PBD matrix used to determine the influence of different variables (A, B, C, D, E, F and G) on spore and enzyme activity in SSF by
The solid-state fermentation (SSF) is the best option to produce spores of biological control agents (BCA), because the spores have a long shelf life, compared with the obtained in liquid cultures. The spore production under SSF conditions using polyethylene bioreactors (bag-type) is a new topic. Only little information mainly about bioreactors design and adequate conditions to spore production is available. The main aim of this study was to use the corn cob as substrate in SSF and produce spores of the fungi BCA Trichoderma asperellum in a polyethylene bioreactor. In the process was added biomass of the phytopathogenic fungi Colletotrichum gloeosporioides and Phytophthora capsici as inducers of hydrolase enzymes (endoglucanases, exoglucanases and chitinases). It is possible to obtain high levels of spores, cellulases and chitinases using a polyethylene bioreactor under SSF conditions by T. asperellum and corn cob as substrate. Under the SSF conditions evaluated, the biomass of C. gloeosporioides has an inducer effect just on the spore production. However, P. capsici have effect on all response variables evaluated. The spore production was twice when used P. capsici as inducer. The most influential factor under SSF was the moisture. Levels of 66 and 50% of this factor increase the yield in all response variables evaluated (sporulation, cellulases and chitinases), C. gloeosporioides and P. capsici, respectively.
- Trichoderma asperellum
- solid-state fermentation
- bag bioreactor
The diseases induced by phytopathogens are the leading cause of losses in the most crops worldwide. It is well known that the control of such diseases through the use of chemical pesticides is not effective, they generate resistant strains phytopathogenic, the wastes are toxic and they have carcinogenic effects [1, 2]. In the last years, the alternative proposed is the use of antagonist microorganism of phytopathogens, which results in an adequate biological control, which is highly effective and environmental friendly . For this, the production of high concentrations of the spores of biological control agents (BCA) is necessary, and so currently there are several production processes of different microorganisms. The solid-state fermentation (SSF) is the best to this aim, because the spores have a long shelf life, compared with the obtained in liquid cultures [3, 4]. In other way, the spore production in SSF is relatively easiest, so it can be realized by personal with no experience, and therefore make possible the technology transference to farmers . The spore production under SSF conditions using polyethylene bioreactors (bag-type) is a new topic. Only little information mainly about bioreactors design and adequate conditions to spore production [1, 5, 6] is available. In SSF, most of the time wastes from other manufacturing process are used; therefore, this potential is commonly investigated in developing countries . Wastes of rice, maize meal, corn cob, rice husk, banana husk, wheat bran and tea leaves, among others have been used as substrate to spore production by SSF [3, 5, 6]. There are some compounds that can be added to the substrate of SSF in little proportions to induce some interest metabolite. For example, there are reports of the addition of casein and gluten to produce proteases, waste shrimp silage to chitinase production, among others [7, 8]. The main aim of this study was to use the corn cob as substrate in SSF and produce spores of the fungi BCA
2. Materials and methods
2.1. Microorganism and culture conditions
2.2. Phytopathogen biomass production
A cornmeal medium (17 g/L) was used to produce phytopathogen biomass. This medium was maintained under shaking for 1 h at 58°C. Then, it was filtrated and sterilized (15 at 115°C). The inoculation of phytopathogens was as follows:
In this work, we evaluate as substrate corn cob (CC) proportioned by the Mexican Institute of Maize, UAAAN Coahuila, México. The material was dried, ground, fractioned (300–1680 μm) and stored under low moisture conditions for further evaluation. This material was used as a substrate on SSF without pretreatment.
2.4. Solid-state fermentation
Polyethylene bags were used as bioreactor in all experiments. Sporulation and cellulase production were evaluated. Plackett-Burman design (PBD) was used in this experiment to determine the most influential factors on spore and enzyme production by
2.5. Enzyme activity determination
After SSF each sample was analyzed to determine cellulase activity , chitinase activity  and reducing sugars . The carboxymethylcellulose activity (CMCA) was carried out at 50°C for 30 min. Sample (1 mL) and substrate (1 mL of carboxymethylcellulose 1%) were the mix reaction. Citrate buffer (1 mL at 50 mM, pH 4.8) and substrate (1 mL) were the substrate control. The enzyme control was the mix of sample (1 mL) and citrate buffer (1 mL).
The filter paper activity (FPA) was carried out at 50°C for 1 h. Sample (1 mL) and substrate (filter paper Whatman No.1 (1 cm×5 cm) in 1 mL of citrate buffer at 50 mM, pH 4.8) were the reaction mix. The control substrate was the mix of citrate buffer (2 mL) and filter paper. Sample (1 mL) and citrate buffer (1 mL) were the enzyme control.
Chitinase activity was carried out at 37°C for 1 h. The reaction mix, enzyme and substrate control were done similar to carboxymethylcellulose activity. In this case, substrate (chitin oligosaccharides) and buffer solution (acetate 50 mM, pH 4.0) were replaced.
Sugar concentration was determined after each enzyme reaction. An enzyme activity (U) was defined as the amount of enzyme that catalyze the release of 1 µmol of glucose per minute.
2.6. Design and statistical analysis
A PBD was used to SSF. Spore and enzyme production were the response variables. Data were analyzed by ANOVA using STATISTICA 7.0 software; when needed mean treatments were compared using Tukey’s multiple range procedure. A
3.1. Screening of significant factors using Plackett-Burman design
Studies were performed in eight runs each one to identify the combination of factors which allow us to obtain a significant level of spore, cellulase and chitinase production by
3.2. Solid-state fermentation with C. gloeosporioides biomass
Table 2 summarizes the results obtained in SSF using the biomass of
|Treatment||Sporulation index (Spores/g)||Enzyme activities|
|A||4.2E+08 ± 6.25E+07cd||1.847 ± 0.02b||1.183 ± 0.17b||3.671 ± 0.46b|
|B||4.0E+08 ± 4.30E+07d||0.132 ± 0.02d||0.165 ± 0.06cd||1.795 ± 0.05f|
|C||4.8E+08 ± 5.10E+07cd||2.582 ± 0.08a||1.549 ± 0.05a||5.118 ± 0.28a|
|D||2.3E+08 ± 6.65E+07e||0.204 ± 0.10d||0.006 ± 0.00d||2.149 ± 0.06ef|
|E||2.3E+08 ± 7.35E+07e||0.529 ± 0.10d||0.443 ± 0.05c||2.996 ± 0.08cd|
|F||7.3E+08 ± 9.55E+07a||1.958 ± 0.17b||1.381 ± 0.23ab||3.692 ± 0.13b|
|G||6.2E+08 ± 8.15E+07ab||1.243 ± 0.02c||0.390 ± 0.01c||2.538 ± 0.01de|
|H||5.2E+08 ± 3.50E+06bc||2.474 ± 0.47a||1.193 ± 0.25b||3.418 ± 0.29bc|
In the first case, the spore production was influenced by the temperature in a negative way. Between the range of the values (24 and 30°C), the study shows that 24°C is the best to produce a better sporulation index and possibly if we reduce the value, the sporulation can be major. The moisture, inoculum and inducer are the other factors that also have influence on spore production, just in a positive way. It means, it is necessary to increase the value of each factor (Figure 1). The moisture, pH and inoculum were the factors more determining endoglucanase production (CMCA). These factors had positive values, which mean that high values allow high enzyme activity. A significant effect was observed with the substrate concentration, but this effect was negative, so low amount of substrate is needed to obtain high enzyme yields (Figure 2).
The exoglucanase (FPS) in the same way to CMCA was influenced by the moisture (Positive). Low levels in the substrate and temperature show the best enzymatic yields (Figure 3). The moisture was the factor with major influence on the chitinase production. The substrate and inducer were also significant, but in negative way, it is necessary to use low values to increase the yield. The time and pH were important, so these factors must be in high levels (Figure 4).
3.3. Solid-state fermentation with P. capsici as inducer
Now, Table 3 shows the results obtained in SSF using the biomass of
|Treatment||Sporulation index (Spores/g)||Enzyme activities|
|A||3.1E+08 ± 2.0E+06d||4.238 ± 0.32c||1.660 ± 0.02d||2.848 ± 0.22b|
|B||1.4E+09 ± 1.5E+08a||4.861 ± 0.13bc||2.466 ± 0.14b||3.216 ± 0.15ab|
|C||2.6E+07 ± 1.0E+06f||0.062 ± 0.02e||0.121 ± 0.00g||1.527 ± 0.25d|
|D||8.1E+08 ± 9.0E+06b||5.012 ± 0.40b||2.272 ± 0.01c||2.978 ± 0.10b|
|E||4.4E+08 ± 6.0E+07c||4.814 ± 0.29bc||2.200 ± 0.02c||3.082 ± 0.04b|
|F||3.7E+08 ± 3.0E+07d||1.538 ± 0.56d||0.696 ± 0.01e||2.144 ± 0.24c|
|G||1.2E+09 ± 1.1E+08a||7.825 ± 0.21a||2.764 ± 0.01a||3.609 ± 0.18a|
|H||1.1E+08 ± 3.0E+06e||1.168 ± 0.21d||0.310 ± 0.13f||1.956 ± 0.22cd|
The sporulation of
There are several studies that report the production of different enzymes under SSF [12, 13]. Currently, the SSF is a commonly used system because the raw materials such as sugarcane bagasse, wheat bran, among others  are cheaper. The control of temperature, pH, moisture, purity of the culture and process time are some factors that difficult the rigorous control of the fermentation process .
Sometimes, it is hard to find one combination of the SSF conditions in which we can obtain high yield in all response variables evaluated (sporulation, cellulases and chitinases). In the case of SSF with biomass of
Now, in the SSF with
In the start of the study, we think that the addition of certain concentration of phytopathogen biomass could generate an induction effect of some hydrolase enzymes. The production of chitinases when were used
In the SSF with
In the case of SSF with
In this study, the moisture and temperature are the two important factors. Among the values evaluated, a level of 66% of moisture and 24°C of temperature shows the best yields in spore and enzymes production on SSF with
This study demonstrated that the biomass addition of any one phytopathogen shows an increment in the spore production by
Currently, there researches are aimed at the high biomass production of the biological control agents using several systems to produce it. Kancelista
There are few works that report the use of polyethylene bioreactors to produce spores in SSF using some biological control. The use of this kind of bioreactor needs to utilize special plastic bags which allow the gas exchange and microorganism respiration . In some cases, we can use a cotton tap on the bag to allow gas exchange. In this study, the maximal spore production obtained was 7.3×108 and 1.4×109 spores/g CS to the SSF with
It is possible to obtain high levels of spores, cellulases and chitinases using a polyethylene bioreactor under SSF conditions by
The authors thank National Council of Science and Technology (CONACYT, Mexico) for the financial support of this research project. Author de la Cruz-Quiroz thanks IMBE personal for all technical support.