Environ. Sci. Technol. 1998, 32, 3789-3792
Rapid Atrazine Mineralization under Denitrifying Conditions by Pseudomonas sp. Strain ADP in Aquifer Sediments N I R S H A P I R , †,‡ R A P H I T . M A N D E L B A U M , * ,† A N D CARSTEN S. JACOBSEN‡ Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50-250, Israel, and Department of Geochemistry, Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark
Atrazine mineralization was studied in sediments taken from a shallow aquifer underlying a cornfield continuously receiving atrazine and terbuthylazine. In contrast to previous publications indicating slow or nonexisting mineralization rates under denitrifying conditions in sediments, we hereby report on the ability of the bacterium Pseudomonas sp. strain ADP to rapidly mineralize atrazine in aquifer sediments under nitrate reducing conditions. When atrazine was present in low concentrations (relevant to nonpoint sources such as agricultural application), the bacterium mineralized 55% and 75% of the atrazine in 2 and 4 days, respectively. When atrazine was present in high concentrations (relevant to spill sites), P. ADP mineralized 48% and 78% in 4 and 15 days, respectively. The present study indicates that bioaugmantation with an effective atrazine mineralizing bacterium such as P. ADP could yield high mineralization rates even under oxygen limited conditions and have a significant implication for bioremediation of atrazine in contaminated aquifers.
Introduction Significant amounts of atrazine [2-chloro-4-(ethylamino)6-(isopropylamino)-s-triazine] leach beneath the aerobic soil layer into lower soil strata and into groundwater where oxygen limited conditions prevail (1, 2). Frequent detection of s-triazine herbicides and their metabolites in subsurfaces (3, 4) and groundwater (3, 5) has increased the importance of elucidating their degradation pathways and rates in the subsurface environment (6). Most studies have reported very slow or nonexistent biological atrazine degradation in aquifers (7-9). Therefore, abiotic degradation was generally considered the dominant dissipation mechanism under aquifer conditions (7, 10). The relatively slow degradation rates of atrazine have been attributed to several factors, including application history, soil nutrient conditions, temperature, oxygen content, redox potential, and the absence of atrazine degrading microorganisms. The presence of nitrate as a frequent co-contaminant in atrazine contaminated aquifers (11) has prompted research * Corresponding author phone: (972)396-83316; fax: (972)39604017; e-mail:
[email protected]. † Agricultural Research Organization, The Volcani Center. ‡ Geological Survey of Denmark and Greenland. 10.1021/es980625l CCC: $15.00 Published on Web 10/13/1998
1998 American Chemical Society
into the effect of atrazine on denitrification (12) and on its own degradation under denitrifying conditions (7-9, 13, 14). The effect of atrazine on the rate of denitrification in soils remains unclear, because atrazine has been reported to enhance (15) or to have no effect on denitrification in soils (16). The slow atrazine degradation rates under anaerobic conditions are reported by several authors: Goswami and Green (8) detected atrazine mineralization only under aerobic conditions. Chung at al. (17, 18) reported 20% atrazine transformation in anaerobic sediments after 30 weeks of incubation. After amendment of basal salt medium, atrazine was removed in 38 weeks with hydroxyatrazine being the main metabolite. No dealkylation of atrazine was observed by Wehtje et al. (7) after 70 days in simulated aquifer conditions. McMahon et al. (9) observed no mineralization of the atrazine ring in 23 days, with some dealkylation to deisopropylatrazine in shallow aquifer sediments; deeper sediments exhibited less dealkylation. Top et al. (19) reported that atrazine was not degraded under anaerobic or denitrifying conditions in sediment samples from two agricultural watersheds and enrichment cultures. Nair and Schnoor (13, 20) showed that denitrifying or oxygen limited conditions in soils reduced atrazine biotransformation and mineralization. Wilber and Parkin (14) reported that second-order rate constants were in the range of (1-3) × 10-5 L/mg VSS for biotransformation of atrazine under aerobic, nitrate-reducing, sulfate-reducing, and methanogenic conditions. Recently three pure bacterial cultures were reported to transform atrazine under reduced oxygen conditions (2123). The isolate M91-3 could mineralize atrazine under aerobic and anaerobic conditions in liquid media (not containing soil). The isolate degraded approximately 50% of the initial atrazine concentration, but mineralization was not reported (22). Since atrazine dealkylation metabolites are still regulated compounds and have a health risk (24), complete atrazine mineralization, or dechlorination is desired. The strain YAYA6 isolated by Yanze-Kontchou and Gschwind was reported to be less efficient under conditions of limited oxygen supply (23). In a later study Stucki et al. (25) reported that this isolate used in continuous liquid reactor under anaerobic conditions (without soil) could mineralize less than 50% of the feed atrazine. Pseudomonas sp. strain ADP (P. ADP) was reported to rapidly mineralize atrazine under both aerobic (26) and oxygen limited conditions (21). In the present study, we report on its atrazine mineralization activity in actual aquifer sediments under denitrifying conditions. The practical value of such a study is best reflected in the requirement to rapidly clean a highly contaminated aquifer in a residential neighborhood in Western Australia (Franzmann, 1998, personal communication). This site is contaminated with up to 2000 µg L-1 (27), which not only disqualifies the water as drinking water but also disables the use of the water for irrigation.
Experimental Section The culture of P. ADP used in this study was grown in a 250 mL shake flask containing 100 mL of liquid atrazine medium (28) at 30 °C on an orbital shaker (150 rpm). Cells were harvested at the late log phase (after 24 h, 8.4 × 108 cells mL-1) by centrifugation (6000g, 10 min), washed three times, and resuspended in sterile phosphate buffer (0.1 M, pH 7.4). Soil above a shallow aquifer and sediments from 30 cm below the water table were collected from a shallow aquifer at Drengsted, Jutland, Denmark. The aquifer underlined a cornfield continuously cultivated and sprayed with either VOL. 32, NO. 23, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Soil and Sediment Characteristics soil depth (cm)
sand silt clay (%)
0-30 87.6 35-55 91.9 210-230 95.5
7.1 3.9 1.5
5.3 4.2 3.0
organic organic total Kd matter carbon nitrogen (%) (%) (%) (L kg-1) 4.1 1.7 0.1
2.41 1.02 0.02
0.15 0.04 0.01
3.06 0.58 0.06
atrazine (20 years, until banned in 1995) or terbuthylazine (after 1995). After collecting the soil samples from an excavated pit (2 m deep, reaching the water table) sediments from the aquifer (210-230 cm below soil surface) were collected using an aluminum borer (7.5 cm wide). Soil analysis was performed by Centrallaboratoriet, Tjele, Denmark, using standard methods. Organic matter content was determined by combusting the sample and measuring the total CO2. Total organic carbon was calculated after subtracting the calcium carbonate CO2, which was measured separately (CO2 release from the sample at low pH). Total nitrogen was measured as described by Hansen (29), and particle size analysis was performed by sedimentation after dispersion using sodiumpyrophosphate. The distribution coefficient, Kd (L kg-1), was calculated from the Freundlich equation x/m ) KdCe1/n. The experimental procedure was carried out according to Shapir and Mandelbaum (30). Soil and sediment characteristics are provided in Table 1. Biometric flasks were used for atrazine mineralization studies. Twenty-gram (equivalent dry weight) wet sediment samples (at water holding capacity) were placed in a capped glass flask (100 mL). Each sample was amended with 100 µL of methanol containing [U-ring-14C]atrazine and nonlabeled atrazine to yield final concentrations of 0, 0.01, and 10 mg of atrazine kg-1 soil and 6 Mbq per sample. Controls (not receiving atrazine) were amended with 100 µL of methanol. After application, the solvent was evaporated in a chemical hood and the sediment thoroughly mixed. Twenty-four h after atrazine application, the samples were amended with 2 mL of phosphate buffer (0.1 M, pH 7.4), containing sodium nitrate and P. ADP cells, to yield 3.5 mM of nitrate (including the preexisting nitrate) and 1 × 106 bacterial cells g-1 sediment. A noninoculated control (with or without atrazine) was prepared with nitrate only. Half of the sediment samples (including control samples not inoculated with P. ADP) were incubated under anaerobic conditions and amended with citrate (as a carbon source) solution in phosphate buffer (0.1 M, pH 7.4) to yield a final concentration of 6.8 µmol sodium citrate g-1 sediment and a final water content of 37.5% [v/w]. Anaerobism was achieved by flashing the samples with high purity nitrogen and introduced into a nitrogen flushed anaerobic hood (Coy, Grass Lake, MI). The absence of oxygen was continuously verified using an oxygen electrode. Both aerobic and anaerobic samples were incubated at a constant temperature of 20 °C. A CO2 trap (3 mL glass tube containing 2 mL of 0.5 N NaOH) was placed in each flask. At predetermined times the accumulated radioactivity in the traps was measured using a liquid scintillation counter (Wallac 1400, Turku, Finland). The extent of denitrification was measured as
FIGURE 1. Gel chromatograpy of MCH-PCR amplified gene region from atzA. Lanes: (M) size marker; (1) 104 P. ADP cells g-1 sediment; (2) 103 P. ADP cells g-1 sediment; (3) 102 P. ADP cells g-1 sediment; (4) uninoculated sediment sample; and (5) positive control using P. ADP cell extract as template. disappearance of nitrate from 50 µL samples taken from the liquid phase. Nitrate in the extract was reduced to NO2 by passing through a column of Cd and measured after addition of sulfanilic acid and 1-naphthylamine by flow injection analysis. All the experiments were carried out in triplicates. Detection of atzA (an atrazine dechlorination gene) in the soils was carried out using magnetic capture hybridization (MCH) to extract template DNA for polymerase chain reaction (PCR). The oligonucleotide probe (96 bp) used for the MCH was prepared to match an internal sequence of the atzA gene (Table 2). The probe was synthesized and biotinilated on a five-carbon spacer arm incorporated on the 5′ end of the probe, by Gibco-BRL (Life Technologies, MD). Conjugation between the probe and the magnetic beads as well as the extraction from soils were performed according to Jacobsen (31). The PCR reaction amplified a 444 bp product from nucleotide 472 to nucleotide 915 in the atzA gene region (Table 2).
Results and Discussion Since atrazine and nitrate are frequent co-contaminants in aquifers underlying agricultural fields (12, 32), it was of interest to study the mineralization of atrazine under denitrifying conditions. Intrinsic atrazine mineralization did not occur in the aquifer sediments sampled from a shallow aquifer underlying a cornfield continuously receiving chlorinated s-triazine herbicides for 20 years, even after the addition of citrate to the sediment (data not shown). This result was in line with many previous reports indicating very slow atrazine degradation by indigenous microflora (8, 9, 13, 19). Moreover, the atzA gene from a major atrazine mineralization pathway was not detected in the sediments (Figure, 1). The absence of atrazine mineralizing activity coupled with the low organic matter content and low values of Kd (relative to the upper soil layers (Table 1)) may contribute to a rapid spread of the contaminant in the groundwater. Inoculation of sediments contaminated with 0.01 ppm atrazine with P. ADP, resulted in rapid mineralization under both aerobic and anaerobic conditions (Figure 2). Four days
TABLE 2. Sequences and Positions of Oligonucleotides Used in the MCH-PCR To Detect atzA in Aquifer Sediment oligonucleotide
position in atzA gene
forward reverse MCH-probe
472-489 897-915 651-746
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sequence 5′GCA-CGG-ACG-TCA-ATT-CTA 5′CGC-ATT-CCT-TCA-ACT-GTC 5′CGA-TGG-CGG-TCT-ATG-GTG-AGG-TGG-GTG-TGA-GGG-TCG-TCTACG-CCC-GCATGT-TCT-TTG-ATC-GGA-TGG-ACG-GGC-GCA-TTC-AAG-GGT-ATG-TGG-ACG-CCT
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FIGURE 2. Mineralization of [U-ring-14C]atrazine applied to aquifer sediments at low concentration (0.01 ppm). after inoculation, 75% (7.5 ng g-1 dw) of the atrazine was mineralized. In sediments containing 10 ppm atrazine only 1.2% (120 ng g-1 dw) removal was recorded (Figure, 3), but the total amount of mineralized atrazine was much larger than in the 0.01 ppm treatment. Citrate amendment, however, significantly increased the mineralization rate to total 48% in 4 days (4800 ng g-1 dw) and 78% (7800 ng g-1 dw) after 15 days of incubation. Interestingly, citrate amendment largely improved atrazine degradation when atrazine was present in high concentration but was not necessary in the low concentration treatment. In fact, when citrate was added to sediments contaminated with 0.01 ppm atrazine, citrate amendment decreased the degradation rate. It is apparent that the inoculation of the sediments with P. ADP was necessary for rapid mineralization to occur, but the need for citrate addition was dependent on the initial atrazine concentration. The rapid mineralization of low concentrations of atrazine occurred mostly within 48 h from inoculation. It is therefore likely that the inoculated bacteria could still use storage polymers as carbon and energy sources and were not dependent on an external source to support the mineralization of the atrazine present in the sediment. Atrazine itself is an inferior energy source as the ring carbons are at the oxidation state of CO2. Since the presence of large amounts of readily available carbon sources in sediments is unlikely, the full degradation of large amounts (10 ppm) of atrazine may requires the addition of an external carbon source. Indeed, our results clearly indicated that the addition of citrate was necessary to achieve 80% mineralization of atrazine in sediments containing 10 ppm of atrazine. Thus, the mineralization pattern of small quantities of atrazine by P. ADP without the addition of citrate as an external carbon source cannot be extrapolated to atrazine mineralization patterns in sediments containing higher levels of atrazine. Citrate amendment was also necessary for full removal of nitrate when atrazine was present in concentrations of 0.01 ppm (Figure, 4). Much slower but significant denitrification occurred in sediments containing 10 ppm of atrazine, in spite of the absence of additional carbon source (Figure 4). This denitrifying activity may be attributed to the use of atrazine alkyl side chains as possible carbon sources. Taken together, citrate serves as an electron donor for stimulating both atrazine mineralization and denitrification, while nitrate serves as an alternative electron acceptor for “anaerobic respiration” (26). Oxygen availability, which is often a limiting factor for degradation in contaminated aquifers, can therefore be alleviated in the presence of nitrate. Previously, Splichal and Schwab (33) suggested that the prolonged persistence of atrazine under aquifer conditions (t1/2 ) 209-648 days) is an indication of less microbial activity or the absence of atrazine degraders. The present data
FIGURE 3. Mineralization of [U-ring-14C]atrazine applied to aquifer sediments at high concentration (10 ppm).
FIGURE 4. Nitrate uptake in aquifer sediments contaminated with low atrazine concentration (full symbols) and in sediments contaminated with high atrazine concentration (empty symbols). Triangles ) anaerobic conditions, circles ) anaerobic conditions with the addition of citrate. indicate that the absence of the atrazine-dechlorinating gene (atzA) may be a limiting factor for intrinsic bioremediation and that a bioaugmantation approach may be needed. P. ADP seems to be a promising bioremediation agent when inoculated into aquifer sediments at a level of about 1 × 106 cells g-1 dw sediment. Its unique ability to rapidly mineralize atrazine is the result of a rapid turnover of three consecutive enzymatic hydrolysis reactions that transform atrazine (a compound only few bacteria can degrade) to cyanuric acid (a compound degraded by many bacteria) without the need for oxygen. Public perception is often against bioaugmentation with exotic bacteria containing rare genes for bioremediation schemes. However, the fact that P. ADP was initially isolated from agricultural soil (26), coupled with recent reports on the global spread of atz A, B, and C genes (34), suggests that the use of P. ADP for bioremediation of atrazine in aquifers is likely to be effective and safe. The overall picture arising from the literature reflects reduced atrazine mineralization rates under anaerobic conditions. These low rates are further reduced in the presence of soils or sediments. The present study indicates that bioaugmentation with P. ADP in the presence of an anvailable carbon source can result in rapid comineralization of atrazine and nitrate in aquifer sediments Further studies are underway to elucidate the performance and survival of P. ADP in microcosms and under in-situ aquifer conditions.
Acknowledgments This work was partially supported by Grant no. 8867197 for strategic research in bioremediation from the Israeli Ministry VOL. 32, NO. 23, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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of Sciences and a grant from Novartis to R.T.M. The Danish Research Council, Committee On Biotechnology Grant no. 9502015 and The Danish Strategic Environmental Research program on Pesticides supported the work of C.S.J., and enabled student exchange. We thank Janis MacFarland for providing labeled atrazine and Anita Jorgensen and Thomas Holst for technical assistance.
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Received for review June 22, 1998. Revised manuscript received August 19, 1998. Accepted August 21, 1998. ES980625L
