# Factorial Design to Stimulate Biomass Development with Chemically Modified Starch
## 2 Materials and Methods  
The chemical modification of starch was realized in order to solubilize the starch molecule. For this reason, the following chemicals were used: propylene oxide (PO) at 99% purity and sodium hydroxide (NaOH) micropearls for analysis, provided by Acros Organics (Noisy-le-Grand, France); dimethyl sulphoxide (DMSO) at 99.5% purity and potato starch, provided by Panreac Quimica SA (Barcelona, Espana). Deionized water was used throughout this work. The chemical reaction was carried out in a flask at a continuous high pressure at 21 \({}^{\circ}\)C. The dried starch was dissolved in DMSO at a concentration of 1 g to 10 mL [18; 19; 20; 21; 22; 23]. After the starch solubilization (at 21 \({}^{\circ}\)C for 48 h), the 1,2-epoxyalkane was added at molar ratios of epoxyalkane to the alcohol function present in starch. The reaction mixture was stirred at 21 \({}^{\circ}\)C for 6 h. As a final reaction, the compound was catalyzed by adding NaOH. The modified starch was neutralized with hydrochloric acid, dialyzed for 4 days using a cellulose membrane (Medicell International, MWCO of 12-14,000 Da), and lyophilized. For biomass development, culture media were used in order to create the sample's experimentation. The quantities were expressed as one liter of medium (distilled water: 1 L); the pH was measured before the sterilization, which was carried out with the autoclave (121 \({}^{\circ}\)C, 20 min). The culture media were composed of the following: malt agar extract (MAE) ( malt extract (Merck)--20 g; yeast extract (Biokar Diagnostics)--2 g; agar (Sigma)--15 g; chloramphenicol--0.2 g) and mineral medium (MM) (KCl--0.250 g; NaH\({}_{2}\)PO\({}_{4}\) x 2H\({}_{2}\)O--6.470 g; Na\({}_{2}\)HPO\({}_{4}\) x 2H\({}_{2}\)O--10.410 g; MgSO\({}_{4}\)--0.244 g; NO\({}_{3}\)NH\({}_{4}\)--1.000 g; solution of oligoelements (10 mL par L) ZnSO\({}_{4}\), MnCl\({}_{2}\), FeSO\({}_{4}\), CuSO\({}_{4}\), CaCl\({}_{2}\), MoO\({}_{3}\)). The inoculum of fungi came from spores harvested from the soil [25]. For the preparation of fungus inoculums, the easily quantifiable spores, the propagates, were used. For each isolate, 2-3 mL of sterile distilled water was deposited on a 5-day pre-culture on the MAE medium out of the Petri box. The suspension of spores, recovered in a test tube, was shaken in a vortex in order to homogenize it well. The concentration of the suspension in spores was estimated using a cell of Malassez and an optical microscope. The estimate biomass of fungus was made on different substrates: native starch and starch modified with propylene oxide. The medium was prepared with the quantities described above by using elements such as the source of carbon glucose (similar to the reference) and the modified starch. In each Erlenmeyer, 25 mL of the medium, prepared without the solution of NO\({}_{3}\)NH\({}_{4}\) to avoid the production of a Maillard reaction during the pressure-sealing, was added. These last samples were sterilized with the autoclave. The solution of NO\({}_{3}\)NH\({}_{4}\) was added, and after, the mediums with the desired number of spores (104 spores/mL) were combined. The fungus cultures were incubated on a rotary shaker at room temperature for 5 days. After 5 days, the cultures were filtered by a vacuum. The filters with biomasses were dried with a drying oven at 110 \({}^{\circ}\)C for 24 h. The biomasses on the filters were measured, and a second weighing was carried out after 48 h in order to confirm that the content of the water was completely evaporated. In order to optimize the conditions for the development of the microorganisms, a factorial model, using the MATLAB program model, was applied [26; 27; 28; 29; 30; 31]. For the realization of this study, 27 tests were carried out, applying the factorial 3\({}^{3}\) design method. In order to optimize the biomasses development of microorganisms, different parameters were used. In Table 1, the varied parameters and the field variation are presented as follows: