http://www.jsrr.in 94 ISSN: 2249-7846 (Online) Science Research Reporter 2(1): 94-103, March 2012 ISSN: 2249-2321 (Print) Malathion Degradation by Azospirillum lipoferum Beijerinck S N Kanade, A B Ade 1 and V C Khilare Botany Research Center, Vasantrao Naik Mahavidyalaya, CIDCO, Aurangabad 431003 (MS) India 1 Department of Botany, University of Pune, Pune 411007 (MS) India [email protected] ABSTRACT Malathion is one of the largest organophosphorus insecticides in the world which has a wide variety of applications in the agriculture industry around the world. Its toxic effects are harmful to the animals ranged from invertebrates to vertebrates including human. For protecting crop against the insects it is sprayed on it, however, leaches in the soil which leads to the soil pollution. Due to this practice there is loss of soil microflora and soil becomes infertile. For its degradation in the soil usage of the microbes in the soil has been proved to be the effective method for controlling soil pollution. In the present investigation therefore attempts have been made to make use of Azospirillum lipoferum for Malathion degradation. Azospirillum lipoferumis the free living nitrogen fixer generally found in the rhizoplane of the crop plants. Malathion was found degraded effectively by it with intracellular degradation mechanisms.Total six degradation products were scored after gas chromatography and mass spectrometry analysis. KEY WORDS: Azospirillumlipoferum, Malathion, organophosphorus, insecticide, degradation INTRODUCTION To feed every mouth, the pressure on agriculture has been increased tremendously. Therefore it is mandatory for the farmers to obtain more yield of the crop at any cost. At the same time the usage of the pesticide is increased to protect the crop from the attack of the pests. Because of the excessive use of the pesticides, the residue level in the soil is increased which leads to the infertility and loss of soil microflora. Pesticide degradation is the breaking down of the toxic pesticides into the non-toxic compounds, in some cases, down to the original elements from where they were derived (Vargas, 1975). There are three types of pesticide degradation, photodegradation, chemical degradation and microbial degradation. Photodegradation is carried out by light, particularly sunlight, and can destroy the pesticides on foliage of the soil surface and even in the air (Kiss and Virag, 2009). Microbial degradation is the breakdown of pesticides by fungi, bacteria and other microorganisms that use pesticides as food source. Soil conditions such as moisture, temperature, aeration, pH and amount of organic matter affects the rate of microbial degradation because of their direct influence on the microbial growth and activity (Fosteret al, 2006). Microbial process plays an important role in the biological transformation or degradation of pesticides (Matsumara, 1974). The major aspect of pesticide degradation is being studied with the various types of saprophytic microbes (Mostafa et al, 1972a); however, the report is available in the rhizobial degradation of pesticides which may serve as nitrogen fixing pesticide degrader as well. Malathion, the organophosphorus insecticide has several applications in the agriculture. Its chemical name is ‘diethyl mercaptosuccinate’ or ‘o, o-dim-ethyl phosphorodithioate’. Malathion is also known as carbophos, maldison and mercaptothion. The toxic effects of Malathion are well known on the wide range of animals. The effects on mammals especially on human being were noted as leukemia, kidney damage, brain damage, lung damage, etc. Its carcinogenic effects include the chromosome defects in human blood cells and gene loss from the human DNA. The birth defects in birds, turtles and frogs are well known. The animals include fishes, annelids, crustaceans, echinoderms, insects, molluscs, nematodes, flatworms and many zooplanktons which showed the toxic effects of Malathion (Buffin et al, 2003). There are enough evidences where the application of chemicals to soil has resulted change in soil microflora (Gangawane and Saler, 1988). Most of the pesticides are administered as spray or dust on plants which ultimately reaches the soil as run off or drift where they influence the microbial balance of the soil. Moreover, the residual effect on the plant and life is known (Alexander, 1969).
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http://www.jsrr.in 94 ISSN: 2249-7846 (Online)
Science Research Reporter 2(1): 94-103, March 2012 ISSN: 2249-2321 (Print)
Malathion Degradation by Azospirillum lipoferum Beijerinck
S N Kanade, A B Ade1 and V C Khilare
Botany Research Center, Vasantrao Naik Mahavidyalaya, CIDCO, Aurangabad 431003 (MS) India 1Department of Botany, University of Pune, Pune 411007 (MS) India
ABSTRACT
Malathion is one of the largest organophosphorus insecticides in the world which has a wide variety of applications in the agriculture industry around the world. Its toxic effects are harmful to the animals ranged from invertebrates to vertebrates including human. For protecting crop against the insects it is sprayed on it, however, leaches in the soil which leads to the soil pollution. Due to this practice there is loss of soil microflora and soil becomes infertile. For its degradation in the soil usage of the microbes in the soil has been proved to be the effective method for controlling soil pollution. In the present investigation therefore attempts have been made to make use of Azospirillum lipoferum for Malathion degradation. Azospirillum lipoferumis the free living nitrogen fixer generally found in the rhizoplane of the crop plants. Malathion was found degraded effectively by it with intracellular degradation mechanisms.Total six degradation products were scored after gas chromatography and mass spectrometry analysis.
KEY WORDS: Azospirillumlipoferum, Malathion, organophosphorus, insecticide, degradation
INTRODUCTION To feed every mouth, the pressure on agriculture has been increased tremendously. Therefore it is mandatory for the farmers to obtain more yield of the crop at any cost. At the same time the usage of the pesticide is increased to protect the crop from the attack of the pests. Because of the excessive use of the pesticides, the residue level in the soil is increased which leads to the infertility and loss of soil microflora. Pesticide degradation is the breaking down of the toxic pesticides into the non-toxic compounds, in some cases, down to the original elements from where they were derived (Vargas, 1975). There are three types of pesticide degradation, photodegradation, chemical degradation and microbial degradation. Photodegradation is carried out by light, particularly sunlight, and can destroy the pesticides on foliage of the soil surface and even in the air (Kiss and Virag, 2009). Microbial degradation is the breakdown of pesticides by fungi, bacteria and other microorganisms that use pesticides as food source. Soil conditions such as moisture, temperature, aeration, pH and amount of organic matter affects the rate of microbial degradation because of their direct influence on the microbial growth and activity (Fosteret al, 2006).
Microbial process plays an important role in the biological transformation or degradation of pesticides (Matsumara, 1974). The major aspect of pesticide degradation is being studied with the
various types of saprophytic microbes (Mostafa et al, 1972a); however, the report is available in the rhizobial degradation of pesticides which may serve as nitrogen fixing pesticide degrader as well. Malathion, the organophosphorus insecticide has several applications in the agriculture. Its chemical name is ‘diethyl mercaptosuccinate’ or ‘o, o-dim-ethyl phosphorodithioate’. Malathion is also known as carbophos, maldison and mercaptothion. The toxic effects of Malathion are well known on the wide range of animals. The effects on mammals especially on human being were noted as leukemia, kidney damage, brain damage, lung damage, etc. Its carcinogenic effects include the chromosome defects in human blood cells and gene loss from the human DNA. The birth defects in birds, turtles and frogs are well known. The animals include fishes, annelids, crustaceans, echinoderms, insects, molluscs, nematodes, flatworms and many zooplanktons which showed the toxic effects of Malathion (Buffin et al, 2003). There are enough evidences where the application of chemicals to soil has resulted change in soil microflora (Gangawane and Saler, 1988). Most of the pesticides are administered as spray or dust on plants which ultimately reaches the soil as run off or drift where they influence the microbial balance of the soil. Moreover, the residual effect on the plant and life is known (Alexander, 1969).
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Kanade et al,
Therefore degradation of pesticide in the environment and soil is desirable as soon as the pests or diseases are controlled. In view of this the present investigation was carried out in which the attempts have been made to find out the Malathion degrading ability of Azospirillumlipoferum.
MATERIAL AND METHODS Rhizoplane of sugarcane was added in Nfb semisolid agar medium (Dobereiner et al., 1976). After incubation at 35oC for 72 hours, the pellicles were seen 2 mm below the upper surface of the medium and was streaked on Nfb medium (containing 2 % Agar and 0.02 g of Yeast Extract) and potato medium (200 g of peeled fresh potatoes were cooked in 1000 ml of Distilled Water and then filtered through sheets of cotton. 2.5 g Malic acid, 2.5 g Sucrose and 20 g Agar was added and pH adjusted to 6.8). Plates were then incubated at 35oC for 72 hours. Small dry curled pinkish colonies were selected for cultural and morphological studies and transferred on new semisolid Nfb medium. This inoculated semisolid Nfb medium after 72 hours was used for biochemical characterization and a loop full was transferred on Nfb medium slants and after incubation stored at 4oC. The isolated Azospirillum lipoferum was identified on the basis of morphological and biochemical characters according to Bergey’s manual of Determinative Bacteriology (Bergey and Holt, 1994).
To check the tolerance limit of Azospirillum lipoferum against the Malathion, it was inoculated on the Nfbagar medium with various concentrations of Malathion. The growth was measured in terms of colony diameter on plates. The concentrations used for testing the tolerance limit were 25, 50, 75, 100, 200, 1000 and 2000 µg/ ml. Sub-lethal concentration of Malathion was used for the degradation. The Azospirillum lipoferumwas inoculated in the sterilized Nfb broth medium supplemented with Malathion. The incubation was done at room temperature with regular shaking conditions at the time interval of one day each. This was done for 10 days. Analysis of Malathion degradation products was done. The culture filtrate was obtained in the Nfb broth. The products were analyzed with thin layer chromatography (TLC). After sonication of the cells the extract was analyzed by gas chromatography and mass spectrometry (GCMS) along with the standard Malathion. RESULTS AND DISCUSSION
The isolated Azospirillum lipoferum showed pinkish, opaque, smooth, star shaped colonies having umbonate elevation. The Azospirillum lipoferum showed positive tests for catalase production, H2S production and ketolactose production. The tests conducted as per the Bergey’s manual of Determinative bacteriology.
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Science Research Reporter 2(1): 94-103, March 2012 ISSN: 2249-2321 (Print)
Fig. 1.GCMS analysis of residual Malathion after degradation by Azospirillum lipoferum
Fig 2: Chromatogram area covered by standard malathion.
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Fig.3: TLC analysis of the culture filtrate from the MPSS medium supplemented with Malathion and Inoculated with Azospirillum lipoferum after incubation of 10 days
Fig. 4: Gas Chromatogram of the Malathion degradation products after degradation by Azospirillum lipoferum
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Science Research Reporter 2(1): 94-103, March 2012 ISSN: 2249-2321 (Print)
Fig. 5.GCMS analysis of malathion degradation product, phosphorothioic acid, o,o,s- trimethyl ester
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6.68 6.72 6.76 6.916.836.81 6.86 6.99
6.86
7.00
6.77
NL:1.16E5
TIC MS Blank
NL:4.41E6
TIC MS sampleno-11180
sampleno-11180 #33 RT: 6.86 AV: 1 SB: 19 6.88-6.96 , 6.69-6.79 NL: 6.55E5T: + c Full ms [30.00-800.00]
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109.76
47.05155.40
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157.42264.12 310.66 439.61412.02 495.22 532.23 577.79 663.52
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7.55 7.60 7.65 7.70 7.75 7.80 7.85
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7.667.587.56
7.81 7.837.76
7.82
7.68
NL:9.47E4
TIC MS Blank
NL:3.21E7
TIC MS sampleno-11180
sampleno-11180 #108 RT: 7.68 AV: 1 SB: 8 7.71-7.74 , 7.58-7.61 NL: 2.57E6T: + c Full ms [30.00-800.00]
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124.9593.01 295.88
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264.12 311.71342.28
174.47344.36 451.23 692.62638.58526.08 722.83561.04
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Fig. 6.GCMS analysis of malathion degradation product, phosphorodithioic acid, o,o,s-trimethyl ester
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7.667.587.56
7.81 7.837.76
7.82
7.68
NL:9.47E4
TIC MS Blank
NL:3.21E7
TIC MS sampleno-11180
sampleno-11180 #108 RT: 7.68 AV: 1 SB: 8 7.71-7.74 , 7.58-7.61 NL: 2.57E6T: + c Full ms [30.00-800.00]
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172.52
124.9593.01 295.88
63.24
264.12 311.71342.28
174.47344.36 451.23 692.62638.58526.08 722.83561.04
(mainlib) Phosphorodithioic acid, O,O,S-trimethyl ester
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 1800
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15 31
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6379
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8.338.288.17 8.21
8.25
8.29
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TIC MS Blank
NL:1.91E6
TIC MS sampleno-11180
sampleno-11180 #159 RT: 8.25 AV: 1 SB: 7 8.29-8.31 , 8.18-8.22 NL: 1.06E5T: + c Full ms [30.00-800.00]
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172.4758.98
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326.03
103.75 413.59
497.17
343.69 498.17414.60173.49 584.72
485.26 500.14270.16 608.12 685.56 713.22 784.92
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Fig. 7. GCMS analysis of malathion degradation product, Butanedioic acid, mercapto-
Fig. 8. GCMS analysis of malathion degradation product, Succinic acid mercapto diethyl ester, s-ester with o-s-dimethyl phosphorodithioate by Azospirillumlipoferum.
(mainlib) Butanedioic acid, mercapto-
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RT: 24.56 - 26.12
24.6 24.8 25.0 25.2 25.4 25.6 25.8 26.0
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25.9825.9225.8225.68
25.6125.5225.37
25.2825.13
25.00
26.09
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NL:3.22E7
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sampleno-11180 #1645 RT: 25.98 AV: 1 SB: 15 26.03-26.12 , 24.56-24.66 NL: 4.57E6T: + c Full ms [30.00-800.00]
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127.01
99.33
282.46
71.45 330.86
180.65281.14 453.32 610.44359.77 509.07 734.56640.11 795.36
(mainlib) Succinic acid, mercapto-, diethyl ester, S-ester with O,S-dimethyl phosphorodithioate
50 70 90 110 130 150 170 190 210 230 250 270 290 310 3300
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5571
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143158
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Fig. 9.GCMS analysis of malathion degradation product, Butanedioic acid [(dimethoxyphosphinothioyl) thio]-, diethyl ester by Azospirillum lipoferum.
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RT: 26.67 - 27.20
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27.1226.68 26.9826.80 26.9326.8626.7826.74
27.09
27.0227.00
26.97 27.15
26.82
NL:3.94E4
TIC MS Blank
NL:1.38E7
TIC MS sampleno-11180
sampleno-11180 #1729 RT: 27.09 AV: 1 SB: 12 27.13-27.20 , 26.70-26.76 NL: 7.78E5T: + c Full ms [30.00-800.00]
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99.21329.73
126.99
93.11142.83
157.67
357.65
79.18 284.07
201.64453.19
421.23254.23 481.06 512.86 611.07 681.06
(mainlib) Butanedioic acid, [(dimethoxyphosphinothioyl)thio]-, diethyl ester
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47 63
73
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O O
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Science Research Reporter 2(1): 94-103, March 2012 ISSN: 2249-2321 (Print)
Fig. 10. GCMS analysis of malathion degradation product, Succinic acid mercapto diethyl ester, s-ester with o,s-dimethyl phosphorodithioate by Azospirillum lipoferum
The tolerance of Azospirillum lipoferum against Malathion was found up to 1000 µg/ml concentration. The estimation of residual Malathion was also calculated on the basis of area occupied in the gas chromatogram obtained under GCMS technique. Here 256 µg/ml Malathion was obtained as residue out of 1000 µg/ml as original concentration. Hence 74.40 % degradation of Malathion was obtained (Fig.1and 2). The TLC
analysis of the culture filtrate from the Nfb broth used for Malathion degradation showed appearance of only one spot of Malathion which indicated that the degradation was not done outside the bacterial cells (Fig.3). The intracellular Malathion degradation products were detected after sonication of the cells as per the chromatogram shown in figure 4. The individual products with different functional groups were as per the figures 5-10.
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42.24
42.4141.87 42.5542.5041.96 42.02 42.13
42.34
42.4941.90
42.07
NL:9.61E4
TIC MS Blank
NL:4.53E6
TIC MS sampleno-11180
sampleno-11180 #2844 RT: 42.34 AV: 1 SB: 33 42.44-42.63 , 41.79-42.06 NL: 3.85E5T: + c Full ms [30.00-800.00]
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172.66
99.00
282.03204.47
237.27
71.06 315.85
367.52441.26
532.98 659.47597.06 760.85
(mainlib) Succinic acid, mercapto-, diethyl ester, S-ester with O,S-dimethyl phosphorodithioate
50 70 90 110 130 150 170 190 210 230 250 270 290 310 3300
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5571
79
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O
O S
O
O
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Total six products were obtained with varied
functional groups as phosphorothioic acid, o,o,s- trimethyl ester , phosphorodithioic acid, o,o,s-trimethyl ester, Butanedioic acid, mercapto- derivative, Succinic acid mercapto diethyl ester, s-
ester with o-s-dimethyl phosphorodithioate, Butanedioic acid [(dimethoxyphosphinothioyl) thio]-,diethyl ester and Succinic acid mercapto diethyl ester, s-ester with o,s-dimethyl phosphorodithioate.
LITERATURE CITED Alexander M, 1969. Microbial degradation and biological effects of pesticides in soil.In: Soil Biol. Reviews of research, UNESCO, Paris, 209-240. Bergey DH and Holt JH, 1994. Bergey’s Manual of Determinative bacteriology Williams and Wilkins, Baltimore, Maryland, USA. Buffin D, Diamand E, McKendry R and Wright L, 2003. Pesticide Action NetworkEurolink Centre, 49 Effra Road, London SW2 1BZ, UK. Dobereiner J, Marriell JE and Nery M, 1976. Ecological distribution of Azospirillum lipoferumBeijerink. Can. J. Microbiol., 22: 1464- 1473. Foster L, John R, Kwan BH and Vancov T, 2006. Microbial degradation of the organophosphate pesticide ethion. FEMS microbiology Letters, 240: 49- 53. Gangawane LV and Saler RS, 1988. A comparative study of tolerance of fungicides by Rhizoctoniabatalicola and other microfungi in the rhizosphere of groundnut. Pesticides, 22: 27- 37. Kiss A and Virag D, 2009. Photostability and photodegradation pathways of distinctive pesticides. J. Environ. Qual., 38: 157-163. Matsumara F, 1974. Microbial degradation of pesticides. Survival in Toxic Environments. (M.A.O. Khan, J.P. Bederka Eds.) Academic Press, New York, 129,154. Mostafa NIY, Bahig MRE, Fakhr IMI and Adam Y, 1972a. Malathion breakdown by soil fungi. Z. Naturforsch. 27 b, 1115- 1116. Vargas JM, 1975. Pesticide degradation. J. Arboriculture, 1(12):232- 233.
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