Chapter 1 INTRODUCTION

Transcription

1 Chapter 1 INTRODUCTION 16

2 Lignocellulose is the predominant component of woody plants and dead plant materials and the most abundant biomass on earth. It is a major renewable natural resource of the world and represents a major source of renewable organic matter. The plant biomass regarded as wastes are biodegradable and can be converted into valuable products such as biofuels, chemicals, cheap sources for fermentation, improved animal feeds and human nutrients. The wastes produced from industries and agricultural farmlands constitute a major problem to our environment. Such wastes include cereal straws, corn cobs, wood pulp, sawdust, cotton wastes and many others. Usually they are burnt in heaps thereby releasing offensive odor and gases to the atmosphere. Some are thrown into rivers and streams thereby endangering aquatic life. These wastes could be put into appropriate use in order to reduce environmental hazard and pollution (Jonathan, 2008). In 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 protein. Lignocellulosics are abundant sources of carbohydrate, continually replenished by photosynthetic reduction of carbon dioxide by sunlight energy (Fan et al., 1987). Thus they are the most promising feedstock for the production of energy, food and chemical (Wu and Lee, 1997). The bioconversion of cellulosic materials has been receiving attention in recent years. It is now a subject of intensive research as a contribution to the development of a large scale conversion process beneficial to mankind. Lignocellulosic biomass has complex structure containing mainly cellulose, lignin, and hemicellulose. It is difficult to separate cellulose and hemicellulose for saccharification without the loss of carbohydrates. Therefore for efficient enzymatic or acid saccharification from lignocellulosic biomass pretreatment is necessary process. 17

3 Pretreatment increases the crystallinity of cellulose while removing lignin and other inhibitors, there by enabling its enzymatic degradation. In addition pretreatment may increase the surface area of the cellulose thereby enhancing its reactivity with the enzyme and thus its transformation. Among the various biomass materials lignocellulosic biomass has been looked on as a promising feedstock because of their abundance, their cheapness and their huge potential availability which could be converted into fermentable sugars (Kewalrami, 1998). Compared to agricultural residues, the lignification degree and cellulose crystallinity are higher which cause more difficult for organisms to attack wood cellulose. The most important step of lignocellulosic biomass bioconversion is cellulose hydrolysis but due to the complex structure of the lignocellulosic substrate the difficulty is how to decompose it to fermentable sugars high efficiently. Therefore, removing the lignin block and making the cellulose contact with cellulose is the key to improve the hydrolysis. So pretreatment to wood before bioconversion appeared to be more necessary. There are many methods for pretreatment including physical, chemical and biological. Cellulose and hemicellulose are macromolecules from different sugars whereas lignin is an aromatic polymer synthesized from phenyl propanoid precursors. The composition and percentage of these varies from one species to another. Cellulose makes up about 45% of the dry weight of wood. This linear polymer is composed of D-glucose subunits linked by β-1, 4 glycosidic bonds forming cellobiose molecules. These from long chains linked together by hydrogen bonds and Vaderwals forces. Biochemical or chemical conversion is required to transform this insoluble polymeric form of glucose into useful materials. Lignocellulose breakdown is catalyzed by secreted enzymes which constitute a major fraction of the extracellular proteins in soil and compost systems. Cellulose is the 18

4 predominant structural constituent in wood fibers. It is the predominant structural constituent in wood fibers. It occurs in nature solely in a semi-crystalline state. Cellulose (C 6 H 10 O 5 ) n is a large chain polymeric polysaccharide carbohydrate of beta-glucose. It is a linear homopolymer consisting of glucose units joined by β-1, 4 bonds (Leschine, 1995) and in most crop residues it is embedded n a matrix of hemicelluloses, lignin, pectin and proteins (Ghosh and Singh, 1993) and is totally insoluble in water and is a linear, unbranched homopolysaccharide consisting of glucose subunit joined together via β, 1-4 glycosidic linkages. Individual cellulose molecules vary widely in length and are usually arranged in bundles or fibrils (Walsh, 2002). Cellulose is probably the most abundant biological compound on terrestrial earth. It is the dominant waste material from agriculture in the form of stalks, stems and husk and is one of the most abundant renewable sources. By means of chemical or bioconversion methods it is possible to transform this insoluble polymer into glucose, an excellent substrate for industrial fermentation. Mainly bacteria, fungi and actinomycetes achieve bioconversion of these materials. Bioconversion or enzymatic hydrolysis of cellulose has been regarded as the ultimate choice in view of the specificity as well as the simpler operating conditions for producing glucose. Hemicellulose is a complex carbohydrate polymer and makes up 25-30% of total wood dry weight. It is polysaccharide with a lower molecular weight than cellulose. Sugars are linked to either β-1, 4 glycosidic bonds and occasionally β-1, 4-glycosidic bonds. The hemicelluloses are associated with cellulose and are branched, low molecular weight polymers composed of several different kinds of pentose and hexose sugar monomers. The component sugars of hemicellulose are of potential interest for conversion into chemical products. The inorganic component of extraneous material generally constitutes 0.2% to 1.0% of wood substance, although greater values are 19

5 occasionally reported. The enzymatic machinery for degrading cellulose, hemicellulose and lignin is possessed only by microorganisms. Lignin along with cellulose is most abundant polymer in nature. It is present in the cellular cell wall which is insoluble in water and optically inactive. The polymer is synthesized by the generation of free different radicals which are released in the peroxidase mediated dehydrogenation of three phenylpropionic alcohols. Coniferyl alcohol (guaiacyl propanol), Coumaryl alcohol (p-hydroxyphenyl propanol), Sinapyl alcohol (syringyl propanol). Coniferyl alcohol is the principal component of softwood lignins whereas guaiacyl and syringyl alcohols are the main constituents of hardwood lignins. Lignins present in the raw materials and releasing fermentable sugars are eliminated by chemical, thermal pretreatment followed by enzymatic or acidic hydrolysis. However, biological treatments have been proposed either to replace the physicochemical treatment or for detoxification or specific removal of inhibitors prior to fermentation. It is very large molecule and completely insoluble materials so the first step in their degradation by environmental microbes must be extracellular. No microbial cell can absorb molecular complexes of the size of a cellulose fiber still less that of lignin molecules. It is a complex heterogeneous polymer of phenlypropanoid units (Ghayal and Khandeparkar, 1994). Structurally lignin is an amorphous heteroploymer, non water soluble and optically inactive it consists of phenyl propane units joined together by different types of linkages. White rot fungi are the microorganisms that most efficiently degrade lignin from wood. Two major families of enzymes are involved in ligninolysis by white rot fungi: peroxidases (Lignin peroxidase; Lip, Manganese dependent peroxidase; Mnp) and laccases. By virtue of such a structure, the lignocellulosic crop materials are relatively refractory to direct bioconversion and hence pretreatment is necessary for the 20

6 effective utilization of lignocellulosic material (Ghosh and Singh, 1993). Theoretically lignin might be converted to a variety of chemical products. The polymers that make up lignocellulose give strength to plant materials and are the dominant component of biomass on the land surface. Lignocellulose is the principal form of fixed carbon in the terrestrial biosphere. It is the world s most abundant natural biopolymer and a potentially important source for the production of industrially useful materials such as fuels and chemicals. The biological degradation of cellulose, hemicellulose, and lignin has attracted the interest of microbiologists and biotechnologists for many years. Fungi are the best known microorganisms capable of degrading these three polymers. Microorganisms have two types of extracellular enzymatic systems: The hydrolytic system, which produces hydrolases and is responsible for cellulose and hemicellulose degradation and a unique oxidative and extracellular lignilolytic system, which depolymerizes lignin. The enzymatic conversion of the carbohydrate part of lignocellulosic material has received considerable interest during recent years. This source of raw material is available in abundance and generally free of cost. Enzymatic hydrolysis of cellulose for sugar production shows many advantages like low energy requirement, mild operating conditions over other chemical conversions and minimal by product formation. It leads to the production of reducing sugars, a substrate for single cell protein and a raw material for industrial fermentation. To reduce the production cost and enhance the formation of cellulases, which are both essential for the utilization of the carbohydrate components of lignocellulosics, different strategies can be applied (Kewalrami, 1998). It is the primary product of photosynthesis in terrestrial environments, and the most abundant renewable bioresource produced in the biosphere (100 billion dry tons/year) (Jarvis, 2003). Cellulose is commonly degraded by an enzyme called cellulase. 21

7 Cellulases have been applied in different sectors of food and cotton industries with prominence to the treatment of agricultural and industrial wastes (Anguair, 2001). Most of the cellulolytic microorganisms belong to eubacteria and fungi. Micro organisms capable of degrading cellulose produce a battery of enzymes with different specificities working together. Cellulases hydrolyze the β-1, 4-glycosidic linkages of cellulose. They are divided into two classes referred to as endoglucanases and cellobiohydrolases. Endoglucanases (endo-1, 4-β-glucanases, EGs) can hydrolyze internal bonds releasing new terminal ends. Cellobiohydrolases (exo-1, 4-β-glucanases, CBHs) act on the existing or endoglucanase generated chain ends. An effective hydrolysis of cellulose also requires β-glucosidases which break down cellobiase releasing two glucose molecules. The release of sugars from cellulose is the main basis of microbial interactions occurring in such environments (Leschine, 1995). Cellulases are consortium of free enzymes which comprise of Endoglucanses (β-1,4-d-glucan-4-glucanohydrolase which hydrolyze cellulose to gluco oligosaccharides, Exoglucanases including cellobiohydrolases release cellobiose from crystalline cellulose Carboxymethylcellulase, Exoglucanse (β-1, 4-D-glucan-4-glucohydrolase, and β- glucosidases which degrade oligosaccharides to glucose (Lynd et al., 2002). The right proportion of these enzymes acts synergistically for maximum saccharification. Endoglucanase cleaves internal β-1, 4-glucan chain links in cellulose randomly and opens the molecules for cellobiohydrolases which hydrolase the bonds at non reducing end of crystalline cellulosic chain producing Cellobiose. Cellobiases split the disaccharide units and convert cellobiose into glucose and thus complete the cellulolysis (Duenas et al., 1995). Cellulases are of great ecological and commercial importance in amelioration and redunting of municipal, forestry and agricultural waste from paper, lumber and textile 22

8 industries to control environmental pollution,biocomposting to produce natural organic fertilizers, production of food and feed supplements for cattle and poultry feed stocks, production of plant protoplast for genetic manipulation, preparations of pharmaceuticals, baking, malting and brewing, extraction of fruit juices and processing of vegetables, botanical extraction for maximum oil yield, processing of starch and fermenting tea and coffee. The key element in bioconversion process is the hydrolytic enzymes mainly cellulases. Cellulase refers to a family of enzymes, which act in concert to hydrolyze cellulose. Cellulase enzyme has been reported by Fan et al. (1987); Wu and Lee, (1997); Kansoh et al. (1999); Ojumu et al. (2003) and Immanuel et al. (2007) for the bioconversion of lignocellulosics to these useful products. Cellulase is the most abundant renewable resource on terrestrial earth, and enzymatic conversion of cellulosic materials to glucose is a very promising process. Enzymatic hydrolysis of cellulose has the advantage of being energy sparing and avoids the use of toxic substances or corrosive acids because of the relatively mild reaction conditions. Since the production of cellulase enzyme is a major factor in the hydrolysis of cellulosic materials, it is important to make the process economically feasible. Much study has been done on the production of cellulase from lignocellulosic substrates (Solomon et al., 1999; Depaula et al., 1999; Kansoh et al., 1999; Milala et al., 2005; Immanuel et al., 2007; Alam et al., 2008). Cellulases are known to be produced by many microorganisms, especially fungi. These cellulase producers are of industrial significance and for maintenance of global carbon cycle. Cellulases are among the industrially important hydrolytic enzymes and are of great significance in present day biotechnology. Cellulases are widely used in food, feed, textile and pulp, paper industries (Nakari and Pentilla, 1996). The cellulase system 23

9 in fungi is considered to comprise three hydrolytic enzymes: Endo-(1, 4)-β-D-glucanase (endoglucanase, endocellulase, CMCase) which cleaves β-linkages at random, commonly in the amorphous parts of cellulose. Exo-(1,4)-β-D-glucanase (cellobiohydrolase, exocellulase, microcrystalline cellulase, avicelase,c1) which releases cellobiose from non-reducing or reducing end, generally from the crystalline parts of cellulose and β- glucosidase (cellobiase) which releases glucose from cellobiose and short chain cello oligosaccharides (Bhat and Bhat,1997). Exoglucanse (EG or Cx), hydrolyses internal β-1, 4 glucan chain of cellulose at random, primarily within amorphous regions and display low hydrolytic activity toward crystalline cellulose (Walsh, 2002; Grassin and Fauquembergue, 1996). Cellulase catalyses the conversion of insoluble cellulose to simple, water-soluble products. Cellulase is multienzyme system composed of several enzymes with numerous isoenzymes, which act in synergy. Cellulases have a wide range of applications. Potential applications are in food, animal feed, textile, fuel, chemical industries, paper and pulp industry, waste management, medical/pharmaceutical industry, protoplast production, genetic engineering and pollution treatment (Tarek and nagwa, 2007; Beguin and Anbert, 1993; Coughlan, 1985; Mandels, 1985). Glucose produced from cellulosic substrate could be further used as substrate for subsequent fermentation or other processes which could yield valuable end products such as ethanol, butanol, methane, aminoacid, single cell protein etc (Walsh, 2002). Cellulase is a synergistic enzyme that is used to break up cellulose into glucose or other oligosaccharide compounds (Chellapandi and Jani, 2008). They are important industrial enzymes and find applications in several industrial processes (Hanif et al., 2004). The recent thrust in bioconversion of agricultural and industrial wastes to chemical 24

10 feedstock has led to extensive studies on cellulolytic enzymes produced by fungi and bacteria (Baig et al., 2004). Solid state fermentation (SSF) is the culture of microorganisms on moist solid substrates in the absence or near absence of free water. Whereas submerged fermentation (SF) consists of the cultivation of microbial cells in liquid media under controlled condition for the production of desirable metabolites. Based on SSF processes, agriculture wastes can readily be used as substrates for the cultivation of numerous microorganisms for the production of various metabolites which are important for industrial applications. Some of these products include enzymes, flavoring compounds, pigments, pharmaceutical products and industrial chemicals. Enzymes are the most frequently reported metabolites produced via SSF some of which include cellulases, xylanases, lipases, mannanases, phytases, proteases, lignin degrading enzymes.pulp and paper industries form the largest consumer of cellulases and hemicellulases (Bajpai, 1999). Cellulases can be either produced via SSF or SF using bacteria, yeast and filamentous fungi. SSF offers advantages over fermentation in liquid broth (SF) like higher product yield, better product quality, cheaper product recovery and cheaper technology. These fermentation systems which are closer to the natural habitats of microbes may prove more efficient in producing certain enzymes and metabolites (Oguntimein et al., 1992). A great variety of fungi and bacteria can fragment these macromolecules by using hydrolytic or oxidative enzymes and use as a carbon source. Cellulolytic enzymes are synthesized by a number of microorganisms. Fungi and bacteria are the main natural agents of cellulose degradation (Lederberg, 1992). The enzymatic degradation of cellulose requires cellulose-cellulase system. Though a large number of microorganisms 25

11 are capable of degrading cellulose only few of them produce significant quantities of cell free cellulase capable of completely hydrolyzing crystalline cellulose in vitro (Kotchoni et al., 2003). The highly productive and diverse microbial community living in tropical and subtropical mangrove ecosystems continuously transforms nutrients from dead mangrove vegetation into sources of nitrogen, phosphorus and other nutrients that can be used by the plants. To conserve the mangrove ecosystems which are essential for the sustainable maintenance of coastal fisheries, maintenance and restoration of the microbial communities should be undertaken. Mangroves represent one of the most productive ecosystems in tropical environments and are characterized by efficient turn over of nutrients (Kothari, 2002).Presence of wide diversity of organisms in mangroves is associated with the degradation of plant litter and this brings about the cycling of carbon and nitrogen in the system. Free living bacteria, fungi, and yeasts have been reported to play important role in the formation of detritus (Von Prahl, 1980; Maria and Sridhar, 2002). Effective pretreatment is needed to reduce the lignin content and crystallinity of the cellulose and to increase the surface area or pore size of materials. Since lignin makes the access of cellulolytic enzymes to cellulose difficult, its removal is especially necessary prior to the enzymatic hydrolysis (Sun and Cheng, 2002). Efforts have been made to find out suitable ways of microbial and enzymatic modifications of cereal straws to improve their biodegradability and to upgrade their feeding value for ruminants (Akin et al., 1995). Among the microorganisms soil fungi received much attention in the production of cellulolytic and lignolytic enzymes in delignification of paddy straw for improving digestibility (Kirk et al., 1980). The availability of highly active cellulose that works effectively in low concentration would 26

12 be of great significance. This requires selection and improvement of suitable strains for large amount of enzyme production which can be done using different mutagenic agents like UV irradiations, Gamma irradiation and Chemical mutagens. Fungi are the chief source for various industrially important enzymes as they are able to synthesize and secrete large amounts of extracellular enzymes. The exponential increase in the application of enzymes in various fields in the last few decades demands extension in both qualitative improvement and quantitative enhancement through strain improvement and medium optimization for higher enzymatic yield. Improvement microbial stains for the over production of industrial products has been the hallmark of all commercial fermentation process. The microorganisms are used as potential biotechnological sources of industrially relevant enzymes have stimulated a renewed interest in the exploration of extracellular enzymatic activity. Screening programmes for the selection of microorganisms able to produce extra cellular enzymes continue to be an important aspect of biotechnology although the advances in genetics and microbial physiology are having strong impact on enzyme production. Wastes and their disposal have become enough substances of environmental concern. The use of biological means have greater advantages over the use of chemicals for degradation because biotechnological synthesized products are less toxic and environmentally friendly (Liu et al., 1998). Large quantities of lignocellulosic wastes are generated through forestry, agricultural practices and industrial processes, particularly from agro-allied industries such as breweries, paper pulp, textile and timber industries. These wastes generally accumulate in the environment thereby causing pollution problem (Abu et al., 2000). 27

13 The bioconversion of cellulosic material is now a subject of intensive research as a contribution to the development of a large- scale conversion process beneficial to mankind (Kumakura, 1997). 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. However, some features of natural cellulosic materials, like the degree of crystallinity, lignification and capillary structure are known to inhibit their degradation or bioconversion The availability of highly active cellulase would be of great significance and hence requires selection and improvement of suitable strains for enzyme production and development of fermentation technology for producing them in large quantity. Besides they are used in animal feeds for improving the nutritional quality and digestibility in processesing of fruit juices and in baking while deinking of paper is yet another emerging application (Tolan and Foody, 1999). A potential challenging area where cellulases would have a central role is the bioconversion of renewable cellulosic biomass to commodity chemicals. The cost of production and low yields of these enzymes are the major problems for industrial application. Therefore the investigations on the ability of the cellulose hydrolyzing microbial strains to utilize inexpensive substrate have been done. Researchers have strong interests in cellulases because of their applications in industries of starch processing, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp and paper industry and textile industry (Gao et al., 2008). Perpetual renewal of plant biomass via the process of photosynthesis ensures an inexhaustible supply of such material. Any process which could efficiently and economically convert cellulolytic material to glucose would be of immense industrial significance. 28

14 Considering the importance and application of the cellulases this study aimed to screen the fungal isolates from mangrove soil for the cellulolytic ability. Also for understanding the condition for the production and activity of cellulases by different fungal cultures. This work focuses on factors relevant for improvement of enzymatic hydrolysis of lignocellulosic material sawdust. To understand the biochemistry of lignocellulose degrading fungi it is needed to optimize various conditions. The purpose of this work was also to investigate the possibility of producing cellulase enzyme from lignocellulosic substrate by using mangrove fungi. As the possibility of the mangrove fungi in the degradation of the lignocellulosic substrate sawdust and production of cellulase enzyme by using mangrove fungi is least studied and reported till now. Work also includes production of valuable enzyme from cheap, renewable raw material to achieve sustainable development. The main objectives of the study were: 1. To isolate, identify and screen cellulase producing fungi from mangrove soil 2. To check the production of cellulase enzyme in SSF and SF 3. To optimize the conditions for maximum production of cellulase 4. To check the effect in enzyme production by UV irradiation 5. To analyze the biodegradation and possibility of product formation 6. To study the nutrient effects and growth curve of fungi 29