P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 1

MICROBIAL CELLULASES AND ITS APPLICTIONS: A

REVIEW

P.Saranraj*, D. Stella and D. Reetha

* P.Saranraj

Department of Microbiology,

Annamalai University,

Annamalai Nagar,

Chidambaram – 608 002, E mail: microsaranraj@gmail.com

Abstract

Numerous agricultural residues generated due to diverse agricultural practices and food

processing such as rice straw, yam peels, cassava peels, banana peels among others represents

one of the most important energy resources. The major components of these are cellulose and

hemicellulose (75-80%) while lignin constitutes only 14%. Yearly accumulation of these

agricultural residues causes deterioration of the environment and huge loss of potentially

valuable nutritional constituents which when processed could yield food, feed, fuel, chemicals

and minerals. Agricultural residues when dumped in open environment constitute health hazard

due to pollution and support for the growth of microorganisms. The present review is focused on

microbial cellulases and its applications. Cellulose is considered as one of the most important

sources of carbon on this planet and its annual biosynthesis by both land plants and marine algae

occurs in many tones per annum. Recycling of agricultural residue can be achieved naturally and

artificially by microorganisms. Aerobic organisms such as fungi, bacteria, and some anaerobic

organisms have been shown to be able to degrade some constituents of these residues. Fungi

play a significant role in the degradation of cellulose under aerobic conditions. Cellulases are

important enzymes not only for their potent applications in different industries, like industries of

food processing, animal feed production, pulp and paper production , and in detergent and textile

industry, but also for the significant role in bioconversion of agriculture wastes in to sugar and

bioethanol. This review assesses the following topics: cellulose in agricultural wastes, cellulases

and its types, cellulolytic microorganisms, microbial degradation of cellulose and cellulase

production, microbial fermentation for cellulase production and application of cellulases.

Keywords: Cellulose, Agricultural wastes, Cellulase, Microorganisms and Fermentation.

Sci

ence

In

stin

ct P

ub

lica

tion

s

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 2

Introduction

espite a worldwide and enormous utilization of natural cellulosic sources, there are still

abundant quantities of cellulose-containing raw materials and waste products that are not

exploited or which could be used more efficiently. The problem in this respect is however to

develop processes that are economically profitable. Cellulose containing wastes may be

agricultural, urban, or industrial in origin, sewage sludge might also be considered a source of

cellulose since its cellulosic content provides the carbon needed for methane production in

the anaerobic digestion of sludge [1]. Agricultural wastes include crop residue, animal

excreta and crop processing wastes slashing generated in logging, saw dust formed in timber

production and wood products in forestry originated activities

Cellulose is earth’s major biopolymer and is of tremendous economic importance around the

globe. Cellulose is the major constituent of raw materials like cotton (over 94%) and wood

(over 50%). Cellulose is the primary structural component of the plant cell wall. It accounts

for over half of the carbon in the biosphere. Approximately 1015 of cellulose were estimated

to be synthesized and degraded annually. Cellulose is predominantly of plant origin, it also

occurs in the stiff outer mantles of marine invertebrates known as tunicates (urochordates).

Cellulose from major land plants as forest trees and cotton is assembled from glucose, which

is produced in the living plant cell from photosynthesis. In the oceans, however, unicellular

plankton produces most cellulose or algae using the same type of carbon-di-oxide fixation

found in photosynthesis of land plants. It is estimated that the amount of carbon assimilated

by plants throughout the year is 200 billion tones. Plants in the form of structural

polysaccharides, which human beings cannot degrade, store most of this energy [2].

Cellulosic biomass offers a possible solution. It is a complex mixture of carbohydrate

polymers known as cellulose, hemicellulose, lignin, and a small amount of compounds

known as extractives. Examples of cellulosic biomass include agricultural and forestry

residues, municipal solid waste, herbaceous and woody plants, and underused standing

forests. Cellulose is composed of glucose molecules bonded together in long chains that form

a crystalline structure [3]. Cellulose is a fibrous, tough, water-insoluble substance.

Hemicellulose is not soluble in water. It is a mixture of polymers made up from xylose,

mannose, galactose, or arabinose. Hemicellulose is much less stable than cellulose. Lignin,

which is present along with cellulose in trees, is a complex aromatic polymer of

phenylpropane building blocks. Lignin is resistant to biological degradation [4].

Cellulose in agricultural wastes Agriculture wastes contain a high proportion of cellulosic matter which is easily decomposed

by a combination of physical, chemical and biological processes. The bunch consists of 70

moisture and 30% solid; of which holocellulose accounts for 65.5, lignin 21.2, ash 3.5, hot

water-soluble substances 5.6 and alcohol-benzene soluble 4-1% [5]. Lignin is an integral cell

wall constituent, which provides plant strength and resistance to microbial degradation [6].

The recognition that environmental pollution is a worldwide threat to public health has given

rise to a new massive industry for environmental restoration. Biological degradation, for both

economic and ecological reasons, has become an increasingly popular alternative for the

treatment of agricultural, industrial, organic as well as toxic waste. These wastes have been

insufficiently disposed leading to environmental pollution.

D

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 3

Plant lignocellulosics as organic substances are subject to attacks by biological agents such

as fungi, bacteria and insects. Acids can breakdown the long chains in cellulose to release the

sugars through hydrolysis reaction, but because of their high specificity, cellulase can achieve

higher yield of glucose from cellulose. A portion of pretreated biomass can be used to feed a

fungus or other organism that produces cellulase that can then be added to pretreated solids to

release glucose from cellulose [7]. Filamentous fungi which use cellulose as carbon source

possess the unique ability to degrade cellulose molecules in plant lignocellulose. Although, a

large number of microorganisms are capable of degrading cellulose, only a few of these

produce significant quantities of cell-free enzymes capable of completely hydrolyzing

crystalline cellulose in vitro [8].

Cellulases

Bioconversion of cellulose containing raw materials is an important problem of current

biotechnology due to the increasing demand for energy, food and chemicals. Cellulases are

enzymes which hydrolyze the β-1,4- glycosidic linkage of cellulose and synthesized by

microorganisms during their growth on cellulosic materials [9]. The complete enzymatic

hydrolysis of cellulosic materials needs different types of cellulase; namely endoglucanase,

(1,4-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exocellobiohydrolase (1, 4-D-glucan

glucohydrolase; EC 3.2.1.74) and glucosidase (D-glucoside glucohydrolase; EC 3.2.1.21).

Enzymatic process to hydrolyze cellulosic materials could be accomplished through a

complex reaction of these various enzymes. Two significant attributes of these enzyme-based

bioconversion technologies are reaction conditions and the production cost of the related

enzyme system. Therefore, worldwide there has been many research works focused on

obtaining new microorganisms producing celluloytic enzymes with higher 105 specific

activities and greater efficiency [10]. Enzymes produced by marine microorganisms can

provide numerous advantages over traditional enzymes due to the wide range of

environments [11].

Cellulases are comprised of independently folding, structurally and functionally discrete units

called domains or modules, making cellulases modular [12]. A typical free cellulase is

composed of a carbohydrate binding domain (CBD) at the C-terminal joined by a short poly-

linker region to the catalytic domain at the N-terminal. There are only two modes of action

for the hydrolysis of cellulose by cellulases, either inversion or retention of the configuration

of the anomeric carbon. At least two amino acids with carboxyl groups located within the

active site catalyze the reaction by acid-base catalysis.

The commonly described mode of action for cellulases on polymers is either exo- or endo-

cleavage, and all cellulases target the specific cleavage of β-1,4-glycosidic bonds. Using this

classification system, cellobiohydrolases (exoglucanases) were classified as exo-acting based

on the assumption that they all cleave β-1,4-glycosidic bonds from chain ends. As well, those

enzymes truly exo-acting often have a tunnel-shaped closed active site which retains a single

glucan chain and prevents it from readhering to the cellulose crystal [13]. While

endoglucanases on the other hand, are often classified as endo-acting cellulases because they

are thought to cleave β-1, 4-glycosidic bonds internally only and appear to have cleft-shaped

open active sites.

Endoglucanase are active on amorphous regions of cellulose and thus their activity can be

assayed using soluble cellulose substrates; i.e., the carboxymethylcellulase assay (CMCase).

However, there is now supporting evidence that some cellulases display both modes of

action, endo- and exo- [14]. Thus classification has changed; cellobiohydrolases

(exoglucanases) are described as active on the crystalline regions of cellulose; whereas,

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 4

endoglucanases are typically active on the more soluble amorphous region of the cellulose

crystal. There is a high degree of synergy seen between cellobiohydrolases (exoglucanases)

and endoglucanases, and it is this synergy that is required for the efficient hydrolysis of

cellulose crystals.

Types of cellulases

Five general types of cellulases based on the type of reaction catalyzed:

1. Endo-cellulase breaks internal bonds to disrupt the crystalline structure of

cellulose and expose individual cellulose polysaccharide chains.

2. Exo-cellulase cleaves 2-4 units from the ends of the exposed chains produced by

endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose.

3. There are two main types of exo-cellulases (or cellobiohydrolases, abbreviate

CBH) - one type working processively from the reducing end, and one type

working processively from the non-reducing end of cellulose.

4. Cellobiase or beta-glucosidase hydrolyses the exo-cellulase product into individual

monosaccharides.

5. Oxidative cellulases that depolymerize cellulose by radical reactions, as for

instance cellobiose dehydrogenase (acceptor).

Cellulose phosphorylases that depolymerize cellulose using phosphates instead of water. In

the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to

beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria in the

ruminating chambers of herbivores. Aside from ruminants, most animals (including humans)

do not produce cellulase in their bodies, and are therefore unable to use most of the energy

contained in plant material. Enzymes which hydrolyze hemicellulose are usually referred to

as hemicellulase and are usually classified under cellulase in general. Enzymes that cleave

lignin are occasionally classified as cellulase, but this is usually considered erroneous. Within

the above types, there are also progressive (also known as processive) and non-progressive

types. Progressive cellulase will continue to interact with a single polysaccharide strand; non-

progressive cellulase will interact once then disengage and engage another polysaccharide

strand. Most fungal cellulases have a two-domain structure with one catalytic domain, and

one cellulose binding domain, that are connected by a flexible linker. This structure is

adoption for working on an insoluble substrate and it allows the enzyme to diffuse two-

dimensionally on a surface in a caterpillar way. However, there are also cellulases (mostly

endoglucanases) that lacks cellulose binding domain. These enzymes might have a swelling

function.

Cellulolytic microorganisms A variety of microorganisms take part in Cellulose hydrolysis with an aid of a multienzyme

system. Among the best-characterized cellulase systems are as follows: White rot fungus

Phanerochaete chrysosporium, Soft-rot fungi, Fusarium solani, Penicillum funiculosum,

Talaromyces emersonii, Trichoderma koningii and Trichoderma reesei. Some of the

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 5

aerobic cellulolytic bacteria which are having best-characterized cellulase systems are as

follows: Cellulomonas sp., Cellvibrio sp., Microbispora bispora and Thermomonospora sp.

Examples of anaerobic cellulolytic bacteria are as follows: Acetivibrio cellulolyticus,

Bacteroides cellulosolvens, Bacteriodes succinogenes, Clostridium thermocellum,

Ruminococcus albus and Ruminococcus flavefaciens. Fungi are the main cellulase producing

microorganisms, though a few bacteria and actinomycetes have also been reported to yield

cellulase activity. Fungal genera like Trichoderma and Aspergillus are known to be cellulase

producers and crude enzymes produced by these microorganisms are commercially available

for agricultural use. The genus Aspergillus species attack cellulose producing significant

amount of cell free cellulase capable of hydrolyzing cellulose into fermentable soluble sugars

such as glucose; an important raw material in chemical industries. Aspergillus and

Trichoderma specie are well known efficient producers of cellulases [15]. Several studies

have been carried out to produce cellulolytic enzymes from biowaste degradation process by

many microorganisms including fungi such as Trichoderma, Penicillium and Aspergillus

species etc., by Mandels and Reese [16].

Microbial degradation of cellulose and cellulase production

Microorganisms bring about most of the cellulose degradation occurring in nature. They meet

this challenge with the aid of a multi-enzyme system. They include fungi and bacteria,

aerobes and anaerobes, mesophiles and thermophiles and occupy a variety of habitats.

Aerobic bacteria produced numerous individual, extra-cellular enzymes with binding

modules for different cellulose conformations. Anaerobic bacteria possess a unique

extracellular multienzyme complex, called cellulosome. Binding to a non-catalytic structural

protein (scaffoldin) stimulates activity of the single components towards the crystalline

substrate. The most complex and best investigated cellulosome is that of the thermophilic

bacterium Clostridium thermocellum.

Cellulase preparations are able to decompose natural cellulose (e.g. filter paper) as well as

modified celluloses such as carboxymethyl cellulose or hydroxyethyl cellulose.

Cellulasehydrolyses 1,4-β-D-glucosidic linkages in cellulose, licheninand cereal β -D-

glucans. The exoglucanases are thought to act primarily on newly generated chain ends

producing mainlycellobiose , β-Glucosidase hydrolyses terminal β-D-glucose residues from

the ends of cellulose molecules. In nature, cellulose is found in association with other

components e.g. hemicellulose, lignin and pectin. SERVA cellulases contain a number of

other activities, which assist in breaking down these components and degrading cell walls. α-

Amylase hydrolyses 1,4- α -D-glucosidic linkages in polysaccharides containing three or

more 1,4- α -linked D-glucose units. Pectinase randomly cleaves 1, 4- α -D-galactosiduronic

linkages in galacturans. These products also contain hemicellulase and protease activities.

Cellulase is used to modify the surface properties of cellulosic fibers and fabric in order to

achieve a desired surface effect [17]. Cellulase has been used to degrade environmental

wastes such as plant wastes (lignocellulosics). Cellulase as an industrial enzyme is imported

for use in Nigeria. Therefore, its production using readily available sources (example plant

residues) will help reduce importation costs. It is against this background, that this study was

carried out to evaluate the cellulase activity of Aspergillus candidus on various agro-forestry

residues as feed substrates and to determine the effects of pH on cellulase activity. Cellulase

production by different organisms in submerged state fermentation has received more

attention and is found to be cost-prohibitive because of high cost of process engineering.

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 6

Microbial fermentation for cellulase production.

Many microorganisms are capable of degrading and utilizing cellulose and hemicellulose as

carbon and energy sources. During composting, the capacity of thermophilic microorganisms

to assimilate organic matter depends on their ability to produce the enzymes needed for

degradation of the substrate [18]. Enzymatic hydrolysis processing of cellulosic materials

could be accomplished through a complex reaction of various enzymes. Cellulases are

inducible enzymes which are synthesized by microorganisms during their growth on

cellulosic materials [19]. Both fungi and bacteria have been heavily exploited for their

abilities to produce a wide variety of cellulases and hemicellulases. Most emphasis has been

placed on the use of fungi because of their capability to produce copious amounts of

cellulases and hemicellulases which are secreted to the medium for easy extraction and

purification. In addition, the enzymes are often less complex than bacterial glycoside

hydrolases and can therefore be more readily cloned and produced via recombination in a

rapidly growing bacterial host such as Escherichia coli.

Most importantly, bacteria inhabit a wide variety of environmental and industrial niches,

which produce cellulolytic strains that are extremely resistant to environmental stresses.

These include strains that are thermophilic or psychrophilic, alkaliphilic or acidiophilic, and,

strains that are halophilic. Not only can these strains survive the harsh conditions found in the

bioconversion process, but they often produce enzymes that are stable under extreme

conditions which may be present in the bioconversion process and this may increase rates of

enzymatic hydrolysis, fermentation, and, product recovery. Researchers are now focusing on

utilizing, and improving these enzymes for use in the biofuel and bioproduct industries.

Cellulose, being an abundant and renewable resource, is a potential raw material for the

microbial production of food, fuel and chemicals. Various bacteria, actinomycetes and

filamentous fungi produce extra cellular cellulases when grown on cellulosic substrates

though many actinomycetes have been reported to have less cellulase activity than moulds.

Investigations on the extracellular cellulases of fungi have been concentrated mainly on

Trichoderma sp. and studies on other mesophilic fungi suggested the possibility that other

cellulase systems could be utilized for the hydrolysis of cellulose [20].

Maulin Shah et al. investigated the ability Phylosticta sp. and Aspergillus sp. to produce

various lignolytic and cellulolytic enzymes such as laccase, lignin peroxidase, xylanase,

endo-1,4-β-d-glucanase (CMCase) and exo-1,4-β-d-glucanase [filter paper activity (FP

activity)] on banana agricultural waste (leaf and pseudostem biomass) biomass under solid

state fermentation (SSF) condition [21]. The production pattern of these enzymes was studied

during the growth on the organisms for a period of 40 days. Very low levels of cellulolytic

enzyme activities were observed compared to lignin degrading enzymes by both the

organisms. Maximum specific activities of studied enzymes were obtained at 20 days of

culture growth.

Narasimha et al. compared the production of cellulase (filter paper activity, endoglucanase

and (glucosidase) by Aspergillus niger on three media in liquid shake culture [22]. The

culture filtrate of this organism exhibited relatively highest activity of all three enzymes and

extracellular protein content at 7 days interval during the course of its growth on Czapek-Dox

medium supplemented with 1.0% (w/v) cellulose. Urea as a nitrogen source and pH 5.0 were

found to be optimal for growth and cellulase production by Aspergillus niger. Among various

soluble organic carbon sources and lignocelluloses tested, carboxymethylcellulose and

sawdust at 1% supported maximum production of all three enzymes by Aspergillus niger.

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 7

Reeta Rani Singhana et al. carried out cellulase production studies using the fungal culture

Trichoderma reesei using four different lignocellulosic residues (both raw and pre-treated) by

solid-state fermentation [23]. The effect of basic fermentation parameters on enzyme

production was studied. Maximal cellulase production obtained was 154.58 U/gds when pre-

treated sugarcane bagasse (PSCB) was used as substrate. The optimal conditions for cellulase

production using PSCB were found to be initial moisture content - 66%, initial medium pH-

7.0, incubation temperature -28°C, NH4NO3 at 0.075 M, and 0.005 M cellobiose. The

optimal incubation time for production was 72 hrs. Results indicate the scope for further

optimization of the production conditions to obtain higher cellulase titres using the strain

under SSF.

Munir khan et al. carried out cellulase production by solid state bioconversion (SSB) method

using rice straw, a lignocellulosic material and agricultural waste, as the substrate of three

Trichoderma sp. and Phanerochaete chrysosporium in lab-scale experiments [24]. The results

were compared to select the best fungi among them for the production of cellulase.

Phanerochaete chrysosporium was found to be the best among these species of fungi, which

produced the highest cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and

2.40 IU/mL of carboxymethylcellulose activity (CMCase). The “glucosamine” and “reducing

sugar” parameters were observed to evaluate the growth and substrate utilization in the

experiments. In the case of Phanerochaete chrysosporium, the highest glucosamine

concentration was 1.60 g/L and a high concentration of the release of reducing sugar was

measured as 2.58 g/L obtained on the 4th day of fermentation.

Acharya et al. focused the factors relevant for improvement of enzymatic hydrolysis of saw

dust by using Aspergillus niger. Different cultural conditions were examined to assess their

effect in optimizing enzyme production [25]. Alkaline pretreated (2 N NaOH) saw dust at

9.6% concentration gave 0.1813 IU/mL cellulase activity. Optimum pH for cellulase

production was between 4.0 and 4.5. Submerged fermentation at 120 rpm at 28°C gave

higher yields of cellulase compared to static condition. Several other parameters like

inoculum size, time duration, nitrogen source and its concentration were also optimized for

the cellulase production by using saw dust as substrate.

Sherif et al. isolated twelve Aspergillus species from some local soil samples [26]. On the

basis of cellulolytic activity, Aspergillus fumigatus was selected and used for production of

exoglucanase , endoglucanase , CMCase, β-glucosidase and xylanase by adopting SSF

condition using mixed substrate of rice straw amended with wheat bran. Effect of Culture

conditions including; incubation period, initial pH, incubation temperature, moisture level,

different nitrogen sources, different lignocelluloses as carbon source and different ratios of

mixed rice straw and wheat bran were evaluated. The fungus expressed high enzyme

production after 4 days incubation at moisture level 75%, initial pH 5-6, at 40°C in presence

of NaNO3 as an inorganic nitrogen source. The recorded activities were 14.71, 8.51, 0.93,

0.68 and 42.7 IU g-1 for CMCase, β-glucosidase, exoglucanase, endoglucanase and xylanase,

respectively.

Milila et al. used rice husk, millet straw, guinea corn stalk and sawdust as fermentation feed

substrate for the evaluation of cellulase activity secreted by Aspergillus candidus [27]. The

substrates were pretreated with 5% NaOH (alkaline treatment) and autoclaved. From the

fermentation studies, rice husk, millet straw and guinea corn stalk feed substrates showed the

highest cellulase activity of 7.50, 6.88 and 5.84 IU, respectively. The effect of pH showed

that optimal pH for maximum cellulase activity varied in each of the substrates used. Rice

husk and millet straw had maximum enzyme activity at pH 5, while guinea corn stalk and

sawdust had maximum activity at pH 3 and 4, respectively.

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 8

Abo-State et al. isolates twenty nine fungal strains from agriculture wastes [28]. Aspergillus

sp. was the predominant genera in these agriculture wastes. The most potent cellulase

producers were selected for studying their cellulase productivities on Wheat Straw (WS),

Wheat Bran (WB), Rice Straw (RS) and Corn Cob (CC) as cheap, renewable agriculture

wastes by solid state fermentation (SSF). Five Aspergillus sp. and standard strain

Trichoderma viride were grown on the agriculture wastes and CMCase, FPase, Avicelase and

soluble protein were determined. Trichoderma viride produces the highest CMCase on WS

(555U/ml), while the highest FPase (141U/ml) and Avicelase (46U/ml) were produced on

WB. The isolated strain Aspergillus MAM-F35 gave the highest CMCase (487U/ml), FPase

(79U/ml) and Avicelase (35U/ml) on WS.

Fatma et al. production of cellulase by Trichoderma reesei cultivated on alkali treated rice

straw using solid state fermentation (SSF) technique [29]. The high cellulase activity was

obtained when the fungus was cultivated on substrate with about 75 % (v/w) moisture, pH 4.8

for 5 days incubation at 28 ± 2ºC, as it gave 16.2 IU/g substrate. The obtained cellulase of 1.2

IU/ ml culture filtrate was applied for saccharification (5% w/v) of alkali treated rice straw, in

0.1M citrate buffer pH 4.8 in shaker water bath of 100 rpm. Sugary solution of 1.07 %

glucose was achieved after 16 hrs. The sugary solution was concentrated to give 10% (w/v)

glucose. Ethanolic fermentation was conducted by Saccharomyces cerevisiae under static

condition giving 5.1% (v/v) ethanol after 24 hrs. The fermented mash contained 3.6 g/L yeast

cell can be utilized as fooder yeast used for animal feeding.

Narmeen El Sersy et al. screened six marine strains of Actinomycetes for their carboxymethyl

cellulase (CMCase) productivity [30]. Streptomyces ruber was chosen to be the best

producing strain. The highest enzyme production (25.6 U/ml) was detected at pH 6 and 40°C

after 7 days of incubation. Plackett-Burman design was applied to optimize the different

culture conditions affecting enzyme production. Results showed that a high concentration of

KH2PO4, and a low concentration of MgSO4 had a significant effect on enzyme production.

Rice straw was used as a low cost source of cellulose. It was found that 30 g/l rice straw was

the suitable concentration for maximum enzyme production. Partial purification of cellulase

enzyme using an anion exchange chromatography resulted in the detection of two different

types of CMCases, type I and II, with specific activity of 4239.697 and 846.752 U/mg,

respectively.

Hafiz Iqbal et al. investigated the potential of a filamentous fungus, Trichoderma harzianum

for hyper-production of third most demanded industrial enzyme carboxymethyl cellulase

using cheap and easily available agro-industrial residue wheat straw as growth supporting

substrate under still culture solid state fermentation technique [31]. Production of

carboxymethyl cellulase was substantially enhanced through media optimization process. To

promote carboxymethyl cellulase production, they evaluated the effect of several kinetic

parameters like pretreatment, substrate concentration, initial moisture content, pH, incubation

temperature and inoculum size on carboxymethyl cellulase production. Samples were

harvested after every 24 hrs to study the profile of cellulase enzyme produced by the fungus

on proximally analyzed wheat straw. By optimizing the SSF medium containing 2 % HCl

pretreated wheat straw; maximum carboxymethyl cellulase activity (480±4.22 μM /mL/min)

was recorded after 7th day of incubation at pH 5.5; temperature, 35°C; moisture, 40 % and

inoculum size, 10 %, using optimum substrate concentration (3%).

Siva Sakthi et al. isolated Aspergillus niger from the spoiled coconut and identified using

LPCB staining based on its morphological and cultural features [32]. Optimization of

cellulase production was done by using various physical (Temperature, pH, Salinity and

Incubation time) and chemical parameters (Carbon sources and Nitrogen sources) which

could influence the enzyme activity. Cellulase production was maximum at the temperature

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 9

20°C and minimum at 40ºC. The optimal pH for the cellulase production was observed

maximum in 6.0 and minimum in 7.0. Cellulase production was maximum at 48 hrs and

minimum at 24 hrs. Cellulase production was maximum with when fructose was used as a

carbon source and minimum with sucrose. Cellulase production was maximum when Malt

extract was used as a nitrogen source and minimum with yeast extract.

Applications of cellulases

Cellulases were initially investigated several decades back for the bioconversion of

biomass which gave way to research in the industrial applications of the enzyme in

animal feed, food, textiles and detergents and in the paper industry. With the shortage

of fossil fuels and the arising need to find alternative source for renewable energy

and fuels, there is a renewal of interest in the bioconversion of lignocellulosic biomass

using cellulases and other enzymes. In the other fields, however, the technologies and

products using cellulases have reached the stage where these enzymes have become

indispensable.

Textile industry

Cellulases have become the third largest group of enzymes used in the industry since

their introduction only since a decade. They are used in the bio- stoning of denim

garments for producing softness and the faded look of denim garments replacing the use of

pumice stones which were traditionally employed in the industry. They act on the

cellulose fiber to release the indigo dye used for coloring the fabric producing the faded

look of denim. Humicola insolens cellulase is most commonly employed in the equally

good cellulases are utilized for digesting off the small fiber ends protruding from the

fabric resulting in a better finish cellulases, used in softening defibrillation , and in

processes for providing localized variation in the color density of fibers.

Laundry and detergent

Cellulases, in particular EG III and CBH I, are commonly used in detergents for

cleaning textiles Several reports disclose that EG III variants, in particular from

Trichoderma reesei are suitable for the use in detergents. Trichoderma viride and

Trichoderma harzianum are also industrially utilized natural sources of cellulases, as

Aspergillus niger. Cellulase preparations, mainly from species of Humicola (Humicola

insolens and Humicola griseathermoidea) that are active under mild alkaline conditions

and at elevated temperatures, are commonly added in washing powders , and in

detergents.

Food and animal feed

In food industry, cellulases are used in extraction and clarification of fruit and

vegetable juices. production of fruit nectars and purees, and in the extraction of olive

oil Glucanases are added to improve the malting of barley in beer manufacturing and in

wine industry, better maceration and color extraction is achieved by use of exogenous

hemicellulases and glucanases. Cellulases are also used in carotenoid extraction in the

production of food coloring agents.

Enzyme preparations containing hemicellulase and pectinase in addition to cellulases are

used to improve the nutritive quality of forages. Improvements in feed digestibility and

animal performance are reported with the use of cellulases in feed processing describes

the feed additive use of Trichoderma cellulases in improving the feed conversion ratio

and increasing the digestibility of a cereal-based feed.

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 10

Pulp and paper industry

In the pulp and paper industry, cellulases and hemicellulases have been employed for

biomechanical pulping for modification of the coarse mechanical pulp and hand sheet

strength properties de-inking of recycled fibers and for improving drainage and run ability

of paper mills. Cellulases are employed in the removing of inks coating and toners

from paper Bio characterization of pulp fibers is another application where microbial

cellulases are employed. Cellulases are also used in preparation of easily biodegradable

cardboard. The enzyme is employed in the manufacture of soft paper including paper

towels and sanitary paper and preparations containing cellulases are used to remove

adhered paper.

Biofuel

Perhaps the most important application currently being investigated actively is in the

utilization of lignocellulosic wastes for the production of biofuel. The lignocellulosic

residues represent the most abundant renewable resource available to mankind but their

use is limited only due to lack of cost effective technologies. A potential application

of cellulase is the conversion of cellulosic materials to glucose and other fermentable

sugars, which in turn can be, used as microbial substrates for the production of single cell

proteins or a variety of fermentation products like ethanol.

Organisms with cellulose systems that are capable of converting biomass to alcohol

directly are already reported. But, none of these systems described are effective alone to

yield a commercially viable process. The strategy employed currently in bioethanol

production from lignocellulosic residues is a multi-step process involving pre-treatment

of the residue to remove lignin and hemicellulase fraction, cellulase treatment at 50°C to

hydrolyze the cellulosic residue to generate fermentable sugars, and finally use of a

fermentative microorganism to produce alcohol from the hydrolyzed cellulosic material.

The cellulose preparation needed for the bioethanol plant is prepared in the premises

using same lignocellulosic residue as substrate, and the organism employed is almost

always Trichoderma ressei. To develop efficient technologies for biofuel production,

significant research has been directed towards the identification of efficient cellulase

systems and process conditions besides studies directed at the biochemical and genetic

improvement of the existing organisms utilized in the process. The use of pure enzymes in

the conversion of biomass to ethanol or to fermentation products is currently

uneconomical due to the high cost of commercial cellulases.

Effective strategies are yet to resolve and active research has to be taken up in this direction.

Overall, cellulosic biomass is an attractive resource that can serve as substrate for the

production of value added metabolites and cellulases as such. Apart from these common

applications, cellulases are also employed in formulations for removal of industrial

slime , in research for generation of protoplast and for generation of antibacterial

chitooligosaccharides, which could be used in food preservation, immune modulation

and as a potent antitumor agent.

Conclusion

In the recent years, one of the most important biotechnological applications is the conversion

of agricultural wastes and all lignocellulosics into products of commercial interest such as

ethanol, glucose and single cell products. The key element in bioconversion process of

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 11

lignocellulosics to these useful products is the hydrolytic enzymes mainly cellulases. The

bioconversions of cellulosic materials are now a subject of intensive research as a

contribution to the development of a large scale conversion process beneficial to mankind.

Such process would help alleviate shortages of food and animal feeds, solve modern waste

disposal problem and diminish man’s dependence on fossil fuels by providing a convenient

and renewable source of energy in the form of glucose. A diverse spectrum of cellulolytic

microorganism mainly fungi and bacteria have been isolated and identified over the years and

this still continue to grow rapidly. Fungi are the main cellulase producing microorganism and

Aspergillus and Trichoderma are the main fungal genera that were used for commercial

production of cellulase. Therefore the present review showing the ability of microorganisms

to synthesize high amount of extra cellular exoglucanase within a relatively short period of

time, utilizing agro wastes that would otherwise cause environmental pollution, could be used

for rapid and commercial production of cellulase

References

[1] Daamwal M, Schraft H and Qin W. Fungal bioconversion of lignocellulosic residues: Opportunities and

perspectives. Int. J. Biol. Sci., 2009; 5: 578-595.

[2] Chandra MS, Viswanath B and Reddy BR. Cellulolytic enzymes on lignocellulosic substrates in solid state

fermentation by Aspergillus niger. Indian J. Microbiol., 2007; 47: 323-328.

[3] Kotchoni OS, Shonukan O and Gachomo WE. Bacillus pumillus, BPCR16, a promising candidate for

cellulase production under conditions of catabolic repression. African Journal of Biotechnology, 2003; 2:

140-146.

[4] Bernfeld P. Amylases, alpha and beta., Methods. Enzymology, 1955; 1: 149-58.

[5] Thambirajah JJ, Zulkafli MD and Hashim MA. Microbiological and biochemical changes during the

composting of oil palm empty fruit bunches. Effect of nitrogen supplementation on the substrate. Biores.

Technol., 1995; 52: 133-134.

[6] Sherif A, Shah AR and Madamwar D. Xylanase production under solid-state fermentation and its

characterization by an isolated strain of Aspergillus foetidus in India. World J. Microbiol. Biotechnol.,

2005; 21: 233-243.

[7] Wyman CE. Cellulosic ethanol: A unique sustainable liquid transportation fuel. MRS Bull., 2008; 33: 381-

382.

[8] Immanuel G, Dhanusa R, Prema P and Palavesam A. Effect of different growth parameters on

endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. Int.

J. Environ. Sci. Technol., 2006; 3: 25-34.

[9] Lee SM and Koo YM. Pilot-scale production of cellulose using Trichoderma reesei Rut C-30 in fed-batch

mode. J. Microbiol. Biotechnol., 2001; 11: 229-233.

[10] Johnvesly B, Virupakshi S, Patil GN, Ramalingam N and Naik GR. Cellulase-free thermostable alkaline

xylanase from thermophillic and alkalophillic Bacillus sp. JB-99.J Microbiol Biotechnol., 2002; 12, 153-

156.

[11] Rasmussnen RS and Morrissey MT. Marine biotechnology for production of food ingredients. Adv Food

Nutr Res., 2007; 52, 237-92.

[12] Henry B, Teeri TT and Warren. A scheme for designating enzymes that hydrolyse the polysaccharides in

the cell walls of plants. FEBS Lett., 1998; (425): 352-354.

[13] Divne C, Stahlberg J, Teeri T and Jones TA. High-resolution crystal structures reveal how a cellulose chain

is bound in the 50Å long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol., 1998; (275):

309-325.

[14] Davies GJ and Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure, 1995; 5 (3):853-

859.

[15] Peij N, Gielkens MMC, Verles RP and Graff LH. 1998. The transcriptional activator xin R regulates both

xylanolytic endoglucanase gene expressions in Aspergillus niger. Applied Environ. Microbiol., 1998; 64:

3615-3617.

[16] Mandels M and Reese ET. Fungal cellulase and microbial decomposition of cellulosic fibres. Dev. Ind.

Microbiol., 1985; 5: 5-20.

P.Saranraj et al.

International Journal of Biochemistry & Biotech Science 2012; 1: 1-12 12

[17] Kotchoni OS, Shonukan O and Gachomo WE. Bacillus pumillus, BPCR16, a promising candidate for

cellulase production under conditions of catabolic repression. African Journal of Biotechnology, 2003; 2:

140-146.

[18] Tuomela M., Vikman M, Hatakka A and Itavaara M. Biodegradation of lignin in a compost environment: A

review. Biores. Technol., 2000; 72: 169-183.

[19] Lee SM and Koo YM. Pilot-scale production of cellulose using Trichoderma reesei Rut C-30 in fed-batch

mode. J. Microbiol. Biotechnol., 2001; 11: 229-233.

[20] Darmwal M, Schraft H and Qin W. Fungal bioconversion of lignocellulosic residues: Opportunities and

perspectives. Int. J. Biol. Sci., 2009; 5: 578-595.

[21] Maulin P. Shah, Reddy GV, Banerjee R and Kothari IL. Microbial degradation of banana waste under solid

state bioprocessing using two lignocellulolytic fungi (Phylosticta spp. MPS-001 and Aspergillus spp. MPS-

002). Process Biochemistry, 2005; 40 (1): 445-451.

[22] Narasimha G, Sridevi A, Buddola Viswanath et al. Nutrient effect of cellulosic enzymes produced by

Aspergillus niger. African Journal of Biotechnology, 2006; 5 (5): 472-476.

[23] Reeta Rani Singhania, Rajeev K Sukumaran, Anu Pillai et al. Solid-state fermentation of lignocellulosic

substrates for cellulase production by Trichoderma reesei NRRL 11460. Indian Journal of Biotechnology,

2006; 5: 332-336.

[24] Munir H, Khan S, Ali A and Alam Z. Use of fungi for the bioconversion of rice straw into cellulase

enzyme. Journal of Environmental science and health, 2007; 42 (4): 381-386.

[25] Acharya PB, Acharya DK and Modi HA. Optimization of cellulase production by Aspergillus niger using

saw dust as substrate. African Journal of Biotechnology, 2008; 7 (22): 4147-4152.

[26] Sherief AA, El-Tanash AB and Atia N. Cellulase production by Aspergillus fumigatus grown on mixed

substrate of rice straw and wheat bran. Res. J. Microbiol., 2008; 5: 199-211.

[27] Milala MA, Shehu BB, Zanna H and Omosioda VO. Degradation of agro-waste by cellulase from

Aspergillus candidus. Asian J. Biotechnol., 2009; 1: 51-56.

[28] Abo-State, MAM, Hameed AI and Gannam RB. Enhanced production of cellulase by Aspergillus niger

isolated from agricultural wastes by solid state fermentation. American Journal of Agriculture and

Environmental Sciences, 2010; 8 (2): 402-410.

[29] Fatma MN, Asquieri ER and Park YK. Production and characterization of extracellular cellulases from a

thermostable Aspergillus sp. Rev. Microbiol., 2010; 23: 183-188.

[30] Narmeen El Sersy, Abdul Hanan, Hassan Ibrahim and Abdul Hameed. Optimization, economization and

characterization of cellulase produced by marine Streptomyces ruber. African Journal of Biotechnology,

2010; 9 (38): 6355-6354.

[31] Hafiz Iqbal, Muhhamed Aghser and Shahbaz Hussain. Media optimization for hyper production of cellulase

by using proximally analyzed agricultural wastes with Trichoderma hazardium by solid state fermentation.

IJAVMS, 2010; 4 (2): 47-55.

[32] Siva Sakthi S, Saranraj P and Rajasekar M. Optimization for cellulase production by Aspergillus niger

using paddy straw as substrate. International Journal of Advanced Scientific and Technical Research, 2011;

1 (1): 69 - 85.