biodegradation of tannery effluentusing fungi trichoderma harzianum

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BIODEGRADATION OF TANNERY EFFLUENT USING FUNGI Trichoderma harzianum Project by MELLOW PAULSON (40906214010) FEENA GEORGE (40906214008) Supervisor : Mr.R.Magesh,M.tech., 1

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Page 1: BIODEGRADATION OF TANNERY EFFLUENTUSING FUNGI Trichoderma harzianum

BIODEGRADATION OF TANNERY EFFLUENT

USING FUNGI Trichoderma harzianum

Project by

MELLOW PAULSON (40906214010)

FEENA GEORGE (40906214008)

Supervisor : Mr.R.Magesh,M.tech.,

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Page 2: BIODEGRADATION OF TANNERY EFFLUENTUSING FUNGI Trichoderma harzianum

INTRODUCTION

Higher chromium level in tannery effluent adversely affect seed germination.

Treatment of tannery effluent using ion exchange, adsorption on to activated carbon are excessively energy consuming.

Selective fungi which are efficient for degradation of pollutants can be isolated from tannery effluent itself.

Treating tannery effluent by using fungi is an efficient biodegradation method.

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INTRODUCTION

Recently tannery effluent contributes one of the major industrial pollution problem.

Chemical precipitation methods for effluent treatment are expensive and will produce solid waste.

Biodegradation using effective microorganisms are economical and easy to use.

Tannery effluent contains some harmful toxic dyes. Higher level of toxic components in the effluent

including chromium, aluminium and dissolved salts are lethal to flora and fauna in the environment.

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Page 4: BIODEGRADATION OF TANNERY EFFLUENTUSING FUNGI Trichoderma harzianum

OBJECTIVES

TANNERY EFFLUENT

1.TO BIODEGRADE USING SELECTIVE

FUNGI FROM TANNERY EFFLUENT

2.TO STUDY THE EEFECT ON

GERMINATION OF

Vigna radiata

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Page 5: BIODEGRADATION OF TANNERY EFFLUENTUSING FUNGI Trichoderma harzianum

MATERIALS REQUIRED

Tannery effluent Vigna radiata seeds Potato dextrose agar Potato dextrose broth Cyclomixer Centrifuge Petriplates UV- spectrophotometer Gas chromatography flame

photometric detector pH meter

Dissolved oxygen probe Electrical conductivity

meter Light microscope Hand lensMass culture of

Trichoderma harzianum Glass slide Lactophenol with cotton

blue 1% streptomycin

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Overview of the study- Biodegradation of tannery effluent using fungi

Isolation of fungi from tannery effluent

Quantification of fungi at different concentrations

Identification of fungi

Massculture of selective fungi

Inoculation of mass cultured fungi

Incubation of inoculated fungi at different concentration of of effluent for 2-4 days

Estimation of physico-chemical parameters including heavy metals after incubation

Analysis of sample in gas chromatography

Page 7: BIODEGRADATION OF TANNERY EFFLUENTUSING FUNGI Trichoderma harzianum

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Overview of the study-Effect of tannery effluent on seed germination

Collection of seeds of Vigna radiata

Incubation of seeds in different concentrations of

tannery effluent

Seeds were grown in distilled water which served as control plate

Average shoot length of seeds were noted at different time

interval

Relative toxicity on germinating seeds were analysed in different

concentrations of both treated and untreated tannery effluent

Page 8: BIODEGRADATION OF TANNERY EFFLUENTUSING FUNGI Trichoderma harzianum

METHODOLOGY

1. Isolation of fungi from tannery effluent

2. Quantification of fungi in different concentrations of tannery effluent

3. Identification of fungi

4. Mass culture of selective fungi Trichoderma harzianum

5. Inoculation and incubation of selective fungi in to different effluent concentrations

6. Determination of physico-chemical parameters

7. Analysis of biodegradation by gas chromatography flame photometric detector(GC-FPD).

8. Estimation of decolourisation percentage

9. Collection of seeds of Vigna radiata and incubation of ten seeds in different concentrations of

tannery effluent

10. Relative toxicity on germinating seeds were analysed in different concentrations by measuring

average shoot length at different time intervals using both treated and untreated tannery

effluent 8

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1.ISOLATION OF FUNGI FROM TANNERY EFFLUENT

• 1mlof effluent was serially diluted on PDA media

• These plates were incubated for 48-96 hours at room temperature

• After incubation period the colony forming units were counted by using the following formula

• C.F.U = Number of colonies *dilution factor / volume plated in ml

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2.QUANTIFICATION OF FUNGI IN DIFFERENT CONCENTRATION OF TANNERY EFFLUENT

• Different concentrations of tannery effluent were prepared .

• All concentrations of the effluent were plated on potato dextrose agar media prepared on sterilized petriplates including control plate.

• The plates were incubated for 48-96 hours at room temperature.

• Cultured fungi was identified.

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3.IDENTIFICATION OF FUNGI

• Identification of culturable molds was done usingtheir macroscopic and microscopic appearances.

• Morphological characteristics of the culture basedon colours, shape, pigmentation, reversepigmentation were studied by using the hand lens.

• Microscopic characters were observed under lightmicroscope by preparing the slides usinglactophenol with cotton blue .

• The characters of conidia bearing structure, shape,septation, colour and were observed.

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4.MASS CULTURE OF SELECTIVE FUNGI

• Fungal culture grown on the petriplates on potato dextrose agar were identified and selective fungi from it was mass cultured.

• Mass culture was done in potato dextrose broth media in small conical flasks.

• 1% sterptomycin was added to the broth medium to prevent bacterial contamination

• Trichoderma harzianum was selectively chosen for mass culturing

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5.INOCULATION OF MASS CULTURED FUNGI IN TANNERY EFFLUENT

• Five gram of Trichoderma harzianum which was mass cultured as selective fungi was inoculated into different concentrations of tannery effluent.

• Fungal culture was incubated in tannery effluent of different concentrations for 48-96 hours at room temperature.

• After incubation period some of the physico-chemical parameters were checked.

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6.PHYSICO- CHEMICAL CHARACTERISATION OF TANNERY EFFLUENT

• Different parameters tannery effluent was tested and tabulated

• The parameters including pH , dissolved oxygen content, conductivity, BOD, optical density were checked.

• Dissolved oxygen content was calculate by D.O probe.

• Conductivity was tested by electrical conductivity meter.

• Optical density was checked in UV Spectrophotometer 14

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7.ANALYSIS OF BIODEGRADATION BY GC-FPD

• Treated and untreated effluent sample was analysed in gas chromatography flame photometric detector.

• 0.5µl of different concentrations of tannery effluent sample was injected to gas chromatography flame photometric detector.

• The amount of toxic pollutants including chromium and sulphidecan be detected and measured from the chromatogram.

• The amount of analyte can be explained by single point extentionmethod.

• Response factor for standard run of chromium and sulphide is calculated as,

• Response factor = peak area / sample amountAmount of analyte is calculated by the following formula

• Amount of analyte = Peak area / Response factor

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8.ESTIMATION OF DECOLOURISATION PERCENTAGE

• 20 ml of the sample was mixed well using cyclomixer for at 1000 rpm. • The supernatant was collected and the optical density value was noted.• 5 gram of Trichoderma harzianum was added to two litre of sample.• After four hours incubation 20ml of the sample was mixed using

cyclomixer for 10 minutes.• After proper mixing of the solution was centrifuged for 2 minutes at

1000rpm.• The supernatant was collected and optical density was noted in uv-

spectrophotometer.• This process was done at regular intervals of time (4 hours) • Decolourisation percentage was calculated using the formula,

Decolourisation % = initial O.D – final O.D/initial O.D *100

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9.COLLECTION AND INCUBATION OF Vignaradiata IN TANNERY EFFLUENT

• Seeds of Vigna radiata were collected and washed with running water to remove contamination of seed coat, prior to germination studies.

• Ten seeds were used in all petriplates keeping one petriplate with distilled water served as control.

• The shoot length were noted at different time interval and the average length were calculated.

• The relative toxicity of both untreated and treated effluent `concentrations on the germinating seeds was calculated .

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10.RELATIVE TOXICITY ASSSESMENT ON GERMINATING SEEDS USING TANNERY EFFFLUENT

• The relative toxicity was calculated after each 24 hours of incubation

• The relative toxicity percentage was calculated using the formula,

R.T.% = (X-Y) / X *100

X is germination percentage or seedling length in control at

particular hour of incubation.

Y is germination percentage or seedling length in the presence

of the tannery effluent at the same hour of incubation.

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RESULTSISOLATION OF FUNGI FROM TANNERY EFFLUENT

SI NO. Concentration No. of colonies C.F.U/ml

1 10-2 400 40*10^4

2 10-3 200 20*10^5

3 10-4 100 10*10^6

4 10-5 80 80*10^6

5 10-6 45 45*10^7

6 10-7 8 8*10^8

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QUANTIFICATION AND IDENTIFICATION OF FUNGI

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Different fungi including Trichoderma viridea, Trichoderma

harzianum and Paecilomyces variotii were obtained

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MASS CULTURED Trichoderma harzianum

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DETERMINATION OF PHYSICO-CHEMICALCHARACTERISTICS

CONCENTRATIO

N OF THE

EFFLUENT

25% 50% 75% 100% UNTREATED

pH 7.14 7.18 7.21 7.27 7.61

CONDUCTIVITY

(µs per cm) 4.4 10.2 14.3 16.8 17.3

DISSOLVED

OXYGEN (mg/lit) 2.08 1.96 1.72 1.23 0.98

BOD (mg/lit)22 28 32 34 35.67

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ESTIMATION OF DECOLOURISATION PERCENTAGE

SI NO Time in hours O.D at 540nmPercentage of

Decolourisation

1 0 .126 0

2 4 .112 11.11%

3 8 .091 27.77%

4 12 .080 36.50%

5 16 .067 46.82%

6 20 .052 58.73%

7 24 .043 65.87%

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EFFECT OF UNTREATED EFFLUENT ON GERMINATING SEEDS OF

Vigna radiata(shoot length in cm)

Concentration of effluent

24hrs 48hrs 96hrs

25% 1 cm 2 cm 2.5 cm

50% 0.5 cm 1 cm 1.5 cm

75% nil 0.5 cm 0.5 cm

100% nil nil 0.5 cm

Control 1 cm 2.5 cm 3.2 cm

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RELATIVE TOXICITY OF UNTREATED TANNERY EFFLUENT WITH TIME

CONCENTRATION

OF THE EFFLUENT 24 HOURS 48 HOURS 96 HOURS

25% 0 20 22

50% 50 60 53

75% 100 80 84

100% 100 100 84

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EFFECT OF TREATED EFFLUENT ONGERMINATING SEEDS OF

Vigna radiata (shoot length in cm)

Concentration of effluent

24hrs 48hrs 96hrs

25% 1.5 2.5 3

50% 1 1.5 2

75% 0.5 1 1.50

100% 0.25 0.5 0.5

Control 1 2.5 3.2

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RELATIVE TOXICITY OF TREATED TANNERY EFFLUENT WITH TIME

CONCENTRATION OF

THE EFFLUENT 24 HOURS 48 HOURS 96 HOURS

25% 33 0 25

50% 66 40 50

75% 66 60 62

100% 83 80 87

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CHROMATOGRAM OF UNTREATED TANNERY EFFLUENT

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CHROMATOGRAM OF 25% TANNERY EFFLUENT

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BIODEGRADATION CURVE OF SULPHIDE

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BIODEGRADATION CURVE OF CHROMIUM

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DISCUSSION Present study focuses on the isolation, quantitation and

incubation of selective fungi in tannery effluent for the effluent treatment

Trichoderma harzianum was the selective fungi inoculated in to the tannery effluent for biodegradation.

The decolourisation percentage of tannery effluent using Trichoderma harzianum was found to be effective.

The relative toxicity of both untreated and treated tannery effluent were tested on germinating seeds of Vigna radiata.

The biodegradation level was estimated by gas chromatography flame photometric detector.

Chemical methods of degrading effluents existed were costly and energry consuming than biodegradation using fungi

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CONCLUSION

Mass cultured fungi Trichoderma harzianumdegraded the toxic components of tannery effluent including chromium and sulphur .

The effect of tannery effluent on germinating seeds was studied and it was proved that the relative toxicity of treated tannery effluent was lower as compared to untreated effluent.

Thus biodegradation using fungi Trichodermaharzianum was proved to be eco- friendly , cheaper and effective method.

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THANK YOU

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