cellulase ssf and application

1985, 49(1):205. Appl. Environ. Microbiol. D. S. Chahal

for Cellulase ProductionreeseiTrichodermaSolid-State Fermentation with

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Vol. 49, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1985, p. 205-2100099-2240/85/010205-06$02.00/0Copyright C 1985, American Society for Microbiology

Solid-State Fermentation with Trichoderma reesei forCellulase Production

D. S. CHAHALBacteriology Research Centre, Institut Armand-Frappier, Laval, Quebec, Canada H7V 1B7

Received 7 May 1984/Accepted 16 October 1984

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentationof wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reeseion a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its laterphase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-statefermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases(17.2 IU/ml), I-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable ofhydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition ofcomplementary enzymes, l-glucosidase, and xylanases.

Solid-state fermentation (SSF) is a process whereby aninsoluble substrate is fermented with sufficient moisture butwithout free water. (The abbreviation SSF is also used for"simultaneous saccharification and fermentation." But SSFis retained here for "solid-state fermentation" because it isalso commonly used [4, 8, 29], and it is an antonym toanother state of fermentation, i.e., "liquid-state fermenta-tion" [LSF].) In liquid-state fermentation (LSF), on theother hand, the substrate is solubilized or suspended as fineparticles in a large volume of water. In most LSF, substrateconcentrations ranging from 0.5 to 6% are used dependingupon the density of the substrate. SSF requires no complexcontrols and has many advantages over LSF (8); however, ithas its own inherent problems (4).A critical analysis of literature on enzymatic hydrolysis

reveals that high cellulase activity per unit volume of fer-mentation broth is the most important factor in obtainingsugar concentrations of 20 to 30% from hydrolysis of cellu-lose for ethanol production from cellulosic materials (3). Ithas also been confirmed that cellulase activity per unitvolume can be increased by increasing the cellulose concen-tration in the medium (16), but it is not possible to handlemore than 6% cellulose in a conventional fermentor becauseof rheological problems. A maximum concentration of sub-strate which can be handled in the conventional fermentor isca. 2% for wood pulp and 6% for crystalline cellulose.Therefore, to increase the cellulose concentration to over6%, SSF seems to be the most attractive alternative (3, 4).

Trichoderma reesei is well known as a cellulase-producingorganism (1-3, 7, 16, 21, 24, 25). The literature on enzymeproduction indicates that various mutants of T. reesei areable to produce 160 to 250 IU/g of pure cellulose under LSF(Table 1). The highest cellulase yield, 290 IU/g of celluloseby mutant Rut-C30 as recorded by Tangnu et al. (25), has notbeen reported again. Therefore, we concluded that thecellulase potential of various mutants of T. reesei rangesbetween 160 and 250 IU/g of pure cellulose in LSF.Development of an economical process for cellulase pro-

duction is hindered because of the high costs of substrate(pure cellulose) and of some of the chemicals, such asproteose peptone, and because of low yields of cellulases perunit of cellulose. To overcome these bottlenecks, we first

used a cheap source of cellulose which requires minimumpretreatment and purification, and we then increased thecellulase yields per unit of cellulose. At present the cheapestcellulose sources are lignocelluloses (crop residues, wood,and wood residues). Peitersen (18) obtained a filter paperactivity equivalent to 0.28 IU/ml by growing T. reeseiQM9123 on alkali-treated barley straw. Tangnu et al. (25)also reported very low activity of cellulases, i.e., 0.12 and0.28 IU/ml on 1 and 2% acid-treated corn stover, respec-tively. Cellulase activity increased to 2.0 IU/ml when washed;acid and base-treated corn stover was used at a 2% concen-tration. Recently, high cellulase activity, 3.7 IU/ml (168 IU/gof cellulose), was reported by Chahal et al. (5) by growing T.reesei (Rut-C30) on 2.2% cellulose from washed, steam-treated wood. Cellulase yields of 168 IU/g of crude cellulosewere as good as those obtained on pure cellulose by others(Table 1).Encouraged by these results, we envisaged a new ap-

proach to producing cellulases on cheap cellulose sourcesand to increasing cellulase yields per unit of cellulose. Thenew approach for the production of a cellulase system withhigh hydrolytic potential was to grow T. reesei on lignocel-lulose in SSF, similar to the Koji process of Toyama (27)except that wheat bran, wheat germ, or rice bran, theexpensive additives, were not used. Moreover, in the newapproach, the lignocelluloses were not delignified sincealmost all hemicelluloses are removed during delignification.Rather than delignification and removal of hemicelluloses,the lignocelluloses were treated with a small quantity ofNaOH to solubilize some of the hemicelluloses and lignin toexpose cellulose. The treated lignocelluloses were notwashed, and all of the solubilized hemicelluloses and ligninwere retained in the medium.

MATERIALS AND METHODSMicroorganisms. T. reesei QM9414, kindly supplied by

Mary Mandels, U.S. Army Natick Development Center,Natick, Mass., was continuously maintained on delignifiedwheat straw (WS) agar medium in petri plates. The WS agarmedium was specially designed for this purpose. The nutri-ents in the medium are described below. In one of thecolonies, a sector showing high hydrolytic activity was no-

205

APPL. ENVIRON. MICROBIOL.

ticed. The mutation occurred due to continuous subculturingon WS medium (usually cultures of T. reesei are maintainedon potato-dextrose agar). The new mutant was transferred tosimilar medium, and in five further transfers a stable mutantwas obtained. This new mutant was tentatively namedQMY-1.Another hyper-cellulase-producing mutant of T. reesei,

NRRL 11460 (Rut-C30), received from J. J. Ellis, NorthernRegional Research Centre, Peoria, Ill., was also used forcomparison. It is referred to below as Rut-C30.

Substrate. WS, ground to 20-mesh powder, was used as asource of cellulose. WS contains (percent dry weight):cellulose, 40; hemicelluloses, 29.2; lignin, 13.6; protein, 3.6;and other materials, 13.6 (23). Therefore, WS is composed ofca. 70% insoluble carbohydrates suitable for the growth of T.reesei and for cellulase production.Aspen pulp was prepared by a chemical-thermomechani-

cal process (10), and the pulp thus prepared was calledchemithermomechanical pulp (CTMP). During this process,the wood was pulverized to fine fibers which still retainedmost of the hemicelluloses and lignin. The chemical compo-sition of CTMP was as follows (percent dry weight): cellu-lose, 63 to 66; hemicelluloses, 15 to 18; and lignin, 9 to 11.

Pretreatment. Powdered substrate (5 g; 20 mesh) wasdispensed into each Erlenmeyer flask of 250-ml capacity.Each substrate was treated with NaOH (4% [wt/wt]) with33.3% moisture at 121C (WS for 0.5 h and CTMP for 1 h).The treated substrate was not washed. All of the solubilizedhemicelluloses and lignin were retained in the fermentationmedium. After the addition of nutrients, the pH was adjusted

TABLE 1. Cellulase production potential of new mutants of T.reesei in LSF

Cellulose CellulaseMutanta (yr developed) concn (IU/g of Renfce-

(%) ~~cellulose)QM6a (parent strain; 6 100 21

1971)QM9414 (1971) 0.75 240 24

2 200 72.5 172 256 166 21

MCG77 (1977) 2 195 76 183 21

NG14 (1977) 2 180 76 250 21

Rut-C30 (1979) 5 290 255 160 26 233 21

15 200 2

L27 (1981) 8 225 22CL-847 (1983) 5 (+1)- 229 28D1-6 (1983) 1 140 17

a Strains QM6a, QM9414, MCG77, and NG14 were from the U.S. ArmyNatick Research and Development Laboratories; Rut-C30 was from CookCollege, Rutgers, N.J.; L27 was from Cetus Corp.; CL-847 was from France;and D1-6 was from Delhi.

b Range, 160 to 250.e +1, 1% glucose.

to ca. 5.8 with H2SO4. Water solubles were obtained bysuspending treated WS in water (1:10) and filtering throughfour layers of cheese cloth. The WS solubles contained 2.5%solids (hemicelluloses, lignin, and other cell solubles).

Nutrients. Nutrients described by Mandels and Weber (15)for cellulase production were supplied in concentrated form,but proteose peptone was replaced with yeast extract (DifcoLaboratories, Detroit, Mich.). The quantity of nutrientsrequired for each substrate was determined at the rate oftheir carbohydrate content. The required amount of concen-trated nutrient salt solution (5 ml for WS and 5.7 ml forCTMP) was added to 5 g of substrate. The concentrated saltsolution contained the following, dissolved in 200 ml ofwater: KH2PO4, 28 g; (NH4)2SO4, 19.6 g; urea, 4.2 g;MgSO4 * 7H20, 4.2 g; CoCl2, 4.2 g; FeSO4 * 7H20, 70 mg;MnSO4 7H20, 21.84 mg; ZnSO4 * 7H20, 19.6 mg; CaC12,28 mg; and yeast extract, 7 g. As referred to below, a fullconcentration of the nutrients means the complete requiredquantity of nutrients as mentioned above, whereas a one-half concentration is one-half of that quantity. All of theflasks were autoclaved at 121C for 20 min, after the nutrientsalt solution was mixed with the substrate.

Moisture. The moisture content of the substrates afterpretreatment and the addition of nutrients and inoculum was80% (wt/wt) in SSF. Sterilized water was added for LSF, toobtain the desired concentration of the substrate in thefermentation medium.

Inocula. Inocula of mutants QMY-1 and Rut-C30 wereproduced on the modified medium as described above butcontaining 1.5% glucose, with the nutrient salt solutiondiluted accordingly. For inoculation of each flask containing5 g of substrate, 5 ml of 2-day-old culture was used. Theinoculum was spread on the surface of the substrate.

Culture conditions. All of the SSF cultures were incubatedat 30C in a humidified incubator (about 80% relative humid-ity), whereas the LSF cultures were incubated at the sametemperature on a shaker at 150 rpm.

Extraction of the cellulase system. The culture of SSF fromeach flask (originally 5 g of substrate) was mixed well withmore water to bring the final weight of the mixture (mycel-ium plus unutilized lignin, cellulose, and hemicelluloses) to100 g. Tween 80 was added at a rate of 0.1%. The mixturewas shaken for 0.5 h and centrifuged. The supernatant wasused for enzyme determination. It was estimated that about7 to 10% cellulases remained adsorbed on the residues(mycelium and unutilized cellulose, hemicelluloses, andlignin) when the residues were suspended in water andTween 80 as before and the supernatant was tested forcellulase titer.

Analyses. Cellulase titer was calculated in internationalunits of enzyme activity (glucose released per min) on filterpaper for 60 min, by the method of Mandels et al. (11).P-Glucosidase titer was measured in international units ofglucose released per min with 1% salicin solution for 30 min.Xylanase titer was measured in international units of xylosereleased per min with 1% xylan for 10 min (9). The cellulaseyield per gram of cellulose was calculated by dividing totalinternational units of cellulase titer in 100 ml of enzymebroth by grams of cellulose present in the substrate suppliedin the flask.

Sugars were estimated by using a Beckman 344 gradienthigh-pressure liquid chromatograph with an Altex 156 refrac-tive index detector and a Spherogel 7.5% carbohydratecolumn with a flow rate of 0.5 ml/min in the mobile phase ofwater at 80C. The sugar samples were appropriately dilutedbefore injection.

206 CHAHAL

SOLID-STATE FERMENTATION WITH T. REESEI 207

RESULTS AND DISCUSSION

Cellulase production in LSF and the effect of differentconcentrations of WS. The highest cellulase titer (1.65 IU/ml)and cellulase yield (412 IU/g of cellulose) in LSF wereobtained with mutant QMY-1 in 1% WS (0.4% cellulose)slurry after 7 days (Table 2). When the concentration of WSwas increased to 5% (2% cellulose), the enzyme productiontime was increased from 7 to 11 days. The enzyme activityincreased to 6.0 IU/ml, but the cellulase yield dropped to 300IU/g of cellulose (Table 2). The drop in cellulase yields mighthave been due to poor mass transfer in the thick slurry of 5%WS. The decrease in cellulase yield by mutant QM9414 withincreases in the concentration of cellulose is also evidentfrom the work of Sternberg (24), Gallo et al. (7), Ryu andMandels (21), and Tangnu et al. (25), as presented in Table 1.

TABLE 2. Cellulase production on WS by QMY-1Concn of Time of CellulaseType of Concn of III .b Cellulase

fermentation WS M cellulose incuba- titer yield(nutrient concn) [wt/wt]) (% tion (IU/ml) (IU/g of[wt/wt]) (days) (l/n cellulose)LSF (full 1 0.4 5 1.44 360concn) 7 1.65 412

5 2.0 7 1.3 6511 6.0 30014 5.5 275

SSF (full 20a 8b 11 6.0 300concn) 14 6.3 385

22 8.6 43022C' 7.4 370

SSF (one-half 20 8 11 5.5 275concn) 14 6.7 335

22 6.7 33522c 8.0 400

5 g of WS + 20 g of water (no free water) = 20%o solids in each flask.b 5 g of WS contains 2 g of cellulose = 8% cellulose in each flask.c Cultures were stirred once after 11 days of incubation and were further

incubated for 11 days without stirring.

Cellulase production in SSF. (i) On WS. SSF was carriedout with a full concentration of nutrients in one set ofexperiments and with a one-half concentration in another setto evaluate the effect of different concentrations of salts inthe medium, since some microorganisms are unable to growin the high osmotic pressure of the medium. T. reeseiQMY-1 was quite tolerant to the high concentrations of thenutrients, as indicated in Table 2. It produced the highestenzyme titer (8.6 IU/ml) and cellulase yield (430 IU/g ofcellulose) in SSF on a full concentration of nutrients, after 22days. The highest cellulase yield, 412 IU/g of cellulose,obtained in LSF, was because that medium contained thelowest cellulose concentration (0.4%), as discussed earlier.However, in the present study, a cellulase yield of 275 to 300IU/g of cellulose in LSF on 2% cellulose concentrations wasconsidered for comparisons between LSF and SSF. Whenthe nutrients were supplied in a one-half concentration, thecellulase titer dropped to 6.7 IU/ml, cellulase yield droppedto 335 IU/g of cellulose, and there was no increase in enzymeyields after an incubation of more than 14 days.But when the cultures were stirred after 11 days of growth

and further incubated for 11 days without any stirring (total

of 22 days of incubation), the cellulase titer decreasedsomewhat for the full concentration of nutrients, whereas itincreased considerably (8.0 IU/ml) for a one-half concentra-tion of nutrients. This indicates that half of the quantity ofrequired nutrients was sufficient to get an optimum cellulasetiter as well as an optimum cellulase yield. This finding couldcontribute to a reduction in the cost of enzyme production.Recently, more expensive media have been developed toincrease cellulase production by new mutants of T. reesei inLSF on pure cellulose (17, 28). Even then, the highest yieldsobtained are quite low, i.e., 140 IU/g of cellulose for mutantD1-6 (17) and 229 IU/g of cellulose for mutant CL-847 (28)(Table 1).

(ii) On WS and CTMP. Cellulase production by strainQMY-1 was compared with that of strain Rut-C30, a hyper-cellulase-producing mutant, on two different lignocellulosicsubstrates, WS and CTMP. QMY-1 produced its highestcellulase titer (over 8 IU/ml) and yield (over 400 IU/g ofcellulose) on treated WS as compared with that of Rut-C30,a 6.2 IU/ml cellulase titer and a yield of 310 IU/g of cellulose.Cellulase production by both of the mutants decreasedconsiderably on untreated WS (Table 3). The mutant Rut-

TABLE 3. Cellulase production by mutants QMY-1 and Rut-C30on WS and CTMP

Mutant

QMY-1 Rut-C30Substrate'~(days) Cellulase Cellulase

Cellulase yed Cellulase yilSubstateadays) titer yield titer yield(IU/mI) (IU/g of (IU/mI) (IU/g of(/m

cellulose) (Um cellulose)Treated WS

9 0.7 35 0.6 3016 4.4 220 1.6 8018 7.8 390 2.6 13020 8.1 405 6.2 31026 8.5 425 5.2 26030 7.2 360 4.6 230

Untreated WS9 1.7 85 1.1 5516 2.3 115 2.6 13018 2.4 120 2.8 14020 2.1 105 3.1 15526 2.2 110 3.2 16030 1.9 95 4.2 210

Treated CTMP9 1.5 45 0.9 2716 4.7 142 5.3 16118 3.9 4.5 13620 6.3 191 6.6 20026 6.0 182 6.5 19730 6.2 188 4.8 145

Untreated CTMP9 2.2 67 1.9 5716 5.3 161 4.1 12418 3.3 4.1 12420 5.0 151 5.8 17626 5.6 170 6.0 18230 7.2 218 5.0 152a Cellulose content of wheat straw, 40%; cellulose content of CTMP, 66%.

There was 5 g (dry weight) of each substrate in each flask. Substrates weretreated with 4% NaOH (wt/wt) at 121'C for 0.5 h (WS) or 1 h (CTMP) with a1:2 (solid to liquid) ratio.

VOL. 49, 1985


TABLE 4. Cellulase production by different mutants of T. reeseion different cellulosic substrates in LSF

Mutant' and Cellulase Cellulasesubstrateb titer" yield

(5%o) (IU/ml) (lU/g of(5%) (lU/mi)~~~~~~~~~~cellulose)

QM-9414Solka Floc 1.85 36.1SE 0.70 25.4SEWA 0.96 21.1

Rut-C30Solka Floc 5.56 111.0SE 1.57 57.1SEWA 2.10 46.1

E.58Solka Floc 1.93 38.6SE 0.62 22.5SEWA 0.51 11.2a Mutants were grown for 11 days at 28C in LSF. E.58 is a mutant from

Trichoderma harzianum (Forintek Culture Collection).bSE, Steam-exploded wood (55% cellulose); SEWA, steam-exploded

wood, water extracted and alkali treated (91% cellulose).C Data from reference 6.

C30 failed to grow in a number of flasks containing WS orCTMP. This finding indicated that this mutant is not welladapted to such conditions of SSF.The mild alkali treatment of CTMP did not affect cellulase

production by either mutant (Table 3). Because the highestcellulase titer and yield obtained on treated CTMP after 20days in SSF were almost comparable to those obtained onuntreated CTMP, CTMP seems to be a good substrate forcellulase production even without any further treatment.Further studies on cellulase production on CTMP are inprogress.The cellulase titer and yield on CTMP with strains QMY-1

and Rut-C30 (Table 3) and on treated WS (Table 2) withstrain QMY-1 in SSF were higher than those of mutantsQM-9414, Rut-C30, and E.58 on steam-exploded wood, onsteam-exploded and alkali-treated wood with water ex-tracted, and even on pure cellulose (Solka Floc; Brown Co.,Berlin, N.H.) in LSF, as reported by others (Table 4). Thetiter and yield of cellulase obtained with QMY-1 in SSF werealso higher than those obtained by other workers who grewvarious mutants of T. reesei on pure cellulose in LSF (Table1). The results clearly indicate that the new approach ofretaining the hemicelluloses and lignin of the alkali-pretrea-ted lignocelluloses (WS, CTMP) in SSF increased signifi-cantly the cellulase titer per unit volume and the cellulaseyield per unit of cellulose.

Role of hemicelluloses and lignin in cellulase production.The increase in cellulase titer was postulated to be due to theuse of hemicelluloses during the initial growth of T. reeseiand then to the use of cellulose during the later phase ofgrowth for production of cellulases. To test this postulate,we grew T. reesei QMY-1 in LSF on pure cellulose (a-cellulose; Sigma Chemical Co., St. Louis, Mo.) in one set ofexperiments, and cellulose was fortified with a mixture ofsolubles obtained from WS. Delayed and slow synthesis ofcellulases during the early phase for WS soluble-fortifiedcellulose was attributed to the presence of hemicelluloses,an easily metabolizable carbon source (Fig. 1). After hemi-celluloses were used, cellulase synthesis increased consid-erably during the later phase of fermentation. This indicatedthat WS solubles which contained mostly hemicelluloses and

lignin were responsible for the high cellulase titer (3.4 IU/ml)and yield (340 IU/g of cellulose). However, further work onthe role of hemicelluloses and lignin, individually and incombination, is in progress.

Composition of the cellulase system. The cellulase systemproduced in SSF contained the following enzymatic activi-ties (international units per milliliter): cellulase, 8.6; 3-glucosidase, 10.6; and xylanase, 270. The xylanase titer wasquite variable (between 190 and 480 IU/ml); however, theratio of cellulases and 3-glucosidase varied between 1:1 and1:1.5 in various cellulase system preparations. These areenzyme activities when 5 g of WS fermented in SSF wassuspended in ca. 100 ml of water to extract the enzymes. Theenzyme titer could be doubled (17.2 IU/ml) by extracting theenzyme in 50 ml of water. The composition of the cellulasesystem indicated that there was no need to add extra3-glucosidase or xylanase for the hydrolysis of pure cellu-

lose or lignocelluloses.Hydrolytic potential of the cellulase system. (i) Cotton. The

cellulases produced in SSF, when used in a substrate-to-en-zyme ratio of 1 g:20 IU, showed saccharification of differentconcentrations of untreated cotton as high as that reportedby Mandels et al. (13) (Table 5). There was also an indicationthat this enzyme system was able to hydrolyze a higherconcentration (10%) of cotton without any reduction in thepercentage of hydrolysis. The high-pressure liquid chroma-tography of the hydrolysate obtained from 10% concentra-tions of cotton showed mostly glucose (92.8%) and very littlecellobiose (3.5%). It is therefore assumed that cellulasesproduced in SSF may be able to hydrolyze a higher concen-tration of cellulose to obtain high concentrations of glucosein the hydrolysate suitable for economical ethanol fermen-tation or for fermentation of any other product.

(ii) Cellulose fiber. Hydrolysis of cellulose fiber withcellulases produced in SSF was faster than that of cotton.There was also an indication that cellulases produced in SSFgave a faster rate and higher percentage of hydrolysis thancellulases produced in LSF (Table 6).

(iii) Delignified WS. WS delignified by the method ofToyama (26) was hydrolyzed with the cellulase system

4

0-LL.

00 50 100 150 200 250

TIME (h)FIG. 1. Effect of hemicelluloses and lignin on cellulase produc-

tion by mutant QMY-1 on 1% cellulose (sigma cell). (The ratio ofcellulose to WS solubles in the medium was 1:0.25.) Symbols: 0,cellulose only (control); A, cellulose plus WS solubles containinghemicelluloses and lignin.

208 CHAHAL

I

SOLID-STATE FERMENTATION WITH T. REESEI 209

TABLE 5. Hydrolysis of cotton with cellulases produced in SSFSubstrate Substrate/enzyme % Saccharification"concn

w] ratio (gIU) 24 h 48 h1 1:20 16.9 17.72 1:20 15.4 17.94 1:20 17.9 22.48 1:20 13.6 22.110 1:37 15.4

5C 1:20 18.0 21.6Saccharification = grams of reducing sugars x 0.9 x 100.

grams of substrateb 15.4% Saccharification = 17.1 g of reducing sugars per liter (15.7 g of

glucose [92.8%] + 0.6 g of cellobiose [3.5%] + 0.4 g of xylose [2.2%] = 16.9 gof sugars per liter, detected by high-pressure liquid chromatography).

C Data derived by using a substrate concentration of 5% (dry weight) arefrom reference 13, with T. Reesei QM9414 cellulases.

produced in SSF at a 10% (wt/vol) concentration. Hydro-lysis was done at pH 6.7, the original pH of the enzymesolution, and also at standard pH 4.8 (Fig. 2 and 3). The rateof hydrolysis of cellulose into glucose was very high for thefirst 20 h of hydrolysis at both pH levels, and ca. 65% of totalhydrolysis was recorded during this period.Almost all of the xylan and arabinan were hydrolyzed

within the first 20 h of hydrolysis.Very little cellobiose accumulated in the hydrolysate. The

maximum concentration of cellobiose accumulated was 7.75g/liter (Fig. 2), which was too low to cause any significantinhibition of cellulose hydrolysis (12, 14). After 20 h ofhydrolysis, the concentration of cellobiose further decreasedto ca. 3 g/liter.

CC0(.)Conx

TABLE 6. Comparison of hydrolysis of cellulose' fiber withcellulases produced in SSF and LSF

System of Substrate/enzyme % Saccharificationfermentaion ratio (g/IU) 24 h 48 h 72 h

SSF 1:20 23.6 32.2 38.6LSF 1:20 19.2 29.4 35.3

a Cellulose fiber (Sigma Chemical Co., St. Louis, Mo.), 5% concentration.

The hydrolysate obtained with pH 6.7, after 96 h ofhydrolysis, contained 99.75 g of sugars per liter (glucose,68.18 g; cellobiose, 3.19 g; xylose, 26.71 g; arabinose, 1.67g), whereas the hydrolysate obtained with pH 4.8, after thesame hydrolysis period, contained 86.14 g of sugars per liter(glucose, 56.49 g; cellobiose, 3.88 g; xylose, 24.31 g; arabi-nose, 1.46 g), giving total saccharifications of 89.7 and77.9%, respectively.

Conclusions. The increase in cellulase yields, from a rangeof 160 to 250 IU/g of pure cellulose to a range of 250 to 430IU/g of crude cellulose from WS and CTMP, was due to theuse of a hemicellulose fraction during the initial growth ofthe organism and to the production of cellulases on acellulose fraction of substrates during the later phase ofgrowth of the organism. The presence of lignin could beanother factor in the increase of cellulase yields. The role oflignin and hemicelluloses in enzyme production is beingfurther evaluated.High cellulase activity of 8.6 IU/ml could be doubled (17.2

IU/ml) by extracting cellulases with half the quantity ofwater. Because high cellulase titer (over 15 IU/ml) is re-quired to obtain high concentrations of glucose for econom-ical ethanol fermentation (4), SSF seems to hold promise forobtaining a high cellulase titer per unit volume of enzymebroth. The cellulase system produced in SSF containedsufficient quantities of ,B-glucosidase and xylanase for com-

100.

90

80

_ 70

CP,, 60c00 50

t 400

20io-

0 20 40 60 80 100T I M E ( h)

FIG. 2. Hydrolysis of delignified WS at pH 6.7. Symbols: A,total sugars; x, glucose; *, xylose; Ol, cellobiose; 0, arabinose.

0 20 40 60TIME ( h )

80 iob)FIG. 3. Hydrolysis of delignified WS at pH 4.8. Symbols are

defined in the legend to Fig. 2.

41x

00 0 oi aK..m IT I 1----T- I I I I I I

VOL. 49, 1985

I


plete hydrolysis of pure cellulose as well as lignocelluloses.It was also assumed that the cellulase system produced inSSF was rich in C1 factor as proposed much earlier by Reeseet al. (20). It was reported by them (20) that the C1 factor wasessential to hydrolyze the crystalline portion of cellulose,and this concept is still maintained by Reese (19). By thenew approach, ca. 100 g of sugars per liter were obtained byhydrolyzing 100 g of delignified WS with the cellulase systemproduced with SSF. Moreover, SSF enables drastic reduc-tions in the cost of enzyme because cellulase yields per unitof cellulose were increased by 72%, crude cellulose (WS)with a minimum of pretreatment and purification could beused as a carbon substrate, the quantity of nutrients could bereduced to one-half, and SSF does not require very complexcontrol systems. However, SSF has its own inherent prob-lems, including maintenance of pH, moisture level, aeration,agitation, etc., when large quantities of solid substrates areused (4).

ACKNOWLEDGMENTSI thank M. Mandels and E. T. Reese, Materials Protection and

Biotechnology Division, Science and Advanced Technology Labo-ratory, U.S. Army Natick Research and Development Laboratories,Natick, Mass., and V. Portelance, Bacteriology Research Center,Institut Armand-Frappier, Laval, Quebec, Canada, for critical read-ing of the manuscript and for their valuable suggestions. I am alsovery grateful to Johanne Lemay for her technical help and to P. S.Chahal for drawing the figures and for technical help.

LITERATURE CITED

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210 CHAHAL

cellulase ssf and application

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