Cellulose is an organic compound with the formula (C6H10O5) and is the most abundant organic polymer on Earth. Cellulose is an important structural component of the primary cell wall of green plants, many forms of algae and the oomycetes.

1. COMMERCIAL PRODUCTION OF
CELLULASE & ITS USES
Submitted to,
Dr. Liza Jacob
Associate Professor
Dept. of Botany
St. Teresa’s College
Ernakulam
Submitted by,
Silpa Selvaraj
II M.Sc. Botany
Roll no:13
St. Teresa’s College
Ernakulam

2. INTRODUCTION
 Cellulose is an organic compound with the formula
(C6H10O5) and is the most abundant organic polymer on
Earth.
 Cellulose is an important structural component of the
primary cell wall of green plants, many forms of algae and
the oomycetes.
 It is a polysaccharide consisting of a linear chain of several
hundred to many thousands of β(1→4) linked D-glucose
units.
 Plant-derived cellulose is usually found in a mixture with
hemicellulose, lignin, pectin and other substances, while
bacterial cellulose is quite pure.
 Cellulose consists of fibrils with crystalline and amorphous
regions.
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CELLULOSE

3.  The number of glucose molecules in one chain can vary from 100 to more than 10,000.
 The smallest unit in the cellulose structure is considered as elementary fibril, a bundle of
30-36 cellulose chains bound by hydrogen bonding.
 Many elementary fibrils are bundled into a unit called microfibril (Fig. 1).
 Cellulose molecules are very tightly packed inside the microfibril, and do not allow
penetration of even small water molecules. So accessibility of enzymes is mainly to surface
molecules only.
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5.  Cellulase is produced chiefly by fungi, bacteria, and protozoans that catalyze the
decomposition of cellulose and some related polysaccharides.
 Cellulases break down the cellulose molecule into monosaccharides such as β-glucose, or
shorter polysaccharides and oligosaccharides.
 The specific reaction involved is the hydrolysis of the 1,4-β-D-glycosidic linkages in
cellulose, hemicellulose, lichenin, and cereal β-D-glucans.
 Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult
compared to the breakdown of other polysaccharides such as starch.
 Most mammals have only very limited ability to digest dietary fibres like cellulose by
themselves.
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CELLULASE

6.  In many herbivorous animals like cattle, sheep and horses, cellulases are produced by
symbiotic bacteria.
 Endogenous cellulases are produced by animals , such as some termites, snails and
earthworms.
 Recently, cellulases have also been found in green microalgae (Chlamydomonas
reinhardtii, Gonium pectorale and Volvox carteri).
 Most fungal cellulases have a two-domains; catalytic domain and cellulose binding
domain, that are connected by a flexible linker.
 However, there are also cellulases (endoglucanases) that lack cellulose binding domains.
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7. TYPES AND MODE OF ACTION
Cellulase is composed of three type of enzyme system based on their mode of action
 Endoglucanase : is also called 1, 4-β-D-glucan Glucanohydrolase or Carboxymethyl cellulase. It cuts
internal amorphous sites and gives rise to different length of oligosaccharide.
 Exoglucanase 1, 4-β-D-Glucan Cellobiohydrolases (Cellodextrinases) :Exoglucanase acts on the
exposed ends of cellulose polysaccharides chains, resulting either glucose or cellobiose as major
product.
 Exoglucanase 1, 4-β-D-Oligoglucan Cellobiohydrolases (Cellodextrinases) : It is another form of
exoglucanase, which acts on the reducing end of cellulose polysaccharide to produce various
disaccharides units.
 Β-Glucosidases or β-D-Glucoside Glucohydrolases :It cleaves at the end of non reducing
polysaccharide and hydrolyzes soluble cellodextrins and cellobiose.
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8. MICROBES USED FOR PRODUCING CELLULASE
MICROBES USED FOR PRODUCING CELLULASE
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10. ADVANTAGES OF USING MICROBIAL SOURCES FOR THE
PRODUCTION OF CELLULASE
 Economical - produced on large scale within limited space and time.
 Can be easily extracted and purified.
 Can grow in a wide range of environmental conditions.
 Can be genetically manipulated to increase the yield of cellulases.
 Generally cheaper to produce.
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11. PRODUCTION OF CELLULASE
 Fermentation technology is widely used in the production of enzymes.
 It is done by two fermentation techniques:
 Solid State Fermentation and Submerged Fermentation.
 The substrates used in the process can be wheat, bran straw, rice, bran, green gram, husk
etc.
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12. Solid State Fermentation (SSF):
 This process requires solid substrate and is more efficient as it has;
 (i) Lower consumption of energy and water.
 (ii) Highly concentrated product.
 (iii) Reduced waste stream.
 It lacks foam build up and reported to be most appropriate process in enzyme production.
 Bacillus sp., Clostridium thermocellum, B. cereus and B. licheniformis are produced by SSF.
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13. Submerged Fermentation (SmF):
 This process utilizes liquid nutrient rich substrates like molasses and broth for the growth of
microorganisms.
 It has higher production rate as well as protein rate.
 Some microorganisms produced by SmF are Aspergillus flavus , Aspergillus niger, Bacillus
amyloliquefaciens UNPDV-22, Bacillus sp. SMIA, Brucella, and B. licheniformis.
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14. Screening and Isolation of microorganism
 Cellulase-producing bacteria were isolated from soils by the dilution pour plate or spread
plate method using CMC agar media.
 The plates were incubated at 45, 50, and 55°C for 24 hours.
 To visualize the hydrolysis zone, the plates were flooded with an aqueous solution of 0.1%
Congo red for 15 min and washed with 1 M NaCl.
 A fungal colony isolate with the highest activity was selected for optimization.
 The isolated fungal colony was subcultured and maintained on Czapek-Dox-agar slants and
stored at 4 °C in a refrigerator, until needed.
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15.  Production medium contained (g/L) glucose 0.5 gm, peptone 0.75 gm, FeSO4 0.01 gm,
KH₂PO, 0.5 gm, and MgSO, 0.5 gm.
 The inoculated medium was incubated at 37°C in shaker incubator for 24 h.
 At the end of the fermentation period, the culture medium was centrifuged at 5000 rpm for
15 min to obtain the crude extract, which served as enzyme source.
 Production medium at pH 7 was inoculated with overnight grown selected bacterial strain.
 The broth was incubated at different temperatures from 35, 40, 45, 50, 55, and 60°C for 24
h.
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16. Submerged fermentation (SmF)
 Submerged fermentation was carried out in 250 ml Erlenmeyer flasks containing 100 ml of
fermentation medium.
 The composition of the medium contained the following: L-Glutamic acid, 0.3; NH4NO2, 1.4;
K2HPO4, 2.0; CaCl₂, 2.0; MgSO4, 0.3; protease peptone, 7.5; FeSO4, 5.0; MnSO4, 1.6;
ZnSO4, 1.4; tween 80, 20% (v/v); coir waste.
 The medium was sterilized by autoclaving at 121°C for 15 min.
 Each flask was inoculated with 1ml of the inoculum. The cultures were incubated on a rotary
shaker (120 rpm) at 30°C for 72 h.
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17. Solid state fermentation (SSF)
 Solid state fermentation was carried out in 250 ml Erlenmeyer flasks that contained 10 g of
coir waste and 15 ml of distilled water (moistening agent).
 The flasks were sterilized at 121°C for 15 min and cooled to room temperature.
 About 1ml of inoculum was added, mixed well and incubated at 30°C in a humidified
incubator for 96 h.
 The flasks were periodically mixed by gentle shaking.
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18. Enzyme extraction
 At the end of the fermentation the culture broth from submerged fermentation was centrifuged
at 6000 rpm for 15 min and the supernatant was used as a source of extracellular enzyme.
 In solid state fermentation (SSF) the enzyme was extracted from the coir waste by mixing the
entire waste with distilled water and agitated on a rotary shaker (120 rpm) at 30 °C for 1hour.
 Dampened cheese cloth was used to filter the extract and they were centrifuged at 6000 rpm
for 15min and the clear supernatant was used as a source of extracellular enzyme.
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19. Cellulose Hydrolysis
 Cellulose can be hydrolyzed to glucose monomers using chemicals (commonly using strong
acid) or biological catalysts.
 Acid hydrolysis is a quick process; however, it suffers from several limitations such as high-
energy requirement, high capital cost, corrosion-resistant equipment, high disposal cost, and
degradation product formation.
 Enzymatic hydrolysis is an alternative to acid hydrolysis that addresses several of these
limitations and has been the focus of research in the last several decades.
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20. MECHANISM OF CELLULOSE HYDROLYSIS
 Cellulolytic microorganisms play an important role in the
hydrolysis of lignocellulosic polymer materials.
 Endoglucanase randomly cleaves at internal site of
cellulose to yield oligosaccharides of different lengths.
 Exoglucanase attacks on the reducing and non-reducing
ends of cellulose to liberate glucose, cellobiose and cello
oligosaccharides hydrolyzed by β-glucosidases.
 The efficient hydrolysis requires presence of β-
glucosidases.
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21. OPTIMIZATION OF PHYSIOLOGICAL FACTORS
A) pH and temperature :
 The pH and temperature play a key role in the enzyme production.
 Their optimization has to be done as it affects the production of enzyme directly.
 The cellulase activity has been recorded at pH value 5.0 at temperature 30°C for Penicillium
and pH value 4 at temperature 30°C for Aspergillus tubingensis .
B) Carbon source :
 The carbon sources such as starch, glucose, maltose, lactose and fructose has been used to
replace glucose (original carbon source).
 Glucose has been documented as the highest cellulase production.
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22. C) Effect of nitrogen :
 The nitrogen source are yeast extract, peptone, urea and ammonium sulphate, ammonium chloride.
 Among nitrogen sources ammonium sulphate has been recorded as good nitrogen source.
 The maximum activity of cellulase has been obtained on yeast extract and combination of peptone and
ammonium molybdate.
D) Effect of incubation period :
 The time taken by microorganism to synthesize enzyme by using medium nutrients is termed as
incubation period.
 It depends on the type of fermentation technique and microorganism used.
 As SSF take longer time in comparison with SmF. Bacteria requires less time than fungi for cultivation.
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23. APPLICATIONS OF CELLULASE
1. Food processing industry
 It is used to improve nutritive quality of fermented food items and in production of low calorie
food ingredient oligosaccharides.
 It is utilized in the production of food colouring agents like carotenoids.
 Macerating enzyme consists of cellulases with pectinase and hemicellulase are used to
improve the cloud stability and texture of nectar.
 It is also used to release antioxidants from fruit and vegetable and helps in controlling
atherosclerosis, chronic heart disease and reduce food spoilage.
 Reduce roughage in dough.
 Clarify fruit juices.
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24. 3. Textile and laundry industry
 It is used to improve the fabric quality by biostoning of denim garments, biopolishing of non-
denim fabrics, defibrillation.
 Removes stain (by breaking down fibres).
 Reduce pilling on fabrics (by increasing fiber smoothness).
 Cellulase is also used in biofinishing as it is rich in endoglucanase activity.
 Cellulase along with protease and lipase in detergents are widely used as household washing
powder.
 Cellulase is applicable to remove excess dye from denim fabrics, soften the cotton fabrics
without damaging the fibre.
 Helps in removing soil from inter fibril spaces of the fabrics. 24

25. 2. Animal feed industry
 Cellulase is utilized to improve feed, body weight and feed gain by ruminants.
 Cellulase degrades certain cereal component and upgrades the nutritional value of feed.
 The dietary fibres present in feed consist of arabinoxylans, cellulose, other plant components
including chitin, β-glucan, waxes, lignin, and oligosaccharides like proteases, amylases and
glucanases.
 Cellulase in addition with proteases is significant to improve the quality of pork meat.
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26. 4. Brewery industry
 Cellulase is used in the production of wine and beverages.
 In wine production, plays a key role by improving colour extraction, filtration and improve the
stability and quality of wine.
 Enhances aroma.
 Accelerated colour release.
 In malting, cellulases produce high quality malt.
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27. 5. Paper and pulp industry
 Cellulase is used for bio-mechanical pulping, biomodification of fibres, removing of ink coating
and toners from paper, improving drainage of paper mills.
 It is also used in the manufacture of soft paper like sanitary paper, paper towels and
biodegradable cardboard.
 Cellulases are also used to enhance the bioleachability of soft wood.
 The cellulase alone or combination with hemicellulase has been used to improve pupil
betability, runnability, paper sheet and trouble free printing process.
 Cellulase in addition with xylanase is considered as most affective for recycling the waste
paper from old books, newspapers, magazines. This process is known as deinking.
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28. 6. Agriculture industry
 It is used for increasing crop growth and in improvement of soil fertility.
 The use of exogenous cellulase is a potential means to straw decomposition and increase
soil fertility.
 Straw incorporation is strategy to improve soil quality and reduce the dependence on mineral
fertilizers.
 Cellulase is also used as biocontrol agents to provide protection against plant pathogens.
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29. 7. Biofuel industry
 The biocoversion of lignocellulosic substrates and other enzymes are used for the commercial
production of biofuel.
 Cellulase is used in the conversion of non food biomass such as agriculture waste and energy
crops into fermentable sugar for renewable fuel and chemical.
 The use of agrowastes such as sugarcane, bagasse, rice straw for bioethanol production are
invaluable and do not create any problem in the environment.
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30. REFERENCES
 Bhati, N., Shreya, & Sharma, A. K. (2021). Cost‐effective cellulase production, improvement
strategies, and future challenges. Journal of Food Process Engineering, 44(2), e13623.
 Cen, P., & Xia, L. (1999). Production of cellulase by solid-state fermentation. Recent progress
in bioconversion of lignocellulosics, 69-92.
 Singhania, R. R., Adsul, M., Pandey, A., & Patel, A. K. (2017). Cellulases. In Current
developments in biotechnology and bioengineering (pp. 73-101). Elsevier.
 Sukumaran, R. K., Singhania, R. R., & Pandey, A. (2005). Microbial cellulases-production,
applications and challenges.
 https://nopr.niscpr.res.in/bitstream/123456789/5375/1/JSIR%2064%2811%29%20832-844.pdf
 https://www.google.com/url?sa=t&source=web&rct=j&opi=89978449&url=https://www.ijrpr.com/
uploads/V2ISSUE7/IJRPR630.pdf&ved=2ahUKEwiK0NanxqSEAxV7RmwGHXytAqIQFnoECC
8QAQ&usg=AOvVaw11J9h7VVkhHK0z78paJtUz
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