Abstract
Clavulanic acid is a specific and irreversible inhibitor of a wide range of bacterial β-lactamases. Clavulanic acid has been used in human therapy for several years in combination with amoxicillin. The effect of inoculum quality on the production of clavulanic acid was first investigated in batch or fed-batch flask cultures with S. clavuligerus. A methodology to obtain an optimal concentration maintaining range for glycerol in the culture was developed to enhance the clavulanic acid production. Furthermore, the effect of intermittently feeding both glycerol and ammonium ion on the biosynthesis of clavulanic acid was also investigated. The inoculum quality and the morphology did influence the clavulanic acid production. The optimal age and size of inoculum for the production of clavulanic acid in batch and fed-batch flask culture was 28 hrs and 10%, respectively. The optimal concentration range of glycerol could be maintained low in the culture, and the concentration range for clavulanic acid biosynthesis were 1~5 g/l. Whatever we try to control the pH of the cultures or initially add ornithine to the cultures, intermittently feeding glycerol of 0.25 g and ammonium ion of 5 mg in shake flask cultures resulted in the maximum production of clavulanic acid that was 15~30% higher than that with only feeding glycerol culture. Subsequently, the biosynthesis of clavulanic acid was inhibited due to the increasing amount of ammonium ion continuously added. The highest maximum production of clavulanic acid could be obtained under the conditions: initially concentration of ornithine of 5 mM, and simultaneously and intermittently feeding glycerol of 0.25 g and ammonium ion of 5 mg with the pH controlled in the culture. It was 2.74-fold higher than batch culture.