Siva Project.1

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Enzymesare large biological molecules responsible for the thousands ofchemical

interconversions that sustain life. They are highly selective catalysts, greatly

accelerating both the rate and specificity of metabolic reactions, from the digestion of

food to the synthesis of DNA. Most enzymes are proteins, although some catalytic

RNA molecules have been identified. Enzymes adopt a specific three-dimensional

structure, and may employ organic (e.g. biotin) and inorganic (e.g. magnesium ion)

cofactors to assist in catalysis.

In enzymatic reactions, the molecules at the beginning of the process, called

substrates, are converted into different molecules, called products. Almost all

chemical reactions in a biological cell need enzymes in order to occur at rates

sufficient for life. Since enzymes are selective for their substrates and speed up only a

few reactions from among many possibilities, the set of enzymes made in a cell

determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (Ea) for a

reaction, thus dramatically increasing the rate of the reaction. As a result, products are

formed faster and reactions reach their equilibrium state more rapidly. Most enzyme

reaction rates are millions of times faster than those of comparable un-catalyzed

reactions. As with all catalysts, enzymes are not consumed by the reactions they

catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do

differ from most other catalysts in that they are highly specific for their substrates

.Enzymes are known to catalyze about 4,000 biochemical reactions.
http://en.wikipedia.org/wiki/Metabolismhttp://en.wikipedia.org/wiki/Metabolismhttp://en.wikipedia.org/wiki/Catalysishttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Ribozymehttp://en.wikipedia.org/wiki/Ribozymehttp://en.wikipedia.org/wiki/Protein_structurehttp://en.wikipedia.org/wiki/Protein_structurehttp://en.wikipedia.org/wiki/Cofactor_%28biochemistry%29http://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Substrate_%28biochemistry%29http://en.wikipedia.org/wiki/Product_%28biology%29http://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Metabolic_pathwayhttp://en.wikipedia.org/wiki/Activation_energyhttp://en.wikipedia.org/wiki/Chemical_equilibriumhttp://en.wikipedia.org/wiki/Chemical_equilibriumhttp://en.wikipedia.org/wiki/Activation_energyhttp://en.wikipedia.org/wiki/Metabolic_pathwayhttp://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Product_%28biology%29http://en.wikipedia.org/wiki/Substrate_%28biochemistry%29http://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Cofactor_%28biochemistry%29http://en.wikipedia.org/wiki/Protein_structurehttp://en.wikipedia.org/wiki/Protein_structurehttp://en.wikipedia.org/wiki/Ribozymehttp://en.wikipedia.org/wiki/Ribozymehttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Catalysishttp://en.wikipedia.org/wiki/Metabolismhttp://en.wikipedia.org/wiki/Metabolism


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Biological function:

Enzymes serve a wide variety of functions inside living organisms. They are

indispensable for signal transduction and cell regulation, often via kinases and

phosphatases.[77] They also generate movement, with myosin hydrolyzing ATP to

generate muscle contraction and also moving cargo around the cell as part of the

cytoskeleton.[78] Other ATPases in the cell membrane are ion pumps involved in

active transport. Enzymes are also involved in more exotic functions, such as

luciferase generating light in fireflies.[79]Viruses can also contain enzymes for

infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release

from cells, like the influenza virus neuraminidase.

An important function of enzymes is in the digestive systems of animals.

Enzymes such as amylases and proteases break down large molecules (starch or

proteins, respectively) into smaller ones, so they can be absorbed by the intestines.

Starch molecules, for example, are too large to be absorbed from the intestine, but

enzymes hydrolyze the starch chains into smaller molecules such as maltose and

eventually glucose, which can then be absorbed. Different enzymes digest different

food substances. In ruminants, which have herbivorous diets, microorganisms in the

gut produce another enzyme, cellulase, to break down the cellulose cell walls of plant

fiber.[80]

Review of literature:
http://en.wikipedia.org/wiki/Function_%28biology%29http://en.wikipedia.org/wiki/Signal_transductionhttp://en.wikipedia.org/wiki/Kinasehttp://en.wikipedia.org/wiki/Phosphatasehttp://en.wikipedia.org/wiki/Enzyme#cite_note-77http://en.wikipedia.org/wiki/Enzyme#cite_note-77http://en.wikipedia.org/wiki/Enzyme#cite_note-77http://en.wikipedia.org/wiki/Myosinhttp://en.wikipedia.org/wiki/Muscle_contractionhttp://en.wikipedia.org/wiki/Cytoskeletonhttp://en.wikipedia.org/wiki/Enzyme#cite_note-78http://en.wikipedia.org/wiki/Enzyme#cite_note-78http://en.wikipedia.org/wiki/Enzyme#cite_note-78http://en.wikipedia.org/wiki/Ion_pump_%28biology%29http://en.wikipedia.org/wiki/Active_transporthttp://en.wikipedia.org/wiki/Luciferasehttp://en.wikipedia.org/wiki/Fireflyhttp://en.wikipedia.org/wiki/Enzyme#cite_note-79http://en.wikipedia.org/wiki/Enzyme#cite_note-79http://en.wikipedia.org/wiki/Enzyme#cite_note-79http://en.wikipedia.org/wiki/HIV_integrasehttp://en.wikipedia.org/wiki/Reverse_transcriptasehttp://en.wikipedia.org/wiki/Influenzahttp://en.wikipedia.org/wiki/Neuraminidasehttp://en.wikipedia.org/wiki/Digestive_systemshttp://en.wikipedia.org/wiki/Amylaseshttp://en.wikipedia.org/wiki/Proteaseshttp://en.wikipedia.org/wiki/Starchhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Maltosehttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Ruminantshttp://en.wikipedia.org/wiki/Herbivoroushttp://en.wikipedia.org/wiki/Cellulasehttp://en.wikipedia.org/wiki/Enzyme#cite_note-80http://en.wikipedia.org/wiki/Enzyme#cite_note-80http://en.wikipedia.org/wiki/Enzyme#cite_note-80http://en.wikipedia.org/wiki/Cellulasehttp://en.wikipedia.org/wiki/Herbivoroushttp://en.wikipedia.org/wiki/Ruminantshttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Maltosehttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Starchhttp://en.wikipedia.org/wiki/Proteaseshttp://en.wikipedia.org/wiki/Amylaseshttp://en.wikipedia.org/wiki/Digestive_systemshttp://en.wikipedia.org/wiki/Neuraminidasehttp://en.wikipedia.org/wiki/Influenzahttp://en.wikipedia.org/wiki/Reverse_transcriptasehttp://en.wikipedia.org/wiki/HIV_integrasehttp://en.wikipedia.org/wiki/Enzyme#cite_note-79http://en.wikipedia.org/wiki/Enzyme#cite_note-79http://en.wikipedia.org/wiki/Fireflyhttp://en.wikipedia.org/wiki/Luciferasehttp://en.wikipedia.org/wiki/Active_transporthttp://en.wikipedia.org/wiki/Ion_pump_%28biology%29http://en.wikipedia.org/wiki/Enzyme#cite_note-78http://en.wikipedia.org/wiki/Cytoskeletonhttp://en.wikipedia.org/wiki/Muscle_contractionhttp://en.wikipedia.org/wiki/Myosinhttp://en.wikipedia.org/wiki/Enzyme#cite_note-77http://en.wikipedia.org/wiki/Phosphatasehttp://en.wikipedia.org/wiki/Kinasehttp://en.wikipedia.org/wiki/Signal_transductionhttp://en.wikipedia.org/wiki/Function_%28biology%29


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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.

Santos et al. (1978) found that microscopic fungus Penicilliumitalicum when

grown in synthetic liquid medium produced at least three enzymes with -1-4

glucanase activity. A suggested characterization of these three enzymes indicated that

they had different mode of action. The first one was on endoglucanase, the second

was an exoglucanase and third probably has both mechanism of action. Glucose had a

repressive effect on all three enzymes. Only small amounts of -1, 4-glucanase-2 and

3 were present in the cells when they were actively growing in the presence of this

sugar. However, when the cell were transferred to a medium low in glucose a

significant increase in the specific activity of -1, 4-glucanase took place this was due

in past to a much more active production of -4, 4-glucanan-2 and 3 and in part to the

appearance of -1, 4-glucanase -1, which could only be detected after more than 12

hours of incubation in this medium.

Stewart and Parry (1981) studied that during growth in liguid culture medium

containing a single cellulosic or non- cellulose carbon source

Aspergillusfumigatusrelised cellulose into the medium. The amount of cellulose

depended on nitrogen source, pH, temperature, type and concentration of carbon

source. Maximum cellulolytic activity was observed after incubation for 60 hours


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with 1% (w/v) CM-cellulose as carbon source, (NH4)2SO4 as nitrogen source and a

starting pH of 7.0.

Fllanes and Schaffeld (1982) found that fermentation of leached beat pulp by the

cellulolytic fungus tricodermareesei QM9414 was under carbon limitation, with

celluloses as the only carbon and energy source. Nitrogen was supplied as ammonium

sulphate and the medium was supplemented with other mineral salts as required for

growth. After 40 to 45 hours of fermentation, approximately 80% of the cellulose and

45% of hemicellulose was degraded. Both ,exogenase and endogenase were induced,

endoglucanse was growth associated, while exogenlucanase appeared later in growth

phase, reaching its maximum activity in the stationary phase.

Sen et al. (1982) produced that cellulose and - glucosidasebe using thermophilic

fungus, Myceliopthrathermophila D-14. The optimumtemperature and incubation

period for extra cellular enzymes acting on different types of cellulosic substrates

were determind. Partial purification of enzymes was carried out by (NH4 )2 SO4

precipitation and gel filtration. Electrophoresis of the fractions obtained from gel

filtration revealed at least eight components in the enzymes.

Tsay (1983) found that cellulases ofCellulomonas sp. Used effectively rice straw

which was delignified with 1% (w/w) sodium hydroxide. The effect of other

conditions such as pH, nitrogen source, substrate concentration and incubation time

on the productions such as pH, nitrogen source, substrate concentration and

incubation time on the production of filter paper and endoglucanse activity wre

studied. Maximum production of endoglucanase and filter paper activity was


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odserved at 28C in 7 days, in the presence of 1% NaOH treated rice straw as

substrate at an initial pH of 3.0 when NaNO3 was used as source of nitogen.

Deshpandeet al. (1984) reported an assay for selective determination of

exoglucanase in enzyme systems also containgendoglucanase and -glucosidase. The

assay was based on the observation that exoglucanase hydrolyzed the gluconic bond

of p-nitrophenl--D-cellobioside to yield cellobiose and -nitrophenol. Free

cellobiose reacted with DNSA reagent from colored complexes to be determined

spectrophotometerically at 540 nm.

Mishra et al. (1984) found that Penicilliumfuniculosom produced a complete

cellulose system having endo--1, 4-glucanase (15-20 U/ml). Thesaccharification of

alkali treated cotton sugarcane bagasse by P.funiculosum enzyme 70 and 63%,

respectively. It was possible to obtain glucose concentration as high as 305 using 50%

bagasse.

Macris et al. (1985) studied the production and cross synergistic action of

cellulolytic enymes from fungal mutant. Significant levels of cellulose and -

glucosidase capable of saccharifying cotton fiber and wheat straw cellulose were

excreted by the selected mutant Aspergillusustus M35 and Trichodermaharsianum

M5 grown on these cellulosic materials. The optimum growth media contained 0.3%

urea, 0.2% KH2PO4,0.03%7H20,0.7% (NH4)2PO4 0.03 % CaCl2. 2H2O and 0.01%

yeast extract, cross synergism was observed between the celluloyticsystem of the

fungi upon hydrolysis of cotton.


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Ortega (1985) studied the effect of pH, temperature and substrate concentration

on the cellulose (-1-4 endogulcanase) activity ofA.candidus. Maximum activites

were obtained when the concentration of substrate (CMC) was 6% at pH 4 and 50 C

temperature. The enzyme retained 85 % of its original activity under optimal

conditions of pH and temperature after 36 hours of incubation.

Szozodark (1988) investigated production of cellulases and zylanases by

Trichodermareesei on wheat straw. Autohydrolyzed and ethanol alkali pulped wheat

straw was examined as candidate feedstock for both cellulose and xylanase

production and enzymatic hydrolysis. Submerged culture supernatant with highest

enzymatic activities whereas, maximal efficiency of enzymatic hydrolysis was

recorded in straw treated with ethanol NaOH mixture.

Rho et al. (1990) investigated the influence of cellulosic and hemicellulosic

substrates on the production of accolade and xylanase complexes in Aspergillusniger.

They reported that culture conditions with substrate exhibited profound efforts on

endoglucanase, -glucosidase, endoxylanase and -xylosidase. Biosynthesis of

cellulose and xylanase complexes in A. nigerwere found to co-ordinarily regulated at

the level 0 induction. Multiple forms of extra cellular cellulose and xylanasecompex

seemed to be outcome of specific gene experession.

Chandrashekarand kaveriappa(1991) studied that production of extracellularby

two aquatic hypsometers,Lunulosporacurvulaand Flagellosporapeicilliodes. Results

showed that CMC (carboxymethylcellulose) was the best source of carbon and

(NH4)2SO4 the best source of nitrogen. An optimum pH of 5.2 and 28C was found to


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favor maximum enzyme activity in 12 days old culures. 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 KH2PO4, 0.2;

MgSO4.7H2O O.O3; CaCl2.2H2O, O.O3 and (NH4)2SO4, O.2% at pH- 5.0

inoculated with 6% inoculum 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 higestactivites 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.

Mohite and Maga(2010) have reported the potential of sorghum straw as a

substrate forcellulaseproduction . In submerged culture conditions Aspergillusniger

strain isolated fromsoil collected from Ankaleshwar-Gujrat the production of

cellulase was 0.77 units/ml. The 5lowest production was recovered on wheat straw

medium i.e. 0.28units/ml. These contrastfinding suggest that the yield of cellulases is

depend on the potential of fungal strains andpretreatment to the media components.

UsamaF.Ali and HalaS.Saad El-Dein (2008) growntwo strains ofAspergillusnigerand

Aspergillusnidulanson water hyacinth (Eichhorniacrassipes,Martin).They prepared


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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 cellulaseenzymes .

P.B.Acharyaet 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 NaOHsolution 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 P.B. Acharya and his group

arecomparatively promising with the earliear work reported by Ojummuetal. (2003).this

groupreported thatA. flavusgrows 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 andthey found that among range of pH values of 4.0

to 6.0 the maximum cellulase yield wasrecorded 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. Their

report shows resemblance with the previous report ofMcCleary and Glennie-Holmes

(1985).


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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 usedAspergillus

heteromorphus strain for their research. Their results shows correlation with the

resultsofNarasimha e.al. (2006) for filter paper assay.Narasimha and his group used

saw dust as asubstrate and inoculated A. niger as a source for cellulase production.

Muthuvelayudhamet al. (2006) used sugarcane buggase as a substrate and got

cellulase production 96 I.U./ml byusing Trichodermareesi 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.

Kang et al.(2004) and Yang et al.(2004) both have reported the potential of wheat

straw as a substrate for cellulose fermentation.

Mohite and Magar(2010) have reported the potential of sorghum straw as a

substrate forcellulaseproduction . In submerged culture conditions Aspergillus niger

strain isolated fromsoil collected from Ankaleshwar-Gujrat the production of

cellulase was 0.77 units/ml.

Mohammad Sohail et al., (2009) collected 128 fungal stains from

nativeenvironment 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.Aspergillusnigergroup shown maximum output for

hydrolytic activity. The production ofendoglucanase and beta glucosidase was

reported moer than 1.5 I.U./ml from those strains.


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The reports of Mohemmad Sohail et al., (1997) . They reported maximum activity

ofAspergillusnigerfoe the production of endoglucanase more than 2 I.U./ml. Sohail

et.al. (2009) have reported that all cellulose overproducing Aspergillusstrains were

from soil origin that indicates the property ofdegradation of biomass of

Aspergillusgroup. Under the soil. Banana pill powder and coirpowder 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.

M. Jayantet al.(2011) and his coworkers tried to get cellulase production by

inoculatingstrains ofA.nigerand Pencilliumchrysogenumsimultaneously that the co-

culturing approachof 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 statefermentation.

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

mediumfor cellulase production. In seven days crude cellulase is used for de-

colorization ofmethylene blue dye on on separate basal medium with 3% sucrose

solution in five days atsubmerged 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 onAspergillusandTrichodermasps , In addition to these , other


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species were also reportedcellulose degradation by brown rot fungi of

Coniophoraceae family(1980).

C. Ravindranet al(2011) reported the potential of Chaetomiumspecies collected

from marine mangroveplants 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 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.

M. Ashgeret al(2003) reported the use ofArachinotusspecies for theproduction 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 deckinstrument 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 andstorage.


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Daniel Klen-Marcuschameret al(2011)discussed this issue, they performeda

sensitivity analysis to study the effect of feedstock prices and fermentation time on

the costcontribution 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.

Ranaet 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 .

Raj et al. (1995) investigated the influence of various processing aids coupling

agents in improving fiber dispersion as well as compatibility between thefiber and the

matrix . Stearic acid and mineral oil were used as additives and maleatedethylene 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

correspondingcomposites. This effect was also reflected in improved mechanical

properties of the composites.

Gatenholmet al.(1991) studied the nature of adhesion in composites of

modified cellulose fibers and polypropylene. Cellulose fibers were surface-modified

withpolypropylene maleic-anhydride copolymer and characterized by contact angle


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measurement, ESCA, FTIR, and SEM techniques. Composites reinforced with

surfacemodifiedcellulose fibers showed significantly improved mechanical properties

comparedto composites with untreated cellulose fibers. This was due to improved

fiber wetting,dispersion and fiber-matrix adhesion as seen in SEM micrographs.

Interfacial interactionsinvolved were covalent and hydrogen bonds that formed across

the fiber-matrix interface.

Raj and Kokta (1989) investigated the influence of using various dispersing

aids (stearic acid and mineral oil) and a coupling agent (maleated ethylene) in

cellulosefiber reinforced polypropylene composites. Tensile strength and modulus of

thecomposites studied were found to increase with fiber content when either strearic

acid ormineral oil (1% by weight of fiber) were added as processing aids during the

compounding. The properties also were found to be affected by the amount of

processingaid used. Maximum increases in the properties were observed when the

processing aid wasadded in 1% concentration (by weight of fiber). A further increase

in the amount ofprocessing aid caused the properties to decline dramatically. Stearic

acid was found toperform better in improving the fiber dispersion compared to

mineral oil.

Deinking review of literature:

Woodward et at. (1994)suggested that catalytic hydrolysis may not be

essential,since enzymes can remove ink under nonoptimal conditions. Mere 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


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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 inconventional

deinking.

Prasad et al.and Rushing et al. (1992)reportedreductions in particle sizefrom 16%

to 37%, depending on inktype. So far, there is no credible explanation for this

reduction in the size of ink particles.

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.

Putzet 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


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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.

Zeyeret al. (1995) studied the performance of enzymesin deinking of ONP. Their

resultsdemonstrated that the arrangementof unit operations is of importance.No

deactivation of enzymes by shearstress was observed. Statistical investigation of

particles on handsheets demonstrated that many ink particles were likely still at their

original location.

Nakano (1993) has reported that an alkaline lipase efficiently removed offset-

printing inks. Enzymes that catalyzethe 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

Phanerochaetechrysosporiumand with lignin-degradingenzymes.

.

Yang et al. (26) reported that the freeness of enzymatically deinked MOW pulp

was 32% higherthan that of control pulp.

Heiseet al.(1996)and Prasad et al. (24) foundthat enzyme treatment

significantlyincreased pulp freeness from 510 to570 mL CSF and 440 to 490 mL

CSF, respectively. Prasad et al.observed that freeness increased in all the enzyme-

treated samples compared with the control. The freeness increase varied from 50%


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for a cellulose treated colored flexo-printed newsprint to 14% for black-and-

whiteprinted newsprint treated with a hemicellulase preparation.

Heiseet 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.

Introduction: Cellulose producing microbes:

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 D et al. 2002). It is the most abundant and renewable

biopolymer on earth and the dominating waste material from agriculture (Bhat M K

& 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.


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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 (Machessaultet 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 (Klemmet 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.


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Cellulaseshydrolyze 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-glucancellobiohydrolase,

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)](Schuleinet 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

togetherto hydrolyze cellulose to small cello-oligosaccharides. The oligosaccharides

(mainly cellobiose) are subsequently hydrolyzed to glucose by a major -glucosidase

(Bguinet al.(1994).

Cellulases are used in the textile industry (Gusakovet al.(2000), in

detergents(Kottwitzet al(2005), pulp and paper industry, improvingdigestibility 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(Himmelet al.(1999). However, in production of

bioethanol, the costs of the enzymes to be used for hydrolysis of the raw material need to


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be reduced and their efficiency increased in order to make the process economically

feasible.

Commercial production of cellulases has been triedby either solid or submerged

culture including batch, fed batch, and continuous flow processes. Media usedin cellulase

fermentations contain cellulose indifferent degrees of purity, or as raw lignocellulosic

substrates (Doppelbaueret al(1991), which is especially truein 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 processcontrol 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


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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 Cellulomonasand 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

Trichodermareesei, 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;

andActinomycetes-Streptomyces, Actinomucor, and Streptomyces.

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 (Hayashidaet 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


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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 Ref

Fungi Aspergillus

Fusarium

Humic

Melanocarpus

Penicillium

Trichoderma

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

40

43

44

46

47

36

42

48

38

37

45

9

41

18

52


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Bacteria

Actinomycete

Acidothermus

Bacillus

Clostridium

Pseudomonas

Rhodothermus

Cellulomonas

Streptomyces

Thermonospore

cellulolyticus

Bacillus sp

Bacillus

subtilis

acetobutylim

thremocellum

P. cellulosa

R. marinus

fimi

C.bioazotea

C.uda

S.drozdowici

S. sp

S. lividans

T. fusca

T. curvata

49

50

54

55

51

53

58

32

59

60

61

62

56

57

Application of cellulose:

(Uhliget al.(1998) and Singh et al.(2007), Kirket al(2002)

Pulp and Paper industry:


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Considering the importance and application of the cellulases, this study was aimed toscreen the indigenous fungal isolates for the cellulytic ability. Furthermore, this studyaims to provide better understanding of condition for the production and activity ofcellulases by different fungal cultures.

Materials and method:

Sample collection:

Sample were collected from Muttukadu Isolation of fungi from infected parts of plant

i.e., leaf and stem of wheat plant was made by directly scraping the fungal growth and

inoculation on SDA.

Isolation and identification of fungus:

Serial dilution:

To the 99ml of distilled water was taken, 1ml of 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.

Plating Technique:

One gm of soil sample was added to 99 ml of sterile distilled water in a 250 ml

Erlenmeyer flask and kept in a mechanical shaker at 120 rpm for 15 mins. Serial

dilutions up to 10 4 was done. Sabourauds dextrose agar (SDA) medium was

prepared, the pH was adjusted to 5.5 and sterilized at 15 lbs for 121C for 15 mins.


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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.

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 dropof mountant with the coverslip edge, and lower gently, avoiding air bubbles. The

preparation is now ready for examination.

Lactophenol Cotton Blue:

This stains chitin, making such structures as spore ornamentation show up muchmore clearly than they do with most other stains (including Congo Red).

A parting shot - well, this one is more to do with avoiding departing. Thechemicals 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.


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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.

Purification of Fungal Isolates by Single Hyphal Tip Method [10]:

Sabourauds dextrose agar was prepared, sterilized at 15 lbs for 121C for 15 mins

and dispensed onto sterile petridishes. After solidification, peripheral mycelia from

the slants were carefully lifted, streaked on to SDA plates and incubated at 30C for 5

days. After incubation, the colonies were observed for hyphal developments. The

peripheral tip of the mycelial growth was taken from the plates, reinoculated onto

SDA medium and incubated at 30C for 5 days.

Preparation of Inoculum:

Purified fungal isolates grown in plates were taken after good growth of mycelial

matt and 0.6 cm diameter mycelial discs were cut with a flame sterilized cork-borer

and used as inoculated

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 (Mandelset al., 1976). Soil samples were supplemented with saw

dust and synthetic medium (modified Mandels medium) containing in( g%):


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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%):

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


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then de-stained with 1M 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 Mandelset 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.

Mediumoptimization for over production of the enzyme is an important step and

involves a number ofphysico-chemical parameters such as the incubation period, pH,

temperature and supplementedSubstrate 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


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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 temperature

Fungal isolates were inoculated in the synthetic medium followed by incubation at

25C, 30C,35C and 40C. Enzyme production was measured on each couple of

days from 2nd to 10th days of incubation which was followed by enzyme assays

using CMC (Soneet al.1999)

Optimization of pH

The optimized media were prepared using the individual substrates and the pH was

set atdifferent levels such as 4.0, 5.0, 6.0, 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 4.0 and 9.0. The

most suitable pH of the fermentation medium was determined by adjusting the pH of

theculture medium at different levels in the range of pH 3 to 9 using different

buffers(Akibaet al.1995)

Effect of carbon sources on enzyme production

Effect of various carbon compounds viz., cellulase, CMC, glucose, sucrose and

maltose wereused for studying. The broth was distributed into different flasks and 0.5

to 3.0 % of eachcarbon sources were then added before inoculation of the strain and


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after culture inoculation,the flasks were incubated for 7 days at 452 0

C.(Kachlishvili et al 2002)

Optimization of nitrogen sources

For optimization proposes nitrogen source like peptone, yeast extract, urea, casein

and wheywere used. All the flasks were incubated at 30C in orbital shaker incubator

at 120 rpm. All these supplements were used in percent concentrations such as 0.25,

0.50, 1.0 and 1.25. The samples were drawn on the 6th day for enzyme assays. .

In the present study, to detect the appropriate nitrogen source for cellulase production

by theTrichodermaviride. The influence of peptone, beef extract, ammonium nitrate,

sodiumnitrate and yeast extract were studied. The fermentation medium was

supplemented withorganic and inorganic compounds at 0.5 to 3.0% level, replacing

the prescribed nitrogensource of the fermentation medium (Moore et al.1990).


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Deinking introduction:

The traditional (chemical) deinking process is widely used and is considered to be

effective for the removal of ink particles. However, the widespread technology using

thermoplastic toner in the printing process presents a special challenge in the

conventional deinking process. Furthermore, the process requires the usage of large

quantities of chemical agents (Shrinath et al. 1991). This makes the process highly

damaging to the environment, and treatment of the effluent to meet environmental

regulations is costly (Prasad et al. 1992). On the contrary, a biological process (using

enzymes) has been evaluated and proven successful in deinking various types of

wastepaper. One of the benefits of using enzymes in the deinking process is the

minimumtreatment of effluent produced; it is also less harmful to the environment. The

effluentfrom an enzymatic deinking has been reported to be lower in COD content than

wastewater from a corresponding chemical deinking process (Putz et al. 1994).

Furthermore, enzymatic deinking can avoid the alkaline environment that is commonly

employed in the chemical deinking process. This consequently will cut down the cost of

chemicals that are used to treat the effluent and also reduce the COD loaded into the

wastewater system.

Enzymes used in the deinking process detach ink particles from fiber by partially

hydrolyzing the cellulose fibers on the fiber/ink inter-bonding regions, which facilitates

ink detachment during the flotation process (Kim et al. 1991). In addition, enzymatic

deinking technology has been found to possess advantages in deinking MOW because

removal of toner used in xerographic and laser printed processes is very difficult using

the alkaline deinking process (Prasad 1993; Jeffries et al. 1994). This is because the


33/43

tonercontains thermoplastic binders that fuse onto the paper fibers during the printing

process.They will remain as flat, large, rigid particles when treated with chemical; such

particlesare poorly separated during the flotation process (Jeffries et al. 1994, 1995; Viesturs et

al.1999). Even a longer pulping process is not very efficient at breaking down the tonerparticles

to a favorable size to be removed by flotation process (Shrinath et al. 1991).

In addition to ink removal, enzymatic deinking may improve the strengthproperties and

increase freeness of the paper sheets, reduce fines content of the recycledfiber, and enhance the

brightness and cleanliness of the pulp. On the other hand, inkparticles sizes larger than 40 m are

visible to the naked eye, and recycled fibercontaining these ink particles size would downgrade

the quality of the final product (Carr1991; Shrinath et al. 1991; Ferguson 1992; Borchardt and

Rask 1994). However, thisproblem can be solved by the enzymatic deinking process. Reduction

in ink particles sizecan be achieved by the enzyme action. Furthermore, small particles are

removed moreeffectively during the flotation process.

Previous studies have found that, based on increment in brightness, deinking of

MOW and ONP were more effective using enzymes compared to the chemical method

(Lee et al. 2011). However, brightness alone cannot represent the overall quality of the

deinked paper. Other pulp and paper properties such as optical properties, mechanical

strength, and cleanliness are also very important but have yet to be examined and

determined, as the previous studies are limited. Therefore, objective of this present workwas to

examine and compare the quality of the deinked paper and the effluent after enzymatic and

chemical deinking process.


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EXPERIMENTAL

Sources of Enzymes

The enzymes used in this study were a mixture of cellulase and xylanase. These

enzymes were produced by a local isolate; A. niger USM AI 1 via solid state fermentation

(SSF) process, using a newly developed SSF bioreactor namely FERMSOSTAT. The

cellulase to xylanase ratio obtained was 1:5, and this enzymes ratio was used in deinking

of MOW and ONP. Cellulase and xylanase activities were determined according to

Gessesse and Gashaw (1999) and Gessesse and Gashe (1997), respectively.

Selection and Preparation of MOW and ONP

The wastepaper samples (MOW and ONP) used in this study were obtained

within the campus of the university. The MOW mainly consisted of photocopier and

computer 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 high

moisture until needed.

Enzymatic and Chemical Deinking Process

Prior to pulping, wastepaper was soaked in tap water for one hour at room

temperature and then transferred into the developed bioreactor system for disintegration.

The disintegration process was carried out for 60 minutes under room temperature at 6%

consistency and 600 rpm. After disintegration, the pulp was recovered by dewatering

before being used in enzymatic deinking process (Pala et al. 2004). Pulp (2 kg on air-dry

basis) 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 and


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replaced by the appropriate amount of diluted enzyme solution in order to maintain the

pulp slurry at 4% consistency. Diluted enzyme solution was used in order to achieve

better dispersion. The reaction of enzymes with pulp occurred at pH 5.5 and 55oC for 45

min with continuous slow mixing, and the total enzyme used was 1.2 U per gram of air-

dry pulp (0.2 U of cellulase and 1.0 U of xylanase). A control was run as described above

except using thermally inactivated enzyme (Gubitz et al. 1998a). After the enzymatic

hydrolysis process, a small volume of the pulp slurry was used to assay for the reducing

sugar being produced. The pulp slurry was diluted with water and transferred to flotation

vessel using a diaphragm pump.

Water was added to the flotation vessel after the pulp suspension had been

transferred into the flotation vessel, and the water addition was controlled by the water

level sensor. The flotation process was carried out at 0.24% consistency with 280 L/min

of supplied air.

After the flotation process, the pulp suspension was transferred into pulp

collecting vessel using a diaphragm pump in order to separate the pulp from pulp water

suspension. Thereafter, the deinked pulp was rinsed with water (3X) and handsheets was

made in order to determine the efficiency of the deinking process.

The conditions used in enzymatic deinking of MOW and ONP were previously

optimized, and the results are summarized in Table 1. Control and sample pulps were

processed in a similar manner to that given in the enzymatic hydrolysis described above

except heat inactivated and active enzymes were used, respectively.

Meanwhile, similar conditions were used in the chemical deinking process except

replacing enzyme with 2% (w/w) NaOH and 2% (w/w) sodium silicate (Pala et al. 2004).


36/43

The pH of the pulp slurry was adjusted to pH 11.4. The control condition was run as

described above with the absence of chemicals. Blank refers to the pulp slurry after

pulping without performing the flotation process. Three trials run were carried out for all

the experiments.

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