site ELSEVIER al bal chology 25 (1999) 244 Cellulase production by Neurospora crassa on wheat straw M.D. Romero*, J. Aguado, L Departamento de ingemerio Qui, Fac: CC. Qi Reccived 1 June Abstract The production of enzymes ef the cellulase complex by the fungus Neurospora crassa on milled and sieved wheat Gonzalez, M. Ladero as Universidad Compiatnse, Madi Spam 9%; recive in revised frm LT Ferry 199% acepsed 2 Marc 199 The effects of suaw concentration, temperature, and intial pH on the production of the f-lucasdase, exoglucanase, and endoglucanase activities, as well as eattcellulr and mycelial prowin, were observed. With vtis forall the enzymes. Two maxima were observed both for glucosidase and endoglucanase, ase ativty was constant from the end ofthe exponential phase. When 9 prot concentration of 5% yielded the higher hile the exoglu of Baucosidive was increas Inika pH Value of 6.5 and a temperature of 20°C, a straw 1 nhinitor was ued. the prdetinn without Huetuations. When the temperature vas 25°C, the enzyines production was lowe, wietes the mycelial protein value was higher. Changes in the inti pH, lowering of raising it. lowered the enzymes production, but favored fungus growth, © 1909 Elsevier Seience Ine, All rights eserved Keywonis-Newospora erat, C introduction Cellulose is the only renewable carbon source that is available i large quantities and can be a solution to the problems of energy, enemicals, and food. Cellulose can be hydrolyzed by acid or enzymatic treatments, yielding sol ble products of low molecular weight such as hexoses and penioses Enzymatie hydrolysis of cellulose is good use of waste cellulose [1.2]. Beeause the production of enzymes is the anajr eost factor ofthe hydrolysis of sary 10 improve the yields of the enzymes to make the process economically attactive [3] A single enzyme eurat accomplish the task of extensive cellulose degradation: multiple enzymes are required. Con sequently. microorganisms tha grow successfully on cellu- lose as a substrate are capable of doing so hy producing a cellulase complex that consist of atleast three extracellular enzyme activities: lulose, itis neces: 1. endoglucanase (EC 32.1.4, endo-I,4-p-sineanase) 2 exoglucanase (EC 3.21.91. 1 4--collobiohyérolase) 3. Peylucosidane (EC 32.121) = Comesposing wor, Tak = 341-38.47185 fine 441-398-4114 Ema adres: salons ouemex sin. ven.se (MLD. Remstor Although the most intensively studied cellulolytic fungus has been Trichoderma reeset and other fungi of the same species [4-6], Eberhart et al, [7] carried out cellulase pro- duction by using Neurospora. and they isolated mutants, altered for the production of the eompicn. ‘included cell-1, a mutant with elevated, uninduced levels oF Doth cellulase and cellabiase Yara etal. [8] have shown that Ncraysa, a well-known, ‘fungus used im yenetie and proteuse studies, is true lolytic fimgus capable of synthesizing and secreting high levels of the cellulase complex enzy These mutants crystalline cellulose, and other carbon sources, as an inducer for the enzymes, ‘On the other bund, in 2 previous work [9], we showed that wheat straw, a very plentiful sgsicultural waste, was an adequate carbon source forthe orodduction of the cellulolytic, ‘complex of the fangus 7. reesei that induced the production, of these enzymes, Wheat straw is an adequate source of cellulose because ofits low lignin content (<20% wt) [2]. Disposing of great quantities of wheat straw after har- vesting creates an environmental problem. Traditional ways ‘of disposing of the wheat straw mcludes fermenting in plastic bags to produce cattle feed and burning, There is a umber of studies on the use of agricultural wastes to produce enzymes of importance in the bioprocessing indus- iy [10-13 141-022990'8 see foot mater © 199% ElsevieeSsiace Ine. Ai sab eeseved Pile 0141-05 9(99)00038-6In this paper. we present a study of the production oF cellulase complex oy N. crass growing on wheat straw as the sole carbon source, as well as the effects that wheat straw concentration, initial pH ofthe grovving medium, and femperature have on the growth and tne production of df= ferent enzymatic activites of the cellulase complex. 2. Ma ials and methods 2 Cuitures N. crassa Fungal Genetics Stock Cemer (University of Kansas) 4335, stain esll-T was aseptically wansferred to Pete dishes wits Vogel's medium, incubated for 7-11 days at 25°C, and stored at 4°C. The basal medium of the fermentation was the same as described by Vogel [14] Milled and sieved wheat straw (parle sue < 0.177 mm) was added at 2 convenient concentration as the cutbon source, snd the medium was adjusted to the desired pH. Inocula were prepared by asep- fi uspeasion of spores (approximately 107 25 ml of the sterilized medium. ‘The cultures were incubated aerobically in 250ml Er. Jenmeyer dasks in an incubation shaker (New Brunswick, Scientific Co. in, Edison NJ, USA) at the temperature of the experiment, At several times, cultures were contifuged at 4000 % g for 10 min, and the extracellular protcin and cuzymatic aetivitios tested in the supernatant previously adjusted to pit 4.8, while the sold mycelia was died to measure the dry weight of mycelial protein, All the exper. iments were carried out in duplicate, and the extracellular protein and enzymatic actvitics of each experiment were also analyzed in duplicate ly acing 2.2, Enzyme assays 2.2.4, Cellalase activity BC 3.2.1 Tneubations were carried out with 50 sil of pernatant, 1.95 ml of citrate buffer. pH 4.8, and 50 mg of Whatman no. 1 iter paper, at S0°C for 60 min. The redue- ing sugars were estimated as described by Miller [15]. 2.2.2, Endoglucanase activity EC 3.214 Culture supematant (50 yl), in 0.95 mil of citrate buffer, pH a8, wa ated at 50°C for 10 min with 1 ml of 2% carboximethylealiulose in the same buffer. The reducing sugars were estimated as described previously 2.2.3, Avicelase activity BC 3.2.1.9 Avicelase activity was determined as described for the endoglucanase activity, but the incubation was carried out 1with 1 ml of 1% Avicel suspension instead of earboximeth- yleetluiose: ne and Microbial Technology 25 (3900) 244 250) as Table 1 Wheat saw Mota prem es 1 065+ 001 034P : = 0.060208) 3 2oi= owes) 5 2a = 00846 ‘ 093 = 0.02 008) "Tho tne wd by orm to grow unl te Stationary phase i | maeatises 2.24, B-Glucosidase (EC 3.21.21) B-Glucosidase activity was determined by incubating, 200 gil of culture, 2.2 ml eitrate buffer, pH 4.8, andl 1.6 mi ‘of 5 mM p-nittophenylylicopyranoside st 50°C for 10 min, stopping the reaction by adding 6 mi of 0.1 M NaOH. The amount of p-nitrophenol produced was estimated by mea suring the absorbance ac 420 nm, Filter paper (Cellulase), endoglucanase, avicelase, and Beglucosidase activities were calculated in. intemationsl units (TU), One an amount of enzyme that produces reducing sugar equivalents 10 I mol min of o-glucose or p-nitrophenol under the assay tions stated. Al assays were performed ind fof enzyme activity is defined ws the 2.3, Protein determination 2.3L. Exiracellular protein Extracellular protein was measured in culture superna tants by the Bradford method [16] by using bovine serum, allurin as a standard 23.1. Mycelial protein Myceliai protein was extracted from the solid with 20 mi of | M NaOH at 50°C for 20 mun and measured as described. for the extracellular protein, 3. Results and discussion Wheat straw has hemieellulosie components that ean nn used as a carbon ns. However, iW as the drawback of low accessibility because of the presence of lignin in the straw, the erystallinity of cellulose, and its low specific surface. In a previous work [17], it was, shown that pretreatment of the siraw was needed for the growth of 7 yeesei. A physical treatment was used: the straw was milled and sieved, yielding higher activities ofthe ‘enzymes ofthe cellulase complex of the fungus. reesei, when, the fraction of d, < 0.177 mm was used. The treatment reduced the crystallinity index by 45%, and the content oF lignin by 9.2%, giving acavities similar to the ones obtained with this Funguss by using pure cellulose as substrate [18] ‘The production of celiulase complex from the fungusne and Microbial Dechnotogy 25 (3000) 248-250) het stawcorersion gi) ol | | ™| | =a ° 0 TES bas eae TTT ti i een F140) Blom nl avin fast ion wh sa cnet, I~ 68,7. apse ~ 3 crassa was carried out with this kind of wheat straw, The influence of the staw concentration, the temperature, and the initial value pH were studied. 3.1 Influence of the straw concentration, Te values of mycelia protein abtaned in the earlier phase are shown in Table | for wheat straw concentrations from 10 6% at an initial pH value of 6.5 and a temperature of 30°C: Higher growth was obtained with a straw concentration of 2% at 20 h of fermentation, whereas higher concentra tions inhibited the fungus growth, usually increasing the exponential phase duration, Phiy mold is able to growth on ‘wheat straw, redueing the concentration of this substrate When a 2% concentration of wheat straw was used in the fermentation. only 15% of the wheat straw remained after the growth phase, a consumption rate of 2.4 mg/ml X h) When a $% concentration of wheat straw was used, 22% of| the straw remained after the growth phase, but the mani sum substrate consumption rate was reached: 9 mg/(ml 1). Inall the fermentation runs, there was a dramatic red tion inthe wheat straw concentration. on a dry weight basis, during the growth phase, while the fungus did not metabo- lize the remaining straw ¢spproximately 15-20% of the ‘wheat straw at the beginning of fermentation) in the sta- tionary phase. eis ti i thal the able to dissolve all the cellulose into the medium during the growth phase and it uses the eelluiose dissolved in the ‘medium during the stationary phase ‘The fungus was able to grow on a 2! wheat straw until ts biomass reached 8 mg/ml, 2s ean be bscrved in Fig. La. The yield biomasssubsrate reached the concentration of ‘maximum (40%) at this concentration of wheat straw Higher concentrations of wheat straw were inhibitory for the growth ofthe fungus. The fungus dissolved some ponents of the wheat saw, mainly cellulogs, into the me= dium, so the wheat straw conversion (on a dry weight oasis) ‘Iuctuated herween 75 and 87%, reaching its maximum at @ 2% concentration of wheat straw. This variation of the ‘conversion of wheat straw, with the initia) concentration of this substrate, is shown in Fig, 1b, "The enzymes were produced both during the growth and stationary phases, so the process of these enzymes" produc- tion and secretion is a growih-assoctated one, The maxima values of cellulase activity (FP) are shown in Tabie 2, joined to the values of protein secreted 10 the medium ané the specific activity expressed ax FP activity per mg of protein at several straw concentrations. As it can be observed, the values oF filter paper activity and extracellular protein were higher for a 5% straw concentration, even though the higher specific uetivity was obtained with a straw concentration of Table 2 Mavinnam voles of ther papor acy, exter prossn, a Wheat sow Calle Extracellular ‘Specie sonccnttion activi protein sai es) ure ‘ey itm) 1 2s + 0m 090 + 0.002 ass 2 260m 322+ 002 our 5 3 = 001 3502003 0380 6 a70* 002 Los + 0.02 0667MED. Romero eal! Easvme and Microbial Technology 25 (3900) 244-250) 2 or: os. (Same om ao ae ore om to am a rein) Fig. 2. Exaymnaticstvives an exacts protein t HPC and inital pl 6.5 at ifren what ta conceuaton: 12M @ 58s, f-Glucoidie activity las dogs att (hk exoacunase actin (eh an extracel pratin The enzyme activities and the extracellular protein ver sus the time of fermentation ave shown in Fig. 2 (for straw concentrations of 2% and 5%). The secretion of the on- zymes was affected in a similar way; the stationary phase started approximately 40h later when a 5% straw concen- ‘ration was used compared to the 2% straw experiments (Table *), B-Glucosidase, endoglucanase, and extracel ‘protein sited important fluctuations withthe fermentation boeing generally more dramatic and with higher values for the 5% straw. ‘The highest value of endoglucanase activity was achieved at 100 h. Later. a steep decrease was observed at 170 h, following another maximum at 240/h, In the case oF Bealucosidase, a low activity was obtained when tne and Microbial Technology 25 (3099) 266 280 coe 3 {> yet tan 0. of oo mor) Fig. 2. p-Oluwonidse activity (4) und cela pte (b) wih (@ and witout °C, oH a a “65, wheat awe ‘was growing, and, when the stationary phase was reached, there was a sudden increase in activity until it reached a maximum at 100 h. A decrease was observed afterwards, as in the case of the endoglucanases, reaching a minimum at 170 A. The highest value of activity was achieved at 240 b, ‘A fast inerease in avicelase activity was observed in the frst wo a0 a sine sis (HP (ba een nil pt sles g eb ae fey )whenyL-methy auf Horde added the medium. T, tempers 20h, and the activity was maintained with slight fluctuations luring fermentation. This fact was indcperdent of the wheat straws concentration, and the production associated with growth seems 10 be the way this fungus produses some enzymes with this activity. The highest values of enzymatic activity: are ‘obtained at 100 h following a deerease until a fermentation time of 170 h when a S0% acuvity for FP was obtained. har paper ac tn) wo tee ©6571. empertire ~ 50°C, whoa a ~ 58%Myc proton tra) time (h) Fig 5, Mycela entein () an FP () aire tepernnnes (AOC: @ 258C a 2 Afterwards, the activity increased until reaching a maximum at 240), even higher than that obiained at 100 h ‘The sudden decrease in enzymatic activities after the frst 100 h of experimentation could be due to protein hydrolysis by proteases secreted by the fungus when there was a lack of any essential nutzient in the mediui, When fermentation is carried aut and adding. a protease inhibitor, phenyl-methyl-sulfonyl fluoride at a concentration of 5 mM, the ductuatioas observed in enzymatic activity ‘were attenuated, especially at Tonger times of fermentation (175 h), when the proteases production was higher because Of nutrient consumption, The B-glucosidase activity obtained in the presence of 3 mM phenyi-methyl-subfony! Muoride is shown in Fig. 3 5.2. Inpluence of pit and temperanire The results obtained when the fermentation was carried out at different initial pH values ata straw concentration of SY are shown in Fig. 4. Itean be observed that 2 mold growth {inycolial protein) was Favored by a starting pH oF 6, whereas the enzyme production was higher ata staring pH of 65. Similarly, a higher level of mycelial protein was ob- served when the expi 1 er twas set at 25°C than when it was set at 30°C temperature, even thougth the enzyme production was higher at this temperature [Fig. 5} Ie had been observed previously in the fermentation of other cellulolytic fungus thar when the temper changed atthe end of the exponential phase, hi of enzyme activity was achieved [13,14], Consequently, a ne and Werobial Technology 25 (3099) 246 280) 29 3 : i 4 3 I coe To 4 run with a starting temperature of 25°C and a final temper= ature of 30°C was carried out. However, in the case of 1 crassa, a lower enzyme production was observed compared, 1 that obtained when using 30°C from the heginning, 4. Conclusions ‘The fungus N. crassa was able to produce cellulolytic enzymes wnen it grew on wheat straw as the sole source oF carbon, dissolving into the medium 75 87% of the compa- nenis of this solid substeate in the growth phase. ‘The B-glucosidase and endoglucanase activities showed ‘luctuations with fermentation time that could be attenuated by adding a prowease inhibitor. such as phenyl-methyl-sule ony! Muoride. It can b¢ concluded that the fungus .V. crassa, when towing on wheat straw, requires different conditions for _nrowth and for enzyme production. “he best conditions for ‘growth ate wheat siraw concentration of 2%, initial pe: 0: 6. anda temperature oF 25°C during the exponential phase and 30°C for the stationary phase, whereas the major cellulasic activities are obtained with wheat straw concentration oF 5%, initial pH of 6.5, and a tomperature of 30°C. References 1H Via SD, Dysva G, Raykovska ¥, Seige, Sellalse pre tion by Lschocerma sp. grown on corn fibre sunste. Proc Biochem wer 3256152st MED. Romero ot al! Kame and Werobial Dechnotogy 25 (3099) 246 280) [P] Zillios C, Deven P. Hydkolysis of wieat saw by 2 thermostable ‘nlaxylansse: Adsorption nd Kina stades. Enzyme Mict® Tee ol 995225803, [5] Sumerrer- Cornea MI, Tengerdy RP. Production of callalse 0 uy cme bagasse by fungal mxod culture solid suastae frmewaton Sioteinol Lez 197 192065-7. [i] Dinuin VS, Gtose TK. 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