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ELSEVIER

Bioresource Technology68 (1999) 173-178

Mixed culture solid substrate fermentation of Trichoderma reesei

with Aspergillus niger on sugar cane bagasse
Marcel Gutierrez-Correa a*, Leticia Portal a, Patricia Moreno ~, Robert P. Tengerdy u
aLaboratorio de Micologia y Biotecnologia, Universidad Nacional Agraria La Molina, Apartado 456 Lima 1, Peru
bDepartment of Microbiology, Colorado State University, Fort Collins, CO 80523, USA

Received 8 April 1998; revised 26 June 1998; accepted 10 July 1998

Abstract
Trichoderma reesei LM-UC4, the parent strain, and its hypercellulolytic mutant LM-UC4E1 were co-cultured with Aspergillus
niger ATCC 10864 in solid substrate fermentation on alkali-treated sugar cane bagasse for cellulolytic enzyme production.

Bagasse was supplemented with either soymeal or with ammonium sulfate and urea, and fermented at 80% moisture content and
30C. Mixed culturing produced better results with the inorganic supplement. The mutant strain was more responsive to mixed
culturing than the parent strain when A. niger was the cooperating partner. In a mixed culture of the mutant with inorganic
N-source, 10% more biomass, but 63% more cellulase, 85% more endoglucanase and 147% more fl-glucosidase was produced
than in single culture. Since co-culturing helped enzyme production more than growth, it appeared that synergistic interactions
not directly related to growth were responsible for increased enzyme production. Although soymeal supplementation increased
biomass production in the same mixed culture by 30%, it did not increase enzyme production. Mixed culturing is thus beneficial
for the economic production of cellulases on nutritionally poor agricultural residues, without the need for supplementation with
expensive organic supplements. 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Cellulase; fl-glucosidase; Trichoderrna reesei; Aspergillus niger; Mixed culturing; Solid substrate fermentation

1. Introduction

Lignocellulosic biomass is the most abundant organic

raw material in the world. Production of ethanol from
renewable lignocellulosic resources may improve
energy availability, decrease air pollution, and diminish
atmospheric CO2 accumulation (Lynd et al., 1991).
Reducing the cost of conversion of lignocellulosics into
ethanol will stimulate new markets for the agriculture
sector, thereby increasing domestic employment while
reducing balance of payments deficits (Wyman, 1994;
Tengerdy and Szakacs, 1998). Bioconversion of lignocellulose into ethanol is feasible, and enzyme-based
technologies for cellulose hydrolysis are being
developed. A critical point in these enzyme-based
technologies is the production cost of an efficient
enzyme system.
Due to its complexity, lignocellulose bioconversion
requires the action of multiple enzymes. The complete
*Corresponding author. Fax: 51 1 3495670; E-mail: mgclmb@
lamolina.edu.pe

hydrolysis of cellulose requires the action of the cellulase system containing cellobiohydrolase, endoglucanase and fl-glucosidase (Kubicek, 1992). Many
microorganisms, both fungi and bacteria can degrade
lignocellulose. In nature, lignocellutolytic microbes
interact in mixed culture to degrade lignocellulose
(e.g., wood decay) (Bayer and Lamed, 1992).
Lignocellulolytic enzymes are produced by both
bacterial and fungal fermentation in submerged or
solid substrate systems (Persson et al., 1991).
Resembling the natural habitat of filamentous fungi
growing on solid lignocellulosic particles, solid
substrate fermentation (SSF) involves the growth of
microorganisms on moist solid substrates in the
absence of free water (Murthy et al., 1993; Tengerdy,
1996). Its advantages are the low capital costs for
equipment, high volumetric productivity and decreased
operational costs. Its disadvantages are lower overall
productivity, high labor intensity and difficult operational control, but these factors are compensated in
developing countries by the low cost of starting
materials and the low cost of labor.

0960-8524/99/$ - - see front matter 1998 Elsevier Science Ltd. All rights reserved.
PII: S0960-85 24(98)00139-4

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M. Gutierrez-Correaet al./Bioresource Technology68 (1999) 173-178

Mixed culture fermentations are those in which the

inoculum consists of two or more organisms. Oriental
food fermentations are good examples of this type of
fermentation. Other examples include mushroom cultivation, composting, anaerobic digestion of organic
matter, dairy fermentation and ensiling (Meers, 1974;
Steinkraus, 1982; Wood, 1984; Hogan et al., 1989;
Fogarty and Tuovinen, 1991). Product or processspecific mixed culture fermentations have been used
for biodelignification and enzyme production (Panda et
al., 1983; Duff, 1985; Duff et al., 1987; Wan Yusoff and
Thayan, 1991; Castillo et al., 1994; Arora, 1995;
Dueflas et al., 1995; Asiegbu et al., 1996; GutierrezCorrea and Tengerdy, 1997, 1998a, 1998b).
A series of experiments with mixed culture SSF have
been reported from this laboratory, indicating that
strain compatibility and nutritional status are determining factors for mixed culturing (Castillo et al., 1994;
Duefias et al., 1995; Gutierrez-Correa and Tengerdy,
1997, 1998a, 1998b). The most important observation
was that synergistic interactions between compatible
partners may overcome nutritional limitations in poor
agricultural residues. In this paper earlier observations
were corroborated, and strain compatibility was reexamined, using a parent-mutant pair of Trichoderma
reesei strains, and a different cooperating partner than
in earlier experiments.

2. Methods

2.1. Substrate treatment

Sugar cane bagasse was kindly provided by Sociedad

Paramonga Ltd (Peru). This material was thoroughly
washed, dried and milled to 1-2 mm particle size using
a Wiley mill. The milled bagasse was mixed with 0.12 g
NaOH/g dry weight (DW) and autoclaved at 121C for
20min. After autoclaving, the treated bagasse was
washed with tap water, then distilled water until
neutrality, and then dried at 80C.
2.2. Microorganisms and inoculum

The strains Trichoderma reesei LM-UC4 (Julca and

Gutierrez-Correa, 1987), its mutant LM-UC4E1
(Gutierrez-Correa and Tengerdy, 1997), and Aspergillus
niger ATCC 10864 (Castillo et al., 1994) were used in
this work. Stock cultures were maintained on potato
dextrose agar slants. Spores were washed from a 7-day
agar slant culture with 10 ml sterile 0.01% Tween 80
solution, and 2 ml aliquots (10 v spores/ml) were added
to each of several 250ml shake flasks containing
100 ml basal salt solution (Reese and Mandels, 1966)
supplemented with 0.2% glucose, 0.2% peptone, 0.4%

treated bagasse and 0.01% Tween 80. The inoculated

flasks were incubated at 30C and 200 rpm for 48 h.
2.3. Solid substrate fermentation

Before fermentation, the C:N ratio of treated

bagasse was adjusted to 10:1 by the addition of an
adequate nitrogen source. Either 0.14g ammonium
sulfate + 0.02 g urea, or 2.48 g soymeal was added to
3 g treated bagasse, and moistened with the basal salt
solution to 80% moisture content. This substrate in a
250 ml flask was autoclaved at 121C for 15 min.
Each flask was then inoculated with 1.5% (w/w) T.
reesei, either LM-UC4 or LM-UC4E1 mycelial
inoculum, and incubated at 30C in a 95% relative
humidity chamber. Based on earlier determination of
optimal inoculation procedure (Castillo et al., 1994;
Duefias et al., 1995), A. niger (1%, w/w) mycelial
inoculum was added to each flask at 36 h for soyl-neal
supplemented bagasse and at 48h for ammonium
sulfate and urea supplemented bagasse. Single cultures
of the three strains were used as control. Triplicate
flasks were set up for each experimental variation.
2. 4. Sample processing

The contents of three flasks for each time point were

thoroughly blended in either 30 ml (ammonium sulfate
supplement) or 50ml (soymeal supplement) distilled
water by shaking for 30 min at 150 rpm, filtered and
the liquid part was used for enzyme determinations.
The solids were collected for dry matter and biomass
determinations. Dry weight loss was determined after
drying the solids in an oven at 80C for 48 h.
2.5. Biomass determination

Fungal biomass was estimated indirectly by determining either the mycelial protein content (inorganic
nitrogen) or the glucosamine content (soymeal).
Mycelial protein was determined as reported by Snyder
and Desborough (1978). Glucosamine was measured
according to Arima and Uozumi (1967) on dried solids
processed as reported by Desgranges et al. (1991). The
glucosamine content was related to mycelial protein by
the following correlation:
y=0.13x-2.38

(r =0.96)

where y is the mycelial protein content (% DW) and x

is the glucosamine content (mg/g DW) (GutierrezCorrea and Tengerdy, 1998a). Finally, fungal biomass
was estimated by multiplying the mycelial protein
content by 2.5 (Duefias et al., 1995).

M. Gutierrez-Correa et al./Bioresource Technology 68 (1999) 173-178

2.6. Enzyme assays

Endoglucanase activity (EG) and filter paper activity
(FPA) for cellulase were measured according to
Mandels et al. (1975). /3-glucosidase activity (BG) was
measured as reported by Kubicek (1983). One international unit (IU) of enzyme activity was defined as the
amount of enzyme that releases 1/zmol product per
min (glucose equivalents for FPA and EG, and
p-nitrophenol for BG).

175

partner). The corresponding specific enzyme activities

improved even more, indicating that apparent synergistic interactions in co-culturing helped enzyme
production more than growth.

0,3

3. R e s u l t s a n d d i s c u s s i o n

(A)

0,2

~o0,1

Since previous research indicated that strain

compatibility is an important consideration in mixed
culturing, in this series of experiments the Trichoderma
strains were co-cultured with A. niger instead of A.
phoenicis.

24

48

72

96

120

144

Hours

3.1. Fungal growth in mixed culture

0,3

Mixed culturing slightly improved fungal growth with

both Trichoderma strains (Fig. 1, Table 1). Soymeal
supplement also gave better growth, both in single and
mixed cultures. The maximum increase (30%) was
obtained in the mixed culture of T. reesei LM-UC4E1
and A. niger with soymeal supplement.

0,2

0,1

3.2. Enzyme production

.o

The benefit of mixed culturing was clearly

manifested only with the inorganic N-source. The
mutant strain was more responsive to mixed culturing
than the parent strain (Fig. 2, Table 1). Although
biomass production increased only by 10% in such a
mixed culture, cellulase activity increased by 63%,
endoglucanase activity by 85% and /3-glucosidase
activity by 147% (the latter largely due to the A. niger

24

48

72

96

Hours

Fig. 1. Growth kinetics of T. reesei LM-UC4 ([], l) or T. reesei

LM-UC4E1 (A, A) with A. niger () in single (open symbols) and
mixed (closed symbols) culture SSF on bagasse supplemented with
ammoniumsulfate and urea (A) or soymeal(B).

Table 1
Maximum growth and enzyme activities in mixed culture solid substrate fermentation on sugar cane bagasse
Cultures

T. reesei LM-UC4
T. reesei LM-UC4E1
A. niger
T. reesei LM-UC4 andA. niger
T. reesei LM-UC4E1 andA. niger

Nitrogen
supplement

Inorganic
Soymeal
Inorganic
Soymeal
Inorganic
Soymeal
Inorganic
Soymeal
Inorganic
Soymeal

Time
(h)

120
72
120
72
120
72
120
72
120
72

Biomass
(g/g DW)

0,17
0,20
0.20
0,21
0,15
0,16
0,20
0.21
0.22
0.27

Enzyme activities

Specific enzyme activities

(IU/g substrate DW)

(IU/g biomass)

FPA

EG

BG

FPA

EG

BG

6.5
10.0
9.0
13.0
5.6
6.4
8.5
10.7
14.7
15.5

57.2
58.7
70.4
61.2
65.8
51.4
89.5
63.2
129.0
73.4

7.7
19.5
8.8
25.0
13.2
27.3
17.2
18.3
21.7
23.4

38.2
50.0
45.0
61.9
37.3
40.0
42.5
50.9
66.8
57.4

336.5
293.5
352.0
291.4
438.7
321.3
447.5
300.9
586.4
271.8

55.0
97.5
44.0
119.0
88.0
170.6
86.0
87,1
98.6
86,7

176

M. Gutierrez-Correa et al./Bioresource Technology 68 (1999) 173-178

The rich organic N-source, soymeal, in contrast,

helped only growth but not enzyme production, with
correspondingly low specific enzyme activities in mixed
cultures. It appears that a nutritionally poorer substrate
is actually more suitable for mixed culturing, at least
for cellulase production.
Soymeal, however, increased xylanase production in
mixed cultures (Gutierrez-Correa and Tengerdy,
1998a). The different nutritional responses of mixed
cultures to cellulase and xylanase production may be
due to different enzyme regulations by nutrient components (Coughlan and Hazelwood, 1993; Haltrich and
Steiner, 1994), and to complex synergistic interactions

i--

in co-culturing (Stahl and Christensen, 1992; White

and Boddy, 1992).
The somewhat conflicting results in the literature on
the effect of nutrients on mixed culturing indicate the
need for a careful scrutiny of nutrients, fermentation
conditions and microbial interactions on a case-by-case
basis (Wan Yusoff and Thayan, 1991; Madamwar and
Patel, 1992; Castillo et al., 1994).
A nutrient sparing effect is evident particularly for
the case of the T. reesei LM-UC4E1 and A. niger interaction in cellulase production. The reason may be
symbiotic associations in mixed cultures that not only
overcome but overcompensate for nutrient limitations

16

16

12
8

(B)

J
0
0

140
13 120
100
80
r=

60

40
20
0

O
"O
fLU

24

48

72 96 120 144
Hours

24

48
Hours

72

i -

96

24

48

72

96

80
70
60

/~--._~

(A)

-- 50

40
30
20
10
0

-r

24

48

72

96

120 144

Hours

Hours

--- 30

~- 25
t-t
20

(B)

~ 20
-o 10

?,

0
0

24

48

72
Hours

96

120 144

24

48
Hours

72

96

Fig. 2. Time course profiles of enzyme production by single and mixed culture SSF on bagasse supplemented with ammonium sulfate and urea
(A) or soymeal (B). Symbolsas in Fig. 1.

177

M. Gutierrez-Correa et aL/Bioresource Technology 68 (1999) 173-178

Table 2
Comparison of maximum growth and enzyme activities in mixed culture solid substrate fermentation on sugar cane bagasse
Cultures

T. reesei LM-UC4 and A. niger

T. reesei LM-UC4E1 and A. niger
T. reesei LM-UC4 and A. phoenicis
T. reesei LM-UC4E1 and A. phoenicis
T. reesei LM-UC4 and A. phoenicis

Nitrogen
supplement

Inorganic
Soymeal
Inorganic
Soymeal
Inorganic
Inorganic
Inorganic
Soymeal

Biomass
(g/g DW)

0.20
0.21
0.22
0.27
0.35
0.29
0.20
0.23

Reference

Enzyme activities
(IU/g substrate DW)
FPA

EG

BG

8.5
10.7
14.7
15.5
13.4
9.7
11.0
9.4

89.5
63.2
129.0
73.4
73.8
20.0
113.2
48.3

17.2
18.3
21.7
23.4
18.1
8.5
18.0
17.9

(1)
(1)
(2)
(2)
(3)

(1) This work; (2) Gutierrez-Correa and Tengerdy (1997); (3) Gutierrez-Correa and Tengerdy (1998b).

in t h e substrate. This n u t r i e n t s p a r i n g effect gives a

solid e c o n o m i c a d v a n t a g e to m i x e d culturing over
single culturing for e n z y m e p r o d u c t i o n on n u t r i e n t limited agricultural waste p r o d u c t s , saving the cost of
expensive o r g a n i c s u p p l e m e n t s .
The maximum biomass production and enzyme
activities o b s e r v e d in p r e v i o u s a n d c u r r e n t e x p e r i m e n t s
are c o m p a r e d in T a b l e 2. T h e n u t r i e n t s p a r i n g effect is
c o m p a r a b l e with e i t h e r A . niger o r A . p h o e n i c i s as
c o o p e r a t i n g p a r t n e r s . Strain compatibility, however, is
an i m p o r t a n t m o d i f y i n g factor. F o r the p a r e n t strain,
A . p h o e n i c i s was a b e t t e r p a r t n e r for the m u t a n t A.
niger. Strain c o m p a t i b i l i t y is a critical f a c t o r in m i x e d
culturing, a n d has to b e e s t a b l i s h e d case by case for
e a c h application.

3.3. Conclusion

It has b e e n e s t a b l i s h e d in this r e s e a r c h that strain

c o m p a t i b i l i t y a n d n u t r i t i o n a l status o f the s u b s t r a t e a r e
d e t e r m i n i n g factors for successful m i x e d c u l t u r e
f e r m e n t a t i o n . T h e m o s t i m p o r t a n t conclusion f r o m
these results is that m i x e d culturing is a d v a n t a g e o u s in
n u t r i e n t - l i m i t e d conditions, w h e r e symbiotic associations m a y o v e r c o m e such limitations. In p r a c t i c a l t e r m s
it m e a n s that c h e a p e r m e d i a m a y b e u s e d for e n z y m e
p r o d u c t i o n by m i x e d cultures t h a n by single cultures,
w i t h o u t sacrificing e n z y m e yields.

Acknowledgements
This r e s e a r c h was s u p p o r t e d by grants f r o m the
Multinational Project of Biotechnology of the Organization o f A m e r i c a n States a n d by N S F g r a n t
INT-9214903. T h e a u t h o r s wish to t h a n k M r M a r i o
G a r c i a for his technical assistance.

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