Process Biochemistry 34 (1999) 909 – 912

Cellulase production by solid state fermentation on lignocellulosic

waste from the xylose industry
Liming Xia *, Peilin Cen
Department of Chemical Engineering, Zhejiang Uni6ersity, Hangzhou 310027, People’s Republic of China

Received 17 September 1998; received in revised form 9 December 1998; accepted 12 December 1998

Abstract

Cellulase production was carried out by solid state fermentation using corncob residue, a lignocellulosic waste from the xylose
industry, as the substrate of Trichoderma reesei ZU-02. The effects of water content, dosage of wheat bran and initial pH value
in solid substrate on cellulase synthesis were studied in shallow tray fermentors. The solid substrate could be reused in at least
three batches and the highest cellulase activity (158 IFPU/g koji) was obtained in the second fermentation batch. To produce
cellulase on a larger scale, a deep trough fermentor with forced aeration was used and 128 IFPU/g koji (  305 IFPU/g cellulose)
was reached after 5 days solid state fermentation. The enzyme koji produced in the present process can be used directly to
hydrolyze corncob residue effectively, when the cellulase dosage was above 20 IFPU/g substrate, the saccharification yield could
be over 84%. © 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Cellulase; Production; Solid-state fermentation; Corncob; Residue; Repeated batch processes; Hydrolysis

1. Introduction In China, xylose is produced from corncobs by dilute

Cellulase production is the most important step in
the economical production of ethanol, single cell
protein and other chemicals from renewable cellulosic
materials. To date, the production of cellulase has been
widely studied in submerged culture processes, but the
relatively high cost of enzyme production has hindered
the industrial application of cellulose bioconversion. It
has been reported that solid state fermentation is an
attractive process to produce cellulase economically due
to its lower capital investment and lower operating
expenses [1,2]. Another approach to reduce the cost of
cellulase production is the use of lignocellulosic materi-
als as substrates rather than expensive pure cellulose. In
prior publications, abundant agricultural residue such
as corn stover, wheat straw, rice straw, bagasse, etc.
were used in cellulase production [3,4]. Although these
raw materials are cheaper, pretreatment is generally
required to improve the utilizability of lignocellulosic
materials and the cost is still considerable.

This work was supported by the National Natural Science Foun-
dation of China (No. 29876036).
* Corresponding author. Fax: +86-571-7951358.
acid hydrolysis. A large amount of waste corncob
residue is produced in the xylose industry and often
causes environmental pollution. It is an important issue
to deal with the residue both for the comprehensive
utilization of lignocellulosic resources and for the pre-
vention of environmental pollution. Since most hemi-
cellulose in corncobs has been hydrolyzed to xylose, the
corncob residue is porous and easy to degrade by
cellulolytic fungi. In this work, corncob residue without
further pretreatment was used as a substrate in solid
state fermentation to produce cellulase.
2. Materials and methods
2.1. Microorganism
The strain Trichoderma reesei ZU-02 (originally from
ATCC56764) was used for cellulase production. Spores
were stored on potato dextrose agar slants at 4°C. For
the seed culture, the spores of T. reesei from slants were
inoculated into the seed medium and cultured under
aerobic conditions at 30°C for 48 h. The mycelium
suspension was used as an inoculum in all experiments.

0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 0 1 5 - 1
910 L. Xia, P. Cen / Process Biochemistry 34 (1999) 909–912
2.2. Lignocellulosic substrate
Corncob residue with 20-mesh size was obtained
from a local xylose manufacturer and had the following
composition: cellulose 58.5%, hemicellulose 11.7%,
lignin 20.2%, and others 9.6%.
2.3. Medium
The seed medium for T. reesei was that of Mandels et
al. [5]. The medium composition (dry weight basis) for
solid state fermentation for cellulase production was
usually the following (%): corncob 66; wheat bran 30;
(NH4)2SO4 2; Urea 0.5; KH2PO4 0.5; MgSO.47H2O 0.5;
CaCl2 0.45; CoCl2 0.05. The initial pH value of the
medium was  4.5 after sterilization at 121°C for 40
min and the water content of the substrate was 75% for
most experiments with the exceptions pointed out in the
text.
2.4. Cellulase production
Small scale experiments were carried out in metal
shallow trays (50× 40 × 5 cm). After inoculation with
10% liquid seeds, the trays were installed in an incuba-
tor in which air humidity and temperature were kept at
92% and 30°C, respectively.
Large scale production of cellulase was performed in
a deep trough fermentor (4×2 × 1.5 m), the thickness
of the solid substrate layer was  30cm and the tem-
perature was controlled at 28 – 30°C, air with over 90%
humidity blew through the bottom of the cultivation
chamber by the forced aeration.
2.5. Hydrolysis
The hydrolysis experiments were performed with 10%
corncob residue as substrate and different enzyme
dosage at 50°C, pH 4.8 for 48 h. The yield of enzymic
hydrolysis was calculated as follows:
Fig. 1. Effect of water content in substrate on cellulase production by
solid state fermentation.
yield (%)
= reducing sugar× 0.9
× 100/polysaccharides in substrate
2.6. Analysis methods
The mouldy substrates (koji) produced by solid state
fermentation were mixed with 40 volumes of water to
extract cellulase, stirred slowly at room temperature for
3 h and filtered. The liquid portion was then used for
the measurement of cellulase activity.
Filter paper activity (FPA) and cellobiase activities
(CB) were measured according to the method recom-
mended by Ghose [6] and expressed as international
units (IU). One international unit of filter paper activity
(IFPU) is the amount of enzyme which forms 1 mmol
glucose (reducing sugars as glucose) per min during the
hydrolysis reaction. One international unit of cellobiase
is the amount of enzyme which forms 2 mmol glucose
per min from cellobiose. The reducing sugar was deter-
mined using the dinitrosalicilic acid (DNS) method [6].
3. Results and discussion
3.1. Cellulase production in shallow tray fermentors
3.1.1. Effect of water content in the substrate
The water content of solid substrates is one of the
key factors in cellulase production. Experiments with
different water contents of substrate were carried out in
shallow tray fermentors. The results after 6 days culti-
vation of Trichoderma reesei are shown in Fig. 1. The
optimal water content in the solid substrate appears to
be 70%. Under these culture conditions, a cellulase
activity of 126 IFPU/g koji was obtained, i.e. 300
FPIU/g cellulose could be produced.
3.1.2. Dosage of wheat bran
Different dosages of wheat bran have been tested in
cellulase production by solid state fermentation with
70% water content of substrate at 28–30°C. The results
of 6 days culture are shown in Fig. 2.
The optimum dosage of wheat bran was 30%. A
smaller dosage of wheat bran will result in poor fungal
growth and low cellulase activity. On the other hand,
higher dosage may cause extensive fungal growth, but
decrease cellulase accumulation.
3.1.3. Effect of initial pH 6alue on cellulase synthesis
To evaluate the effects of initial pH value in solid
substrate on cellulase synthesis, the initial pH values
were adjusted by the addition of lime slurry to 4.5, 5.0,
5.5 and 6.0, respectively. The corncob residue with an
original pH value 2.8 was also used in solid state
 L. Xia, P. Cen / Process Biochemistry 34 (1999) 909–912 911

cellulase protein was harvested, the solid substrate with

mycelia and spores was used for the next batch fermen-
tation, but neither fresh substrate nor inoculum was
added. This process was repeated several times (Table
2).
This process could be repeated for at least three
batches (Table 2). The culture time for each batch
fermentation was shortened in turn and the cellulase
activity obtained in the second batch was higher than
that in the first batch. It is also of interest to note that
in the first three batches, the CB increased with the
number of batches.

3.2. Solid state fermentation in deep trough fermentor

The time course of cellulase synthesis in a deep

Fig. 2. Effect of wheat bran dosage on cellulase production.
trough fermentor by Trichoderma reesei is shown in
Table 1
Fig. 3. Compared with the shallow tray fermentor, the
Filter paper activity (IU/g) obtained by solid state fermentation with mass transfer in the deep trough fermentor with forced
a different initial pH value of the substrate aeration can be effectively enhanced. After 5 days of
solid state fermentation, the cellulase activity of 128
Batch pH IFPU/g koji (  305 IFPU/g cellulose) was reached.
2.8 4.5 5.0 5.5 6.0
The deep trough fermentor may therefore produce cel-
lulase on a large scale and with high productivity and
1 18 110 121 118 123 uniform quality.
2 26 126 115 124 116
3 22 156 152 158 155
3.3. Hydrolysis of corncob residue by cellulase

Table 2 The enzyme koji produced by Trichoderma reesei in

Cellulase production under repeated batch process in solid state solid state fermentation was used directly to hydrolyze
fermentation corncob residue. The results of enzymic hydrolysis are
shown in Fig. 4.
Batch Culture time (h) FPA (IU/g) CB(IU/g)
When the enzyme dosage was changed from 10
1 144 124 10 IFPU/g substrate to 15 IFPU/g substrate, the yield of
2 120 158 15 enzymic hydrolysis was raised sharply from 52.4 to
3 108 102 23 74.5%. Further increase in the cellulase dosage did not
4 96 40 11 produce a corresponding increase in the hydrolysis

fermentation. The results of three batch solid state

fermentation with a different initial substrate pH value
are listed in Table 1. The cultivation period for each
batch was 6 days.
The original corncob residue of pH 2.8 was unsuit-
able for cellulase production since the low pH value
resulted in poor growth. In contrast, if the initial pH
value of the substrate was between 4.5 and 6.0, no
significant effect was observed on cellulase production.

3.1.4. Repeated batch processes

Cellulase production was undertaken in solid state
fermentation by repeated batch processes. When one
batch fermentation was completed (cellulase activity
reached the maximum), the enzyme koji was soaked
and stirred slowly in 20 volumes of clean water for 3 h Fig. 3. Time course of cellulase production in a deep trough fermen-
to extract the cellulase. After the liquid containing tor.
912 L. Xia, P. Cen / Process Biochemistry 34 (1999) 909–912
Fig. 4. Effect of cellulase dosage on the hydrolysis of corncob residue.
yield. According to these results, 1 kg of cellulase koji
produced in the present process can hydrolyze more
than 4 kg of corncob residue with a saccharification
yield of  90%. This process therefore has a high
potential for comprehensive utilization of renewable
lignocellulosic resources.
References
[1] Deschamps F, Giuliano C, Asther M, Huet MC, Roussos S.
Cellulase production by Trichoderma harzianum in static and
mixed solid-state fermentation reactors under nonaseptic condi-
tions. Biotechnol Bioeng 1985;27:1385 – 8.
[2] Chahal DS. Production of Trichoderma reesei cellulase system
with high hydrolytic potential by solid-state fermentation. En-
zymes in biomass conversion. ACS Symp Ser 1991;460:111–22.
[3] Rao MNA, Mithal BM, Thakkur RN, Sastry KSM. Solid-state
fermentation for cellulase production by Pestalotiopsis 6ersicolor.
Biotechnol Bioeng 1983;25:869 – 72.
[4] Chahal PS, Chahal DS, Le GBB. Production of cellulase in
solid-state fermentation with Trichoderma reesei MCG80 on
wheat straw. Appl Biochem Biotechnol 1996;57/58:433–42.
[5] Mandels M, Medeiros JE, Andreotti RE, Bissett FH. Enzymatic
hydrolysis of cellulose: evaluation of cellulase culture filtrates
under use condition. Biotechnol Bioeng 1981;23:2009 – 26.
[6] Ghose TK. Measurement of cellulase activities. Pure Appl Chem
1987;59(2):257– 68.