Batch, ammonium‐controlled and simultaneous glucose and ammonium controlled fermentations were compared for the production of protease by Bacillus subtilis NCIB 8054. During fermentations of B. subtilis, maximum protease production was obtained in the stationary phase. Protease production in fermentations controlled at 5 mmol dm−3 ammonium was 1.5 times greater than in uncontrolled batch fermentations. Simultaneous control of ammonium at 5 mmol dm−3 and glucose at 0.15 g dm−3 using controllers based on an ammonium electrode and an oxygen electrode, doubled protease production compared with fermentations having only an ammonium control and tripled protease production compared with uncontrolled batch fermentations. The protease yield on glucose and protease yield on ammonium was increased in fermentations with simultaneous glucose and ammonium control.

A control system has been devised for the maintenance of stable ammonium concentrations throughout a fedbatch fermentation. The control system is based on an ammonia gas-sensing electrode that monitors a pH-adjusted effluent stream from the fermentor. To overcome the time lag between the fermentor and the electrode, feedback control included metered flows of ammonium to both the fermentor and the electrode vessel. The system was used to study the growth of Escherichia coli B (ATCC 11303) at controlled ammonium concentrations of 5 to 200mM. Apparent specific growth rates, biomass and protein production, and glucose yields were essentially constant from 5 to 170mM. Above 170mM ammonium growth was inhibited. As ammonium concentration decreased from 170 to 5mM, ammonium yields increased from 1 to 24 g cell dry wt/g ammonium utilized. The results demonstrate that control of ammonium concentrations at levels so low that ammonium would be exhausted in batch fermentations can significantly increase overall ammonium yields.

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Aims:  To further improve the soy‐based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale‐up the Clostridium tetani process into small fermentors. Methods and Results:  Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l‐tyrosine and l‐cysteine could be eliminated from our soy‐based medium without effect. Seven carbon sources were compared with glucose in the soy‐based medium, but none was found to be superior to glucose. The process was successfully scaled‐up into 250‐ml bottles, 1‐l bottles and 1‐l fermentors. Conclusions:  Quite remarkably, when comparing the tetanus production process in our soy‐based medium with the traditional animal/dairy‐containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l‐tyrosine and l‐cysteine components could be eliminated, making the medium (Hy‐Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale‐up from test tubes into 1‐l fermentors allows us to predict that further scale‐up into large fermentors will be successful. Significance and Impact of the Study:  Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow’s disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable‐based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.