When stationary culture was replaced by submerged cultivation in a fermente
r, a significant increase in the yield of diphtheria toxin in a short culti
vation time (less than 48 h as against 7-8 d) was noted. It was found that
under optimal conditions of temperature, vortex mixing and surface aeration
, an alkaline pH favoured toxin release. Furthermore, to enhance the produc
tion volume, two-and three-step semicontinuous batch cultivations were carr
ied out. The toxin produced was of good titre with an adequate antigenic pu
rity. Under optimal conditions, the variation in the Limes of flocculation
(Lf titre) was likely due to the quality of the production medium, which in
turn depended on the quality of the raw materials used. The process was al
so optimized in a pilot-scale fermenter.