Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

Article (PDF Available)inAnnals of Microbiology 63(2) · July 2012with168 Reads
DOI: 10.1007/s13213-012-0502-z
Abstract
The effects of cultural parameters such as carbon and nitrogen source and environmental factors including tem-perature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO 3 , peptone and NH 4 SO 4 as the nitrogen sources in fed-batch and batch cultures . Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.
ORIGINAL ARTICLE
Comparative mycelial and spore yield by Trichoderma viride
in batch and fed-batch cultures
Abiodun A. Onilude & Bukola C. Adebayo-Tayo &
A. Olubusola Odeniyi & Damilola Banjo &
Emmanuel Oluwaseun Garuba
Received: 22 December 2011 / Accepted: 20 June 2012
#
Springer-Verlag and the University of Milan 2012
Abstract The effects of cultural parameters such as carbon
and nitrogen source and environmental factors including tem-
perature and pH were investigated on spore and mycelial yield
of Trichoderma viride, which has potential as a biocontrol agent
against species of Fusarium in batch culture and fed-batch
culture where there was limiting nutrient. The results obtained
indicated tha t growth and sporulat ion of T. viride were greatly
influenced by various carbon and nitrogen sources, and by
environmental factors such as pH and temperature. Mannitol,
wheat bran and rice bran as sole carbon sources appear to
stimulate high mycelial growth and spore yield in fed-batch
culture.GrowthandsporulationwerealsofavouredbyNaNO
3
,
peptone and NH
4
SO
4
as the nitrogen so urces in fed-batch and
batch cultures
.
Maximum growth and sporulation was between
pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good
for mycelium growth of T. viride while temperatures between
30 to 45 °C were good for sporulation. The amoun t of spore and
mycelium produced and the time required for attainment of
maximum spore yield increased with increasing carbon and
nitrogen source in ba tch culture. The final spore yield obtained
in fed-batch culture was two times higher than the apparent
spore-carrying capacity of batch culture. These results show
that T. viride is capable of growing and sporulating with varied
nutritional and environmental conditions, and, therefore, this
strain of T. viride may be useful as a biocontrol agent under
diverse physiological and environmental conditions.
Keywords Trichoderma viride
.
Spore yield
.
Biocontrol
agent
.
Mycelial growth
.
Carbon
.
Nitrogen
Introduction
The use of biological control agents (BCAs) in which organ-
isms play an important role is increasingly replacing chemical
means of disease control (Whipps and Lumsden 2001). Of the
various groups of organisms used, the fungal-based BCAs
have gained wide acceptance next to bacteria (mainly
Bacillus thuringiensis) primarily because of their broader
spectrum in terms of disease control and high production yield
(Coppings and Menn 2000). Of the various fungi used,
Trichoderma sp. have been the cynosure of many researchers
who have been contributing to the pursuit of biological control
through the use of fungi (Ahmed and Baker 1987;Benhamou
and Chet 1993). This is because it can easily establish itself in
different pathosystems, has moderate effects on soil balance
and does not harm beneficial organisms that contribute to
pathogen control. Furthermore, it has no known harmful effect
on humans, wildlife and other beneficial organisms (Whipps
and Lumsden 2001).
In the use of the various BCAs (Trichoderma sp. inclusive),
spores are the most useful propagule (Churchill 1982). These
must then be processed in large quantities quickly, inexpen-
sively and efficiently, if BCAs are to be able to compete
favourably with chemical control agents (Lisansky and Hall
1983). Fungal spores are normally mass-produced in large
liquid culture fermentation (Churchill 1982), and information
on the effects of manipulating liquid culture conditions to
maximize the efficiency of spore production is of potential
value. Reduction of the mycelium in the liquid culture would
also be desirable, since it creates separation and disposal
problems (Cascino et al. 1990).
A. A. Onilude
:
B. C. Adebayo-Tayo
:
A. O. Odeniyi
:
D. Banjo
Microbial Physiology and Biochemistry Laboratory, Department
of Microbiology, University of Ibadan,
Ibadan, Nigeria
E. O. Garuba (*)
Department of Biological Sciences, Bowen University,
Iwo, Nigeria
e-mail: oluwaseungaruba@live.com
Ann Microbiol
DOI 10.1007/s13213-012-0502-z
There is, however, a paucity of information on the studies
regarding culture conditions like fed-batch culture in a fer-
menter that allo w finer control of substrate concentration,
solids concentration, C:N ratio, the proportion of total bio-
mass produced as spores (the relative spore yield) and
single/multiple nutrient addition. Investigation of these
parameters in relation to sporulation is thus necessary in
order to maximize spore production by Trichoderma sp.
for use as a BCA. This paper therefore reports on limiting
conditions which affect spore formation in batch and fed-
batch cultures and which may be adapted t o large-scale
fermentations.
Materials and methods
Microorganism
Trichoderma viride with antagonistic effect on species of
Fusarium, especially Fusarium solani (unpublished data)
used in this study was obtained from The Culture
Collection C entre of The Department of Microbiology
University of Ibadan, Nigeria.
Inoculum preparation
Spores used as inoculum were prepared according to the
method of Nahar et al. (2008).
Batch culture fermentat ion
Batch fermentation was in 250-ml Erlenmeyer flasks con-
taining 50 ml of the liquid medium described by Al-Taweil
et al. (2009), which contains (g/l) ammonium chloride (2.0),
sodium potassium tartrate (2.0), MgSO
4
7H
2
O (4.0),
K
2
HPO
4
(14.0), CaCl
2
(0.2), KH
2
PO
4
(4.0), yeast extract
(4.5), trace element (2.0 ml), [ZnSO
4
.7H
2
O (0.0014),
FeSO
4
.7H
2
O (0. 005), MnSO
4
(0.0016), CoCl
2
(0.002)],
glucose (7.5), NaNO
3
(6.0), and corn steep liquor (5 .0).
The liquid medium was adjusted to pH 5.5 using citrate
buffer. The medium was inoculated with 1 ml suspension
of the spores of Trichoderma viride and incubated at ambi-
ent temperature in static mode for 7 days.
Fed-batch experiments
Fed-batch experiments were done using the fermentation
medium above and inoculated with 1 ml of the spore sus-
pension and incubation carried out at ambient temperatures
in the dark for 7 days. Then, 4 ml of the limiting nutrient,
which was yeast extract (0.05 mg/ml), was added every
12 h. Each fed-batch culture was sampled periodical ly for
spore counting by aseptically removing 4 ml of the culture
with a sterile syringe every 12 h. At the final harvest, the
spore concentrations of 50 ml subsamples were determined.
Determination of spore concentration
For the determination of the spore concentration, the content
of the flasks was filtered through a sterilized double-layer
muslin cloth to separate th e harvestable spores from the
mycelium. The stock su spension was kept in a Rotary
Shaker Flask for 2 min, and 3 ml of the suspension was
added into a cuvette. The equipment was calibrated with
3 ml of blank solution (liquid medium). The spore count
was determined at a wavelength of 550 nm using a Perkin
Elmer Lambda 25 UV Spectrophotometer.
Determination of mycel ia weight
The modified method of Al-Taweil et al. ( 2009) was used to
determine the fung al biomass with the my celium being
filtered through a pre-weighed muslin cloth. It was washed
two times with distilled water. The washed mycelium was
dried at 80 °C to constan t mass, and dry weight was calcu-
lated by difference.
Effect of different carbon sources on fungal mycelium
and spore yield
The carbon sources used in this study were glucose, mannitol,
starch, wheat bran and rice bran. The fermentation medium
was supplemented with each of the carbon sources at the rate of
(g/l) 2.5, 5.0, 7.5, 10.0, and 15.0 in 250-ml Erlenmeyer flasks
separately before autoclaving. The pH of the liquid medium in
each flask was adjusted using citrate buffer to pH 5.5.
Thereafter, sterilisation was carried out and the liquid medium
was inoculated with 1 ml of spore suspension of Trichoderma
viride in both batch and fed-batch cultures. The flasks were
incubated at room temperature for 7 days after which the
mycelium weight and spore concentration was determined.
Effect of different nitrogen sources on fungal mycelium
and spore yield
The nitrogen sources investigated were NaNO
3
,NH
4
SO
4,
peptone, soy meal preparation and casein. The nitrogen
sources were supplemented at the rate of (g/l) 1.0, 3.0, 5.0,
7.0, and 9.0 in sets of 250-ml Erlenmeyer flasks containing
50 ml of liquid medium. The pH of the liquid medium was
adjusted as de scribed e arlier. Thereafter, sterilisation was
carried out and the liquid medium was inoculated with
1 ml of sp ore suspension of Trichoderma viride in both
batch and fed-batch cultures. Flasks were incubated at room
temperature for 7 days after which the spore concentration
and mycelial weight were determined.
Ann Microbiol
Effect of pH on fungal mycelium and spore yield
Different pH levels selected for the study were 3.0, 3.5, 4.0,
5.0, 5.5 and 6.0, and 50 ml of liquid medium were prepared in
sets of 250-ml Erlenmeyer flasks. The pH of the medium was
adjusted with citrate buffer in triplicate sets before autoclav-
ing. After autoclaving, the cooled medium was inoculated and
incubated as described earlier. Mycelial weight and spore
concentration were determined as previously described.
Effect of temperature on the spore yield and mycelium
growth
Fifty millilitres of the liquid medium adjusted to pH 5.5 using
citrate buffer was dispensed into 250-ml Erlenmeyer flasks
and sterilized. The flasks were then each inoculated with 1 ml
of spore suspension of Trichoderma viride. Four different
incubation temperatures, 25, 30, 37 and 45 °C, were used to
cultivate the Trichoderma viride for spore production in both
batch and fed-batch cultures for 7 days so as to study the effect
of temperature on the spore yield and mycelium growth.
Results
The results of the effects of different carbon sources on spore
yield and mycelia production in batch and fed-batch experi-
ments are presented in Figs. 1 and 2. The effects of different
carbon sources on spore formation in batch cultures revealed
that mannitol at a concentration of 15.0 g/l supported the
highest spore yield of 0.69±0.15 SEM) followed by starch
at 7.5 g/l with 0.52±0.08 and glucose with 0.48±0.01 at 5.0 g/
l. The lowest spore yield of 0.15±0.03 was recorded when
2.5 g/l starch concentration was used as the sole carbon source
(Fig. 1a). Similarly, mannitol at 15.0 g/l gave the highest
mycelial weight of 19.63±0.43 g/l closely followed by
10.0 g/l mannitol concentration (15.51 g/l mycelial weight)
and starch (14.81±0.72 g/l mycelial weight) at 15.0 g/l con-
centration. The lowest mycelial weight of 6.78 g/l recorded in
this study was from 2.5 g/l wheat bran concentration (Fig. 1b).
Results of Fed-batch experiments are presented in Fig. 2a, b
and revealed that wheat bran at 7.5 g/l supported the highest
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Glucose Mannitol Starch Rice bran Wheat bran
Absorbance (550nm)
Carbon sources
Glucose Mannitol Starch
Rice branWheat bran
Carbon sources
a
2.5g/l
5.0g/l
7.5g/l
10.0g/l
15.0g/l
0
5
10
15
20
25
Mycelium growth (g/l)
b
2.5g/l
5.0g/l
7.5g/l
10.0g/l
15.0g/l
Fig. 1 Effect of carbon sources on spore yield (a)andmycelium
growth (b)byTrichoderma viride after 7 days in batch culture. Data
are given as means ± SEM, n0 3
Glucose Mannitol Starch Wheat
bran
Ricebran
Carbon sources
0
0.5
1
1.5
2
2.5
3
Glucose Mannitol Starch Wheat
bran
Ricebran
Absorbance (550nm)
Carbon sources
a
2.5g/l
5.0g/l
7.5g/l
10.0g/l
15.0g/l
0
2
4
6
8
10
12
Mycelium growth (g/l)
b
2.5g/l
5.0g/l
7.5g/l
10.0g/l
15.0g/l
Fig. 2 Effect of carbon sources on spore yield (a)andmycelium
growth (b)byTrichoderma viride after 7 days in fed-batch culture.
Data are given as means ± SEM, n0 3
Ann Microbiol
spore yield of 2.48±0.01 closely followed by glucose (2.37±
0.07 at 2.5 g/l). Glucose is followed by starch (2.15±0.12 at
5.0 g /l) while the least sp ore produced (0.64±0.04) was
recorded in mannitol-containing medium at a concentration
of 15.0 g/l (Fig. 2a). Rice bran at 15.0 g/l stimulated the
highest mycelial weight of 10.41 g/l followed by rice again
at a concentration of 5.0 g/l (10.22±0.13 g/l). Wheat bran
extract at 15.0 g/l also gave a high mycelial weight of 9.95 g/
l while a 2.5 g/l starch concentration stimulated the least
amount (3.26 g/l) of mycelia in this study (Fig. 2b).
Effect of nitrogen sources
Results of the investigation of the effects of different nitrogen
sources on spore yield and mycelia production by T. viride
used in this study are presented in Fig. 3a, b for batch cultures
and revealed that the medium containing NaNO
3
as the
nitrogen source appears to stimulate the highest spore yield
at all concentrations relative to all other nitrogen sources
investigated. At 1 %, NaNO
3
gave a spore yield of 1.81±
0.05 followed by 0.65±0.06 at 5 %, and 0.52±0.03 at 7 %.
Following NaNO
3
is the soy meal preparation, which gave a
spore yield of 0.48±0.07 at 5 % concentration and 0.44±0.02
at 7 %. The lowest sporeyield (0.14±0.04) was, however,
recorded in a medium contai ning NH
4
SO
4
(3 %) as the
nitrogen source. The best mycelial growth (22.08±0.45 g/l)
was produced in the medium that contained NH
4
SO
4
as the
nitrogen source, followed by peptone which gave a mycelial
growth of 22.06±0.37 g/l in batch culture (Fig. 3b). Fed-batch
experiments showed that T. viride gave the best spore yield
(3.13±0.06) in the medium that contained NaNO
3
as the
nitrogen source, followed by casein which gave a spore yield
of 1.78±0.02. The best mycelial growth (12.89±0.47 g/l) was
produced in the medium that contained peptone as the nitro-
gen source, followed by NaNO
3
whichgaveamycelial
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Casein NaNO
3
NaNO
3
NH
4
SO
4
NH
4
SO
4
SMP Peptone
Absorbance (550nm)
Nitrogen sources
Casein SMP Peptone
Nitro
g
en sources
a
1.0g/l
3.0g/l
5.0g/l
7.0g/l
9.0g/l
0
5
10
15
20
25
Mycelium growth (g/l)
b
1.0g/l
3.0g/l
5.0g/l
7.0g/l
9.0g/l
Fig. 3 Effect of different nitrogen sources on spo re yield (a) and
mycelium growth (b)byTricho derma viride after 7 days in batch
culture. Data are given as means ± SEM, n0 3
0
0.5
1
1.5
2
2.5
3
3.5
Casein NaNO
3
NH
4
SO
4
SMP Peptone
Aabsorbance (550nm)
Nitrogen sources
Casein NaNO
3
NH
4
SO
4
SMP Peptone
Nitro
g
en sources
a
1.0g/l
3.0g/l
5.0g/l
7.0g/l
9.0g/l
0
2
4
6
8
10
12
14
Mycelium growth (g/l)
b
1.0g/l
3.0g/l
5.0g/l
7.0g/l
9.0g/l
Fig. 4 Effect of different Nitrogen sources on spore yield (a) and
mycelium growth (b)byTrichoderma viride after 7 days in fed-batch
culture. Data are given as means ± SEM, n0 3
Ann Microbiol
growth of 9.32±0.29 g/l in fed-batch culture. Soy meal prep-
aration gave the least spore yield of 1.04±0.03 and mycelial
growth of 6.25±0.62 g/l (Fig. 4a and b).
Effect of medium pH on spore yield and mycelia formation
Five different initial pH levels, 3.5, 4.0, 5.0, 5.5, and 6.0,
were established in the liquid medium and the fermentation
was carried out for 7 days in both batch and fed-batch
cultures. Results obtained are shown in Fig. 5a, b.
Optimum pH recorded in this study for spore yield in batch
culture by T. viridae wa s pH 4 (0.80±0.10). This was
followed by pH 5 which gave a spore concentration of
0.58±0.07 while the least spore yield was recorded (0.19±
0.09) at pH 3 and 3. 5. The highest mycelial yield of 15.13±
0.02 g/l was obtained at pH 5.0 followed by a mycelial
weight of 14.43±0.09 g/l at pH 4, while the lowest mycelial
weight of 10.4±0.10 g/l was recorded at pH 6 (Fig. 5b).
However, in fed-batch culture, pH 6 was the optimum pH
for spore yield (1.62±0.06) followed by pH 4 (1.46±0.03),
and the lowest spore yield (0.28±0.05) was recorded at pH
3.0 (Fig. 5a), while pH 4 was the optimum pH for mycelium
growth (13.79±0.04 g/l) followed 11.85±0.04 g/l at pH 5.T.
viride gave the lowest mycelial weight of 4.49±0.03 g/l at
pH 6.0 (Fig. 5b).
Effect of incubation temperature on spore yield and mycel ial
growth
The results of the investigation into the effects of t emper-
ature on mycelial growth and soprulation by T.viride in
both batch and fed-batch cultures are presented in Fig. 6a,
b. The results showed that, in batch cultures, 25 ° C was
optimum for spore production with a spore yield of 0.54±
0.02. This is closely followed by 30 °C which gave a
spore yield of 0.50±0.06, while the least spore yield (0.34
±0.0 6) was r ecorded at 45 °C (Fig. 6a). The highest
mycelial weight (12.61±0.08 g/l) was obtained at 30 °C
followed by 11.96±0.08 g/l at 37 °C, and the least myce-
lial we ight of 5.66±0.03 g/l was obtaine d at 45 °C
(Fig. 6b). In fed-batch cultures, the optimum temperature
for spore production was f ound to be 45 °C with a spore
yield of 1.84±0.06 followed by 0.98±0.01 at 37 °C, while
the least spore yield (0.56±0.05) was recorded at 25 °C
(Fig. 6a). Mycelia growth in fed-batch cultures revealed
that a temperature of 30 °C is optimum for mycelia
production ( 13.27±0.01 g/l) followed by 37 °C (11.70±
0.06 g/l) while temperature of 45 °C stimulated the lowest
mycelial growth of 8.51±0.2 g /l (Fig. 6b).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
123456
(
Absorbance (550nm)
pH
a
Batch cultures
Fed-batch cultures
0
2
4
6
8
10
12
14
16
33.54 55.56
Mycelium growth (g/l)
p
H
b
Batch cultures
Fed-batch cultures
Fig. 5 Effect of initial pH on spore yield (a) and mycelial growth (b)
by Trichoderma viride after 7 days in batch and fed-batch cultures.
Data are given as means ± SEM
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
25 30 37 45
Absorbance (550nm)
Temperature (°C)
a
Batch cultures
Fed-batch cultures
0
2
4
6
8
10
12
14
25 30 37 45
Mycelium weight (g/l)
Tem
p
erature (°C)
b
Batchcultures
Fed-batch cultures
Fig. 6 Effect of incubation temperature on spore yield (a) and myce-
lial growth (b)byTrichoderma viride after 7 days in batch and fed-
batch cultures. Data are given as means ± SEM
Ann Microbiol
Discussion
The results of this study showed that this Trichoderma viride
has the ability to use a variety of carbon and nitrogen sources
for mycelial growth and spore production at different levels.
This ability has also been reported by Papavizas (1995). This
ability has been suggested as the main reason for the ubiqui-
tous nat ure of Trichoderma sp. (Roussos et al. 1991).
Mannitol stimulating the highest mycelial weight and spore
yield by T. viride in this study has also been reported by
Jayaswal et al. (2003) in batch culture. This could be as a
result of it being easily transported across the cell membrane
and oxidizing to generate energy (Schlegel 2002). Abundant
mycelial growth and sporulation on medium containing wheat
bran and rice bran in fed-batch cultures observed in this study
are in agreement with the reports of Roussos et al. (1991),
Ibrahim and Low (1993) and Sharma et al. (2002). Wheat bran
and other agro-industrial residues contain an adequate amount
of other nutrients like protein, fats, fibre, ash, Ca, Mg, P, K,
etc. with various amino acids and porosity for oxygen supply
which also help in growth and sporulation.
For optimum spore and mycelial production, NaN O
3
and
NH
4
SO
4
were found to be appropriate relative to other
nitrogen sources investigated in batch cultures, while
NaNO
3
and peptone were found appropriate for spore and
mycelium, respectively, in fed-batch cultures. This could be
as a result of the ease with which these compounds diffuse
quickly into the cells (Nicholas 1 965). Better growth of T.
viride with ammonium sulphate and other forms of inorgan-
ic nitrogen could also be due to the fact that uptake of
ammonium nitrogen reduces the pH of the surroundings,
thereby creating a sli ghtly acidic pH which is ideal for
fungal growth (MacNish 1988). Ammonium sulphate and
sodium nitrate supporting sporulation and mycelial growth
may be important when considering the use of this T. viride
as biocontrol agent in relation to agricultural practices, be-
cause of the use of ammonium and other inorganic fertil-
izers. The use of peptone which also supported high
mycelial yield has been reported by Esan and Oancea
(2010). This could be attributed to it being a complex
mixture of peptides and amino acids containing some
water-soluble vitamins (Cochrane 1958).
The results of the study carried out on the effect of initial
pH on spore yield of T. viride in batch cultures showed that
pH 4 was the optimum pH for spore yield while pH 5 gave
the maximum mycelial growth, and pH 6 was optimum for
spore yield and pH 4 was optimum for mycelial growth in
fed-batch. Generally, T. viride has been reported to grow and
sporulate well between pHs 4 and 6 (Aube and Gagnon
1969; Lewis and Papavivaz 1983; Bastos 2001; Steyaert et
al. 2010). The pH of the growth medium has been identified
as a factor which affects the permeability of the cell wall.
Hence, maximum growth and sporulation at the optimum
pH could be because the permeability of the cell wall
reaches its optimum allowing the easy diffusion of nutrients
needed for growth into the cell Grffin (1994)
Investigation of the effect of temperature showed that this
T. viride has a broad range of temperature tolerance as
regards growth and sporulation. A similar observation was
recorded by Jayaswal et al. (2003). Maximum spore yield
and mycelial growth at the optimum temperatures in both
batch and fed-batch cultures could be because it also affects
their metabolic activity especially the production of volatile
antibiotics and enzymes (Tronsmo and Dennis 1978).
In this present study, the fed-batch cultures were initiated
with a high s pore density as a convenient experim ental
starting condition, and were established to meet the require-
ment for a continuous flux of subst rates through a limiting
substrate (yeast extract) pool, by adjusting the rate of sub-
strate input to be lower than the approximately maximum
(substrate-unlimited) rate of substrate demand by the fungal
population. The yeast extract of the medium composition
has been reported to be essential for microbial cultivat ion as
it offers some additional growth factors like vitamins and
amino acids, as well as some organic nitrogen compounds
with high bioavailability (Esan and Oancea 2010). Mycelial
growth and sporulation observed in both batch (when avail-
able nutrient was non-limiting) and fed-batch cultures (when
available nutrient was limiting) in this study have also been
reported by Morton (1961). According to him, the most
general condition for induction of sporulation is the reduc-
tion or exhaustion of assimilable nitrogen while carbohy-
drate is still available. Andrew and Harris (1997), explained
that the sporulation initiat ed response to nutrient limitation
involves reorganization of the endogenous resource as well
as the use of exogenous substrate.
Conclusively, this work demonstrated the possible produc-
tion of spores of T. viride in fed-batch cultures using cheap and
readily available raw materials. This is of great importance
when considering the production of T. viride for use as a
biocontrol agent. However, scale-up trials using small- and
large-scale bioreactors under the conditions we have found
optimal is recommended to determine the suitability of the
organism (in terms of quantity) for industrial application.
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