Optimization of biomass production of pseudomonas fluorescens in a stirred tank bioreactor

Abstract

Pseudomonas fluorescens (P. fluorescens) is a denitrification bacterium that able to convert nitrate compounds into nitrogen gas. This process is always slow due to limitation of organic sources that are present in wastewater treatment plant. The efficiency of P. fluorescens as a nitrate removal strongly depends on the physiological status of cells and the amount of microorganism added to the treatment plant. Therefore, the objectives of this study are to optimize the culture medium in shake flask and to develop cultivation strategy for the biomass production of P. fluorescens in a pilot scale 16-L bioreactor. The medium composition was first optimized using one factor at time (OFAT) and response surface methodology (RSM) methods, which the Box-Behnken experimental design was employed. Analysis of variance (ANOVA) showed significance of findings for each factors with high coefficient of determination (R2) of 95.58 %. The optimum medium composition of biomass production was composed of: sucrose, 8.0 g L-1; yeast extract, 3.0 g L-1; di-potassium phosphate, 2.0 g L-1; and magnesium sulfate heptahydrate, 1.5 g L-1. This medium gave biomass of 3.28 g L -1 (about 57.6 % higher compared to un-optimized medium). After this step, the optimized medium was used to cultivate the cells in batch mode with and without pH control in a 16-L stirred tank bioreactor. It was found that controlling the culture pH at 7.2 during cultivation increased biomass by 39.56 %. In addition, a series of constant feeding strategy in combination with a control pH 7.2 was carried out to increase the biomass production. Both sucrose and full medium feeding were applied, and both yielded biomass of 8.46 g L -1 and 14.98 g L -1, respectively. Under constant feeding strategy of full medium, nutrients were consumed after 10 hours of feeding. Therefore, a gradual increase of medium feeding rate was applied to increase the biomass. The highest biomass obtained using increased rate feeding strategy was 33.5 g L -1. In conclusion, the medium optimization accompanied by bioprocess optimizations in terms of pH control and applying fed batch cultivation strategy in the 16-L bioreactor enhanced the growth rate and biomass production of P. fluorescens.