Montague et a l (1989) described physical measurements as volume of the bioreactor, temperature, pH, pressure, agitation, viscosity, and chemical measurements such as volumetric gas flow rates, liquid flow rates (acid, base, antifoam and nutrient feeds ), dissolved oxygen transfer, nutrient concentration, redox potential, product and biomass concentration, and rheological measurements. Some fermentation variables can be measured on a continuous basis with some degree of reliability and are used to maintain the desirable environmental conditions of the process. Conditions an be maintained reasonably close to the the desired values (e.g. ±0.1 °C, ±0.1 pH ). However, the major problem is knowing the most desirable operating condition. In order to optimise the performance of the process, operating conditions are often chosen based on past experience and experimentation approaches.
Microorganisms respond differently to fermentation variables. The optimum measured value of these variables for growth rate may be different from that for growth yield and entirely different from the optimum value for product formation. For penicillin production a higher penicillin yield is obtained by starting at a temperature of 30®C, followed by operating at a constant, low, temperature of 20°C to 25®C compared to a controlled fermentation at 25°C (McCann and Calam, 1972). Andreyeva et a l, (1973) discussed the effect of pH on growth and penicillin production and stated that the optimum pH profile should not be constant as a rule, since the optimum value is likely to be different for growth and product formation. Different pH values for a maximum specific oxygen
uptake rate for 66 hours (pH=6.7) and 90 hours (pH=7.0) was observed with growth on synthetic media.
The difficulty arises in that changing one variable would lead to a change in a number of other variables. A change in pH can affect the evolution of CO2 which affects the mass balances and biomass estimations (Royce, 1993). Montague et a l, (1989) demonstrated the major interaction that can take place between variables within a fermentation (Figure 1.9), and stated that other interdependences could also be postulated. Many of these interactions have been modelled mathematically.
Agitation Aeration Pressure
Oxygen Supply
Carbon Dioxide removal Carbon and energy sources Temperature PR Rheology Morphology Growth rate concentration ■^Production
Figure 1.9 Typical Bioprocess Interactions (Montague et a l ,1989).
The reproducibility of bioprocesses is mainly dependent on the reproducibility of environmental conditions for the cells, and therefore on the quality of the equipment. In a batch culture, a strict control of the physical parameters like pH, temperature, pressure, or aeration is not sufficient to obtain reproducible results due to the continuous change in biomass, substrate, and product concentrations. Control over the medium composition, inoculum (state, size, and volume), and other environmental variables are also important. A good reproducibility of fermentation results has been reported from repetitive aerobic batch fermentations of yeast S.cerevisiae by Locher et a i (1991).
Effect of environmental variables on growth and optimisation of yeast S.cerevisiae has been extensively studied by many authors. Yeast grows quite well at temperatures between 28°C to 32®C and at pH levels between 3.6 and 6.5 (Chen and Chiger, 1985). Changes in environmental variables such as oxygen or substrate concentration may alter the yeast's metabolism from oxidative to oxido-reductive (Auberson and Stocker, 1992).
Nutrient concentration (Kappeli, 1986) can also effect the metabolism. Kalle and Naik (1987) studied the effect of controlled aeration on glycerol production in S.cerevisiae. Increasing aeration (1-1.4 vvm), improved the ability of the strain to metabolise higher concentrations of sugar in the medium (400-465 g/L), which led to a better growth rate and higher productivity of glycerol by threefold. While at aeration rates greater than 1.4 vvm productivity was reported to decline due to the general inhibition of the fermentation.
Ahmad et al., (1991) studied the effect of some environmental variables on growth characteristics of Candida utilis. The lag phase of the growth curve was reported to increase with initial sugar concentration, and to decrease with an increase in inoculum dosage and agitation speed. Increasing inoculum dosage, air flow and agitation speed was reported to increase the overall yield (0.27 at 200 rpm and 0.57 at 700 rpm; 0.28 at 21 L/min and 0.45 at 1.2 L/min).
Abel et al. (1994) investigated the effects of variations in dissolved oxygen concentration in the micro environment of yeast cells on their physiological behaviour. The gas composition was changed periodically by varying the flowrate ratios of the air to nitrogen gas, keeping the flowrate of the gas mixture constant. For the batch cultivation on glucose the results showed that, cell growth rate on glucose, and the final cell concentrations were higher for the experiment with a higher aeration frequency (0.79 min*^) and dissolved oxygen concentration between 30 and 2% than experiments with an aeration frequency of 0.56 min'* and dissolved oxygen concentration between 55 and 5%. The conclusion was made that the influence of periodical changes of dissolved oxygen concentration decreased with increasing frequency, due to the slow response of the cells to variations in the environment. Abel et al. (1994) indicated that as a consequence of the periodically changing gas composition and dissolved oxygen concentration, aerobic and anaerobic phases interchange. During the aerobic phase the respiratory capacity is high and glucose is metabolised oxidatively due to a subcritical glucose flux. The residual respiratory capacity is used for consumption of ethanol. The researchers concluded that at a high frequency of the gas consumption change, the respiratory capacity of the cells might not be able to respond to the dissolved oxygen concentration changes, thus, an intermediate respiratory capacity is followed according to the average dissolved oxygen concentration. At low frequencies of dissolved oxygen variation, however, the respiratory capacity changes periodically causing the growth rate on ethanol and the yield coefficient of the growth on ethanol as well as cell concentration to decrease.
The applied environmental conditions have a direct impact on the properties of the yeast as a product of fermentation. For example higher temperatures tend to give drier y e a st, and at lower pH value a cream coloured instead of a white yeast (Burrows, 1970) is produced.