Jiyoung Seo*, Junho Jeon*, Sang-Don Kim**, Suil Kang***, Jaehong Han**** and Hor-Gil Hur** *Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), Oryong-dong, Buk-gu, Gwangju 500-712, Korea **Center for Water Research of GIST, Oryong-dong, Buk-gu, Gwangju 500-712, Korea (E-mail:
[email protected]) ***International Environmental Research Center, Gwangju Institute of Science and Technology, Gwangju, Korea ****Metalloenzyme Research Group, BET Research Institute and Department of Biotechnology, Chung-Ang University, Anseong, Korea Abstract The fungus Mucor ramannianus was tested to elucidate the biological fate of a class of N-methyl carbamate pesticide carbofuran and its hydrolysed form carbofuran phenol. The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column showed that two peaks occurred after incubation of M. ramannianus with which 1 mM carbofuran was combined as a final concentration. In culture of M. ramannianus with 1 mM carbofuran phenol, it produced two other metabolites compared to metabolites of carbofuran. LC/MS analysis suggested that two of the metabolites produced from carbofuran phenol were most likely to be 2-hydoxy-3-(3-methylpropan-2-ol)phenol or 7a(hydroxymethyl)-2,2-dimethylhexahydro-6H-furo[2,3-b]pyran-6-one and 3-hydroxycarbofuran-7-phenol. Keywords Biodegradation; carbofuran; carbofuran phenol; fungus; Mucor ramannianus
Water Science & Technology Vol 55 No 1–2 pp 163–167 Q IWA Publishing 2007
Fungal biodegradation of carbofuran and carbofuran phenol by the fungus Mucor ramannianus: identification of metabolites
Introduction
Carbofuran (2,3-dihydro-2,2-dimethylbenzofurna-7-ylmethylcarbamate), a class of Nmethyl carbamate pesticides, is highly toxic (LD50 ¼ 2 mg kg21 in mice) and an inhibitor of acetylcholinesterase, an enzyme vital for functioning the central nervous system (Fahmy et al., 1970). Formulations of carbofuran are used in agricultural applications worldwide, and in 1995, over five million pounds of carbofuran were applied to fields in United States (Karns et al., 1986). As a consequence of extensive and diverse use and unintentional discharge into the environment, carbofuran has become one of the most frequently detected pesticides in water resources. Accumulation of carbofuran and its metabolites in the environment could potentially pose health hazards (Chaudhry, 2002). Microbial metabolites of carbofuran have been reported previously (Felsot et al., 1981; Ramanand et al., 1988; Chapalmandugu and Chaudhry, 1992) and include 7-phenol (2,3dihydro-2,2-dimethyl-7-hydroxybenzofuran), 3-hydroxycarbofuran (2,3-dihydro-2,2dimethyl-3-hydroxy-benzofuran-7-yl methylcarbamate), 3-hydroxycarbofuran-7-phenol (2,3-dihydro-2,2-dimethyl-3,7-dihydroxybenzofuran), 3-ketocarbofuran (2,3-dihydro-2,2dimethyl-3-keto-benzofuran-7-yl methylcarbamate), 3-ketocarbofuran-7-phenol (2,3-dihydro-2,2-dimethyl-3-keto-7-hydroxybenzofuran) (Chapalmandugu and Chaudhry, 1992; Chaudhry, 2002) and 5-hydroxycarbofuran (2,3-dihydro-2,2-dimethyl-5-hydroxybenzofuran-7-yl–methylcarbamate) (Behki and Topp, 1993).With improved environmental protection policies related to clean up and disposal of the large-scale wastewater from doi: 10.2166/wst.2007.051
163
J. Seo et al.
pesticide industries, the handling and disposal of pesticide rinse water generated by individual farmers or commercial agrochemical applicators has become a significant waste management issue (Wang and Lemley, 2003). Unlike prokaryotes, eukaryotic fungi has shown diverse metabolic potential, resulting in metabolites very similar to those produced from mammalian metabolism (Ferris et al., 1973; Zhang et al., 1996; el Sayed et al., 2000; Cha et al., 2001; Park et al., 2002, 2003). This metabolic pathway helps, indirectly to elucidate metabolic fates of organic compounds occurring in mammalian liver cells instead of using mammalian microsomal fractions or live organisms. Fungal metabolism also provides an easy preparative method for metabolites in large quantities. In this study, we studied biodegradation of carbofuran and carbofuran phenol by Mucor ramannianus, common soil fungus, to better understand fungal role in the degradation of carbofuran and carbofuran phenol. Materials and methods Reagents and chemicals
Carbofuran (2,3-dihydro-2,2-dimethylbenzofurna-7-benzofuran N-methylcarbamate, 98%, C12H15NO3) and 2,3-dihydrobenzofuran (99%) was purchased from Aldrich Chemical Company (Milwaukee, WI). Acetonitrile, ethyl acetate and methanol (HPLC grade) were purchased from Fisher (Fair Lawn, NJ, USA).
Fungal culture conditions
Cultures were grown in 100 ml of potato dextrose broth (PDB) medium containing carbofuran and carbofuran phenol in a final concentration of 1 mM at 27 8C in a shaking incubator with 200 rpm for 10 d.
High pressure liquid chromatography (HPLC)
Analytical HPLC was performed using a Varian ProStar HPLC (Walnut Creek, CA), which was equipped with a Spherisorb ODS-2 C18 column (5 mm particle size, 4.6 mm £ 25 cm, Waters Corporation, Milford, MA) and a photo diode array (PDA) detector. For analytical scale, the mobile phase, which was composed of water-acetonitrile containing 1% formic acid, was a linear gradient programmed as follows; 10% acetonitrile at 0 min, 40% acetonitrile at 15 min, 90% acetonitrile at 25 min, 90% acetonitrile at 40 min. The flow rate was 1 mL/min and the UV detection was performed at 270 nm. For large preparation of metabolites, a Varian prep-HPLC equipped with a Rainin C18 ODS column (10 mm particle size, 21.4 mm £ 25 cm, Varian Associates, Walnut Creek, CA) and a photo diode array (PDA) detector was used. The mobile phase, which was composed of water-methanol containing 1% formic acid, was programmed linearly as follows; 20% methanol at 0 min, 40% methanol at 10 min, 70% methanol at 20 min, 90% methanol at 50 min. The flow rate was 15 mL/min and UV detection was performed at 270 nm.
Liquid chromatography/mass spectrometry (LC/MS)
164
LC/MS was performed by coupling a HP 1100 system to a Quattro LC triple quadruple tandem mass spectrometer (Micromass, Manchester, UK) in electrospray ionisation (ESI þ ) mode. The source temperature, desolvation temperature, cone voltage and capillary voltage were kept at 60 8C, 220 8C, 26 V, and 3.99 kV, respectively. An electron multiplier voltage of 640 V was used. The nebuliser gas and desolvation gas were ultra-pure nitrogen set at 94 and 562 L/h, respectively.
Results and discussion Degradation of carbofuran and carbofuran phenol by fungus
J. Seo et al.
Elution profiles of HPLC equipped with a reverse phase C-18 column showed three peaks and two peaks produced from carbofuran phenol and carbofuran, respectively, by M. ramannianus. LC/MS analysis suggested that two of the metabolites produced from carbofuran phenol were most likely to be 2-hydoxy-3-(3-methylpropan-2-ol)phenol and 3-hydroxycarbofuran-7-phenol. M. ramannianus R-56 produced two metabolites (I) and (II) at 12.5 and 14.7 min, respectively, on the HPLC elution profile (Figure 1), and metabolite (II) was also present in the incubation of M. ramannianus with carbofuran phenol. In addition, M. ramannianus produced two more metabolites (III) at 8 and 22 min respectively (Figure 1). All the metabolites, however, were not produced in the control experiments of both soil fungus alone and target compounds alone. Analysis of metabolites of carbofuran and carbofuran phenol
Metabolites produced from carbofuran by M. ramannianus were analysed by UV/visible spectrometry and LC/mass spectrometry at positive mode. Metabolite (I) had a molecular weight of 219 with a fragment ion (m/z) at 162.8. Metabolite (II) had a molecular weight of 152.8. Metabolite (III) had a molecular weight of 183.3 with a fragment ion m/z 164.8. Molecular weights of the metabolite (III) clearly implicated the molecular structures 2-hydroxy-3-(3-methylpropan-2-ol) phenol or 7a-(hydroxymethyl)-2,2-dimethylhexahydro-6H-furo[2,3-b]pyran-6-one by molecular (M þ H)þ; peak at m/z 182 (Figure 2), which was one of the accumulated metabolites, suggesting that the M. ramannianus could degrade carbofuran-7-phenol by hydrolysis at furanyl ring. The molecular structures of metabolites (I) and (II) were not yet identified. A nuclear magnetic resonance (NMR) analysis regarding those metabolites will be attempted. We observed an unusual metabolite (IV) in the degradation of carbofuran phenol by M. ramannianus. LC/MS analysis identified this unusual intermediate by a (M þ H)þ peak at m/z 343.4 (data not shown). This metabolite was observed as the most predominant metabolite among the metabolites of carbofuran phenol found in this study, based on HPLC analysis. As this AU 2.0 1.5
AU
(II) Carbofuran (I)
1.5
1.0
(A)
Absorbance at 270 nm
0.5
0
(II) 276.80nm
0 5
10
15
20
25
30
AU 1.25
Carbofuranphenol
350
450
550 nm
(Carbofuranphenol) 279.82 nm
1.00
(II)
(III)
250
35
(IV) 400
(I) 278.26nm
1.0
0.5
mAU 600
(Carbofuran) 270.73 nm
2.0
0.75
(B)
(II) 277.19nm
0.50
200
0.25 0
(III) 274.87nm
0 5
1 10
15
20
25
30
35
250
350
450
550
nm
Retention time (min)
Figure 1 HPLC elution profiles of the extracts of the M. ramannianus with carbofuran and carbofuran phenol in potato dextrose medium for 7 d. A UV/Visible spectrum of each extract is presented at the right captions
165
219.9
10 100
(I)
J. Seo et al.
Relative abundance
162.8 80 60
180.8 237.9
40 254.9
20 0 1 100
200
300
400
500
152.8
100
(II)
Relative abundance
80 60 40 153.8 20
134.8
0 100
200
300
400
500
183.3
100
(III) Relative abundance
80 60
164.8
40 150.7 20 0 100
200
300
400
500
m/z Figure 2 LC/MS spectra of metabolites produced from carbofuran (I, II) and carbofuran phenol (III) by M. ramannianus culture in 10
metabolite appeared in the cultures during the incubation, the cultures were changed to a red-brown colour. This metabolite would be the result of condensation of some metabolites. Conclusions 166
The metabolite produced from carbofuran had a different retention time, as compared to those of metabolites from carbofuran phenol. Based on the metabolites, M. ramannianus
J. Seo et al.
may adopt different tracks of metabolism for carbofuran and carbofuran phenol, although the two compounds have similar structures. Metabolites of carbofuran by microorganisms include carbofuran phenol, 3-hydroxycarbofuran, 3-ketocarbofuran, 3-hydroxycarbofuranphenol and 3-ketocarbofuranphenol. Carbofuran is currently reported to degrade to 4hydroxycarbofuran. However, no study has been carried out to identify microbial metabolites of carbofuran resulting from cleavage of aromatic or furanyl rings. In the present study, we suggest a possible metabolic intermediate of either 2-hydroxy-3-(3-methylpropan-2-ol)phenol or 7a-(hydroxymethyl)-2,2-dimethylhexahydro-6H-furo[2,3-b]pyran-6one, resulting from fungal degradation of carbofuran phenol. The results, which are the first reports of the metabolism of carbofuran phenol by fungus, will provide valid information for the environmental risk assessment related with pesticide carbofuran. Acknowledgements
This research was supported by a grant from the Sustainable Water Resources Research Center of 21st Century Frontier R&D program through the Center for Water Research at Gwangju Institute of Science and Technology.
References Behki, R. and Topp, E. (1993). Metabolism of carbofuran by Rhodococcus TEI, abstr.113, 93rd General Meeting of the American Society for Microbiology. Cha, C.J., Doerge, D.R. and Cerniglia, C.E. (2001). Biotransformation of malachite green by the fungus Cunninghamella elegans. Microbiology, 67(9), 4358– 4360. Chapalmandugu, S. and Chaudhry, G.R. (1992). Microbial and biotechnological aspects of metabolism of carbamates and organophosphates. Crit. Rev. Biotech., 12(5), 357 – 389. Chaudhry, G.R. (2002). Induction of carbufuran oxidation to 4-hydroxycarbofuran by Pseudomonas sp. 50432. FEMS Microbiol. Lett., 214(2), 171 – 176. el Sayed, K.A., Halim, A.F., Zaghloul, A.M., Dunbar, D.C. and McChesney, J.D. (2000). Transformation of jervine by Cunninghamella elegans ATCC 9245. Phytochemistry, 55, 19 –22. Fahmy, M.A., Fukuto, T.R., Meyers, R.O. and March, R.B. (1970). The selective toxicity of Nphosphorothioyl carbamate esterase. J. Agric. Food Chem., 18(5), 793 –796. Felsot, A., Maddox, J.V. and Bruce, W. (1981). Enhanced microbial degradation of carbofuran in soils with histories of furadan use. Bull. Environ. Contamin. Toxicol., 26(6), 781 – 788. Ferris, J.P., Fasco, M.J., Stylianopoulou, F.L., Jerina, D.M., Daly, J.W. and Jeffrey, A.M. (1973). Monooxygenase activity in Cunninghamella bainieri: evidence for a fungal system similar to liver microsomes. Arch. Biochem. Biophy., 156, 97– 103. Karns, J.S., Mulbry, W.W., Nelson, J.O. and Kearney, P.C. (1986). Metabolism of carbofuran by a pure bacterial culture. Pest. Biochem. Physiol., 25, 211 – 217. Park, M.K., Hur, H.G., Liu, K.W., Lee, Y.H., Lee, Y.S. and Kim, J.H. (2002). In vitro metabolism of ethaboxam by rat liver microsomes. Agric. Chem. Biotechnol., 45(2), 94 –98. Park, M.K., Liu, K.W., Lim, Y., Lee, Y.H., Hur, H.G. and Kim, J.H. (2003). Biotransformation of a fungicide ethaboxam by soil fungus Cunninghamella elegans. J. Microbiol. Biotechnol., 13(1), 43 – 49. Ramanand, K., Sharmila, M. and Sethunathan, N. (1988). Mineralization of carbofuran by a soil bacterium. Appl. Environ. Microbiol., 54(8), 2129– 2133. Wang, Q. and Lemley, A.T. (2003). Oxidative degradation and detoxification of aqueous carbofuran by membrane anodic Fenton treatment. J. Hazardous Materials B., 98, 241 – 255. Zhang, D.L., Freeman, J.B. and Walker, Y. (1996). Fungal biotransformation of the antihistamine azatadine by Cunninghamella elegans. Appl. Environ. Microbiol., 62(9), 3477– 3479.
167
