Bioresource Technology 169 (2014) 143148

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus

strains facilitates efcient hydrolysis of pretreated wheat straw and
shows promises for on-site enzyme production
Marta Kolasa a, Birgitte Kir Ahring a,b, Peter Stephensen Lbeck a, Mette Lbeck a,
a
b

Section for Sustainable Biotechnology, Aalborg University Copenhagen, A. C. Meyers Vnge 15, DK-2450 Copenhagen SV, Denmark
Bioproducts, Sciences and Engineering Laboratory (BSEL), Washington State University Tri-Cities, 2710 Crimson Way, Richland 99354, WA, USA

h i g h l i g h t s
 Co-cultivation of Trichoderma reesei with novel Aspergillus saccharolyticus.
 80% efciency of hydrolysis using crude enzymes from mixed cultures.
 Solid-state fermentation being well-suited for co-cultivation of selected strains.

a r t i c l e

i n f o

Article history:
Received 14 April 2014
Received in revised form 23 June 2014
Accepted 24 June 2014
Available online 1 July 2014
Keywords:
Mixed culture
Trichoderma
Aspergillus
Hydrolysis
On-site enzyme production

a b s t r a c t
Co-cultivation of fungi may be an excellent system for on-site production of cellulolytic enzymes in a
single bioreactor. Enzyme supernatants from mixed cultures of Trichoderma reesei RutC30, with either
the novel Aspergillus saccharolyticus AP, Aspergillus carbonarius ITEM 5010 or Aspergillus niger CBS
554.65 cultivated in solid-state fermentation were tested for avicelase, FPase, endoglucanase and
beta-glucosidase activity as well as in hydrolysis of pretreated wheat straw. Around 30% more avicelase
activity was produced in co-cultivation of T. reesei and A. saccharolyticus than in T. reesei monoculture,
suggesting synergistic interaction between those fungi. Fermentation broths of mixed cultures of T. reesei
with different Aspergillus strains resulted in approx. 80% efciency of hydrolysis which was comparable
to results obtained using blended supernatants from parallel monocultures. This indicates that
co-cultivation of T. reesei with A. saccharolyticus or A. carbonarius could be a competitive alternative for
monoculture enzyme production and a cheaper alternative to commercial enzymes.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
The use of lignocellulosic plant biomass for production of biofuels and high value added biomaterials in a biorenery rely on enzymatic hydrolysis of pretreated biomass to produce fermentative
sugars for further processing. A complete set of hydrolytic enzymes
for synergistic lignocellulosic biomass conversion includes especially cellobiohydrolases, endoglucanases, beta-glucosidases as
well as the newly described oxidative enzymes (Horn et al.,
2012). The biorenery plant for commercial production of bioethanol using enzymatic conversion of biomass has been established
recently (Beta Renewables S.p.A), however, the cost of enzymatic
hydrolysis is still considered high. The potential solution could
Corresponding author. Tel.: +45 99402589; fax: +45 99402594.
E-mail addresses: mk@bio.aau.dk (M. Kolasa), bka@tricity.wsu.edu (B.K. Ahring),
psl@bio.aau.dk (P.S. Lbeck), mel@bio.aau.dk (M. Lbeck).
http://dx.doi.org/10.1016/j.biortech.2014.06.082
0960-8524/ 2014 Elsevier Ltd. All rights reserved.

be to produce enzymes on-site in a biorenery plant in order to

eliminate or reduce the expenses of enzyme delivery from external
suppliers as well as of addition of preservatives and stabilizers that
are normally added to reduce enzyme degradation during storage
(Kazi et al., 2010). On-site enzyme manufacturing can be achieved
either by co-cultivation of compatible fungal strains in a single bioreactor, cultivation of single strain genetically modied with several cellulolytic genes, or cultivation of multiple monocultures,
followed by enzyme blending. Strain engineering is limited by
the number of genes that can be inserted in a single host (Bryant,
2010) resulting in an appropriate balanced enzyme cocktail, while
separate production of enzymes by monocultures increases the
cost of double equipment needed (Duff and Cooper, 1985), thus
co-cultivations are designed to produce Trichoderma reesei
cellulases together with Aspergillus sp. beta-glucosidases in a single
process (Gutierrez-Correa and Tengerdy, 1997; Wen et al., 2005).
Among those, T. reesei and Aspergillus niger, being well-known

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M. Kolasa et al. / Bioresource Technology 169 (2014) 143148

commercial enzyme producers, are one of the most studied model

co-cultivation pair both in liquid and solid-state fermentation
(Ahamed and Vermette, 2008; Castillo et al., 1994; Fang et al.,
2010). Mixed cultures of multiple fungal species producing cellulolytic enzymes are found in nature for example in wood degradation
(Bayer and Lamed, 1993). Thus, co-cultivation could be even more
successful in solid-state fermentation, resembling the natural solid
substrates (Gutierrez-Correa and Tengerdy, 1997). For effective
co-cultivation, strain compatibility is a very important factor;
shown by the fact that the right compatibility is able to overcome
substrate scarcity (Gutierrez-Correa et al., 1999).
On-site separate production of T. reesei cellulases and the novel
Aspergillus saccharolyticus beta-glucosidases and their use in the
hydrolysis of pretreated biomass was reported recently (Rana
et al., 2014). The aim of the present work was to screen selected
promising fungal strains in co-cultivation for production of an efcient set of hydrolytic enzymes in a single ask at laboratory scale
and to compare them against co-cultivation of T. reesei and A. niger
as background reference. The hypothesis was that co-cultivation of
the good cellulase producer, T. reesei with the promising betaglucosidase producers, Aspergillus carbonarius and A. saccharolyticus, increases overall enzyme production and facilitates synergistic
enzymatic conversion of cellulose.
2. Methods
2.1. Fungal strains
This study comprises four fungal strains T. reesei RutC30, A. saccharolyticus AP, A. carbonarius ITEM 5010 and A. niger CBS 554.65.
The strains were maintained in 10% glycerol at 80 C.
2.2. Spore suspension
For spore generation, strains were grown on potato dextrose
agar (PDA) (SigmaAldrich) plates prepared according to the manufacturers instructions. Spores were harvested from 7 days old
plate cultures by addition of sterile Milli-Q water with 0.1%
Tween-80 (SigmaAldrich). Spore suspension was ltered through
the Miracloth (Merck), followed by spore counting with a FuchsRosenthal counting chamber (Hecht-Assistent).
2.3. Compatibility tests
To investigate compatibility of each co-cultivation, pairwise
combinations of strains were grown on the same glucose
(SigmaAldrich), Avicel PH-101 (SigmaAldrich), carboxymethylcellulose sodium salt (CMC) (SigmaAldrich) or wheat bran (WB)
(Finax) agar plate, prepared as follows: 2% (w/v) of either glucose,
avicel, CMC or WB were added to the basic Czapek (Cz) agar medium (3 g/l NaNO3, 1 g/l K2HPO4, 0.5 g/l KCl, 0.5 g/l MgSO4
7H2O,
0.01 g/l FeSO4
7H2O, 15 g/l agar) (Samson et al., 2004). Additionally, avicel and CMC were supplemented with 0.1% (w/v) glucose
to induce mycelial growth. The pH was adjusted to 4.8 and 1 ml
of trace metal solution (TMS: 1 g ZnSO4
7H2O, 0.5 g CuSO4
5H2O
in 100 ml water) per liter medium was added just before autoclaving at 121 C for 20 min. Spore suspensions (5  105 spores/ml)
were inoculated in three different ways (A, B, C) as described by
Hu et al. (2011). Inoculation A was done by placing 2 ll of each
spore suspension from a fungal pair with 3 cm distance from each
other. Inoculation B was done by mixing 2 ll of each spore suspension and pipetting mixture in the center of the plate. Inoculation C
was done by mixing 2 ll of each spore suspension and spreading it
over whole plate. Plates were incubated at 25 2 C for 7 days (or
until certain colony size allowing for interaction between fungi

was reached). Fungal compatibility was assessed by observing colony shapes, radial expansion and pattern of growth with reference
to Porter (1924) and Hu et al. (2011). Observation was done with
naked eye and microscope (Olympus SZ2-ILST LED Illuminator
Stand). Sample duplicates were assessed.
2.4. Solid-state fermentation (SSF)
Solid medium comprised 25.6% (w/v) wheat bran and 15.4% (w/
v) sphagnum peat in Milli-Q water. A total of 40 g of medium was
added to 250 ml Erlenmeyer ask, closed with wadding and gaze.
The medium was autoclaved twice at 121 C for 60 min. Medium
was inoculated with 1 ml of 5  106/ml spore suspension and incubated as stationary culture at 25 C for 10 days. Flasks were shaken
by hand twice a day for equal distribution of spores. Both mixed
cultures, inoculated with 1:1 spore ratio (2.5  106 spore/ml of
each fungus) and corresponding monocultures (5  106/ml spore
concentration) were set up in duplicates. In mixed cultures, strains
were inoculated either at the same time (at 0 h) or 48 h apart.
2.5. Preparation of enzyme supernatants
Enzymes from solid-state cultures were extracted by adding
sterile Milli-Q water up to 100 ml and shaking asks at 25 C,
200 rpm, overnight. The slush was then squeezed through Miracloth and centrifuged at 4 C, 8000g for 20 min. The supernatants
were transferred to fresh 50 ml Falcon tubes and centrifuged again
as above. Claried supernatants were used in enzyme assays and
hydrolysis of pretreated wheat straw.
2.6. Enzyme assays
All enzyme activities (U/ml) are dened as units of activity per
1 ml of enzyme preparation. Sample duplicates were tested in
enzyme assays. Due to high concentration of wheat bran proteins
in the samples, the content of exclusively fungal proteins could
not be calculated and protein content as a basis for adjusting the
enzyme load for comparative studies would not be valid.
Endoglucanase activity was measured using Azo-CM (carboxymethyl)-Cellulose as a substrate, according to manufacturers protocol (Megazyme) (Wood and Bhat, 1988). The mixture of enzyme
extract (50 ll) and substrate (50 ll) made in 100 mM sodium acetate buffer, pH 4.5, was incubated at 40 C, 700 rpm (Eppendorf
Thermomixer comfort) for 30 min. The reaction was terminated
by addition of 250 ll precipitant solution (100 ml of sodium acetate trihydrate (20 g) and zinc acetate (2 g) Milli-Q water solution,
pH 5.0 in 400 ml of 96% ethanol), vortexing briey and measuring
the absorbance at 590 nm. A standard curve was obtained by activity measurement of different concentrations of pure A. niger cellulase (0.84 U/mg) (SigmaAldrich) at the above assay conditions.
One unit of endoglucanase activity was dened as the amount of
enzyme that liberates 1 lmol glucose from carboxymethylcellulose per minute at pH 4.5 and 40 C.
Beta-glucosidase activity measurement was done with 5 mM pnitrophenyl b-D-glucopyranoside (pNPG) (SigmaAldrich) as a substrate. The assay was carried out according to the method of
Flachner et al. (1999) in a microtiter format (Srensen et al.,
2011b). Briey, 10 ll of enzyme extract was added to 100 ll of
substrate in 50 mM sodium citrate buffer, pH 4.8, and incubated
at 50 C for 10 min. The assay was ended by withdrawing 60 ll
of reaction volume and adding it to 100 ll of 1 M Na2CO3 (stop
reagent), prior to absorbance reading at 405 nm. A standard curve
was constructed with different concentrations of p-nitrophenol
(pNP) (SigmaAldrich). One unit of beta-glucosidase activity was
dened as the amount of enzyme that liberates 1 lmol pNP from
pNPG per minute at pH 4.8 and 50 C.

M. Kolasa et al. / Bioresource Technology 169 (2014) 143148

Avicelase activity assay was based on hydrolysis of avicel and

determination of released reducing sugar by dinitrosalicylic acid
(DNS) method (Ghose, 1987; Miller, 1959). The assay was carried
out similarly to the method described by Momeni et al. (2013).
Ten ll enzyme extract was added to 140 ll 1% Avicel PH-101
(SigmaAldrich) in 10 mM sodium acetate, pH 5.0, and incubated
at 37 C, 1400 rpm (Eppendorf Thermomixer comfort) for 2 h.
Then, the solids were spun down, 100 ll supernatant was mixed
with 200 ll DNS, incubated at 100 C for 10 min and cooled down
on ice, followed by absorbance measurement at 540 nm in a microtiter plate. A standard curve was obtained with different glucose
concentrations (100, 250, 500, 750, 1000, 1250, 1500 lg/ml); thus
the amount of released reducing sugar was reported as the glucose
equivalents. One unit of avicelase activity was dened as the
amount of enzyme that liberates 1 lmol glucose equivalents of
reducing sugar from avicel per minute at pH 5.0 and 37 C.
2.7. Filter paper assay
Filter paper assay was carried out in a similar way as presented
before (Busk and Lange, 2013; Coward-Kelly et al., 2003). A piece
(5 mm  5 mm) of lter paper (Frisenette Aps), 80 ll of McIlvaine
Buffer (McIlvaine, 1921), pH 5.0, and 20 ll of crude enzyme
extracts were added to 1.5 ml Eppendorf tubes. The mixtures were
incubated for 24 h at 45 C. Then, the excess of Novozyme 188
(Novozymes A/S, Bagsvrd, Denmark) was added and the samples
were incubated for 2 h at 45 C to convert any cellobiose to glucose. The solids were spun down at 16,000g for 2 min. A volume
of 90 ll supernatant was mixed with 180 ll DNS, incubated at
100 C for 10 min and cooled down on ice, followed by absorbance
measurement at 540 nm in a microtiter plate. A standard curve
was obtained with different glucose concentrations (100, 250,
500, 750, 1000, 1250, 1500 lg/ml) thus the amount of released
reducing sugar was reported as the glucose equivalents. Filter
paper activity was dened as the amount of glucose equivalents
of reducing sugar released from substrate per milliliter enzyme
extract per minute at pH 5.0 and 45 C. Sample duplicates were
tested.
2.8. Enzyme synergy and efciency of cellulose hydrolysis
To examine synergistic action, enzyme mixtures were analyzed
for efciency of hydrolysis of the solid fraction of steam exploded
dilute-acid pretreated wheat straw (kindly donated by Biogasol
ApS, Denmark). The batch of pretreated wheat straw with cellulose
content of 36% was used for analysis of solid-state fermentation.
Hydrolysis was carried at 6% total solids (TS) similar to bagasse
hydrolysis carried out by Srensen et al. (2011b). A total volume
of 600 ll of crude enzyme extracts was added to pretreated wheat
straw in 700 ll 0.1 M succinic acid buffer, pH 5.0 and incubated at
45 C, 1400 rpm (Eppendorf Thermomixer comfort), for 3 days.
After incubation, the samples were heated at 100 C for 10 min,
mixed with 45 ll of 10% sulfuric acid and centrifuged at maximum
speed, for 10 min; then the supernatants were tested using Ultimate 3000 HPLC (Dionex). Synergy tests included: (1) enzyme
extracts from mixed cultures, (2) enzyme extracts from monocultures used alone, (3) enzyme extracts from monocultures blended
together in 1:1 volumetric ratio post-culture, and nally (4) blend
of Celluclast 1.5 L (40 ll) and Novozyme 188 (10 ll) (Novozymes
A/S, Bagsvrd, Denmark). Sample duplicates were tested in hydrolysis. The efciency of hydrolysis was calculated as a percentage of
the theoretical (maximum) amount of glucose produced in cellulose hydrolysis, given the cellulose content 36% of the TS. Celluclast
1.5 L and Novozyme 188 were used in optimal 4:1 volumetric ratio
corresponding to the loading of 0.04 U/ml of avicelase activity,

145

3.9 U/ml of endoglucanase activity and 2.4 U/ml of betaglucosidase activity in the hydrolysis experiment.
3. Results and discussion
3.1. Compatibility tests
Compatibility, dened as the ability of fungi to grow in mixed
cultures, without inhibiting each other, was tested by co-cultivating them pairwise on agar plates with different carbon sources:
glucose, avicel, CMC, and wheat bran. The experiments were performed in three different ways: by inoculating fungi separately
3 cm apart from each other (Inoculation A), together as mixed
spore suspension either in the center (Inoculation B) or spread over
the whole plate (Inoculation C) (Hu et al., 2011). Porter (1924)
dened ve types of interactions observed for fungi inoculated at
a distance from each other, and later Hu et al. (2011) added the
description of growth patterns when fungal pairs were inoculated
as mixed spore suspension. Based on those reports, the following
growth patterns were classied in this study: compatible growth,
inhibition at contact or distance, and overgrowth. Furthermore,
compatibility was dened for each inoculation type because the
growth patterns turned out to be dependent on initial plating of
spores. Compatible strains inoculated 3 cm apart (Inoculation A),
were expected to grow in the direction of one another without
any negative effect on each other and to intermix in the narrow
(0.5 cm) border at the contact of the colonies. In case of strains
inoculated as mixed spores (Inoculation B and C), compatibility
was described as the ability to share a common area of the plate
by two strains, for example by growing around or among each
other.
In general, growth of each strain was somehow affected by the
presence of another strain on the same plate. None of fungal pairs
was able to grow without inhibition on all tested carbon sources
and in all inoculation setups, indicating that tested strains were
partially compatible. The co-cultivation of the reference pair,
T. reesei and A. niger, resulted in the highest number of compatible
interactions. Among the tested carbon sources, the highest number
of compatible growth patterns was observed on avicel, whereas the
lowest was found on CMC.
Colonies of T. reesei and each of the three black Aspergilli, A. saccharolyticus, A. carbonarius or A. niger, were able to grow in proximity of each other and intermix on the narrow border between them
when avicel or wheat bran was used as a carbon source. All of them
were capable of growing around each other from the same inoculation spot on glucose, while only T. reesei and A. niger followed this
pattern also on avicel. Interestingly, when inoculated as mixed
spores over the whole plate of avicel media, development of
T. reesei was inhibited by the fast-growing A. saccharolyticus and
A. carbonarius, whereas it was less affected by A. niger. In contrast,
on glucose, CMC and wheat bran plates T. reesei was overgrown by
each of the black Aspergilli when inoculated as mixed spore
suspension over whole plate.
3.2. Enzyme production in solid-state fermentation
In this work, the fungal pairs were selected for co-cultivation
studies based on the enzymatic variety among them, thus they
were supposedly supplementing each other. Wheat bran, being a
complex substrate rich in non-starch carbohydrates, starch,
proteins, fat and B-vitamin (Haque et al., 2002), was chosen for
co-cultivation of fungal pairs in solid-state fermentation, as this
growth substrate is known to induce production of a broad range
of hydrolytic enzymes (Smits et al., 1996; Yamane et al., 2002).
Moreover, pretreated wheat straw, being an attractive biomass

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M. Kolasa et al. / Bioresource Technology 169 (2014) 143148

for bioethanol production (Talebnia et al., 2010), was used as a substrate in hydrolysis experiment to study the potential industrial
application of enzymes produced by co-cultivation of selected
strains.
Supernatants from 7 days old mixed cultures and monocultures
of T. reesei and black Aspergilli cultivated in solid-state fermentation were tested for avicelase, FPase, endoglucanase and betaglucosidase activity. Strains were inoculated either simultaneously
(at 0 h) or 48 h apart.
T. reesei monoculture yielded high avicelase, FPase and endoglucanase activities, while Aspergilli produced high beta-glucosidase
activity, with A. saccharolyticus, A. carbonarius and A. niger producing rst, second and third highest respectively (Figs. 1 and 2).
In co-cultivations, enzyme activities varied generally as a function of inoculation time. Remarkably, mixed cultures of T. reesei
and A. saccharolyticus, inoculated 48 h apart, had the highest avicelase activity, even higher than activity produced when T. reesei
grew as monoculture. This might indicate that A. saccharolyticus
has potential positive effect on enhanced T. reesei total cellulase
production in solid-state fermentation, showing the promises of
co-cultivating those fungi for higher enzyme yields. This should
be further investigated by determination of specic enzyme activities. Similarly, the reference mixed cultures of T. reesei and A. niger
produced higher beta-glucosidase activity than A. niger monoculture. As for the FPase activity, mixed cultures, inoculated 48 h
apart, resulted in as high glucose release from lter paper as
T. reesei single culture (Fig. 2).
In the mixed cultures of T. reesei together with either A. saccharolyticus, A. carbonarius or A. niger inoculated 48 h apart, the ratios
of beta-glucosidase to endoglucanase activity were as follows 0.8,

Fig. 2. Filter paper assay. Filter paper activity measured as the amount of glucose
equivalents of reducing sugar released from substrate per milliliter of enzyme
preparation per minute at pH 5.0 and 45 C. Tr = T. reesei, As = A. saccharolyticus,
Ac = A. carbonarius, An = A. niger. 0 h, 48 h = delay in inoculation of Aspergillus strain
to the mixed culture. Error bars indicate standard deviation of mean for sample
duplicates.

1.1, 1.5, respectively. The corresponding ratios of beta-glucosidase

to avicelase activity were equal to 11.0, 15.9 and 24.3. The respective ratios were equal to 0.6 and 54.5 for the mixture of Celluclast
1.5 L and Novozyme 188. In contrast, the respective ratios were
close to 0 in T. reesei monoculture due to very low beta-glucosidase
activity of this strain. The accurate ratio of enzymes is required for
optimal hydrolysis which is discussed further.

Fig. 1. Activities of avicelase, endoglucanase (EG), and beta-glucosidase (BG) produced in mixed cultures and monocultures in solid-state fermentation. Enzyme activity is
shown in units per ml enzyme preparation. 1 U of avicelase = the amount of enzyme that liberates 1 lmol glucose equivalents of reducing sugar from avicel per minute at pH
5.0 and 37 C, 1 U of EG = the amount of enzyme that liberates 1 lmol glucose from CMC per minute at pH 4.5 and 40 C, 1 U of BG = the amount of enzyme that liberates
1 lmol pNP from pNPG per minute at pH 4.8 and 50 C. Tr = T. reesei, As = A. saccharolyticus, Ac = A. carbonarius, An = A. niger. 0 h, 48 h = delay in inoculation of Aspergillus
strain to the mixed culture. Error bars indicate standard deviation of mean for sample duplicates.

M. Kolasa et al. / Bioresource Technology 169 (2014) 143148

3.3. Synergy and hydrolytic potential of enzymes produced in solidstate fermentation

Mixed cultures and monocultures were compared with each
other based on the enzyme synergy in the hydrolysis of pretreated
wheat straw. The synergy between Celluclast 1.5 L and Novozyme
188 was used as a benchmark. Those enzyme extracts are produced by single cultures of T. reesei and A. niger, respectively, therefore, they were used in this study as a reference commercial
enzyme preparations, although they have been recently substituted by an advanced enzyme cocktail, Cellic CTec3, that combines
cellulase, beta-glucosidase, hemicellulase and accessory enzyme
activities (Novozymes A/S, 2012). In order to compare the enzyme
extracts from mixed cultures with the monocultures, the cultures
were normalized with respect to the same amount of inoculum
used and the equal volumes of crude enzyme supernatants were
applied in hydrolysis.
Enzyme supernatant from T. reesei monoculture resulted in cellobiose accumulation after hydrolysis due to low beta-glucosidase
activity of this strain (Fig. 3). Blending T. reesei monoculture
enzymes with beta-glucosidase rich enzyme supernatants from
Aspergillus sp. monocultures resulted in depletion of cellobiose
and increase in glucose concentration as a result of enzyme
synergy. Similar synergies were observed for co-cultivations of T.
reesei and any of the Aspergilli strains, when fungi were inoculated
48 h apart. Importantly, the glucose yields in those mixed cultures
were nearly as high as the ones produced by enzyme blends

25

cellobiose

glucose

20

g/l

15
10
5
An

Ac

Tr

As

C+N

Tr+An

Tr An (48h)

Tr+Ac

Tr An (0h)

Tr Ac (48h)

Tr+As

Tr Ac (0h)

Tr As (0h)

Tr As (48h)

Fig. 3. Synergies of enzymes for hydrolysis of pretreated wheat straw. Concentration of glucose and cellobiose released in hydrolysis. Enzymes produced in mixed
cultures and monocultures in solid-state fermentation. Tr = T. reesei, As = A.
saccharolyticus, Ac = A. carbonarius, An = A. niger, C = Celluclast 1.5 L, N = Novozyme
188, + = blend of extracts from monocultures or commercial enzymes. (0 h,
48 h) = delay in inoculation of Aspergillus strain to the mixed culture. Error bars
indicate standard deviation of mean for sample duplicates.

147

prepared by mixing two corresponding monoculture extracts (less

than 1 g/l difference) as well as a mixture of Celluclast 1.5 L and
Novozyme 188. Hence, by reducing the cost of equipment and
eliminating the necessity of enzyme blending, those mixed cultures show great promises for on-site production of enzymes that
can be directly used in the hydrolysis of biomass without prior
purication.
The synergy proles of mixed cultures with simultaneous
inoculation of both strains were similar to the synergies of black
Aspergilli monocultures, showing that low concentration of glucose
released in hydrolysis was probably due to low total cellulase
activity in those mixed cultures. This indicates that time of inoculation of co-cultivated strains is important to produce an accurate
ratio of enzymes for optimal hydrolysis.
Consequently, hydrolytic potentials of co-cultivations inoculated 48 h apart, blended monoculture enzyme extracts, as well
as mixture of Celluclast 1.5 L and Novozyme 188 were around
80% (Fig. 4). Partial growth compatibility of tested strains did not
hinder the enzyme synergy. This observation was also made by
Gutirrez-Correa and Villena (2012). Hydrolysis efciencies of the
recently identied A. saccharolyticus strain (Srensen et al.,
2011a) and of A. niger were previously reported (Dekker, 1986;
Srensen et al., 2011b), whereas the potential of A. carbonarius in
hydrolysis of pretreated wheat straw was found in this study.
Several studies were published, where optimal ratio of betaglucosidase to lter paper activity ranged from 0.12 to 1.5 depending on enzyme assays, source of enzymes and substrates used (Duff
et al., 1987; Sternberg, 1976). However, in this work avicelase
activity instead of lter paper activity was used for the measurement of ratios. Mixed culture of T. reesei and A. saccharolyticus,
inoculated 48 h apart, gave the most efcient ratios of both betaglucosidase to endoglucanase and beta-glucosidase to avicelase
activity, evidenced by the highest hydrolysis efciency of 84%. This
beta-glucosidase to endoglucanase ratio was very close to the corresponding ratio calculated for a mixture of Celluclast 1.5 L and
Novozyme 188. In contrast, beta-glucosidase to avicelase ratio
was much lower as compared to other co-cultivation broths and
mixture of commercial enzymes, indicating that relatively low
loading of A. saccharolyticus beta-glucosidases are enough to
degrade cellobiose released by highly effective T. reesei cellulases.
This conrms the incredibly high efciency of A. saccharolyticus
beta-glucosidases (Srensen et al., 2011b) and demonstrates the
potential of producing an accurate ratio of A. saccharolyticus betaglucosidases and T. reesei cellulases in solid-state co-cultivation;
thus facilitating the development of lower-cost single-tank
enzyme production.

Fig. 4. Efciency of hydrolysis of pretreated wheat straw. Enzymes produced in mixed cultures and monocultures in solid-state fermentation. Tr = T. reesei, As = A.
saccharolyticus, Ac = A. carbonarius, An = A. niger, C = Celluclast 1.5 L, N = Novozyme 188, + = blend of extracts from monocultures or commercial enzymes. 0 h, 48 h = delay in
inoculation of Aspergillus strain to the mixed culture. Error bars indicate standard deviation of mean for sample duplicates.

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M. Kolasa et al. / Bioresource Technology 169 (2014) 143148

In case of tested strains, solid-state fermentation was shown to

be a more effective method for production of a well-balanced lowcost enzyme cocktail than submerged fermentation (data not
shown). Moreover, solid-state fermentation resulted in higher
enzyme yields which can further reduce the cost of enzyme production. Solid-state fermentation was previously described as
being more suited for mixed cultures, by resembling natural environment and allowing each strain to grow in its own niche and
produce enzymes that can further work synergistically in biomass
conversion (Gutierrez-Correa and Tengerdy, 1997). However, as
shown in the recent studies submerged fermentation using mixed
cultures could be improved by applying repeated batch fermentation (Gutirrez-Correa and Villena, 2012).
Further advancement to the mixed cultures could be achieved
by using lamentous fungal biolms that resemble solid-state fermentation with respect to productivity, while the setup is similar
to submerged fermentation, thus being more feasible to scale up
than solid-state fermentation (Gutirrez-Correa et al., 2012).
4. Conclusions
Enzyme cocktails from mixed cultures of T. reesei and either A.
carbonarius or the recently described A. saccharolyticus were shown
to compete with blended extracts from corresponding monocultures, therefore they can lower the cost of on-site enzyme manufacture by integrating production to a single bioreactor and by
eliminating an enzyme blending step. Enzyme cocktail produced
in co-cultivation of T. reesei and A. saccharolyticus was the most
competitive to mixture of Celluclast 1.5 L and Novozyme 188.
Future work is aimed at the scale-up of the mixed culture solidstate fermentation in order to further show its application for
industrial enzyme production.
Acknowledgements
Authors would like to thank Kristian Hansen and Gitte
Hinz-Berg for technical support in the laboratory. This work was
nancially supported by the Danish Council for Strategic Research
(project no. 2101-08-0041 and grant number 11-116803).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2014.
06.082.
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