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Isolation, characterization and diuron transformation capacities of a bacterial strain Arthrobacter sp. N2
Chemosphere 46 (2002) 527–534 www.elsevier.com/locate/chemosphere
Isolation, characterization and diuron transformation capacities of a bacterial strain Arthrobacter sp. N2 Pascale Widehem a, Selim A€ıt-A€ıssa a,1, Celine Tixier b, Martine Sancelme b, Henri Veschambre b, Nicole Truffaut a,* a
b
Laboratoire de G en etique Microbienne, UMR 6022, Universit e de Technologie de Compi egne, BP 20529, 60205 Compi egne Cedex, France Synth ese Et Etude de Syst emes a Int er^ et Biologique, UMR 6504, Universit e Blaise Pascal, 63177 Aubi ere Cedex, France Received 14 November 2000; received in revised form 2 August 2001; accepted 23 August 2001
Abstract A bacterial strain able to transform diuron was isolated from a soil by enrichment procedures. Strain isolation was realized by plating on minimal-agarose medium spread with this herbicide and selecting the colonies surrounded by a clear thin halo. One strain was characterized and identified as an Arthrobacter sp. It metabolized diuron and the final transformation product, 3,4-dichloroaniline, was produced in stoichiometric amounts. The transformation of diuron at different concentrations was more efficient in the presence of alternative sources of carbon and nitrogen. The bacterial activity was also evaluated in soil microcosms with a consequent disappearance of diuron and concomitant appearance of 3,4-dichloroaniline, of which the concentration decreased thereafter. Bacterial cells inoculated in the microcosms survived as viable but eventually nonculturable cells. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Arthrobacter; Phenylurea herbicides; Diuron; Biodegradation; Soil microcosms
1. Introduction The fate of pesticides in edaphic ecosystems is dependent on various abiotic mechanisms such as photochemical degradation, adsorption to soil elements, absorption by plants or leakage, but is also governed to a large extent by the degradation activities of resident microorganisms (Aislabie and Lloyd-Jones, 1995). Hence, the knowledge of catabolic pathways of pesticide-degrading microorganisms may help to solve some problems of agricultural pollution. Herbicide biodegradation processes were studied especially within the wide
*
Corresponding author. E-mail address: nicole.truff[email protected] (N. Truffaut). 1 Present address: Service d’ Ecotoxicologie, INERIS, Parc Technologique ALATA BP2, 60550 Verneuil-en-Halatte, France.
phenylamide group including phenylcarbamates, acylanilides and phenylureas (Aislabie and Lloyd-Jones, 1995). One phenylurea herbicide, diuron (1-(3,4-dichlorophenyl)-3,3-dimethylurea), has been largely used since 1960 as a specific or total herbicide now mainly used on noncrop areas. It is very persistent at soil surface partly due to its low solubility. However, its degradation was observed in soils and was attributed to biological activity (Dalton et al., 1966; Geissbuhler, 1969). It can be degraded or transformed by fungi (Fusarium oxysporum, Kaufman and Blake, 1973; Cunninghamella echinulata, Tillmanns et al., 1978), by mixed bacterial cultures (Ellis and Camper, 1982) or by isolated bacteria (Shelton et al., 1996; Cullington and Walker, 1999). The microbial degradation of the substituted phenylureas usually leads to the corresponding aniline resulting from the aliphatic chain hydrolysis (H€ aggblom, 1992). Dalton et al. (1966) have proposed an aerobic
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 1 9 2 - 8
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degradation pathway for diuron involving two successive N-demethylation reactions followed by the cleavage of the amide bond leading to 3,4-dichloroaniline. This was confirmed for several fungi and demethylated metabolites were isolated (Kaufman and Blake, 1973; Tillmanns et al., 1978; Ellis and Camper, 1982). Less is known about the transformation of diuron by bacteria, some of which can transform diuron into the corresponding aniline. However, no degradation pathway has been elucidated until now, and no other intermediate compound isolated. In this study, we report the isolation and the characterization of a bacterial strain, Arthrobacter sp. N2, from a soil treated over several years with diuron. In the present paper, we studied the characteristics of this strain and its ability to aerobically transform diuron into dichloroaniline in pure culture, either alone or in the presence of alternative carbon sources. In microcosms, we also followed the bacteria survival in soil and showed the transformation of diuron at different concentrations.
2. Materials and methods 2.1. Chemicals Diuron (purity: P 98%) was given by Rh^ onePoulenc Agrochimie (Lyon, France). 3,4-dichloroaniline (3,4-DCA, purity: P 99%) was purchased from Sigma Aldrich Chimie (Saint-Quentin-Fallavier, France). Due to the poor solubility of diuron (42 mg l1 at 25 °C) and 3,4-DCA in water, stock solutions were prepared in solvents (purity: > 99:5%) not metabolized by this strain, acetone (20 mg ml1 ) or dimethylsulfoxide (DMSO), (100 mg ml1 ) depending on the mode of culture. Preliminary experiments indicated that a final concentration inferior to 0.1% (v/v) DMSO in the medium did not affect the bacterial growth. The structure of chemicals is shown in Fig. 1. 2.2. Media Bacteria were cultured in LB medium or in mineral media devoid of carbon or/and nitrogen sources. The mineral salt solution (MMO), without carbon source
and containing NH4 Cl (1 g l1 ) as a nitrogen source, was described by Palleroni and Doudoroff (1972). The mineral salt solution (MMK) contained no nitrogen or carbon source and had the following composition in g l1 : KH2 PO4 , 1; K2 HPO4 , 1; MgSO4 7H2 O, 0.5; FeCl3 6H2 O, 0.004. The final pH value was 6.5. This medium was eventually supplemented with pyruvate or succinate as the carbon source. All media were sterilized by autoclaving at 121 °C for 20 min. The increase in biomass was measured by monitoring absorbance at 570 nm: A570 . 2.3. Soil sampling and enrichment procedures Four different soils were tested for the presence of degrading bacteria: two soils, cultivated and not cultivated, not treated with diuron, from an experimental farm (C^ ote Saint-Andre), an untreated soil (Compiegne), and a soil from a garden path annually treated with diuron (2 g m2 ) for 3 years. The samples were collected from the first 20 cm below the surface, aseptically transferred to sterile vials and stored at 4 °C until use. In a pre-enrichment step, 50 g of the soil samples were homogenized in a 1l Erlenmeyer flask containing 200 ml of MMO supplemented with diuron (solubilized in DMSO as previously indicated) at 0:1 g l1 , and incubated at 30 °C on a rotary shaker for seven days. In the enrichment step, 4 ml of the pre-enrichment culture were suspended in 200 ml MMO supplemented with the same quantity of diuron. Samples were periodically plated onto MMO-agarose supplemented with diuron in order to isolate strains able to degrade this herbicide.
2.4. Isolation and bacterial characterization MMO-agarose plates were spread with 0.1 ml of a diuron solution (20 g l1 in acetone). Acetone was airevaporated, forming an opaque crystal layer of diuron on the agar surface. The plates were then exposed for 10 min to UV light for sterilization. Bacterial strains able to degrade diuron were isolated by plating 10-fold dilutions of the enrichment culture onto these plates and incubated at 30 °C for 8 days. The clones able to degrade diuron appeared as small colonies surrounded by a light halo. This selection method was used for pure strain isolation using atrazine as unique nitrogen source (Mandelbaum et al., 1995). 2.5. Study of diuron transformation
Fig. 1. Chemical structure of diuron and 3,4-DCA.
2.5.1. Inoculum preparation The bacteria were cultured in 100 ml Erlenmeyer flasks containing 20 ml LB medium supplemented by
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40 mg l1 diuron. At the exponential phase, this suspension was used to inoculate the medium: the bacteria were pelleted by centrifugation (5 min, 10 000g). The pellet was washed twice with MMK, resuspended in the same medium and used to inoculate the different media for degradation tests at a final concentration of 106 cells ml1 . 2.5.2. Preparation of samples for analysis 2 ml of a bacterial culture were pelleted. 1.5 ml of the supernatant were placed in 2 ml microtubes and treated with 0.3 ml of ethylacetate (purity > 99:5%). The mixture was agitated during 3 min and centrifuged for 1 min at 12 000g. The organic phase was collected and analyzed by gas chromatography (GC). 2.5.3. Analytical procedure GC analysis was performed with a model 5890 gas chromatograph (Hewlett-Packard) equipped with a flame ionization detector, and a dimethylpolysiloxane capillary column (film thickness, 0:17 lm; length, 25 m; inner diameter, 0.2 mm; HP 1, Hewlett-Packard). The operating conditions were as follows: injector temperature, 250 °C; detector temperature, 300 °C; oven temperature, 110 °C (3 min) to 150 °C at 8 °C min1 ; carrier flow rate, 1:3 ml min1 ; sample, 1 ll. The metabolites were identified by comparison of retention times of authentic reference compounds. Retention times of diuron and 3,4-DCA in ethylacetate were, respectively, 4 and 5.9 min. The detection limits were 1 mg l1 for diuron and 5 mg l1 for 3,4-DCA. 2.5.4. Diuron transformation To study the transformation of diuron, MMO medium (400 ml) was supplemented with diuron (40 mg l1 ) added as the sole carbon source and MMK medium was supplemented either with succinate (75 mg l1 ) and diuron (40 mg l1 ) as the sole nitrogen source or with diuron as sole nitrogen and carbon source. Each flask was inoculated with the washed cell suspension already described and incubated at 30 °C under agitation. A control experiment without bacteria was carried out under the same conditions. 2 ml of the culture were periodically sampled for assay. 2.5.5. Diuron transformation in the presence of alternative carbon sources Glucose was added to MMO to give final concentrations of 0.25 and 0:5 g l1 . The Erlenmeyer flasks (2 l) contained 400 ml of MMO supplemented with diuron (40 mg l1 ) and glucose or 400 ml MMK supplemented with diuron (40 mg l1 ), and with succinate or pyruvate at different concentrations. The flasks were inoculated with the washed cell suspension and incubated at 30 °C on a rotary shaker. Samples were collected at regular intervals and analyzed by GC. A control experiment was
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carried out under the same conditions without sugar. In parallel, cell numbers were determined by plating on LBagar. Standard errors were given when experiments were performed in duplicate. 2.5.6. Isolation and characterization of 3,4-dichloroaniline To isolate 3,4-dichloroaniline, bacterial resting cell assays and quantitative biodegradation assays were carried out as described in Tixier et al. (2002). The culture supernatants were extracted and concentrated, and the residue purified by column chromatography. After isolation, the structure of the metabolite was identified by NMR and mass spectrometry as indicated elsewhere (Tixier et al., 2002).
3. Microcosms 3.1. Soil samples The soil used to test the bacterial survival was a standard soil from an experimental farm of C^ ote SaintAndre (Isere, France). Its characteristics (granulometry: 41% sand, 38% silt, 19% clay; pH, 5.8; organic matter, 0:018 g1 of soil; humidity, 0:188 g1 ) were previously described (Hallier-Soulier et al., 1996; Ducrocq et al., 1999). Soil samples were sterilized by threefold autoclaving during 1.5 h at 121 °C. To study the fate of bacteria, 100 g of this soil were collected and placed in 500 ml Erlenmeyer flask under sterile conditions. 3.2. Inoculum preparation The bacterial cells were cultured in LB medium and pelleted at stationary phase by 15 min centrifugation at 5000g. The cells were washed three times in MMK and resuspended in different solutions: sterile 0.9% saline, MMK, MMK supplemented with LB (1/5 : v/v), MMK supplemented with succinate (0:075 g l1 ), MMK supplemented with succinate (0:075 g l1 ) and ammonium sulfate (1 g l1 ). The different bacterial suspensions were used to rehydrate 100 g of soil to 20% hydration and to add cells at 107 –109 cells g1 of soil. The soil samples were incubated at 18 °C. 3.3. Diuron addition to soil Two concentrations of diuron were tested: 0.8 and 2 g kg1 . Diuron was solubilized in acetone as indicated and aliquots (4 ml and 10 ml) were added to 100 g of soil contained in 500 ml Erlenmeyer flasks. The flasks were agitated and acetone was air-evaporated during 24 h under sterile conditions. The bacterial suspensions were added afterwards.
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3.4. Bacterial extraction Aliquots of 1 g of soil were collected at intervals and suspended in phosphate buffer (pH 7.6) containing the following in g l1 : NaCl, 8.5; Na2 HPO4 , 1.2; NaH2 PO4 , 0.8; Na4 P2 O7 , 10H2 O, 1. This mixture was vortexed for 2 min and after 10 min settling, the supernatant was collected and plated in triplicate on LB-agar for cells enumeration. Bacteria in the presence of diuron were also enumerated by acridine orange direct counting performed according to Hobbie et al. (1977). This indicates the total number of cells ml1 . The detection limits were 103 cells g1 for both methods, epifluorescence and LB-agar enumeration. 3.5. Diuron and metabolite extraction Aliquots of 2.5 g of soil were collected at intervals and dried at 65 °C (below the melting point of 3,4-DCA) for 1 h. Two ml of cold acetone were added to 2 g of dried soil and this solution was mixed for 1 h at 4 °C to limit acetone volatilization. The liquid phase was collected and centrifuged at 12 000g for 5 min. The supernatant was taken to determine diuron and 3,4-DCA concentrations. Diuron concentration was evaluated by GC analysis, under the conditions already described except that oven temperature programmation was different: 100–150 °C at 6 °C/min. The presence of 3,4DCA was evaluated by GC analysis and also detected by a rapid colorimetric method, on the basis of the production of a diazo color complex, which has been described previously (Walker, 1987). The detection limits were 30 mg kg1 for diuron, 5 mg kg1 by GC analysis and 1 mg kg1 by Walker’s method, respectively, for 3,4DCA.
4. Results 4.1. Isolation and characterization of diuron-transforming bacteria Four soil samples were tested for the presence of diuron degrading bacterial strains. The initial culturable microflora in the soil samples determined by plating on LB-agar was about 108 CFU g1 of soil, and did not significantly increase during the 8-days enrichment step (data not shown). In only one sample, a garden soil treated for several years with 2 g diuron m2 , platings of the 10-fold diluted enrichment mix on diuron-MMOagarose allowed to recover small size translucent bacterial colonies after 5 days at 30 °C. Among these, five clones were surrounded by a clear area free of diuron crystals, indicating a degradation of the herbicide by the colony. A first characterization of three clones showed no major differences between them and one isolate,
designated N2, was used for further experiments. On LB solid medium, strain N2 formed convex, cream colored, opaque and shiny colonies. On LB-agar plates spread with diuron, the colonies were larger, surrounded with a light halo. Microscopic examination of cultures in liquid LB medium displayed different morphological aspects of the bacteria depending on the growth stage: cells were nonspore-forming, nonmotile, gram-positive, aggregated irregular rods at exponential phase becoming coccoid at stationary phase. In MMK medium supplemented with the herbicide, bacterial morphology was the same, but cells appeared more aggregated. Biochemical and physiological tests were used for the identification of strain N2: catalase, b-glucuronidase, bgalactosidase, a-glucosidase tests were positive, b-glucosidase and phosphatase alkaline negative. Bacteria grew on glucose, citrate, succinate, pyruvate, not on saccharose and were resistant to metronidazole (4 lg). In anaerobic conditions the strain was urease-negative and no fermentation occurred on the hydrocarbons tested (glucose, ribose, xylose, mannitol, maltose, lactose, saccharose, glycogen). Finally, according to its morphological, fatty acid analysis and biochemical characteristics, the N2 strain was identified by Institut Pasteur-Paris as an Arthrobacter sp., close to A. globiformis and deposited at the Institut Pasteur Collection under number CIP105365. 4.2. Diuron transformation The biotransformation of diuron was monitored by GC analysis. Only two peaks were detected during the experiments, corresponding to the diuron itself, and to 3,4-DCA. Fig. 2 shows diuron disappearance and 3,4DCA formation as a function of time, at 30 °C, in three different culture media (MMO, MMK and MMK supplemented with succinate). Whatever the medium, 3,4-DCA concentrations increased while diuron concentration decreased. In contrast, control media without bacteria did not show any variation of diuron concentration which remained stable, and no 3,4-DCA was detected. Diuron completely disappeared after 30 h as it was the unique nitrogen source (MMK+succinate), after 50 h as it was the unique carbon source (MMO), and after 70 h as it was the only carbon and nitrogen source (MMK). The rate of disappearance of diuron was therefore improved in the presence of additional sources of carbon and nitrogen. The final concentration of 3,4-DCA (MW 162) was about 27 mg l1 , corresponding to 0:17 mmole l1 of medium. This molarity was in adequation with the number of moles of diuron initially introduced (equivalent to 40 mg l1 ), meaning that all molecules of diuron (MW 233) were transformed into 3,4-DCA. Moreover, these results indicate that the 3,4-DCA was not further degraded by the bacteria under these conditions. No
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mental conditions described elsewhere (Tixier et al., 2002), led to the formation of one metabolite detected by HPLC. After isolation, the structure of the metabolite was identified by NMR and mass spectrometry. The commercially available 3,4-dichloroaniline had the same retention time (6.7 min) and presented the same spectral data as the isolated metabolite. This confirmed the identification of 3,4-dichloroaniline as the terminal metabolite in diuron degradation by Arthrobacter sp. N2. 4.4. Effect of a cosubstrate on transformation The transformation of diuron was studied in the presence of different carbon cosubstrates, at 30 °C, in MMK medium supplemented with succinate (75 mg l1 or 500 mg l1 ) or pyruvate (500 mg l1 ), or in MMO medium supplemented with glucose (250 mg l1 or 500 mg l1 ). The results listed in Table 1 are expressed in terms of transformation rate, where Va is the average rate from the beginning of the culture to the time until diuron had totally disappeared, and Vmax is the maximal rate attained during the culture. At low concentration of succinate (75 mg l1 ), both Va and Vmax were increased by a factor of 1.9 and 2.3, respectively. At higher concentration of succinate (500 mg l1 ), Va was not significantly improved, but Vmax was 3.2-fold higher than the control experiment lacking the cosubstrate, and 4.1 higher if pyruvate 500 mg l1 was used as a cosubstrate. In contrast, when glucose 500 mg l1 was used in MMO medium containing nitrogen, Va remained quite constant while Vmax was only slightly increased by a factor 1.6 compared with the same medium without cosubstrate. Generally, these results show that the average transformation rate was improved by the addition of cosubstrate, even at a low concentration (results with
Fig. 2. Transformation of diuron in different media: (a) MMO (diuron as the sole source of carbon); (b) MMK supplemented with succinate 75 mg l1 (diuron as the sole source of nitrogen); (c) MMK (diuron as the sole source of carbon and nitrogen); and (d) Sterile control was MMO without bacteria. The concentration of diuron ( ) and 3,4-DCA ( ) were determined after GC analysis.
other metabolite could be observed during the transformation process by this procedure. 4.3. Isolation of 3,4-dichloroaniline Isolation of the unique metabolite as 3,4-dichloroaniline helped to confirm this result. Diuron degradation by resting cells of Arthrobacter sp. N2, under experi-
Table 1 Effect of a cosubstrate on diuron transformation at 30 °C diuron concentration 40 mg l1 Va a (mg l1 h1 )
Vmax b (mg l1 h1 )
MMK medium No cosubstrate 7:5 mg l1 succinate 500 mg l1 succinate 500 mg l1 pyruvate
0:57 0:03 1:11 0:05 1:18 0:16 1:67 0:27
0:72 0:05 1:62 0:28 2:35 0:39 2:97 0:41
MMO medium No cosubstrate 250 mg l1 glucose 500 mg l1 glucose
1:26 0:13 1.48 1.62
1:89 0:26 2.83 3.05
Media and cosubstrates
a b
Va : average rate of diuron disappearance. Vmax : maximal rate of diuron disappearance.
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succinate), together with the maximal transformation rate (especially with succinate or pyruvate), in the presence (MMO medium) or in the absence (MMK medium) of nitrogen. In parallel, a substantial cell growth was observed in the presence of different cosubstrates, correlated to the change of physiological state during the transformation process.
5. Microcosms 5.1. Effect of inoculum preparation Standard soil microcosms were incubated at 18 °C. Preliminary experiments in liquid MMO medium have shown that bacterial cells were able to rapidly degrade diuron at this temperature. Diuron completely disappeared in 30 h at 18 °C with Va and Vmax values of 1.3 and 2:1 mg l1 h1 comparable to the values obtained at 30 °C. Bacterial samples resuspended in different media were used to inoculate soil microcosms at 18 °C. At intervals the cells were extracted and counted by plating on LB agar. When bacteria were resuspended in sterile 0.9% saline or in MMK, no colony was observed on LBagar (data not shown). However, in other media: MMK supplemented with LB (1/5), succinate (75 mg l1 ) or succinate (75 mg l1 ) and ammonium sulfate (1 g l1 ), colonies were observed on LB-agar (Fig. 3). In all three conditions, the bacterial cells were able to survive in soil during more than 35 days after reinoculation. In the presence of succinate and ammonium sulfate, the number of cells was stable for the time of the experiment. In the presence of LB, the number of cells decreased during 4 days and increased after this period.
The bacterial number decreased after 12 days in soil. When the inoculum medium was supplemented with succinate, the number of colonies was stable for 10 days and progressively decreased to attain 105 cells g1 of soil after 35 days. Thus the bacteria were able to survive in soil under certain conditions, though they need mineral salts and carbon source to be culturable after reinoculation of soil. However, the absence of colony after plating on LB-agar does not indicate that bacteria were not able to survive but that they rather lost their culturability in synthetic medium, entering into a ‘‘viable but nonculturable’’ (VNC) state (Trevors et al., 1994; Ducrocq et al., 1999) where they cannot divide and form colonies on solid medium. 5.2. Bacterial survival in the presence of diuron We studied the fate of bacteria in microcosms in the presence of diuron at two different concentrations (0.8 and 2 g kg1 of soil) at 18 °C during 30 days. The cells were enumerated by plate counts and by a direct epifluorescence method. In the absence of diuron, the results obtained with both methods were comparable despite unexplained differences, indicating that the number of bacteria was stable in soil (Table 2). In the presence of diuron 0.8 g kg1 , the two profiles were similar and showed that bacteria were able to survive at this concentration. Diuron began to decrease from day 16 concomitantly with the appearance of 3,4DCA. Diuron concentration decreased to 65% after 30 days. 3,4-DCA was detected at day 16, and increased at day 30. In the presence of diuron 2 g kg1 , the profiles obtained by epifluorescence counts were very stable while no cells were detected after day 4 by plating on LB-agar. However, cells were active since the diuron concentration decreased and 3,4-DCA was detected at day 16 by Walker’s method and also by GC analysis. The concentration of 3,4-DCA was low (12–16 mg g1 ) and decreased thereafter (1–5 mg kg1 at day 30).
6. Discussion and conclusions
Fig. 3. Effect of inoculum conditions on bacteria survival: MMK þ LB ( ); MMK ( ) supplemented with succinate (75 mg l1 ) and ammonium sulfate (1 g l1 ); MMK ( ) supplemented with succinate (75 mg l1 ).
By soil enrichment and after plating on MMOagarose spread with diuron, we isolated several clones distinguishable by the presence of a clear halo. One isolate, N2, was able to metabolize diuron in pure culture and completely transformed it in 3,4-DCA. By morphological and biochemical characteristics, strain N2 was identified as an Arthrobacter sp., likely A. globiformis. Diuron was transformed by Arthrobacter sp. N2 into its corresponding aniline, 3,4-dichloroaniline as was expected according to previous reports. The corresponding aniline has been demonstrated to be a terminal metabolite in the biotransformation of various phe-
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Table 2 Number of cells in microcosms determined by plating (CFU g1 of soil) or by epifluorescence, in the absence or in the presence of diuron at 0.8 or 2 g kg1 of soil Diuron (g kg1 )
Days
1
4
0
CFU g1 of soil (107 ) Epifluorescence (107 )
17 3.9
24 4.1
2.8 5.1
3.1 1.5
4.6 8.3
0.8
CFU g1 of soil (107 ) Epifluorescence (107 ) Diuron (g kg1 ) 3,4-DCAa (mg kg1 ) 3,4-DCAb (mg kg1 )
15 2.2 0.8 – –
26 3.2 nd nd nd
18 6.2 0.8 – –
4.8 2.9 0.6 – 2.2
30 7.6 0.3 17.7 14.8
2
CFU g1 of soil (107 ) Epifluorescence (107 ) Diuron (g kg1 ) 3,4-DCAa (mg kg1 ) 3,4-DCAb (mg kg1 )
11.9 3.9 2.0 – –
– 1.7 2.0 – –
– 3.3 1.4 16.9 11.9
– 1.6 1.3 5.0 1.0
8
0.01 0.59 2.0 – –
16
30
Determinations of diuron and 3,4-DCA (a ) were performed by GC analysis or 3,4-DCA (b ) by Walker’s method. (nd) Not determined, (–) absence of compounds or bacteria.
nylamides by fungi (Blake and Kaufman, 1975; H€ aggblom, 1992) or bacteria (Athiel et al., 1995; Cullington and Walker, 1999). 3,4-DCA was not further transformed by strain Arthrobacter sp. N2. One study reports its degradation as unique carbon, nitrogen and energy source by Pseudomonas diminuta (Surovtseva et al., 1985). By co-metabolism, 3,4-DCA could be degraded in vitro in the presence of aniline or propioanilide (You and Bartha, 1982) or in the presence of monochloroaniline (Reber et al., 1979; Zeyer and Kearney, 1982). Moreover, 3,4DCA can be degraded in soil slurry systems (Brunsbach and Reineke, 1993). To determine factors influencing bacteria survival in soil (as reviewed by van Veen et al., 1997), the experiments were conducted in microcosms. To overcome the difficulty in estimating the population in soil, two complementary techniques of cell detection were used. Bacterial viability was evaluated by plating on LB-agar. The presence and the physiological state of bacteria were checked by epifluorescence. The microcosms were incubated at 18 °C as Arthrobacter sp. N2 was able to degrade diuron at this temperature. The presence of carbon source and mineral salts in the medium was necessary to obtain bacterial colonies on LB-agar. The strain was able to survive in soil after reinoculation in the presence or in the absence of diuron. Bacteria inoculated in soils polluted by high concentrations of diuron (2 g kg1 of soil) became nonculturable but their presence was detected by epifluorescence counts. This technique has the major advantage to observe the bacteria in situ. However, epifluorescence count only gives an estimation of the cell number in soil. We have noted that cells were generally associated with soil particles, which possibly hampers the extraction of bacteria.
Trevors et al. (1994) have discussed this phenomenon of nonculturability. It is sometimes difficult to evaluate whether bacteria are dead or are in a viable but nonculturable state. However, in the present case the bacteria were active since the disappearance of diuron and the appearance of 3,4-dichloroaniline were both observed by GC analysis. This was confirmed by detection of 3,4-DCA by Walker’s method. In all conditions, in soil microcosms no quantitative relation was observed between the amount of disappeared diuron and the amount of the appeared 3,4DCA, since the concentration of 3,4-dichloroaniline extracted from microcosms is at the highest 17–18 mg g1 , which represents at best 3% of what would be expected from the total transformation of diuron. This may result either from the formation of condensation products of 3,4-DCA (Bartha et al., 1968; Chisaka and Kearney, 1970; Hill et al., 1981) or rather from the chemical attachment of the chloroaniline to humic acids present in soil (Brunsbach and Reineke, 1993). The transformation of different phenylureas by resting cells of Arthrobacter sp. N2 is also reported (Tixier et al., 2002) and confirmed by HPLC analysis. Experiments are described to evaluate the toxicity of metabolites evolved by the action of degradative fungal strains on 3,4-DCA. As 3,4-DCA or its derivatives are considered to be much more toxic than diuron itself to microbial and vegetal flora and may have detrimental effects on the environment, it is thus of interest to know the evolution of this molecule and of other phenylamide herbicides and to evaluate the impact of their metabolites in complex ecological systems such as soils where bacteria and fungi are active in metabolizing organic molecules.
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Acknowledgements This work was supported by a GDR-CNRS ‘‘Exosols’’ (Groupe de Recherches-Centre National de la Recherche Scientifique), and by a grant from Conseil Regional de Picardie.
References Aislabie, J., Lloyd-Jones, G., 1995. A review of bacterial degradation of pesticides. Aust. J. Soil Res. 33, 925–942. Athiel, P., Alfizar, Mercadier, C., Vega, D., Bastide, J., Davet, P., Brunel, B., Cleyet-Marel, J.C., 1995. Degradation of iprodione by a soil Arthrobacter-like strain. Appl. Environ. Microbiol. 61, 3216–3220. Bartha, R., Linke, H.A.B., Pramer, D., 1968. Pesticide transformations: production of chloroazobenzene from chloroanilines. Science 161, 582–583. Blake, J., Kaufman, D.D., 1975. Characterization of azylanilides-hydrolysing enzyme(s) from Fusarium oxysporium Schlecht. Pest. Biochem. Physiol. 5, 305–313. Brunsbach, F.R., Reineke, W., 1993. Degradation of chloroanilines in soil slurry by specialized organisms. Appl. Microbiol. Biotechnol. 40, 402–407. Chisaka, H., Kearney, P.C., 1970. Metabolism of propanil in soil. J. Agric. Food Chem. 18, 854–858. Cullington, J.E., Walker, A., 1999. Rapid biodegradation of diuron and other phenylurea herbicides by a soil bacterium. Soil Biol. Biochem. 31, 677–686. Dalton, R.L., Evans, A.W., Rhodes, R.C., 1966. Disappearance of diuron from cotton fields soils. Weeds 14, 14–31. Ducrocq, V., Hallier-Soulier, S., Pandard, P., Thybaud, E., Truffaut, N., 1999. Contribution of quantitative PCR, plant and earthworm bioassays to monitor 4-chlorobiphenyl biodegradation in soil microcosms. Appl. Soil Ecol. 12, 15–27. Ellis, P.A., Camper, N.D., 1982. Aerobic degradation of diuron by aquatic microorganisms. J. Environ. Sci. Health B 17, 277–289. Geissbuhler, H., 1969. The substituted ureas. In: Kearney, P.C., Kaufman, D.D. (Eds.), Degradation of Herbicides. Marcel Dekker, New York, pp. 79–111. H€ aggblom, M.M., 1992. Microbial breakdown of halogenated aromatic pesticides and related compounds. FEMS Microbiol. Rev. 103, 29–72. Hallier-Soulier, S., Ducrocq, V., Mazure, N., Truffaut, N., 1996. Detection and quantification of degradatives genes in soils contaminated by toluene. FEMS Microbiol. Ecol. 20, 121–133.
Hill, R.H., Rollen, Z.J., Kimbrough, R.D., Groce, D.F., Needham, L.L., 1981. Tetrachloroazobenzene in 3,4-dichloroaniline and its herbicidal derivatives: propanil, diuron, linuron, and neburon. J. Environ. Health 36, 11–14. Hobbie, J.E., Daley, R.J., Jasper, S., 1977. Use of nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33, 1225–1228. Kaufman, D.D., Blake, J., 1973. Microbial degradation of several acetamide, acylanilide, carbamate, toluidine and urea pesticides. Soil Biol. Biochem. 5, 297–308. Mandelbaum, R.T., Allan, D., Wackett, L.P., 1995. Isolation and characterization of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Appl. Environ. Microbiol. 61, 1451–1457. Palleroni, N.J., Doudoroff, M., 1972. Some properties and taxonomic subdivisions of the genus Pseudomonas. Ann. Rev. Phytopathol. 10, 73–100. Reber, H., Helm, V., Karanth, N.G.K., 1979. Comparative studies on the metabolism of anilines and chloroanilines by Pseudomonas multivorans strain an 1. Eur. J. Appl. Microbiol. 7, 181–189. Shelton, D.R., Khader, S., Karns, J.S., Pogell, B.M., 1996. Metabolism of twelve herbicides by Streptomyces. Biodegradation 7, 129–136. Surovtseva, E.G., Ivoilov, V.S., Karasevich, Y.N., Vassil’eva, G.K., 1985. Chlorinated anilines, a source of carbon, nitrogen, and energy for Pseudomonas diminuta. Mikrobiologiya 54, 948–952. Tillmanns, G.M., Walln€ offer, P.R., Engelhardt, G., Olie, K., Hutzinger, O., 1978. Oxidative dealkylation of five phenylurea herbicides by the fungus Cunninghamella echinulata Thaxter. Chemosphere 1, 59–74. Tixier, C., Sancelme, M., A€ıt-A€ıssa, S., Widehem, P., Bonnemoy, F., Cuer, A., Truffaut, N., Veschambre, H., 2002. Structure, ecotoxicity and fate of metabolites with soil fungi. Chemosphere 46 (4), 519–526. Trevors, J.T., Kuikman, P., van Elsas, J.D., 1994. Release of bacteria into soil: cell numbers and distribution. J. Microbiol. Meth. 19, 247–259. van Veen, J.A., van Overbeek, L.S., van Elsas, J.D., 1997. Fate and activity of microorganisms introduced into soil. Microbiol. Mol. Biol. Rev. 61, 121–135. Walker, A., 1987. Enhanced degradation of iprodione and vinclozolin in soil: a simple colorimetric test for identification of rapid degrading soils. Pest. Sci. 21, 233–240. You, I.S., Bartha, R., 1982. Stimulation of 3,4-dichloroaniline mineralisation by aniline. Appl. Environ. Microbiol. 44, 678–681. Zeyer, J., Kearney, P.C., 1982. Microbial metabolism of propanil and 3,4-dichloroaniline. Pest. Biochem. Physiol. 17, 224–231.
Isolation, characterization and diuron transformation capacities of a bacterial strain Arthrobacter sp. N2 Pascale Widehem a, Selim A€ıt-A€ıssa a,1, Celine Tixier b, Martine Sancelme b, Henri Veschambre b, Nicole Truffaut a,* a
b
Laboratoire de G en etique Microbienne, UMR 6022, Universit e de Technologie de Compi egne, BP 20529, 60205 Compi egne Cedex, France Synth ese Et Etude de Syst emes a Int er^ et Biologique, UMR 6504, Universit e Blaise Pascal, 63177 Aubi ere Cedex, France Received 14 November 2000; received in revised form 2 August 2001; accepted 23 August 2001
Abstract A bacterial strain able to transform diuron was isolated from a soil by enrichment procedures. Strain isolation was realized by plating on minimal-agarose medium spread with this herbicide and selecting the colonies surrounded by a clear thin halo. One strain was characterized and identified as an Arthrobacter sp. It metabolized diuron and the final transformation product, 3,4-dichloroaniline, was produced in stoichiometric amounts. The transformation of diuron at different concentrations was more efficient in the presence of alternative sources of carbon and nitrogen. The bacterial activity was also evaluated in soil microcosms with a consequent disappearance of diuron and concomitant appearance of 3,4-dichloroaniline, of which the concentration decreased thereafter. Bacterial cells inoculated in the microcosms survived as viable but eventually nonculturable cells. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Arthrobacter; Phenylurea herbicides; Diuron; Biodegradation; Soil microcosms
1. Introduction The fate of pesticides in edaphic ecosystems is dependent on various abiotic mechanisms such as photochemical degradation, adsorption to soil elements, absorption by plants or leakage, but is also governed to a large extent by the degradation activities of resident microorganisms (Aislabie and Lloyd-Jones, 1995). Hence, the knowledge of catabolic pathways of pesticide-degrading microorganisms may help to solve some problems of agricultural pollution. Herbicide biodegradation processes were studied especially within the wide
*
Corresponding author. E-mail address: nicole.truff[email protected] (N. Truffaut). 1 Present address: Service d’ Ecotoxicologie, INERIS, Parc Technologique ALATA BP2, 60550 Verneuil-en-Halatte, France.
phenylamide group including phenylcarbamates, acylanilides and phenylureas (Aislabie and Lloyd-Jones, 1995). One phenylurea herbicide, diuron (1-(3,4-dichlorophenyl)-3,3-dimethylurea), has been largely used since 1960 as a specific or total herbicide now mainly used on noncrop areas. It is very persistent at soil surface partly due to its low solubility. However, its degradation was observed in soils and was attributed to biological activity (Dalton et al., 1966; Geissbuhler, 1969). It can be degraded or transformed by fungi (Fusarium oxysporum, Kaufman and Blake, 1973; Cunninghamella echinulata, Tillmanns et al., 1978), by mixed bacterial cultures (Ellis and Camper, 1982) or by isolated bacteria (Shelton et al., 1996; Cullington and Walker, 1999). The microbial degradation of the substituted phenylureas usually leads to the corresponding aniline resulting from the aliphatic chain hydrolysis (H€ aggblom, 1992). Dalton et al. (1966) have proposed an aerobic
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 1 9 2 - 8
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degradation pathway for diuron involving two successive N-demethylation reactions followed by the cleavage of the amide bond leading to 3,4-dichloroaniline. This was confirmed for several fungi and demethylated metabolites were isolated (Kaufman and Blake, 1973; Tillmanns et al., 1978; Ellis and Camper, 1982). Less is known about the transformation of diuron by bacteria, some of which can transform diuron into the corresponding aniline. However, no degradation pathway has been elucidated until now, and no other intermediate compound isolated. In this study, we report the isolation and the characterization of a bacterial strain, Arthrobacter sp. N2, from a soil treated over several years with diuron. In the present paper, we studied the characteristics of this strain and its ability to aerobically transform diuron into dichloroaniline in pure culture, either alone or in the presence of alternative carbon sources. In microcosms, we also followed the bacteria survival in soil and showed the transformation of diuron at different concentrations.
2. Materials and methods 2.1. Chemicals Diuron (purity: P 98%) was given by Rh^ onePoulenc Agrochimie (Lyon, France). 3,4-dichloroaniline (3,4-DCA, purity: P 99%) was purchased from Sigma Aldrich Chimie (Saint-Quentin-Fallavier, France). Due to the poor solubility of diuron (42 mg l1 at 25 °C) and 3,4-DCA in water, stock solutions were prepared in solvents (purity: > 99:5%) not metabolized by this strain, acetone (20 mg ml1 ) or dimethylsulfoxide (DMSO), (100 mg ml1 ) depending on the mode of culture. Preliminary experiments indicated that a final concentration inferior to 0.1% (v/v) DMSO in the medium did not affect the bacterial growth. The structure of chemicals is shown in Fig. 1. 2.2. Media Bacteria were cultured in LB medium or in mineral media devoid of carbon or/and nitrogen sources. The mineral salt solution (MMO), without carbon source
and containing NH4 Cl (1 g l1 ) as a nitrogen source, was described by Palleroni and Doudoroff (1972). The mineral salt solution (MMK) contained no nitrogen or carbon source and had the following composition in g l1 : KH2 PO4 , 1; K2 HPO4 , 1; MgSO4 7H2 O, 0.5; FeCl3 6H2 O, 0.004. The final pH value was 6.5. This medium was eventually supplemented with pyruvate or succinate as the carbon source. All media were sterilized by autoclaving at 121 °C for 20 min. The increase in biomass was measured by monitoring absorbance at 570 nm: A570 . 2.3. Soil sampling and enrichment procedures Four different soils were tested for the presence of degrading bacteria: two soils, cultivated and not cultivated, not treated with diuron, from an experimental farm (C^ ote Saint-Andre), an untreated soil (Compiegne), and a soil from a garden path annually treated with diuron (2 g m2 ) for 3 years. The samples were collected from the first 20 cm below the surface, aseptically transferred to sterile vials and stored at 4 °C until use. In a pre-enrichment step, 50 g of the soil samples were homogenized in a 1l Erlenmeyer flask containing 200 ml of MMO supplemented with diuron (solubilized in DMSO as previously indicated) at 0:1 g l1 , and incubated at 30 °C on a rotary shaker for seven days. In the enrichment step, 4 ml of the pre-enrichment culture were suspended in 200 ml MMO supplemented with the same quantity of diuron. Samples were periodically plated onto MMO-agarose supplemented with diuron in order to isolate strains able to degrade this herbicide.
2.4. Isolation and bacterial characterization MMO-agarose plates were spread with 0.1 ml of a diuron solution (20 g l1 in acetone). Acetone was airevaporated, forming an opaque crystal layer of diuron on the agar surface. The plates were then exposed for 10 min to UV light for sterilization. Bacterial strains able to degrade diuron were isolated by plating 10-fold dilutions of the enrichment culture onto these plates and incubated at 30 °C for 8 days. The clones able to degrade diuron appeared as small colonies surrounded by a light halo. This selection method was used for pure strain isolation using atrazine as unique nitrogen source (Mandelbaum et al., 1995). 2.5. Study of diuron transformation
Fig. 1. Chemical structure of diuron and 3,4-DCA.
2.5.1. Inoculum preparation The bacteria were cultured in 100 ml Erlenmeyer flasks containing 20 ml LB medium supplemented by
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40 mg l1 diuron. At the exponential phase, this suspension was used to inoculate the medium: the bacteria were pelleted by centrifugation (5 min, 10 000g). The pellet was washed twice with MMK, resuspended in the same medium and used to inoculate the different media for degradation tests at a final concentration of 106 cells ml1 . 2.5.2. Preparation of samples for analysis 2 ml of a bacterial culture were pelleted. 1.5 ml of the supernatant were placed in 2 ml microtubes and treated with 0.3 ml of ethylacetate (purity > 99:5%). The mixture was agitated during 3 min and centrifuged for 1 min at 12 000g. The organic phase was collected and analyzed by gas chromatography (GC). 2.5.3. Analytical procedure GC analysis was performed with a model 5890 gas chromatograph (Hewlett-Packard) equipped with a flame ionization detector, and a dimethylpolysiloxane capillary column (film thickness, 0:17 lm; length, 25 m; inner diameter, 0.2 mm; HP 1, Hewlett-Packard). The operating conditions were as follows: injector temperature, 250 °C; detector temperature, 300 °C; oven temperature, 110 °C (3 min) to 150 °C at 8 °C min1 ; carrier flow rate, 1:3 ml min1 ; sample, 1 ll. The metabolites were identified by comparison of retention times of authentic reference compounds. Retention times of diuron and 3,4-DCA in ethylacetate were, respectively, 4 and 5.9 min. The detection limits were 1 mg l1 for diuron and 5 mg l1 for 3,4-DCA. 2.5.4. Diuron transformation To study the transformation of diuron, MMO medium (400 ml) was supplemented with diuron (40 mg l1 ) added as the sole carbon source and MMK medium was supplemented either with succinate (75 mg l1 ) and diuron (40 mg l1 ) as the sole nitrogen source or with diuron as sole nitrogen and carbon source. Each flask was inoculated with the washed cell suspension already described and incubated at 30 °C under agitation. A control experiment without bacteria was carried out under the same conditions. 2 ml of the culture were periodically sampled for assay. 2.5.5. Diuron transformation in the presence of alternative carbon sources Glucose was added to MMO to give final concentrations of 0.25 and 0:5 g l1 . The Erlenmeyer flasks (2 l) contained 400 ml of MMO supplemented with diuron (40 mg l1 ) and glucose or 400 ml MMK supplemented with diuron (40 mg l1 ), and with succinate or pyruvate at different concentrations. The flasks were inoculated with the washed cell suspension and incubated at 30 °C on a rotary shaker. Samples were collected at regular intervals and analyzed by GC. A control experiment was
529
carried out under the same conditions without sugar. In parallel, cell numbers were determined by plating on LBagar. Standard errors were given when experiments were performed in duplicate. 2.5.6. Isolation and characterization of 3,4-dichloroaniline To isolate 3,4-dichloroaniline, bacterial resting cell assays and quantitative biodegradation assays were carried out as described in Tixier et al. (2002). The culture supernatants were extracted and concentrated, and the residue purified by column chromatography. After isolation, the structure of the metabolite was identified by NMR and mass spectrometry as indicated elsewhere (Tixier et al., 2002).
3. Microcosms 3.1. Soil samples The soil used to test the bacterial survival was a standard soil from an experimental farm of C^ ote SaintAndre (Isere, France). Its characteristics (granulometry: 41% sand, 38% silt, 19% clay; pH, 5.8; organic matter, 0:018 g1 of soil; humidity, 0:188 g1 ) were previously described (Hallier-Soulier et al., 1996; Ducrocq et al., 1999). Soil samples were sterilized by threefold autoclaving during 1.5 h at 121 °C. To study the fate of bacteria, 100 g of this soil were collected and placed in 500 ml Erlenmeyer flask under sterile conditions. 3.2. Inoculum preparation The bacterial cells were cultured in LB medium and pelleted at stationary phase by 15 min centrifugation at 5000g. The cells were washed three times in MMK and resuspended in different solutions: sterile 0.9% saline, MMK, MMK supplemented with LB (1/5 : v/v), MMK supplemented with succinate (0:075 g l1 ), MMK supplemented with succinate (0:075 g l1 ) and ammonium sulfate (1 g l1 ). The different bacterial suspensions were used to rehydrate 100 g of soil to 20% hydration and to add cells at 107 –109 cells g1 of soil. The soil samples were incubated at 18 °C. 3.3. Diuron addition to soil Two concentrations of diuron were tested: 0.8 and 2 g kg1 . Diuron was solubilized in acetone as indicated and aliquots (4 ml and 10 ml) were added to 100 g of soil contained in 500 ml Erlenmeyer flasks. The flasks were agitated and acetone was air-evaporated during 24 h under sterile conditions. The bacterial suspensions were added afterwards.
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3.4. Bacterial extraction Aliquots of 1 g of soil were collected at intervals and suspended in phosphate buffer (pH 7.6) containing the following in g l1 : NaCl, 8.5; Na2 HPO4 , 1.2; NaH2 PO4 , 0.8; Na4 P2 O7 , 10H2 O, 1. This mixture was vortexed for 2 min and after 10 min settling, the supernatant was collected and plated in triplicate on LB-agar for cells enumeration. Bacteria in the presence of diuron were also enumerated by acridine orange direct counting performed according to Hobbie et al. (1977). This indicates the total number of cells ml1 . The detection limits were 103 cells g1 for both methods, epifluorescence and LB-agar enumeration. 3.5. Diuron and metabolite extraction Aliquots of 2.5 g of soil were collected at intervals and dried at 65 °C (below the melting point of 3,4-DCA) for 1 h. Two ml of cold acetone were added to 2 g of dried soil and this solution was mixed for 1 h at 4 °C to limit acetone volatilization. The liquid phase was collected and centrifuged at 12 000g for 5 min. The supernatant was taken to determine diuron and 3,4-DCA concentrations. Diuron concentration was evaluated by GC analysis, under the conditions already described except that oven temperature programmation was different: 100–150 °C at 6 °C/min. The presence of 3,4DCA was evaluated by GC analysis and also detected by a rapid colorimetric method, on the basis of the production of a diazo color complex, which has been described previously (Walker, 1987). The detection limits were 30 mg kg1 for diuron, 5 mg kg1 by GC analysis and 1 mg kg1 by Walker’s method, respectively, for 3,4DCA.
4. Results 4.1. Isolation and characterization of diuron-transforming bacteria Four soil samples were tested for the presence of diuron degrading bacterial strains. The initial culturable microflora in the soil samples determined by plating on LB-agar was about 108 CFU g1 of soil, and did not significantly increase during the 8-days enrichment step (data not shown). In only one sample, a garden soil treated for several years with 2 g diuron m2 , platings of the 10-fold diluted enrichment mix on diuron-MMOagarose allowed to recover small size translucent bacterial colonies after 5 days at 30 °C. Among these, five clones were surrounded by a clear area free of diuron crystals, indicating a degradation of the herbicide by the colony. A first characterization of three clones showed no major differences between them and one isolate,
designated N2, was used for further experiments. On LB solid medium, strain N2 formed convex, cream colored, opaque and shiny colonies. On LB-agar plates spread with diuron, the colonies were larger, surrounded with a light halo. Microscopic examination of cultures in liquid LB medium displayed different morphological aspects of the bacteria depending on the growth stage: cells were nonspore-forming, nonmotile, gram-positive, aggregated irregular rods at exponential phase becoming coccoid at stationary phase. In MMK medium supplemented with the herbicide, bacterial morphology was the same, but cells appeared more aggregated. Biochemical and physiological tests were used for the identification of strain N2: catalase, b-glucuronidase, bgalactosidase, a-glucosidase tests were positive, b-glucosidase and phosphatase alkaline negative. Bacteria grew on glucose, citrate, succinate, pyruvate, not on saccharose and were resistant to metronidazole (4 lg). In anaerobic conditions the strain was urease-negative and no fermentation occurred on the hydrocarbons tested (glucose, ribose, xylose, mannitol, maltose, lactose, saccharose, glycogen). Finally, according to its morphological, fatty acid analysis and biochemical characteristics, the N2 strain was identified by Institut Pasteur-Paris as an Arthrobacter sp., close to A. globiformis and deposited at the Institut Pasteur Collection under number CIP105365. 4.2. Diuron transformation The biotransformation of diuron was monitored by GC analysis. Only two peaks were detected during the experiments, corresponding to the diuron itself, and to 3,4-DCA. Fig. 2 shows diuron disappearance and 3,4DCA formation as a function of time, at 30 °C, in three different culture media (MMO, MMK and MMK supplemented with succinate). Whatever the medium, 3,4-DCA concentrations increased while diuron concentration decreased. In contrast, control media without bacteria did not show any variation of diuron concentration which remained stable, and no 3,4-DCA was detected. Diuron completely disappeared after 30 h as it was the unique nitrogen source (MMK+succinate), after 50 h as it was the unique carbon source (MMO), and after 70 h as it was the only carbon and nitrogen source (MMK). The rate of disappearance of diuron was therefore improved in the presence of additional sources of carbon and nitrogen. The final concentration of 3,4-DCA (MW 162) was about 27 mg l1 , corresponding to 0:17 mmole l1 of medium. This molarity was in adequation with the number of moles of diuron initially introduced (equivalent to 40 mg l1 ), meaning that all molecules of diuron (MW 233) were transformed into 3,4-DCA. Moreover, these results indicate that the 3,4-DCA was not further degraded by the bacteria under these conditions. No
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531
mental conditions described elsewhere (Tixier et al., 2002), led to the formation of one metabolite detected by HPLC. After isolation, the structure of the metabolite was identified by NMR and mass spectrometry. The commercially available 3,4-dichloroaniline had the same retention time (6.7 min) and presented the same spectral data as the isolated metabolite. This confirmed the identification of 3,4-dichloroaniline as the terminal metabolite in diuron degradation by Arthrobacter sp. N2. 4.4. Effect of a cosubstrate on transformation The transformation of diuron was studied in the presence of different carbon cosubstrates, at 30 °C, in MMK medium supplemented with succinate (75 mg l1 or 500 mg l1 ) or pyruvate (500 mg l1 ), or in MMO medium supplemented with glucose (250 mg l1 or 500 mg l1 ). The results listed in Table 1 are expressed in terms of transformation rate, where Va is the average rate from the beginning of the culture to the time until diuron had totally disappeared, and Vmax is the maximal rate attained during the culture. At low concentration of succinate (75 mg l1 ), both Va and Vmax were increased by a factor of 1.9 and 2.3, respectively. At higher concentration of succinate (500 mg l1 ), Va was not significantly improved, but Vmax was 3.2-fold higher than the control experiment lacking the cosubstrate, and 4.1 higher if pyruvate 500 mg l1 was used as a cosubstrate. In contrast, when glucose 500 mg l1 was used in MMO medium containing nitrogen, Va remained quite constant while Vmax was only slightly increased by a factor 1.6 compared with the same medium without cosubstrate. Generally, these results show that the average transformation rate was improved by the addition of cosubstrate, even at a low concentration (results with
Fig. 2. Transformation of diuron in different media: (a) MMO (diuron as the sole source of carbon); (b) MMK supplemented with succinate 75 mg l1 (diuron as the sole source of nitrogen); (c) MMK (diuron as the sole source of carbon and nitrogen); and (d) Sterile control was MMO without bacteria. The concentration of diuron ( ) and 3,4-DCA ( ) were determined after GC analysis.
other metabolite could be observed during the transformation process by this procedure. 4.3. Isolation of 3,4-dichloroaniline Isolation of the unique metabolite as 3,4-dichloroaniline helped to confirm this result. Diuron degradation by resting cells of Arthrobacter sp. N2, under experi-
Table 1 Effect of a cosubstrate on diuron transformation at 30 °C diuron concentration 40 mg l1 Va a (mg l1 h1 )
Vmax b (mg l1 h1 )
MMK medium No cosubstrate 7:5 mg l1 succinate 500 mg l1 succinate 500 mg l1 pyruvate
0:57 0:03 1:11 0:05 1:18 0:16 1:67 0:27
0:72 0:05 1:62 0:28 2:35 0:39 2:97 0:41
MMO medium No cosubstrate 250 mg l1 glucose 500 mg l1 glucose
1:26 0:13 1.48 1.62
1:89 0:26 2.83 3.05
Media and cosubstrates
a b
Va : average rate of diuron disappearance. Vmax : maximal rate of diuron disappearance.
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succinate), together with the maximal transformation rate (especially with succinate or pyruvate), in the presence (MMO medium) or in the absence (MMK medium) of nitrogen. In parallel, a substantial cell growth was observed in the presence of different cosubstrates, correlated to the change of physiological state during the transformation process.
5. Microcosms 5.1. Effect of inoculum preparation Standard soil microcosms were incubated at 18 °C. Preliminary experiments in liquid MMO medium have shown that bacterial cells were able to rapidly degrade diuron at this temperature. Diuron completely disappeared in 30 h at 18 °C with Va and Vmax values of 1.3 and 2:1 mg l1 h1 comparable to the values obtained at 30 °C. Bacterial samples resuspended in different media were used to inoculate soil microcosms at 18 °C. At intervals the cells were extracted and counted by plating on LB agar. When bacteria were resuspended in sterile 0.9% saline or in MMK, no colony was observed on LBagar (data not shown). However, in other media: MMK supplemented with LB (1/5), succinate (75 mg l1 ) or succinate (75 mg l1 ) and ammonium sulfate (1 g l1 ), colonies were observed on LB-agar (Fig. 3). In all three conditions, the bacterial cells were able to survive in soil during more than 35 days after reinoculation. In the presence of succinate and ammonium sulfate, the number of cells was stable for the time of the experiment. In the presence of LB, the number of cells decreased during 4 days and increased after this period.
The bacterial number decreased after 12 days in soil. When the inoculum medium was supplemented with succinate, the number of colonies was stable for 10 days and progressively decreased to attain 105 cells g1 of soil after 35 days. Thus the bacteria were able to survive in soil under certain conditions, though they need mineral salts and carbon source to be culturable after reinoculation of soil. However, the absence of colony after plating on LB-agar does not indicate that bacteria were not able to survive but that they rather lost their culturability in synthetic medium, entering into a ‘‘viable but nonculturable’’ (VNC) state (Trevors et al., 1994; Ducrocq et al., 1999) where they cannot divide and form colonies on solid medium. 5.2. Bacterial survival in the presence of diuron We studied the fate of bacteria in microcosms in the presence of diuron at two different concentrations (0.8 and 2 g kg1 of soil) at 18 °C during 30 days. The cells were enumerated by plate counts and by a direct epifluorescence method. In the absence of diuron, the results obtained with both methods were comparable despite unexplained differences, indicating that the number of bacteria was stable in soil (Table 2). In the presence of diuron 0.8 g kg1 , the two profiles were similar and showed that bacteria were able to survive at this concentration. Diuron began to decrease from day 16 concomitantly with the appearance of 3,4DCA. Diuron concentration decreased to 65% after 30 days. 3,4-DCA was detected at day 16, and increased at day 30. In the presence of diuron 2 g kg1 , the profiles obtained by epifluorescence counts were very stable while no cells were detected after day 4 by plating on LB-agar. However, cells were active since the diuron concentration decreased and 3,4-DCA was detected at day 16 by Walker’s method and also by GC analysis. The concentration of 3,4-DCA was low (12–16 mg g1 ) and decreased thereafter (1–5 mg kg1 at day 30).
6. Discussion and conclusions
Fig. 3. Effect of inoculum conditions on bacteria survival: MMK þ LB ( ); MMK ( ) supplemented with succinate (75 mg l1 ) and ammonium sulfate (1 g l1 ); MMK ( ) supplemented with succinate (75 mg l1 ).
By soil enrichment and after plating on MMOagarose spread with diuron, we isolated several clones distinguishable by the presence of a clear halo. One isolate, N2, was able to metabolize diuron in pure culture and completely transformed it in 3,4-DCA. By morphological and biochemical characteristics, strain N2 was identified as an Arthrobacter sp., likely A. globiformis. Diuron was transformed by Arthrobacter sp. N2 into its corresponding aniline, 3,4-dichloroaniline as was expected according to previous reports. The corresponding aniline has been demonstrated to be a terminal metabolite in the biotransformation of various phe-
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533
Table 2 Number of cells in microcosms determined by plating (CFU g1 of soil) or by epifluorescence, in the absence or in the presence of diuron at 0.8 or 2 g kg1 of soil Diuron (g kg1 )
Days
1
4
0
CFU g1 of soil (107 ) Epifluorescence (107 )
17 3.9
24 4.1
2.8 5.1
3.1 1.5
4.6 8.3
0.8
CFU g1 of soil (107 ) Epifluorescence (107 ) Diuron (g kg1 ) 3,4-DCAa (mg kg1 ) 3,4-DCAb (mg kg1 )
15 2.2 0.8 – –
26 3.2 nd nd nd
18 6.2 0.8 – –
4.8 2.9 0.6 – 2.2
30 7.6 0.3 17.7 14.8
2
CFU g1 of soil (107 ) Epifluorescence (107 ) Diuron (g kg1 ) 3,4-DCAa (mg kg1 ) 3,4-DCAb (mg kg1 )
11.9 3.9 2.0 – –
– 1.7 2.0 – –
– 3.3 1.4 16.9 11.9
– 1.6 1.3 5.0 1.0
8
0.01 0.59 2.0 – –
16
30
Determinations of diuron and 3,4-DCA (a ) were performed by GC analysis or 3,4-DCA (b ) by Walker’s method. (nd) Not determined, (–) absence of compounds or bacteria.
nylamides by fungi (Blake and Kaufman, 1975; H€ aggblom, 1992) or bacteria (Athiel et al., 1995; Cullington and Walker, 1999). 3,4-DCA was not further transformed by strain Arthrobacter sp. N2. One study reports its degradation as unique carbon, nitrogen and energy source by Pseudomonas diminuta (Surovtseva et al., 1985). By co-metabolism, 3,4-DCA could be degraded in vitro in the presence of aniline or propioanilide (You and Bartha, 1982) or in the presence of monochloroaniline (Reber et al., 1979; Zeyer and Kearney, 1982). Moreover, 3,4DCA can be degraded in soil slurry systems (Brunsbach and Reineke, 1993). To determine factors influencing bacteria survival in soil (as reviewed by van Veen et al., 1997), the experiments were conducted in microcosms. To overcome the difficulty in estimating the population in soil, two complementary techniques of cell detection were used. Bacterial viability was evaluated by plating on LB-agar. The presence and the physiological state of bacteria were checked by epifluorescence. The microcosms were incubated at 18 °C as Arthrobacter sp. N2 was able to degrade diuron at this temperature. The presence of carbon source and mineral salts in the medium was necessary to obtain bacterial colonies on LB-agar. The strain was able to survive in soil after reinoculation in the presence or in the absence of diuron. Bacteria inoculated in soils polluted by high concentrations of diuron (2 g kg1 of soil) became nonculturable but their presence was detected by epifluorescence counts. This technique has the major advantage to observe the bacteria in situ. However, epifluorescence count only gives an estimation of the cell number in soil. We have noted that cells were generally associated with soil particles, which possibly hampers the extraction of bacteria.
Trevors et al. (1994) have discussed this phenomenon of nonculturability. It is sometimes difficult to evaluate whether bacteria are dead or are in a viable but nonculturable state. However, in the present case the bacteria were active since the disappearance of diuron and the appearance of 3,4-dichloroaniline were both observed by GC analysis. This was confirmed by detection of 3,4-DCA by Walker’s method. In all conditions, in soil microcosms no quantitative relation was observed between the amount of disappeared diuron and the amount of the appeared 3,4DCA, since the concentration of 3,4-dichloroaniline extracted from microcosms is at the highest 17–18 mg g1 , which represents at best 3% of what would be expected from the total transformation of diuron. This may result either from the formation of condensation products of 3,4-DCA (Bartha et al., 1968; Chisaka and Kearney, 1970; Hill et al., 1981) or rather from the chemical attachment of the chloroaniline to humic acids present in soil (Brunsbach and Reineke, 1993). The transformation of different phenylureas by resting cells of Arthrobacter sp. N2 is also reported (Tixier et al., 2002) and confirmed by HPLC analysis. Experiments are described to evaluate the toxicity of metabolites evolved by the action of degradative fungal strains on 3,4-DCA. As 3,4-DCA or its derivatives are considered to be much more toxic than diuron itself to microbial and vegetal flora and may have detrimental effects on the environment, it is thus of interest to know the evolution of this molecule and of other phenylamide herbicides and to evaluate the impact of their metabolites in complex ecological systems such as soils where bacteria and fungi are active in metabolizing organic molecules.
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Acknowledgements This work was supported by a GDR-CNRS ‘‘Exosols’’ (Groupe de Recherches-Centre National de la Recherche Scientifique), and by a grant from Conseil Regional de Picardie.
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