Solid-State Fermentation with Trichoderma

reesei for Cellulase Production

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

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Vol. 49, No. 1

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1985, p. 205-210

0099-2240/85/010205-06$02.00/0
Copyright C 1985, American Society for Microbiology

Solid-State Fermentation with Trichoderma reesei for

Cellulase Production
D. S. CHAHAL

Bacteriology 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 fermentation
of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160
to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17
IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei
on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later
phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state
fermentation. 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 of
hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of
complementary enzymes, l-glucosidase, and xylanases.

used a cheap source of cellulose which requires minimum

pretreatment and purification, and we then increased the
cellulase yields per unit of cellulose. At present the cheapest
cellulose sources are lignocelluloses (crop residues, wood,
and wood residues). Peitersen (18) obtained a filter paper
activity equivalent to 0.28 IU/ml by growing T. reesei
QM9123 on alkali-treated barley straw. Tangnu et al. (25)
also reported very low activity of cellulases, i.e., 0.12 and
0.28 IU/ml on 1 and 2% acid-treated corn stover, respectively. Cellulase activity increased to 2.0 IU/ml when washed;
acid and base-treated corn stover was used at a 2% concentration. Recently, high cellulase activity, 3.7 IU/ml (168 IU/g
of cellulose), was reported by Chahal et al. (5) by growing T.
reesei (Rut-C30) on 2.2% cellulose from washed, steamtreated wood. Cellulase yields of 168 IU/g of crude cellulose
were as good as those obtained on pure cellulose by others
(Table 1).
Encouraged by these results, we envisaged a new approach to producing cellulases on cheap cellulose sources
and to increasing cellulase yields per unit of cellulose. The
new approach for the production of a cellulase system with
high hydrolytic potential was to grow T. reesei on lignocellulose in SSF, similar to the Koji process of Toyama (27)
except that wheat bran, wheat germ, or rice bran, the
expensive additives, were not used. Moreover, in the new
approach, the lignocelluloses were not delignified since
almost all hemicelluloses are removed during delignification.
Rather than delignification and removal of hemicelluloses,
the lignocelluloses were treated with a small quantity of
NaOH to solubilize some of the hemicelluloses and lignin to
expose cellulose. The treated lignocelluloses were not
washed, and all of the solubilized hemicelluloses and lignin
were retained in the medium.

Solid-state fermentation (SSF) is a process whereby an

insoluble substrate is fermented with sufficient moisture but
without free water. (The abbreviation SSF is also used for
"simultaneous saccharification and fermentation." But SSF
is retained here for "solid-state fermentation" because it is
also commonly used [4, 8, 29], and it is an antonym to
another state of fermentation, i.e., "liquid-state fermentation" [LSF].) In liquid-state fermentation (LSF), on the
other hand, the substrate is solubilized or suspended as fine
particles in a large volume of water. In most LSF, substrate
concentrations ranging from 0.5 to 6% are used depending
upon the density of the substrate. SSF requires no complex
controls and has many advantages over LSF (8); however, it
has its own inherent problems (4).
A critical analysis of literature on enzymatic hydrolysis
reveals that high cellulase activity per unit volume of fermentation broth is the most important factor in obtaining
sugar concentrations of 20 to 30% from hydrolysis of cellulose for ethanol production from cellulosic materials (3). It
has also been confirmed that cellulase activity per unit
volume can be increased by increasing the cellulose concentration in the medium (16), but it is not possible to handle
more than 6% cellulose in a conventional fermentor because
of rheological problems. A maximum concentration of substrate which can be handled in the conventional fermentor is
ca. 2% for wood pulp and 6% for crystalline cellulose.
Therefore, to increase the cellulose concentration to over
6%, SSF seems to be the most attractive alternative (3, 4).
Trichoderma reesei is well known as a cellulase-producing
organism (1-3, 7, 16, 21, 24, 25). The literature on enzyme
production indicates that various mutants of T. reesei are
able to produce 160 to 250 IU/g of pure cellulose under LSF
(Table 1). The highest cellulase yield, 290 IU/g of cellulose
by mutant Rut-C30 as recorded by Tangnu et al. (25), has not
been reported again. Therefore, we concluded that the
cellulase potential of various mutants of T. reesei ranges
between 160 and 250 IU/g of pure cellulose in LSF.
Development of an economical process for cellulase production is hindered because of the high costs of substrate
(pure cellulose) and of some of the chemicals, such as
proteose peptone, and because of low yields of cellulases per
unit of cellulose. To overcome these bottlenecks, we first

MATERIALS AND METHODS

Microorganisms. T. reesei QM9414, kindly supplied by
Mary Mandels, U.S. Army Natick Development Center,
Natick, Mass., was continuously maintained on delignified
wheat straw (WS) agar medium in petri plates. The WS agar
medium was specially designed for this purpose. The nutrients in the medium are described below. In one of the

colonies, a sector showing high hydrolytic activity was no205

206

CHAHAL

APPL. ENVIRON. MICROBIOL.

ticed. The mutation occurred due to continuous subculturing

on WS medium (usually cultures of T. reesei are maintained
on potato-dextrose agar). The new mutant was transferred to
similar medium, and in five further transfers a stable mutant
was obtained. This new mutant was tentatively named
QMY-1.
Another hyper-cellulase-producing mutant of T. reesei,
NRRL 11460 (Rut-C30), received from J. J. Ellis, Northern
Regional Research Centre, Peoria, Ill., was also used for
comparison. It is referred to below as Rut-C30.
Substrate. WS, ground to 20-mesh powder, was used as a
source 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 of
ca. 70% insoluble carbohydrates suitable for the growth of T.
reesei and for cellulase production.
Aspen pulp was prepared by a chemical-thermomechanical process (10), and the pulp thus prepared was called
chemithermomechanical pulp (CTMP). During this process,
the wood was pulverized to fine fibers which still retained
most of the hemicelluloses and lignin. The chemical composition of CTMP was as follows (percent dry weight): cellulose, 63 to 66; hemicelluloses, 15 to 18; and lignin, 9 to 11.
Pretreatment. Powdered substrate (5 g; 20 mesh) was
dispensed into each Erlenmeyer flask of 250-ml capacity.
Each substrate was treated with NaOH (4% [wt/wt]) with
33.3% moisture at 121C (WS for 0.5 h and CTMP for 1 h).
The treated substrate was not washed. All of the solubilized
hemicelluloses and lignin were retained in the fermentation
medium. After the addition of nutrients, the pH was adjusted
TABLE 1. Cellulase production potential of new mutants of T.
reesei in LSF
Mutanta (yr developed)

Cellulose
concn
(%)

Cellulase
(IU/g of

Renfce-

~~cellulose)

QM6a (parent strain;

1971)

100

21

QM9414 (1971)

0.75

240

24

2
2.5
6

200
172
166

7
25
21

MCG77 (1977)

2
6

195
183

7
21

NG14 (1977)

2
6

180
250

7
21

5
5
6
15

290
160
233
200

25
2
21
2

L27 (1981)

225

22

CL-847 (1983)

5 (+1)-

229

28

D1-6 (1983)

140

17

Rut-C30 (1979)

a Strains QM6a, QM9414, MCG77, and NG14 were from the U.S. Army
Natick Research and Development Laboratories; Rut-C30 was from Cook
College, 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 by

suspending treated WS in water (1:10) and filtering through
four 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 (Difco
Laboratories, Detroit, Mich.). The quantity of nutrients
required for each substrate was determined at the rate of
their carbohydrate content. The required amount of concentrated nutrient salt solution (5 ml for WS and 5.7 ml for
CTMP) was added to 5 g of substrate. The concentrated salt
solution contained the following, dissolved in 200 ml of
water: 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 full
concentration of the nutrients means the complete required
quantity of nutrients as mentioned above, whereas a onehalf concentration is one-half of that quantity. All of the
flasks were autoclaved at 121C for 20 min, after the nutrient
salt solution was mixed with the substrate.
Moisture. The moisture content of the substrates after
pretreatment and the addition of nutrients and inoculum was
80% (wt/wt) in SSF. Sterilized water was added for LSF, to
obtain the desired concentration of the substrate in the

fermentation medium.
Inocula. Inocula of mutants QMY-1 and Rut-C30 were
produced on the modified medium as described above but
containing 1.5% glucose, with the nutrient salt solution
diluted accordingly. For inoculation of each flask containing
5 g of substrate, 5 ml of 2-day-old culture was used. The
inoculum was spread on the surface of the substrate.
Culture conditions. All of the SSF cultures were incubated
at 30C in a humidified incubator (about 80% relative humidity), whereas the LSF cultures were incubated at the same
temperature on a shaker at 150 rpm.
Extraction of the cellulase system. The culture of SSF from
each flask (originally 5 g of substrate) was mixed well with
more water to bring the final weight of the mixture (mycelium plus unutilized lignin, cellulose, and hemicelluloses) to
100 g. Tween 80 was added at a rate of 0.1%. The mixture
was shaken for 0.5 h and centrifuged. The supernatant was
used for enzyme determination. It was estimated that about
7 to 10% cellulases remained adsorbed on the residues
(mycelium and unutilized cellulose, hemicelluloses, and
lignin) when the residues were suspended in water and
Tween 80 as before and the supernatant was tested for
cellulase titer.
Analyses. Cellulase titer was calculated in international
units of enzyme activity (glucose released per min) on filter
paper for 60 min, by the method of Mandels et al. (11).
P-Glucosidase titer was measured in international units of
glucose released per min with 1% salicin solution for 30 min.
Xylanase titer was measured in international units of xylose
released per min with 1% xylan for 10 min (9). The cellulase
yield per gram of cellulose was calculated by dividing total
international units of cellulase titer in 100 ml of enzyme
broth by grams of cellulose present in the substrate supplied
in the flask.
Sugars were estimated by using a Beckman 344 gradient
high-pressure liquid chromatograph with an Altex 156 refractive index detector and a Spherogel 7.5% carbohydrate
column with a flow rate of 0.5 ml/min in the mobile phase of
water at 80C. The sugar samples were appropriately diluted
before injection.

VOL. 49, 1985

SOLID-STATE FERMENTATION WITH T. REESEI

RESULTS AND DISCUSSION

Cellulase production in LSF and the effect of different

concentrations of WS. The highest cellulase titer (1.65 IU/ml)
and cellulase yield (412 IU/g of cellulose) in LSF were
obtained with mutant QMY-1 in 1% WS (0.4% cellulose)
slurry after 7 days (Table 2). When the concentration of WS
was increased to 5% (2% cellulose), the enzyme production
time was increased from 7 to 11 days. The enzyme activity
increased to 6.0 IU/ml, but the cellulase yield dropped to 300
IU/g of cellulose (Table 2). The drop in cellulase yields might
have been due to poor mass transfer in the thick slurry of 5%
WS. The decrease in cellulase yield by mutant QM9414 with
increases in the concentration of cellulose is also evident
from the work of Sternberg (24), Gallo et al. (7), Ryu and
Mandels (21), and Tangnu et al. (25), as presented in Table 1.

TABLE 2. Cellulase production on WS by QMY-1

Type of
fermentation
(nutrient concn)

LSF (full
concn)

SSF (full
concn)

[wt/wt])

Concn
III of
cellulose
(%

0.4

2.0

Concn of
WS M

20a

[wt/wt])

8b

Time
.b of
incubation

(days)
5
7
7
11
14
11
14
22
22C'

Cellulase
titer
(IU/ml)
(l/n

1.44
1.65
1.3
6.0
5.5

Cellulase
yield

(IU/g of
cellulose)
360
412
65
300
275

6.0
6.3
8.6

7.4

11
5.5
SSF (one-half
14
6.7
concn)
22
6.7
22c
8.0
=
+
5 g of WS 20 g of water (no free water) 20%o solids in each
20

300
385
430
370
275
335
335
400

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 carried

out with a full concentration of nutrients in one set of
experiments and with a one-half concentration in another set
to evaluate the effect of different concentrations of salts in
the medium, since some microorganisms are unable to grow
in the high osmotic pressure of the medium. T. reesei
QMY-1 was quite tolerant to the high concentrations of the
nutrients, as indicated in Table 2. It produced the highest
enzyme titer (8.6 IU/ml) and cellulase yield (430 IU/g of
cellulose) in SSF on a full concentration of nutrients, after 22
days. The highest cellulase yield, 412 IU/g of cellulose,
obtained in LSF, was because that medium contained the
lowest cellulose concentration (0.4%), as discussed earlier.
However, in the present study, a cellulase yield of 275 to 300
IU/g of cellulose in LSF on 2% cellulose concentrations was
considered for comparisons between LSF and SSF. When
the nutrients were supplied in a one-half concentration, the
cellulase titer dropped to 6.7 IU/ml, cellulase yield dropped
to 335 IU/g of cellulose, and there was no increase in enzyme
yields 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

207

of 22 days of incubation), the cellulase titer decreased

somewhat for the full concentration of nutrients, whereas it
increased considerably (8.0 IU/ml) for a one-half concentration of nutrients. This indicates that half of the quantity of
required nutrients was sufficient to get an optimum cellulase
titer as well as an optimum cellulase yield. This finding could
contribute to a reduction in the cost of enzyme production.
Recently, more expensive media have been developed to
increase cellulase production by new mutants of T. reesei in
LSF on pure cellulose (17, 28). Even then, the highest yields
obtained are quite low, i.e., 140 IU/g of cellulose for mutant
D1-6 (17) and 229 IU/g of cellulose for mutant CL-847 (28)
(Table 1).
(ii) On WS and CTMP. Cellulase production by strain
QMY-1 was compared with that of strain Rut-C30, a hypercellulase-producing mutant, on two different lignocellulosic
substrates, WS and CTMP. QMY-1 produced its highest
cellulase titer (over 8 IU/ml) and yield (over 400 IU/g of
cellulose) 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 decreased
considerably on untreated WS (Table 3). The mutant Rut-

TABLE 3. Cellulase production by mutants QMY-1 and Rut-C30

on WS and CTMP
Mutant

Substrate'~(days)
Substateadays)

QMY-1
Cellulase
titer
yield
Cellulase
yed

(IU/mI)
(/m

Rut-C30
Cellulase
titer
yield
Cellulase
yil

(IU/g of

(IU/mI)
(Um

cellulose)

(IU/g of

cellulose)

Treated WS
9
16
18
20
26
30

0.7
4.4
7.8
8.1
8.5
7.2

35

0.6

30

220
390
405
425
360

1.6
2.6
6.2
5.2
4.6

80
130
310
260
230

Untreated WS
9
16
18
20
26
30

1.7
2.3
2.4
2.1
2.2
1.9

85
115
120
105
110
95

1.1
2.6
2.8
3.1
3.2
4.2

55
130
140
155
160
210

Treated CTMP
9
16
18
20
26
30

1.5
4.7
3.9
6.3
6.0
6.2

45
142

0.9
5.3
4.5
6.6
6.5
4.8

27
161
136
200
197
145

Untreated CTMP
9
16
18
20
26
30

2.2
5.3
3.3
5.0
5.6
7.2

67
161

1.9
4.1
4.1
5.8
6.0
5.0

57
124
124
176
182
152

191
182
188

151
170
218

a Cellulose content of wheat straw, 40%; cellulose content of CTMP, 66%.

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

208

APPL. ENVIRON. MICROBIOL.

CHAHAL

TABLE 4. Cellulase production by different mutants of T. reesei

on different cellulosic substrates in LSF
Mutant' and
substrateb
(5%)
(5%o)

Cellulase
titer"
(lU/mi)
(IU/ml)

Cellulase
yield
~ ~(lU/g
~ ~ ~ cel ulofose)

QM-9414
Solka Floc
SE
SEWA

1.85
0.70
0.96

36.1
25.4
21.1

Rut-C30
Solka Floc
SE
SEWA

5.56
1.57
2.10

111.0
57.1
46.1

E.58
Solka Floc
SE
SEWA

1.93
0.62
0.51

38.6
22.5
11.2

a 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 or

CTMP. This finding indicated that this mutant is not well
adapted to such conditions of SSF.
The mild alkali treatment of CTMP did not affect cellulase
production by either mutant (Table 3). Because the highest
cellulase titer and yield obtained on treated CTMP after 20
days in SSF were almost comparable to those obtained on
untreated CTMP, CTMP seems to be a good substrate for
cellulase production even without any further treatment.
Further studies on cellulase production on CTMP are in
progress.
The cellulase titer and yield on CTMP with strains QMY-1
and Rut-C30 (Table 3) and on treated WS (Table 2) with
strain QMY-1 in SSF were higher than those of mutants
QM-9414, Rut-C30, and E.58 on steam-exploded wood, on
steam-exploded and alkali-treated wood with water extracted, and even on pure cellulose (Solka Floc; Brown Co.,
Berlin, N.H.) in LSF, as reported by others (Table 4). The
titer and yield of cellulase obtained with QMY-1 in SSF were
also higher than those obtained by other workers who grew
various mutants of T. reesei on pure cellulose in LSF (Table
1). The results clearly indicate that the new approach of
retaining the hemicelluloses and lignin of the alkali-pretreated lignocelluloses (WS, CTMP) in SSF increased significantly the cellulase titer per unit volume and the cellulase
yield per unit of cellulose.
Role of hemicelluloses and lignin in cellulase production.
The increase in cellulase titer was postulated to be due to the
use of hemicelluloses during the initial growth of T. reesei
and then to the use of cellulose during the later phase of
growth for production of cellulases. To test this postulate,
we grew T. reesei QMY-1 in LSF on pure cellulose (acellulose; Sigma Chemical Co., St. Louis, Mo.) in one set of
experiments, and cellulose was fortified with a mixture of
solubles obtained from WS. Delayed and slow synthesis of
cellulases during the early phase for WS soluble-fortified
cellulose was attributed to the presence of hemicelluloses,
an easily metabolizable carbon source (Fig. 1). After hemicelluloses were used, cellulase synthesis increased considerably during the later phase of fermentation. This indicated
that 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 on
the role of hemicelluloses and lignin, individually and in
combination, is in progress.
Composition of the cellulase system. The cellulase system
produced in SSF contained the following enzymatic activities (international units per milliliter): cellulase, 8.6; 3glucosidase, 10.6; and xylanase, 270. The xylanase titer was
quite variable (between 190 and 480 IU/ml); however, the
ratio of cellulases and 3-glucosidase varied between 1:1 and
1:1.5 in various cellulase system preparations. These are
enzyme activities when 5 g of WS fermented in SSF was
suspended in ca. 100 ml of water to extract the enzymes. The
enzyme titer could be doubled (17.2 IU/ml) by extracting the
enzyme in 50 ml of water. The composition of the cellulase
system indicated that there was no need to add extra
3-glucosidase or xylanase for the hydrolysis of pure cellulose or lignocelluloses.
Hydrolytic potential of the cellulase system. (i) Cotton. The
cellulases produced in SSF, when used in a substrate-to-enzyme ratio of 1 g:20 IU, showed saccharification of different
concentrations of untreated cotton as high as that reported
by Mandels et al. (13) (Table 5). There was also an indication
that this enzyme system was able to hydrolyze a higher
concentration (10%) of cotton without any reduction in the
percentage of hydrolysis. The high-pressure liquid chromatography of the hydrolysate obtained from 10% concentrations of cotton showed mostly glucose (92.8%) and very little
cellobiose (3.5%). It is therefore assumed that cellulases
produced in SSF may be able to hydrolyze a higher concentration of cellulose to obtain high concentrations of glucose
in the hydrolysate suitable for economical ethanol fermentation or for fermentation of any other product.
(ii) Cellulose fiber. Hydrolysis of cellulose fiber with
cellulases produced in SSF was faster than that of cotton.
There was also an indication that cellulases produced in SSF
gave a faster rate and higher percentage of hydrolysis than
cellulases produced in LSF (Table 6).
(iii) Delignified WS. WS delignified by the method of
Toyama (26) was hydrolyzed with the cellulase system

0LL.

I
0

50

150
100
TIME (h)

200

250

FIG. 1. Effect of hemicelluloses and lignin on cellulase production by mutant QMY-1 on 1% cellulose (sigma cell). (The ratio of
cellulose to WS solubles in the medium was 1:0.25.) Symbols: 0,
cellulose only (control); A, cellulose plus WS solubles containing
hemicelluloses and lignin.

VOL. 49, 1985

SOLID-STATE FERMENTATION WITH T. REESEI

TABLE 5. Hydrolysis of cotton with cellulases produced in SSF

% Saccharification"
Substrate
Substrate/enzyme

TABLE 6. Comparison of hydrolysis of cellulose' fiber with

cellulases produced in SSF and LSF

concn

ratio

w]

(gIU)

24 h

48 h

System of
fermentaion

Substrate/enzyme
ratio (g/IU)

24 h

17.7
17.9
22.4
22.1

SSF
LSF

1:20
1:20

23.6
19.2

21.6

1
2
4
8
10

1:20
1:20
1:20
1:20
1:37

16.9
15.4
17.9
13.6
15.4

5C

1:20

18.0

Saccharification = grams of reducing sugars x 0.9 x 100.

grams of substrate
b 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 g
of sugars per liter, detected by high-pressure liquid chromatography).
C Data derived by using a substrate concentration of 5% (dry weight) are
from reference 13, with T. Reesei QM9414 cellulases.

produced in SSF at a 10% (wt/vol) concentration. Hydrolysis was done at pH 6.7, the original pH of the enzyme
solution, and also at standard pH 4.8 (Fig. 2 and 3). The rate
of hydrolysis of cellulose into glucose was very high for the
first 20 h of hydrolysis at both pH levels, and ca. 65% of total
hydrolysis 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 7.75
g/liter (Fig. 2), which was too low to cause any significant
inhibition of cellulose hydrolysis (12, 14). After 20 h of
hydrolysis, the concentration of cellobiose further decreased
to ca. 3 g/liter.

209

% Saccharification
48 h
72 h

32.2
29.4

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

38.6
35.3

5% concentration.

The hydrolysate obtained with pH 6.7, after 96 h of

hydrolysis, contained 99.75 g of sugars per liter (glucose,
68.18 g; cellobiose, 3.19 g; xylose, 26.71 g; arabinose, 1.67
g), whereas the hydrolysate obtained with pH 4.8, after the
same hydrolysis period, contained 86.14 g of sugars per liter
(glucose, 56.49 g; cellobiose, 3.88 g; xylose, 24.31 g; arabinose, 1.46 g), giving total saccharifications of 89.7 and
77.9%, respectively.
Conclusions. The increase in cellulase yields, from a range
of 160 to 250 IU/g of pure cellulose to a range of 250 to 430
IU/g of crude cellulose from WS and CTMP, was due to the
use of a hemicellulose fraction during the initial growth of
the organism and to the production of cellulases on a
cellulose fraction of substrates during the later phase of
growth of the organism. The presence of lignin could be
another factor in the increase of cellulase yields. The role of
lignin and hemicelluloses in enzyme production is being
further evaluated.
High cellulase activity of 8.6 IU/ml could be doubled (17.2
IU/ml) by extracting cellulases with half the quantity of
water. Because high cellulase titer (over 15 IU/ml) is required to obtain high concentrations of glucose for economical ethanol fermentation (4), SSF seems to hold promise for
obtaining a high cellulase titer per unit volume of enzyme
broth. The cellulase system produced in SSF contained
sufficient quantities of ,B-glucosidase and xylanase for com-

100.
90
80
_ 70
CP
,, 60
c
0

C0

(.)

50

Co

nx

t 40
0

41
x

20

io-

100
80
60
40
TI 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

a I
I
I
80
60
iob)I
( h)
FIG. 3. Hydrolysis of delignified WS at pH 4.8. Symbols
defined in the legend to Fig. 2.
T

0
K..m

20

1----T-

oi

40
TIME

are

210

APPL. ENVIRON. MICROBIOL.

CHAHAL

plete hydrolysis of pure cellulose as well as lignocelluloses.

It was also assumed that the cellulase system produced in
SSF was rich in C1 factor as proposed much earlier by Reese
et al. (20). It was reported by them (20) that the C1 factor was
essential to hydrolyze the crystalline portion of cellulose,
and this concept is still maintained by Reese (19). By the
new approach, ca. 100 g of sugars per liter were obtained by
hydrolyzing 100 g of delignified WS with the cellulase system
produced with SSF. Moreover, SSF enables drastic reductions in the cost of enzyme because cellulase yields per unit
of cellulose were increased by 72%, crude cellulose (WS)
with a minimum of pretreatment and purification could be
used as a carbon substrate, the quantity of nutrients could be
reduced to one-half, and SSF does not require very complex
control systems. However, SSF has its own inherent problems, including maintenance of pH, moisture level, aeration,
agitation, etc., when large quantities of solid substrates are
used (4).
ACKNOWLEDGMENTS
I thank M. Mandels and E. T. Reese, Materials Protection and
Biotechnology Division, Science and Advanced Technology Laboratory, U.S. Army Natick Research and Development Laboratories,
Natick, Mass., and V. Portelance, Bacteriology Research Center,
Institut Armand-Frappier, Laval, Quebec, Canada, for critical reading of the manuscript and for their valuable suggestions. I am also
very grateful to Johanne Lemay for her technical help and to P. S.
Chahal for drawing the figures and for technical help.
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