Modeling of poly-β-hydroxybutyrate production by Bacillus subtilis and its use for feed-forward bioreactor studies.

Abstract

Successful scale-up of Bacillus subtilis culture for poly-β-hydroxybutyrate (PHB) production was performed in 5-l stirred-tank reactor using batch, fed-batch, and two-stage culture strategies. The kinetics of biomass production, substrate consumption, and PHB production were established in the stirred tank bioreactor in all the studies. A mathematical model was developed to investigate the role of limiting substrate on overall culture metabolism. A fed-batch strategy was predicted on the basis of computer simulations, for maximum PHB production. This was performed by extrapolation of batch model for predicting the feeding rate and suitable time of feeding. Substrate inhibition was studied and the substrate inhibition terms were incorporated in the model. The maximum cell biomass concentration in batch culture (24h) and fed-batch culture (30h) was 1.79 ± 0.03g/l on dry cell weight (DCW) basis and 1.66 ± 0.050g/l on DCW basis and the corresponding PHB content was 68.71% and 85.54% of DCW, respectively. Glucose was found to be the major limiting nutrient during the bioreactor culture. A two-stage culture, where cells were first grown in stage I in LBG media containing excess carbon and thereafter in stage II in OM media, showed biomass production of 1.95 ± 0.045g/l at 4h and PHB production of 93.33% of DCW at 16h. A 9% increase in growth and 25% increase in PHB yield were obtained using two-stage culture with computer-simulated feeding strategy in the 5l reactor. Oxygen limitation was overcome in modified two-stage culture to obtain a PHB production of 98% at 30h. KEY POINTS: • Polyhydroxybutyrate production was studied in a 5-l stirred-tank bioreactor using HPLC • Mathematical model-assisted fed-batch strategy was implemented in bioreactor • Two-stage fed-batch cultivation was implemented and PHB production was 93% of dry weight in Gram-positive bacteria.