P.Saranraj et al.
*
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 .
*
P.Saranraj
Department of Microbiology,
Annamalai University,
Annamalai Nagar,
Chidambaram – 608 002, E mail: microsaranraj@gmail.com
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Introduction
D 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.
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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 polylinker 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 endocleavage, 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,
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P.Saranraj et al. 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; nonprogressive 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 twodimensionally 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
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P.Saranraj et al. 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 -Dglucans. 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.
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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.
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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 pretreated 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.
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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
KH
2
PO
4
, and a low concentration of MgSO
4
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
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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.
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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
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P.Saranraj et al. 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
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