Influence of assay medium on degradation of malathion by Serratia marcescens

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1 Indian Journal of Biotechnology Vol. 4, April 2005, pp Influence of assay medium on degradation of malathion by Serratia marcescens V Kannan* and V Vanitha Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai , India Received 23 December 2003; revised 12 May 2004; accepted 25 May 2004 Serratia marcescens isolated from degraded cattle bone was able to grow on high concentrations of malathion, a nonsystemic wide spectrum organophosphorus pesticide. The test organism tested under three different assay conditions very effectively catalyzed the breakdown of malathion. Degradation potential of the test organism was high in nutrient broth than in mineral salts medium and distilled water. Decline in the ph during growth on malathion and assay period indicated the formation of acidic intermediates. Keywords: pesticides, degradation, malathion, Serratia marcescens IPC Code: Int. Cl. 7 A01N63/02; C12R1:43 *Author for correspondence: Tel: extn. 204: Fax: kannanavo@yahoo.co.in; vkannan@unom.ac.in Introduction Increased and persistent use of pesticides to boost crop productivity has resulted in the entry of large amounts of pesticides into various ecosystems with actually only a small portion reaching the targeted sites. Today, pesticides are extensively used not only in agriculture but also for many diverse purposes such as human and animal health protection, pest control, in forest and aquatic environments and protection of buildings and other structures. Contamination of surface waters and ground water by pesticides is a major environmental concern. Organophosphorus pesticides like malathion are among the most widely used pesticides in non-crop areas as well as in food crops. Given the potential carcinogenic risk from these pesticides there is a serious need to develop remediation processes to eliminate or minimize contamination in the environment. Biodegradation could be a reliable and cost effective technique for pesticide abatement 1. Malathion-S-1,2 bis (ethoxycarboxyl) ethyl O,O-dimethyl phosphorodithioate (C 10 H 19 O 6 PS 2 ) is reported to affect central nervous system, immune system, adrenal glands, liver and blood. Walker and Stojanovic 2 isolated an Arthrobacter sp. from soil that degraded malathion to malathion monoacid, malathion diacid, dimethyl phosphorodithioate and dimethyl phosphorothioate. Numerous bacteria, isolated from a salt marsh environment, have been found capable of degrading malathion cometabolically 3. Dimethyl phosphoro-dithioic acid and diethyl fumarate as the degradation products of malathion have also been identified 4. However, organisms isolated from paper mill effluents formed mono carboxylic acid as breakdown product 5. Number of bacteria capable of degrading malathion increased in the sediments with increasing frequency of application and increasing level of treatment 6. Fungal mediated slow malathion degradation has also been reported in Egypt 7. Thus, the mechanisms of degradation of malathion by microorganisms differ considerably. In this communication, the authors report the results of degradation of malathion by Serratia marcescens isolated from a source, which had no previous history of pesticide application, using three different assay media. Materials and Methods Southern Insecticide and Fertilizers, Ambattur, Chennai supplied malathion, (commercial grade). A strain of Serratia marcescens isolated from partially decayed cattle bone collected from M/s Subbaraj and Co, Koyambedu near Chennai was used for the degradation of malathion. Serratia marcescens was grown in both nutrient broth and mineral salts medium 8 with ph 7.2 at 30 C. Growth was measured as turbidity at 540 nm in a DU- 40 Spectrophotometer at 2 hrs interval for 24 hrs. The

2 278 INDIAN J BIOTECHNOL, APRIL 2005 capability of the test organism in the degradation was followed in the above media amended with malathion at various concentrations (0.3, 0.6, 0.9, and 1.2 m mol ml -1 ) and ph was measured in digital model Vorion analyzer at 24 hrs interval for 10 days. The degradation assay was performed with whole cells of test organism grown in nutrient broth as bulk cultures. The 24-hr-old culture ( cfu ml -1 ) was centrifuged at 10,000 g for 15 min and the pellet resuspended in a known volume of nutrient broth, mineral salts medium and distilled water and the cell density in suspensions adjusted to cfu ml -1. To 1 ml each of the cell suspension (cell suspension in: i. nutrient broth, ii. mineral salts medium and iii. distilled water), 1 ml of potassium phosphate buffer (0.1 M) ph 7.2 and malathion at 0.3, 0.6, 0.9 and 1.2 m mol ml -1 were added and incubated for 3 hrs. Of the three sets maintained for each of the assay mixture, one set from each concentration was centrifuged at 1 hr interval and the clear supernatant was used for spectral analysis. The clear supernatant was scanned from nm in a DU-40 spectrophotometer, analyzed for specific absorption regions in the spectrum, since malathion has a specific absorption maximum at 210 nm in water. Thin layer chromatography was used in the separation of degradation by-products of malathion 9. Culture filtrate (100 ml) of the test organism grown in nutrient broth for 144 hrs with 0.9 m mol ml -1 malathion was concentrated by lyophilization and the concentrated extract was dissolved in 1 ml ethanol. Ethanol extract (100 µl) was loaded on 0.5 mm thick silica gel plates and the chromatogram was developed with a mobile phase containing Hexane:Acetic acid: Diethyl ether (2:2:1 V/V). Then the plate was air dried and visualized under UV at 3560Å for spots of breakdown products. The spot observed was scrapped, extracted with an aliquot of ethanol, scanned from nm and the spectral properties were analysed. Protein was measured by Coomassie brilliant blue dye binding method 10. Results The test organism, S. marcescens showed good growth both in nutrient broth and mineral salts medium, though it was recorded higher in nutrient broth at all age levels (Fig. 1). S. marcescens also showed good growth both in nutrient broth and in mineral salts medium when amended with various concentrations of malathion. Increase in cell density was observed even from 24 hrs in flasks containing Fig. 1 Growth kinetics of Serratia marcescens 0.3 and 0.6 m mol of malathion, there was a lag of 48 hrs in growth observed in flasks containing 0.9 and 1.2 m mol of malathion (Fig. 2). Thus, at low concentrations, the test organism was able to grow without much lag phase, while at high concentrations it showed an extended lag phase of hrs and between the two media tested, nutrient broth supported very good growth of the test organism than mineral salts medium. Whole cells of S. marcescens, when incubated with various concentrations of malathion amended in nutrient broth, showed remarkable breakdown action on malathion during the three hours of incubation. The characteristic absorption maximum of malathion at 210 nm in water was found altered when amended with nutrient broth. The spectral analysis showed 2 characteristic absorption regions, one near nm, which was very sharp and a broader second at 320 nm. The absorption at regions was characteristically decreasing with increase in incubation time while in contrast the absorption at 320 was gradually increasing with the time of incubation and thus confirming the breakdown of malathion and the reduction in the concentration of malathion (Fig. 3). With varying concentrations of malathion; i.e m mol ml l -1 in nutrient broth as the assay medium, degradation by whole cells ranged from m mol h -1 mg -1 protein (Table 1). The assay with cells incubated in mineral salts medium amended with various concentrations of malathion also resulted in the breakdown of malathion, however, the absorption peaks characteristically exhibited were different when compared to the absorption peaks observed with nutrient broth. Here again the specific absorption peak of malathion at 210 nm was not observed, instead

KANNAN & VANITHA: MALATHION DEGRADATION BY SERRATIA MARCESCENS 279 - -- Fig.

3 KANNAN & VANITHA: MALATHION DEGRADATION BY SERRATIA MARCESCENS Fig.2-Growth of Serratia marcescens in nutrient broth (NB) and Minimal salts medium amended with various concentrations of malathion [Co-Control (unanended), A-0.3, B-0.6, C-0.9 and D- 1.2 m mol mr'] three specific absorption peaks that extended from UV to visible region, at , 350 and 374 nm, respectively were observed (Fig. 4). The spectrum obtained after 3 hrs of incubation showed only 2 clear absorption peaks, one at nm and the other at nm, with the total disappearance of absorption peak at 350 and 374 nm. The quantity of. malathion degraded by the test organism in mineral salts medium amended assay mixture varied between and m mol h mg' protein (Table 1). In the degradation assay, with distilled water as the medium, malathion exhibited its characteristic absorption region at nm (Fig. 5). Degradation of malathion was clearly observed as decreasing in specific absorption with increase in incubation period. The degradation capability was calculated to be varying between 3.33 and 7.49% during the experimental period and the quantity of malathion degraded was between and 0.09 m mol h'l mg' protein (Table 1). Thus, the malathion degradation

4 280 INDIAN J BIOTECHNOL, APRIL 2005 Fig. 3 Malathion degradation by cells of S. marcescens incubated in nutrient broth potential of cells of S. marcescens was highest when incubated in nutrient broth compared to other two incubation media though the number of cells incubated being the same in three assay media tested. The change in ph during growth and whole cell assay monitored closely and the results were supportive. The ph in the growth medium kept at 7.2 at the start of the experiment was gradually decreased to 3.2 and 3.0 in mineral salts medium and nutrient broth, respectively (Fig. 6a). Similarly, the ph 7.2 kept at the start of the assay decreased to 6.3, 6.1 and 6.0 in distilled water, mineral salts medium and nutrient broth, respectively at the end of 3 hrs (Fig. 6b). All the results were subjected to one way ANOVA and Duncan s multiple comparison tests. The results of the analysis of degradation of malathion by cells incubated in nutrient broth varied significantly at 0.05 level when compared to the Fig. 4 Malathion degradation by cells of S. marcescens incubated in mineral salts medium Fig 5 Malathion degradation by cells of S. marcescens incubated in distilled water Table 1 Degradation of malathion by cells incubated in three different assay media Incubation medium Concentration of malathion (m mol) Nutrient broth Degradation %/hr Quantity degraded (m mol -1 hr -1 mg -1 protein) Mineral salts medium Degradation %/hr Quantity degraded (m mol -1 hr -1 mg -1 protein) Distilled water Degradation %/hr Quantity degraded (m mol -1 hr -1 mg -1 protein) CD (0.05) Note: The results shown in the Table are mean value of 3 replications

5 KANNAN & VANITHA: MALATHION DEGRADATION BY SERRATIA MARCESCENS 281 Fig. 6a ph change during growth with malathion at 0.9 m mole Fig. 7 Absorpgtion spectra of TLC separated spot Fig. 6b ph change during whole cell assay with malathion at 0.9 m mole results of analysis with mineral salts medium and in distilled water. In addition, there was no significant variation between the analyses done with mineral salts medium and distilled water as the assay medium. TLC analysis of 144-hr-old culture filtrate of S. marcescens grown in nutrient broth amended with 0.9 m mol malathion showed a single separated spot. The spot, when extracted with ethanol and analyzed spectrally, showed the formation of a new absorption region at 238 nm and reduction in peak height and volume at 260 nm region, which was absent in the authentic sample indicating the breakdown of malathion (Fig. 7). Discussion The results indicated that the test organism, S. marcescens isolated from a source, which had no history of pesticide application, is able to grow well in both the nutrient broth and mineral salts medium amended with various concentrations of malathion and thus supporting the view of Lowe et al 11 that microbes from different environments with no history of target chemicals also possess potential for degrading variety of compounds by producing highly stable enzymes. The extended lag phase observed in the present study at higher concentrations of malathion (Fig. 2) had also been reported earlier in the case of malathion degradation by Pseudomonas putida 12. Hence, this could be the fact that at lower concentrations the cell number versus toxic molecules ratio would have been in favour of cells. While at higher concentrations, there could be more number of toxic molecules per cell, which resulted in the instant death of certain cells and the tolerant cells would have taken more time to withstand the concentration of the toxic molecules and then established themselves. Barth et al 13 have also reported similar observations in the degradation of Trichloroethylene by Burkholderia cepacia where the cell density of 0.8 (at 540 nm) had shown more degradation at the cell density of 0.5 (at 540 nm), which is in agreement with our observations. Hydrolytic cleavage of organophosphate bond is considered as the initial step in the metabolism of organophosphate 14. But the hydrolytic reaction did not supply the energy required for the growth of the organisms and only the degradation products from these pesticides appear to serve as energy for growth and proliferation of microorganisms 15. The alteration of ph in the medium from 7.2 to 3.0 during the growth of the test organism in malathion amended medium proved not only the breakdown of malathion but also the formation of acidic intermediates. Similarly, the ph of the assay mixtures also showed the change of ph from 7.2 to at the end of 3 hrs, which confirmed the breakdown of malathion and the formation of acidic intermediates, also reported by Matsumara and Boush 16. Formation of hydrolytic

6 282 INDIAN J BIOTECHNOL, APRIL 2005 acidic products during the breakdown of malathion had also been reported earlier 3,17. The comparative account of degradation of malathion by cells of S. marcescens using three different assay media reported here showed remarkable variation on the absorption property of malathion and the intermediates, which have not been reported so far, since earlier workers used a single assay medium. Thus, this study exposed the inadequacy in the method of assay of malathion degradation. The assay mixture containing mineral salts medium at the end of 3 hrs showed 2 characteristic absorption regions i.e. one at nm and the other one at nm, which was well supported by the reports of Lakshmi Rani 12 who also reported 2 kinds of intermediates of malathion degradation by Pseudomonas putida showing specific absorptions at 232 and 242 nm. Further, the TLC analysis of culture filtrate of S. marcescens grown in malathion amended medium showed the separation of one spot with R f value of 0.6 and the spot eluted when analyzed for spectrum showed an absorption peak at 238 nm. This observation is also very similar to that of Lakshmi Rani 12, who showed an intermediate compound with absorption maximum at 242 nm in the culture filtrate in P. putida. Though organophosphate hydrolase mediated breakdown of malathion has been reported earlier 18, Horne et al 19 could not substantiate this observation even though they have identified a gene opd (organophosphorus degradation) in an Agrobacterium strain. The protein produced by this organism was well suited for bioremediation of dimethyl organophosphorus compounds but could not hydrolyze aliphatic organophosphorus malathion. Wang et al 20 also successfully demonstrated the expression of two functional moieties on the surface of E. coli cells for cellulose and organophosphate hydrolase binding but hydrolase mediated degradation of malathion was not proved. In the present work, the authors showed that S. marcescens was able to breakdown malathion using hydrolytic process evidenced by the decrease in ph during growth as well as in whole cell assay. This, together with the results of TLC analysis of culture filtrate, possibly conclude that S. marcescens isolated from an environment that had no previous history of pesticide application is able to grow on malathion by degrading it through hydrolytic process. Further, the degradation capability increased with incubation in nutrient broth to show that some amount of growth factor or a nutrient in the assay mixture is essential for effective degradation of malathion by the test organism. The latest report of Gyaneshwar et al 21 exposing the nitrogen fixing capability of an endophytic strain of S. marcescens stimulate a new line of thinking in use of this bacterium as a potential bioremediation tool for cleansing our environment. Thus, similar to the suggestions of Hashmi et al 22 using the strain Pseudomonas, capable of degrading malathion, for treating wastewater containing high content of hazardous pesticide, S. marcescens could also be tried for the effective removal of hazardous chemicals in wastewater. 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M-3, Bull Environ Contam Toxicol, 42 (1989) Bourquin A W, Microbial malathion interaction in artificial salt marsh ecosystem Effect of degradation, Appl Environ Microbiol, 40 (1975) Omar S S, Availability of phosphorus and sulphur of pesticide origin by fungi, Biodegradation, 9 (1999) Kilobane J J, Chatterjee D K, Karns J S, Kellor S T & A M Chakrabatty, Biodegradation of 2,4,5-trichlorophenoxy acetic acid by a pure culture of Pseudomonas cepacia, Appl Environ Microbiol, 44 (1982) Jaglan P S & Gunther F A, A thin layer chromatographic procedure for separating desmethyl parathion (O-methyl-O- P-nitrophenyl phosphoro-thionate) and its S isomer (Smethyl-O-P-nitrophenyl phosphorothionate), Bull Environ Contam Toxicol, 5 (1970) Bradford M M, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye binding, Anal Biochem, 72 (1976) Lowe S E, Biology, ecology and biotechnological applications of anaerobic bacteria adapted to environmental stresses in temperature, ph, salinity or substrates, Microbiol Rev, 57 (1993) Lakshmi Rani N, Catabolic versatility of Pseudomonas putida in organo phosphorus pesticides degradation, Ph D Thesis, University of Madras, Chennai, Barth J A C, Kalin R M, Clarke D, Larlein M, Schuth C et al, Changes of the carbon isotopic composition of trichloroethylene during aerobic biodegradation- A new tool

7 KANNAN & VANITHA: MALATHION DEGRADATION BY SERRATIA MARCESCENS 283 to estimate removal efficiencies, in Ground water research, edited by Rosbjerg et al (Balkema, Rotterdam, The Netherlands) Munnecke D M, Johnson L M, Talbot H W & Bharik S, Microbial metabolism and enzymology of selected pesticides, in Biodegradation and detoxification of environmental pollutants, edited by A M Chakrabarty (CRC Press, Boca Raton, FL) 1982, Sethunathan N, Adhiya T K, Bharik S & M Sharmila, Degradation products of commonly used insecticides in Indian rice soils, ACS Symp Ser, 459 (1991) Matsumara F & Boush G M, Malathion degradation by Trichoderma viridae and a Pseudomonas sp., Science, 53 (1966) Mostafa I Y, Fakhir I M I, Bahing M R E & El Zawahry Y A, Metabolism of organo phosphorus insecticides. XII. Degradation of malathion by Rhizobium sp., Arch Microbiol, 36 (1972) Lai K, Stolowich N J & Wild J R, Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase, Arch Biochem Biophys, 318 (1995) Horne I, Sutherland T D, Harcourt R L, Russell R J & Oakeshot J G, Identification of an opd (organophosphorus degradation) gene in an Agrobacterium isolate, Appl Environ Micribiol, 68 (2002) Wang A A, Munchandanni A & Chen M W, Specific adhesion to cellulose and hydrolysis of organophosphorus nerve agents by a genetically engineered E. coli strain with a surface expressed cellulose-binding domain and organophosphorus hydrolase, Appl Environ Microbiol, 68 (2002) Gyaneshwar P, James E K, Mathan N, Reddy P M, Reinhold- Hurek B & Ladha J K, Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens, J Bacteriol, 183 (2001) Hashmi I, Shaukel S S, Khan M A & Khan M A, An application of principle component analysis to the study of activated sludge treatement system Performance efficiency for the degradation of malathion, Pak J Biol Sci, 2 (1999)

Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples:

Dr. Sanjeeva Srivastava IIT Bombay Work-flow: protein sample preparation Precipitation methods Removal of interfering substances Specific examples: Sample preparation for serum proteome analysis Sample