilution series was performed by transferring 1 mL of soil suspension from the 10-1 dilution to a second vial containing 9 mL of MS medium (10-2 vial); this vial was then vortexed and 1 mL of solution transferred to the 10-3 vial. The process was repeated to generate a dilution series of eight vials for each replicate sample. The MPN vials were incubated at room temperature on a rotary shaker at 150 rpm for 21 days. After incubation, 1-mL aliquots were centrifuged at 12 000 rpm for 10 min. The supernatant was then analyzed by reverse-phase HPLC. Vials were scored positive if 90%). There was no change in atrazine concentration in sterile soil controls prepared as a 10-1 dilution vial over a 21-day period. The soil contained 0.05) using the statistical program SigmaStat (Jandel Scientific Software, San Rafael, CA).
Results Atrazine Mineralization. After 5 weeks in soil without the degrader consortium, 11% of ethyl-chain-labeled atrazine was N-dealkylated and converted to CO2, while only 2.4% of the ring-labeled atrazine was mineralized to 14CO2 (Figure 1). There was no significant difference in N-dealkylation or mineralization of atrazine in planted versus nonplanted soils. Introduction of the atrazine-mineralizing consortium into this soil (experiment 2) significantly increased the rate of mineralization of ring-labeled atrazine to 71% in inoculated, nonplanted soils and 84% in inoculated soil planted with corn (Figure 2). As in experiment 1, plants had no statistically significant effect on the rate of mineralization in either the inoculated or noninoculated soils. In the absence of the degrader consortium, only 1-3% of the atrazine was mineralized by the indigenous microflora, with the exception of the treatment planted with corn and amended with nitrogen, in which the indigenous microflora mineralized 18% of the atrazine. Most of the mineralization activity in this treatment occurred in only one of the three replicate microcosms; thus this treatment was not significantly different at the 95% confidence level. Addition of nitrogen to the inoculated treatments had no significant effect on atrazine mineralization, irrespective of the presence of plants (Figure 2). Corn plants in both experiments did not show any visual symptoms of nutrient stress other than their small size at the end of the 5-week growth period. Nonsterile plants grown in the greenhouse had an average dry mass of 1.74 g versus 1.65 g when grown in the growth chamber. The standard deviations for dry mass of nonsterile plants grown in the greenhouse versus the growth chamber were 0.1 and 0.08 g, respectively. Sterile plants
FIGURE 1. Mineralization of ring-labeled [14C]atrazine and Ndealkylation of [14C]ethyl-chain-labeled atrazine in planted and nonplanted soils after 5 weeks. Each column represents the mean of five replicates. Error bars represent the standard error of the mean.
had an average dry mass of 1.2 g with a standard deviation of 0.09. At the end of experiment 2, an MPN of the atrazine degraders was performed for both the planted and nonplanted soils. The MPN was based on the least abundant member of the consortium required for atrazine disappearance from solution. No atrazine-degrading population was detected in the noninoculated soil treatments despite the occurrence of at least some low mineralization activity as indicated by the evolution of 14CO2. The data did show, however, that the final population size of the degrader population in the inoculated soils was 30-fold greater in planted soils as compared to the nonplanted soils. Actual consortium unit numbers were 8.1 × 104 g-1 in planted soil with a 95% confidence interval from 2.45 × 104 to 2.67 × 105 versus 2.7 × 103 g-1 in nonplanted soil with a 95% confidence interval of (0.8-8.9) × 103. In the vials that scored positive, there was no measurable accumulation of atrazine metabolites. Metabolite Analysis. The parent compound atrazine comprised >50% of the extractable residue in all treatments in both experiments, except in the nonsterile, planted treatment in experiment 1, where atrazine comprised only 29% of the extractable fraction (Figure 3). The second most common residue in both experiments was hydroxyatrazine. In experiment 1, sterile and nonsterile microcosms were compared to determine the relative importance of the plant versus the soil microflora for conversion of atrazine to hydroxyatrazine. However, by the end of the experiment, all of the sterile microcosms were contaminated as determined by plating soil directly on tryptone-soy agar. Only one or two colony morphologies were observed, and it may be presumed that microbial diversity (i.e., atrazine Ndealkylating bacteria) was considerably lower than in the nonsterilized soil. In planted microcosms, hydroxyatrazine comprised 57% and 41% of the extractable residues under nonsterile and initially sterile conditions, respectively, in comparison to 18% and 10% of extractable residues in
FIGURE 2. Mineralization of ring-labeled [14C]atrazine in inoculated soil with and without plants. Each point represents the mean of four replicates. Error bars represent the standard error of the mean. The symbols labeled with an (a) identify treatments that were significantly different from treatments with the symbols labeled with a (b). Treatments with the same letter are not significantly different from each other. Statistical differences between treatment means were determined by all pairwise multiple comparison procedures (Student-Newman-Keuls method) at the 0.05 level.
nonplanted soil (Figure 3). All metabolites other than hydroxyatrazine, including dealkylhydroxy metabolites and ring fission products, made up 10% or less of the extractable radioactivity (Figure 3). A similar trend was observed in experiment 2, in which hydroxyatrazine comprised ca. 20% of the total remaining atrazine in planted soil as compared to
