Enzyme and Microbial Technology 50 (2012) 311–317

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Enzyme and Microbial Technology

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Promoting pellet growth of Trichoderma reesei Rut C30 by surfactants for easy
separation and enhanced cellulase production
Nicholas V. Callow, Lu-Kwang Ju ∗
Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906, United States

a r t i c l e i n f o a b s t r a c t

Article history: It is desirable to modify the normally filamentous Trichoderma reesei Rut C-30 to a pellet form, for easy
Received 30 November 2011 biomass separation from the fermentation medium containing soluble products (e.g., cellulase). It was
Received in revised form 17 February 2012 found in this study that this morphological modification could be successfully achieved by addition of the
Accepted 23 February 2012
biosurfactant rhamnolipid (at ≥0.3 g/L) and the synthetic Triton X-100 (at ≥0.1 g/L) to the fermentation
broth before the cells started to grow actively. Thirteen other surfactants tested were not as effective.
Keywords:
Furthermore, the added rhamnolipid and Triton X-100 increased the maximum cellulase activity (Fil-
Trichoderma reesei
ter Paper Units) produced in the fungal fermentation; the increase was 68 ± 7.8% for rhamnolipid and
Morphology
Cellulase
73 ± 12% for Triton X-100. At the concentrations required for pellet formation, rhamnolipid had negative
Rhamnolipid effect on the cell growth: with increasing rhamnolipid concentrations, the growth rate decreased and
Triton X-100 the lag-phase duration increased linearly. Triton X-100 caused no significant differences in growth rate
or lag phase.
© 2012 Elsevier Inc. All rights reserved.

1. Introduction filamentous morphology of T. reesei causes a significant increase in

Microorganisms have been used to produce various useful mate-
rials, including the cultivation of fungi for the purpose of food,
antibiotic and enzyme production [1,2]. One of the well-studied
fungi is Trichoderma reesei Rut C-30, a hyper-cellulase producing
strain. The Rut C-30 strain is known to produce the highest cellulase
activity at pH 4.5, while the optimal pH for xylanase production and
growth is 6.0 [3,4]. Various enzyme inducing agents have been stud-
ied, with cellulose, oligosaccharides (hydrolysis intermediates) and
sophorose providing the greatest expression potential and lactose
to a lesser extent [5–8]. The use of cocultures of Candida bombi-
cola and T. reesei to produce sophorolipids in situ, as the source of
sophorose for inducing cellulase production, has also been reported
[9].
Aside from process conditions and inducing agents, some surfac-
tants (e.g., Tween-80, Tween-60, and Tween-20) have been found to
improve the release of extracellular proteins and increase activities
of cellulase and xylanase enzymes from T. reesei and Trichoderma
viride [10–12]. It is believed that these surfactants can increase the
enzyme transport across cell membrane [11], [13–15].
While many studies have provided insight into the optimal
growth conditions, one industrially important aspect for separa-
tion operations is the mycelial biomass morphology. The naturally
∗ Corresponding author. Tel.: +1 330 972 7252; fax: +1 330 972 5856.
E-mail address: LukeJu@UAkron.edu (L.-K. Ju).
broth viscosity and the biomass becomes difficult to separate from
the liquid medium. Therefore, formation of compact mycelial pel-
lets is desirable for the purpose of easy cell separation. Many factors
can affect fungal morphology in submerged fermentation but gen-
eralization and prediction of behavior caused by these factors on
different species at different culture conditions is difficult. It has
been demonstrated that when spore suspensions are used as inoc-
ula, the inoculation conditions can affect the morphology in some
species, with generally lower inoculation of spores promoting the
formation of pellets and higher inoculation concentrations yielding
to flocs [16]. For instance, this effect was well studied for Aspergillus
niger confirming that the pellet forming tendency decreases with
increasing inoculum spore concentration [17]. Yet, regardless of
the spore concentration used for inoculation, the addition of 0.5 g/L
of Tween-80 could inhibit the formation of pellets of T. reesei Rut
C-30 [14]. The carbon sources/additives used in the medium can
also affect pellet formation. For example, various strains of A. niger
were shown to grow as free mycelia on lactose as a substrate but
the same species could form pellets when glucose was added to
the medium [18]. Similarly, the addition of sorbose with T. ree-
sei QM9414 was found to induce a clear shift from filamentous to
compact pellet form [19]. The addition of carboxypolymethylene
or carboxymethylcellulose to cultures of Aspergillus fumigatus also
promoted smaller discrete pellets over larger globular formations
[20].
Surfactants are a special group of additives that can also affect
fungal morphology. Previous work has described the role of

0141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2012.02.006
312 N.V. Callow, L.-K. Ju / Enzyme and Microbial Technology 50 (2012) 311–317
surfactants on the morphological structure of Penicillium, particu-
larly about how the added compounds could cause or inhibit pellet
formation [21–23]. It has also been reported that the addition of
Tween-40 to a Trichoderma harzianum shake flask culture might
promote pellet formation [24]. For T. reesei Rut C-30 a study has
indicated that Tween-80 might inhibit its pellet formation [14].
Although Tween-80 may inhibit pellet formation, it was shown that
a 50 mM pH 4.8 buffer of citrate or phthalate will promote T. reesei
to form compact pellets while succinate and formate buffers pro-
duces soft pellets, and an unbuffered system yields only dispersed
morphology. In all cases the pellet formation was diminished with
100 mM buffer concentration or non-optimal pH, indicating that
the concentration, chemical type and pH can all be significant
factors to pellet formation [25]. There have been no other known
studies on the surfactant effects on the morphology of T. reesei.
In this work, pellet formation of T. reesei Rut C-30 (in the
common medium containing 0.2 g/L Tween-80) is reported to be
effectively promoted by the addition of some surfactants. The
effects of these surfactants on cell growth and cellulase production
in a submerged fermentation process are also discussed.
2. Materials and methods
2.1. Surfactants
Triton X-100 (Triton), Tween-80, Tween-20 and Span-80 were obtained from
Sigma–Aldrich (St. Louis, MO, USA). A concentrated solution of rhamnolipid mixture
was purchased from EchoChem (Delia, AB, CA). Pluronic® surfactants were obtained
from BASF (Parsippany, NJ, USA).
2.2. Microorganism
T. reesei Rut-C30 (NRRL 3469) was obtained from the U.S. Department of Agri-
culture (USDA) Agricultural Research Service (ARS) Culture Collection (also known
as the NRRL Collection). The culture was maintained on slants of potato dextrose
agar (30 g/L, Sigma, P2182) at 4 ◦ C and sub-cultured every 4 weeks. Inoculum was
prepared by aseptically adding three loops of cells from a mature slant to potato
dextrose broth (24 g/L, Sigma P6685), and incubating for 3 days at room tempera-
ture on a magnetic stir plate set to approximately 300 rpm. This provided an entirely
filamentous culture, at the late exponential growth phase, for inoculation of the test
systems. All systems were inoculated at 10% (v/v). This inoculation technique pro-
vides a high concentration of actively growing hypha in an attempt to avoid pellet
formation caused when inoculating with a low concentration of spores.
2.3. Fermentation medium
Unless otherwise specified, the chemicals used were from either Fisher Scientific
(Fairlawn, NJ, USA) or Sigma–Aldrich (St. Louis, MO, USA). All cultures were grown
with a modified Mandels and Webber medium containing (g/L): (NH4 )2 SO4 1.4,
KH2 PO4 2.0, MgSO4 ·7H2 O 0.3, CaCl2 ·2H2 O 0.4, urea 0.3, proteose peptone (Remel)
1.0, Tween-80 0.2, FeSO4 ·7H2 O 0.005, MnSO4 ·H2 O 0.0016, ZnSO4 ·7H2 O 0.0014, CoCl2
0.002, and lactose 10.0 [10]. The unadjusted pH was 5.5 after autoclaving.
2.4. Fermentation setup
Test cultures were grown in 50 mL volume in 250 mL flask at 28 ◦ C in a Que
Orbital shaker (Model 4703, Parkersburg, WV, USA) at 250 rpm without pH control,
initially at 5.5, for 7 days. Triton or rhamnolipid was tested at the following concen-
trations (g/L): 0.0, 0.1, 0.3, 0.6, 0.8, 1.0 and 1.5. Cultures were followed for 7 days and
visually checked for bulk morphology. Periodic samples (2 mL) were taken asepti-
cally in a laminar flow hood (Forma Scientific Model 1839, Marietta, OH) every 24 h
to measure mycelial biomass, cellulase enzyme activity, sugar concentration and
residual surfactant concentration.
2.5. Mycelial biomass estimation
The intracellular protein concentration was measured and then converted to
the mycelial dry-weight concentration according to a previously established corre-
lation [26]. The procedure is briefly described in the following: The culture flask was
manually mixed to suspend the biomass and simultaneously a 1.0 mL sample was
collected. The sample was centrifuged at 9300 × g for 10 min to obtain a pellet. The
supernatant was collected for further processing. The pellet was re-suspended and
washed twice with de-ionized water. After each wash step, the biomass was cen-
trifuged and the water discarded. Following the last wash step, all samples were
frozen at −20 ◦ C while waiting testing. To release the intracellular proteins, an
unfrozen pellet was suspended in 1.0 mL of 0.2 M sodium hydroxide and heated
at 100 ◦ C for 20 min. Following cooling, the digested sample was centrifuged at
5900 × g for 10 min to remove cell debris; the supernatant was determined for pro-
tein concentration by using the Bio-Rad Protein Assay Kit (Bio-Rad, Kit# 500-002,
Hercules, CA, USA). The protein assay was calibrated with bovine serum albumin
solutions as standards. The calculated protein concentration was converted to equiv-
alent mycelium dry weight by a previously established correlation [26].
2.6. Sugar concentration and cellulase activity estimation
Reducing sugar concentrations were measured using a non-specific 3,5-
dinitrosalicylic acid (DNS) test method [27]. Cellulase activities, in terms of Filter
Paper Units (FPU), were estimated using the method proposed by Ghose for dilute
concentrations [26,27]. For the FPU activity determination, 100 ␮L of supernatant
was mixed in 1.4 mL of a pH 4.8 and 0.05 M sodium citrate buffer in a tall 25 mL
test tube and incubated at 50 ◦ C for 1 h in an orbital reciprocating water bath
(Boekel Grant, ORS 200) set at 50 rpm. In this test, the enzyme sample contained a
1 cm × 6 cm strip of Whatman #1 filter paper, while the blank was incubated with-
out the filter paper substrate. The reaction was stopped with the addition of 3 mL
DNS reagent and heated to 100 ◦ C for 5 min for color development. Absorbance at
595 nm was correlated to a standard glucose concentration curve.
2.7. Triton X-100 concentration measurement
Triton concentrations were determined by the reverse phase high-performance
liquid chromatography (HPLC) using a Hewlett-Packard LC1100 series HPLC (Palo
Alto, CA, USA) fitted with a G1315A diode array detector, a temperature controlled
auto-injector, and a Waters Symmetry C18 column (Milford, MA, USA) operating
at 24 ◦ C. The sample injection volume was 20 ␮L. A quaternary pump provided the
following water–acetonitrile sequence to isolate the Triton: 4 min hold at 40:60,
3 min ramp to 10:90, 2 min hold at 10:90, 6 min ramp to 40:60 and a 15 min hold
at 40:60. The flow rate of mobile phase was 0.5 mL/min. The Triton concentration
was correlated to the height of a 200-nm absorbance peak at the retention vol-
ume of 8.05 mL. Samples were prepared for analysis by diluting the supernatant
from centrifuged culture broth with equal volume of methanol and then centrifug-
ing at 10,000 × g to remove insoluble components [28]. The methanol–supernatant
mixture was stored at 4 ◦ C while in the auto-injector to prevent microbial growth.
Standards were prepared in a similar method, neglecting the final centrifugation.
2.8. Rhamnolipid concentration measurement
Rhamnolipid concentrations were determined by a modified Orinoco method
[29]. In a disposable polypropylene micro centrifuge tube, a 50 ␮L sample was mixed
with 100 ␮L of 10 mM phosphate buffer, pH 2.5, and extracted at room temperature
with 1.0 mL of ethyl acetate for 24 h in a shaker at 250 rpm (Que orbital shaker,
Model 4703, Parkersburg, WV, USA). The ethyl acetate-aqueous mixture was cen-
trifuged at 8000 rpm for 10 min to accelerate phase separation. Into a clean glass
test-tube, 0.5 mL of the ethyl acetate phase was collected and allowed to evapo-
rate. The dry rhamnolipid residue was re-dissolved in 0.85 mL of a 0.05 M sodium
bicarbonate solution added into the glass test tube. The tube and solution was then
cooled in an ice water bath before addition of 1.65 mL refrigerated anthrone reagent
(2 g/L anthrone in concentrated sulfuric acid at 4 ◦ C) [30]. Vials were sealed using
polytetrafluoroethylene lined caps and heated at 95 ◦ C for 16 min. After cooling to
room temperature, absorbance was measured at 635 nm using a spectrophotometer
(Model UV-1601, Shimadzu Corporation, Columbia, MD). Calibration standards were
obtained with pure rhamnose dissolved in the sodium bicarbonate solution and
processed by the same procedure following the addition of the anthrone reagent. A
correlation factor of 2.87 was used to convert the measured rhamnose concentration
to the equivalent rhamnolipid concentration.
3. Results
3.1. Surfactant screening
A series of surfactants were screened for their effects on the mor-
phology of T. reesei Rut C-30. In a sequence of shake flask studies,
1.0 g/L of each tested surfactant was added to the fresh medium
before inoculation. A visual observation of cell morphology was
used to estimate the response. These visual observations are sum-
marized in Table 1. At 1 g/L Brij-52, Tween-80, and a majority of
the non-ionic Pluronic surfactants did not affect the morphology.
However, Span-80 and Pluronic L31, F77, and a 50/50 mixture of F77
and L61 increased the filamentous nature of the fungus. Tween-20,
Pluronic P65 and F88 showed slight tendencies to increase floccula-
tion of the cells. Yet, these surfactants were unable to produce true
“pellets.” The sodium dodecyl sulfate (SDS) was inhibitory to the
cell growth at the 1 g/L concentration tested. However, as shown by
 N.V. Callow, L.-K. Ju / Enzyme and Microbial Technology 50 (2012) 311–317 313

Table 1
Results of surfactant screening on pellet formation of T. reesei Rut C-30. Surfactants were tested at 0.5 g/L and 1 g/L to visually assess the tendency for pellet formation.

Surfactant Effect Comments

None None No pellet formation, generally filamentous morphology

Pluronic® F77 None No apparent effects on bulk morphology
Pluronic® F68 None No apparent effects on bulk morphology
Pluronic® L61 None No apparent effects on bulk morphology
Pluronic® L35 None No apparent effects on bulk morphology
Brij 52 None No apparent effects on bulk morphology
TWEEN® 80 None No apparent effects on bulk morphology
F77/L61 Blend − Slightly lower flocculation than without surfactant, an apparently
more uniform distribution of mycelia
Pluronic® L31 − Producing a slightly more uniform mycelia distribution with less
natural flocculation
Span® 80 − Slightly lower natural agglomeration of mycelia than without
surfactant
TWEEN® 20 + Formation of some flocs when compared to the surfactant free control
Pluronic® P65 + While similar to a surfactant free control, surfactant addition showing
a tendency for flocculation
Pluronic® F88 ++ Increasing surfactant concentration causing a tendency for increased
flocculation with a slight reduction in free mycelia and larger
agglomerates
Rhamnolipid +++++++ Nearly complete smooth pellet formation at concentrations greater
than 0.3 g/L, the solution being otherwise light brown in color and free
of filamentous growth
Triton X-100 ++++++ Formation of nearly uniform ∼1 mm pellets without many free mycelia
SDS Sporadic low growth (tested at 1 g/L)

the pictures in Fig. 1, rhamnolipid and Triton were both successful
in producing a complete “pellet” growth when tested at concen-
trations greater than 0.3 g/L. At 96 h, pellets in the systems added
with 1.0 g/L surfactants had the average sizes of 1.7 ± 0.3 mm and
1.1 ± 0.3 mm, respectively, for the Triton and rhamnolipid systems.
3.2. Rhamnolipid
3.2.1. Fermentation profiles
Following the screening process, rhamnolipid additive was
demonstrated to significantly modify the morphology and produce
stable pellets. To further investigate this attribute, shake flask cul-
tures were grown at varying concentrations of rhamnolipid. For
example, the profiles for reducing sugar concentration, cell con-
centration, cellulase (FPU) activity and surfactant concentration are
shown in Fig. 2 for the system initially with 1 g/L rhamnolipid. The
rhamnolipid concentration did not remain constant during the fer-
mentation; instead, it decreased along the process time (Fig. 2).
In some experiments, the decrease was clearly more significant
during the period of active growth (data not shown).
As shown in Fig. 2, the addition of 1 g/L rhamnolipid caused a
substantially prolonged lag phase. In Fig. 3, the lag-phase duration
(estimated up to the beginning of active growth, with the accuracy
allowed by the 12 h sampling period) was plotted against the ini-
tial concentration of rhamnolipid added. The lag-phase duration
increased linearly (R2 = 0.97) with increasing rhamnolipid concen-
trations.
To further evaluate if rhamnolipid had an effect on the growth
rate beyond the lag phase, the doubling time during the period
of fastest growth was determined. Fig. 4 shows the doubling time
as a function of the initial rhamnolipid concentration. When the
initial concentration of rhamnolipid was 0.3 g/L or less, there was no
significant difference in the mean doubling times, with an ANOVA
p-value of 0.61. For concentrations of 0.6–1.0 g/L, there was also
no significant difference in the doubling times, with a p-value of
0.44. However, the ANOVA results on the entire data set gave a p-
value of 0.01, suggesting that the rhamnolipid concentration had
significant effects on the mean doubling time. As shown in Fig. 4,
the effects of rhamnolipid on the cell growth rate occurred at the
initially added concentrations between 0.3 and 0.6 g/L; in general,
the effects were to increase the doubling time, i.e., slow down the
growth. Why the growth rate did not further decrease at higher
rhamnolipid concentrations remained unknown.
3.2.2. Cellulase production
Fig. 5 indicates the maximum cellulase activities obtained in
the cultures with different initial rhamnolipid concentrations. In
otherwise the same medium, T. reesei produced higher cellulase
activities with higher concentrations of rhamnolipid added. The

Fig. 1. Morphology comparison of T. reesei Rut C-30: 5 mL samples transferred from shake flasks to Petri dishes, samples collected at 96 h from systems with (a) 1.0 g/L initial
rhamnolipid, (b) 1.0 g/L initial Triton X-100, and (c) control with no surfactant added.
314 N.V. Callow, L.-K. Ju / Enzyme and Microbial Technology 50 (2012) 311–317

Triton X 100 Rhamnolipid

25

20

Doubling Time (h)

15

10

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Surfactant Concentraon (g/L)

Fig. 4. Doubling time for growth of T. reesei Rut C-30 as a function of the initial
surfactant (rhamnolipid or Triton X-100) concentration in the medium.

enzyme activity was increased up to 68 ± 7.8%, in the culture added

with 1.5 g/L of initial rhamnolipid.
The possibility that the higher cellulase activities observed
were caused by the effects of rhamnolipid on the FPU analysis
itself was considered, since it was known that surfactants could
enhance cellulase performance by reducing the irreversible bind-
Fig. 2. Typical fermentation profiles observed for the systems with 1.0 g/L rhamno- ing of the enzymes on solid biomass [31,32]. A cellulase-containing
lipid in the initial medium, compared with those for the rhamnolipid-free control.
supernatant sample, collected by centrifugation of broth grown in
140 medium without rhamnolipid (or Triton X-100), was divided into
multiple tubes and added with different concentrations of rhamno-
120 lipid (or Triton). These samples were then subjected to FPU analysis
y = 74.24x + 5.819 (data not shown). ANOVA analysis on these enzyme activities mea-
R² = 0.97
Observed Lag Time (h)

100 sured with different rhamnolipid concentrations has a p-value of

0.10, indicating there is not a significant difference among the mean
80 enzyme activity values (FPU) at these surfactant concentrations.
Therefore, the observed increases in enzyme activity in Fig. 5 were
60
indeed due to increased extracellular enzyme production, not the
effects of surfactant on FPU analysis.
40

20 3.3. Triton X-100

0 3.3.1. Fermentation profiles

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60
Similar to rhamnolipid, Triton X-100 effectively caused T. reesei
Inial Rhamnolipid Concentraon (g/L)
Rut C-30 to form pellets. However, Triton had noticeably milder
Fig. 3. Rhamnolipid prolonged lag phase. The observed lag duration increased lin- effects than rhamnolipid on the fungal cell growth. When com-
early with increasing concentration of rhamnolipid in the initial medium (R2 = 0.97). pared to the surfactant free system, which did not have a lag-phase;
2
Maximum Cellulase Obtained (FPU/mL)

Rhamnolipid Triton X 100

1.5

0.5
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Inial Surfactant Concentraon (g/L)

Fig. 5. Maximum cellulase activity (FPU) obtained in fermentation with different initial concentrations of either Triton X-100 or rhamnolipid.
 N.V. Callow, L.-K. Ju / Enzyme and Microbial Technology 50 (2012) 311–317 315

14.0
1.0 g/L Triton X-10 0 0.0 g/L Triton X-10 0
surfactants, such as Pluronic F-68, were reported to reduce the
12.0
Cell Concentraon

apparent shear forces exerted on shear-sensitive cells, presumably

10.0
8.0 by forming an extracellular protective coating [33]. In the current
(g/L)

6.0 study a majority of the Pluronic surfactants tested did not alter the
4.0 bulk morphology of the fungus, while the Pluronic L31 and F77/L61
2.0
blend tended to decrease the cell flocculation and the P65 and F88
0.0
10.0
increased the apparent agglomeration. The addition of Tween-80
and Span-80 also did not increase pellet formation, in agreement
Lactose (g/L)

8.0
6.0 with the existent literature [14]. Providing no consistent results or
4.0 trends, the data did not support the shear reduction by surfactant
2.0 addition as the primary cause of pellet formation.
0.0 The two surfactants that successfully modified the cell mor-
1.0
phology were rhamnolipid and Triton X-100. Rhamnolipid is a
0.8
group of bio-surfactants synthesized best known by Pseudomonas
Triton X-100

0.6
aeruginosa, commonly overproduced during the stationary phase
(g/L)

0.4 [34,35]. Rhamnolipid is considered an antifungal agent for use in

0.2 controlling pathogenic zoospores, reported to do so by damaging
0.0 the cell membrane [36]. Triton X-100 is commonly used for cell
lysis [37]. Therefore, the two surfactants found effective in modi-
1.6
Cellulase Acvity

1.4 fying T. reesei morphology are both known to cause cell membrane
1.2
(FPU/mL)

1 damage. On the other hand, sodium dodecyl sulfate (SDS), caused

0.8
0.6
0.4
0.2
0 while Triton X-100 and rhamnolipid are not [38–40]. It is uncer-
0 20 40 60 80 100
Time (h)
Fig. 6. Typical fermentation profiles observed for the systems with 1.0 g/L Triton
X-100 in the initial medium, compared with those for the Triton-free control.
Triton produced a constant one-day lag when added at concentra-
tions above 0.1 g/L. Following the lag phase, the Triton-influenced
growth maintained a consistent doubling rate irrespective of the
additive concentration. The measured doubling times during the
active growth phase for varying initial concentrations of Triton
are also shown in Fig. 4. A single factor ANOVA indicates this
data set has a p-value of 0.74 and the differences in growth rates
are not large enough to indicate a significant difference between
the tested additive concentrations. The global average doubling
time is 9.1 ± 1.8 h. The typical fermentation profiles indicating the
reducing sugar consumption, cell growth, cellulase production and
change of Triton concentration are shown in Fig. 6. Note that the
Triton concentration again decreased along with the active cell
growth, but the decrease was less than that of rhamnolipid (Fig. 2)
and the Triton concentration remained relatively constant after the
culture entered the stationary phase.
3.3.2. Cellulase production
Higher cellulase production by T. reesei Rut C-30 was observed
in the presence of Triton X-100 at initial concentrations of at least
0.1 g/L. The increase in cellulase activity did not appear to be a func-
tion of the initial Triton concentration added, as shown in Fig. 5. For
Triton X-100 concentrations in excess of 0.1 g/L, there was an aver-
age 73 ± 12% increase in the maximum cellulase activity produced,
comparable to the highest increases obtained in the systems added
with 1.0 and 1.5 g/L of initial rhamnolipid. As discussed earlier for
rhamnolipid, the increases were not due to the effects of surfactants
on the FPU analysis.
4. Discussion
A number of factors can significantly alter the growth and mor-
phology of T. reesei Rut C-30. One of the most prevalent causes for
morphological changes in a submerged fermentation is the shear
imparted onto the growing cells by mechanical agitation: lower
shear environments favor the development of pellets [23]. Some
significant growth reduction but failed to successfully modify the
morphology. SDS is considered a protein-denaturing surfactant
120 140 160 tain, though possible, that the protein-denaturing property of SDS
prevented this surfactant from being a more suitable morphology
modifier.
It is understandable that the cells could exhibit higher enzyme
productivity due to an increased cell permeability caused by the
surfactant-membrane interactions [14,15,41]. At lower concentra-
tions Triton X-100 caused higher increases in the cellulase activities
obtained than rhamnolipid did (Fig. 5). It is possible that Triton
X-100 is more effective in causing membrane leakage. It is also
possible that the much higher frequency of branching, due to the
weakening of cell membrane, shortened the mean hypha length
and caused the pellet formation. This increase of branching fre-
quency has been reported for other fungi and correlated to their
tendency of pellet formation [17,42].
While both Triton X-100 and rhamnolipid could induce a lag
phase in growth, they produced different cellular responses on
other aspects. Presumably, during the lag phase the cells were
attempting to adjust themselves (e.g., by strengthening their cell
membrane) to overcome the destabilizing effects of the surfac-
tants. When measuring the concentration of Triton X-100 during
the growth period, the surfactant appeared to be adsorbed onto or
absorbed into the growing cells. In the later stages when cell lysis
occurred, Triton was partially released from the cells, indicated by
the slight incline in concentration (Fig. 6). However, in the case of
rhamnolipid, the concentration began to decrease before the cells
began to grow actively (Fig. 2). It is possible that the cells coped
with the addition of rhamnolipid by cleaving the sugar moiety from
the lipid. As shown in Fig. 2, the release in rhamnose might have
been the cause for the slight increase in reducing sugar concen-
tration just before active cell growth (during 48–120 h, somewhat
mirroring the decrease in rhamnolipid concentration). Trichoderma
has been reported to contain relatively poor systems necessary to
digest rhamnose [43]. In either case, the tested surfactant concen-
trations did not permanently inhibit the cell growth. However, if
the surfactants are significantly degraded it may pose a cost issue
for larger scale production. At the end of 2011 Triton is generally
selling for $2.50/kg; rhamnolipids are currently a specialty product
that requires a fermentation and purification process that would
significantly increase operating costs for maintaining the pellet
formation.
Application of the pellet forming technology to large-scale
fermentors remains to be studied. At the same cell concentration
316 N.V. Callow, L.-K. Ju / Enzyme and Microbial Technology 50 (2012) 311–317
and operation conditions, pellet formation would reduce broth
viscosity significantly and increase the rate of gas-liquid interfacial
oxygen transfer, resulting in a higher dissolved oxygen concentra-
tion (DO) in the bulk liquid phase. But, how much higher the bulk
DO would be and if this higher DO would be sufficient to overcome
the resistance of oxygen diffusion into the pellets are unknown and
depend strongly on the pellet size. Assuming for perfectly spher-
ical pellets, the critical pellet radius (Rcrit) at which DO would be
exhausted at the center of a pellet can be derived mathematically,
as given by Metz and Kossen [16]. As expected, Rcrit would depend
on the specific oxygen consumption rate of the species, packing
density of cells in the pellet (e.g., g cells/cm3 pellets), and the
bulk DO (on pellet surface). Experimentally, the critical radii were
reported to range from 0.12 mm (for A. niger with high packing
density, up to 0.2 g/cm3 ) to 2.5 mm (for Aspergillus nidulans with
a low packing density in the range of 0.006–0.015 g/cm3 ) [16].
Apparently, the critical pellet radius is strongly species and culture
condition dependent. The critical pellet radius for T. reesei is
not available in the literature. The T. reesei pellets observed in
this current study made in shake flasks had the average sizes
(diameters) of 1.7 ± 0.3 mm and 1.1 ± 0.3 mm, respectively, when
1 g/L of Triton or rhamnolipids were included in the medium.
Compared with the aforementioned critical radii reported for
other fungi, most of the pellets observed in this study were poten-
tially not too large to incur severe oxygen limitation in the pellet
core.
In addition to the surfactant type and concentration, other
factors such as shear, impeller design, and growth medium com-
position (particularly C:N or C:P ratios, with N or P being the
limiting nutrient) are expected to also affect the pellet size
and packing density in typical stir-tank fermentors [44]. Future
studies are warranted to evaluate these factors for T. reesei in
fermentors equipped with good monitoring and/or control fea-
tures for temperature, pH, DO, agitation speed, and aeration
rate. Nevertheless, under the given pellet forming conditions, the
modest increase in enzyme production coupled with the pellet
formation could offer significant cost savings because of the con-
venient separation of cells from the enzyme-containing medium
for downstream processing and the potential retention (by sim-
ple settling) of the cell pellets for repeated use for enzyme
production.
5. Conclusion
T. reesei Rut C-30 morphology was successfully altered from fil-
aments to rapidly settling pellets by addition of Triton X-100 (at
≥0.1 g/L) or rhamnolipid (at ≥0.3 g/L). Rhamnolipid caused a lag
phase (up to 6 days, by 1.5 g/L rhamnolipid) and the increase in
lag phase was linearly proportional to the initial rhamnolipid con-
centration added. Triton X-100 (≥0.1 g/L) also induced a lag phase,
which was however constant at about 1 day, independent of the
surfactant concentration. Triton did not affect the cell doubling time
during the active growth phase while rhamnolipid increased the
doubling time at higher concentrations. Both surfactants increased
the maximum extracellular cellulase activity and showed no lasting
negative effects.
Acknowledgements
This work was supported by the U.S. Department of Agriculture
under the Biomass Research and Development Initiative (award #
2009-10001-05112). Dr. Narayanan Srinivasan, a previous student
in our group, made the initial observation of pellet formation of T.
reesei Rut C-30 in a rhamnolipid-added medium, which led to this
systematic study.
References
[1] Archer DB. Filamentous fungi as microbial cell factories for food use. Curr Opin
Biotechnol 2000;11(5):478.
[2] Ghorai S, Banik SP, Verma D, Chowdhury S, Mukherjee S, Khowala S. Fungal
biotechnology in food and feed processing. Food Res Int 2009;42(5-6):577.
[3] Xiong H, von Weymarn N, Leisola M, Turunen O. Influence of pH on the
production of xylanases by Trichoderma reesei Rut C-30. Process Biochem
2004;39(6):731.
[4] Juhasz T, Szengyel Z, Szijarto N, Reczey K. Effect of pH on cellulase production
of Trichoderma reesei RUT C30. Appl Biochem Biotechnol 2004;113:201–11.
[5] Shin CS, Lee JP, Lee JS, Park SC. Enzyme production of Trichoderma reesei Rut
C-30 on various lignocellulosic substrates. Appl Biochem Biotechnol 2000;(84-
86):237–45.
[6] Ilmén M, Saloheimo A, Onnela ML, Penttila ME. Regulation of cellulase gene
expression in the filamentous fungus Trichoderma reesei. Appl Environ Micro-
biol 1997;63(4):1298–306.
[7] Lo C-M, Zhang Q, Callow NV, Ju L-K. Cellulase production by continuous culture
of Trichoderma reesei Rut C30 using acid hydrolysate prepared to retain more
oligosaccharides for induction. Bioresour Technol 2010;101(2):717.
[8] Ju L-K, Afolabi OA. Wastepaper hydrolysate as soluble inducing substrate for
cellulase production in continuous culture of Trichoderma reesei. Biotechnol
Prog 1999;15(1):91.
[9] Lo C-M, Ju L-K. Sophorolipids-induced cellulase production in cocultures of
Hypocrea jecorina Rut C30 and Candida bombicola. Enzyme Microb Technol
2009;44(2):107.
[10] Tangnu SK, Harvey WB, Charles RW. Enhanced production of cellulase, hemi-
cellulase, and beta-glucosidase by Trichoderma reesei (Rut C-30). Biotechnol
Bioeng 1981;23(8):1837–49.
[11] Panda T, Gruber H, Kubicek CP. Stimulation of protein secretion in Tricho-
derma reesei by Tween surfactants is not correlated with changes in enzyme
localization or membrane fatty acid composition. FEMS Microbiol Lett 1987;
41(1):85.
[12] Liu J, Yuan X, Zeng G, Shi J, Chen S. Effect of biosurfactant on cellulase and
xylanase production by Trichoderma viride in solid substrate fermentation. Pro-
cess Biochem 2006;41(11):2347.
[13] Pardo AG. Effect of surfactants on cellulase production by Nectria catalinensis.
Curr Microbiol 1996;33(4):275–8.
[14] Domingues FC, Queiroz JA, Cabral JMS, Fonseca LP. The influence of culture
conditions on mycelial structure and cellulase production by Trichoderma reesei
Rut C-30. Enzyme Microb Technol 2000;26(5-6):394–401.
[15] Reese ET, Maguire A. Surfactants as stimulants of enzyme production by
microorganisms. Appl Microbiol 1969;17(2), 242-&.
[16] Metz B, Kossen NWF. Biotechnology review the growth of molds in the form of
pellets—a literature review. Biotechnol Bioeng 1977;19:781–99.
[17] Papagianni M, Mattey M. Morphological development of Aspergillus niger in
submerged citric acid fermentation as a function of the spore inoculum level.
Application of neural network and cluster analysis for characterization of
mycelial morphology. Microbial Cell Factories 2006;5(1):3.
[18] Réczey K, Stålbrand H, Hähn-Hagerdal B, Tjerneld F. Mycelia-associated B-
glactosidase activity in mocrobial pellets of Aspergillus and Penicillium strains.
Appl Microbiol Biotechnol 1992;38:393–7.
[19] Nanda M, Bisaria VS, Ghose TK. Effect of l(−)sorbose on cellulase activity in
Trichoderma reesei QM9414. J Gen Microbiol 1986;132:3201–7.
[20] Yang W, Hartwieg EA, Fang A, Demain AL. Effects of carboxymethylcellulose
and carboxypolymethylene on morphology of Aspergillus fumigatus NRRL 2346
and fumagillin production. Curr Microbiol 2003;46:24–7.
[21] Nielsen J, Johansen CL, Jacobsen M, Krabben P, Villadsen J. Pellet formation
and fragmentation in submerged cultures of Penicillium chrysogenum and its
relation to Penicillin production. Biotechnol Prog 1995;11(1):93–8.
[22] Vansuijdam JC, Kossen NWF, Paul PG. An inoculum technique for the production
of fungal pellets. Eur J Appl Microbiol Biotechnol 1980;10(3):211–21.
[23] Suijdam JC, Kossen NWF, Paul PG. An inoculum technique for the production
of fungal pellets. Appl Microbiol Biotechnol 1980;10(3):211.
[24] Lucatero S, Galindo E, Larralde-Corona CP. Quantitative characterisation of the
morphology of Trichoderma harzianum cultured in shake-flasks and containing
Tween 40. Biotechnol Lett 2004;26(1):41–4.
[25] Ferreira SMP, Duarte AP, Queiroz JA, Domingues FC. Influence of buffer systems
on Trichoderma reesei Rut C-30 morphology and cellulase production. Electron
J Biotechnol 2009;12:3.
[26] Zhang Q, Lo C-M, Ju L-K. Factors affecting foaming behavior in cellulase fer-
mentation by Trichoderma reesei Rut C-30. Bioresour Technol 2007;98(4):753.
[27] Ghose TK. Measurement of cellulase activities. Pure Appl Chem
1987;59(2):257–68.
[28] Karlsson G, Hinz AC, Henriksson E, Winge S. Determination of Triton X-100
in plasma-derived coagulation factor VIII and factor IX products by reversed-
phase high-performance liquid chromatography. J Chromatogr A 2002;946(1-
2):163–8.
[29] Bailey RW. The reaction of pentoses with anthrone. Biochem J
1958;68(4):669–72.
[30] Ludwig TG, Goldberg JV. The anthrone method for the determination of carbo-
hydrates in foods and in oral rinsing. J Dent Res 1956;35(1):90–4.
[31] Wu J, Ju L-K. Enhancing enzymic saccharification of waste newsprint by sur-
factant addition. Biotechnol Prog 1998;14(4):649–52.
[32] Qing Q, Yang B, Wyman CE. Impact of surfactants on pretreatment of corn
stover. Bioresour Technol 2010;101(15):5941–51.
 N.V. Callow, L.-K. Ju / Enzyme and Microbial Technology 50 (2012) 311–317
[33] Sowana DD, Williams DRG, O’Neill BK, Dunlop EH. Studies of the shear pro-
tective effects of Pluronic F-68 on wild carrot cell cultures. Biochem Eng J
2002;12(3):165.
[34] Mulligan CN, Gibbs BF. Correlation of nitrogen metabolism with biosur-
factant production by Pseudomonas aeruginosa. Appl Environ Microbiol
1989;55(11):3016–9.
[35] Chayabutra C, Wu J, Ju L-K. Rhamnolipid production by Pseudomonas aeruginosa
under denitrification: effects of limiting nutrients and carbon substrates.
Biotechnol Bioeng 2001;72(1):25–33.
[36] Stanghellini ME, Miller RM. Biosurfactants: their identity and potential
efficacy in the biological control of zoosporic plant pathogens. Plant Dis
1997;81(1):4–12.
[37] Schnaitm CA. Solubilization of cytoplasmic membrane of Escherichia coli by
Triton X-100. J Bacteriol 1971;108(1):545.
[38] Sigma-Aldrich. X100—Product Information Sheet; 1999. Available from:
http://www.sigmaaldrich.com/etc./medialib/docs/Sigma-Aldrich/Product
Information Sheet/x100pis.Par.0001.File.tmp/x100pis.pdf [cited: 4/21/2000].
317
[39] Tanford C. Protein denaturation. In: Advances in protein chemistry. Academic
Press; 1968. p. 211–217.
[40] Sánchez M, Aranda FJ, Espuny MJ, Marqués A, Teruel JA, Manresa Á, et al.
Thermodynamic and structural changes associated with the interaction
of a dirhamnolipid biosurfactant with Bovine Serum Albumin. Langmuir
2008;24(13):6487.
[41] Ahamed A, Vermette P. Effect of culture medium composition on Tri-
choderma reesei’s morphology and cellulase production. Bioresour Technol
2009;100(23):5979–87.
[42] Terra ND, Tatum EL. A relationship between cell wall structure and colonial
growth in Neurospora crassa. Am J Bot 1963;50(7):669–77.
[43] Druzhinina IS, Schmoll M, Seiboth B, Kubicek CP. Global carbon utilization pro-
files of wild-type, mutant, and transformant strains of Hypocrea jecorina. Appl
Environ Microbiol 2006;72(3):2126–33.
[44] Yu L, Chao Y, Wensel P, Chen S. Hydrodynamic and kinetic study of cellulase
production by Trichoderma reesei with pellet morphology. Biotechnol Bioeng
2012, doi:10.1002/bit.24433.