CHAPTER-02

REVIEW OF LITERATURE

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The production of cellulase and other lignocellulosic enzyme have been widely studied in submerged culture process in the laboratory, anging from shake flask to fermentations. A stirred tanker reactor, widely used for the production of cellulose and other lignocellulosic enzymes, is known to have shear problem which rupture mycelial cells and deactivate the enzymes (Anita and Namita 2009). Alternative bioreactors such as the air –lift or bubble –column, which have a lover shear stress , seem to produce better result. For eg. Studied on cellulase and xylanase production by Aspergillus niger in various bioreactors showed that in general , better yield and productivity were shown in a bubble –column and an airloopair – lift than in the stirred –tank reactor, however the relative high cost of enzyme production has hindered the industrial application of the enzymatic process. It has been reported that the solid state fermentation (SSF) is an attractive altenative process to produce fungal microbial enzymes using lignocellulosic material from agricultural waste due to its lover capital investment and lover operating cost (Anita and Namita 2009). The productivities of all enzymes except cellulase were higher in media with this Bagasse sample than that of pure cellulose powder. The saccharification of alkalitreated cotton and sugar cane Bagasse by P. funciculosum enzyme were 70 and 63 %, respectively. A glucose concentration of 30% was obtained using 50% Bagasse. Several fungi and bacteria are capable of producing multiple groups of enzymes, collectively known as cellulases, acting synergistically to hydrolyze β1,4- glycosidic bonds within the cellulose molecules. Filamentous fungi, particularly Aspergillus species have been reported as efficient producers of cellulase. The members of Aspergillus species are major agents of decomposition and thus possess the ability to produce a range of enzymes like cellulases (Berka et al., 1992; Peij et al., 1998; Coral et al., 2002). The recent thrust in bioconversion of lignocellulosic biomass to chemical feedstock has led to extensive studies on cellulolytic enzymes produced by bacteria and fungi. Though the growth period of bacteria is shorter than that of fungi , their life half –backed cellulsae system makes them less useful in the
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industrial production of cellulase. However high cost of cellulases production hindered use of this enzyme in industry. For the utilization of lignocellulosic biomass, it is necessary step to enhance the cellulose production and reduces its production cost .The use of purified cellulosics as substrate is uneconomical for large scale production of cellulases. Therefore cheaply available waste like newspaper and cotton waste and Baggase was tested to find out whether it could support th production of cellulases by Aspergillus species under submerged fermentation and Penicillium species. When Baggase waste incubated with the cellulolytic enzyme complex, sugar is released. The degree of saccharification was assayed on the basis of released of reducing sugar. Saccharification was affected by many factors like pH, temperature and incubation time. Maximum amount of reducing sugar released on 5th day of incubation period until the end of the saccharification period when it was not decreased. The optimum pH and temperature for release of reducing sugar was 5 and 30ᵒC respectively. The optimum pH, temperature and incubation yime saccharification were optimum for conditions for the synthesis of cellulolytic enzymes.(Baig et al.,2004). The utilization of cellulosic waste with biosynthesis of cellulaseis also of great importance. Cotton was pretreated in sereral ways and then used as a fermentation substrates for the production of cellulase by Trichoderma viridei. Thermomechanical treatment in dil H2SO4 or NaOH followed by fermentation gave the best yield of Cl celluloase . The effect of pretreatment on enzyme hydrolysis of cellulose in agricultural waste and industrial waste is of great importance .Celluosic waste from wine and tea industries were subjected to hydrolysis . Prior treatment of cellulosic waste with NaOH enhance the solubilization of lignin and cellulose components and significantly increased the yield of hydrolysis products i.e. glucose and total reducing sugars. (Khokhashvili & Sikharulidze ,1989) . The comparative reactivity of different cellulosic substrates in the enzymic hydrolysis with Trichderma viride and A. niger & P. chrysogenum. Dissolving cellulase pulp , Newspaper cellulose , Avicel and cellpphane (David and Thirty 1982) .

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The first attempt to isolate fungi from soil was made by Adametz(1866) after which various investigators studied the soil fungus flora by various methods.Among these investigators the names of Lender(1908) ,Hagem(1910) ,Bachwith (1911) , Jensen (1912), Goddard (1913) , Dale(1914), werkenthin (1916) ,Paine (1927) , Wajid(1985) ,are more prominent. But the work of Waksman (1922) and wakssam(1927) gave the soil dilution plates method to isolates soil fungi , which is still being extensively used. Both quantitative studies were made by these investigators. They found that number and kind of soil , soil rejection (pH), depth of soil , moisture , season of year, tillage and manuring practices. They also influenced by methods of investigation, nature of medium and material employed. The work has been carried out on the isolation and identification of A. niger & P. chrysogenum species from soil sample which determine the antagonistic properties of the mold cultures (Dennis, 1970; Dennis & Webster , 1971; Rifai.964, 1969) . The decrease in cellulase activity shown by the fungus after attaining its maximum peak period of enzyme secretion could be attributed to so many other factors. Products of cellulase action on cellulose (cellobiose and glucose) inhibite enzyme secretion. Depletion of carbon and nitrogen source cause starvation and hence the fungus may not grow and cellulase activity is growing . Linder and Terri, 1996: The effect of pH on cellulose secretion shows that each substrate supports a particular pH for maximum enzyme secretion. Aspergillus species grow and metabolize well in acidic pH medium between pH 3-5 .(Linder and Terri, 1996 ).It was also proposed that cellulases produced by filamentous fungi rely on several aromatic amino acids for high binding capacity surface and this might enhance enzyme activity(Dosoretz et al, 1990). A method for cellulase production by mixed fungi in solid substrate fermentation (SSF). They noted that higher activities af all enzymes of cellulase complex were achieved in 4 day of mixed culture, than in single culture Trichoderma ressei . The highest filter paper cellulase and β -glucosidase , representing approximately 3 and 6 fold increase over the activities attained in single culture .(Duenas et al.
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1995). The mixed culture soid substrate fermentation (SSF) process also enriched the product with 13% fungal protein. The biomass production was 0.29g l-h-1. Gadgil et al. (1995) worked on enhanced cellulase production by a mutant of Trichoderma ressei. They reported that the need for renewable energy sources have focused attention on the renewable polysaccharide cellulose , which can be enzymetically hydrolyzed to yield sugar Trichoderma ressie .The cellulase production by certain fungi and found that micro-organism have the ability to degrade native cellulose . Since the 1st observation of the action of conversion of cellulosic biomass to fermentable sugar needs economical process from the production of cellulases .soil is inhabitated by a large number of micro-oganisms like bacteria, actinomycetes, algae and fungi, which form the major component of soil. Eggin and Lloyed (1968), Malic and Eggins 1970 The utilization and protein enrichment of cellulose containing industrial waste by way of fungal cultivation is of great use. (Zetelki et al. 1983):. the effect of new microbial strain of A. niger to produce extracellular glycol protein enzyme such as a cellulase , useful in the hydrolysis of biomass , such as steam –exploded wood to simpler components. (Montencourt and Bland 1988). the enzymatic saccharification of municipal wastes with the help of cellulase from bacteria or fungi to produce sugars. The waste were separated from glass and metal components, air dried, ground and shaken with enzyme preparation in citrate buffer. The product was glucose with smaller amounts of xylose and cellobiose. (Clanet et al. 1989) there are three main types of enzyme found in cellulse system that can degrade crystalline cellulose .Exocellobiohydrolase (β-1 ,4 –Dglucancellobiohydrolase) which release cellobiose units from crystalline cellulose , endo-β-1,4-D glucanohydrolase which degrades regions of amorphous cellulose and β-glucosidase (D-glucosidase glucohydrase or cellobiase ) which hydrolyse short oligosaccaharides such as cellobiose and cellotriose to glucose. (Gilks et al. 1984). More recently the problem of effluent from processing operation and their disposal has gained public recognition. Each waste contains its unique quality and
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characteristics, which then suggest the type of treatment required. The two divisons of waste –Domestic and Industrial effluent have different make –ups and often require various treatment process. Though, waste treatment is generally classified into four levels: primely, secondly, tertiary and quarternary treatment with each treatment level aimed at removing a more specific class of contaminants. More efflicient technologies like steam explosion pretreatment must be applied to completely degrade lignin ang gain access to the cellulose molecule s. Although, these susbtrate were pretreated to delignify them, only a significant percentage of the total lignin content was removed. The organism through cascades of enzymatic action must therefore degrade the remaining to enable its unlimited access to the cellulose molecule. (Gharpuray et. al,1983). Bioconversion of lignocellulosic material to usefull, higher value products normally require multi-step process which include –(1) Pretreatment (mechanical ,chemical or biological, ) (2) hydrolysis of the polymers to produce readily metabolizable molecules (eg. Hexane or pentose sugars), (3) bio-utilization of these molecule to support microbial growth or to produce lignocellulosic enzymes have been widely studied in submerged culture processes in the laboratory, ranging from shake flask to fermentations. A stirred –tank reactor ,widely used for the production of cellulose and other lignocellulosic enzymes, is known to have shear problems which rupture mycelium cells and may deactivate the enzymes. Alternative bio-reactor such as the air-lift or bubble column, which have a lower shear stress, seem to produce better results. For example, studies on cellulose and xylanase production by aspergillus niger in various bioreactors showed that in general, better yield and productivity were shown in a bubble –column and an air loop air-lift than in the stirred tank reactor. However, the relatively high cost of enzyme production has hindered the industrial application of the enzymatic process. Lignocellulose consists of lignin, hemicelluose and cellulose and comiled from the typical compostions of the three components in various lignocellulosic materials. Because of the difficulty in dissolving lignin without destroying it and some of its submits, its exact chemical structure is difficult to ascertain. In general lignin contains three aromatic alcohol (coniferyl alcohol, sinapyl and pcounmaryl
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). In addition ,grass and dicot lignin also contain large amounts of phenolics acids such as p-coumaric ferulic acid, which are esterified to alcohol groups of each other and to other alcohol such as sinypl and pcoumaryl alcohols. Lignin is further linked to both hemicelluloses and cellulose forming a physical seal around the latter two components that is an impenetrable barrier preventing penetration of solution and enzymes. Hemicellulose macromolecules are often polymers of pentose, hexoses (mostly mannose ) and a number of sugar acids while cellulase is a homogeneous polymer of glucose. Lignin is the most recalcitrant to degradation wheras cellulose, because of the its highly ordered crystalline structure, is more resistant to hydrolysis than hemicelluloses (Howard and Abotsi, 2003). Carboxymethyl cellulase, one of the members of cellulase complex, cleaves the internal glycosidic bonds of cellulosic chains and acts synergistically with exoglucanases and β-D-glucosidases during the solublization of cellulosic material. It has versatile applications in various fields; widely used in textile industry and in laundry detergents; used in the pulp and paper industry for various purposes; facilitates fermentation of biomass into biofuels and even to treat Phytobezoars, a form of cellulose bezoar found in human stomach. It has great potential for utilization in the food industry; for coffee processing, extraction and clarification of juices (Galante et al., 1998; Grassin and Fauquembergue, 1996a; Uhlig, 1998), extraction of oils from the oilseeds and olive plant (Dominguez et al., 1995; Sosulski and Sosulski, 1993 ), Bread production, Brewery and wine Biotechnology (Harada et al., 2005; Uhlig, 1998; Galante et al., 1998; Grassin and Fauquembergue, 1996b; Caldini et al., 1994; Gunata et al., 1990) production of fruit nectars and purees and to alter the sensory 3 properties of fruits and vegetables (Humpf and Schrier, 1991; Krammer et al., 1991; Marlatt et al., 1992; Pabst et al., 1991). Cellulase has been used to degrade environmental waste such as plant waste (lignocellulosics).cellulase as an industrial enzyme is imported for use. Therefore , Its production using readily available sources will help reduce importation costs.
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The combination of biological and chemical agents enable the complete degradation of lignin. Organic acids such as oxalate produce by the organism inhibit lignin peroxidase, a lignin degrading enzyme and this could also be responsible for the differences in the cellulase activity (Kirk et al., 1980). The ability of microbes to degrade native and modified cellulose so far has revealed that only a few fungi posses ability to degrade native cellulose. A majority of microbes can however degrade modified cellulose. The cost of carbohydrate raw material influences the economy of many fermentation processes, hence the cost play a decisive role in future ans scope of industries employing fermentation processes. A lot of emphasis had been given to screening of the agriculture wastes for release of sugars produced by hydrolysis of lignocellulosics. Thus, the reducing sugars released following the saccarification of available agriculture waste hydrolysis can be used for production of alcohol and other chemicals. The recent thrust in bioconversion of agriculture and industrial wastes to chemical feedstock has led to extensive studies on cellulolytic enzymes produced by fungi and bacteria. Cellulose is a potentially valuable resource for fibre, fuel and feed. Cellulase is used to modify the surface properties of cellulosic fibres and fabric in the order to achieve a desired surface effect (Kotchoni et al.) The isolation of cellulase from Trichoderma viride and Aspergillus niger and its uses for degradation of newspaper. When milled newspaper (particle size 96 µm) at a concentration of 7.58 was the cellulase a glucose concentration of 2.568 was achieved in 27.5 hours, when the saccharification was carried out at 50⁰C and pH 4.8 with stirring. (Lucio et al.1984). The hydrolysis of lignocelluloses by Penicillium funiculosum was also reported. It was reported that enzymatic hydrolysis of cellulose is a promising methed for conversion of waste cellulase to glucose. During the past few years , the decelopment of this technology has procede rapidly with significant advances made in enzyme production , pretreatment and hydrolysis. A variety of fungi were reported to produce cellulases but among these Trichoderma ressei and its
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mutants are powerful proucers of cellulases. However , the search of new and possibly better source of cellulases was continued due to the low levels of ᵦglucosidase of T. ressei, Penicillium chrysogenum produces a complete cellulases having endo-β-1,4-glucanse, and high β-glucosidase. The saccharifiction of alkali –treated cotton and Baggase by p. chrysogenum enzyme was 70% and 63% , respectively . It was possible to obtain glucose concentration as high as 30% using 50% Bagasse. It was of interst that the percent saccharification of cellulosic substrates with the Penicillium enzyme was comparable to that of T. ressei cellulase when the same amount of filter paper activity was used. (Mishra et al. 1984) Generally, the production of cellulases has been shown to be inducible ans was affected by the nature of the substrate used in fermentation. Therefore, the choice of an appropriate inducing substrate is of importance. However, the values of CMC activity were always higher than the filter paper activity during the same interval of the fermentation period. This holds true for all the yields in different studies can be attributed to use of different substrates fermentation . Differnce in titres of enzyme yields in different studies can be attributed to use of different materials as solid matrix, different cultural practices and different organism. The decresed in both cellulase activities may be due to the accumulative effect of cellulosic. Cellulobiose is a dimer of glucose which is known to inhibit both endoglucanse and glucosidase. The time of the highest cellulase activity depends upon the substrate and fungus. But in time, it decreased until the end of the fermentation period. (Ojumu et al.,2003). One of the largest cellulosic agro-industrial byproducts is sugar cane bagasse, fibrous residue of cane stalks left over after the crushing and extraction of juice. It is a lignocellulosic residue (by-product) of the sugar industry and is used by the sugar mills as fuel for boilers. In the recent era, there is an increasing trend towards more efficient utilization of agro-industrial byproducts/wastes, including sugar cane Bagasse and one of the significant applications is the production of enzymes (Pandey et al., 2000). Although the economy of such processes is
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affected by the high cost of product, the utilization of cheaper and indigenous substrates has contributed somewhat to the economical recovery ( Pandey et al., 2000). Sugar cane Bagasse may be chemically treated to generate different bagasse samples with varying quantities of lignin and hemicelluloses, keeping the cellulose fraction intact. Pretreatment affects the structure f biomass by solubilizing hemicellulose , reducing crystalline and increase the available surface area and pore volume of the substrate . There are numerous pretreatment method or combination of pretreatment methods available. In general, pretreatment techniques can be grouped into three categories, physical, chemical and biological. Physical pretreatment methods include combination, steam explsion etc. The most common chemical pretreatment methods used for cellulosic feedstck are dilute acid, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide or other chemical to make the biomass more digestible by the enzyme. Biological pretreatment are sometimes used in combination wit chemical treatment to solubilize the lignin in order to make cellulose more accessible to hydrolysis and fermentation. Pretreatment of cellulosic biomass in a cost effective manner is a major change of cellulose –ethanol technology research and development (Rye and Lee 1983). There a strong influence of intial pH of the medium on the enzyme production . Low and high pH is not suitable for the reducing sugar as for the enzyme production. The Incubation temperature is also an important factor for enzyme production .Proper cultivation time was also significant for growth and production . Reducing sugar yield is not a linear function of the quantity of in enzyme in assay mixture as discussed by Ghose. Twice as much enzyme will give equal sugar in half the life, while twice the amount of enzyme would not be expected to yield twice the reducing sugar in equal time. Assay mixtures may, in some cases, contain reduced sugar unrelated to hydrolysis of substrate glucosidic bonds by the enzymes. Release of Cellulolytic enzyme will lead to initiation of attack on

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cellulosic component of lignocelluloses. Maximum enzyme activity was observed on the 8th day incubation at 37ᵒC. Lignocellulosics are abundant sources of carbohydrate, continually replenished by photosynthetic reduction of carbon dioxide by sunlight energy. Thus they are the most promising feedstock for the production of energy, food and chemical. The bioconversion of cellulosic material has been receiving attention in recent years. It is now subject of intensive research as a contribution to the development of the 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 (Smith et al., 1987). An endoglucanase produced by Baccilus species grown on glucose which showed activity towards crystalline from of cellulose such as cotton and avicell. It was stable in pH range of 4-10 and temperature upto 60 ˚C. The enzyme activity was completely inhibited by Hg++, while other metal ions chelator , various oxidizing agents and repeated freezing and thawing had no marked effects on activity. (Sharma et al. 1990) A growth medium was developed and the fermentation parameters for maximal production of extracellular xylanase and β -oxlosidase by Aspergillus species derived from wild strain was studied. The optimal pH for the production of xylanase and β-xylosidase was 4.0 at 30˚C and 35˚C, respectively. It was founded that oat spelt xylan produced the highest activities of xylanase and β-xylosidase than corn steep liqur, protease peptone and yeast extract. (Smith and Wood ,1991) The bacterial systems have also been investigated for saccharification of Lignocellulosic biomass and might have advantage because of the fast growth rate of bacteria . It ahs also been reported that the enzyme preparation from the cellulolytic bacteria can effectively saccharify different cellulosic substrates
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(Choudhury et al. 1980 , 1981; Lynd and Grethlein, 1987; Rajoka and Malic , 1984; Clostridium thermocellum, a thermophylic anaerobic bacterium, has attracted increased interest for conversion of Lignocellulosic biomass. cellulase system of Pseudomonas spp. , has been fairly well studied . Yamane et al. (1970), Yoshikawa et al. (1974) studied the Biogenesis of multiple cellulase components of P. fluoresenes var. cellulosa with special emphasis on effect of culture conditions of the multiplicity of cellulase titers produce by the organism.( Waldron and Eveleigh, 1986 ) The cellulase activity is the production of non-specific by products other than glucose by unspecified side reaction. These by-product promote glucose degradation and reduce its yield. The submerged fermentation culture method used may also contribute to the difference in cellulase activity. A comparison of solid state fermentation and submerged fermentation has shown that submerged method has shearing forces which rupture mycelia cells and deactivate enzyme, thus enzyme activity is decreased (Wase et al. 1985).

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