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The involvement of 48 and 51 was, however, concluded by trapping experiments in special cases 96 For example, the ene reaction proceeds stereoselectively in a suprafacial manner; specifically, oxygen attack and hydrogen removal take place on the same side of the double bond 84,397 As a result, neither racemization of optically active compounds387 ,398 nor cis-trans isomerization394 occurs. Diradical or dipolar intermediates are not consistent with these observations. The lack of a Markovnikov-type directing effect also rules out dipolar intermediates 60,399 Isotope effects observed in the reaction of different deuterium-labeled 2,3-dimethyl-2-butenes indicated that not all hydrogens (deuteriums) are equally competitive in the transformation 84,397 This led to the suggestion of a symmetric transition state in which oxygen interacts only with the cis grouped C-H(D) bonds 84,397 Although 1,2-dioxetanes are excluded as intermediates in the formation of allylic hydroperoxides, they may be formed as primary products in the reaction of singlet oxygen with alkenes not possessing allylic hydrogen: 36o,363,366,368-37o,374

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due to its low solubility in the liquid phase, a continuous supply is necessary from the gas phase Oxygen gradients can occur as a result of the interaction between oxygen transfer, long circulation times, and microbial kinetics Since oxygen consumption occurs in segregated uid elements circulating in the fermenter, the time constant for oxygen consumption is often the same order of magnitude as the liquid circulation Depletion of the oxygen may occur at long circulation times, whereas the oxygen concentration remains relatively high at short circulation times The time scales for circulation in large scale bioreactors may be comparable to the time scales for certain metabolic processes and adjustments (Roels, 1982) Bailey and Ollis (1986) assumed that the circulation time in a stirred vessel is lognormally distributed and that oxygen consumption rate follows a zero-order reaction.

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They showed that the exposure of cells to starvation conditions increases as the mean circulation time and the standard deviation of circulation times increases This may also indicate that the kinetic models developed under the very different mixing conditions in a small reactor may not apply when greater mixing times and environmental uctuations are encountered at the large scale Several authors have indicated the presence of dissolved oxygen concentration gradients, resulting from insuf cient mixing and mass transfer Their existence has been inferred from regime analysis (see Section 18-26) (Oosterhuis, 1984; Sweere et al, 1986) and in some cases by physical measurement (Carilli et al, 1961; Steel and Maxon, 1966; Manfredini et al, 1983; Oosterhuis and Kossen, 1983, 1984).

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Oosterhuis and Kossen (1984) reported the existence of dissolved oxygen concentration gradients in a 25 m3 production scale fermenter equipped with two Rushton impellers, using a low viscosity broth (refer to Figure 18-1) The values of dissolved oxygen were corrected for hydrostatic pressure Relatively high concentrations were measured in the impeller region (8 to 15% of air saturation at ambient pressure) and low values (0 to 6% of saturation) away from the vicinity of the impeller region in the macromixed zones A strong radial gradient was also observed at the level of the agitator Therefore, cells circulate in agitated vessels from the well-mixed impeller region (active zone) to the relatively poorly mixed regions (quiescent zones) and will experience uctuations in dissolved oxygen concentration In some fermentations, performance may be governed by the ef ciency with which nutrients such as glucose are mixed.

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Gradients in glucose concentration and their effects on microbial metabolism are discussed further in Section 18-271 Inhomogeneities also occur when the addition or removal of a component in a system is made in a nonuniform manner Thus, the addition of concentrated acid or base for pH control will raise the local pH to a high value that will persist for longer if the mixing rate is slow Solution pH is a fundamental parameter in the regulation of cellular metabolism, and the effects of spatial variations in pH can be important for successful scale-up The effect of pH on cell metabolism is discussed in Section 18-211 The viscosity of the broth will in uence the bulk mixing, air dispersion, and power draw by the agitator The rheological behavior of fermentation broths have been reviewed (Metz, 1976; Charles, 1978; Riley et al, 2000) and will generally.

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