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Cephalosporin C

Fig. 9.4. The biosynthesis of cephalosporin C, indicating the oxygen consuming steps:

(i) isopenicillin-N-synthase,

(ii) deacetoxycephalosporin C synthase (commonly called expandase),

(iii) deacetyl cephalosporin C synthase (commonly called hydroxylase).

,il the gas. The method for provision of a culture with a supply iur varies with the scale of the process:

(i) Laboratory-scale cultures may be aerated by means of the shake-flask technique where the culture (50 to 100 cm3) is grown in a conical flask (250 to 500 cm3) shaken on a platform contained in a controlled environment chamber.

(ii) Pilot- and industrial-scale fermentations are normally carried out in stirred, aerated vessels, termed fermenters, of the type described in Chapter 7. However, it is often advantageous to culture relatively small volumes (1 dm3) in a stirred, aerated vessel as this enables the cultural conditions to be better monitored and controlled, and facilitates the addition of supplements and the removal of samples. Some fermenters are so designed that adequate oxygen transfer is obtained without agitation and the design of these systems (termed bubble columns and air-lift fermenters) is also discussed in Chapter 7.

Bartholomew et al. (1950) represented the transfer of oxygen from air to the cell, during a fermentation, as occurring in a number of steps:

(i) The transfer of oxygen from an air bubble into solution.

(ii) The transfer of the dissolved oxygen through the fermentation medium to the microbial cell.

(iii) The uptake of the dissolved oxygen by the cell.

These workers demonstrated that the limiting step in the transfer of oxygen from air to the cell in a Strep-tomyces griseus fermentation was the transfer of oxygen into solution. These findings have been shown to be correct for non-viscous fermentations but it has been demonstrated that transfer may be limited by either of the other two stages in certain highly viscous fermentations. The difficulties inherent in such fermentations are discussed later in this chapter.

The rate of oxygen transfer from air bubble to the liquid phase may be described by the equation:

where CL is the concentration of dissolved oxygen in the fermentation broth (mmoles dm""3), t is time (hours), dCL/At is the change in oxygen concentration over a time period, i.e. the oxygen-transfer rate (mmoles 02 dm~3 h 1), Kl is the mass transfer coefficient (cm h-1), a is the gas/liquid interface area per liquid volume (cm2 cm \), C* is the saturated dissolved oxygen concentration (mmoles dm Kl may be considered as the sum of the reciprocals of the resistances to the transfer of oxygen from gas to liquid and (C* - CL) may be considered as the 'driving force' across the resistances. It is extremely difficult to measure both KL and 'a' in a fermentation and, therefore, the two terms are generally combined in the term KLa, the volumetric mass-transfer coefficient, the units of which are reciprocal time (h_1). The volumetric mass-transfer coefficient is used as a measure of the aeration capacity of a fermenter. The larger the KLa, the higher the aeration capacity of the system. The KLa value will depend upon the design and operating conditions of the fermenter and will be affected by such variables as aeration rate, agitation rate and impeller design. These variables affect 'KL' by reducing the resistances to transfer and affect 'a' by changing the number, size and residence time of air bubbles. It is convenient to use KLa as a yardstick of fermenter performance because, unlike the oxygen-transfer rate, it is unaffected by dissolved oxygen concentration. However, the oxygen transfer rate is the critical criterion in a fermentation and, as may be seen from equation 9.1, it is affected by both KLa and dissolved oxygen concentration. The dissolved oxygen concentration reflects the balance between the supply of dissolved oxygen by the fermenter and the oxygen demand of the organism. If the KLa of the fermenter is such that the oxygen demand of the organism cannot be met, the dissolved oxygen concentration will decrease below the critical level (Ccrit). If the K, a is such that the oxygen demand of the organism can be easily met the dissolved oxygen concentration will be greater than Ccrit and may be as high as 70 to 80% of the saturation level. Thus, the KLa of the fermenter must be such that the optimum oxygen concentration for product formation can be maintained in solution throughout the fermentation.

DETERMINATION OF K, a VALUES

The determination of the KLa of a fermenter is essential in order to establish its aeration efficiency and to quantify the effects of operating variables on the provision of oxygen. This section considers the merits and limitations of the methods available for the determination of KLa values. It is important to remember at this stage that dissolved oxygen is usually monitored using a dissolved oxygen electrode (see Chapter 8) which records dissolved oxygen activity or dissolved oxygen tension (DOT) whilst the equations describing oxygen transfer are based on dissolved oxygen concentration. The solubility of oxygen is affected by dissolved solutes so that pure water and a fermentation medium saturated with oxygen would have different dissolved oxygen concentrations yet have the same DOT, i.e. an oxygen electrode would record 100% for both. Thus, to translate DOT into concentration the solubility of oxygen in the fermentation medium must be known and this can present difficulties.

The sulphite oxidation technique

Cooper et al. (1944) were the first to describe the determination of oxygen-transfer rates in aerated vessels by the oxidation of sodium sulphite solution. This technique does not require the measurement of dissolved oxygen concentrations but relies on the rate of conversion of a 0.5 m solution of sodium sulphite to sodium sulphate in the presence of a copper or cobalt catalyst:

The rate of reaction is such that as oxygen enters solution it is immediately consumed in the oxidation of sulphite, so that the sulphite oxidation rate is equivalent to the oxygen-transfer rate. The dissolved oxygen concentration, for all practical purposes, will be zero and the KLa may then be calculated from the equation:

(where OTR is the oxygen transfer rate).

The procedure is carried out as follows: the fermenter is batched with a 0.5 m solution of sodium sulphite containing 10 3 m Cu2+ ions and aerated and agitated at fixed rates; samples are removed at set time intervals (depending on the aeration and agitation rates) and added to excess iodine solution which reacts with the unconsumed sulphite, the level of which may be determined by a back titration with standard sodium thiosulphate solution. The volumes of the thiosulphate titrations are plotted against sample time and the oxygen transfer rate may be calculated from the slope of the graph.

The sulphite oxidation method has the advantage of simplicity and, also, the technique involves sampling the bulk liquid in the fermenter and, therefore, removes some of the problems of conditions varying through the volume of the vessel. However, the method is time consuming (one determination taking up to 3 hours, depending on the aeration and agitation rates) and is notoriously inaccurate. Bell and Gallo (1971) demonstrated that minor amounts of surface-active contaminants (such as amino acids, proteins, fatty acids, esters, lipids, etc.) could have a major effect on the accuracy of the technique and apparent differences in aeration efficiency between vessels could be due to differences in the degree of contamination. Also, the rheology of a sodium sulphite solution is completely different from that of a fermentation broth, especially a mycelial one so that it is impossible to relate the results of sodium sulphite determinations to real fermentations. To quote Van't Riet and Tramper (1991) "It can safely be said that the application of this method should be strongly discouraged".

Gassing-out techniques

The estimation of the KLa of a fermentation system by gassing-out techniques depends upon monitoring the increase in dissolved oxygen concentration of a solution during aeration and agitation. The oxygen transfer rate will decrease during the period of aeration as CL approaches C* due to the decline in the driving force (C* — CL). The oxygen transfer rate, at any one time, will be equal to the slope of the tangent to the curve of values of dissolved oxygen concentration against time of aeration, as shown in Fig. 9.5.

To monitor the increase in dissolved oxygen over an adequate range it is necessary first to decrease the oxygen level to a low value. Two methods have been employed to achieve this lowering of the dissolved oxygen concentration — the static method and the dynamic method.

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