There are various types of agricultural residues that are not useful to man and thus constitute a nuisance to the environment. About 147.2 million metric tons of fiber sources are found in the world while the global output of wheat straw residue and rice straw were estimated at 709.2 and 673.3 million metric tons respectively in the 1990’s. Additionally the total global output of non wood fibers was put at about 2.5 million metric tons. About 61 million metric tons of crop residue available in Nigeria, out of these only 21% are consumed by sheep and goat and only if they are processed into acceptable and digestible forms.
Lignocellulose are the major structural component of woody plant and non woody plant such as grasses and represent a major source of renewable organic matter. Lignocelluloses waste refers to plant biomass wastes that are composed of cellulose, hemicelluloses and lignin. The chemical properties of the component of lignocellulosics make them a substrate of enormous biotechnological value (Malherbe and Cloete, 2003). Large amounts of lignocellulosic waste are generated through forestry and agricultural practice and they pose an environmental pollution problem. They may be grouped into different categories such as wood residues (including sawdust and paper mill discards), grasses, waste paper, agricultural residues (including straw, stover, peelings, cobs, stalks, nuts, shells, non-food seeds, bagasse, domestic waste lignocelluloses garbage and sewage), food industry residues, municipal solid waste, (Qi, et al., 2005; Roig, et al., 2006; Rodriguez, et al., 2008). However the huge amount of residual plant biomass considered as waste can potentially be converted into various different value added products. Bioconversion offers a cheap and safe method of not only disposing the agricultural residues but also it has the potential to convert lignocellulosic waste into usable forms such as reducing sugars that could be used for the production of other value added products. The biomass feedstock in Nigeria can provide enough potential of bioconversion of cellulosic materials is frequently considered because of their high availability and low cost.
1.1 Groundnut shells
Groundnut (Arachis hypogea) is an important oil seed crop of Nigeria. The pulp or dry pericarp contains about 25-40 percent shell (Dey, et al., 2002). The chemical composition of groundnut shell is as follows; cellulose 65.7; protein 7.3; mineral, 4.5; and lipids 1.2%. (Masenda et al., 2004). Groundnut shell is used in mushroom cultivation (Reneau, et al; 1980) and the production of extracellular enzymes by prodigious cellulolytic fungi. These groundnut shells waste have been insufficiently disposed leading to environmental concern (Fabiyi and Ogunfowora, 2011). Plant lignocellulosics as organic substance are subjects to attacks by biological agents such as fungi, bacteria and insects (Highleey, et al., 1987) breakdown the long chain in cellulose to release the sugar through hydrolysis reaction, but because of their ability, they can achieve higher yield of glucose from cellulose (Wyman, 2004). A portion of pretreated biomass can be used by organisms that produce cellulase that can then be added to pretreated solids to release glucose from cellulose. Filamentous fungi which use cellulose as carbon source possess the unique ability to degrade cellulose molecule. Fungi are the main cellulase producing microorganism though a few bacteria have also been shown to have cellulase activity. Fungal genera like Tricoderma and Aspergillus are known to be cellulase producers. The genus Aspergillus species attack cell wall to free little amount of the cell free cellulase capable of hydrolyzing cellulose to fermentable soluble sugars such as glucose in chemical industries.
Enormous amount of agricultural, industrial and municipal cellulosic waste have been accumulating or used inefficiently due to the high cost of their utilization processes (Lee et al., 2002).
1.2 Statement of problem
One of the limitation to the use of groundnut shells which constitute a nuisance in our environment is the fact that organism that can break the barrier the lignin structure holding up the cellulose content of the groundnut shell to be accessible has not been identified.
This work was aimed at biodegradation of groundnut shells to fermentable sugar using fungal isolates.
1.3.1 Objectives of the study
The objective of this study includes:
- To isolate fungal from steeped groundnut shell.
- To determine the best suited culture conditions for degradation of the groundnut shells to fermentable sugar.
- To determine the fungal isolate best suited for the degradation.
- To compare the action of the fungal in monoculture and in co-culture
1.4 Literature Review
Lignin is a complex cross-linked polymers of aromatic rings (phenolic monomers). A highly branched macromolecule composed of several types of aromatic acids that has an important role in the physical characteristics of wood as well as in preventing lignocellulosic materials against biological attacks, e.g. against cellulose degrading microorganisms. The term lignin was introduced in 1819 and derived from latin word lignum ( Sjostrom, 1993).
Cellulose a polymer of glucose residues connected by 1-4 linkage being the primary structural materials of plant cell wall is most abundant carbohydrate in nature (Saha, et al; 2005). Cellulose, a widely distributed long-chain polymeric and skeletal polysaccharide carbohydrate, of beta-glucose, is the most abundant renewable natural product that can be obtained in the biosphere. It forms the primary structural component of green plant, and represent about 50% of cell wall material of plants. The primary cell wall of plant is made of cellulose, the secondary wall contains cellulose with variable amount of ligin. Cellulose has been used by man for ages. However, its enormous potential as a renewable energy source was recognized only after cellulose degrading enzymes (Cellulases) had been identified (Bhat and Bhat, 1997). The cellulose molecules composed of longer slander bundles of long chains of β-D-glucopyranose residues liniked by β-1,4-glycosidic bonds, called elementary fibrils, by condensation. This is in contrast to the α-1, 4- glycosidic bonds present in starch: cellulose is a straight chain polymer. The molecule adopts an extended rod like conformation. In microfibrils, the multiple hydroxyl group on the glucose residues hydrogen bond with each other, holding the chains firmly together and contributing to their high tensile strength. This strength is important in cell wall, where they are meshed into a carbohydrate matrix, helping keep plant cells rigid (Lee, et al.,2002)
The molecule have two regions; one of this which is called crystalline cellulose’ is composed of highly-oriented molecules and another is called amorphous cellulose’ which comprise less oriented molecules. Many of this elementary fibrils form together in microfibril, and furthermore several microfibrils joined together form a macro fibril (Zarnea, 1994).
Compared to starch, cellulose is also much more crystalline. Given a cellulose material, the portion that does not dissolve in 17.5% solution of sodium hydroxide at 20 c is a-cellulose, which is true cellulose; the portion that dissolves and then precipitates upon acidification is B-cellulose; and the proportion that dissolves but does not precipitate is y-cellulose. The crystalline region is very difficult to break or hydrolyze because of strong bonds and also the tight packing of the cellulose structure. The region is always in orderly arrangement and waterproof. The amorphous region is solid and randomly arranged (Spano, et al.,1975).
The hydroxyl groups of cellulose can be partially or fully reacted with various chemicals to provide derivates with useful properties. Cellulose esters and cellulose ethers are the most important commercial materials. In principles, though not always in current industrial practice, cellulosic polymers are renewable resources. Among the esters are cellulose acetate and cellulose triacetate, which are film-forming and fiber-forming materials that find a variety of uses. The inorganic ester nitrocellulose was initially used as an explosive and was an early film forming material.
Annual cellulose production is estimated to be 1.5×10 tonnes. Proportion of cellulose in plant tissues are about 20 to 40% dry weight and about 90% in cotton fiber. Most cellulose in the nature exist as waste paper. Its potential as an alternative energy source has stimulated researches on converting cellulose to soluble sugars. One of the methods is pyrolysis to biological methods such as the application of cellulase enzyme (Bharadwaj, et al., 2004). Therefore, it has become considerable interest to develop processes for effective utilization of cellulosic waste as inexpensive carbon source. Cellulose, a widely distributed long-chain polymeric and skeletal polysaccharide carbohydrate, of beta-glucose, is the most abundant renewable natural product that can be obtained in the biosphere. It forms the primary structural component of green plant, and represents about 50% of cell wall material of plants. The primary cell wall of green plants is made of cellulose; the secondary wall contains cellulose with variable amounts of lignin. Cellulose has been used by man for ages. However, its enormous potential as a renewable energy source was recognized only after cellulose grading enzymes (Cellulases) had been identified (Bhat and Bhat,1997) The cellulose molecules composed of longer slander bundles of long chains of ß-D-glucopyranose residues linked by β-1, 4-glycosidic bonds, called ‘elementary fibrils’ by condensation. This is in contrast to the α-1, 4-glycosidic bonds present in starch, Cellulose is a straight chain polymer. The molecule adopts an extended rod-like conformation. In microfibrils, the multiple hydroxyl groups on the glucose residues hydrogen bond with each other, holding the chains firmly together and contributing to their high tensile strength. This strength is important in cell walls, where they are meshed into a carbohydrate matrix, helping keep plant cells rigid. (Lee et al., 2002).
The cellulose molecules composed of longer slander bundles of long chains of ß-D-glucopyranose residues linked by β-1, 4-glycosidic bonds, called ‘elementary fibrils’ by condensation. This is in contrast to the α-1, 4-glycosidic bonds present in starch, Cellulose is a straight chain polymer. The molecule adopts an extended rod-like conformation. In microfibrils, the multiple hydroxyl groups on the glucose residues hydrogen bond with each other, holding the chains firmly together and contributing to their high tensile strength. This strength is important in cell walls, where they are meshed into a carbohydrate matrix, helping keep plant cells rigid. (Lee et al., 2002)
126.96.36.199 Cellulose degradation (Cellulose breakdown)
The ability to breakdown cellulose is not possessed by mammals. Typically, this ability is possessed only by certain bacteria (which have specific enzymes) like Cellulomonas, which are often the flora on the gut walls of ruminants like cows and sheeps, or by fungi, which in nature are responsible for cycling of nutrients. The enzymes utilized to cleave the glycosidic linkage in cellulose are glycoside hydrolases including endo-acting cellulases and exo-actingglucosidases. Such enzymes are usually secreted as part of multienzyme complexes that may include dockerins and cellulose binding modules.
The ability of cellulolytic microorganisms to degrade cellulose vary greatly with the physio-chemical charactericstics of the substrate, such as the size and permeability of cellulolytic enzymes and other reagent molecules, which are involved in relation to the size and surface properties of the grown fibrils and the space between microfibrils and cellulose molecules from amorphous region. Degree of crystallinity of cellulose, the unit cell dimensions of cellulose, stereoscopic conformation and rigidity of the anhydrous glucose unit, degree of polymerization of cellulose molecules, the nature of components with which cellulose is associated and the nature, concentration and distribution of substituted groups are the other physio-chemical characteristics.
The crystallinity degree of cellulose is one of the most important structural parameters, which affect the rate of enzymatic degradation by hydrolysis. Therefore, the rate of degradation should be a function of the surface properties of cellulose, which makes possible the access of enzymes to polymeric molecules (Lee, 1982; Kit, 1987; Dhillion, 1984 .)
188.8.131.52 Cellulase and sub-classes
Cellulase enzymes exist in complex system in order to efficiently harvest energy produced by polysaccharides. These enzymes are complex modular proteins that are comprised of one or more catalytic domain and substrate binding domain, which hydrolyze a particular substrate. This enzymes complex breaks down cellulose to β-glucose. Cellulase enzyme complex system consist of three major components that are endoglucanase, C (EC 184.108.40.206), exoglucanase or cellobiohydrolase, C (EC 220.127.116.11) and cellobiase or β-glucosidase (EC 18.104.22.168). Members of all these classes are necessary to degrade cellulose (Bhat, 2009; Gielkens, 2003).
Endocellulase (β-1, 4-D glucan hydrolase) breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains. C’s substrate are cellulose (nature), cellodextrin (particularly hydrolyse), phosphoric acid hydrated(swollen) cellulose, CM cellulose (substrate normally used in cellulose assay system) and hydroxyl ethyl cellulose. The catalytic reaction by C involves a few steps. First, the cellulose must be activated through enzymatic oxidation, which is postulated to be catalyzed by cellbiose oxidase thatoxidizes glucose into uronic acid. This enzyme uses cellobiose (quinine oxidoreductase) as substrate. Cellbiose will be active only when cellulose fiber is associated with lignin. Endoglucanase attack the amorphous region of cellulose that is exposed. It cannot penetrate cellulose layer to hydrolyze amorphous region found in the centre of the bundle (Spano et al., 1975).
Exocellulase or known as β-1,4- D glucans cellobiohydrolasse scientifically, cleaves 2-4 unit from the exposed chains produced by endoglucanase, resulting in the tetrasaccharides or disaccharide such as cellobiose. C, has strong affinity towards cellulose and able to hydrolyse the crystalline cellulose up to 80%. Both endogluconase (c) and cellobiohydrolyse (C) will act synergistically to hydrolyse activated cellulose. Endoglucanase will act first on the amorphous region, which will be starting point for cellobiohydrolase to act on cellulose. Synergistic association is not strong when substrate is amorphous in nature (Wood and Philips 1969). Cellobiase or β-glucosidase (β-D-glucosidase glucohydrolase) hydrolyses the endocellulose product into individual monosaccharides known as β-glucose. Mainly symbiotic bacteria in the ruminating chambers of herbivores produce it. Besides the ruminants, most animals and humans do not produce cellulose. Therefore unable to use most of the energy contained in plant material. Secretion of β-glucosidase is highly dependent on the culture conditions. A relatively high pH can enhance β-glucosidase production. (Juhasz, et al., 2004).
For a complete hydrolysis of cellulose to glucose, the cellulose system must contain the following enzymes; Endoglucanase (1,4-β-glucan glucanohydrolase, E.C. 22.214.171.124), exoglucanase (1, 4-β-glucan cellulobiohydrolase, E.C 126.96.36.199), and β-glucosidase (β-glucoside glucohydrolase or cellobiase E.C 188.8.131.52), (Ryu, 1980, Sandhu, 1992, Wood, 1987 and 1987). Only the synergy of these enzymes makes it possible for cellulose to hydrolyze to glucose. In addition to C1 activity (Cotton hydrolyses activity) is necessary for splitting of the elementary fibrils from the crystalline cellulose (Fan, 1983, Woodward,1983, Nakanishi,1983).
184.108.40.206 Mechanism of Cellulase Activity
To penetrate into the crystalline cellulose region, nevertheless, C1 activity must be augmented by endoglucanase to break the 1,4-β linkage in the cellulose molecules (glucose chains). As soon as the two broken ends of the cellulose molecules are lifted, C1 activity enter beneath the cellulose molecules to release the glucose chain from the rest of the cellulose by its swelling and H-bond-breaking effects. The newly released long glucose chain (cellulose molecules) is broken down by endoglucanase into shorter chains (oligosaccharides, cellotetraoses, cellotrioses) (Woodward and wiseman, 1983).
Direct physical contact between enzyme and the surface of cellulose molecules is a preliminary requirement to hydrolysis (Robson, 1989; Beguim, 1990; Armstrong, 1983). Since the cellulose is an insoluble and structurally complex substrate, this contact can be achieved only by diffusion of the enzymes into the complex structural matrix of the cellulose (Nakanishi, et al, 1983). There are three types of reaction catalyzed by cellulose. The first step is the breakage of the non-covalent interactions present in the crystalline structure of cellulose (endo-cellulase). It is then followed by hydrolysis of the individual cellulose fibers to break it into smaller sugar (exo-cellulase) finally the disaccharides and tetra-saccharides are hydrolyzed into glucose by β-glucosidase (Champagne et al., 2007).
The first step in bioconversion of lignocellulosics to order value added useful product is size reduction and pretreatment (Gao, et al., 2008). The goal of any pretreatment technology is to alter or remove structural and compositional impediments to hydrolysis in order to improve the rate of enzyme hydrolysis and increase yields of fermentable sugars from cellulose or hemicelluloses (Mosier, et al., 2005). Pretreatment is an important tool for practical cellulose conversion processes. It is required to alter the structure of cellulose biomass to make more accessible to the enzymes that convert the carbohydrate polymer into fermentable sugars and to cellulose producing microorganisms (Patel, et al., 2007). A successful pretreatment must meet the following requirements:
- Improve formation of sugars or the ability to subsequently form sugars by hydrolysis.
- Avoid degradation or loss of carbohydrate.
- Avoid formation of byproducts inhibitory to subsequent hydrolysis and fermentation processes.
- Be cost effective (Silverstein, et al., 2004).
The pre-treatment stage promotes the physical disruption of the lignocellulosic matrix in order to facilitate acid or enzyme-catalyzed hydrolysis. It can have significant implications on the configuration and efficiency of the rest of the process and ultimately also the economics (Mabee, et al., 2006).
There are different methods of pre-treatment; they include mechanical, physical, chemical and biological.
1.5.1 Mechanical Pre–treatment
Mechanical based pre-treatment technologies are aimed at reducing the size of lignocelluloses waste to facilitate subsequent treatment. Reduction of biomass, size below sieves shows the best mechanical performance (de souse, et al., 2004). It increases the digestibility of cellulose and hemicelluloses in the lignocelluloses biomass. The use of mechanical chopping (De souse, et al., 2004), hammer milling (Iniguez Covarrubias, et al, 2001, Mani, et al., 2004), grid milling (Mtui and Nakamura, 2005), roll milling (Qi, et al., 2005), vibratory milling (Guerra, et al., 2006) and ball milling (Inoue, et al., 2008) have proved success as a low cost pre-treatment strategy. The pulverized materials with increased surface area have been found to facilitate the subsequent physiochemical and bio-microbial pre-treatment of groundnut shell and other lignocelluloses waste. They result to improved digestibility of cellulose and lignocelluloses to low enzyme loads. Mechanical uncondensed-arylether linkages (Inoue, et al., 2008) solubility and fermentation efficiency of the natural lignocellulosic residues leading to value-added utilization of these residues (Qi, et al., 2005).
1.5.2 Biological pre–treatment
Biological pre-treatment use fungi to solubilize the lignin. Bio-deligninfication is the biological degradation of lignin by microorganisms. It employs wool degrading microorganism, including white-brown, soft-rot fungi, and bacteria to modify the chemical composition and structure of the lignocellulosic biomass so that the modified biomass is more amenable to enzyme digestion (Lee, J. W. et al., 2007). Fungi have distinct degradation characteristics on lignocellulosic biomass. In general, brown and soft rots mainly attack cellulose while imparting minor modifications to lignin, and white-rot fungi are more actively degrading the lignin component (Schurz, J. 1978).
1.6 SUBMERGED FERMENTATION
Submerged substrate fermentation has been widely used in the production of cellulose and other products (Haltrich et al, 1996., Kim et al., 1997) Chahal et al.,(1996). Mixing in submerged processes can be by mechanical agitation or by air circulation. The submerged fermentation is widely used for it is estimated that 80% of the degradation of groundnut shell is obtained by submerged fermentation (Jose et al 2010., Sociol et al, 2006). This has the advantage of lower investment and maintenance cost compared with surface cultures. The disadvantages are the high cost of energy and a more sophisticated technology, which in turn requires specialized staff. Submerged fermentation can be carried out in batch, fed-batch or continuous systems, although the batch mode is more frequently used. Compared with surface culture, submerged culture are somewhat less sensitive to changes in the composition of media, which is an advantage when using molasses having a highly variable composition on the other hand, a typical problem of submerged culture is the formation of foam which can be avoided using anti-foam agents and chambers with volume of up to one third of the total fermentation volume. The common anti-foams are animal and vegetable fats or mechanical systems.
Co-culture can be defined as the growth of more than one distinct cell type in a combined culture which can be employed to monitor inter cellular communication and cell migration.