SCREENING

AND

OPTIMIZATION

OF

FUNGAL

CELLULASES

ISOLATED FROM THE NATIVE ENVIRONMENTAL SOURCE AND ITS DEINKING ACTIVITY

INTRODUCTION:

Cellulose is the most common organic polymer, representing about 1.510 tons of the total annual biomass production through photosynthesis especially in the tropics, and is considered to be an almost inexhaustible source of raw material for different products (Klemm et al., 2002). It is the most abundant and renewable biopolymer on earth and the dominating waste material from agriculture (Bhat and Bhat., 1992). A promising strategy for efficient utilization of this renewable resource is the microbial hydrolysis of lignocellulosic waste and fermentation of the resultant reducing sugars for production of desired metabolites or biofuel.

Cellulose is a crystalline polymer, an unusual feature among biopolymers. Cellulose chains in the crystals are stiffened by inter and inter chain hydrogen bonds and he adjacent which overlie one another are held together by weak Van-der Waals forces. In nature, cellulose is present in a nearly pure state in few instances whereas in most cases, the cellulose fibers are embedded in matrix of other structural biopolymers, primarily hemicelluloses and lignin (Machessault et al., 1997). An important feature of this ceystalline array is the relative impermeability of not only large molecules like enzymes but in some cases even small molecules like water. There are crystalline and amorphous regions, in the polymeric structure and in addition there exists several types of surface irregularities (Cowling et al., 1975). This heterogeneity makes the fibers capable of swelling when partially hydrated, with the result that the micro-pores and cavities become sufficiently large enough to allow penetration of glucose composed of anhydoglucose units coupled to each other by -1-4 glycosidic bonds. The number of glucose units in the cellulose molecules varies and degree of polymerization ranges from 250 to well over 10,000 depending on the source and treatment method (Klemm et al., 2005). Though lignocellulosic biomass is generally recalcitrant to microbial action, suitable pretreatments resulting in the disruption of lignin

structure and increase accessibility of enzymes have been shown to increase the rate of its biodegradation (Lynd et al., 2002).

Microbial degradation of lignocellulosic waste and the downstream products resulting from it is accomplished by a concerted action of several enzymes, the most prominent of which are the cellulases, which are produce by a number of microorganisms and comprise several different enzyme classifications. Cellulases hydrolyze cellulose (-1,4-D-glucan linkages) and produce as primary products glucose, cellobiose and cellooligosaccharides. There are three major types of cellulose enzymes [cellobiohydrolase (CBH or 1,4--D-glucan cellobiohydrolase, EC 3.2.1.91), Endo--1,4-glucanase (EG or endo-1,4--D-glucan 4-glucanohydrolase, EC3.2.14) and -glucosidase (BG-EC 3.2.1.21)] (Schulein et al., 1988). Enzymes within these classifications can be separated into individual components, such as microbial cellulose compositions may consist of one or more CBH components, one or more EG components and possibly -glucosidases. The complete cellulose system comprising CBH, EG and BG components synergisticallyact to convert crystalline cellulose to glucose. The exo-cellobiohydrolases and the endoglucanases act together to hydrolyze cellulose to small cellooligosaccharides. The oligosaccharides (mainly cellobiose) are subsequently hydrolyzed to glucose by a major -glucosidase (Bguin et al., 1994).

Cellulases are used in the textile industry (Gusakov et al., 2000), in detergents (Kottwitz et al., 2005), pulp and paper industry, improving digestibility of animal feeds, in food industry, and the enzymes account for a significant share of the world enzyme market. The growing concerns about short age of fossil fuels, the emission of green housegases and air pollution by incomplete combustion offossil fuel has also resulted in an increased focus on production of bioethanol from lignocellulosics and especially the possibility to use cellulases and hemicellulases to perform enzymatic hydrolysis of the lignocellulosic material (Himmel et al., 1999). However, in production of bioethanol, the costs of the enzymes to be used for hydrolysis of the raw material need to be reduced and their efficiency increased in order to make the process economically feasible.

Commercial production of cellulases has been tried by either solid or submerged culture including batch, fed batch, and continuous flow processes. Media used in cellulase fermentations contain cellulose indifferent degrees of purity, or as raw lignocellulosic substrates (Doppelbauer et al., 1991), which is especially true in the case of solid-state fermentation. Cellulases are inducible enzymes and the most problematic and expensive aspect of industrial cellulase production is providing the appropriate inducer for cellulases. Cellulase production on a commercial scale is induced by growing the fungus on solid cellulose or by culturing the

organism in the presence of a disaccharide inducer such as lactose. However, on an industrial scale, both methods of induction result in high costs. Since the enzymes are inducible by cellulose, it is possible to use cellulose containing media for production but here again the process is controlled by the dynamics of induction and repression. At low concentrations of cellulose, glucose production may be too slow to meet the metabolic needs of active cell growth and function. On the other hand, cellulase synthesis can be halted by glucose repression when glucose generation is faster than consumption. Thus, expensive process control schemes are required to provide slow substrate addition and monitoring of glucose concentration. Moreover, the slow continuous delivery of substrate can be difficult to achieve as a result of the solid nature of the cellulosic materials. The challenges in cellulase production involve developing suitable bioprocesses and media for cellulase fermentation, besides identification of cheaper substrates and inducers. Genetic modification of the cellulose producers to improve cellulase activity has gone along way to give better producers with high enzyme titers (Fowler et al., 2000), but still cellulase production economics needs further improvement for commercial production of ethanol from biomass.

MICROORGANISMS PRODUCING CELLULASES:

Cellulolytic microbes are primarily carbohydrate degraders and are generally unable to use proteins or lipids as energy sources for growth. Cellulolytic microbes notably the bacteria Cellulomonas and Cytophaga and most fungi can utilize a variety of other carbohydrates in addition to cellulose (Poulsen et al., 1988), while the anaerobic celluloytic species have a restricted carbohydrate range, limited to cellulose and or its hydrolytic products. The ability to secrete large amounts of extracellular protein is characteristic of certain fungi and such strains are most suited for production of higher levels of extracellular cellulases. One of the most extensively studied fungi is Trichoderma reesei, which converts native as well as derived cellulose to glucose. Most commonly studied cellulolytic organisms include: Fungal species-Trichoderma, Humicola, Penicillium, Aspergillus; Bacteria-Bacilli, Pseudomonads, Cellulomonas; and Actinomycetes-Streptomyces, Actinomucor andStreptomyces.

While several fungi can metabolize cellulose as an energy source, only few strains are capable of secreting a complex of cellulase enzymes, whichcould have practical application in the enzymatic hydrolysis of cellulose. Besides T. reesei, other fungi likeHumicola, Penicilliumand Aspergillushave the ability to yield high levels of extracellular cellulose (Hayashida et al., 1988). Aerobic bacteria such as Cellulomonas, Cellovibri and Cytophagaare capable of cellulose degradation in pure cultures (Lynd et al., 2002). However, the microbes

commercially exploited for cellulase preparations are mostly limited to T. reese H .insolens, A. niger, Thermomonosporafusca, Bacillus sp, and a few other organisms (table 1).

Major microorganisms employed in cellulase production Microorganism Major group Genus Species A.niger Fungi Aspergillus A. nidulans A. oryzae (recombinant) Fusarium F. solani F. oxysporum Humicola H. insolens H. grisea Wood et al., (1977) Ortega et al., (1990) Schulein et al., (1997) Takashima et al., (1998) Reference Ong et al., (2004) Kwon et al., (1992) Takashima et al., (1998)

Melanocarpus

M. albomyces P. brasilianum

Oinonen et al., (2004) Jorgensen et al., (2003) Chaabouni et al., (1995) Mo et al., (2004) Schulein et al., (1988) Fowler et al., (1990) Gusakov et al., (2000)

Penicillium

P. occitanis P. decumbans T. reesei

Trichoderma

T. longibracm T. harzianum

Bacteria Acidothermus Bacillus

A. cellulolyticus Bacillus sp Bacillus subtilis C. acetobutylim Clostridium C. thremocellum

Tucker et al., (1989) Mawadza et al., (2000) Heck et al., (2002) Lopez et al., (2004) Nochure et al., (1993)

Pseudomonas Rhodothermus Cellulomonas

P. cellulose R. marinus C. fimi C.bioazotea C.uda S.drozdowici S. sp

Yamane et al., (1970) Hreggvidsson (1996) Shen et al., (1996) et al.,

Streptomyces Actinomycete Thermonospore S. lividans T. fusca T. curvata Fennington et al.,(1982) Okeke et al., (1992)

APPLICATION OF CELLULOSE: PULP AND PAPER INDUSTRY: Coadditive in pulp bleaching; biomechanical pulping; improved draining; enzymatic deinking; reduced energy requirement; reduced chlorine requirement; improved fiber brightness, strength properties, and pulp freeness and cleanliness; improved drainage in paper mills; production of biodegradable cardboard, paper towels, and sanitary paper .

AGRICULTURE INDUSTRY:

Plant pathogen and disease control; generation of plant and fungal protoplasts; enhanced seed germination and improved root system; enhanced plant growth and flowering; improved soil quality; reduced dependence on mineral fertilizers.

BIOCONVERSION INDUSTRY:

Conversion of cellulosic materials to ethanol, other solvents, organic acids and single cell protein, and lipids; production of energy-rich animal feed; improved nutritional quality of animal feed; improved ruminant performance; improved feed digestion and absorption; preservation of high quality fodder.

DETERGENTS INDUSTRY:

Cellulase-based detergents; superior cleaning action without damaging fibers; improved color brightness and dirt removal; remove of rough protuberances in cotton fabrics; antire deposition of ink particles.

FERMENTATION INDUSTRY:

Improved malting and mashing; improved pressing and color extraction of grapes; improved aroma of wines; improved primary fermentation and quality of beer; improved viscosity and filterability of wort; improved must clarification in wine production; improved filtration rate and wine stability.

FOOD INDUSTRY:

Release of the antioxidants from fruit and vegetable pomace; improvement of yields in starch and protein extraction; improved maceration, pressing, and color extraction of fruits and vegetables; clarification of fruit juices; improved texture and quality of bakery products; improved viscosity fruit purees; improved texture, flavor, aroma, and volatile properties of fruits and vegetables; controlled bitterness of citrus fruits.

TEXTILE INDUSTRY: Biostoning of jeans; biopolishing of textile fibers; improved fabrics quality; improved absorbance property of fibers; softening of garments; improved stability of cellulosic fabrics; removal of excess dye from fabrics; restoration of colour brightness OTHERS INDUSTRY: Improved carotenoids extraction; improved oxidation and colour stability of carotenoids; improved olive oil extraction; improved malaxation of olive paste; improved quality of olive oil; reduced risk of biomass waste; production of hybrid molecules; production of designer cellulosomes. Considering the importance and application of the cellulases, this study was aimed to screen the indigenous fungal isolates for the cellulytic ability. Furthermore, this study aims to provide better understanding

of condition for the production and activity of cellulases by different fungal cultures (Uhlig et al., 1998 and Singh et al., 2007, Kirk et al., 2002). The traditional (chemical) deinking process is widely used and is considered to beeffective for the removal of ink particles. However, the widespread technology usingthermoplastic toner in the printing process presents a special challenge in theconventional deinking process. Furthermore, the process requires the usage of largequantities of chemical agents (Shrinath et al., 1991). This makes the process highlydamaging to the environment, and treatment of the effluent to meet environmentalregulations is costly (Prasad et al., 1992). On the contrary, a biological process (usingenzymes) has been evaluated and proven successful in deinking various types ofwaste paper. One of the benefits of using enzymes in the deinking process is the minimum treatment of effluent produced; it is also less harmful to the environment. The effluent from an enzymatic deinking has been reported to be lower in COD content thanwaste water from a corresponding chemical deinking process (Putz et al., 1994).Furthermore, enzymatic deinking can avoid the alkaline environment that is commonlyemployed in the chemical deinking process. This consequently will cut down the cost ofchemicals that are used to treat the effluent and also reduce the COD loaded into thewaste water system. PULP PREPARATION: The wastepaper samples used in this study were obtainedwithin the book shop. The mainly consisted of newspaper andcomputer printout paper. The wastepaper was manually sorted by hand to remove non-paper objects. The sorted wastepaper was kept in a room away from sunlight and highmoisture until needed. Prior to pulping, wastepaper was soaked in tap water for one hour at roomtemperature and then transferred into the developed bioreactor system for disintegration.The disintegration process was carried out for 60 minutes under room temperature. After the pulp was recovered by dewateringbefore being used in enzymatic deinking process . Pulp (2 kg on air-drybasis) was suspended in water and pulped for 60 minutes at 4% consistency and 400 rpm. After the pulping process the appropriate volume of water was removed andreplaced by the appropriate amount of diluted enzyme concentration such as(0.5-3.0) solution in order to maintain thepulp slurry at 4% consistency. The reaction of enzymes with pulp occurred at pH 5.5 and 55C for 24 hrs with continuous slow mixing. A control was run as described aboveexcept using thermally inactivated enzyme (Gubitz et al., 1998). Contents of the tube were mixed and absorbance was noted at 540 nm.

REVIEW OF LITERATURE:

The production and properties of cellulases have been extensively studied during recent years. Microorganisms are well suited for production of cellulases through fermentation of inexpensive and nonconventional sources like agro-industrial wastes. The development to the achievement of high levels of extra cellular accumulation of cellulose for subsequent applications is useful industrial processes.

Rho et al., (1990) investigated the influence of cellulosic and hemicellulosic substrates on the production of accolade and xylanase complexes in Aspergillus niger. They reported that culture conditions with substrate exhibited profound efforts on endoglucanase, -glucosidase, endoxylanase and -xylosidase. Biosynthesis of cellulose and xylanase complexes in A. niger were found to co-ordinarily regulated at the level 0 induction. Multiple forms of extra cellular cellulose and xylanase compex seemed to be outcome of specific gene experession. Gatenholm et al., (1991) studied the nature of adhesion in composites of modified cellulose fibers and polypropylene. Cellulose fibers were surface-modified with polypropylenemaleic-anhydride copolymer and characterized by contact angle measurement, ESCA, FTIR, and SEM techniques. Composites reinforced with surface modified cellulose fibers showed significantly improved mechanical properties compared to composites with untreated cellulose fibers. This was due to improved fiber wetting, dispersion and fiber-matrix adhesion as seen in SEM micrographs. Interfacial interactions involved were covalent and hydrogen bonds that formed across the fiber-matrix interface. Chandrashekar and kaveriappa (1991) studied that production of extracellular by two aquatic

hypsometers, Lunulosporacurvula and Flagellosporapeicilliodes. Results showed that CMC (carboxy methylcellulose) was the best source of carbon and (NH 4)2SO4 the best source of nitrogen. An optimum pH of 5.2 and 28C was found to favor maximum enzyme activity in 12 days old cultures. Glucose and sucrose were found to suppress the activity of cellulose in both the organisms. Singh et al., (1992) investigated the production of cellulases by A. niger as 101 grown on 2% alkali treated corn cobs under various cuture and environmental conditions. The fungus gave maximum production of celluloses when grown for 7 days in the growth medium containing 1.0% substrate along with KH 2PO4, 0.2%; MgSO4.7H2O, O.O3%; CaCl2.2H2O, O.O3% and (NH4)2SO4, O.2% at pH- 5.0 inoculated with 6% inoculums and kept under continous shake conditions.

Duenas et al., (1995) subjected ammonia treated bagasse 5% to mixed culture fermentation with T. reesi LMUC 4 and A. phoenicis QM 329 in pot fermenter at significantly higest activites of all the enymes of the cellulose complex were achived in 4 days of mixed culture fermentation than in single culture ( T. reesei). The higestfpase and - glucosidase activities seen in mixed culture fermentation were 18.7 and 38.6 lu/g dry weight respectively, representing approximately 3 and 6 fold increase over the activities attained in single culture fermentation. Raj et al., (1995) investigated the influence of various processing aids coupling agents in improving fiber dispersion as well as compatibility between the fiber and the matrix. Stearic acid and mineral oil were used as additives and maleated ethylene as a coupling agent. The results showed that the addition of stearic acid duringthe compounding greatly improved the fiber dispersion in the polymer matrix compared tountreated fibers as seen in SEM micrographs of fracture surfaces of the corresponding composites. This effect was also reflected in improved mechanical properties of the composites. The reports of Mohmmad sohail et al., (1997). They reported maximum activity of Aspergillus nigerfor the production of endoglucanase more than 2 I.U/ml. Sohail et.al. (2009) have reported that all cellulose overproducing Aspergillus strains were from soil origin that indicates the property of degradation of biomass of Aspergillus group. Under the soil. Banana pill powder and coir powder both biowastes used as substrates by M.UshaKiranmayi and her group (2011). They used solid substrate method for cellulase production the higher values reported by them were0.072 I.U./ml for banana pill powder and 0.046 I.U./ml for coir powder. Ashger et al., (2003) reported the use of Arachinotus species for the production of endoglucanase and they reported higher activity of endoglucanase was 1.13I.U./ml, when the fungus was grown 7.5% corn cob as substrate . Stephen Decker et al., (2003) has developed an automated filter paper assay technique for the determination of 8 cellulases . That filter paper assay method is based on a Cyberlab C400 robostick deck instrument equipped with customized incubation, reagent storage and plate reading capabilities that allow rapid evaluation of cellulases acting on enzymes of 84 different samples, development of such technologies is the proof of importance of cellulase production research in biotechnology. Cellulases are used in various industries , their applications in production of biofuels still far away due high cost of production of enzyme fermentation , recovery and storage. Kang et al., (2004) and Yang et al., (2004) both have reported the potential of wheat straw as a substrate for cellulose fermentation.

Anita Singh and Bishnoi (2006) reported highest caboxy methyl cellulose activity of 83 I.U. /ml and the filter paper activity was 3.2I.U./ml. the used Aspergillus heteromorphus strain for their research. Their results shows correlation with the results of Narasimha et al., (2006) for filter paper assay. Narasimha and his group used saw dust as a substrate and inoculated A. niger as a source for cellulase production. Muthuvelayudham et al., (2006) used sugarcane buggase as a substrate and got cellulase production 96 I.U./ml by using Trichoderma reesi strain, Their result s are similar to the finding of Amita Singh and hergroup for CMCase assay. They further suggest that the highest cellulases were produced on wheat straw and lowest on corn cob. Rana et al., (1997) studied the effect of acetylation on jute fibersat different reaction times and reaction temperatures. The modified fibers werecharacterized by FTIR, DSC, TGA and SEM studies. The extent of moisture regain and thermal stability was reported. From the study, the authors found that the thermal stability of acetylated jute is higher than that of untreated jute . Acharya et al., (2008)found in different culter conditions the hydrolysis of saw dust, they foundthat in alkaline pretreated conditions (2 N NaOH) saw dust at 9.6% concentration gave theoptimum yield value 0.1813 IU/ml. cellulase activity. In their research Acharya and his coworkers , have collected saw dust from saw mill near Gandhinagar, Gujarat, India. It was sieved by mesh no. 60. To make uniform particle size. That saw dust was pretreated with NaOH solution of variable concentration of range 1-5 N. solution incubated for 12 hours . They gotmaximum cellulase activity at 2N, NaOH (0.1813 I.U./ml). Later on 2N NaOH pretreated saw dust at different dust concentration 9 range (2.4-12%) in wet weight conditions were used and among these range the maximum activity was recorded at 9.6% that was 0.1813 I.U./ml. under submerged conditions at 120 rpm. The finding of Acharya and his group arecomparatively promising with the earliear work reported by Ojummu et al., (2003).This group reported that A. flavus grows on saw dust produced highest cellulase activity 0.0743

I.U./ml.at 12 hours treatment of 3% saw dust. Acharya and his group also worked on pH optimization for cellulase production and they found that among range of pH values of 4.0 to 6.0 the maximum cellulase yield was recorded at pH 4.0(0.0925 I.U.). Akiba et al.,(1995) also reported the optimal pH values from A. niger in the range of 6.0 to 7.0. Mohammad Sohail et al., (2009) collected 128 fungal stains from native environment of Karachi, Pakistan, from different sources like soil, plant material , spoiled juice. They screened these isolates for hydrolytic enzymes production and found that among these 128 strains of different genera of fungi majority of strains had shown the hydrolytic activity. Aspergillus nigergroup shown maximum output for hydrolytic

10

activity. The production of endoglucanase and beta glucosidase was reported more than 1.5 I.U./ml from those strains. Mohite and Maga (2010) have reported the potential of sorghum straw as a substrate for cellulose production . In submerged culture conditions Aspergillus niger strain isolated from soil collected from Ankaleshwar-Gujrat the production of cellulase was 0.77 units/ml. The lowest production was recovered on wheat straw medium i.e. 0.28units/ml. These contrast finding suggest that the yield of cellulases is depend on the potential of fungal strains and pretreatment to the media components. Usama F.Ali and HalaS.Saad El-Dein (2008) grown two strains of Aspergillusniger and Aspergillus nidulanson waterhyacinth

(Eichhorniacrassipes,Martin).They prepared various blends of water , Fortified with Czapeck-Dox in different ratios . It was found that Water hyacinth blend with Czapeck-Dox mediumwith 4:1 ratio reach its maximum concentration of cellulose enzymes . Mohite and Magar (2010) have reported the potential of sorghum straw as a substrate for cellulose production. In submerged culture conditions Aspergillus niger strain isolated from soil collected from Ankaleshwar-Gujrat the production of cellulase was 0.77 units/ml. Jayant et al., (2011) and his to get cellulase production by inoculating strains of A.niger and Pencillium chrysogenum simultaneously that the co-culturing approach of inoculation. In this new method for cellulase production they got cellulase production with 7 maximum cellulaseactivity at solid state fermentation of 3.5 I.U/ml on newspaper waste. This co-culturing approach gave higher production from the previous reports of solid state fermentation. Talekar et al., (2011) and his research group tried to get cellulase production from local isolates

of A.niger , A. nidulansfrom water hyacinth heaps of Rankala lake near Kolhapur (Maharashtra). The cultures further inoculated on water hyacinth blended medium for cellulase production. In seven days crude cellulase is used for de-colorization of methylene blue dye on on separate basal medium with 3% sucrose solution in five days at submerged culture condition. The total de-colorization of flasks in five days. The decolorizationprotocol followed by Talekar and coworkers from the research of Jothimani and Prabhakaran (2003).In the production of cellulases there are many reports on Aspergillus and Trichoderma sps. In addition to these , other species were also reported cellulose degradation by brown rot fungi of Coniophoraceae family. C. Ravindran et al., (2011) reported the potential of Chaetomium species collected from marine mangrove plants for cellulase production. They further suggested that activity 6.5I.U/ml. and stability of cellulase enzyme between neutral to alkaline pH and high temperature. It is useful to industry and different

11

biotechnological applications. Rhizopusoryzae grown on the substrate of water hyacinth blend reported by Moumita Karmakar and RinaRaniRay (2011). They reported highest endoglucanase activity of 450I.U./ml. at substrate concentration 1.5% at pH 7.5. Daniel Klen-Marcuschamer et al., (2011) discussed this issue, they performed a sensitivity analysis to study the effect of feedstock prices and fermentation time on the cost contribution of ethanol price . They concluded that a significant effort is still required to lower the contribution of enzymes to bio fuel production costs. To summarize this review we can justify that there is great potential. Kim et al., (1991) have reported that newspaper pulps bleached after being deinked by enzymatic and conventional means had similar brightness values. In the case of conventional deinking, hydrogen peroxide is used in the pulping as well as the bleaching step, but in the case of enzymatic deinking, hydrogen peroxide is used only in the bleaching process. Enzymatically deinked pulps were thus easier to bleach and required half as much hydrogen peroxide. In a similar study with letterpress- printed newspaper, enzymatically deinked pulps had lower initial brightness values than conventionally deinked pulps. However, subsequent bleaching with hydrogen peroxide produced similar brightness values, with peroxide usage lowest for the enzymatic process. Prasad et al., and Rushinget al., (1992) reported reductions in particle size from 16% to 37%, depending on ink type. So far, there is no credible explanation for this reduction in the size of ink particles. Prasad et al., (1992)observed that freeness increased in all the enzyme-treated samples compared with the control. The freeness increase varied from 50% for a cellulose treated colored flexo-printed newsprint to 14% for black-and-white printed newsprint treated with a hemicellulase preparation. Nakano et al., (1993) has reported that an alkaline lipase efficiently removed offset- printing inks. Enzymes that catalyze the removal of surface lignin may hold promise for deinking of newsprint that contains a proportion of lignin-rich mechanical pulp. This approach has been evaluated using white-rot fungi Phanerochaete chrysosporiumand with lignin-degrading enzymes. Woodward et at., (1994) suggested that catalytic hydrolysis may not be essential, since enzymes can remove ink under non optimal conditions. Near cellulase binding alone may be enough to disrupt the fiber surface to an extent sufficient to release ink during pulping. It is also reported that cellulases peel fibrils from fiber surfaces, thereby freeing ink particles for dispersal in suspension. Enzymatic effects may be indirect, removing microfibrils and fines size varied with pulping time in the presence of cellulases; overall reduction was greater than that noted in conventional deinking.

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Putz et al., (1994) reported that brightness levels obtained after bleaching enzymatically deinked offsetprinted newspaper pulp were slightly higher than for pulp produced by conventional deinking with the same quantity of hydrogen peroxide applied during pulping. The benefits of neutral cellulose for deinking MOW wasexploited by a French group. Using a neutral cellulase as a post-treatment to a standard alkaline chemical treatment,they reported additional brightness and greater ink removal. Zeyer et al., (1995) studied the performance of enzymes in deinking of ONP. Their results demonstrated that the arrangement of unit operations is of importance. No deactivation of enzymes by shear stress was observed. Statistical investigation of particles on hand sheets demonstrated that many ink particles were likely still at their original location. Heise et al., (1996) reported the results of three industrial-scale trial runs to evaluate enzymatic deinking of nonimpact-printed toners. Increased ink removal was achieved using a low level of a commercially available enzyme preparation in combination with a surfactant. The brightness of enzymatically deinked pulp was two points higher than that of the control pulp. The enzyme trials also displayed improved drainage and comparable strength when compared with the control. No significant differences in the quality and treatability of the process water were noted, although the effluents from these trials had lower oxygen demand and toxicity than the effluents from the control.

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AIM AND OBJECTIVE

The soil sample was collected from Muttukadu mangrove forest, ECR road, Chennai. The 14 fungus were isolated from collection area Muttukadu, ECR road, Chennai. The screening 14 fungus for cellulase activity. Selected fungus was assayed for deinking activity.

MATERIALS AND METHOD:

SAMPLE COLLECTION: Soil sample were collected from Muttukadu mangrove forest, ECR road, Chennai used for isolation and characterization of fungi. The samples was collected in sterile polythene bag and preserved at 4C in refrigerator and samples were tested within 24 hrs of collection. STUDY AREA: Muttukadu mangrove forest, ECR road, Chennai.

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ISOLATION AND IDENTIFICATION OF FUNGUS: SERIAL DILUTION: In 500 ml conical flask, the 99ml of distilled water was taken, 1gm of soil sample was added and mixed. Seven test tubes were taken and marked as 10, 10, 10, 10, 10, 10 and 10. And then each test tubes was added to 9ml of distilled water. 1ml of sample from the 100ml was transferred to 10 dilution. From 10 test tube 1ml was transferred to 10 and simultaneously to 10 dilution transferring was done. Then the test tubes 10, 10, 10 was used for fungal isolation.

SABOURAUDS DEXTROSE AGAR MEDIUM: INGREDIENTS Peptone : Dextrose : Agar : Distilled water : pH : g/I 10.0 g 40.0 g 20.0 g 1000 ml 5.50.2

PLATING TECHNIQUE: Sabourauds dextrose agar (SDA) medium was prepared, the pH was adjusted to 5.5 and sterilized at 15 lbs for 121C for 15 mins. To this, filter sterilized Streptomycin solution (50 mg/L of streptomycin) was added and thoroughly mixed to avoid bacterial growth. One ml aliquots of 10 4 dilution was pipetted out into sterilized petridishes and about 15 ml of Sabourauds dextrose agar medium was pour plated in duplicate. The

15

dishes were then rotated clockwise and anti-clockwise for uniform distribution of the samples. The solidified plates were incubated at 30C in an incubator for five days.

Identification of fungi: Identified fungi on the basis of morphology and growth patternaccording to the methods recommend by Harrigan (1998). It was based on generalexamination of growth pattern of mycelia and spores under microscope after staining. The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls. LACTOPHENOL COTTON BLUE: This stains chitin, making such structures as spore ornamentation show up much more clearly than they do with most other stains (including Congo Red). A parting shot - well, this one is more to do with avoiding departing. The chemicals mentioned above include some seriously caustic, acidic and toxic substances, and so they really must be stored where children cant get hold of them. The fumes from ammonia, for example, can burn eyes in fact some tests merely require ammonia vapour to pass over fungal tissue to invoke a colour change. A secure safe could save a life. I have one bolted to the wall next to the bench where I keep my microscope, and it is ideal for storing chemicals, razorblades, glass slides etc. PROCEDURE FOR CORNEAL SCRAPE MATERIAL:

Place a drop of 70% alcohol on a microscope slide. Immerse the specimen/material in the drop of alcohol. Add one, or at most two drops of the lactophenol/cotton blue mountant/stain before the alcohol dries out.

Holding the coverslip between forefinger and thumb, touch one edge of the drop of mountant with the coverslip edge, and lower gently, avoiding air bubbles. The preparation is now ready for examination.

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SCREENING OF FUNGAL STRAINS FOR CELLULOSE ACTIVITY: The isolated strain were screened for cellulose activity on agare medium containing, either amorophous or crystalline cellulose. The screeing medium used is that of in a mineral salt medium (Mandels et al., 1976). Soil samples were supplemented with saw dust and synthetic medium. INGREDIENTS (g/%) Peptone (0.1%), Urea (0.03%), MnSO. HO (0.0016%), ZnSO.7HO (0.0014%), (NH)SO (0.14%), MgSO.7HO (0.03%), FeSO.7HO (0.05%), CaCl (0.01%), CoCl.6HO (0.0029%), KHPO(0.2%).

CELLULOSE PRODUCING FUNGAI WERE SCREENED ON SELECTIVE CARBOXYMETYL CELLULOSE CONTAINING(1%): INGREDIENTS (g/%) NaNO (2.0), KH PO (1.0), MgSO. 7HO (0.5), KCl (0.5), carboxymethyl cellulose sodium salt (10.g), Peptone (0.2g), Agar (70.0),

Plates were spot inoculated with spores suspension of pure culture and incubation at 30 c. after 3 days, plates were flooded with 1% congo red solution for 15 minutes then de-stained with 1M

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NaCl solution for 15 minutes. The diameter of decolorization around each colony was measured the fungal colony showing largest zone of decolorization was selected for cellulose production.

ENZYME ASSAYS : FILTER PAPER ASSAY (FOR TOTAL CELLULOSE ACTIVITY): cellulose activity was determined by a method of Mandels et al., 1976). An aliquot of 0.5 ml of cellfree culture supernatant was transferred to a clean test tube and 1ml of sodium citrate buffer (pH 4.8) was added. Whatman #1 filter paper stirp (6 cm 1 cm) was added to each tube. Tubes were vortexed to coil filter paper in bottom of tube. Tubes were incubated in a water bath at 50 C for 1 hour followed by an addition of DNS reagent (3 ml). tubes were then placed in a boiling water to each tube. Contents of the tube were mixed and absorbance was noted at 540 nm. Cellulase activity was expressed in term of filter paper unit (FPU) per ml of undiluted culture filtrate. A filter paper unit (FPU) is defined as mg of reducing suger liberated in one hour under standard assy conditions. Reducing sugar produced in one hour was calculated by comparing A with that of standard curve. OPTIMIZATION OF PRODUCTION OF CELLULASE ENZYME: Cellulase production depends upon the composition of the fermentation medium. Medium optimization for over production of the enzyme is an important step and involves a number of physico-chemical parameters such as the incubation period, pH, temperature and supplemented Substrate in submerged fermentation. For the initial optimization of the medium, the traditional method of one variable at a time approach was used by changing one component at a time while keeping the others at their original level. The selected cellulolytic strains were grown in selected media consisting of selected substrates for enzyme production. Studies were performed in shake flasks to optimize different fermentation conditions for hyper cellulase production.

OPTIMIZATION OF PH: The optimized media were prepared using the individual substrates and the pH was set at different levels such as 5.0, 7.0 and 9.0 with 1% NaOH and concentrated HCl. Maximum cellulase activity was seen at pH 5. However it was observed that the cellulose activity has a broad pH range between 5.0 and 9.0. The most suitable pH of the fermentation medium was determined by adjusting the pH of the culture medium at different levels in the range of pH 3 to 9 using different buffers (Akiba et al., 1995).

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OPTIMIZATION OF TEMPERATURE: Fungal isolates were inoculated in the synthetic medium followed by incubation at 25C, 30C and 35C. Enzyme production was measured on each couple of days from 2nd to 10th days of incubation which was followed by enzyme assays using CMC (Sone et al.,1999).

EFFECT OF CARBON SOURCES ON ENZYME PRODUCTION: Effect of various carbon compounds viz., glucose, sucrose and maltose were used for studying. The broth was distributed into different flasks and 1 to 3.0 % of each carbon sources were then added before inoculation of the strain and after culture inoculation, the flasks were incubated for 7 days at 452 C. (Kachlishvili et al., 2002) EFFECT OF NITROGEN SOURCES ON ENZYME PRODUCTION: For optimization proposes nitrogen source like peptone, sodium nitrate and yeast extract were studied. The fermentation medium was supplemented with organic and inorganic compounds at 1 to 3.0 % level, replacing the prescribed nitrogen source of the fermentation medium (Moore et al., 1990).

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RESULTS AND DISCUSSION: Cellulase enzyme of microbial origin possess considerable industrial potential due to their biochemical diversity and wide applications in pulp and paper industry , fertilizers prodution, detergents and processes like waste treatment, textile industry.The present study was aimed at producing cellulase from three fungal species and optimization and deinking activity of cellulase have also been carried out. The result obtained has been summarized in this chapter. ISOLATION OF CELLULASE PRODUCING STRAINS: ISOLATION AND IDENTIFICATION A total 6 fungal strains were isolated from the sediment samples. The total number of species included 2 members of Zygomycotina i.e., Mucor racemosus and Rhizopus stolonifer and 3 species in 3 genera of Deuteromycotina represented only by Hypomycetes such as Aspergillus niger, A. flavus, Fusarium sp, and Trichoderma sp. All the 6 fungal strains isolated belonging to five genera were qualitatively assayed by well diffusion assay method in water agar medium supplemented with cellulose. Based on the diameter of zone of clearance they were classified in to two categories viz., strains with enzyme activity (+), and non enzyme producers (-). The zone of clearance A, B and C. CMC-Congo red plate assay of Trichoderma sps, Aspergillus niger, Fusarium sps.

20

Plate: (A)Trichoderma sps

Plate: (B) Aspergillus niger

Plate: (C) Fusarium sps Among the 6 strains, only 3, i.e., Aspergillus niger, Fusarium sp and Trichoderma sp belonged to the enzyme activity (+) categoryand another 3 strains didnt show any cellulolytic activity. Hence, the Aspergillus niger, Fusarium sp and Trichoderma spwere selected for cellulose enzyme production and different growth conditions like temperature, pH, carbon and nitrogen sources were optimized.

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CELLULASE PRODUCTION AND ASSAY: The cellulase production medium was found to be the most suitable medium as Aspergillus nigerproduced 8.100.07 IU/mL of cellulase on five days of incubation compared other two species tested. The result was shown in table (1) and fig (1). MANDELS MEDIUM: Table. 1: The cellulase production medium and most suitable medium as Aspergillus niger s.no Organisms Cellulase

Aspergillus niger

8.100.07 IU/mL

Fusarium sps

5.300.06 IU/mL

Trichoderma sps

3.800.08 IU/mL

Fig.1. Cellulase production medium and most suitable medium as Aspergillus niger.

Cellulase
Enzyme activity IU/mL
10 8 6 4 2 0 Aspergillus niger Fusarium sps Trichoderma sps

Organisms
Cellulase

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MEDIUM OPTIMIZATION FOR CELLULASE PRODUCTION: OPTIMUM pH: Among the various pH ranges studied for the detection of optimum pH of the cellulose enzyme production, the enzyme production of Aspergillus niger, Fusarium sp and Trichoderma sp were very high at the pH 7 (8.10 IU/ml, 4.60 and 3.80 IU/ml respectivrly). The results are presented in table and the optimum pH is depicted in table (2) and fig (2). Effect of pH on cellulase production by these fungi supports the findings of Lee et al., (2002) who reported that CMCase, Avicelase and FPase activities exhibit a pH optimum of approximately 4, while the pH optimum of glucosidase was between pH 5 and 6. Table. 2: Optimum pH on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps. s.no 1 2 3 Organisms Aspergillus niger Fusarium sps Trichoderma sps pH-5 8.100.07 4.600.11 3.800.08 pH-7 6.300.05 3.850.07 2.600.11 pH-9 3.800.08 2.800.03 1.500.06

Fig. 2: Optimum pH on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps. 9 8 7 6 5 4 3 2 1 0 pH-5 pH-7 pH-9

Enzyme activity IU/mL

pH
Aspergillus niger Fusarium sps Trichoderma sps

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OPTIMUM TEMPERATURE: In the different temperature range used to detect the optimum temperature for cellulose production the cellulose activity of Aspergillus niger, Fusarium sp and Trichoderma sp were very high at 300C (8.200.08, 6.400.11 and 3.550.11 IU/ml). The results are presented in table (3) and fig (3). Many workers have reported different temperatures for maximum cellulose production either in flask or in fermentor studies using Trichoderma sp. suggesting that the optimal temperature for cellulase production also depends on the strain variation of the microorganism (Murao et al., 1988, Lu al et., 2003).

Table. 3: Optimum temperature on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps. s.no 1 2 3 Organisms Aspergillus niger Fusarium sps Trichoderma sps 25C 7.500.06 4.900.09 2.700.08 30C 8.200.08 6.400.11 3.550.11 35C 5.600.11 2.700.06 2.400.11

fig. 3: Optimum temperature on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps.

9 8 7 6 5 4 3 2 1 0 25C 30C 35C

Enzyme activity IU/mL

Temperature
Aspergillus niger Fusarium sps Trichoderma sps

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EFFECT OF DIFFERENT CARBON SOURCES: Among the different carbon sources studied for the detection of optimum carbon source for cellulose production, the cellulose activity was very high for sucrose (6.200.12, 4.900.09 and 2.980.08 U/ml). The results are presented in table (4) and fig (4). Mendals and Reese (1957) also reported that maximum yields of cellulase were obtained on 1% different carbon substrate using T. viride. Cellulase production commended on reaching nitrogen limiting conditions and the yield of cellulase decreased when excess peptone was presented, various inorganic nitrogen sources have been optimized by different workers for cellulase production (Sherief et al., 2010; Solomon et al., 1997; Lee etal., 2010).

Table. 4: Effect of carbon sources on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps. s.no 1 2 3 Organisms Aspergillus niger Fusarium sps Trichoderma sps Glucose 5.100.07 3.400.11 2.210.09 Sucrose 6.200.12 4.900.09 2.980.08 Maltose 4.800.05 2.700.06 1.830.09

Fig. 4: Effect of carbon sources on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps.

Enzyme activity IU/mL

7 6 5 4 3 2 1 0 Glucose Sucrose Carbon sources Fusarium sps Maltose

Aspergillus niger

Trichoderma sps

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EFFECT OF DIFFERENT NITROGEN SOURCES: Among the different nitrogen sources studied for the detection of optimum nitrogen source for cellulose production, the cellulose activity was very high for peptone (6.880.14, 4.670.06 and 3.550.11 IU/ml). The results are presented in table (5) and fig (5). Though the addition of organic nitrogen sources such as beef extract and peptone resulted in increased growth and enzyme production, was reported before, they were not an effective replacement for inorganic nitrogen sources because of their higher cost (Sun et al., 1999).

Table. 5: Effect of nitrogen sources on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps.

s.no

Organisms

Peptone

Beef extract

Sodium nitrate

1 2 3

Aspergillus niger Fusarium sps Trichoderma sps

6.880.14 4.670.06 3.550.11

6.200.09 3.800.12 1.850.10

3.500.0 2.820.14 1.760.07

Fig. 5: Effect of nitrogen sources on the cellulase production using Aspergillus niger, Fusarium sps, Trichoderma sps.

Enzyme activity IU/mL

7 6 5 4 3 2 1 0 Peptone Beef extract Sodium nitrate

Nitrogen sources
Aspergillus niger Fusarium sps Trichoderma sps

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Deinking activity (%): Among the different enzyme concentration studied for the detection of deinking activity, the maximum deinking activity is found to be Aspergillus niger 83% in 2ml enzyme concentration. The minimum deinking activity is found to be Trichoderma sps 48% in 1.5ml concentration. Enzymatic deinking was found to have high potential as an alternative to the chemical deinking process, which showed several problems and disadvantages compared to enzymatic deinking process. The results are presented in table (6) and fig (6). Table. 6: Enzymatic deinking activity. s.no 1 Organisms Aspergillus niger 0.5ml 64 1ml 60 1.5ml 75 2ml 83 2.5ml 68 3ml 55

2 3

Fusarium sps Trichoderma sps

54 61

74 56

71 48

75 65

66 50

69 50

90 80 70 60

Ink removel (%)

50 40 30 20 10 0 0.5ml 1ml 1.5ml 2ml 2.5ml 3ml Enzymatic Deinking Aspergillus niger Fusariumsps Trichoderma sps

Fig. 6: Enzymatic deinking activity.

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Deinking activity

28

Summary and conclusion:

Cellulose is worlds most abundant organic substance (Ruttloff., 1987) and comprises a major storage form of photosynthesized glucose. It is the major component of biomass energy (Scott et al., 1987). Because a large proportion of vegetation added to soil is cellulose therefore, decomposition of cellulose has a special significance in the biological cycle of carbon (Lederberg, 1992). In industry, these enzymes have found novel application in production and processing of chemicals, food and manufactured goods such as paper, rayon etc., and extraction of valuable components from plants and improvement of nutritional values of animal feed (Wiseman, 1995). Fungi are well known agents of decomposition of organic matter in general and of cellulosic substrate in particular as reported by Lynd et al., (2002). Sone et al., (1999) reported maximum enzyme production by Aspergillus species at 37C. The results of the present study partly confirms the finding that higher enzyme yield was noted at 37C than at 30C. Some Aspergillus strains however, showed higher enzyme yield at 30C as reported by Philippidis (1994). The properties of cellulytic enzyme like those of all proteins are modified by prevailing physical condition such as temperature and pH. Enzyme exhibits its catalytic activity with in these ranges of physical conditions. Beside this, concentration, composition and quality of substrate along with enzyme concentration and reaction time are also important factors that determine the rate of hydrolysis and final yield of the product (Godfrey, 1996; Philippidis, 1994). Enzymes have an optimum pH with in which their activity is maximum and at higher or lower pH values, their activity decreases (Lehinger, 1993). The present study showed that optimum pH for many Aspergillus cellulases was found to be near pH 4.8 as also reported by Vries & Visser (2001). An induction in the enzyme production/activity was noted when fungal isolate (Aspergillus niger) was grown on cellulose as a sole carbon source while a very low rate of enzyme activity/production was observed when glucose was used as sole carbon substrate. These results are in agreement with the ones obtained by other workers, where residual enzyme activity was noted when cellulytic fungi were grown in presence of glucose and many fold increase in enzyme yield were reported in the presence of cellulosic substrate (Lederberg, 1992; Lynd et al., 2002). The production of cellulase for the utilization of cellulose is induced only in the presence of specific substrate (or product there of) but suppressed when easily utilizable sugars such as glucose are available (Lynd et al., 2002). Although cellulases are inducible, but there is a low level of constitutive production of these enzymes suggesting that there may be isozymes, some of them remain repressed in absence of inducer and

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presence of inducer greatly affect the enzyme yield (Yalpani, 1987). However, cellulase production is also influenced by several other factors, such as carbon, nitrogen and phosphorus sources, the ratio of carbon to nitrogen provided, trace elements, pH and aeration rate (Philippidis, 1994). It is therefore, of great importance for enzymology in general and for applied enzymology like industrial enzymology in particular, to be able to identify and define these optimas, as outside these ranges, enzyme performance is either considerably reduced or completely obliterated (Godfrey, 1996). The cellulase activity of fungal cultures, Aspergillus sp. and Penicillium sp. was confirmed by Congo Red Dye decoloration andalso quantitatively with dinitro-salicylic acidreagent method. The results thus obtained bythe above methods were very much the sameand matched with earlier reports of Sazci et al., (1986). Narasimha et al., (1998) isolated Aspergillus and Penicillium sps. from soil contaminatedwith cotton ginning effl uents and made similaridentifi cation reports. Sadaf Jahangeer et al., (2005) reported that majority of Aspergillus and Penicillium sps. were found to posses cellulolytic activity. Aspergillus niger isolated from soil contaminatedwith effluents of cotton ginning industry showed highest cellulase activity (Narasimha et al., 1998). A wide range of Aspergillus sp. Has been identified to possess all components of cellulases complex (Vries & Visser 2001). The biological option, using cellulases and esterases, is now a well-documented approach for deinking and recycling of waste paper. Many research papers and a few patents in recent past have been published in this regard. Most of initial studies on deinking have been carried out with a view to replace chemicals with enzymes. The cellulases that have mainly been tested are the commercially available sources of multi/mono-component enzyme preparations of Trichoderma reseei, H. insolens supplied by Novo-Nordisk, IOGEN, Genencor, etc (Pala et al., 2004; Jefferies et al., 1994). In addition a few of the recent studies have also tested cellulases (with xylanase activities) from the wild type isolates of A. terreus, Aspergillus L22, Trichoderma pseudokoningii, Gleophyllum sp, Orpinomyces sp, Fusarium sp., etc. (Marques et al., 2003; Gubitz et al., 1998; Geng and Li 2003; Vyas and Lachke 2003). In summary, the pulp and paper propertied after enzymatic and chemical deinking processes were characterized. The results obtained are shown in table (6) and fig (6). Based on the results obtained, it can be concluded that the overall pulp and paper properties obtained from enzymatic deinking process were better compared to the chemical deinking process.

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