Because of the need for renewable energy resources, cellulose, which can be enzymatically hydrolyzed to glucose, has drawn lot of attention during the past decade. However, the process of cellulose conversion using cellulase is not yet economically feasible because of the high cost of enzymes. Factors influencing the cellulase production of Trichoderma koningii using both acid and steam treated sugar cane bagasse and rice straw as carbon sources were investigated. The highest levels of cellulase activities were obtained using a culture medium containing urea and (NH4)2SO4 together as nitrogen sources at 0.217% and 0.241% for both carbon sources. When the culture medium was supplemented either with 0.5% Tween 60 or Tween 80, the rate of cellulase production was increased considerably. Maximum levels of both filter paper and CMC-ase activities produced on both media were obtained at 25 °C and 100 r.p.m., while the highest level of â -glucosidase production was obtained at 30 °C and 200 r.p.m

El-hawary, F. I.

Mostafa, Y. S.

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Acta Alimentaria, Vol. 30 (1), pp. 313 (2001)FACTORS AFFECTING CELLULASE PRODUCTIONBY TRICHODERMA KONINGIIF. I. EL-HAWARY and Y. S. MOSTAFAMicrobiology Department, Faculty of Agriculture, Mansoura University, Mansoura. Egypt(Received: 22 November 1999; revision received: 5 June 2000; accepted: 29 August 2000)Because of the need for renewable energy resources, cellulose, which can be enzymaticallyhydrolyzed to glucose, has drawn lot of attention during the past decade. However, the process ofcellulose conversion using cellulase is not yet economically feasible because of the high cost ofenzymes. Factors influencing the cellulase production of Trichoderma koningii using both acidand steam treated sugar cane bagasse and rice straw as carbon sources were investigated. Thehighest levels of cellulase activities were obtained using a culture medium containing urea and(NH4)2SO4together as nitrogen sources at 0.217% and 0.241% for both carbon sources. When theculture medium was supplemented either with 0.5% Tween 60 or Tween 80, the rate of cellulaseproduction was increased considerably. Maximum levels of both filter paper and CMC-aseactivities produced on both media were obtained at 25 °C and 100 r.p.m., while the highest levelof β-glucosidase production was obtained at 30 °C and 200 r.p.m.Keywords: cellulase production, sugar cane bagasse, rice straw, pretreatmentThere has been a continuously increasing interest towards the saccharification ofcellulosic waste materials by enzymes in recent years (ENAYATI & PARULEKAR, 1995).The enzymatic conversion of native cellulose is catalyzed by the synergistic action ofenzymes including endo-β-1,4-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91),and β-glucosidase (EC 3.2.1.21). These enzymes are synthesized by a large variety ofmicroorganisms including fungi and bacteria as well. However, fungal species havebeen more intensively studied because they produce extracellular cellulases.Due to the low specific activity of cellulases, a large quantity of enzyme per unitamount of substrate is required to obtain high conversion yield of cellulose. Animportant obstacle in the exploitation of lignocellulosics is the rather costly productionof cellulases contributing as much as 50% of the overall cost of saccharification process(MANDELS, 1985). Furthermore, microbial synthesis of cellulase enzymes is subjected tocatabolite repression, and it has to be induced by cellulose. In the present study, theproduction of cellulolytic enzyme system of T. koningii using two different carbon0139-3006/2001/$ 5.00  2001 Akadémiai Kiadó, Budapest4 EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGIIsources, i.e. acid and steam treated sugar cane bagasse and rice straw was examined.Various factors, such as quality and concentration of nitrogen source; addition ofsurfactants; incubation temperature as well as agitation speed, were studied in order tooptimize the cellulase production of T. koningii.1. Materials and methods1.1. MicroorganismFreeze-dried spore preparation of Trichoderma koningii 2691 was obtained fromthe National Collection of Agricultural and Industrial Microorganisms (NCAIM),Budapest, Hungary. The fungal stock culture was maintained on PDA slants medium at4 °C.1.2. Enzyme productionThe growth and production medium (TÜRKER & MAVITUNA, 1987) contained7 g l1 (NH4)2SO4, 10 g l1 KH2PO4, 1.5 g l1 MgSO4.7H2O, 1.5 g l1 CaCl2.2H2O,1.5 g l1 urea, 1 g l1 glucose. The medium was supplemented with trace elements:25 mg l1 FeSO4.7H2O, 10.3 mg l1 MnSO4.4H2O, 7 mg l1 ZnSO4.7H2O and18.3 mg l1 CoCl2.6H2O. Steam pretreatment of both sugar cane bagasse and rice strawwas carried out according to CARRASCO (1994) at 120 °C for 100 min in the presence of1% sulfuric acid. Pretreated sugar cane bagasse and rice straw were used as carbonsources. The production medium contained 1.5% of carbon source. A 3 ml sporesuspension obtained from 7-d PDA slant culture was used to initiate growth in a shakeflask containing 50 ml of sterile culture medium at pH 5.0. Cultures were incubated on arotary shaker at 100 r.p.m. and 30 °C. The fermentation broth was harvested thenfiltered and analyzed for various enzyme activities.1.3. AssaysFilter paper activity (FPA) was measured according to MANDELS and co-workers(1976) procedure. The reaction mixture containing 0.5 ml of 0.05M acetate buffer (pH4.8) and 0.5 ml of culture filtrate was incubated together with a 1×3 cm (25 mg) strip ofWhatman No. 1 filter paper at 50 °C for 60 min. The enzymatic reaction was terminatedby addition of 1 ml DNS reagent (MILLER, 1959). After 5 min of boiling and addition of10 ml distilled water the absorbance was measured at 540 nm.Acta Alimentaria 30, 2001EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGII 5Carboxyl-methyl-cellulose degrading capacity (CMC-ase) was determined byincubating 0.5 ml of enzyme sample together with 0.5 ml of 1% carboxy-methyl-cellulose in 0.05 mol l1 acetate buffer (pH 4.8) at 50 °C for 30 min. The hydrolysis wasstopped by addition of 1 ml DNS reagent. After boiling for 5 min and dilution with 10ml of distilled water the absorbance was read at 540 nm.For β-glucosidase activity determination 10 µl of culture filtrate was incubatedtogether with 1 ml of 0.67 mmol l1 (0.02%) p-nitrophenyl-β-D-glucopyranoside in0.05 mol l1 acetate buffer (pH 4.8) ad 50 °C for 10 min. The enzymatic reaction wasterminated by addition of 3 ml 0.1 mol l1 NaOH solution. The absorbance wasmeasured at 400 nm (RÉCZEY et. al., 1990).2. Results and discussion2.1. The effect of nitrogen sourceThe effect of alternative nitrogen sources on the production of cellulolyticenzymes using two different media was investigated. Ammonium sulfate and urea ofbasal medium were replaced with urea or ammonium sulfate separately or with otherorganic and inorganic nitrogen sources is such amounts that the final nitrogenconcentration in the media remained unchanged. The results are summarized in Fig. 1. Itcan be seen that the highest enzyme activities were obtained when the original culturemedium was used in which both urea and (NH4)2SO4 were added. With all othernitrogen sources considerably lower activities were obtained.These findings were in good agreement with the results obtained by MANDELSand WEBER (1969) and TÜRKER and MAVITUNA (1987). It was also observed that usingurea or (NH4)2SO4 separately resulted in much lower cellulase production, which couldbe due to pH changes during the cultivation. The final pH of the culture broth was moreacidic when ammonium containing compounds were added to the medium compared tourea or other organic nitrogen sources. This observation was in good agreement withHAAPALA and co-workers (1994, 1996). They showed that the deamination of urearesulted in increasing pH in the culture medium.2.2. The effect of nitrogen source concentrationThe effect of various concentrations of urea and ammonium sulfate as nitrogen sourceson the cellulase production was also investigated. The results are plotted in Fig. 2.Acta Alimentaria 30, 20016 EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGIIFig. 1. The effect of nitrogen source on the cellulase production of T. koningii using various carbon sources.A: pretreated sugar cane bagasse, B: pretreated rice straw. Filter paper activity (FPA): white bars,CMC-ase activity:black bars, β-glucosidase activity: grey barsActa Alimentaria 30, 2001EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGII 7Fig. 2. The effect of nitrogen source concentration on the cellulase production of T. koningii using variouscarbon sources. A: pretreated sugar cane bagasse, B: pretreated rice straw. Filter paper activity(FPA): n, β-glucosidase activity:s, CMC-ase activity: lActa Alimentaria 30, 20018 EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGIIIt can be seen that 0.217% nitrogen from urea and (NH4)2SO4 containing medium wasoptimal for cellulase production by T. koningii. However, maximum CMC-ase andβ-glucosidase activities were obtained at a lower nitrogen source concentration (seeFig. 2). At 0.194% nitrogen concentration, the CMC-ase and β-glucosidase activitiesreached 96% of the activities obtained on the control medium (0.217% nitrogen source).It is clear from Fig. 2/A that applying the original amount of ammonium sulfate(7 g l1), while increasing the urea from 1.5 g l1 to 2.0 g l1 (which increase the totalnitrogen source concentration from 0.217% to 0.241%) caused the increase in theenzyme production of T. koningii by about 11.18% in the case of FPA. The increase inthe CMC-ase due to increasing the amount of urea was about 7.2%, while significantlyhigher, 42.10%, β-glucosidase activity was obtained.These results were in line with WEBB and co-workers (1986) and KHALAF ALLAHand co-workers (1993).2.3. The effect of surfactantsThe effect of Tween 40, 60, 80 on the cellulase production of T. koningii wasstudied at two different concentrations i.e. 0.1 and 0.5% surfactant in the culturemedium. The results are shown in Fig. 3. The highest enzyme production rates wereobtained when 0.5% Tween 60 was applied in the medium.The rate of enzyme production of T. koningii using acid-steam treated sugar canebagasse was increased about 1.3 times compared to the control medium, in which therewas no surfactant added. However, using acid-steam treated rice straw as the carbonsource 0.5% Tween 80 added to the medium was optimal for cellulase production (seeFig. 3/B).Cellulase production was 3.43%, 2.49% and 13.24% higher for FPA, CMC-aseand β-glucosidase compared to the control medium. The final pH of the medium wasslightly affected by Tween 80. The effect of Tween 80 on the cellulase production byvarious fungi was observed by HUNG and co-workers (1988). LONG and KNAPP (1991),and STUTZENBERGER (1987) reported the mechanism of enhancement in the enzymesecretion by Tween. Surfactants can increase the permeability of the cell membrane,facilitating more rapid secretion of extracellular enzymes, which in turn leads to greaterenzyme synthesis.Acta Alimentaria 30, 2001EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGII 9Fig. 3. The effect of surfactants on the cellulase production of T. koningii using various carbon sources.A: pretreated sugar cane bagasse, B: pretreated rice straw. Filter paper activity (FPA): white bars,CMC-ase activity: black bars, β-glucosidase activity: grey barsActa Alimentaria 30, 200110 EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGIIFig. 4. The effect of incubation temperature on the cellulase production of T. koningii using various carbonsources. A: pretreated sugar cane bagasse, B: pretreated rice straw. Filter paper activity(FPA): n, β-glucosidase activity:s, CMC-ase activity: lActa Alimentaria 30, 2001EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGII 11Fig. 5. The effect of agitation on the cellulase production of T. koningii using various carbon sources.A: pretreated sugar cane bagasse, B: pretreated rice straw. Filter paper activity (FPA): n,β-glucosidase activity:s, CMC-ase activity: lActa Alimentaria 30, 200112 EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGII2.4. The effect of incubation temperatureTo account for the effect of incubation temperature on the cellulase production ofT. koningii using two different carbon sources, cultivation was run at 20, 25, 30 and35 °C. The results are shown in Fig. 4. It can be seen that cultivation temperatureof 25 °C was optimal for cellulase production (FPA, and CMC-ase), while 30 °C wasfavorable for β-glucosidase production for both acid-steam treated bagasse andrice straw.Higher temperature resulted in progressive decline in cellulase production. Theenzyme activities obtained at 35 °C were about 5060% lower than that of obtained at25 °C using both acid-steam treated bagasse and rice straw as carbon source. Theseresults were in agreement with those obtained by KNAPP and LEGG (1986),DOPPELBAUER and co-workers (1987), BASTAWDE (1992) and CHAUDHURI and SAHAI(1993).2.5. The effect of agitationThe effect of agitation on the cellulase production using two different carbonsources was investigated. The results showed that the enzyme production could beincreased with increasing agitation (Fig. 5), however increasing the agitation speedabove 100 r.p.m. resulted in a decreased cellulase enzyme production. Although, theFPA and CMC-ase activities were decreased when the agitation speed was increasedfrom 100 to 200 r.p.m, the β-glucosidase production was increased considerably.The high concentration of carbon source resulted in increased viscosity of themedium that may affect the availability of oxygen needed for β-glucosidase production.These results were in agreement with those reported by MAGNELLI and co-workers(1996), BUSWELL and CHANG (1994), SILVA and co-workers (1995) and RÉCZEY andco-workers (1996).3. ConclusionsAcid-steam pretreatment of both sugar cane bagasse and rice straw proved to bean effective method to increase accessibility for cellulase production with T. koningii2691. Urea and other organic nitrogen compounds were better N-sources than inorganicammonium containing salts. Supplementing the culture medium with surfactants, suchas Tween, increased the cellulase production considerably. The effect of incubationtemperature and agitation was also investigated. The results obtained were similar tothose of reported earlier using other Trichoderma strains. It can be concluded thatT. koningii is an effective cellulase producing microorganism showing good yields ofenzymes on both pretreated bagasse and rice straw.Acta Alimentaria 30, 2001EL-HAWARY & MOSTAFA: CELLULASE PRODUCTION BY TRICHODERMA KONINGII 13ReferencesBASTAWDE, K. B. (1992): Cellulolytic enzymes of thermotolerant Aspergillus terreus strain and their actionon cellulosic substrates. World J. Microb. Biotechnol., 8, 4549.BUSWELL, J. A. & CHANG, S. (1994): Biomass and extracellular hydrolytic enzyme production by sixmushroom species grown on soybean waste. Biotechnol. 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Factors affecting cellulase production by trichoderma koningii

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Factors affecting cellulase production by trichoderma koningii

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