screening and optimization of fungal cellulases isolated from the native environmental source and it

7/27/2019 Screening and Optimization of Fungal Cellulases Isolated From the Native Environmental Source and It

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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


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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 cello-

oligosaccharides. 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 cello-

oligosaccharides. 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


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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 (Fowleret 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


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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 Reference

Fungi

Bacteria

Aspergillus

Fusarium

Humicola

Melanocarpus

Penicillium

Trichoderma

Acidothermus

Bacillus

Clostridium

A.niger

A. nidulans

A. oryzae

(recombinant)

F. solani

F. oxysporum

H. insolens

H. grisea

M. albomyces

P. brasilianum

P. occitanis

P. decumbans

T. reesei

T. longibracm

T. harzianum

A. cellulolyticus

Bacillus sp

Bacillus subtilis

C. acetobutylim

C. thremocellum

Ong et al., (2004)

Kwon et al., (1992)

Takashima et al., (1998)

Wood et al., (1977)

Ortega et al., (1990)

Schulein et al., (1997)

Takashima et al., (1998)

Oinonen et al., (2004)

Jorgensen et al., (2003)

Chaabouni et al., (1995)

Mo et al., (2004)

Schulein et al., (1988)

Fowleret al., (1990)

Gusakov et al., (2000)

Tuckeret al., (1989)

Mawadza et al., (2000)

Hecket al., (2002)

Lopez et al., (2004)

Nochure et al., (1993)


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Actinomycete

Pseudomonas

Rhodothermus

Cellulomonas

Streptomyces

Thermonospore

P. cellulose

R. marinus

C. fimi

C.bioazotea

C.uda

S.drozdowici

S. sp

S. lividans

T. fusca

T. curvata

Yamane et al., (1970)

Hreggvidsson et al.,

(1996)

Shen et al., (1996)

Okeke et al., (1992)

Fennington et al.,(1982)

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.


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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


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of condition for the production and activity of cellulases by different fungal cultures (Uhlig et al., 1998 and

Singh et al., 2007, Kirket 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.


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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 non-

conventional 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 inAspergillus niger. They reported that culture conditions with

substrate exhibited profound efforts on endoglucanase, -glucosidase, endoxylanase and -xylosidase.

Biosynthesis of cellulose and xylanase complexes inA. nigerwere 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. nigeras 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.


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Duenas et al., (1995) subjected ammonia treated bagasse 5% to mixed culture fermentation with T.

reesi LMUC 4 andA. 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 ofAspergillus nigerfor

the production of endoglucanase more than 2 I.U/ml. Sohail et.al. (2009) have reported that all cellulose

overproducingAspergillus 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.

Ashgeret al., (2003) reported the use ofArachinotus 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.


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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 inoculatedA. nigeras 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. nigerin

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


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activity. The production of endoglucanase and betaglucosidase 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 ofA.nigerandPencillium

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.

Talekaret al., (2011) and his research group tried to get cellulase production from local isolates

ofA.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 ofConiophoraceae 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


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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-Marcuschameret 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 offset-

printed 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 isolationand characterization of fungi.

The samples was collected in sterile polythene bag and preserved at 4C in refrigerator andsamples 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 addedand 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 dilutiontransferring was done.

Then the test tubes 10, 10, 10 was used for fungal isolation.SABOURAUDS DEXTROSE AGAR MEDIUM:

INGREDIENTS g/I

Peptone : 10.0 g Dextrose : 40.0 g Agar : 20.0 g Distilled water : 1000 ml pH : 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


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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 mountantwith 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 30c. 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 cell-

free 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 50C 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.


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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 asAspergillus niger

Fig.1. Cellulase production medium and most suitable medium asAspergillus niger.

0

2

4

6

8

10

Aspergillus niger Fusarium sps Trichoderma spsEnzymea

ctivityIU/mL

Organisms

Cellulase

Cellulase

s.no Organisms Cellulase

1 Aspergillus niger 8.100.07 IU/mL

2 Fusarium sps 5.300.06 IU/mL

3 Trichoderma sps 3.800.08 IU/mL


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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 ofAspergillus 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 usingAspergillus niger,Fusarium sps, Trichoderma sps.

s.no Organisms pH-5 pH-7 pH-9

1 Aspergillus niger 8.100.07 6.300.05 3.800.08

2 Fusarium sps 4.600.11 3.850.07 2.800.03

3 Trichoderma sps 3.800.08 2.600.11 1.500.06

Fig. 2: Optimum pH on the cellulase production usingAspergillus niger,Fusarium sps, Trichoderma sps.

0

1

2

3

4

5

6

7

8

9

pH-5 pH-7 pH-9

E

nzymeactivityIU/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 ofAspergillus 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 usingAspergillus niger,Fusarium sps, Trichoderma

sps.

s.no Organisms 25C 30C 35C


2 Fusarium sps 4.900.09 6.400.11 2.700.06

3 Trichoderma sps 2.700.08 3.550.11 2.400.11

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

sps.

0

1

2

3

4

5

6

7

8

9

25C 30C 35C

EnzymeactivityIU/mL

Temperature



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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 (Sheriefet 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 Organisms Glucose Sucrose Maltose


2 Fusarium sps 3.400.11 4.900.09 2.700.06

3 Trichoderma sps 2.210.09 2.980.08 1.830.09

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

sps.

0

1

2

34

5

6

7

Glucose Sucrose Maltose

EnzymeactivityIU/mL

Carbon sources



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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


2 Fusarium sps 4.670.06 3.800.12 2.820.14

3 Trichoderma sps 3.550.11 1.850.10 1.760.07

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

Trichoderma sps.

0

1

2

3

4

5

6

7

8

Peptone Beef extract Sodium nitrate

EnzymeactivityIU/m

L

Nitrogen sources



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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 niger83% 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.

Fig. 6: Enzymatic deinking activity.

0

10

20

30

40

50

60

70

80

90

0.5ml 1ml 1.5ml 2ml 2.5ml 3mlInkremovel(%)

Enzymatic Deinking

Aspergillus niger Fusariumsps Trichoderma sps

s.no Organisms 0.5ml 1ml 1.5ml 2ml 2.5ml 3ml

1 Aspergillus niger 64 60 75 83 68 55

2 Fusarium sps 54 74 71 75 66 69

3 Trichoderma sps 61 56 48 65 50 50


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Deinking activity


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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 byAspergillus 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 manyAspergillus 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. andPenicillium 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 ofAspergillus and

Penicillium sps. were found to posses cellulolytic activity. Aspergillus nigerisolated 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 ofA. 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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