Optimizing of Trichoderma Viride Cultivation in Submerged State Fermentation
Optimizing of Trichoderma Viride Cultivation in Submerged State Fermentation
ISSN 1546-9239
© 2009 Science Publications
Abstract: Problems statement: A study in Malaysia had shown a strain of Trichoderma viride was
isolated from the soil. Questions were raised whether Trichoderma viride submerged state
fermentation affected by parameters and which of them is effective? Approach: To investigate the
effects of the submerged state fermentation parameters; concentrations of carbon g L−1; (10, 45 and
80), glucose, nitrogen g L−1; (0.10, 0.35 and 0.60) ammonium sulfate, temperature; (20, 30 and 40),
PH; (4.0, 6.0 and 8.0) rpm. Experiments were performed in triplicate and the results were statistically
analyzed using computer software Response Surface Methodology (RSM) using a Box-Behnken
design was applied to batch cultures of T. viride, for identifying the effects of process variables (carbon
sources, nitrogen sources, temperature, RPM and PH). The fermentation was carried out in shake
flasks using a complex medium fungal biomass the mycelium was filtered through filter paper
(Whatman No. 40). It was washed first with distilled water tow times. The washed mycelium was dried
at 105±1°C to constant mass. It was placed in the desiccators and then the mass was determined.
Results: The fermentation pattern was studied to investigate the effects of the submerged state
fermentation parameters; concentrations of carbon g L−1; (10, 45 and 80) glucose, nitrogen g L−1; (0.10,
0.35 and 0.60) ammonium sulfate, temperature; (20, 30 and 40), PH; (4.0, 6.0 and 8.0) rpm; for
Trichoderma biomass production for biotechnological uses (biocontrol agent). Optimum parameters
and maximum biomass production were studied. The maximum biomass production of 13.6 g mL−1
mycelium was noted after 5 days. Although maximum fungal biomass presented maximum growth rate
that observed between the 3rd and 4th days of fermentation. At 3rd day 13.2 g L−1 fungal dry mass was
present, after that there was a slight decrease in the mycelial dry mass. A Box-Behnken experimental
design was used to investigate the effects of five factors; concentrations of carbon g L−1; (10, 45 and
80) glucose, nitrogen g L−1; (0.10, 0.35 and 0.60) ammonium sulfate, temperature; (20, 30 and 40), PH;
(4.0, 6.0 and 8.0), rpm; (100, 175 and 250) on the concentrations of biomass produced in batch cultures
of Trichoderma viride. Optimal medium for maximizing the production of biomass in batch cultures of
T. viride should contain 45 g L−1 C, 0.35 g L−1 N, 30 Temp, 175 rpm and Ph 6 for 5 days fermentation.
Optimization of Trichoderma cultivation in submerged state fermentation to produce the optimum
biomass as stage of biocontrol agent and biofertilizer production which made the production line more
significant. Conclusion: Based on a statistically designed search, results indicated that an optimal
medium for maximizing the production of biomass in batch cultures of T. viride should contain
45 g L−1 C, 0.35 g L−1 N, 30 Temp, 175 rpm and Ph 6. This composition can yield the optimum
biomass 5 days of culture. The identified optimal medium is rich in carbon but provided a limiting
level of nitrogen.
Corresponding Author: Mohammad Bin Osman, School of BioSciences and BioTechnology, Faculty Science and Technology,
University Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia Fax: +603-89293808
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counterparts (virus, bacteria, nematodes and Medium composition, namely Tween 80, also
protozoa[3,4]. Their capability to synthesize antagonistic influences the morphology of T. reesei Rut C-30 and
compounds (proteins, enzymes and antibiotics) and enzyme production. The presence of Tween 80 in
micro-nutrients (vitamins, hormones and minerals) fermentation medium inhibited the pellet formation of
enhance their biocontrol activity. this strain [2]. The optimum conditions for cellulase
Like other fungal BCAs, conidial mass of production were (NH4)2SO4, 0.5 g L−1 as nitrogen
Trichoderma is the most proficient propagule, which source, pH (5.0), temperature (28°C) and incoulum size
tolerates downstream processing (e.g., air drying). (108 spores mL−1). Tween 80 (0.1%) improved the
Despite the advantages, mass production of overall cellulase production as under these optimized
Trichoderma BCAs is less prevalent, owing to high- conditions[7].
cost raw materials like Mendel’s medium, molasses, Trichoderma viride, a mould of family
corn steep liquor and other[8,9]. Hypocreaceae, order Hypocreales and class
Trichoderma spp. Have gained wide acceptance as Ascomycetes, is well known for the biological control
effective BCAs against several commercial and the production of cellulose and chitinase[1]. In this
phytopathogens. These antagonistic fungi are most study we reported the existence of biological agent
common among fungal biocontrol agents because of strain of T. viride.
their multiple BCA characteristics, namely, antagonism The aim of this study is to evaluate and investigate
and plant-growth stimulation[10]. Thus, mass-scale the effects of the submerged state fermentation
production of Trichoderma spp. would have great parameters; concentrations of Carbon g L−1; (10, 45 and
potential for commercial use. Micropropagules of 80) Glucose. Nitrogen g L−1; (0.10, 0.35 and 0.60)
Trichoderma spp. In the form of conidia are preferred Ammonium Sulfate. Temperature; (20, 30 and 40). PH;
over chlamydospores and mycelial biomass because of (4.0, 6.0 and 8.0). Rpm; to obtain optimal submerged
the viability and stability in field application. Therefore, culture conditions for bio-by production from
there are several BCA products of Trichoderma spp. In Trichoderma, to the best of environmental conditions
the market containing conidia of Trichoderma spp. As for submerged culture of Trichoderma viride.
active ingredients. Multiple BCA action renders the The purpose of this study is to optimize the
production of Trichoderma spp. Conidia of commercial submerged culture conditions to produce the suspension
and environmental interest. There is abundant literature by Trichoderma with respect to several operating
on the use of conventional synthetic media like glucose, variables in shake flask fermentation for industrial
cellulose, soluble starch and molasses to produce biotechnological uses as biocontrol agent on large scale.
Trichoderma spp.[5,6]. However, the cost of these raw
materials for commercial production of BCAs is one of MATERIALS AND METHODS
the major limitations behind the restricted use. To
overcome the cost limitation, many researchers have Isolation of producer microorganism: Fungal species
successfully used substrates like corn fiber dry mass, was isolated from soil samples by using a selective
sewage sludge compost and cranberry pomace. Despite medium (PDA). Samples were dusted over the plates and
the use of alternate sources, the cost of production was the plates were incubated at 30±1°C for four days. The
still high, as these raw materials need to be microbial colonies were developed which were picked up
supplemented by othernutrients[10]. Dominguesa et al.[2] and purified by streaking and incubated at 30±1°C for
found that the influence of the size binoculum and the 3 days. A green colonies forming culture with was
composition of the fermentation medium on the selected and identified to be Trichoderma viride [1]. The
morphology and cellulase production were studied. culture was maintained on potato dextrose agar slants.
Different inoculums sizes were studied but the
significative change in fungus morphology was Fermentation: The fermentation was carried out in
observed for spore’s concentration between 105 and shake flasks using a complex medium consisting of
107 spores mL−1 (i.e., 102 and 104 spores mL−1 in pre- (g L−1) ammonium tartrate, 2.0; magnesium msulphate
culture medium). In the medium without Tween 80, at heptahydrate, 4.0; dipotassium phosphate, 14.0;
low inoculum size, the majority of the pellets were calcium chloride, 0.2, NaHPO4,4.0, yeast extract, 3.0,
large and well individualized, in contrast, at higher trace elements, 2.0 mL and glucose 6.0 The flasks
inoculation densities small flocs were obtained, with containing 200 mL fermentation medium were
higher production of soluble protein and higher filter inoculated by 6 days old vegetative inoculums. The
paper activity. It was found that the average pellet size vegetative inoculums were developed from spore
seems to be inversely proportional to the inoculum size. suspension prepared from 6 days old culture slants.
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Table 1: Factor values and the responses of box design runs • Temperature : (20, 30 and 40)
Carbon Nitrogen Biomass • PH: (4.0, 6.0 and 8.0)
Run g L−1 Temperature Rpm PH g L−1 g L−1
1 10 20 250 4 0.10 7.550
• Rpm: (100, 175 and 250)
2 80 20 250 4 0.10 8.210
3 80 20 250 8 0.60 7.800 Dry cell mass determination: To determine the fungal
4 45 30 175 6 0.35 12.50
5 10 20 100 4 0.60 7.650 biomass the mycelium was filtered through filter paper
6 45 30 175 6 0.35 13.50 (Whatman No. 40). It was washed first with distilled
7 80 20 100 8 0.10 8.800 water tow times. The washed mycelium was dried at
8 10 40 100 8 0.60 8.900 105±1°C to constant mass. It was placed in the
9 80 40 100 8 0.60 13.40
10 45 30 175 8 0.35 10.50 desiccators and then the mass was determined.
11 45 30 100 6 0.35 11.32
12 10 20 250 8 0.10 7.230 Specific growth rate: To determine the specific growth
13 45 30 175 6 0.35 13.60
14 80 40 250 8 0.60 8.200 rate (m), natural log of biomass (ln X) was plotted
15 10 20 250 4 0.60 7.800 against time (t). The slope of the line at any moment
16 10 20 100 4 0.10 7.500 gives the specific growth rate at that moment.
17 45 30 175 4 0.35 13.40
18 10 20 100 8 0.60 7.000
19 45 30 175 6 0.60 13.20 Statistical analysis: Experiments were performed in
20 45 40 175 6 0.35 13.20 triplicate and the results were statistically analyzed
21 80 20 250 4 0.60 12.90 using computer software Response Surface
22 10 40 250 8 0.10 7.890
23 80 40 250 8 0.10 7.800 Methodology (RSM) using a Box-Behnken design was
24 10 40 100 4 0.60 8.200 applied to batch cultures of T. viride, for identifying the
25 45 20 175 6 0.35 9.200 effects of process variables (Carbon Sources, Nitrogen
26 10 20 250 8 0.60 9.100
Sources, Temperature, RPM and PH). And the
27 10 20 100 8 0.10 7.800
28 80 40 100 8 0.10 7.500 significant difference among replicates has been
29 45 30 175 6 0.35 13.00 presented as least significant tests in the form of
30 10 40 100 4 0.10 8.320 probability values (Table 1).
31 80 20 100 8 0.60 12.90
32 80 20 100 4 0.10 7.800
33 80 40 250 4 0.60 7.500 RESULTS
34 10 30 175 6 0.35 13.00
35 45 30 250 6 0.35 13.20
36 80 30 175 6 0.35 12.89 The rate of growth and biomass production by
37 45 30 175 6 0.35 11.00 microorganisms is quite important in understanding
38 80 20 250 8 0.10 8.200 their fermentation pattern. (Table 2) shows the rate of
39 80 40 100 4 0.60 12.90
40 10 40 100 8 0.10 7.500
production of fungal biomass during liquid media
41 10 40 250 4 0.60 8.100 fermentation by T. viride at 30±1°C for 5 days of
42 45 30 175 6 0.35 13.00 incubation. Maximum growth rate was observed
43 10 40 250 4 0.10 9.500 between 3 and 4 days of fermentation. At 3d 13.2 g L−1
44 45 30 175 6 0.35 12.80
45 45 30 175 6 0.35 13.00 fungal dry mass was present. After 3 d, there was a
46 10 40 250 8 0.60 9.600 slight decrease in the mycelial dry mass.
47 80 20 100 4 0.60 12.89 The experimental runs and results for the Box-
48 80 40 100 4 0.10 7.200
49 45 30 175 6 0.10 9.000
Behnken design are shown in Table 1. The 50 runs in a
50 80 40 250 4 0.10 7.220 single block were used to study the effects of five
factors on one response. Biomass concentration ranged
The inoculums development and the fermentation were from 7.00-13.60 g L−1. The ANOVA tables (Table 3)
carried out at 30 ±1°C, pH 7.0 with orbital shaking at give the statistical significance of the effects for
l50 rpm. biomass. With regard to biomass concentration, four
effects had P-values of less than 0.05 (Table 3),
Parameters: indicating that they were significantly different from
zero at the 95% confidence level. These effects were
• Carbon g L−1 : (10, 45 and 80) Glucose the nitrogen concentration, carbon concentration, the
• Nitrogen g L−1: (0.10, 0.35 and 0.60) ammonium quadratic effect of carbon concentration and the
sulfate interaction between carbon and nitrogen concentrations.
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