2.7 Analytical methods and calculations
The cell density was measured spectrophotometrically as optical density (OD) at 600 nm and transformed to biomass concentration (g/L) using a linear relationship between cell dry weight (CDW, after cells dehydration at 80°C / 3 days) and OD600 (Eq. 1). OD600 was measured using a Genesys 10 UV Scanning photometer (Thermo Scientific, Germany) with 1-mm cuvettes. For samples with OD > 0.8, the sample was diluted with 0.1 % HCl milli-Q water solution. The specific growth rate (μ in h-1) was calculated using Eq. (2), where X1 and X2 are the biomass concentrations at time points t1 and t2, respectively.
CDW (g/L) = 0.44\(\ \times\ \)OD600; r2=0.987 (1)
\begin{equation} \text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }u=\frac{\text{Ln}X_{2}-\text{Ln}X_{1}}{t_{2}-t_{1}}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (2)\ \ \nonumber \\ \end{equation}
Glycerol, PDO, butanol, methanol, ethanol, 1-propanol, acetate, butyrate, formate, lactate, pyruvate, MA and MB were quantified using high-performance liquid chromatography (HPLC, Kontron Instruments, Switzerland) with an Aminex HPX-87H 300 mm x 7.8 mm column (Bio-Rad Laboratories, USA) as the separation column at an operation temperature of 30°C. The eluent for the HPLC detection was 0.1% trifluoroacetate and the flow rate was maintained at 0.6 mL/min. The detection was assessed by a UV-detector (Shimadzu, Japan) at a wavelength of 210 nm and a differential refractometer RI-detector (Kontron Instruments, Switzerland). The volumetric productivity of PDO (QPDO, in g/L/h) was calculated using Eq. (3):
\begin{equation} \text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }Q_{\text{PDO}}=\frac{C_{2}-C_{1}}{t_{2}-t_{1}}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (3)\ \ \nonumber \\ \end{equation}
where C1 and C2 are the PDO concentrations at time points t1 and t2, respectively.
From the HPLC data, the conversion of acetic acid (XA) or butyric acid (XB) was calculated as follow:
\begin{equation} \text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ X}=\frac{C_{0}-C_{e}}{C_{0}}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (4)\nonumber \\ \end{equation}
Where C0 is the initial concentration of acetic acid or butyric acid; Ce is the concentration of acetic acid or butyric acid after esterification.
The content as weight fraction in percentage (wt %) of each component (C ) in each purification step was calculated from Eq. (5):
\begin{equation} \text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ C}=\frac{c\times d\times v}{w}\times 100\%\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (5)\ \ \nonumber \\ \end{equation}
Where c is the concentration (g/L) determined by HPLC, dthe dilution factor, v the volume (L) and w the weight (g) of the sample taken for analysis.