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Studies on the production and application of cellulase
from Trichoderma reesei QM-9414
S. Hari Krishna, K. C. Sekhar Rao, J. Suresh Babu, D. Srirami Reddy
Abstract High yielding mutant strain, Trichoderma reesei
QM-9414, was employed for the cellulase enzyme pro-
duction. Enzyme production conditions (pH, inoculum
age and concentration, and organic supplements) were
optimized. The ability of partially puri®ed enzyme to hy-
drolyze various regionally abundant lignocellulosic raw
materials was studied. Enzymatic hydrolysis conditions
(temperature, pH, enzyme and substrate concentrations)
were optimized. Temperature 50 °C, pH 4.5, enzyme con-
centration 40 FPU/g substrate and substrate concentration
2.5% were found to be optimum for the maximum yields
of sugars. b-glucosidase supplementation was found to
increase both the sugar yield and hydrolysis rate, and
shorten the reaction time signi®cantly.
1
Introduction
The potential of biotechnical processes based on enzymatic
hydrolysis of cellulosic materials is enormous. As the limits
of non-renewable resources come closer, cellulose must
become major raw material for food, energy, fuel and other
products. Cellulose is a very important renewable raw
material produced in large amounts and is primarily as-
sociated with lignin to result lignocellulose in plants [1].
Pretreatment of lignocellulose is a necessary step to en-
hance its susceptibility to enzyme attack by removing lig-
nin barrier. The ®rst step to produce biochemicals from
lignocellulosics is to convert them into sugars by hydro-
lysis. Enzymatic hydrolysis is superior, in several aspects,
to acid hydrolysis (Table 1). The enzymatic degradation of
cellulose requires cellulose±cellulase system.
Most of the previous studies [2±5] on enzymatic hy-
drolysis directly employed cellulase-producing organisms,
which releases cellulase into medium and found that the
yields were very low. Hence, better alternative will be
employing cellulase directly. Since the commercial cellu-
lases are relatively costly, it was attempted to produce
cellulase ®rst, purify partially and utilize it for hydrolysis
studies. Many microorganisms are shown to produce cel-
lulase. However, the most promising one is Trichoderma
reesei QM-9414, which produce large quantities of extra-
cellular cellulase.
The present investigation was aimed at obtaining opti-
mum conditions for the cellulase production and appli-
cation of the enzyme for hydrolysis of zero-cost
lignocellulosic raw materials to design an economically
feasible hydrolysis process.
2
Materials and methods
2.1
Materials
Trichoderma reesei QM-9414 was procured from NCIM,
National Chemical Laboratories (Pune, India). Antigonum
leptopus (L.) and banana leaves were collected in and
around Andhra University (Visakhapatnam, India). Sugar-
cane leaves were obtained from sugarcane growers in East
Godavari District (Andhra Pradesh, India). Microcrystal-
line cellulose (Solka ¯oc SW-40); a 40 mesh, hammer
milled, ®brous and pure cellulose; was employed as a
substrate of reference. b-glucosidase from Aspergillus ni-
ger was a gift from Novo Nordisk (Bagsvaerd, Denmark).
2.2
Methods
2.2.1
Cellulase production
T. reesei was maintained on potato dextrose agar slants for
6 days at 28 °C before using in enzyme production ex-
periments. The basal medium for the growth of T. reesei
and production of cellulase is as follows (g/l): (NH
4
)
2
SO
4
:
1.4, KH
2
PO
4
: 2.0, Urea: 0.3, CaCl
2
: 0.3, MgSO
4
á7H
2
O: 0.3
and (mg/l): FeSO
4
á7H
2
O: 5.0, MnSO
4
áH
2
O: 1.6, ZnSO
4
á7-
H
2
O: 1.4, CoCl
2
: 2.0. In addition microcrystalline cellulose
(1%), Difco peptone (0.1%) and Tween 80 (Polyoxyethyl-
ene sorbitan monooleate, 0.1%) were added to the medium
to induce cellulase production. pH was controlled using
2N HCl and 2N NaOH. Medium was autoclaved for 30 min
and seeded with a suspension of T. reesei spores, to a ®nal
Bioprocess Engineering 22 (2000) 467±470 ÓSpringer-Verlag 2000
467
Received: 16 August, 1999
S. Hari Krishna, K. C. Sekhar Rao, J. Suresh Babu,
D. Srirami Reddy
Biotechnology Division,
Department of Chemical Engineering,
Andhra University, Visakhapatnam 530 003, India
S. Hari Krishna (&)
Fermentation Technology & Bioengineering Department,
Central Food Technological Research Institute,
Mysore 570 013, India
Authors are thankful to the Andhra University, Visakhapatnam
and the University Grants Commission, New Delhi for supporting
the project. We are grateful to Prof. C. Ayyanna, Head,
Department of Chemical Engineering, Andhra University for
helpful discussions.
concentration of 2 ´10
5
spores/ml. The submerged cul-
ture was run for 6 d at 28 °C and at 3.5 pH on rotary
shaker at 220 rpm.
2.2.2
Partial purification of cellulase
Culture ®ltrate of the production was concentrated ®rst by
precipitating with 20±90% ammonium sulfate (NH
4
)
2
SO
4
saturation. Precipitates were separated by centrifugation
and redissolved in citrate buffer (0.05 M). This prepara-
tion was dialyzed at 4 °C for 24 h in cellophane tubing
against distilled water and subjected for the further de-
salting with Sephadex G-20 powder. Concentrated enzyme
solution was separated from Sephadex by centrifugation
and used in the hydrolysis experiments.
2.2.3
Pretreatment of substrates
Alkaline hydrogen peroxide (H
2
O
2
) treatment was found
to be comparatively superior over autoclaving and alkali
cooking in our earlier studies [6] and was used in this
study. Substrate leaves were cut into small pieces and were
soaked in distilled water for 4 h to remove any soluble
materials. The residues were ®ltered, dried and stored in
polyethylene containers. The residues were treated with
H
2
O
2
by incubating a suspension of residue (1 g in 50 ml)
in distilled water containing 1% H
2
O
2
. NaOH was added to
bring the pH of the suspension to 11.5 and the mixture was
stirred gently for 16 h at room temperature. Insoluble
fraction was vacuum ®ltered, washed repeatedly with dis-
tilled water until the ®ltrate becomes neutral and dried in a
vacuum oven at 50 °C.
2.2.4
Enzymatic hydrolysis (saccharification)
The standard hydrolysis experiments were carried out in
100 ml stoppered conical ¯asks in presence of 0.01%
sodium azide. The pH was adjusted to 4.8 with 0.05 M
sodium citrate buffer. To the pretreated substrates (2.5%
dry basis) was added the T. reesei cellulase (8 FPU/g
substrate) in a total volume of 50 ml. The ¯asks were
incubated at 50 °C on rotary shaker at 150 rpm. Samples
were withdrawn periodically, centrifuged and the super-
natants were analyzed for reducing sugars. The percentage
sacchari®cation was calculated as:
% Saccharification
Reducing sugars 0:9
Total carbohydrates in substrate 100
2.2.5
Analytical methods
Estimation of reducing sugars was carried out by
dinitro salicylic acid (DNS) method [7]. Cellulase
activity was measured as Filter Paper Units (FPU) as
per Mandels et al. [8].
3
Results and discussions
Thousands of microorganisms have the ability to grow on
cellulose. Many of them grow quite rapidly, but only few
produce extracellular cellulase that is capable of convert-
ing the native crystalline cellulose to sugars in vitro [9].
Trichoderma strains, particularly mutants QM-9123 and
QM-9414, are the excellent sources of cellulase suitable for
practical applications. Cellulase is an inducible enzyme in
Trichoderma, with highest yields obtained when the fun-
gus is grown on cellulose rich medium.
3.1
Cellulase production
The shake ¯asks, with nutrients and inoculum, were ad-
justed and controlled at different pHs (3.5, 4.0, 4.5 and 5.0)
and incubated for 6 d. During incubation, samples were
withdrawn for every 24 h and analyzed for the enzyme
levels. Maximum activity was reached at pH 3.5 (Fig. 1).
The ®lter paper (FP) activity increased with increasing
incubation time.
Enzyme activities were found to be higher with the
mycelial inoculum compared to the spore inoculum
(Fig. 2). Inoculum age was also found to be important
(Table 2). The yield of cellulase in a cellulose culture is
reduced unless a second more readily metabolized sub-
Table 1. Comparison of acid and enzymatic hydrolysis processes
Parameter Acid hydrolysis Enzymatic hydrolysis
Pretreatment May be necessary Necessary
Rate of hydrolysis Fast (min) Slow (h)
Temperature High (200 °C) Low (45 °C)
Pressure High Atmospheric
Yield Depends on material and process details Depends on material and process details
Formation of by-products Probably formed Not likely
Industrial processes Yes (in USSR and USA) No (pilot plant only)
Fig. 1. Effect of pH on cellulase production
Bioprocess Engineering 22 (2000)
468
strate is added. The peptone was an excellent additive with
an optimum concentration of 0.075±0.1% (Fig. 3).
3.2
Enzymatic hydrolysis
The hydrolysis of cellulose should yield high sugar content
per enzyme unit. Many factors affect this yield viz., pre-
treatment, inhibition of enzyme by heat or degradation
products, enzyme and substrate concentrations, adsorp-
tion of cellulase to cellulose, speed of enzyme action and
agitation. Optimization of these factors play important
role in the economy of hydrolysis process. To maximize
the sugar yield from chemically modi®ed substrate, the
basic hydrolytic variables (temperature, pH and enzyme
and substrate concentrations) were optimized.
Fig. 2. Effect of inoculum concentration on cellulase production.
(s) spore inoculum 104 spores/ml; (n) 1% mycelial inoculum
3 d old, (m) 5% mycelial inoculum 3 d old
Table 2. Effect of inoculum age on cellulase production
Cultivation
age (h)
Optimum
temperature (°C)
Cellulase
(FPU/ml)
0±30 28.0 0.04
30±120 28.0 5.00
120±160 28.0 6.43
Fig. 3. Effect of peptone (organic supplement) concentration on
cellulase production. (s) 0.05% peptone, (n) 0.1% peptone
Fig. 4. Effect of temperature on sacchari®cation. Reaction con-
ditions: substrate 2.5%, cellulase 8 FPU/g substrate, 4.8 pH, 48 h.
(s)A. leptopus leaves, (n) sugarcane leaves, (m) banana leaves,
(h) microcrystalline cellulose
Fig. 5. Effect of pH on sacchari®cation. Reaction conditions:
substrate 2.5%, Cellulase 8 FPU/g substrate, 50 °C, 48 h. (s)
A. leptopus leaves, (n) sugarcane leaves, (m) banana leaves, (h)
microcrystalline cellulose
Fig. 6. Effect of cellulase concentration on sacchari®cation.
Reaction conditions: substrate 2.5%, 50 °C, 4.5 pH, 48 h. (s)
A. leptopus leaves, (n) sugarcane leaves, (m) banana leaves, (h)
microcrystalline cellulose
469
S. Hari Krishna et al.: Production and application of T. reesei cellulase
Optimum conditions for hydrolysis were arrived at by
carrying out experiments with the celluloses from all se-
lected sources. It was apparent that 50 °C, 4.5 pH, 120 FPU
cellulase/g substrate, 2.5% substrate were optimum for all
substrates tested (Figs. 4±7). Although 120 FPU cellulase/g
substrate was optimum, use of such a high enzyme content
per gram substrate is not economically feasible. In this
regard, it was observed that only a negligible enhancement
in hydrolysis ef®ciency with the increase in enzyme
quantity from 40 to 120 FPU/g substrate. Hence, an enzyme
concentration of 40 FPU/g substrate would be the adequate
concentration. Increase in the substrate quantity (5±25%)
in the reaction medium limited the hydrolysis, due to dif-
®culties in stirring and product inhibition. Extending hy-
drolysis time to 72 h had no signi®cant effect. Accordingly,
48 h period was considered as the optimal reaction time.
Results obtained from the hydrolysis of all the pre-
treated lignocellulosic materials at optimum conditions
were represented in Table 3. It is clear that microcrystal-
line cellulose (MCC) gave maximum conversion in shorter
time followed by celluloses from A. leptopus, banana and
sugarcane leaves. However, zero-cost raw materials should
obviously be the `materials of choice' for enzymatic hy-
drolysis, due to cost effectiveness over MCC. A. leptopus
and banana celluloses were less crystalline than that of
sugarcane and therefore got converted to sugars in a
shorter time compared to sugarcane.
T. reesei cellulase enzyme complex was known to con-
tain low quantities of b-glucosidase, which is mainly re-
quired for the conversion of cellobiose to glucose.
Cellobiose, a well known cellulase inhibitor, will be pro-
duced from cellulose as one of the by-product. So, in order
to compensate the low activity of b-glucosidase of T. reesei
cellulase complex, it was supplemented externally. It was
found that b-glucosidase supplementation not only
increased the sugar yield but also shortened the duration
of hydrolysis time (Table 3).
4
Conclusions
The present investigation enlightened some of the im-
portant variables of enzymatic hydrolysis of pretreated
lignocellulose. Trichoderma strain improvement to pro-
duce b-glucosidase rich cellulase enzyme, and employing
fermenting organism (Saccharomyces cerevisiae/Zymomo-
nas mobilis etc.) to overcome product inhibition are under
study.
References
1. Ghose, T.K.; Ghosh, P.: Bioconversion of cellulosic substances.
J. Appl. Chem. Biotechnol. 28 (1978) 309±320
2. Han, Y.W.; Callihan, C.D.: Cellulose fermentation: Effect of
substrate pretreatment on microbial growth. Appl. Microbiol.
27 (1974) 159±165
3. Updegraff, D.M.: Utilization of cellulose from waste paper
by Myrothecium verrucaria. Biotechnol. Bioeng. 13 (1971)
77±97
4. Ghose, T.K.: Continuous enzymatic sacchari®cation of cellu-
lose with culture ®ltrates of Trichoderma viride QM 6a.
Biotechnol. Bioeng. 11 (1969) 239±261
5. Mandels, M.; Hontz, L.; Nystrom, J.: Enzymatic hydrolysis of
waste cellulose. Biotechnol. Bioeng. 16 (1974) 1471±1493
6. Hari Krishna, S.; Prabhakar, Y.; Rao, R.J.: Sacchari®cation
studies of lignocellulosic biomass from Antigonum leptopus
(Linn). Indian J. Pharma. Sci. 59 (1997) 39±42
7. Miller, G.L.: Use of dinitro salicylic acid reagent for the
determination of reducing sugars. Anal. Chem. 31 (1959)
426±428
8. Mandels, M.; Andreotti, R.; Roche, C.: Measurement of
saccharifying cellulase. Biotechnol. Bioeng. Symp. 6 (1976)
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9. Mandels, M.; Weber, J.: The production of cellulases. Adv.
Chem. Ser. 95 (1969) 391±414
Fig. 7. Effect of substrate concentration on sacchari®cation. Re-
action conditions: cellulase 40 FPU/g substrate, 50 °C, 4.5 pH,
48 h. (s)A. leptopus leaves, (n) sugarcane leaves, (m) banana
leaves, (h) microcrystalline cellulose
Table 3. Enzymatic hydrolysis of different substrates
Raw material % Sacchari®cation
24 h 48 h 24 h48 h
Microcrystalline cellulose
a
94 96 98 98
Antigonum leptopus (L.) 86 88 90 90
Banana 80 90 90 92
Sugarcane (Saccharum
of®cinarum)
76 92 86 94
Conditions: 2.5% substrate, 40 FPU cellulase/g substrate, 50 °C,
4.5 pH
b-glucosidase added 50 U/g substrate
a
No pretreatment applied
470
Bioprocess Engineering 22 (2000)
