Flow cytometry for quantitation of polyhydroxybutyrate production by Cupriavidus necator using alkaline pretreated liquor from corn stover.

Alkaline pretreated liquor (APL) from lignocellulosic feedstock pretreatment is a lignin-rich stream. Polyhydroxybutyrate (PHB), a biodegradable polymer, has been previously synthesized from APL. It is of interest to monitor PHB production and cell number from APL rapidly for process control. However, APL has insoluble substances and is dark, which makes quantitation of cells by visible light absorbance difficult. A sample preparation method was developed using Nile Red staining and flow cytometry to quantify bacterial cells and PHB concentration. A linear model with good fitness (R = 0.9939) was constructed to predict PHB concentration (0.2-2.1 g/L) based on fluorescence intensity acquired from a flow cytometer. A linear model (R = 0.8614) to predict cell number based on fluorescence intensity was also established. The good correlation between PHB concentration and fluorescence intensity indicates the potential of applying flow cytometry for quantitation of PHB from APL and other media that is dark and/or contains insoluble particles.