Optimization of nutritional requirements and ammonium feeding strategies for improving vitamin B₁₂ production by <i>Pseudomonas denitrificans</i>

Abstract

Statistical experiment design and data analysis were used to establish the major factors in a chemically defined medium and to develop an ammonium control strategy to optimize the specific vitamin B₁₂ production rate (Yp) of Pseudomonas denitrificans. Through Plackett-Burman design, the major factors of glucose, ammonium sulfate and KCl were selected as the significant factors affecting vitamin B₁₂ biosynthesis and these were further optimized by central composite design with response surface methodology. The maximum Yp of 34.2 μg/gDCW/h was obtained in batch cultivation under the estimated optimal initial composition of glucose (93.6 g/l), (NH₄)₂SO₄ (7.93 g/l) and KCl (1.24 g/l). Ammonium control strategies in fed-batch fermentation showed that when ammonium concentration was maintained at 40 mmol/l, the maximum Yp reached 36.0 ± 1.31 μg/gDCW/h, which was 57.2% higher than that of the control (22.9 ± 0.83 μg/gDCW/h). This ammonium control strategy successfully enhanced the industrial production, resulting in a stable high vitamin B₁₂ production of 212.02 ± 3.03 mg/l and Yp of 37.1 μg/gDCW/h.

Key words: Statistical designs, Pseudomonas denitrificans, chemically defined medium, ammonium controlling strategy, vitamin B₁₂.