Abstract
The variability of the yield of the intracellular enzyme cytochrome P-450 in Saccharomyces cerevisiae NCYC 754, in closed batch fermentation using a 5-litre computer-controlled stirred-tank fermenter, was investigated using two control strategies: a) Conventional constant set-point control, using previously determined optimal control parameters; b) time-profiled control of three fermenter control parameters, pH, temperature and stirrer speed varied with time according to pre-calculated profiles, computed from a single empirical model using Pontryagin's continuous maximum principle. Cytochrome P-450 was assayed using the reduced carbon monoxide spectrophotometric procedure. The sample treatment and carbon monoxide gassing rate having been optimised before. The accuracy of the fermenter control variables was assessed. The sum of the squared differences (SSD) between the set and actual control values was used. The values of the SSD were related to the enzyme yield. It was found that the enzyme yield was strongly affected by the accuracy of the control, as expected in a near optimal system. It was noted that inaccurate control always gave low enzyme yields, while accurate control gave a range of enzyme yields. It was concluded that yield variability was caused by more than one factor, including the accuracy of control, but that the accuracy of control was overriding. It was found that the accuracy of control of the individual variables depended on the control strategy used. It was concluded that this provides a useful practical method of assessing operational control efficiency, as opposed to analysis of individual control loops, in analysing process efficiency. Analysis of batch to batch variation in components of the complex growth-medium showed that the enzyme yield was affected. Changes in the batch of yeast extract were found to have most effect on the enzyme yield, mycological peptone less so and the glucose and salt virtually none at all. It was concluded that the saving in cost of growth medium, by partially replacing mycological peptone with yeast extract, may be offset by the costs arising from the resulting greater variability in yield. The effect on enzyme yield of the method of sterilising the growth medium indicated that the complex medium contained material which inhibited enzyme production. This material was believed to affect the rheology and the mass-transfer properties of the growth-medium. Analysis of the time interval between the occurrence of the biomass and the enzyme concentration peaks during the fermentation showed that, in this system, the biomass peak always preceded the enzyme peak. In "slower" fermentations the time interval was smaller. This also explained an apparent discrepancy in behaviour between shake-flask and stirred-tank fermentations. In general, the time interval was slightly shorter under time-profile control than with set-point control fermentations. Exhaust gas analysis data showed that the maximum carbon dioxide concentration occurred at the same time as the minimum oxygen concentration. The turning point consistently preceded the enzyme concentration peak by a virtually constant time interval, this time interval was found to be 69 minutes under set-point control and 56 minutes under time-profiled control. This is potentially valuable in enabling the occurrence of the peak enzyme concentration to be predicted accurately, with consequent saving in process time and enzyme yield.