Ability of a microbial consortium to remove pesticide, carbendazim and 2,4-dichlorophenoxyacetic acid

Anchana Pattanasupong1,3, Hiroyasu Nagase1,*, Mariko Inoue1, Kazumasa Hirata1, Katsuji Tani2, Masao Nasu2 and Kazuhisa Miyamoto1 1 Environmental Bioengineering Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan 2 Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan 3 Department of Biotechnology, Thailand Institute of Scientic and Technological Research, 196 Phahonyothin Road, Chatuchak, Bangkok 10900, Thailand *Author for correspondence: Tel.: +81-6-6879-8237, Fax: +81-6-6879-8239, E-mail: [email protected]
Received 9 September 2003; accepted 16 December 2003

Keywords: Biodegradation, carbendazim, consortium, 2,4-D, paddy eld, pesticide

Summary A bacterial consortium capable of simultaneously degrading the fungicide, carbendazim, and the herbicide, 2,4dichlorophenoxyacetic acid (2,4-D) was obtained by enrichment of soil samples collected from paddy elds in Japan. This consortium was acclimated in a continuously fed culture with 20 lM carbendazim and 2 mM 2,4-D as sole carbon sources using a glass column reactor. By denaturing gradient gel electrophoresis, we observed changes in the bacterial population following the degradation of the both pesticides. This acclimated consortium completely degraded up to 100 lM carbendazim and 3 mM 2,4-D within 36 and 24 h, respectively, in batch culture, but a lag time was observed after precultivation in a rich medium. The immobilization of the consortium on a polyester support enhanced the degradation ability of this consortium compared with the use of free cells. This microbial consortium could be useful for bioremediation at sites contaminated with these pesticides.

Introduction Rice is the staple food for many people in the world. To increase the yield of rice in paddy elds, farmers continuously and excessively use many types of pesticide to control pests (insects, weeds and diseases) coupled with crop rotation, leading to the simultaneous accumulation of several types of pesticide in paddy elds. Since rice is an irrigated crop, the use of pesticides directly aects the surrounding aquatic environment. These chemicals can be removed from the environment by physicochemical (e.g., evaporation, volatilization, hydrolysis, oxidation, and photolysis) and biological processes. Bioaugmentation is an important process for the removal of these pesticides (Alexander 1981). Many pollutant-degrading bacteria have been isolated from natural environments. In many cases, however, the bacteria introduced to contaminated sites fail to degrade the pollutants due to their poor survival or low activity in the environment. This is because they are exposed to abiotic (e.g., pH, temperature, and inorganic nutrients) and biotic (e.g., predation and competition) stresses that they never encounter in the laboratory environment. On the other hand, the introduction of a microbial consor-

tium has a higher possibility of success in biodegradation than that of a single strain because such a consortium has a higher ability to adapt to these stresses. Moreover, a microbial consortium contains both the degraders of target compounds and strains that can utilize the metabolic intermediates of the target compounds. Toxic intermediates sometimes remain when using a single degrader strain. However, a microbial consortium can degrade both the toxic pesticide and its intermediates. The biodegradation of pesticides, such as 4-nitrophenol (Laha & Petrova 1998), 1,3-dichloropropene (Ou et al. 2001), dichlorodiphenyltrichloroethane (Bidlan & Manonmani 2002), and endosulfan (Awasthi et al. 2000) by a microbial consortium has been reported. Furthermore, the biodegradation of herbicide mixtures such as those of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (McGhee & Burns 1995), and (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) and (RS)-2-(2,4-dichlorophenoxy)propionic acid (dichlorprop) (Zipper et al. 1999) has also been studied. As described above, various types of pesticide (e.g., herbicides, insecticides, and fungicides) are supplied to each crop. Therefore, a microbial

consortium capable of simultaneously degrading dierent types of pesticide is useful for cleaning up pesticide contaminants. In this study, the fungicide, carbendazim (methyl-2-benzimidazole carbamate, MBC), and the herbicide, 2,4-D, were selected as target pesticides. They are used extensively throughout the world to control pests in paddy elds, and their detectable residues in paddy elds have been reported (Chalam et al. 1996; Readman et al. 1997). In Asia, the Ministry of the Environment in Japan reported that MBC and 2,4-D are frequently detected in environmental water. These pesticides were thought to be endocrine disrupters, which have toxic eects to animals even in the low concentrations (http://www.env.go.jp/chemi/end/ref4/ ref4-1.pdf). Jungbluth (1997) also reported that both of them could be found in paddy soil and runo water in the central region of Thailand. Therefore, it is necessary to remove these pesticides from polluted water completely. MBC is widely used as an active ingredient in the benzimidazole carbamate class of fungicides. It is a systemic fungicide with both protective and curative activities against a wide range of fungal diseases (Boudina et al. 2003). Moreover, MBC is the main degradation product of two other systemic fungicides, benomyl and thiophanate-methyl (Mazellier et al. 2002). It has been documented that MBC has mutagenic and teratogenic eects on mammals at low doses (Holtman & Kobayashi 1997). 2,4-D is a herbicide that has been widely used to control dicotyledonous weeds in cereal and grass crops for over 40 years. Its half-life in soil is 16 weeks, but adverse conditions, such as acidic pH and low temperatures, are known to increase its persistence (Thompson et al. 1984; Sandmann et al. 1988; McGhee & Burns 1995). In this study, continuously fed culture was carried out to obtain a microbial consortium capable of simultaneously degrading these two pesticides. The change in microbial population during the biodegradation process was analysed by denaturing gradient gel electrophoresis (DGGE). The inuence of the consortium immobilization on degradation ability was also investigated.

Tesque, Inc., Japan) as sole carbon sources at a ow rate of 2 ml h)1. The TM contained 2.0 g of (NH4)2SO4, 0.2 g of MgSO4 7H2O, 0.01 g of CaCl2 2H2O, 0.001 g of FeSO4 7H2O, 1.5 g of Na2HPO4 12H2O and 1.5 g of KH2PO4 per litre of deionized water. The pH was adjusted to 7.0. Biodegradation tests After 3.5-months incubation, 10 bacterial supports from the reactor were transferred to a 300-ml ask containing 100 ml of tryptic soy broth medium (TSB; glucose is the major carbon source). The ask was shaken at 150 rev min)1 at 30 C. After overnight cultivation, the culture was centrifuged at 15,000 rev min)1 for 15 min and the cell pellet was washed twice with TM. The cells were resuspended in TM and the optical density at 660 nm (OD660) was adjusted to 1.0. Then 1 ml of the cell suspension was added to 60 ml of TM in a 300-ml ask containing 20 lM MBC, 2 mM 2,4-D, or a mixture of 20 lM MBC and 2 mM 2,4-D. The cells were then incubated under the same conditions for 21 days. All treatments were conducted in triplicate. A control without cells was run in parallel. High performance liquid chromatography (HPLC) To measure the concentration of MBC and 2,4-D, 10 ll of the culture supernatant was analysed using HPLC (D-7000series; Hitachi, Japan) with a u.v. detector (L-4500; Hitachi) at 230 nm. A reversed phase column, Inertsil ODS-3V (4.6 250 mm, 5 lm) was used, and the liquid phase consisting of acetronitrile/0.12 M sodium phosphate buer (pH 3) (1:1, v/v) was supplied at a ow rate of 0.5 ml min)1. Denaturing gradient gel electrophoresis (DGGE) Cells were collected from 1 ml of the sample for DNA extraction, and DGGE analysis was performed as described previously (Kawai et al. 2002). Eect of culture conditions on MBC degradation

Materials and methods Enrichment of microbial cultures able to degrade a mixture of MBC and 2,4-D Soil samples were collected from eight dierent paddy elds in Japan and mixed. Supports, Fabios, made of polyester bre (8 mm diameter, 6 mm height, Unitika Ltd., Japan), were soaked in the soil suspension overnight to allow microbial self-adhesion. These supports were packed in a glass column reactor (3 cm diameter, 18 cm height) and then incubated at 30 C with a continuous supply of air and a mineral medium, Tmedium (TM), containing 20 lM MBC (Aldrich Chemical Company, Inc., USA) and 2 mM 2,4-D (Nacalai

The bacterial supports were taken from the reactor and precultivated by two methods. In one method, the supports were cultivated in TSB as described above. For the other, the supports were repeatedly incubated for four times in the TM added with 20 lM MBC, until cell turbidity was observed. For free cell culture, the cells from each preculture were inoculated into 10 ml of TM containing 20 lM MBC in a 50-ml (approximately 106 c.f.u. ml)1). It was cultivated under the same conditions for 21 days, and MBC concentration was measured. The cells precultivated by these two methods were also cultivated with or without supports. For the immobilized cell culture, 10 Fabios supports were added to each of two types of free cell preculture for 30 min. Then, one support (approximately 107 c.f.u./

support) from each preculture was incubated in 10 ml of TM added with 20 lM MBC at the same conditions of free cell culture. Degradation ability of immobilized consortium The ability of the immobilized consortium to degrade MBC and 2,4-D was investigated. Bacterial supports were taken from the glass column reactor and incubated twice in TM containing 20 lM MBC, 2 mM 2,4-D or a mixture of 20 lM MBC and 2 mM 2,4-D. Then, one bacterial support from each culture was incubated in 10 ml of TM containing each or both pesticides in a 50ml ask and cultivated under the same conditions for 72 h. The eects of MBC and 2,4-D concentrations on the degradation ability of the consortium were also investigated. A bacterial support was incubated in 10 ml of TM containing MBC (20, 30, 40, or 50 lM) or 2,4-D (2, 3, 4, and 5 mM) for 72 h; 0.2 ml of the culture was then sampled every 12 h, and pesticide degradation ability was analysed.

Table 1. Ability of the consortium to degrade MBC and/or 2,4-D. DGGE lane numbera Pesticide addedb Incubation Time (day) Remaining (%) MBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
a

2,4-D 100 0 0 0 0 0 0 0 0 0 0 0 0

Initial MBC MBC MBC MBC MBC MBC MBC MBC MBC MBC MBC MBC 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D and and and and and and 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D

0 3 7 10 14 17 21 3 7 10 14 17 21 3 7 10 14 17 21

100 94.9 25.7 1.9 0.3 0.2 0 96.6 90.7 20.2 4.2 0.7 0.2

Results and discussion Enrichment of microbial consortia able to degrade a mixture of MBC and 2,4-D We attempted to obtain a bacterial consortium that can simultaneously degrade MBC and 2,4-D by enrichment of soil samples collected from paddy elds in Japan. The acclimation of soil microorganisms was carried out in a continuously fed culture supplied with 20 lM MBC and 2 mM 2,4-D as sole carbon sources in the reactor. After 3.5 months incubation, we got a stable bacterial consortium having the ability to degrade MBC and 2,4-D.

DGGE lane numbers are corresponding to those shown in Figure 1. The TM contained 20 lM MBC, 2 mM 2,4-D, or a mixture of 20 lM MBC and 2 mM 2,4-D.
b

Using batch cultures, we analysed the population changes and degradation ability of the microbial consortium obtained from the continuously fed culture. Ten bacterial supports in the reactor were transferred to TSB and incubated overnight. Then, bacterial cells were inoculated in TM containing 20 lM MBC, 2 mM 2,4-D or a mixture of MBC and 2,4-D. As shown in Table 1, this consortium could completely degrade 2 mM 2,4-D within 3 days in cultures containing only 2,4-D and both

Figure 1. Population change in the consortium during biodegradation, followed by DGGE analysis. Lane 1 illustrates the initial population of the consortium; lanes 27 illustrate the bacterial population in the 20 lM MBC culture (a); lanes 813 illustrate the bacterial population in the 20 lM MBC and 2 mM 2,4-D culture (b); and lanes 1419 illustrate the bacterial population in the 2 mM 2,4-D culture (c), during 21 days cultivation. The DGGE lane numbers are shown in Table 1.

pesticides; approximately 99% of 20 lM MBC was degraded within 10 days in the culture containing only MBC, and within 17 days in the culture containing both pesticides. Figure 1 shows the results of DGGE analysis following biodegradation (Table 1) by this consortium. We could observe changes in the bacterial population during 21 days of cultivation. Several bands that dier from the initial bands were observed after the addition of the pesticides into the culture. These DGGE bands that distinctly appeared during the pesticide degradation period seemed to be those of the primary strains, which can utilize the target pesticides. On the other hand, dierent bands appeared after the target compound was completely degraded. It is likely that these bacteria are secondary strains and are maintained in the community because of their capacity to utilize metabolites produced by the primary strains. For example, in Figure 1(a), bands S1 and S2 were observed during the MBC degradation period and then became weak when the MBC was almost completely degraded. They could be considered as those of primary strains. On the other hand, band S3 appeared after the MBC was almost completely degraded, indicating that this strain seemed to be a secondary strain. Richard & Vogel (1999) described a similar phenomenon in which three of seven isolates (primary strains) of a soil microbial consortium were able to degrade petroleum hydrocarbons. Four other bacterial isolates (secondary strains) did not grow on any of the hydrocarbons tested nor did they cooxidize them. The presence of these bacteria in the initial microbial consortium suggests that they might contribute to the complete degradation of diesel fuel. Eect of culture conditions on MBC degradation The eect of precultivation condition and immobilization on MBC degradation was evaluated. As shown in

Figure 2. Eect of preculture condition and immobilization on MBC degradation. Free cells precultivated in TM (n) or precultivated in TSB (m), and cells immobilized in polyester supports precultivated in TM (s) or precultivated in TSB (d), were used to inoculate in TM containing 20 lM MBC followed by cultivation.

Figure 2, the cells precultivated in TSB had lag times of 5 days with the immobilization support and 7 days without the support before the MBC began to be degraded. On the other hand, the cells repeatedly incubated in TM containing MBC did not have a lag time in cultures both with and without the support. This may be explained by a higher acclimation of the microbial consortium that had been enriched under similar conditions (Bastos et al. 2002), while the precultivation in TSB might have increased the population of nondegraders rather than that of microbes utilizing MBC. As shown in Figure 1(a), it is possible that a bacterial strain (band S1), which might be the MBC degrader strain, participated in the microbial consortium but was undetected on the DGGE gel at the initial period of incubation. After this strain acclimated and MBC began to be degraded, the cell numbers of this strain increased to a detectable value after 7 days of incubation. On the other hand, there is another possible explanation for this phenomenon. Madigan et al. (2003) suggested that a lag time is observed when cells are transferred from a rich culture medium to a poor one. When the substrate in the medium is changed, some enzymes are induced to metabolize the new substrate, which was not present in the previous medium. It was also reported that the addition of readily utilizable carbon sources, such as glucose or amino acids, inhibits, decreases or has no signicant inuence on the mineralization of xenobiotic substrates (Swindoll et al. 1988; Rhee et al. 1996; Carmichael & Pfaender 1997; Awasthi et al. 2000). The eect of the presence of supporting materials on MBC degradation was also investigated. As shown in Figure 2, a higher degradation rate was achieved by immobilized cells than by free cells in both precultivation conditions. In the case of the TSB preculture, more than 99% of the MBC was degraded within 10 days by the immobilized microbial consortium and within 14 days by the free microbial consortium. On the other hand, by repeatedly cultivated in TM containing MBC, the period for complete degradation was reduced to only 3 days under the immobilized condition, for 10 days under the free condition. It appears, therefore, that immobilization accelerates the ability of the microbial consortium to degrade MBC. Several studies showed that immobilized cells have a higher degradation ability than free cells. Fava et al. (1996) found that an immobilized ECO3 consortium can aerobically degrade and dechlorinate dichlorobiphenyls more extensively than suspended cells. Diaz et al. (2002) reported that the immobilization of the bacterial consortium MPD-M onto polypropylene bres signicantly enhances the biodegradation rate of crude oil compared with the use of free cells. Winson & Bradley (1996) reported the successful use of immobilized bacteria to enhance the degradation of diesel oil, and suggested that the bacterial support absorbs diesel oil, bringing the cells into closer contact with the carbon source, and thus increasing the bioavailability of the oil.

Figure 3. Degradation ability of immobilized consortium. The immobilized cells were directly cultivated in TM containing 20 lM MBC (s), 2 mM 2,4-D (n) and a mixture of 20 lM MBC (d) and 2 mM 2,4-D (m).

Figure 4. Degradation ability of the immobilized consortium at dierent MBC concentrations. One bacterial support was cultivated in TM containing 20 (), 30 (d), 40 (m), or 50 (j) lM MBC.

Degradation ability of immobilized microbial consortium The degradation ability of the immobilized microbial consortium was investigated in TM containing 20 lM MBC, 2 mM 2,4-D, or a mixture of 20 lM MBC and 2 mM 2,4-D. As shown in Table 1, 2,4-D was completely degraded within 3 days by the culture containing only 2,4-D and both pesticides. Thus, in this experiment, samples were obtained from the culture every 6 h in the initial period of incubation to determine degradation rate. Figure 3 shows that after 18-h cultivation, this consortium could degrade more than 96 and 56% of 2 mM 2,4-D in the 2,4-D enriched culture and in the both pesticides enriched culture, respectively. 2,4-D in both cultures was completely degraded after 24 h. On the other hand, approximately 96% of 20 lM MBC was degraded after 18 h in the culture containing only MBC, and after 60 h in the culture containing both pesticides. The degradation rate in the culture with both pesticides was lower than that in the culture with either of the pesticides. Both pesticides combined might be more toxic than each one action single. However, the degradation ability of this immobilized bacterial consortium was maintained when batch culture with pesticides was repeated several times (data not shown). Rhee et al. (1996) has also reported that it is possible to reuse 16 times pyridine-degrading immobilized bacteria without a loss of their degradation ability within the entire culture period. Degradation of dierent concentrations of MBC and 2,4-D The eects of MBC and 2,4-D concentrations on the biodegradation ability of the immobilized microbial consortium were investigated. All of the MBC at concentrations of 20, 30, 40, or 50 lM was degraded within 36 h (Figure 4). A high degradation ability was also maintained at 100 lM (data not shown). Figure 5 shows that 2 and 3 mM 2,4-D in the cultures were completely degraded within 24 h. At 4 mM, the remain-

Figure 5. Degradation ability of the immobilized consortium at dierent 2,4-D concentrations. One bacterial support was cultivated in TM containing 2 (), 3 (d), 4 (m), or 5 (j) mM 2,4-D.

ing 2,4-D was 60, 30, and 10% after 12, 24, and 72 h, respectively. Nearly 50% of the 5 mM 2,4-D remained after 72 h and the concentration negligibly decreased until 120 h of incubation (data not shown). It is possible that some unidentied intermediate products inhibited the degradation ability. In conclusion, a bacterial consortium isolated from paddy soil samples that had the ability to degrade a mixture of MBC and 2,4-D was obtained by continuously fed culture. A rapid degradation of MBC was observed in the absence of a second organic substrate. Several reports have also demonstrated the MBC degradation ability of bacteria and fungi at high levels (Yarden et al. 1990; Holtman & Kobayashi 1997) or 2,4D (Greer et al. 1990; Mangat & Elefsiniotis 1999; Vroumsia et al. 1999). In this study, our microbial consortium had the ability to degrade these pesticides at concentrations reaching the solubility limit. Moreover, the degradation ability has been maintained in the reactor more than 1 year, and this ability can be kept by freeze-drying preservation. Haugland et al. (1990) reported that a mixed culture of a 2,4-D degrader and a 2,4,5-T degrader does not eectively metabolize a mixture of these two herbicides because 2,4-D inhibits 2,4,5-T metabolism by the 2,4,5-T degrader. In our experiment, some intermediates (not identied yet) were

detected by HPLC analysis during biodegradation of MBC and 2,4-D. The mutual degradation inhibition also occurs in the culture with both MBC and 2,4-D. However, both of them were completely degraded. Moreover, the toxicity of the intermediates was found to decrease by biological toxicity test (Daphtoxkit F). The high potential of this consortium to degrade both pesticides was claried in this paper. The degradation ability of this consortium found to increase by immobilization. The use of immobilized consortium will be advantageous over free cell culture due to keeping adhesion of cells on supports when it is applied for bioremediation of the contaminated water with these pesticides. To develop the pesticide degradation system using immobilized consortium, the eect of environmental parameters, such as temperature, pH, and nutrients concentration, on degradation ability are under investigation.

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