Chapter 2 Literature review
Lignocellulose: Nature’s tightly wrapped gift
Lignocellulose are renewable energy resources that are abundant and found naturally (Perez et al., 2002). They are a natural part of all plants and are generated as waste products of forestry, agriculture and agro-industry. Their accumulation tends to create a problem of waste disposal. Therefore it can be utilised as an inexpensive feedstock in the production of chemicals and biofuels without placing undue stress on scarce food resources that could lead to food prices increase particularly as they are considered to be inedible materials (Balan, 2014; Searchinger Heimlich, 2015). Moreover, the utilization of lignocellulosic agricultural residues has the potential to provide intermediate building blocks chemicals and second generation biofuels without negative competition for land (Walford, 2008; Searchinger & Heimlich, 2015).
Lignocellulosic materials do not contain readily accessible monosaccharides. Considerable efforts in researches are focused on the bioconversion of lignocellulosic materials into platform sugars that can be readily fermented to fuels and chemicals (Mahajan, 2011). One major limitation to its use in the production of bioethanol is its recalcitrant nature to degradation; this means that unlike starch and sugar, lignocellulose require pre-treatment before fermentation. In addition, the structural compositions of lignocellulose are complex and variable and the likelihood of formation of inhibitors to fermenting microorganisms further increases technical challenges (Mosier et al., 2005a; Petersson, et al., 2007; Jönsson & Martín, 2016) and production cost involved in fermentation processes towards the production of bioethanol and biogas alike. Therefore, working with lignocellulosic biomass makes it necessary to factor in the digestibility of the main constituent because it is only with pre-requisite treatment can the cellulose in the plant fibres become exposed for degradation (Kumar et al., 2009).
Composition of Lignocellulose
Plant cell are composed mainly of lignocellulose which comprise of three major groups of polymers – cellulose, hemicellulose and lignin (Mussatto & Teixeira, 2010). The cellulose framework is surrounded by hemicellulose and lignin.
The composition of these constituents can vary from one plant species to another (Table 1) (Kumar et al., 2009; Sorek et al., 2014). Furthermore, the ratios between constituents within a single plant vary with age, stage of growth and other environmental conditions (Perez et al., 2002). Except for cellulose, the other polymers are synthesized within the cell and extruded to cell membrane where they are organized in a matrix-like arrangement; with the primary cell deposited first. This is characterized by relatively amorphous cellulose structure, followed by the synthesis of the secondary cell wall after cell differentiation characterized by cellulose microfibrils with higher crystallinity and altered hemicellulose content (Ding & Himmel, 2006).
The plant cell wall is primarily composed of cellulose which is synthesized by a cellulose synthase complex that is found within the cytoplasmic membrane of plant cells (Taylor et al., 2000; Li et al., 2014). It is a linear homopolysaccharide with repeated units of cellobiose (two anhydrous glucose rings joined via a β-1, 4 glycosidic linkages) (Beguin & Aubert, 1994; Klemm et al., 1998; Li et al., 2014).
These cellobiose units are linked by hydrogen and van der Waals bonds which cause the cellulose to be packed into microfibrils that is made up of highly organized crystalline regions and unorganized amorphous regions (Ha et al., 1998; Khazraji & Robert, 2013) intertwined with both hemicellulose and lignin. Crystalline cellulose is less soluble and less degradable and makes up the larger ratio as compared to amorphous cellulose. The arrangement of crystalline and amorphous cellulose gives plant the unique properties of dual rigidity and flexibility; the hydrogen bond promotes insolubility in most solvents and is partly responsible for the resistance of cellulose to microbial degradation (Jørgensen et al., 2007).
Crystalline cellulose can be degraded to fermentable D-glucose using acids or enzymes that break the glycosidic bonds; however the amorphous form of cellulose is more susceptible to enzymatic degradation (Beguin & Aubert, 1994; Yang et al., 2011). The enzymes for cellulose degradation belong predominantly to hydrolases; cellulose is hydrolyzed by cellulase (endoglucanase), 1,4-β-cellobiosidase and β-glucosidase (Schmidt, 2006).
Hemicellulose is a general term used to represent a group of complex heterogenous polysaccharides characteristically made up of different sugars, such as D-glucose, D-mannose, D-galactose, D-xylose, L-arabinose, as well as other components such as acetic, glucoronic and ferulic acids.
Hemicellulose is similar to cellulose in that – their basic structural compositon is primarily β-1, 4-linked sugars, although, the particular sugar composition of hemicellulose is dependent on the source of polysaccharide. This also means that the main sugar residue in its basic structural composition determines the classification of hemicellulose, for example, xylans, mannans, glucans, glucuronoxylans, arabinoxylans, glucomannans, galactomannans, galactoglucomannans, β-glucans, and xyloglucans (Fengel & Wegener, 1989). Hayashi and Kaida (2011) describes this aptly using xyloglucan which is the main hemicellulose of primary cell walls and thus consists of a β-1, 4-linked glucose backbone that is surrounded by xylose, galactose and in some instances fructose branching sugars. By contrast the main hemicellulose in secondary cell walls of hardwoods and softwoods is xylan and galactoglucomannan respectively. Xylan is composed of β-1, 4-linked xylose that can be substituted by arabinose and glucuronic acid; xylan can also be acetylated. Xylan can be degraded by endo-1, 4-β-xylanase and 1, 4-β-xylosidase to xylose (Jørgensen et al., 2003). Furthermore, wood-derived mannans are composed of β-1, 4-linked mannose and glucose that can be substituted by galactose (Stenius & Vuorinen, 1999; Liepman et al., 2007).
Fengel & Wegener (1989) summarize the difference between hemicellulose and cellulose in terms of sugar-units composition by stating that – hemicelluloses possess shorter chains consisting of different sugars that branch off the main chain molecules; this makes them amorphous and thus makes the structure easier to hydrolyze than cellulose. These monosaccharide chains include pentoses (xylose, rhamnose and arabinose), hexoses (glucose, mannose and galactose) and uronic acids (4-omethylglucuronic, D-glucuronic, and D-galactouronic acids) linked by -1, 4-glycosidic bonds and sometimes by -1, 3-glycosidic bonds (Kuhad et al., 1997).
Hemicelluloses have a lower degree of polymerization (DP 100 – 200) and a lower crystallinity than cellulose (Rowell, 2005). While cellulose is synthesized by cellulose synthases located within the cytoplasmic membrane, hemicelluloses are synthesized in the Golgi complex, and then secreted to the plant cell wall (Keegstra, 2010). The heterogenous nature of hemicellulose compared to cellulose, implies that a complex mixture of enzymes is required for its degradation, such as endoxylanases, β-xylosidases, endomannanases, β-mannosidases, α-L-arabinofuranosidases and α-galactosidases (Jørgensen et al., 2005).
The hydrolysis of hemicellulose portions of the lignocellulosic biomass produces not only hexose sugars but pentose sugars as well as potential microbial inhibitors such as uronic, ferulic and acetic acids which exerts undue stress on the fermenting microorganisms, leading to poor cell growth and low ethanol yields. Although progress has been made to ameliorate the effects of these inhibitors and increase the sugar level and enhance overall fermentability of lignocellulosic hydrolysate, it still remains a challenge in exploring lignocellulosic biomass as fermentation substrates (Blaschek & Ezeji, 2010; Parawira & Tekere, 2011).
Lignin is a complex macromolecule that is structurally composed of three phenyl propane units [p-coumaryl alcohol (p-hydroxyphenyl propanol), coniferyl alcohol (guaiacyl propanol) and sinapyl alcohol (syringyl alcohol)] linked together by carbon-to-carbon (C–C) and ether (C– O–C) linkages (Humphreys & Chapple, 2002; Mussatto & Teixeira, 2010). Its structure is believed to be the result of radical polymerization (Blanchette et al., 1997; Ralph et al., 2004).
The structure of lignin varies widely within species. There is a general consensus that plants such as grasses have the lowest contents of lignin, whereas soft woods have the highest lignin contents. In grasses p-coumaryl alcohol is found predominantly causing the formation of H-lignin, while in soft wood coniferyl alcohol is the main monolignol forming G-lignin, and in hardwood G/S-lignin contains both sinapyl and coniferylmonolignols (Perez et al., 2002; Humphreys & Chapple, 2002).
Lignin is present in the cell wall and is tightly bound to cellulose and hemicellulose; it confers rigidity and structural cohesion to the cell wall, it also acts as a reinforcing component to connect cells and harden xylem vessels, thus, conferring water impermeability but also improves transportation of water from roots to leaves. It forms an amorphous complex with hemicellulose enclosing cellulose and thus prevents the microbial degradation of accessible carbohydrates within wood cell wall (Perez et al., 2002; Barcelo et al., 2004; Schmidt, 2006; Ruiz‐Dueñas & Martínez, 2009).
In contrast to cellulose and hemicellulose; the three-dimensional configuration of lignins makes it highly hydrophobic causing extreme resistance to chemical and enzymatic degradation (Palmqvist & Hahn-Hägerdal, 2000).The degradation of cellulose and hemicellulose portions of lignocellulosic materials produces a significant percentage of fermentable sugars for ethanol production, but the contrary is the case with lignin degradation which is recognized as a potential source of microbial inhibitors (Ezeji et al., 2007); but it can be used as an ash free solid fuel for production of heat and electricity (Galbe & Zacchi, 2002).
Lignocellulosic Agricultural residues used in this study
The understanding that lignocellulosic agricultural waste potentially contain significant concentrations of soluble carbohydrates and inducers that promote the growth of fungi and the efficient production of ligninolytic enzymes has made these waste residues an attractive option as platform ingredients in fermentation technology (Sun et al., 2004; Rosales et al., 2005; Kachlishvili et al., 2006; Winquist et al., 2008; Elisashvili et al., 2009). The implication of exploiting agricultural residues could potentially reduce production cost and serve as an effective method of agro-industrial waste recycle. Furthermore, most ligninolytic fungi have been observed to demonstrate increased enzyme activity when growing on these agricultural residues as compared to the low enzyme activity on defined medium (Bollag & Leonowicz, 1984; Elisashvili et al., 2006; Songulashvili et al., 2007; Magan et al., 2010).
Three of these agricultural residues were the focus of this study because of their preponderance in South Africa. Furthermore it is necessary to consider more than one substrate to accommodate the regional differences in substrate availability. This is necessary because each region cultivates a particular crop based on its climatic conditions and soil differences. The elucidation of physical and chemical properties of commonly found lignocellulose wastes in South Africa is essential for the future design of subsequent processing operations schemes.
The largest agricultural biomass in South Africa comes from corn production, with the total area planted per year varying between 3.8 and 4.8 million hectares which represents approximately 25% of the country’s total arable land (National Department of Agriculture, n.d). The direct implication of corn production is the generation of waste biomass (corn plant residue), which in South Africa alone, accounts for 8, 900 thousand metric tonnes of waste annually (Nation Master, 2005). These waste products are potential source of feedstock for bio-fuels.
Approximately 50% of the weight of the total corn plant is residue with the above ground corn plant, stover, consisting of stalk, leaf, cob and husk that are potentially useful as biomass feedstock (Graham et al., 2007). Approximately 66% of fermentable sugars can be obtained from corn stover, of which approximately 38% and 28% come from cellulose and hemicellulose portions, respectively. Producing ethanol from corn stover hydrolysates will increase the possibility of ethanol production on a larger scale (Blaschek & Ezeji, 2010).
Chapter 1 Overview
Chapter 2 Literature Review
2.1 Lignocellulose: Nature’s tightly wrapped gift
2.2 Composition of Lignocellulose
2.3 Lignocellulosic Agricultural residues used in this study
2.4 Pre-treatment of Lignocellulosic Biomass
2.5 Ligninolytic Enzymes
2.6 Other enzymes involved in lignocellulose degradation – Cellulolytic enzymes
2.7 Factors that influence enzyme production by ligninolytic fungi
2.8 Fungal Interspecific Interactions in Mixed Cultures
2.9 Application of antagonistic interspecific interactions
2.10 Aims and Objectives of Study
Chapter 3 Isolation, Screening and Molecular Characterization of Ligninolytic Fungi
3.2 Literature Review
3.3 Materials and Method
Chapter 4 Macroscopic and Microscopic changes during antagonistic interaction of fungi
4.2 Materials and Method
Chapter 5 Comparative evaluation of enzyme production efficiency of monocultures and paired interactions of fungi on different agricultural substrates
5.2 Literature Review
Chapter 6 Synthesis and Conclusion
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