INTRODUCTION

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Use of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) in the United States was estimated in 1991 to be 34 to 41 million kilograms annually (30). Contamination of soil from pesticide mixing, loading, storage, and rinsing at agricultural chemical dealerships in the Midwest is a concern due to potential contamination of surface water and groundwater. The U.S. Environmental Protection Agency (EPA) reported that atrazine has been found in the groundwater of approximately 25 states due to both point and nonpoint sources. In a 1987 study of Iowa public water systems, 16 of the 18 wells with detections of pesticides, including atrazine, were located within 1,000 feet of an agricultural chemical dealership (12). High levels of pesticides and nitrate in soils, surface water, and groundwater were found at 28 dealerships in Iowa, with maximum atrazine concentrations of 1,100 µg g¹ in soil, 16 µg liter¹ in surface water, and 1,500 µg liter¹ in groundwater (14), which are well above the U.S. EPA maximum contaminant level of 3 µg liter¹ for drinking water. Low-cost strategies for bioremediation of atrazine and other pesticides in soil and groundwater at agrichemical dealership sites are needed.

The degradation of atrazine occurs predominantly by biological processes, including N-dealkylation, dechlorination, and ring cleavage. Atrazine biodegradation can be initiated by N-dealkylation of the ethyl or isopropyl side chains to produce deethylatrazine (DEA) or deisopropylatrazine (DIA) (3, 6, 22-24). Dechlorination has been reported as an early step in atrazine metabolism (6, 20), and two different s-triazine hydrolase enzymes (11, 25) have been characterized. In some microorganisms, complete biodegradation of atrazine to ammonia and CO₂ has been obtained (19, 28, 32). These previous studies show a wide variation in the kinetics and extent of atrazine degradation and the ability of bacteria to grow on atrazine. In addition, the stimulatory or inhibitory effects of additional carbon or nitrogen sources on atrazine degradation varies among the bacteria described in the literature. An understanding of these factors is necessary in order to improve the effectiveness of these bacteria in bioremediation applications.

Previous studies have shown that atrazine-degrading bacteria applied as single strains or as consortia can increase degradation of atrazine in soil, but these treatments vary in their effectiveness. Soil contaminated with 1,500 µg of atrazine g¹ was inoculated with the Pseudomonas strain ADP, resulting in the degradation of approximately 17% of the atrazine (19). Addition of citrate to the soil increased the degradation to 70% of the aged atrazine. Addition of a Pseudomonas strain resulted in mineralization of over 60% of 10 µg of atrazine g¹ soil in 49 days and an atrazine half-life of 1 day (33). However, the addition of the same strain to another low-pH soil and a soil with a high organic-matter content resulted in much slower atrazine degradation, with half-lives of 19 and 22 days, respectively. Mixed cultures of microorganisms have also increased the biodegradation of atrazine when added to soils containing relatively small amounts of atrazine (3, 4).

The objectives of the present study were to determine the factors that govern the atrazine biodegradation by a newly isolated bacterium and to determine its potential effectiveness in degrading atrazine in soil. We describe here the isolation and characterization of Agrobacterium radiobacter J14a, a strain which is capable of utilizing atrazine as a sole nitrogen source. Experiments were also conducted to determine the effects of secondary carbon and nitrogen substrates on atrazine degradation in culture and in soils contaminated with atrazine.

	MATERIALS AND METHODS

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Chemicals. Atrazine, DEA, DIA, deethyldeisopropylatrazine (DEDIA), simazine, and cyanazine were purchased from ChemService, West Chester, Pa. Propazine was obtained from Riedel-de Haen, Seelze, Germany. Ametryne and prometon were obtained from the EPA, Research Triangle Park, N.C. All chemicals exceeded 96% purity and were used without further purification. Hydroxyatrazine (HA), deethyl-hydroxyatrazine (DEHA), and deisopropyl-hydroxyatrazine (DIHA) were obtained from Robert Lerch (USDA-ARS Cropping Systems and Water Quality Research Unit, University of Missouri, Columbia, Mo.). The [¹⁴C-U-ring]atrazine (18.96 mCi mmol¹, 98% radiochemical purity) and [N-ethyl-¹⁴C]atrazine (13.18 mCi mmol¹, 98% radiochemical purity) were purchased from Sigma Chemical Co., St. Louis, Mo.

Enrichment and isolation. Surface soil collected from an atrazine-treated corn field near Sheldon, Nebraska, served as the inoculum for nitrogen-limited enrichment cultures. This enrichment medium, designated BMA, contains a basal minimal salts medium supplemented with 1 g each of sodium citrate and sucrose, 20 ml of vitamin solution (5 mg of thiamine, 2 mg of d-biotin, 2 mg of folic acid, 10 mg of nicotinamide, and 10 mg of pyridoxine per liter), 20 ml of trace elements solution (21), and 50 mg of atrazine in 1 liter of deionized water. After several transfers into fresh BMA liquid medium, a mixed culture was obtained. Due to production of large amounts of polysaccharide on BMA and 0.5 strength tryptic soy agar (TSA) (Difco Laboratories, Detroit, Mich.) plates, it was difficult to obtain a pure culture by streaking for isolation. We used the Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.) density centrifugation method (27) to separate the organisms in the culture. Isolates from each of three bands were screened for the ability to degrade atrazine, and the atrazine-degrading isolate J14a was used in further experiments. A second atrazine-degrading bacterium was also isolated, which we do not describe in this report.

Isolate identification. Fatty acid profile analysis of the isolate was performed by Microcheck, Inc., Northfield, Vt. Microcheck subcultured the isolate onto TSA, incubated the plates overnight at 28°C, harvested and extracted the cells, and analyzed the cellular fatty acids of the isolate by high-resolution gas chromatography. The isolate's profile was compared for similarity to the profiles of their 1,700-strain database. The substrate utilization of J14a was examined by using Biolog plates (Biolog, Inc., Hayward, Calif.). The inoculum for the Biolog plates was grown on TSA overnight and, after inoculation, the plates were incubated for 24 h in the dark at 30°C. The substrate utilization patterns were determined by absorbance measurements with an automated microplate reader and compared with the Biolog strain database. The results obtained from the fatty acid and Biolog identification system analysis were confirmed by tests for 3-ketolactose production, carrot tumorigenesis, Kovac's oxidase, catalase, and urease. Flagellum numbers and position were observed under a transmission electron microscope by the phosphotungstic acid negative-staining technique.

Inoculum preparation. Unless otherwise stated, the inoculum for all of the experiments was prepared by growing bacteria in 50 ml of either BMA or 0.5-strength tryptic soy broth (TSB) for 3 days at 28°C on a rotary shaker at 120 rpm. Cultures were pelleted by centrifugation at room temperature at 7,000 × g for 10 min. Cells were rinsed twice with 20-ml aliquots of sterilized 0.0125 M phosphate buffer (pH 7.2) and quantified by plate count techniques.

Mineralization and degradation. Mineralization of [¹⁴C-U-ring]atrazine was used to confirm the atrazine-degrading ability of the isolate. We prepared triplicate biometer flasks (Bellco, Vineland, N.J.) containing 50-µg ml¹ and 1,500 Bq of [¹⁴C-U-ring]atrazine in 50 ml of BMA. Three uninoculated controls were also monitored for ¹⁴CO₂ production. At each sampling time the entire 10-ml volume of NaOH in the biometer sidearm was removed and replaced with fresh NaOH. A 3-ml aliquot of the NaOH was transferred to a scintillation vial with 15 ml of Ultima Gold scintillation cocktail (Packard Instrument Company, Meriden, Conn.) and analyzed with a Packard 1600 liquid scintillation counter (LSC). The radioactivity counted by the LSC was corrected for quenching and background radioactivity. The final degrader population in each flask was determined by plating. Additional flasks of BMA containing 50 µg of unlabeled atrazine ml¹ were monitored for atrazine concentration and metabolite production by high-pressure liquid chromatography (HPLC) and J14a cell counts. The Monod and logistic growth models (29) were used to describe atrazine mineralization: where S₀ is the initial substrate concentration (in micrograms per milliliter), X₀ is the amount of substrate (in micrograms per milliliter) required to produce the initial population density equal to B₀ (the initial biomass), and X₀ = B₀/Y (yield). The rate constant k₄ is equivalent to µ_max/K_s, where µ_max is the maximum specific growth rate (per hour) and K_s is the half-saturation constant (i.e., the substrate concentration [in micrograms per milliliter] when µ is half of µ_max). An additional parameter, , is needed to estimate S from measures of ¹⁴CO₂ respired and ¹⁴C in the medium;  is the constant fraction of radiolabeled substrate incorporated into the cells. Parameters for these models were estimated by nonlinear regression analysis (29).

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Substrate range. Triplicate flasks containing minimal salts medium were treated with vitamins, carbon sources, trace elements, and one of the following herbicides: atrazine, 50 µg ml¹; ametryne, 5 µg ml¹; cyanazine, 50 µg ml¹; prometon, 5 µg ml¹; propazine, 5 µg ml¹; or simazine, 1 µg ml¹. Each flask was inoculated with approximately 5 × 10⁷ cells ml¹ of J14a and incubated on the rotary shaker for 120 h. Initial and final herbicide concentrations were determined by HPLC.

HPLC analysis. HPLC was performed with a Waters HPLC system with a model 490E UV detector operated at 220 nm and a Nova-Pak C₁₈, 10-cm, radially compressed column. Deionized water and acetonitrile (ACN) were used to separate atrazine, DEDIA, DIA, and DEA starting at 25% ACN, followed by a linear gradient to 75% ACN at 10 min, then returning to 25% ACN by 13 min, and holding those conditions until 15 min at 1.8 ml min¹. Calibration standards contained DEDIA, DIA, DEA, and atrazine with retention times of 1.4, 2.5, 4.1, and 9.3 min, respectively. A modified gradient was used to determine the concentrations of the additional s-triazines used in the substrate range experiment. The mobile-phase flow rate was increased to 2 ml min¹. The gradient started at 25% ACN, reached 75% ACN at 8 min, held at those conditions for 4 min, ramped down to 25% ACN-75% water by 14 min, and held at those conditions for 5 min. Retention times for simazine, cyanazine, atrazine, prometon, ametryne, and propazine were 7.0, 7.1, 7.6, 9.5, 9.9, and 10.0 min, respectively. The [¹⁴C]atrazine HPLC method starts with 25% ACN-75% water for 3 min, changing to 75% ACN by 11 min and back to 25% ACN by 16 min, and holds those conditions until 20 min at a flow rate of 1.0 ml min¹. Atrazine and metabolites were quantified by using a Packard RAM detector. Counting efficiency and background were determined with standards prepared from [¹⁴C]atrazine solutions.

Inoculation and degradation in soil. Soil was collected at two agricultural chemical dealerships in Iowa by removing the top 5 cm of soil from locations which appeared to be impacted by the dealerships' pesticide spills and runoff. These dealerships are code-named Alpha and Bravo, and the soils are referenced by the same names. The soil from Alpha had the following characteristics: a sandy loam texture with 75% sand, 17% silt, and 8% clay; 3.2% organic C and 0.07% total N; and a pH of 7.9 (2:1 slurry). The soil from Bravo was a loamy sand with 78% sand, 18% silt, and 4% clay; 2.4% organic C and 0.05% total N; and a pH of 6.5 (2:1 slurry). Soil at both sites consisted of mixtures of soil, sand, and limestone that had been used to maintain a surface at the site. Soils were passed through a 4-mm sieve and 50-g (dry weight basis) portions of soil were adjusted to 10% moisture in Bellco biometer flasks. Atrazine-mineralizing populations were determined with [¹⁴C-U-ring]atrazine as an N source in a most-probable-number (MPN) technique (17) prior to the start of the experiment. Each biometer was treated with atrazine solutions providing 50 or 200 µg of atrazine g¹ of soil and 3,337 Bq of [¹⁴C-U-ring]atrazine. After a thorough mixing, flasks were incubated at 25°C in the dark. After 3 days of incubation to allow for herbicide sorption to the soil, three flasks at each herbicide concentration were treated with 1% (wt/wt) sucrose, 10⁵ J14a cells g¹ of soil, or both sucrose and J14a and incubated for 60 more days. The ¹⁴CO₂ produced was measured by LSC analysis of the NaOH traps. Additional flasks treated with atrazine (but no ¹⁴C), sucrose, and J14a were prepared as described and incubated in a similar way to monitor the atrazine-mineralizing populations by the MPN technique.

	RESULTS

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Identification. J14a is a gram-negative, motile, rod-shaped bacterium with several peritrichous flagella visible under transmission electron microscopy. Fatty-acid analysis of J14a showed an excellent match (similarity index = 0.629) with A. radiobacter and a good match (similarity index = 0.450) with A. rubi. J14a also matched the Biolog profile for A. radiobacter. When grown on TSA medium, J14a produced generally round, beige, mucoid, opaque, smooth-edged colonies that became rough edged with age. J14a showed positive reactions for catalase, 3-ketolactose production, urease, and Kovac's oxidase test. The 3-ketolactose production is unique to two biovars in the genus Agrobacterium, and this positive reaction by J14a is fairly strong proof that the isolate belongs to the genus Agrobacterium. J14a was negative for the carrot tumorigenesis test.

Metabolism and growth. J14a mineralized approximately 94% of the [¹⁴C-U-ring]atrazine in BMA medium after a lag time of approximately 12 h (Fig. 1). Atrazine disappearance and cell growth (Fig. 1) coincide with ¹⁴CO₂ appearance. Cell growth levels off at approximately 72 h, which coincides with atrazine concentration decreasing below the HPLC detection limit of 25 ng ml¹. Mineralization of [¹⁴C-U-ring]atrazine in an experiment similar to that reported in Fig. 1 (data not shown) was described slightly better by the Monod model than by the logistic model, based on residual sums of squares of 1.87 and 21.1, respectively. Parameter estimates for both models are presented in Table 1. Ring mineralization was nearly complete (94%) with little carbon incorporation into microbial biomass (estimated to be 6% at maximum), while the model value of 0.03 µg of C µg¹ substrate for  is equivalent to 1.5% C incorporated. Differences between the observed and predicted estimates of ¹⁴C in the biomass may be due to incomplete metabolism of atrazine or experimental error (measurement error or incomplete mass balance), because incorporation of the triazine ring C would not be expected. Other microorganisms metabolize the triazine ring to urea, then to CO₂ and NH₄⁺ (10, 28). The Monod parameter K_s was estimated to be 38 µg ml¹, but there is significant uncertainty in this estimate. This may be due to the use of supersaturated solutions of atrazine (50 µg ml¹), which exceed the water solubility of 33 µg ml¹. Above this concentration atrazine forms microprecipitates and resolubilization may limit biodegradation. However, the initial concentration estimates from the models (S₀) were near the measured value of 50 µg ml¹, which suggests that the entire amount of atrazine is available to the organisms.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Degradation of 50 µg of [14C-U-ring]atrazine ml1 in N-limited medium by strain J14a. Atrazine remaining in the medium (A) is shown in relation to cell density (B) and 14CO2 production (C).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Structure of atrazine, atrazine metabolites, and related triazines degraded by strain J14a.

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View larger version (25K): [in this window] [in a new window]   FIG. 3.   Mineralization and biomass incorporation of [ethyl-14C]atrazine by strain J14a in N-limited medium with or without additional carbon sources, expressed as the percentage of added 14C.

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Inoculation and degradation in soil. The addition of J14a to soils from the Alpha site resulted in two to five times more atrazine mineralization (significant at P  0.05) than that by the indigenous microbial community (Fig. 4). Sucrose addition had no effect on mineralization. The soils treated with 200 µg of atrazine g¹ and inoculated with J14a mineralized the atrazine continuously throughout the 63-day period, which is in contrast to the mineralization in inoculated soils treated with 50 µg of atrazine g¹ of soil, where mineralization is substantially slower after 35 days. Atrazine-mineralizing populations in the Alpha soil 63 days after atrazine addition were similar to those in the initial populations, despite the addition of 10⁵ cells g of soil¹ at day 3 of the experiment (Table 3). Inoculation with J14a decreased both extractable atrazine and nonextractable bound residues remaining in the Alpha site soil treated with 50 µg g of atrazine¹, but it did not affect the distribution of ¹⁴C in soils treated with the higher concentration of atrazine (Table 3). Despite the differences in the fractional amount (%) of [¹⁴C]atrazine mineralized in the 50- and 200-µg g¹ treatments amended with J14a, the actual mass of atrazine mineralized was very similar in all four treatments, ranging from 27 to 38 µg g¹.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Mineralization of 50 (A) or 200 (B) µg of [14C-U-ring]atrazine ml1 added to soil from the Alpha site. Soils were amended with strain J14a (105 cells g of soil1), sucrose, or both J14a and sucrose 3 days after atrazine treatment.

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View larger version (30K): [in this window] [in a new window]   FIG. 5.   Mineralization of 50 (A) or 200 (B) µg of [14C-U-ring]atrazine ml1 added to soil from the Bravo site. Soils were amended with strain J14a (105 cells g of soil1), sucrose, or both J14a and sucrose 3 days after atrazine treatment.

	DISCUSSION

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In N-limited media with sucrose, J14a was capable of relatively rapid growth on atrazine, with a doubling time of 8.85 h at µ_max. The µ_max/K_s ratio is a general indicator of substrate use efficiency. For the Monod model this ratio is 3.0 × 10³, compared to the 9.8 × 10⁴ estimated by the logistic model (k₄). For comparison, the analogous V_max/K_m ratios for purified s-triazine hydrolase enzymes from Rhodococcus corallinus NRRL B15444R and Pseudomonas strain ADP are 1.25 × 10² and 2.93 × 10², respectively (11, 25). In contrast, µ_max/K_s ratios for 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria ranged from 8.89 × 10³ to 0.1 due to generally smaller K_s values (calculated from data in reference 15). These comparisons indicate that J14a is slightly less competitive for atrazine than the purified bacterial enzymes and much less competitive than the 2,4-D-degrading bacteria are for 2,4-D. Furthermore, the value of 37.5 µg ml¹ for K_s indicates that atrazine concentrations to support µ_max are unlikely to occur in soil systems due to solubility constraints.

Degradation of atrazine and simultaneous production of ¹⁴CO₂ from [¹⁴C-U-ring]atrazine proceeded without significant accumulation of metabolites in the medium. The simultaneous production of small quantities of HA and DEA indicates that J14a produces enzymes that perform N-dealkylation and dechlorination concomitantly. DEHA is produced by the sequential action of these enzymes. The lack of DIA and DIHA accumulation indicates either that J14a preferentially removes the ethyl over the isopropyl side chain, which is consistent with other reports (5, 24) or that removal of the isopropyl side chain is the rate-limiting step of the J14a atrazine metabolic pathway and these metabolites are utilized immediately after production. Strain J14a is similar to strains M91-3 (28) and Pseudomonas strain ADP (19) in terms of dechlorination to produce HA followed by complete mineralization of the triazine ring, but DEA production was not reported for these other strains. The complete mineralization of the triazine ring by this bacterium is more extensive than with M91-3, Pseudomonas strain ADP, or the Pseudomonas strain YAYA6 (32), which mineralized between 40 and 80% of the atrazine. The identification of J14a as an Agrobacterium species is also different from most other triazine-degrading bacteria, which have been identified as Klebsiella (10, 16), Pseudomonas (6, 19, 32), and Rhodococcus (7, 10, 25, 31) species.

Strain J14a grew in media with atrazine as a sole N source, a finding which is similar to previous reports describing s-triazine ring cleavage to NH₄⁺ and CO₂ (10, 28). J14a is also capable of degrading and utilizing atrazine under conditions of simultaneous C and N limitations. However, under the conditions of these experiments growth did not occur, despite the incorporation of the [ethyl-¹⁴C]atrazine into the cellular biomass. The Rhodococcus TE1 strain dealkylates atrazine without concurrent growth (7), but the M91-3 and Pseudomonas YAYA6 strains grow on atrazine under C-limited conditions (28, 32). Under C-limited conditions, strain J14a incorporated approximately 20% of the ethyl side chain, but very little was mineralized as ¹⁴CO₂. The ability to use C in the N-ethyl and N-isopropyl groups of atrazine for population maintenance or growth would confer an advantage to atrazine-degrading bacteria under conditions where atrazine concentrations are high, such as contaminated agrichemical dealership sites.

We determined the effect of several factors on the expression of triazine-degrading activity in strain J14a. Long-term culture in the absence of atrazine and degradation in the presence of chloramphenicol show that the degradation enzymes are produced constitutively. Mandelbaum et al. (21) reported that NH₄NO₃ suppressed atrazine degradation by a mixed culture, and Gan et al. (13) reported that NH₄ suppressed atrazine mineralization in soil. These studies suggested that exogenous N might affect degradation in J14a. However, strain J14a degrades atrazine in the presence of the inorganic nitrogen sources NH₄NO₃, KNO₃, and (NH₄)₂SO₄, indicating that the J14a atrazine-metabolizing enzymes are not regulated by inorganic N concentrations in the environment. Degradative activity is not completely lost upon exposure to organic N sources, such as the organic components of TSB. However, mineralization of atrazine in TSB was incomplete (only about 63%), and greater concentrations of HA were produced in TSB-grown cells relative to BMA-grown cells. We do not know the mechanisms for this effect, and other studies have not addressed the effect of secondary substrates containing organic N; however, these types of compounds would be present in soil and may have analogous effects on the biodegradation process.

J14a also demonstrated a wide substrate range among a variety of s-triazine substrates in N-limited medium, unlike the Pseudomonas sp. isolated by Yanze-Kontchou and Gschwind (32), which could not degrade cyanazine or ametryne. J14a was capable of degrading every s-triazine substrate tested, although each of the triazines tested shares either an N-ethyl, an N-isopropyl, or a Cl functional group with atrazine (Fig. 2). The broad substrate range, constitutive enzyme production, insensitivity to inorganic N, and rapid dealkylation and dechlorination capabilities of J14a indicate that this organism would be ideal for addition to a spill site with a mixture of s-triazine herbicides.

We added strain J14a to soils collected from agricultural chemical dealership sites treated with atrazine at concentrations which are representative of those found at a wide range of sites. Our studies were conducted under conditions that constitute a rigorous test of bioaugmentation with J14a. We used soils from sites nearly devoid of vegetation and thus likely to be low in easily decomposable C. Furthermore, the addition of atrazine and water 3 days prior to J14a inoculation allowed the reestablishment of competing indigenous microbial populations and the sorption of atrazine. Initial sorption reactions are complete within 24 h, although sorption increases slowly thereafter (26). Other researchers (3, 33) simultaneously applied atrazine and bacterial inoculum into air-dried agricultural soils, which may have increased atrazine availability and reduced competing microbial populations.

Strain J14a augmentation was successful in increasing biodegradation of the herbicide at both concentrations in soil from the Alpha site, but it increased biodegradation only slightly in the Bravo soil. The different responses to J14a inoculation in these two soils appear to be related to the presence of indigenous atrazine degraders and to the competitiveness of strain J14a. In soil from the Alpha site, the indigenous atrazine degrader population was low and J14a was able to increase atrazine mineralization. However, the J14a populations declined from 10⁵ cells g of soil¹ at inoculation to below 10² cells g¹ by 60 days after inoculation. The addition of sucrose did not appear to consistently increase either biodegradation or inoculum survival. Other bioaugmentation studies used inoculum levels 20 to 10,000 times larger than the 10⁵ cells g of soil¹ used here, and the fate of those inoculated cells was not determined (3, 19, 33). The enhanced degradation obtained from J14a in the Alpha soil suggests that effective bioaugmentation can be achieved at lower inoculum levels, particularly if survival of the inoculated strains can be promoted.

The Bravo soils had larger initial populations of atrazine-degrading microorganisms, which were effective in mineralizing atrazine, and J14a addition increased the total mineralization above these levels in only one treatment. J14a addition increased mineralization over noninoculated treatments during the 7- to 35-day period, which suggests an initial impact of inoculation. However, by the end of the experiment, the total atrazine-degrading populations (J14a and other atrazine degraders) were below the 10⁵ J14a cells g of soil¹ added initially. We cannot distinguish between J14a populations and other atrazine degraders in the Bravo soils, but some decline in the J14a population occurred.

Irrespective of our experimental treatments, the atrazine residues remaining in soils indicate that bioremediation was incomplete at 63 days. Estimated concentrations of atrazine in the soil water are well below the K_s for J14a measured in culture. It is uncertain how much of the solvent-extractable atrazine remaining in the soil is available to the J14a cells or other atrazine degraders. As residence time in soil increases, the concentration of atrazine in the solution and water-desorbable phases will decrease, while the proportion of atrazine bound to soil organic matter or clays increases and becomes much less bioavailable (1, 2). Preincubation of atrazine in a soil with a high organic-matter content (36%) lowered the bioavailability of the atrazine and limited degradation by a Pseudomonas strain (33).

Greater amounts of bound residue were formed in the Bravo soil, especially in the 200-µg g¹ treatments, than in the soil from the Alpha site. One possible explanation of this finding is that the microorganisms indigenous to Bravo soil may produce greater extracellular concentrations of HA than the Alpha site microorganisms or J14a. Sorption of HA (log K_om = 2.0 to 2.8) to soil organic matter is greater than that of atrazine (log K_om = 1.6 to 2.0), which could result in an accumulation of unavailable radiolabeled material in the bound residue fraction (9).

Agrobacterium strain J14a was isolated from soil and is capable of rapid atrazine metabolism. The constitutive expression of degradative enzymes and activity on a range of triazine herbicides suggest that strain J14a should be an effective bioaugmentation agent. The addition of this bacterium into a soil with an indigenous population that was ineffective in degrading atrazine resulted in significant increases in biodegradation. However, the biodegradation process was not complete, and the poor survival of J14a was a contributing factor. Long-term competition and survival of the inoculum and bioavailability of the chemical are important factors affecting the effectiveness of introduced microorganisms.