Enhanced degradation of carbofuran by Pseudomonas cepacia and Nocardia sp. in ... the formation of coloured, water-soluble and non-extractable metabolites.
Plant and Soil 8 4 , 4 4 5 - 4 4 9 (1985). Ms. 5994 9 1985 Martinus Ni/hoff/Dr W. Junk Publishers, Dordrecht. Printed in the Netherlands.
E n h a n c e d degradation of carbofuran b y Pseudomonas cepacia and Nocardia sp. in the presence of growth factors K. VENKATESWARLU* and N. SETHUNATHAN
Laboratory o f Soil Microbiology, Central Rice Research Institute, Cuttack-753 006, Orissa, India Received 11 July 1984. Revised October 1984
Key words Carbofuran Degradation Nocardia sp. Pseudomonas cepaeia Soil extract Yeast extract Summary Pseudomonas cepacia and a Nocardia sp., isolated from flooded alluvial soil amended with carbofuran, metabolized ring- ~4C-carbofuran fairly rapidly in mineral salts medium or soil extract, stlpplemented with yeast extract. When the two isolates were incubated with soil extract together with 500vg ml -~ yeast extract, about 40 to 62% of the radioactivity was recovered as water-soluble products. The degradation of carhofuran by Nocardia sp. led to the formation of coloured, water-soluble and non-extractable metabolites.
Introduction Recent build-up of brown planthopper (Nilaparvata lugens Stal.), a major pest of rice necessitated the extensive and intensive use of Fnradan 3G (3% carbofuran; 2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate) in rice culture. Perusal of the literature on carbofuran reveals only indirect involvement of microorganisms in its metabolism in soils2'3'*'* . Apart from our earlier study on the bacterial degradation of earbofuran, after an initial tag of about 20 days, in mineral salts medium s , virtually no information is available on the metabolic fate of this insecticide by pure cultures of microorganisms. This report describes the increased metabolism of ring- t4C-carbofuran in the presence of soil extract and yeast extract by Pseudomonas eepaeia and Nocardia sp., isolated from flooded alluvial soil.
Materials and methods Chemicals Uniformly ring-labelled 14C-carbofuran (specific activity, 2.20mCi/mmol; 98% pure), technical grade earbofuran, earbofuran phenol (2,3-dihydro-2,2-dimethyl-7-hydroxybenzofuran) and 3-hydroxyearbofuran (2,3-dihydro-2,2-dimethyl-7-hydroxybenzofuran) and 3-hydroxycarbofuran (2,3 - dihydro- 2,2- dimethyl- 3 -hydroxy- 7-benzofuranyl methylcarbamate) were gifted by FMC Corporation, Middleport, NY. Stock solution was prepared by dissolving the labelled carbofuran in 100ml acetone. The solvent from aliquots of this stock solution was evaporated completely at room temperature and the residues were then equilibrated with distilled water for 8 h before being sterilized by membrane filtration.
Incubation studies An enrichment culture was prepared in test tubes (25 • 200 mm) by adding 1 mg of the technical grade carbpfuran to 20 g alluvial soil samples under flooded conditions (25 ml distilled water) at 15-day intervals. After four additions of the insecticide, a bacterium capable of * Department of Botany, Nagarjuna University, Nagarjuna Nagar-522 510, India. 445
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degrading carbofuran was isolated as described earlier 6 and was identified as Pseudomonas cepacia through the courtesy of Dr J F Bradbury, CMI, Kew, England. A carbofuran-degrading actinomycete, tentatively placed in the genus Nocardia was also isolated from the simulated oxidized surface of the flooded alluvial soil (10g soil with 12.5 ml of distilled water taken in 250 ml Erlenmeyer flasks) treated with Furadan 3G. The abillity of the above organisms, maintained on agar medium, to degrade carbofuran as sole source of carbon or energy in the presence of growth factors was tested as follows. Millipore-filtered aqueous solutions of ring- 14C-carbofuran and technical carbofuran were introduced into 25 ml portions of sterile media in 100 ml Erlenmeyer flasks to give a final concentration of about 40t~gm1-1. The media employed included (a) mineral salts 6, (b) mineral salts + 500/~g ml -~ yeast extract, (c) soil extract (obtained by autoclaving 1 kg alluvial soil and 1.51 distilled water, and filtering the supernatant suspension) alone and (d) soil extract + 500~tg m1-1 yeast extract. Chemical hydrolysis of carbofuran was precluded by initially adjusting the pH of the media to 6.2 although it changed to 7.6 during incubation in the medium with yeast extract. The media were then inoculated with aliquots of cell suspensions of the two isolates prepared in sterile distilled water. Uninoculated media served as controls. After 10 and 20 days of incubation at 36~ duplicate samples were analysed for carbofuran and its predicted metabolites. Estimates of viable cells of P. cepacia in the media were made after 20 days of incubation by the dilution plating method.
Extraction and residue analysis The residues from the media were extracted thrice with chloroform-diethyl ether (1:1) as described previously~. After evaporating the solvent from pooled fractions of the extract, the residues were dissolved in two ml methanol and analysed after separation by thin-layer chromatography (TLC). The residues in methanol were spotted on TLC plates coated with silica gel G, 300~m thick, along with the standards and the plates were developed with etherhexane (3:1) for a distance of 15era and air-dried. The radioactivity in the silica gel areas on the developed TLC plates opposite to the authentic compounds, carbofuran and its predicted metabolites, carbofuran phenol and 3-hydroxycarbofuran, was determined in liquid scintillation spectrometer Model LSS 20, Electronics Corporation of India Ltd., Hyderabad, as outlined earlier s. Total radioactivity in the methanol extract and water phase remaining after solvent extraction was also assayed. The per cent recovery of carbofuran immediately after its application to the growth media ranged from 92 to 96 and the data values were not corrected to account for the recovery loss.
Results and discussion
The culture isolated from simulated oxidized surface of the flooded alluvial soil possessed the following characteristics. The colonial texture soft, pasty and the colour orange, mycelium production limited to the substrate, branching not profuse, short bacillary and coccoid cells predominant in older cultures. This carbofuran-degrading organism was thus identified as Nocardia sp. as all the above characteristics correspond with the description given in Bergey's Manual I . The results on the degradation of l*C-carbofuran in the presence of soil extract and/or yeast extract by P. cepacia and Nocardia sp., isolated from flooded alluvial soil are presented in Table 1. In all uninoculated media, no appreciable degradation of carbofuran occurred during 20-day incubation period. Again, in mineral salts medium alone, degradation of carbofuran was not much even after inoculation with P. cepacia. But the bacterium effected more rapid degradation of carbofuran in mineral salts medium plus yeast extract. Carbofuran in inoculated media decreased to 81% in mineral salts medium alone, 48% in mineral salts medium plus yeast extract, 64% in soil extract alone and 24% in soil extract plus yeast extract. Thus, most rapid degradation of carbofuran by the bacterium occurred in soil extract plus yeast extract followed by mineral salts medium plus yeast extract and soil extract, in that order. Viable counts of the bacterium after 20 days of inoculation showed a population (• 108/ml
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medium) of 5.5 in mineral salts medium, 13.0 in mineral salts medium plus yeast extract, 4.7 in soil extract alone and 27.0 in soil extract plus yeast extract. These results would indicate that soil extract and/or yeast extract supplied factors for the growth of P. cepacia so that cell numbers increased as a consequence of which the rate of carbofuran degradation was fairly rapid. This increased metabolism of carbofuran is analogous to the reported ehancement in degradation of p-nitroaniline by a strain of Pseudomonas in culture medium having 20flug m1-1 yeast extract 9. Moreover, P. cepacia was shown to degrade carbaryl in the presence of soil extract and/or yeast extract 7. Thin-layer chromatographic analysis of the residues in organic solvent extract of the media revealed that in bacterial degradation, carbofuran was converted to carbofuran phenol (Rf, 0.76), an unidentified product (Rf, 0.00) and certain polar products (not extracted by the solvent used). However, 3-hydroxycarbofuran, formed in negligible quantities, was not quantified. The addition of yeast extract to the soil extract or the mineral salts medium accelerated the bacterial conversion of carbofuran to carbofuran phenol as the major hydrolysis product and an unidentified product (Rf, 0.00). Carbofuran phenol was metabolized further since its concentration decreased between 10 and 20 days. The degradation of carbofuran by the bacterium appeared to be more extensive in soil extract plus yeast extract with the formation of polar products in substantial quantities since more than 67% of the degraded carbofuran was recovered in the water phase remaining after organic solvent extraction. Although carbofuran decreased considerably in inoculated media especially supplemented with yeast extract, there was no appreciable decrease in the total radioactivity recovered in organic solvent phase and water phase remaining after solvent extraction. This would indicate that bacterial conversion of carbofuran to volatile and gaseous products such as carbon dioxide is rather unlikely. As with P. cepacia, carbofuran degradation was enhanced by Nocardia sp. in the presence of soil extract and/or yeast extract and the rate of degradation in different media also followed the same order. But, the rate of degradation of carbofuran by Nocardia sp. was more pronounced when compared to the rate of decomposition by P. cepacia. Within 20 days after inoculation with Nocardia sp., the concentration of carbofuran decreased to 7, 35, 66 and 73% of its original levels in soil extract plus yeast extract, mineral salts medium plus yeast extract, soil extract and mineral salts medium, respectively. Clearly, the addition of yeast extract to the soil extract or mineral salts medium accelerated the loss of carbofuran considerably. During the same period, the insecticide loss from uninoculated media was negligible. The degradation of carbofuran by Nocardia sp. in the media supplemented with yeast extract was accompanied by the change in the colour of the medium from light yellow (due to yeast extract) to reddish brown. Analysis of residues in the organic solvent fraction by thin-layer chromatography and liquid scintillation counting showed that carbofuran phenol and an unidentified metabolite (Rf, 0.00) were formed during the degradation of carbofuran by Nocardia sp. but the accumulation of these products was not proportional to the decrease in the concentration of carbofuran. Interestingly, most of the degraded carbofuran was accounted for as watersoluble metabolites. The coloured product(s) of carbofuran degradation appeared to remain in the water phase even after acidification followed by organic solvent extraction and was responsible for the increase in the radioactivity in the water phase with incubation. The radioactivity in the water phase increased in proportion to the decrease in carbofuran levels reaching to more than 64% of the degraded compound. As with the bacterium, conversion of carbofuran by Nocardia sp. to volatile end products was negligible. No attempt was made to characterise the water-soluble product(s) of carbofuran degradation. The present study clearly indicates that the two microorganisms inhabiting the alluvial soil utilize carbofuran as sole or supplemental source of carbon or energy. Also, this report of the accumulation of significant amounts of coloured, water-soluble and non-extractable metabolites of carbofuran by Nocardia sp. is probably new to the literature.
Acknowledgements We are grateful to Prof A S Rao, Head of the Botany Department, Nagarjuna University for helpful comments in the preparation of this manuscript. This study was supported, in part, by funds from the International Atomic Energy Agency, Vienna, Austria
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(Contract No. 2089/SD), Department of Science and Technology, Government of India and Indian Council of Agricultural Research, New Delhi, India.
References 1 2 3 4 5 6 7 8 9
Buchanan R E and Gibbons N E, Eds. 1974 Bergey's Manual of Determinative Bacteriology. 8th Ed., William and Wilkins, Baltimore. Felsot A S e t al. 1981 Bull. Environ. Contam. Toxicol. 24,778-782. Getzin L W 1973 J. Environ. Entomol. 2, 461-467. Kaufman D D and Edwards D F 1983 Proc. 5th Int. Congr. Pestic. Chem. 4 , 1 7 7 - 1 8 2 . Venkateswarlu K and Sethunathan N 1978 J. Agric. Food Chem. 26, 1148-1151. Venkateswarlu K e t al. 1977 J. Agric. Food Chem. 2 5 , 5 3 3 - 5 3 6 . Venkateswarlu K e t al. 1980 J. Environ. Sci. Health 15B, 421-429. Williams I H e t al. 1976 Bull. Environ. Contam. Toxicol. 15,244-249. Zeyer J and Kearney P C 1983 J. Agric. Food Chem. 3 1 , 3 0 4 - 3 0 8 .
