# Increase of _Trichoderma harzianum_ Production Using Mixed-Level Fractional Factorial Design
## 1 Introduction
### Characteristics Factor for the Production of Spores of the Trichoderma harzianum  
The most important factors influencing fungal production are temperature, pH, type of substrate, inoculum concentration, incubation period, aeration, and moisture content [34]. For the successful growth of biological control agents during production, it is necessary to know the specific and optimal value of the physiological and environmental parameters [35]. Table 2 shows the operation range as well as the optimal values for the factor levels that have been documented in other investigations. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Factor** & **Operation Range** & **Optimal Value** & **References** \\ \hline \multirow{3}{*}{Air} & Yes-No & Yes & [5] \\  & Yes–No & Yes & [32] \\  & Yes–No & Yes & [36] \\ \hline \multirow{3}{*}{Humidity} & 50\%–80\% & 50\% & [6] \\  & 30\%–60\% & 50\% & [37] \\  & 50\%-70\% & 70\% & [38] \\ \hline \multirow{3}{*}{Temperature} & 25\({}^{\circ}\)C-35\({}^{\circ}\)C & 25\({}^{\circ}\)C & [6] \\  & 25\({}^{\circ}\)C-28\({}^{\circ}\)C & 26\({}^{\circ}\)C & [35] \\ \cline{1-1}  & 24\({}^{\circ}\)C-27\({}^{\circ}\)C & 25\({}^{\circ}\)C & [37] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Factor, operation range, and optimal value. A statistical analysis was carried out to determine the influence of _air_ on the growth of the fungus; the results showed that the presence of air increases the production of the Trichoderma fungus [37]. Several authors [5;36] agree with what was reported by the author [37]; a 30% increase in spore production was reported due to the presence of air in the incubation process. Humidity is important in the production of the fungus; an optimal level of humidity must be sought since humidity values less than 30% reduce the growth of microorganisms, and the same happens with values greater than 90%; it is recommended to keep humidity at a range of 50%-70% [38]. The authors [6;39] achieved the highest spore production using 50% humidity. Fungi need a certain temperature to develop and carry out their activities [40]. Through several studies [6;23;41], it has been possible to verify that the optimal range for the development of the fungus is situated at 24 \({}^{\circ}\)C-27 \({}^{\circ}\)C. Obtaining an optimal value in their studies of 25 \({}^{\circ}\)C [6;41] and 26 \({}^{\circ}\)C [23]. Hydrogen potential is a critical parameter in the viability of the fungus [42]. The authors examined the effect of pH on the in vitro activities of the extracellular enzymes of Trichoderma. Most of the Trichoderma strains examined were able to grow in a wide pH range from 2.0 to 6.0 with an optimum of 4.0. However, some of the examined pathogenic fungi had optimal pH at alkaline values of 8-10 [22]. A seven-factor design with two levels was used, and it was possible to determine an optimal pH in filamentous fungi. It was found that it develops better in a range of 3.8 to 6, locating the optimum at 6. Additionally, when the pH takes values greater than 8 and less than 3, destabilization is generated, causing a decrease in spore production [4]. A five-factor design with two levels determined that the maximum spore production was achieved using a pH of 5.8 [6]. For fungal spores' growth, it is necessary to provide them with adequate nutrition (carbon, phosphorus, and nitrogen), so the choice of substrate is key for large-scale production. Different types of substrates such as tomato husk, rice husk, garlic husk, cocoa husk, sesame husk, peanut husk, coffee husk, bean husk, soybean and corn husk, corn cob, rice grains, sorghum, birdseed, broken corn, rice straw, wheat straw, agricultural manure, banana pseudo stem, and dry banana leaves, provide this nutrition, the highest spore production of \(3.1\times 10^{6}\) CFU.g\({}^{-1}\) was obtained using rice grain as a substrate [43]. In another study [44], the substrates rice grain, wheat grain, sorghum grain, and sesame husk were considered, with the highest production of \(3.7\times 10^{8}\) CFU.g\({}^{-1}\) was obtained with rice grain.