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Biodegradation of Carbofuran pesticide by saline soil actinomycetes.

Introduction

Soil containing excessive soluble salts like sodium as exchangeable base is called saline soil. Saline soils are often recognized by the presence of white encrustations or white fluffy deposits of salts on the surface predominantly of chlorides and sulphates of sodium, calcium and magnesium. Soil affected by salinity is basically fertile and productive but have gone out of cultivation resulting in to decline in microbial activity. Actinomycetes are filamentous bacteria. They are mostly gram positive few are acid-fast; Actinomycetes population in soil is lower than true bacteria but higher than fungi. The population of Actinomycetes ranges from 105 to 108 per gram of soil, Actinomycetes is prominent in the decomposition of pesticides and other recalcitrant compounds. Pesticides are chemicals used for killing the pests. They persist for long time in soils. Major consequences pesticides and their accumulation in body of plants, animals and human. Pesticides are also detrimental to non target organisms. They damage the crops and they are toxic to animals and human. Chemical pesticides are considered as pollutant.

Materials and Methods

Resistance of Actinomycetes to carbofuran

319 actinomycetes isolated from saline soil were inoculated on sterile nutrient agar supplement with 20[micro]g /ml carbofuran. The plates were incubated at ambient temperature for 7 days. Development of colony was considered as resistance to pesticide.

Biodegradation ability of carbofuran resistant actinomycetes

Seven carbofuran resistant Actinomycetes were tested for their carbofuran biodegradation ability. Two sets were prepared.

Set A:Biodegradation by co-metabolism:

50ml of glycerol aspargine broth supplemented with 20[micro]g/ml carbofuran was taken in 150ml conical flask.

Set B: Biodegradation by using carbofuran as a sole carbon source:

50ml of minimal broth supplemented with 20[micro]g/ml carbofuran was taken in 150ml conical flasks. Both flasks were sterilized at 121xc for 30min and were inoculated by carbofuran resistant actinomycetes. The flasks were incubated at room temperature for 10 days. After 10 days the medium was filtered. The filterate was treated with equal quantity of chloroform layer. Chloroform layer was separated and subjected for gas chromatography. Residual carbofuran concentration was determined with the help of gas chromatography manual.

Identification of carbofuran resistant Actinomycetes

Identification of Actinomycetes was carried out as per Bergey's manual of Systematic Bacterology volume 4:William et al (1984).

Following criteria were used for the identification of Actinomycetal isolates.

* Colony character

* Morphological characters

* Chemical composition of cellwall

* Whole cell sugar composition

* Biochemical characters

Colony characters

In order to study colonial characteristics, the isolates were inoculated on glycerol asparagine agar and the plates were incubated for 7 days at ambient temperature. A colonial character of the well isolated colony was then recorded.

Morphological characters

In order study the morphological characteristics with reference to aerial mycelium, substrate mycelium and sporulation, the isolated colonies were cultivated on glycerol asparagines agar by simple "Inclined cover slip culture technique" (Gibbs & shapton, 1968) as detailed below.

A sterilized cover slip was carefully inserted at an angle of about 45- into a solidified medium in a petridish so that half of the coverslip was in the medium.

An inoculum from a slope culture was then spread along the line, where the upper surface of the coverslip meets the agar, with a fine wire needle and plates were incubated at ambient temperature for 7 days. The microorganism grows on both, the medium and along the coverslip. Then coverslip was carefully withdrawn from the medium and observed directly under microscope to differentiate between substrate and aerial mycelium.

Following observations were also recorded,

* Presence or absence of aerial mycelium.

* Fragmentation of substrate mycelium.

* Presence of sclerotia.

* Sporulation on substrate mycelium

* Spore chain morphology whether: rectiflexibiles, retinaculiaperti, or spirales.

* Colour of spore mass: Blue, grey, red, violet, white or yellow.

In case of Nocardia species, the growth by coverslip culture technique was studied at 24hrs interval for the fragmentation of the mycelium.

Cell wall analysis

Preparation of biomass

The isolates were cultivated in bulk by shake culture method using glycerol asparagine broth. The biomass was obtained and then washed three times with distilled water, dried at 50 to 55xc for 12 hrs and used for determination of aminoacids (Becker et al. 1964, 1965 & Yamaguchi 1965) and whole cell sugar pattern (Lechevalier,1968) as detailed below.

Amino acid pattern

10mg aliquot of dried cell mass was hydrolyzed with 1.5ml of 2N H2SO4 in a sealed Pyrex tubes held at 100xc for 18 hrs. The tube was then cooled and broken to collect the sample which was then filtered through wattman filter paper No. 1. The filtrate was collected, enough distilled water was added and dried twice at 50xc. The extract was collected in enough distilled water and stored 4xc. This sample was then analyzed for determination of amino acids by paper chromatographic technique by using solvent system as butanol: Acetic acid : Water (25:6:25).

Whole cell sugar pattern

10mg aliquot of dried cell mass was hydrolyzed with 1ml of 2 NH2SO4 in a sealed pyrex tube at 100xc for 2hrs. The tube was then cooled and broken to collect the sample. The hydrolyzed material was treated with saturated solution of barium hydroxyzed with methyl red as an internal indicator till the pH become 5.5. The precipitate of barium sulphate thus produced was separated by centrifugation at 10,000 rpm. The supernatant was collected and filtered. Enough distilled water was added to filtrate and dried at 50xc. Extract was collected in little distilled water and stored at 40xc. Then this sample was analyzed for determination of sugar pattern by paper chromatography by using solvent system as a butanol: acetic acid: water (4:1:5).

Biochemical studies:

In order to study the isolates for activities given below, different methods were used. Following bio chemicals were carried out for the identification of isolates.

* Enzyme activity: Amylase, Protease, (Caseinase, Gelatinase), lipase, Lecithinase, & Tyrosinase

* Utilization of carbon substrate: D-glucose, D-xylose, L-rhamnose, Dgalactose, D-arabinose, lactose, cellulose, sucrose, D-mannitol and sodium citrate.

* Utilization of nitrogen sources: Potassium nitrate and L-phenyl alanine, L-aspargine.

Results and Discussion

All 319 Actinomycetes isolates obtained from saline soils of Sangli District (Maharashtra) were tested for the resistance to 20[micro]g ml 1 concentration of carbofuran. It was found that out of 319 Actinomycetes isolates, only seven actinomycetal isolates were able to resist the pesticide and showed growth.

Actinomycete species showing resistance to carbofuran were identified up to species level. They were Streptomyces alanosinicus, Streptoverticillium album, Nocardia farcinia, Streptomyces atratus, Nocardia vaccini, Nocardia amarae and Micromonospora chalcea. Biochemical

These seven actinomycete isolates were further tested for the degradation of carbofuran (20[micro]g ml 1) by co-metabolism and carbofuran as the sole carbon source. The results are presented in table

It was found that Streptomyces alanosinicus, Streptomyces atratus, Streptoverticillium album, Nocardia farcinia, Nocardia vaccine, Nocardia amarae & Micromonospora chalcca were able to degrade 65.55. 64.85. 34.8, 28.1, 25.2, 12.25 & 6.7% of carbofuran as the sole carbon source respectively and 95.35, 95.1, 83.75, 83.7, 80.2, 79.55, and 69.85% of carbofuran as the sole carbon source respectively.

Thus it seems that actinomycetes isolates obtained from salt affected soil can degrade carbofuran.

It is seen from the table that S. alanosinocus among all is most active in biodegradation of carbofuran. This isolate was also found to show good growth at 12 % salt concentration. This salt tolerant actinomycete isolate can be exploited further for pesticide degradation in saline soil. Soudamini et al. (1996) identified two cultures Pseudomonas stutzeri and Bacillus pumilis, which enhanced carbofuran degradation, resulting in more than 98% loss of the applied carbofuran in 30 days. Other cultures enhanced the degradation up to 70% within the same period. Venkateshwarlu et al (1985) isolated Pseudomonas cepacia and Nocardia sp., from flooded alluvial soil amended with carbofuran. These cultures metabolized ring-14c-carbofuran rapidly in mineral salts medium or soil extract, supplemented with yeast extract. When the two isolates were incubated with soil extract together with 500[micro]g ml-1 yeast extract, about 40 to 62 % of the radioactivity was recovered as water-soluble products. The degradation of carbofuran by Nocardia species led to the formation of coloured, water-soluble and non-extractable metabolites. Williams et al (1976) found that soil microorganisms involve in degradation of carbofuran. Venkateshwarlu (1984) studied degradation of carbofuran carried out by Azospirillium lipoferum and Streptomyces species. Isolated from flooded alluvial soil. Chaudhry et al (1988), Del Carmen et al (1989), Rule et al, (1994), wang et al, (2005), Zhang et al, (2005) have also studied carbofuran degradation by microorganisms and have reported that carbofuran is degraded by various microorganisms in soil.

References

[1] Becker, B., Lechevalier, M.P. and Lechevalier, H.A.1965. Chemical composition of cell-wall preparations from strains of various form genera of aerobic actinomycetes .Appl Microbiol.13; 236-243.

[2] Choudhary, G.R. 1988. Bacterial metabolism of carbofuran. Appl. Environ microbial. 54 (6): 1414-19

[3] Del Carmen, Quintero, M., silva, M. and Perez-Bendito.D.1989.Analysis of carbofuran residues in salt by the stopped flow technique pub Med, 14 (4); 497-500.

[4] Goel, P.K. 2006. Water pollution-causes, effect and control, New Age Interntional Publishers Mumbai

[5] Gibbs, B.M, and Shapton, D.A. 1968. Identification Methods for microbiologist. Part B. Academic Press, New York.

[6] Lechevalier, M.P. 1968. Identification of aerobic actinomycetes of clinical importance, j. Lab, clin.Med. 71; 934-944.

[7] Rule, G.S., mordehai, A.V. and Henion, J. 1994. Determination of carbofuran by on-line immunoaffinity chromatography, Pub, Med.66 (2); 230-235,

[8] Goudamini, Mohapatra and Awasthi, M, D.1996.Enhancement of carbofuran Degradation by soil Enrichment Cultures and by Synergistic Interaction among Bacterial cultures 49 (2); 164-168

[9] Shirling, E.B. and Gottlieb, D. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16; 313-340.

J. Jayabarath *, S. Asma Musfira, R. Giridhar, S. Shyam sundar and R. Arulmurugan

Department of Biotechnology, Pavendar Bharathidasan College of Engineering and Technology, Tiruchirappalli (Affiliated to Anna University-Chennai) Corresponding Author Mail ID-barath_bio@yahoo.co.in
Characters of these isolates are given in table 1.

s.n Actinomycetal F L G S X M A C L L U
o isolate r a l u y a r e e i r

1. Streptomyces - + + V + + + + - + +
 alanosinicus
2. S.atratus + + + + - - + - + +
3. Streptoverticillium - - + - + + + + - + +
 album
4. Nocardia farcinia + _ + + - - + + + + +
5. N.vaccinia + + - - - + - + - +
6. N.amarae + + + - + - + - - -
7. Micromonospora - + + + + - - + + - +
 chalcea

s.n A L G N H2S Cat Cell Wall
o m a e i Amino Sugar
 acid

1. + + + + + + G N.C

2. + + + + + + LL N.C
3. + G N.C

4. + - + + + + M Gal
5. + - - + + - M Ara
6. + + + + + - M Ara
7. + + + - + + M Ara

F:Fragmentation, L:Lactose, G:glucose, S:sucrose, X:xylose,
M:mannitol, A:arginine, C:cellulose, L:lecithinase,P:lipase,
U:urease, A:amylase, C:caseinase, G:gelatinase, N:nitrate,
Cat:catalase, NC:no characteristic sugar pattern, Gal:galactose,
Ara:arabinose, M:mosoDap, G:glycine, LL:LLDAP
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Author:Jayabarath, J.; Musfira, S. Asma; Giridhar, R.; sundar, S. Shyam; Arulmurugan, R.
Publication:International Journal of Biotechnology & Biochemistry
Date:May 1, 2010
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