Supervised By Dr.

Manisha Patnaik
 CONTENTS

• INTRODUCTION
• OBJECTIVES OF STUDY
• METHODOLOGY
• RESULTS
• CONCLUSION
 INTRODUCTION

• Cellulose is a linear polysaccharide which is constructed from monomers of glucose bound together with β-1,4 glucosidal
linkage.

• Cellulose hydrolysis is the key process for biological conversion of cellulosic materials. Cellulase is the enzyme that hydrolyzes
the β-1,4-glycosidic bonds in the polymer to release glucose units.
• Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, protozoans, and catalyzes the cellulolytic (hydrolysis)
of cellulose. Cellulose is commonly degraded by the enzyme cellulase.
• Cellulases are a group of enzymes that catalyse the degradation of cellulose, a polysaccharide built of β-1,4 linked glucose units.
This group consists of endo-1,4-glucanase (EC 3.2.1.4), exo-1,4-glucanase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21).
• Microbial cellulases have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel
production, food and feed industry, brewing, and agriculture.
 OBJECTIVES OF STUDY

The present work is carried with the following objectives.

• Isolation of cellulose degrading bacteria from over ripe Malus domestica (apple).
• Characterization of cellulose degrading bacteria isolated.
• Analysis and optimization of cellulase degrading activity of cellulases .
 METHODOLOGY
1. Fruit sample collection

•Over ripe apples were collected from a fruit shop of Saileshree vihar fruit market, Bhubaneswar.

2. Isolation of cellulose degrading bacteria

•To isolate cellulose degrading bacteria samples were taken from overripe parts, added to autoclaved distilled
water and serially diluted to 10 times spread on carboxy methyl cellulose(CMC) agar medium containing :(g/l):
NH2H2PO4- 1g , KCL- 0.2g, , mgcl2.7H20- 1g, yeast extract -1g, carboxymethylcellulose- 26g, agar- 3g; pH
adjusted to 7.0). The plates were incubated at 37o C for 24 hours.
•Bacteria from the resultant plates were individually sub cultured on CMC media for 24 hours.
•To visualize the hydrolysis zones, the plates were flooded with 0.1% Congo red solution and washed with 1M
NaCl.
•Cellulase-producing activities of the isolates were estimated by carboxymethyl cellulose hydrolysis capacity (HC
value) on the cellulose Congo red agar, i.e. Ratio of diameter of clearing zone and colony following the method
and those with high HC values were selected and sub cultured on nutrient agar plates and slants and stored at 4 oc
for the further study.
Phenotypic and biochemical characterisation and identification of bacteria
•The bacteria were analysed for different morphological and biochemical parameters using different tests
like Gram stain, Oxidase test, catalase test, urease test, indole test, MR and VP test etc.
•Then 16S rRNA was amplified and sequenced.
•Using the morphological, biochemical and sequencing results, the bacteria were identified.
Cellulase activity analysis
•The bacteria were subcultured on the CMC broth medium and incubated at 37 °C for 24 h.
•The supernatant of the culture medium were taken out by centrifugation at 10,000 rpm for 10 minutes at
4°C.
•This was used for assay of cellulase activity using dinitro salicylic (DNS) method ( Ghose, 1987).
•Filter paper was used as substrate and glucose was used as standard.
•FPU units were calculated using the formula

•Further optimization of the enzymatic activity was carried out. The optimum temperature, pH, inoculum
size and rotation velocity were found out.
 RESULTS
Cellulose degrading bacteria were isolated from over ripen apple using CMC agar at 37°C of incubation and flooded with
Congo red solution. After Congo red flooding and observation of zones, two bacterial were selected and named A1 and
A2.
Sampl Zone diameter Colony diameter HC value
e
A1 4 cm 2.4 cm 1.66

A2 3.5 cm 1.5 cm 2.33

The HC values for A1 and A2 were found to be 1.66 and 2.33 respectively.
Table-1 HC values of a) A1 and b) A2
a.Gram staining result of A1(+) & A2(+) b. citrate utilizing test result of A1(+) & A2 (-) c. Voges-Proskauer (acetoin production) test
of A1(-) and A2(-) d. MR test result of A1(+) and A2 (+) e. urease test of A1 and A2 f. Indole test of A1(-) and A2(-) g. Nitrate
reduction test A1(+) and A2(+) h. triple sugar test of A1(K/A) and A2(K/A) i. Lipase test of A1(-) and A2(-) j. chitinase test of A1(-)
and A2(-) k. Amylase test of A1(+) and A2(+) l. Gelatinase test of A1 & A2 m. Casein hydrolysis (proteinase)A1(+) A2(-)
 Results of Biochemical tests for A1 and A2

Sample A1 A2
White raised glossy smooth White raised glossy smooth
Morphology
colonies colonies
Gram staining test + +

Oxidase test + +

Catalase test + +

Urease test + +

Indole Production test - -

MR test + +

VP test - -

Nitrate reduction test + +

Citrate utilization Test - -

Carbohydrate utilization test Glucose Glucose

Caseinase assay + +

Chitinase assay - -

Amylase assay + +

Lipase assay - -
 A1 A2 A1 A2

Isolated DNA Amplified fragment of 16S rRNA ( around 1.5kb)

From the morphological, biochemical and sequencing data , the bacteria were identified to be two species of Bacillus
5.5. Cellulase activity analysis
A1 1.26
For analysis of cellulase production and activity, first units/ml
glucose standard curve was constructed.
A2 2.32
Units/ml

OD

Glucose concentation

Using the standard graph, glucose released for

each sample was calculated, using the amount
of glucose released and the enzyme
concentration, filter paper units were obtained.
 5.6. Optimization of parameters for cellulase production and activity.

Graph representing the effect of temperature on cellulase activity Graph representing the effect of pH on cellulase activity

Graph representing the effect of inoculum size on cellulase activity Graph representing agitation rate on cellulase activity
 Conclusion

 Two cellulolytic bacteria were isolated from Malus domestica.

 Upon Congo red flooding, their HC zones were found to be 1.66 and 2.33, respectively.
 Form, morphological, biochemical and sequencing analysis, the bacteria were identified to be two species of
Bacillus.
 The cellulase activities of the bacteria were found to be 1.26 and 2.32 respectively.
 The optimum parameters for cellulose production and activity were found to be 35°C, pH7, 100μl/50ml inoculum
size, 125rpm agitation rate for A1 and 100rpm agitation rate for A2..
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