## 2 Materials and Methods
### Evaluation of Process Parameters on Yield of Bacterial Nanocellulose Biosynthesis  
In this research the PBD was employed to determine the main effects of individual process parameters on BNC production. The Plackett-Burman design is a useful tool for screening numerous factors in terms of their infectivity on the process without study of interactions among variables. This statistical method is based on the first order model equation:  
\[Y=\beta_{0}+\sum\beta_{i}X_{i}, \tag{1}\]  
where \(Y\) represents the studied response, \(\beta_{0}\) is the model intercept, \(\beta_{i}\) is the regression coefficient, and \(X_{i}\) is the factor. The impact of nine input factors, namely bioreactor area, surface to volume ratio, air flow ratio, humidity of air, glucose concentration, pH, age and volume of inoculum, and cultivation time, was  
Figure 1: The surface air-flow (SAF) bioreactor. The experimental setup contains 3 glass trays with an area of 2070 or 190 cm\({}^{2}\), depending on the culture variant. Air is fed by a compressor and passed through the automatic rotameter, which allows measurement and regulation of the air flow ratio. Between rotameter and bioreactors, two safety valves cutting off the air supply are located. Air is purified on sterile filters (PTFE, \(0.22\) μm) and is introduced into the bioreactors by two spigots placed in the side walls. Air leaves the bioreactors by two outlets placed in the top cover. studied. Process parameters were tested at two levels (low and high, \(-1\) and \(+1\), respectively) in 12 trials (Table 1). Additionally, two dummy factors were considered, which were imaginary variables. The change between levels is not supposed to cause any effects on the studied responses. The use of dummy factors allows evaluation of systematic errors or determination of the unknown significant variables. Variables and dummy factors were arranged randomly in a matrix. The dry weight of cellulose, the glucose conversion into BNC, and the yield, calculated as a dry weight per surface unit, were taken as a response. All experiments were done in the SAF bioreactor. The main effects were calculated as a difference between the average measurements at the high and low level of each variable. The significance of each factor was assessed using Student's test and a low \(p\)-value (\(<\)0.05) determines a significant effect. The PBD matrix (Table 2) was generated using MiniTab18(r)software (Minitab18, Minitab LLC, State College, Pennsylvania, USA, 2018). All experiments were carried out in three replicates. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Factor Symbol** & **Factor** & **Low Level (\(-1\))** & **High Level (\(+1\))** \\ \hline \(X_{1}\) & Bioreactor area (cm\({}^{2}\)) & 190 & 2070 \\ \hline \(X_{2}\) & Cultivation time (days) & 4 & 6 \\ \hline \(X_{3}\) & Age of inoculum (days) & 1 & 5 \\ \hline \(X_{4}\) & Surface to volume ratio (cm\({}^{-1}\)) & 0.71 & 0.33 \\ \hline \(X_{5}\) & Glucose concentration (g/L) & 10 & 40 \\ \hline \(X_{6}\) & Dummy factor 1 & \(-1\) & +1 \\ \hline \(X_{7}\) & Air flow ratio (L/min-m\({}^{2}\)) & 0.94 & 18.80 \\ \hline \(X_{8}\) & Dummy factor 2 & \(-1\) & +1 \\ \hline \(X_{9}\) & Inoculum volume (\%) & 1 & 10 \\ \hline \(X_{10}\) & pH & 4.0 & 6.5 \\ \hline \(X_{11}\) & Humidity of air & dry & wet \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Factors assigned at two levels in the Plackett–Burman design.