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

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.