Biochemical Engineering Journal 40 (2008) 399–407

Culture-based strategies to enhance cellulase enzyme production from

Trichoderma reesei RUT-C30 in bioreactor culture conditions
Aftab Ahamed, Patrick Vermette ∗
Laboratoire de Bioingénierie et de Biophysique de l’Université de Sherbrooke, Department of Chemical Engineering,
Université de Sherbrooke, 2500, blvd de l’Université, Sherbrooke, Québec, Canada, J1K 2R1
Received 28 February 2007; received in revised form 15 November 2007; accepted 17 November 2007

Abstract
Filamentous fungi Trichoderma reesei are considered to be one of the most efficient hyper producers of cellulase that is used in industry.
Cellulase production by T. reesei was carried out using high concentration of cellulose to substitute glucose with the aim to improve cellulase
production while trying to reduce production costs. The experiments were conducted separately as fed batch growth with T. reesei cultured using
four media in a 7 L stirred tank bioreactor. A mixture of lactose and lactobionic acid was added into the bioreactor as cellulase inducers. The use
of a cellulose–yeast extract culture medium yielded the highest enzyme and cell production with a volumetric enzyme activity of 69.8 U L−1 h−1 , a
filter paper activity of 5.02 U mL−1 , a CMCase activity of 4.2 U mL−1 , and a fungal biomass of 14.7 g L−1 . The biomass concentration as a function
of time was constant with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second
slower growth phase due to hydrolysis of cellulose, which follow cellulase concentration augmentation. The costs to produce 1 L of production
medium in laboratory-scale experiments were calculated to compare the tested media.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Cellulose; Lactose; Medium composition; Trichoderma reesei; Cellulase enzyme

1. Introduction
Cellulase produced by filamentous fungi, Trichoderma ree-
sei, is the most efficient enzyme system for the complete
hydrolysis of cellulosic substrates into its monomeric glucose
component, which is a fermentable sugar. Cellulase is an impor-
tant commercial enzyme, widely used in food, animal feed,
textile, pulp and paper, grain alcohol fermentation, starch pro-
cessing, pharmaceutical, malting and brewing industries [1–3].
The extracellular cellulolytic system of T. reesei is composed
of 60–80% cellobiohydrolases or exogluconases, 20–36% of
endogluconases and 1% of ␤-glucosidases, which all act syn-
ergistically in the conversion of cellulose into glucose [4–6].
The synergistic reaction occurs as a result of sequential, cooper-
ative action between the three enzyme components in a complex
in which the product of one enzyme reaction becomes the sub-
strate for another [7]. Strictly speaking, ␤-glucosidase is not
considered as a cellulase because this enzyme does not act on
water insoluble cellulose whereas it shares a common feature
∗ Corresponding author. Tel.: +1 819 821 8000x62826; fax: +1 819 821 7955.
E-mail address: Patrick.Vermette@USherbrooke.ca (P. Vermette).
with the endo and exoglucanases, namely the specificity towards
␤-1-4-glucosidase bonds [8].
Despite the efforts of many laboratories, no commercially
efficient enzyme complex has been produced. The high cost of
enzyme production limits the industrial use of the enzymes in
the production of soluble sugars [9]. Zaldivar et al. [5] have
clearly indicated that the T. reesei cellulase system is deficient
in cellobiase, causing the accumulation of the disaccharide cel-
lobiose, which produces repression and end product inhibition of
the enzyme, both of which limit enzyme synthesis and activity.
In the near future one of the important potential applications
of cellulases and ␤-glucosidases will be the production of fuel
ethanol from lignocellulosic biomass [10,11], which is a good
substitute for gasoline in internal combustion engines. The most
promising technology for conversion of lignocellulosic biomass
to fuel ethanol is based on the enzymatic breakdown of cellulose
using cellulase enzyme [12,13].
The cellulase is an inducible enzyme system in which several
carbon sources have been tested to find the best inducer [14–16].
Cellulose itself has been recognized as one of the best inducer
for the complete cellulase complex and the other most impor-
tant inducers include saphorose and lactose [15,17,18]. Ryu and
Mandels [7] have stated that cellulose, cellobiose and lactose

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2007.11.030
400 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407
are effective inducers only at high concentrations. Muthuve-
layudham et al. [19] have demonstrated that the biosynthesis of
cellulase in T. reesei QM9414 increased by using a mixture of
cellulose and lactose in the culture medium. Similarly, a mixture
of lactose (0.5%) and lactobionic acid (0.5%) has been proved
to be a good inducer for cellulase production in T. reesei M-
7 [20]. Some studies indicated that the cellulase biosynthesis is
repressed by a glucose catabolite [21], when glucose is pulse-fed
to the culture in which cellulase biosynthesis is in progress, until
glucose is exhausted or its residual concentration falls below a
critical level of 0.1 mg mL−1 [22]. Recent studies revealed that
industrial production of cellulase enzyme was carried out by
using Trichoderma reesei RUT-C30 catabolite-resistant mutants
[23]. However, the cellulase produced by T. reesei with cellulose
or lactose as the sole carbon source contains a more complete
and more balanced cellulase complex whereas, some other car-
bon sources like saphorose alone give a less complete array of
cellulase proteins [24]. T. reesei grows rapidly with simple sug-
ars like glucose and fructose and with rich nitrogen sources like
peptone but, more slowly with a lag phase with lactose or cel-
lulose media and ammonium salts. It can also assimilate a wide
range of carbon sources and it grows well on ammonium sul-
fate, ammonium phosphate and ammonia but, it is unable to use
nitrate [7].
Trichoderma reesei RUT-C30 is known to be one of the best
hyper producing cellulolytic fungi. Several factors are known to
influence enzyme production and these include: concentration
and quality of the carbon source, genetically improved mutant
strains, growth conditions, aeration, temperature and pH, only to
name a few [15,25,26]. Cellulose is a linear polymer of glucose
units, which can be hydrolyzed by the action of ␤-glucosidases,
cellobiohydrolases and endogluconases. Various substrates are
able to induce secreted enzymes suited to degrade very precisely
particular combination of polysaccharides and chemical bonds
found in the carbon source. The high molecular weight substrates
are not able to enter the cell and therefore, cannot themselves,
generate such a precise response. It has been suggested that
the regulatory systems respond to characteristic low molecu-
lar weight molecules liberated from a given substrate through
the action of small amount of constitutively secreted enzymes.
T. reesei does not normally produce cellulases when grown in
media containing glucose, except in some mutant strains which
carries a cre1 gene, that normally conveys glucose repression
[27].
The aim of the present study was to improve cellulase pro-
duction by T. reesei RUT-C30 while trying to reduce production
costs by using different culture media with cellulose as a main
substrate and lactose as a fed batch to substitute glucose. The
effect of medium composition in T. reesei RUT-C30 cultures on
cell growth and enzyme production was investigated.
2. Materials and methods
2.1. Strain and spore counting
Trichoderma reesei RUT-C30 strain was a gift from IOGEN
Corporation (Ottawa, Canada). The dry powder spores were
suspended in sterile 20% (v/v) glycerol and the suspension inoc-
ulated aseptically on potato dextrose agar (PDA) Petri plates
(Difco Laboratories, USA). The PDA plates were incubated
at 30 ◦ C for 7 days (until good sporulation occurred) and then
stored at 4 ◦ C. The spores were harvested by washing the Petri
plate with 10 mL of sterile water containing 1% Triton X-100
(Fischer Scientific, Orangeburg, NJ). The spore concentrates
were transferred into 1-mL vial containing 20% (v/v) glycerol
and kept in −80 ◦ C for further use.
The spore concentration in aqueous-1% Triton X-100 sus-
pensions was determined using a hemocytometer with a
phase-contrast microscope.
2.2. Pre-culture media
T. reesei spores were cultured using the following four media.
2.2.1. Cellulose–yeast extract (modified Mohagheghi et al.
[28] culture medium)
10 g L−1 cellulose (Sigma–Aldrich, St. Louis, MO, USA),
10 g L−1 yeast extract (Fischer Biotech, Fair Lawn, NJ, USA),
10 g L−1 glucose (Fischer Biotech), 1.4 g L−1 (NH4 )2 SO4 ,
2 g L−1 KH2 PO4 , 0.4 g L−1 CaCl2 ·2H2 O, 0.3 g L−1
MgSO4 ·7H2 O, 0.005 g L−1 FeSO4 ·7H2 O, 0.0037 g L−1
CoCl2 ·6H2 O, 0.0016 g L−1 MnSO4 ·H2 O, 0.0014 g L−1
ZnSO4 ·7H2 O.
2.2.2. Corn steep–glucose
10 g L−1 glucose, 5.1 g L−1 corn steep solids
(Sigma–Aldrich), 1.4 g L−1 (NH4 )2 SO4 , 2 g L−1 KH2 PO4 ,
0.5 g L−1 CaCl2 ·2H2 O, 0.3 g L−1 MgSO4 ·7H2 O, 0.005 g L−1
FeSO4 ·7H2 O, 0.0016 g L−1 MnSO4 ·H2 O, 0.0014 g L−1
ZnSO4 ·7H2 O.
2.2.3. Cellulose–yeast extract–peptone (modified Mandels
culture medium [15])
7.5 g L−1 cellulose, 0.3 g L−1 yeast extract, 0.8 g L−1 pro-
teose peptone (Sigma–Aldrich), 3 g L−1 urea (Sigma–Aldrich),
1.4 g L−1 (NH4 )2 SO4 , 2 g L−1 KH2 PO4 , 0.3 g L−1
CaCl2 ·2H2 O, 0.3 g L −1 MgSO4 ·7H2 O, 0.005 g L−1
−1
FeSO4 ·7H2 O, 0.02 g L CoCl2 ·6H2 O, 0.0016 g L−1
−1
MnSO4 ·H2 O, 0.0014 g L ZnSO4 ·7H2 O.
2.2.4. Cellulose–yeast nitrogen base–CMC (modified
Ahamed et al. [29] culture medium)
10 g L−1 glucose, 6.7 g L−1 yeast nitrogen base (YNB)
(Sigma–Aldrich), 4 g L−1 KH2 PO4 , 20 g L−1 carboxymethyl-
cellulose (sodium salt, average MW, Acros Organics, NJ, USA).
2.3. Production media
The compositions of production media were the same as
those of the corresponding pre-culture media, except that cel-
lulose concentration was adjusted to 50 g L−1 and 10 g L−1
in cellulose–yeast extract and cellulose–yeast extract–peptone
media, respectively. On the other hand, Tween 80 was added
 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407
only in the cellulose–yeast extract production medium. Glu-
cose was not added in cellulose–yeast extract, cellulose–yeast
extract–peptone and cellulose–yeast nitrogen base–CMC pro-
duction media. Cellulose–yeast extract production medium
contains higher concentration of cellulose. Corn steep–glucose
production medium is generally used in industry as sustain-
able natural resources. Corn steep liquor is a plant juice
often low in carbohydrates and considered as a by-product
rich in proteins (47%) and suitable as a replacement for
yeast extract in fermentation media. In order to obtain high
yields in enzyme production, a carbohydrate source is needed.
Cellulose–yeast extract–peptone medium has been used in the
fermentation industry for over three decades. The source of
nitrogen in this medium was peptone, urea, yeast extract, and
ammonium sulphate. Cellulose–yeast nitrogen base–CMC pro-
duction medium is basically constituted with soluble form
of cellulose as a main substrate along with preferential use
of inorganic or non-peptide nitrogen rather than peptides
containing nitrogen, which inhibits the majority of protease
activity and maintains filamentous morphology [29]. Yeast
nitrogen base is composed of non-peptide nitrogen and min-
erals.
The 7-L stirred tank bioreactor was pre-filled with 4 L of
production medium. In order to yield final lactose and lacto-
bionic acid concentrations of 15% and 0.1%, respectively, all the
four production media were first autoclaved and supplemented
with 3 L of a mixture of 350 g L−1 of lactose (Lynn Proteins,
Granton WI) and 2.3 g L−1 of lactobionic acid (Sigma–Aldrich).
This mixture was injected into the bioreactor at a flow rate of
5.6 mL min−1 following the first 48 h of fermentation. One liter
per day of this mixture was fed for 3 days into the bioreactor (con-
taining 4 L of production medium) at three regular intervals/day;
2 h of gap between each interval followed by an approximately
16-h interval until the next day.
2.4. Inoculum in shake flask culture
Concentrated aqueous spore inocula (1.8 × 108 per mL), kept
at −80 ◦ C in 20% (v/v) glycerol, were used to inoculate 750-mL
Erlenmeyer flasks containing 200 mL of pre-culture medium and
incubated at 30 ◦ C on a rotary shaker (175 rpm). The initial pH
value was approximately 5 and not controlled during the pre-
cultures. After 72 h of growth, the production media were made
from the pre-culture media, which corresponded to 5% (v/v) of
the total production media volume.
2.5. Stirred tank bioreactor cultures
The cellulolytic enzyme production was carried out in fed
batch culture conditions in a 7-L stirred tank bioreactor (New
Brunswick, BioFlo 110 Autoclavable Fermentor & Bioreactor)
with an operating volume of 4 L and equipped with an automatic
monitoring system and controlling facilities for agitation, pH,
aeration, temperature, antifoam, and lactose as fed batch. Tem-
perature was maintained at 30 ◦ C and the agitation at 250 rpm.
The dissolved oxygen (DO) concentration was monitored as a
function of time and the aeration rate was adjusted to 5 LPM.
401
With these levels of agitation and aeration and with the use of
a fortified mixture of lactose and lactobionic acid as specific
inducers, the dissolved oxygen did not drop below 20% of air
saturation in all cultures. The pH was set to 4.8 ± 0.2 and there-
after it was controlled by the automatic addition of 2 M HCl or
2 M NaOH. The foam was controlled by automatic addition of
silicone antifoaming agent (cat. # 10794, Sigma–Aldrich). The
total culture period was 6 days.
2.6. Determination of cellulose content
Cellulose content of cultures was determined by a shortened
procedure of the method of Updegraff [30]. Ten (10) milliliters
of the total culture was centrifuged (3000 × g for 20 min) and the
supernatant was carefully removed with a Pasteur pipette [31].
The pellets were suspended in acetic acid–nitric acid reagent
(3 mL: 150 mL of 80% acetic acid with 15 mL of pure nitric
acid) and boiled for 30 min in a water bath. After cooling and
centrifuging (3000 × g for 20 min), the pellets were washed with
distilled water (10 mL), and the residual cellulose was dried
at 40 ◦ C under reduced pressure until constant weight. Each
measured value of residual cellulose content is an average of
six replications from each culture sample. The average error
percentage associated with the measurement of residual cellu-
lose varied among the tested media. The averaged percentage
errors associated with cellulose–yeast extract, cellulose–yeast
extract–peptone and cellulose–yeast nitrogen base–CMC media
were 5.7%, 17.9%, and 10.2%, respectively. The average error
percentage associated with the pure cellulose content measure-
ment over also six replications of pure cellulose was 3.2%. The
relatively large error percentage obtained with cellulose–yeast
extract–peptone and cellulose–yeast nitrogen base–CMC media
can be related to an incomplete removal of the solid fungal
biomass.
2.7. Determination of dry biomass
The dry fungal biomass was determined by measuring the
solid dry weight [31]. By this method, the mycelial weight was
calculated from the difference between the total dry weight of
the solids (comprising mycelium and residual cellulose) and
that of the residual cellulose (Section 2.6). The dry weight of
the solids was determined by centrifuging the culture (20 mL;
9600 × g for 20 min), washing the pellets three times with water
(10 mL), and drying at 40 ◦ C under reduced pressure until con-
stant weight.
2.8. Glucose and protein content and viscosity
measurements
Glucose was measured in culture supernatants as reducing
sugars using dinitrosalicylic acid and glucose as standard [32].
The protein concentration was assayed with the Quick Start
Bradford Protein Assay (BioRad Protein assay kit) with BSA
as standard [33]. Viscosity was measured by using a Brookfield
DV-III Ultra programmable Rheometer (Brookfield Engineer-
ing Laboratories, Inc. II Commerce Blvd, Middleboro, MA
402 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407

02346-1031 USA) using a spindle-62 operated at a speed of
250 rpm and 30 ◦ C. Given the non-Newtonian behavior of the
fermentation broths and their heterogeneous composition due
to the suspended fungi, the measured viscosity should only be
used for comparison purposes between the different fermenta-
tions.
2.9. Determination of enzyme activities
The cellulase activity was measured using the filter paper
activity (FPA) assay, expressed in filter paper units (FPU)
according to the method of Ghose [34]. This method mea-
sures the release of reducing sugar produced in 60 min from
a mixture of enzyme solution (0.5 mL) and of citrate buffer
(0.05 M, pH 4.8, 1 mL) in the presence of 50 mg Whatman
No. 1 filter paper (1 × 6 cm strip) and incubated at 50 ◦ C. The
released sugars were analyzed by dinitrosalicylic acid method.
One unit of enzyme activity was defined as the amount of
enzyme releasing 1 ␮mol of reducing sugars in 1 min. All
samples were analyzed in triplicate and mean values were cal-
culated.
The volumetric enzyme productivity was based on the
amount of FPU L−1 h−1 . The specific enzyme productivity was
based on volumetric enzyme production divided by dry fun-
gal biomass (g L−1 ) and expressed as FPU g cell−1 h−1 . The
amount of cellulose used by T. reesei was calculated by sub-
tracting the concentration of residual cellulose with that of the
initial cellulose.
The costs to produce 1 L of production media at the laboratory
scale were calculated based on Canadian dollars (as on August
2006) and included only chemicals, as it is sufficient to compare
the culture media.
2.10. Experimentation and analysis
All the values presented in graphs and the table are the means
of three replications. For clarity purposes, standard deviations
have not been added in figures. Data were statistically analyzed
by using the analysis of variance (ANOVA) and these analyses
are presented in Table 1. Statistical tests demonstrated that the
data were highly significant (p < 0.01).
3. Results
Four medium compositions were used to study the produc-
tion level of cellulase enzyme in T. reesei RUT-C30 strain,
during fed batch cultures in a 7 L stirred tank bioreactor. The
maximum cellulolytic enzyme production parameters were sta-
tistically analyzed and listed in Table 1. The process parameters,
fungi growth and cellulase production in T. reesei cultures as a
function of time (6 days) are shown in Figs. 1–3.
In the present study, T. reesei strain grew very well
in cellulose–yeast extract medium with lactose as an
inducer and produced a maximum filter paper activ-
ity of 5.0 U mL−1 , which was 2–3 times significantly
higher (p < 0.01) than those obtained with the other media;
corn steep–glucose (1.4 U mL−1 ), cellulose–yeast extract–
peptone (2.3 U mL−1 ), and cellulose–yeast nitrogen base–CMC
(1.4 U mL−1 ) (Fig. 1A). Similarly, the volumetric enzyme pro-
ductivity in cellulose–yeast extract medium was approximately
2–3 times higher and the specific enzyme productivity was
approximately 1-fold higher than those obtained with corn
steep–glucose medium (3.4 U g cell−1 h−1 ) (Table 1).
The specific filter paper and carboxymethyl cellulase activ-
ities were significantly (p < 0.05) higher in cellulose–yeast
extract and cellulose–yeast extract–peptone media when com-
pared to those of the other two media (Fig. 1B and C). Fig. 1D
reveals the complete depletion of the initial glucose from the
corn steep–glucose medium. Lactose was supplemented after
48 h as an inducer, which was associated with an increase level
of reducing sugars as the culture period increased for all the
four tested media. On the contrary, in cellulose–yeast extract
medium the reducing sugar level was slightly lower than those
of the other three tested media, though a significant increase in
enzyme activity (FPU mL−1 ) was noticed.
Consumptions of cellulose as a function of time in T. ree-
sei strain grown in the four media are depicted in Fig. 2.
Cellulose consumption was maximal in T. reesei grown using

Table 1
The extent of cellulolytic enzyme production in T. reesei RUT-C30 strain grown in different culture media: cellulose–yeast extract (A); corn steep–glucose (B);
cellulose–yeast extract–peptone (C); and cellulose–yeast nitrogen base–CMC (D) with lactose fed at regular intervals in a 7 L New Brunswick stirred tank bioreactor
maintained at 30 ◦ C, an agitation of 250 rpm, and culture pH of 4.8
Parameters Production media Critical difference

A B C D p < 0.05 p < 0.01

Filter paper activity (U mL−1 ) 5.02a ± 1.5 1.4b ± 0.3 2.3b ± 0.6 1.4b ± 0.3 1.12 1.58
Volumetric enzyme productivity (U L−1 h−1 ) 69.8a ± 20.5 19.9b ± 3.6 31.2b ± 8.3 18.9b ± 4.0 15.68 22.13
Specific enzyme productivity (U g cell−1 h−1 ) 7.6a ± 2.0 3.4b ± 0.6 7.6a,c ± 2.0 5.7a,c ± 2.2 2.28
Specific FP activity (U mg cell−1 ) 0.6a ± 0.1 0.2b ± 0.04 0.6a,c ± 0.1 0.4d ± 0.2 0.17
Carboxymethyl cellulase activity (U mL−1 ) 4.2a ± 0.6 2.0b ± 0.3 3.8a,c ± 0.9 2.1b ± 0.1 0.7 0.99
Amount of glucose produced (g L−1 ) 54.3a ± 15.9 15.5b ± 2.8 24.3b ± 6.5 14.7b ± 3.1 12.17 17.18
Cellulose utilized (%) 99.0a ± 0.8 0.0b ± 0.0 37.3c ± 3.3 74.4d ± 2.7 2.92 4.12
Dry biomass (g L−1 ) 14.7a ± 1.5 9.2b ± 1.4 4.5c ± 0.4 7.3d ± 0.5 1.57 2.22
Soluble proteins (mg mL−1 ) 1.0a ± 0.2 0.7b ± 0.05 0.6c ± 0.03 0.6c ± 0.1 0.09 0.12
a The data were compared by ANOVA test.
b Mean values ± standard deviations.
c Means in a row followed by different letters are significant at p < 0.05.
 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407 403

Fig. 1. Extracellular cellulolytic enzyme production as a function of time in T. reesei RUT-C30 strain grown in cellulose–yeast extract (♦); corn steep–glucose ();
cellulose–yeast extract–peptone (); and cellulose–yeast nitrogen base–CMC () culture media with lactose fed at regular intervals in a 7 L New Brunswick stirred
tank bioreactor maintained at 30 ◦ C, an agitation of 250 rpm, and culture pH of 4.8. (A) Filter paper activity (U mL−1 ), (B) specific filter paper activity (U mg cell−1 ),
(C) carboxymethyl cellulase activity (U mL−1 ), and (D) concentration of reducing sugars (g L−1 ) in culture supernatant.

the cellulose–yeast extract medium (Fig. 2B), which was also
evident from Fig. 2A in which a drastic decrease in resid-
ual cellulose content was noticed at 48 h. Direct determination
of the cell dry weight was a problem because of the pres-
ence of insoluble substrate, such as cellulose. Hence, it is
important to know the residual cellulose content to estimate
the mycelium biomass from the bioreactor sample. The dry
biomass was significantly (p < 0.01) higher and reached a
maximum of 14.7 g L−1 in cellulose–yeast extract medium
when compared with the other media; corn steep–glucose
(9.2 g L−1 ), cellulose–yeast extract–peptone (4.5 g L−1 ), and
cellulose–yeast nitrogen base–CMC (7.3 g L−1 ) (Fig. 2C). The

Fig. 2. Growth parameters represented by consumption and use of cellulose as a function of time in T. reesei RUT-C30 strain grown in cellulose–yeast extract (♦);
corn steep–glucose (); cellulose–yeast extract–peptone (); and cellulose–yeast nitrogen base–CMC () culture media with lactose fed at regular intervals in a
7 L New Brunswick stirred tank bioreactor maintained at 30 ◦ C, and agitation of 250 rpm, and culture pH of 4.8. (A) Residual cellulose (g L−1 ), (B) percentage of
cellulose consumed, (C) dry biomass accumulation (g L−1 ), and (D) soluble proteins (mg mL−1 ) in culture supernatant.
404 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407

Fig. 3. Patterns of process parameters as a function of time and cost of culture media for T. reesei RUT-C30 strain grown in cellulose–yeast extract (♦); corn
steep–glucose (); cellulose–yeast extract–peptone (); and cellulose–yeast nitrogen base–CMC () culture media with lactose fed at regular intervals in a 7 L
New Brunswick stirred tank bioreactor maintained at 30 ◦ C, an agitation of 250 rpm and culture pH of 4.8. (A) Viscosity measurement (cP), (B) cultivation pH, (C)
percentage of air saturation, and (D) costs in Canadian dollars to produce 1 L of production media.

total protein content linearly increased with increasing fermen-
tation time in the four tested media but, it was significantly
higher in cellulose–yeast extract medium reaching a maximum
of 1.0 mg mL−1 (Fig. 2D).
The initial viscosity in cellulose–yeast nitrogen base–CMC
medium was 20 cP and gradually decreased to 7 cP over a period
of fermentation (Fig. 3A). This trend was not observed in corn
steep–glucose and cellulose–yeast extract–peptone media for
which the viscosities were initially 2.9 cP and 5 cP, respectively,
and gradually increased to 17.2 cP and 13.1 cP, respectively. The
viscosity of cellulose–yeast extract medium was found to be ini-
tially 5.7 cP and was not stable over time; it reached a maximum
of 29.8 cP at 48 h. The pH was maintained between 4.8 ± 0.2 by
automatic addition of acid and base in all cultures (Fig. 3B). The
DO was maintained to 20% of air saturation (Fig. 3C) in all cul-
tures, though the concentration of cell mass and the amount of
cellulose content were higher in cellulose–yeast extract medium.
With the selected levels of agitation and aeration and with the
use of a fortified mixture of lactose and lactobionic acid as spe-
cific inducers, it was possible to maintain the DO to the desired
level.
Costs for making 1 L of each culture medium were different
from each other (Fig. 3D). The cost estimates are based only on
chemicals used to prepare production media in laboratory-scale
experiments.
4. Discussion
Many microorganisms have been classified as cellulolytic
but, only few possess a complete cellulase complex capable of
efficient depolymerization of crystalline cellulose. Trichoderma
reesei RUT-C30 is a common soil fungus of the Deuteromycete
family which produces cellulolytic enzymes. To achieve maxi-
mum cellulase enzyme, researchers have studied various process
parameters during fermentations that use T. reesei. The influence
of inoculum size and media composition on T. reesei RUT-C30
morphology and enzyme activity were studied [35,36]. Cellu-
lose is abundantly available on this planet and it could be used
to a better extent by using enzymatic action of microorganisms
to convert it into soluble sugars [11]. Cellulose consists of long
insoluble chains of covalently bonded glucose molecules, which
are too large to be transported through cell walls [15]. How-
ever, through the use of microorganisms such as, T. reesei cells,
enzymes known as cellulases are secreted and these enzymes
hydrolyze or depolymerise the cellulose into its monomeric glu-
cose components, which can be readily transported through cell
wall and subsequently metabolized. The cellulase production is
the most expensive step during ethanol production from cellu-
losic biomass, in that it accounted for approximately 40% of
the total cost [9]. In the present study, different concentrations
of soluble and insoluble cellulose were used to know the effi-
ciency of cellulase enzyme production by T. reesei by trying to
minimize production cost in order to enhance the commercial
viability of cellulase production technology.
It has been suggested that the association of higher concen-
tration (50 g L−1 ) of cellulose and lactose (150 g L−1 , in fed
batch) in enzyme production may led to technical and economi-
cal benefits as a result of sequential cooperative action between
the three enzyme components in the complex [37]. The cel-
lulases of T. reesei are inducible enzymes and the maximum
yields are being obtained when cellulose is used as a substrate
but, its commercial success still depends on further improve-
 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407
ments [38]. In the present study, the lower cellulase activity
(1.4 FPU mL−1 ) was obtained when glucose, corn steep and low
concentrations of soluble and insoluble cellulose were added to
the production media, suggesting some synergetic effect as the
enzyme activity was lower than that obtained with the media
using higher concentrations of cellulose. This may be the result
of the produced enzymes being adsorbed on the solid cellulose
surface [39] at the same rate than that of cellulase synthe-
sis. On the contrary, Domingues et al. [35] have reported that
higher cellulase enzyme activity (2.8 FPU mL−1 ) was obtained
with glucose (30 g L−1 ) and lactose (15 g L−1 ) in the produc-
tion medium. It is evident that the enzyme activity (obtained
from the filter paper activity assay) begins to increase in the
cellulose–yeast extract medium at 24 h and reached maximum
at 120 h when the substrate consumption was almost negligible
but, it is not true in the other tested medium compositions in
which the activity (0.7 U mL−1 ) begins at 24 h and maintained
more or less the same activity throughout the fermentation. At
144 h, the cellulase activity slightly decreased in all the tested
media and this may be due to protease formation towards the
end. It is not clear as to why cellulase synthesis was initiated
after most of the cellulose and lactose were consumed. It could
be hypothesized that the biosynthesis of cellulase in the latter
phase takes place due to the availability of nutrients by endoge-
nous metabolism. Also, secretion of cellulases can be delayed
by decreased permeability of the cell membrane, which in turn
leads to lower enzyme synthesis [40]. Lactose is a disaccha-
ride that consists of ␤-d-galactose and ␤-d-glucose molecules
bonded through a ␤1–4 glycosidic linkage. Lactose is the only
soluble and economically viable carbon source for the produc-
tion of cellulases or heterologous proteins regulated by cellulase
expression signals in T. reesei. Also, lactose is a very good
inducer of cellulase enzymes in T. reesei. In cellulose–yeast
extract medium, the concentration of cellulose was 50 g L−1
compared to 10 g L−1 in cellulose–yeast extract–peptone and
20 g L−1 in cellulose–yeast nitrogen base–CMC (soluble form
of cellulose). No cellulose was added in the corn steep–glucose
medium. The lower concentration of reducing sugars in culture
supernatant of the cellulose–yeast extract medium, compared to
those found in the other media, can be explained by a slower
conversion of cellulose into its monomer whereas, the lactose
seems to be metabolized faster and might be quickly consumed
by T. reesei cells. The rapid lactose uptake can enhance the
release of more enzymes in the culture medium, which will fur-
ther contribute to hydrolyze cellulose for subsequent growth. In
other words, the sugars liberated from the cellulose as a result of
enzymatic attack were rapidly used by the growing mycelia. Fur-
thermore, in the other three tested media, the enzymes quickly
convert the available small amount of cellulose along with a
faster lactose metabolism into monomers hence the reducing
sugar level was slightly higher than that of the cellulose–yeast
extract medium, resulting in lower enzyme activity. Wen et al.
[41] have reported similar results showing that reducing sugar
concentration increased as the substrate concentration increased.
Mohagheghi et al. [28] have reported the highest enzyme
productivity of 91.7 IFPU L−1 h−1 and a total productivity of
51 IFPU L−1 h−1 , which were achieved with cellulose concen-
405
tration (as the sole carbon source) of 50 g L−1 [42]. The same
general trend was observed in another study [11]. Wen et al. [43]
have reported a CMCase activity of 12.22 IU mL−1 and cellu-
lase activity of 1.74 IU mL−1 by Trichoderma reesei grown in
agricultural wastes. On the contrary, in the present investiga-
tion, a lower CMCase activity (4.2 U mL−1 ) was obtained and
this could be directly related to the substrate concentration of
the culture media. Similarly, the total volumetric enzyme pro-
ductivity (69.8 U L−1 h−1 ) by T. reesei strain reported by Wen
et al. [43] was higher than those reported elsewhere [11,28,42].
However, in the present study the maximum filter paper activ-
ity and soluble proteins were 5.0 U mL−1 and 1.0 mg mL−1 ,
respectively, in the cellulose–yeast extract medium. Similar
observations of enzyme activity (4.5 FPU mL−1 ) and protein
concentration (0.7 mg mL−1 ) were obtained with T. reesei RUT-
C30 grown using a mixture of 15 g L−1 lactose and 40 g L−1
glucose as substrates in the production medium [35]. Our find-
ings are closely supported by Dien et al., [44] who reported a
maximum cellulase activity of 8.3 U mL−1 and a protein concen-
tration of 1.42 mg mL−1 , when T. reesei RUT-C30 was grown in
20 g L−1 of corn steep and 40 g L−1 of de-starched corn fiber. In
another study, the cellulase activity was 1.9 FPU mL−1 and the
protein concentration was 1.8 g L−1 when T. reesei was grown
in 45 g L−1 of Avicel PH101 [45]. In the present investiga-
tion, maximum cellulase activity (5.02 FPU mL−1 ) and enzyme
productivity (69.8 U L−1 h−1 ) were observed at 120 h, corre-
sponding to a substrate (cellulose) concentration of 50 g L−1 .
However, in other studies, the maximum cellulase activity
was 5.25 IU mL−1 and the maximum enzyme productivity was
31.3 IU L−1 h−1 at 168 h, corresponding to a substrate (corn
cob residues) concentration of 40 g L−1 [46]. Similarly, the
enzyme production was 15.8 FPU L−1 h−1 in T. reesei when
10 g L−1 of cellulose substrate was used [13]. Given the above,
our study reveals that the cellulose–yeast extract medium can
achieve enhanced cellulase activity compared to cellulase activ-
ities reported in the literature for wild type T. reesei.
Cellulose–yeast extract is the only medium in which Tween
80 was added into the production medium during T. reesei cul-
tures. Reese and Maguire [40] have suggested that the addition
of Tween 80 in the medium improved the cellulase yield in Tri-
choderma strains. The mechanism behind this increased yield
may be related to the increased permeability of the cell mem-
brane, allowing more rapid secretion of enzymes, which in turn
leads to higher enzyme synthesis. Also, the addition of yeast
in this medium can induce enzyme production, as it is a good
source of naturally occurring vitamin B complex. It has been pre-
viously reported that yeast extracts stimulate both cell growth
and cellulase production in T. reesei [7]. During fermentation
in bioreactor culture conditions, the reaction formed foam on
the broth surface and into the head space of the fermentor. This
foaming occurred following approximately 20 h of fermenta-
tion. A silicone antifoam agent was used during the bioreactor
cultures, but in cultures involving the cellulose–yeast extract
medium, it was hardly used because almost no foam was formed.
This may be due to lower agitation used in our study compared
to other investigations and to the use of Tween 80, which sup-
presses the foam during fermentation. In general, the bubbling
406 A. Ahamed, P. Vermette / Biochemical Engineering Journal 40 (2008) 399–407
of poorly soluble gases into fermentation broths, either used for
the replenishment of dissolved oxygen or for removal of excess
of CO2 , can lead to severe foaming problems and cause the
reduction of enzyme activity.
The reduction in specific activity (U mg cell−1 ) in corn
steep–glucose medium is possibly due to post-translational inac-
tivation of cellulases by glucose and its derivatives and/or partial
inhibition by the free glucose resulting from the hydrolysis
of lactose. Glucose concentration (i.e., between 0.4 mg mL−1
and 0.6 mg mL−1 ) present in the medium is sufficient for
such catabolite inhibition [35]. Although the strain is capa-
ble to produce cellulases using soluble lactose, it gives only
a non-proportional increase in volumetric enzyme productiv-
ity at higher lactose concentration. Duff and Murray [10] have
reported that most of the T. reesei mutants produce cellulases
with a specific filter paper activity between 0.5 FPU per mg and
1 FPU per mg of proteins and the highest reported activity was
3.6 FPU per mg of proteins using VTT-D-79125 strain.
One of the other important reason to explain the low enzyme
yields is catabolite repression of enzyme biosynthesis due to
cellobiose. In fact, T. reesei are deficient in ␤-glucosidase (cel-
lobiose), therefore cellobioses accumulate and repress enzyme
biosynthesis [5]. Muthuvelayudham and Viruthagiri [6] reported
that the biosynthesis of cellulases in T. reesei QM9414, 97.177
and Tm3 was found to be very high in media with cellulose
and lactose as sole carbon source. On the other hand, enzyme
activity was found to be very low when glucose was used as
carbon source because of inhibition whereas the use of media
containing multiple substrates resulted in an increase enzyme
production [6]. Similarly, lactose and lactobionic acid (1:1
ratio) were proved to be good inducers for cellulase produc-
tion by T. reesei [20]. Vaheri [47] has demonstrated in culture
filtrates of T. reesei, the presence of cello bionic acid and
cellobiono-1, and 5-lactone. These compounds can be formed
following oxidation of end chains of cellulose by cellobiohy-
drolases action. Moreover, the Tween 80 helps to increase the
permeability of cell membrane allowing more rapid secretion of
enzymes.
It appears that fungal cell growth proceeded in two stages:
during the first two days, dry cell mass showed a rapid rise that
was then followed by a slower growth. A possible reason for
this biphasic cell growth is that the presence of the initial yeast
extract in the medium was quickly converted to cell mass and
accounted for the rapid growth during the first 2 days of culture.
The subsequent slower growth phase could then be explained by
the hydrolysis and consumption of cellulose [48]. At the highest
cell mass concentration, cellulose appeared to be exhausted and
heavy sporulation was initiated. Furthermore, during the first
growth phase, the oxygen demand was high and the concen-
tration of DO was slightly lower. When growth slowed down
in the second phase, the DO increased until it reached equi-
librium. Thus, oxygen limitation occurs only in the phase of
rapid growth. The enzyme activity and fungal biomass accu-
mulated over a period of time suggesting that there is a strong
relationship between cellulase synthesis and the amount of total
biomass formation. Finding from this study tends to point out
that the larger biomass accumulation found with T. reesei cul-
tured in the cellulose–yeast extract medium could be associated
with an increased cellulase enzyme production.
As a result, the production cost of cellulose–yeast extract
medium was slightly higher but was, on the other hand, compen-
sated with the enhanced production of cellulase enzyme when
compared with the other tested media. The yield factors for
enzyme production in T. reesei with respect to different medium
compositions in stirred tank bioreactor were estimated on the
basis of specific feed rate and volumetric enzyme productivity.
5. Conclusions
The successful use of cellulosic materials as renewable
carbon sources is dependent on the development of economi-
cally feasible processes for cellulase production and enzymatic
hydrolysis of cellulosic materials to lower molecular weight
products such as hexose and pentose.
The results of fed batch culture in a stirred tank bioreac-
tor with different media compositions exhibited the economical
and technical advantages of enzyme production process using
mixtures of cellulose and lactose as carbon sources. T. ree-
sei RUT-C30 is capable of synthesizing cellulase enzyme
using higher concentrations of cellulose (50 g L−1 ) and lactose
(150 g L−1 as fed batch) and generate 2–3-fold higher enzyme
activity than the other tested media. In the present study, dif-
ferent concentrations of soluble and insoluble cellulose were
used to know the efficiency of cellulase enzyme production by
T. reesei by trying to minimize production costs to enhance the
commercial viability of cellulase production technology.
Further investigations will be required to achieve a better
understanding of how the resulting enzyme activity quan-
titatively relate to the mixture of carbon sources and the
morphological characteristics of the cultures.
Acknowledgements
This study was supported by the Université de Sherbrooke
and NSERC (Strategic projects, project # STPGP 307410-04).
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