Fermentation of renewable organic substrates and evaluation of biomass processing methods for microbial products

This work investigates the controlled production and fractionation of Cupriavidus necator biomass as an integrated strategy to obtain both intracellular polymers (PHA/PHB) and microbial proteins (single-cell protein, SCP) from renewable substrates at laboratory scale. An upstream–downstream workflow was developed using fed-batch fermentations on mixtures of volatile fatty acids (VFAs) with pH-stat and DO-stat (Oxygen Demand) feeding strategies, and by testing growth on a novel CO2-derived substrate, “Compound X”. The collected biomass was mechanically disrupted by bead-milling and subjected to solubility tests in order to achieve partial separation of protein-rich and PHA-rich fractions. The first fermentation was characterized by an inhibitory VFA accumulation and limited biomass production (dry cell weight, DCW ≈ 3 g/L). The implementation of a more uniform feeding profile in the second fermentation avoided overfeeding, resulting in biomass concentrations above 50 g/L, providing sufficient material for downstream processing. Bead-milling experiments showed that the majority of the proteins were released (evaluated as nitrogen released) in the first ~10 min at neutral pH‚ while PHA remained insoluble‚ facilitating fractionation (under optimal conditions‚ nitrogen release reached 40-50%). Shake-flask experiments confirmed that C. necator can effectively use “Compound X” as a carbon source‚ with a less favourable growth yield than that obtained with acetate‚ and the process was examined further in a fed-batch run․ These results show not only the feasibility of an integrated lab-scale approach to the production and fractionation of C. necator biomass‚ but also the valorisation of renewable feedstocks and the optimization of feeding and cell disruption conditions․