Studies on Comparative efficacy of Submerged and Solid state fermentation for

production of fungal α-Amylase

Name : Irfan Supervisor : Dr Farhana Maqbool

Salim
CO-Supervisor : Dr Sidra Pervez
Roll No : 45716
External Supervisor :Dr Saima Jabeen

Department of Microbiology, Faculty of Health Sciences

Hazara University Mansehra
2016-2018
 INTRODUCTION

FERMENTATION

 Metabolic process
 Carbohydrates alcohol + carbon dioxide
 Anaerobic process
 Fermentum stands for boiling
 Zymology
 Louis Pasteur first zymologist
 Economic and Environmental advantages
 Development of new machinery and processes.

2
FERMENTATION TECHNIQUES

 Solid State Fermentation (SSF)

 Submerged Fermentation (SmF).
 SOLID STATE FERMENTATION

 Absence or near-absence of free water.

 Employs natural raw materials as carbon source.
 i.e cassava, barley, wheat bran, soybean cake, ground nut oil cake, fruit. pulps,
saw dust, seeds tea waste, spent brewing.

 Alternative method to subermerged fermentation.

a) High volumetric productivity.
b) Relatively high concentration of product.
c) Less effluent generate.
d) Simple fermentation equipment.

4
SUBMERGED FERMENTATION
 Substrate used for fermentation is always in liquid state
 Used for a variety of purposes, mostly in industrial manufacturing.

 Advantageous because of
a) The ease of sterilization.
b) Process control is easier.

6
 ENZYME

FUNGAL ENZYMES

 Xylanse
 Pectinase
 Lactasess
 Celluloses
INDUSTRIAL PRODUCED ENZYMES

Name of Enzymes Source Applications

Diastase Malt Digestive aid, Supplement to
bread, syrup

Takaddiastase A. Oryzae Digestive aid

Rhizopus niveus
Amylo glucosidase Glucose production
A.niger

Confectionaries to prevent
Invertase S.Cervisiae crystallization of sugar,
Chocolate, High test molasses
 AMYLASE

a) Alpha amylase
b) Beta amylase
c) Gamma amylase

ALPHA AMYLASES

• EC no is (E.C.3.2.1.1)
• Hydrolysis of interior alpha-1, 4-glycosidic bond
• Products like glucose and maltose
• Acts quicker then beta amylase
• Isolated from plants, fungi and bacteria
FUNGAL ALPHA AMYLASES
 COMMERCIAL IMPORTANCE

 Starch Industry
 Detergent Industry
 Food industry
 Textile Industry
 Paper industry
 Pharmaceutical Industry

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 OBJECTIVES

 Production of α-amylase
 Utilization of different agricultural biomass
 Optimization of fermentation conditions
 MATERIAL AND METHODS

 Sample Collection
 Isolation of Fungal Strains
 Selection of Maximum α-amylase Producing Strain
 Preparation of Agricultural Biomass as a Feedstock for Microorganisms
 Optimization of Conditions for α- amylase Production
 Enzyme Assay and Total Protein
 RESULTS
FUNGAL STRAINS

 A. Niger, A. flavus, A. fumigatus and A. terrus.

 Aspergillus flavus showed maximum activity of 452.79 µ/ml on enzyme assay.

500
450
400
E.A U/ml/mint

350
300
250
200
150
100
50
0

A.niger A.niger(New A.fumigatus A.terrus A.flavus A.flavus(New

strain) Aspergillus strains strain)
SSF
From the results of SSF substrate of banana give maximum value 1956.85 µ/ml and in SmF
substrate Orange give optimum result 1992.22 µ/ml.
Comparing the maximum values of both techniques submerged fermentation were selected for
optimization parameters.

2500

2000
E.A U/ml/mint

1500

1000

500

0
Apple Banana Potato Maiz orange
SSF ( type of substrate)
SmF

2500

2000
E.A U/ml/mint

1500

1000

500

0
Apple Banana Orange Maize Potato
SmF ( type of substrate)
 OPTIMIZATION OF RESULTS

Time
 Placing flasks at different time for 3 days, 4 days, 5 days and 6 days.
 maximum growth 216.85 µ/ml occur on flask of 4 days

.
250

200
E.A U/ml/mint

150

100

50

3 4 5 6
Days
 pH

 For observing the optimal pH flasks were place on different pH 5.0, 6.0, 7.0 and
8.0 for 4 days.
 Maximum growths on flask were 572.77 µ/ml having pH 7.0 flask

700

600
E.A U/ml/mint

500

400

300

200

100

5 6 7 8
pH
Temperature
 Placing flasks at temperature 25°C, 30°C, 35°C and 40°C for 4 days at pH 7.0
respectively.
 Maximum assay of 169.81 µ/ml obtained on flask having temperature 30°C.

.
180

160

140
E.A U/ml/mint

120

100

80

60

40

20

0
25°C 30°C 35°C 40°C
Temperature
 Concentration of substrate

 Different concentration of substrate 0.5 %, 1.0 %, 1.5 % and 2.0 % were add.
 1.0% giving maximum value 280.18 µ/ml, which means that it is the familiar
value on which high growth obtained
300

250
E.A U/ml/mint

200

150

100

50

0
0.50% 1.00% 1.50% 2.00%
Concentration of Substrate
Type of Nitrogen Source
 different Nitrogen substances used.
 Peptone, Yeast and both combined
 Give maximum value of enzyme assay 169.81 µ/ml when both nitrogen sources
are used in combination instead of individually.

175

170

165
E.A U/ml/mint

160

155

150

145

140

135

130

Peptone Yeast Peptone + Yeast

Type of Nitrogen Sources
Concentration of Nitrogen Source
 Different concentration of nitrogen source 0.5 %, 1.0 %, 1.5 % and 2.0 % were
add.
 2.0% giving maximum value 433.7 µ/ml.

500

450

400
E.A U/ml/mint

350

300

250

200

150

100

50

0.50% 1.00% 1.50% 2.00%

Concentration of Nitrogen Sources
 Mineral Salts

 Different mineral salts K2HPO4.7H20, MgSO4, K2HPO4.7H20 +MgSO4 and

NaCl were used
 MgSO4 giving maximum value 544.25 µ/ml

600

500
E.A U/ml/mint

400

300

200

100

K2HPO4.7H20 +MgSO4 MgSO4 K2HPO4.7H20 NaCl

Mineral Salts