ISOLATION & SCREENING OF METHYL PARATHION DEGRADING ASPERGILLI FROM SOIL

A Project Report

Submitted In Part Fulfilment Of The Requirement

For The Degree Of Masters Of Science

In Applied Microbiology & Biotechnology

By

Akanksha Khare

Department of Applied Microbiology and BiotechnlogyDr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P) 470 003

•Pesticides are Xenobiotics i.e,man made compound with structure that microorganism have never been exposed to. Many of these are recalcitrant i.e remaining unchanged in environment.

• Methyl parathion (O,O-dimethyl, O-p nitrophenol phosphorothioate) is a broad spectrum organophosphorus insecticides has molecular formula of C8H10NO5PS, with molecular mass of 263.23 daltons.

•Pure methyl parathion is white crystalline, solid powder which solubility 55 – 60 mg/ liter at 25˚C in water.

•Soil microbes convert MP into dimethyl thiophosphoric acid and p – nitrophenol (pnp) by hydrolysis.

IntroductionIntroduction

Mechanism of toxicity

•The primary effect associated with high level exposure to OP insecticides is inhibition of acetylcholinesterase (AChE), resulting in acetylcholine accumulation.

•Resulting in excessive nervous stimulation culminating in respiratory failure & death. Hypotension, Bradycardia, Bronchoconstriction & Bronchial fluid accumulation, symptoms that results from the inability of respiratory muscles to work.

• Clinical symptoms of poisoning with MP include pallor, sweating, dizziness, vomiting, diahorrhea, abdominal cramps, headache, blurred vision, convulsion, dialation of pupils, tears, & cardia arrest.

• It is easily absorbed via all routes of exposure (oral, dermal or inhalation) and is easily distributed to the tissues of body.

•p – nitrophenol, the other hydrolysis product of MP is toxic to plant, animals and human. It is also a suspected carcinogen

Mechanism of Toxicity

Biodegradation of MPBiodegradation of MP

•Hydrolysis is the principle mechanism of biodegradation of pesticides. Complete degradation of which occurs when its hydrolysis products enters into krebs cycle.

•Many bacterial species like Pseudomonas, Alkaligenes, Acinetobacter, and Arthrobacter has been reported to detoxify most of pesticides, but little is known about role of fungi for the same.

•White rot fungi like Trametes versicolor, Phanerochaete chrysosporium etc are known for partial degradation of pesticides.

•The whole process is carried out by Methyl parathion hydrolase (MPH).

•MPH belongs to metallolactamase superfamily and has high catalytic activity towards Organophosphates among all organophosphorus hydrolase (OPH)

Mode of action of MPH and further metabolic activitiesMode of Action of MPH & further metabolism of MP

-

•Bioremediation – In biodegradation of organophosphorus pesticides. A company in Australia now sells carrier based OPH enzymes for removal of OP from Sheep-dip water before it can be applied soil.

•Biotehnological – It is succesfully used to develop and evaluate biosensors and Organophosphorus contamination.

Applications

 

To isolate & screen Methyl parathion Hydrolase producing Aspergilli.

To compare the potential of seven different Aspergillus niger towards Methyl Parathion degradation and Methyl parathion Hydrolase production.

To quantify the production of methyl parathion hydrolase, by determining enzyme activity.

Key objectives of the study

Isolation of Fungi

For isolation of fungi, Saboraud Dextrose Agar media was used.

Composition

Dextrose - 40.0 gm Peptone - 10.0 gm Agar – agar - 20.0gm Distilled water - 1000ml

pH - 7.0

• Method used - Direct plate method

Materials & Methods

S.No. Samples Number of fungi Isolate number Fungus Identified

1. Garden soil(Dept. of

microbiology)

2 GSM1 A.niger

GSM2 A.fumigatus

2. Garden soil(Botany

Dept.)

3 GSB1 A.niger

GSB2 A.fumigatus

GSB3 A.terreus

3. Chemistry Dept.(Near

lab. discharge)

3 CLS1 A.flavipes

CLS2 A.niger

CLS3 A.fumigatus

4. Compost soil 2 CS2 A.versicolor

CS3 A.niger

5. Agricultural

soil(Makroniya),sagar

3 ASM1 A.niger

ASM2 A.versicolor

ASM3 A.fumigatus

6. Agricultural

soil(Kanera),Sagar

3 ASK1 A.flavus

ASK2 A.terreus

ASK3 A.fumigatus

7. Agricultural

soil(Pathriya),sagar

4 ASP4 A.niger

ASP1 A.tamari

ASP2 A.glaucus

ASP3 A.melleus

8. Road side soil 2 RS2 A.ustus

RS3 A.niger

Primary screening

Aspergillus versicolor Aspergillus glaucus

Aspergillus niger Aspergillus melleus

Aspergillus ustus Aspergillus terreus

Screenig for MPH Production in Broth.• Media- Czapek’s Dox Broth(With out sucrose)

NaNO3 - 2.0 gm KCl - 0.5 gm

MgSO4.H2O - 0.5 gm FeSO4.7H2O - Trace K2HPO4 - 1 gm Tween 80 - 4 ml

Distilled water - 1000 ml•Vishniac Solution (gm/ltr) – Contain EDTA (10), ZnSo4.7H2O (4.40), CaCl2.2H2O (1.47). It is used particularly to enhance the growth of Aspergilli.

•Concentration of MP(As carbon Source)Concentration of Methyl parathion were prepared in ppm.Four concentrations i.e., 15 ppm(0.15 mg in 100 ml Distilled water) ,10 ppm (0.1 mg in 100 ml distilled water),20ppm(0.20mg in 100ml distilled water) & 30 ppm.

Screeening of MPH in Broth

Aspergillus versicolor Aspergillus niger

Aspergillus terreus

10 ppm

Aspergillus terreus Aspergillus versicolor

Aspergillus niger

15 ppm

-

•50 ml of media were taken in well labelled 150 ml Erlenmeyer flask each for 10 ppm & 15 ppm concentration of Methyl parathion.

•Three disc of each fungal isolate were inoculated in respective flasks.

•One – one control for each concentration was also prepared having only media but no fungal discs.

•Flasks now kept in shaker at 28˚C, 120 rpm for 7 days.

Assay of Methyl Parathion Hydrolase(MPH Activity)

•Release of para - nitrophenol,an indication of hydrolysis of methyl parathion was taken as a criterion for the estimation of production of methyl parathion hydrolase ,which was assayed spectrophotometrically at 405 nm(Absorbance of PNP).

Methodology

•Citrate buffer (0.05 M,pH 5)-1.05 gm of citric acid was dissolved in 100 ml of distilled water. Adjust pH 5 with 0.2 M NaOH.

•Substrate solution-50 mg of methyl parathion were dissolved in 50 ml distilled water.

•Stopping reagent (1.0 M Na2CO3)-Dissolve 10.6 gm of Na2CO3 in 100 ml of deionized water.

•Standard solution (Stock)-0.0695 pnp (0.01 M) taken in 50 ml of volumetric flask & filled up to mark with citrate buffer.

•Preparations of dilutions :– Dilutions Concentration(µmol/ml) Concentration(nkats/ml)•1:20 0.50 0.833•1:50 0.20 0.333•1:100 0.10 0.167•1:200 0.05 0.083

Preparations For MPH Activity

Blank Standards Enzyme Blank Reaction Tube

Add 1.8 ml of substrate solution

Add 1.8 ml of substrate solution

Add 1.8 ml of substate solution

Add 1.8 ml of substrate solution

0.2 ml Buffer 0.2 ml dilutions

Incubate for 60 min. at 50˚C 0.2 ml Cultural

Filterate

Incubate for 60 min. at

50˚C

Incubate for 60 min. at 50˚C 0.2 ml stopping

ReagentIncubate for 60 min. at 50˚C

0.2 ml stopping Reagent

0.2 ml stopping Reagent 0.2 ml stopping

Reagent

0.2 ml Cultural Filterate

Vortex & take Absorbance at 405 nm

Dilutions Concentration(µmol/ml)

Absorbance(405nm)

1:200 0.050.057

1:100 0.10.135

1:50 0.20.246

1:20 0.50.631

Table- Readings for Standard Curve of PNP

0 0.1 0.2 0.3 0.4 0.5 0.60

0.1

0.2

0.3

0.4

0.5

0.6

0.7

f(x) = 1.26266666666667 x − 0.00106666666666688R² = 0.999217665604327

Standard Curve Of para-nitrophenol

Absorbance(405nm)Linear (Ab-sorbance(405nm))

Concentration(µmol/ml)

Ab

sorb

an

ce(4

05

nm

)

ORGANISM ENZYME ACTIVITY

  10PPM 15 PPM

A.flavus NIL NIL

A.flavipes NIL NIL

A.fumigatus O.145 NIL

A.glaucus 0.736 0.503

A.melleus 1.213 0.937

A.nidulans NIL NIL

A.niger 4.021 3.869

A.penicilloides NIL NIL

A.tamari NIL NIL

A.terreus 3.546 3.286

A.ustus 3.676 1.866

A.versicolor 0.296 0.198

A.flavus

A.flavipes

A.fumigatus

A.glaucus

A.melleus

A.nidulans

A.niger

A.penicilloides

A.tamari

A.terreus

A.ustus

A.versicolor

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

FUNGAL ISOLATES

EN

ZYM

E AC

TIVI

TY MPH ACTIVITY OF AS-PERGILLI

•After determining Enzyme activity in culture filtrate of Test Aspergilli, percentage of Methyl Parathion was also calculated.

• The calculation was done by using following formula:

Percentage of Degradation= [1- Absorbance of test sample/Absorbance of control] × 100

Percentage of MP degradation

ORGANISM PERCENTAGE OF MP DEGRADATION(%)

  10PPM 15PPM

A.flavus NIL NIL

A.flavipes NIL NIL

A.fumigatus NIL NIL

A.glaucus 43.5 38.3

A.melleus 66.2 39.4

A.nidulans NIL NIL

A.niger 85.6 67.1

A.penicilloides NIL NIL

A.tamari NIL NIL

A.terreus 75.3 63.6

A.ustus 72 65.7

A.versicolor 43.5 5.84

A.flavus

A.flavipes

A.fumigatus

A.glaucus

A.melleus

A.nidulans

A.niger

A.penicilloides

A.tamari

A.terreus

A.ustus

A.versicolor

0

10

20

30

40

50

60

70

80

90

PERCENTAGE OF MP DEGRADATION

10PPM 15PPM

FUNGAL ISOLATES

PERC

ENTA

GEO

F D

EGRA

DA

TIO

N

•After determining the MPH activity & percentage of MP degradation in different Aspergilli, it was noted that only A.niger gave maximum enzyme activity & percentage of MP degradation in both concentrations.

•Thus it was supposed to be valuable to screen different A.niger isolated from different sources.

Screening of Different A.niger for MPH activity

Source of Isolation Isolate number Enzyme Activity(nkats/ml)

30 ppm 20ppm

Ground nut seed A.N-1 2.083 3.670

Compost soil A.N-2 2.117 4.221

Botanical Garden soil A.N-3 1.733 3.369

Agricultural soil (Kanera) A.N-4 4.912 6.542

Agricultural soil 

(Makroniya) A.N-5 2.467 3.503

Chemistry Deptt. Soil A.N-6 2.413 3.119

Agricultural soil (Pathriya) 

                             A.N-7 3.72O 3.787

A.N-1 A.N-2 A.N-3 A.N-4 A.N-5 A.N-6 A.N-70

1

2

3

4

5

6

7

MPH Activity of different A.nigers

30 ppm20 ppm

Isolate numbers

En

zym

e a

cti

vit

y

•On screening the different Aspergilli, maximum MPH activity showed by A.niger in both 15ppm &10ppm concentration.

•While A.terreus, A.versicolor, A.melleus, A.ustus & A. glaucus, showed less MPH activity in 15 ppm & more activity in 10 ppm. A.fumigatus gave less activity in 10 ppm but no activity in 15 ppm.

•Where as no activity was found in A.flavus, A.flavipes ,A.penicilloides, A.nidulans & A.tamarii.

•From all the above results, Aspergillus niger was found as potent MPH producer even at Higher concentration(20 & 30 ppm) of MP.

Discussion

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