The document describes the isolation and screening of cellulolytic bacteria from different environmental sources and optimization of cellulase production. 34 bacterial isolates were obtained, with isolates CDB27 and CDB30 showing the highest cellulase activity. CDB27 and CDB30 were identified as Pseudomonas sp. and Bacillus sp. respectively based on morphological and biochemical characterization. Specific enzyme activity of the crude samples of CDB27 and CDB30 were 6.0 U/mg and 8.4 U/mg respectively. Parameters like temperature, pH, incubation period and substrate concentration were optimized to improve cellulase production.

1. Isolation and Screening of Cellulolytic
Bacteria Inhabiting Different Environment
and Optimization of Cellulase Production
Guided by : Prof.Pachorkar ma’am,
Dept. Microbiology,
KTHM College, Nashik
Submitted By
Chaphekar Mrunal Sharad
MSc – 1
Dept – Microbiology
KTHM College Nashik
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2. Abstract
 Isolation Of Bacteria
 Primary Screening Technique Obtained 34 Isolates
 Maximum Cellulase Activity Shown By 11 Isolates
 Secondary Screening For Enzyme Production.
 Most Efficient Enzyme Producers Cdb27 And Cdb30
 Specific Enzyme Activity In The Crude Sample 6.0U/Mg And 8.4 U/Mg Respectively.
 Partially Purified Sample O Be 6.97 U/Mg And 9.3 U/Mg Respectively
Characterization Of Isolates :
1.Cultural
2.Morphological
3.Biochemical
 Pseudomonas Sp = CDB27
 Bacillus Sp = CDB30
 Partial Purification Of The Cellulase Enzyme Ammonium Sulfate Precipitation
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3. Introduction
A. Value Of Cellulose - Renewable Source Of Energy
B. Cellulose Hydrolysis -Intense Research And Industrial Interest .
C. Primary Product Of Photosynthesis In Terrestrial Environments,
D. The Most Abundant Renewable Bioresource Produced In The Biosphere
 (100 Billion Dry Tons/Year) Approx.
 70% Of Plant Biomass Is Locked Up In 5- And 6-carbon Sugars
 Found In Lignocellulosic Biomass -Cellulose, Lesser Hemicelluloses And Lignin.
 Cellulose The Largest Component Of Plant Residues -Terrestrial Ecosystems And --Huge Source Of Energy For
Microorganisms, Indirectly Responsible For Soil Organic Matter Decomposition .
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4. SAMPLES AND THEIR SITES
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5.  The cellulosome was first discovered in 1983 from the anaerobic, thermophilic spore-
forming Clostridium thermocellum.
 The production of cellulase depends on growth parameters

 metal ions - activators and inhibitors
1.Inoculum size,
2.pH value,
3.Temperature
4. Presence of inducers,
5.Medium additives
6.Aeration
7. Growth
8.Time
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6.  Applications Of Cellulases
 Textile Industry
 A.Biopolishing’ Of Fabrics
 B.Stonewashed Look Of Denims
 C.In Household Laundry Detergents For Improving Fabric Softness And Brightness
 In Food, Leather, Paper/Pulp Industries
 In The Fermentation Of Biomass For The Boifuel Production.
 Cellulases In Ruminant Nutrition For Improving Digestibility,
 In Fruit Juices Processing And Another Emerging Application Is De-inking Of Paper
 Erthwormgut -Responsible For Decomposition Of Organic Matter And Composting
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7. OBJECTIVES
 ISOLATION AND CHARACTERIZATION OF CELLULASE PRODUCING BACTERIA IS
IMPORTANT ASPECT OF BIOFUEL RESEARCH, BIODEGRADATION AND
BIOREMEDIATION.
 OBJECTIVES OF STUDY:
 TO ISOLATE AND SCREEN CELLULOLYTIC BACTERIA FROM DIFFERENT
ENVIRONMENTAL SOURCES.
 PARTIAL PURIFICATION OF CELLULASE AND DETERMINATION OF ITS ENZYME
ACTIVITY AND SPECIFIC ACTIVITY.
 OPTIMIZATION OF DIFFERENT PARAMETERS FOR BETTER CULTIVATION AND
PRODUCTION PROCESS.
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8. Method
1. Sample Collection, Isolation And Primary Screening For Cellulase Producing Bacteria:
2. Collected Sample - In Sterile Container And Stored At 4ºc Until Used.
3. Tenfold Serial Dilutions Of Each Soil Sample Were Prepared In Sterilized Distilled Water
4. 0.1 Ml Of That Diluted Sample Was Spread On CMC Medium
5. Composition Of CMC (G/L)
Carboxymethylcellulose (CMC), Tryptone, KH2PO4, Na2hpo4, Mgso4.7H2O, Cacl2 .2H2O, Feso4.7H2O, Agar,
 Incubation At 370 C For 3-5 Days.
 Cellulolytic Activity Of Isolated Strain.
 Clear Zone Of Hydrolysis -Cellulose Degradation.
 Selection For The Highest Cellulase Producer
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9.  Maintananceof Pure Culture:
 The Colonies Significant Clear Zone - Plated On Minimal Agar Medium
 Analysis For Colony Characteristics
 Subcultured On To The Minimal Medium Containing 1% CMC
 And Incubated At 37˚for 24h And Then Stored At 4˚C
 Secondary Screening And Production Of Cellulase Enzyme:
 The Potential Isolates Were Then Evaluated For Enzyme Productivity.
 Those Isolates Showing Maximum Cellulase Production Were Then Considered For The Further Study.
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10.  Preparation Of Crude Enzyme:
 After Incubation, The Cultures Were Centrifuged At 1600 RPM For 20 Min At 4°C
 Supernatant - As A Source Of Crude Enzyme.
 The Crude Enzyme Solution Was Utilized For Determination Of Enzyme Activities.
 Cellulase Enzyme Assay:
1. Carboxymethylcellulase( Cmcase) Activity Was Estimated Using A 1 % Solution Of Carboxymethlycellulose
(CMC) In 0.05 M Citrate Buffer (Ph 4.8) As Substrate.
2. The Reaction Mixture Contained 1 Ml Citrate Buffer, 0.5 Ml Of Substrate Solution And 1ml Of Crude Enzyme
Solution. The Reaction Was Carried Out At 45°C For 30 Min.
3. The Amount Of Reducing Sugar Released In The Hydrolysis Was Measured By Dnsa Method.
4. The Enzyme Unit (Eu) Was Determine As The Amount Of Cmcase Required To Release 1μmole Of Reducing
Sugar Per Ml Per Minute Under Above Assay Condition.
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11.  2.7 Identification of cellulase producing bacteria:
1. morphological,
2. cultural
3. biochemical characterization.
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12. OPTIMIZATION OF CELLULASE
PRODUCTION
 Effect of temperature:
 Effect of pH
 Different values of pH range from 5-8 were chosen for studying their effect on
cellulase enzyme production
 Incubation period :
 Effect of substrate concentration
 Inoculum size
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13. Optimization of cellulase production: The optimum parameters were determined for cellulase production
from the efficient isolates. After fermentation at the different parameters the crude enzyme product was
collected for dertermination of enzymes activity . Enzymes activitywas determined by dnsa method. The
enzyme activity was determined by dnsa method the enzymes activity of cdb27 and cb30 at differtent parameter
is tabulated
Different parameter5 Different values Enzymes activity of cdb27 Enzyme activity of cdb30
tempeture 35 0.98 1.2
40 2.56 1.89
45 2.1 2.6
50 1.86 3.5
55 1.11 3.0
ph 6 1.0 2.7
7 3.11 3.5
7.5 2.6 4.9
8 1.11 3.7
Incubation period 48h 1.34 1.87
72h 2.12 3.46
96h 3.2 5.7
120h 2.89 4.32
Subtrate conc(cmc) 0.2% 1.56 1.11
0.5% 3.4 2.78
1.0% 3.0 5.2
1.5% 2.5 4.6
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14. 0
1
2
3
4
35 40 45 50 55
Effect of temperature on
cellulase production
CDB27 CDB30
0
2
4
6
6 7 7.5 8
Effect of ph on cellulase
production
CDB27 CDB30
1.34
2.21
3.2
1.87
3.46
5.7
0
1
2
3
4
5
6
48h 72h 96h
INCUBATION PERIOD
CDB27 CDB30
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15. RESULS AND DISCUSSION
 3.0 Result and Discussion:
 Isolation and primary screening for cellulase producing bacteria:
 Total 15 samples were collected from 5 different sites and total 34 isolates were obtained
 as shown in Table-1. From these, 20 out of 34 isolates were removed due to similar colonial and
morphological characteristics.
 The resulting 14 isolates were then tested on CMC agar for cellulase activity.
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16. ZONE OF
HYDROLYSIS OF
DIFFERENT ISOLATES
SR
NO
ISOLATE NO COLONY
DIAMETER(
n) (mm)
Zone of
diameter (z)
(mm)
(Z/n)
1 CDB7 8.7 10.0 1.3
2 CDB11 7.5 9.0 1.5
3 CDB14 5.6 7.0 1.4
4 CDB16 10.6 12.0 1.3
5 CDB17 8.0 10.0 2.0
6 CDB19 6.0 7.0 1.0
7 CDB20 6.0 8.0 1.4
8 CDB22 10.0 14.0 1.5
9 CDB24 6.7 7.5 0.8
10 CDB25 9.3 11.0 1.7
11 CDB27 5.3 8.0 2.7
12 CDB30 8.5 11.0 2.5
13 CDB33 6.2 7.5 1.3
14 CDB34 7.4 9.0 1.6
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17. DIAGRAME SHOWING ZONE OF HYDROLYSIS
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18. Secondary screening and production of cellulase enzyme
Specific Activity Of Enzyme From Different Isolate: On The Basis Of Primary Screening The Potential Isolate Were Then
Evaluate For Their Enzymes Productivity In Submerge Fermentation Process .
For The Enzyme Activity Study Both Crude Enzyme Sample Were Assayed By Cellulase Enzyme Activity .
Enzyme Activity Assay : The Protein Concentration In Crude Samples Was Determined With Bovine Serum Albumin As
Standard .
The Enzyme Unit (Eu) Of Both Crude And Partially Purified Enzyme Was Determined By Using Dnsa Method .
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19. ENZYME ACTIVITY ASSAY
Isolate no Specific activity (u/mg) protein
Crude enzyme Partially purified enzymes
CDB27 5.95 6.3
CDB30 6.0 6.97
CDB34 5.0 5.2
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20. IDENFICATION OF MOST EFFICIENT CELLULASE PRODUCING BACTERIA
 Isoates Were Tentatively Identified On The Basis Of Their Morphological, Cultural And Biochemical
Characteristics Following Bergey’s Manual Of Determinative Bacteriology . And Methods Given By
Cappuccino And Sherman (1993). CDB27 And CDB30 Were Identified To Be Pseudomonas Spand Bacillus Sp
Respectively. Their Colonial, Morphological, And Biochemical Characteristics Are Tabulated In Table- 4, 5.
Colony And Morphological Characteristics Of Most Efficient Isolates.
Isolate No 1 Cdb27 : Colony Characteristic On Nutrinet Agar Plate : Small , Circular, Entire, Raised, Smooth Moist
,Translucent Green Color .
On Maconkeys Agar Pinpoint Circular Entire Raised Moist Lactose Non Fermenter Pale Yellowin Color
Growth Characterictic On Specific Media On Cetrimide Agar Growth Obserwed With Pin Point Circular Smooth Colonies
Morphology : Gram Neagative Short Rod To Long Rods Singly Arranged . Activetly Motile
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21.  Isolate NO CDB30: COLONY CHARACTER ON NUTRIENT AGAR PLATE : LARGE , IRREGULAR ENTIRE
RAISED DRY CREAMY OPAQUE NON PIGMENTED
 GROTH CHARACTREISTIC ON SPECIAL MEDIA :ONPOTATO SLICE MEDIUM : CREAMY WHITE DRY
GRWOTH OBSERVED
 MORPHOLOGY GRAM POSITIVE THICK LONG RODS ARRANGED IN CHAIN OF 3TO4 CELLS MOTILE.
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22. BIOCHEMICAL TESTS
BIOCHEMICAL TEST CDB27 CDB30
INODOLE TEST - +
CATALASE + +
OXIDASE
+
+
METHYL RED + +
VOGUS PROSKAUER + +
CITRATE UTILIZATION + +
NITRATE REDUCTION - +
GELATIN LIQUFICATION + +
SUGAR FERMENTATION
GLUCOSE + +
LACTOSE + +
MALTOSE - +
MANITOLE - +
SUCROSE - -
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23. CONCLUSION
 Conclusion : The Present Work Was Carried Out For Isolation Of Potential Cellulase Producing Bacterial Strain And For
That Different Environment Which Were Selected As Sources For Isolation
 TWO ISOLATE’S CDB27 AND CDB30 WERE SELECTED FOR THE DETERMINATION OF POTENTIAL
CELLULASE ACTIVITY AND THE SOURCES FOR BOTH THE ISOLATE WAS PAPER INDUSTRY WASTE THESE
CDB27AND CDB30 WERE CHARACTERISTICS FROM THEIR MORPHOLOGICAL, CULTURALAND
BIOCHEMICAL ANALYSIS AND IDENTIFIED AS PSEUDOMONAS Sp AND BACILLUS SP
 PARTIAL PURIFICATION OF CELLULASE WAS DONE AND THE ENZYME ACTIVITY AND SPECIFIC
ACTIVITY WAS DETERMINED
 THE OPTIMUM PARAMETER REQUIRED FOR THE STABILITY AND BETTER ACTIVITY OF ENZYME WERE
ALSO STUDIED .
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24. THANK
YOU …….
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