Biebl (4) studied the inhibition potentials of 1,3-PD, acetic acid, butyric acid and glyce­rol on the growth of C. butyricum in a pH-auxostat culture and found that all these sub­stances are toxic to C. butyricum. The critical concentrations of these substances, i. e., concentrations above which cells cease to grow, were found to be: 27 g/1 for acetic acid (0.49 g undissociated acetic acid/1); 19 g/1 for butyric acid (0.39 g undissociated acid/1), 64.0 g/1 for 1,3-PD, and 97.0 g/1 for glycerol at pH 6.5 in this study with externally ad­ded substances. Zeng et al. (34) further examined the inhibition potentials of substrate and products both self-produced and externally added on C. butyricum and K. pneumo­
niae with the help of a mathematical model. Whereas the inhibition potential of exter­nally added and self-produced 1,3-propanediol reveals to be essentially the same, buty­ric acid produced by the culture is found to be more toxic than that externally added. The same seems to apply for acetic acid. Furthermore, the inhibitory effect of butyric acid is shown to be due to the total concentration instead of its undissociated form.

The inhibition effects of products and substrate in the glycerol fermentation are essentially irrespective of the strains. Thus, a common growth model could be proposed for the two strains anaerobically grown on glycerol at different pH value:

where p is the specific growth rate; H+ is the hydrogen ion concentration; p*max, KH, and KqH are constants; K* is the saturation constant; , C*hbu, C*Et0H> C*PD , C*GIy

are the critical concentrations of acetic acid, butryic acid, ethanol, 1,3-PD and glycerol respectively. In the above model the parameters p*max, KH, and KoH are strain-spe­cific and can be estimated from experimental data for each strain (34). The critical con­centrations of acetic acid, 1,3-PD and glycerol are assumed to be identical for both C butyricum and K. pneumoniae. The best estimates of the critical concentrations (C*pi) are as follows: 0.35 g/1 for undissociated acetic acid, 10.1 g/1 for total butyric acid, 16.6 g/1 for ethanol, 71.4 g/1 for 1,3-PD, and 187.6 g/1 for glycerol. Eq. (13) describes the product and substrate inhibition of both C. butyricum and K. pneumoniae in different types of continous cultures over a pH range of 5.3-8.5 satisfactorily.

Zeng and Deckwer (55) and Zeng (36) studied also the kinetic of substrate con­sumption and product formation of K. pneumoniae. They found that in order to achieve a high substrate uptake rate and a high production rate of 1,3-PD a certain level of gly­cerol excess in the culture is necessary. The dependence of increase of substrate uptake rate and product formation rate on the excess concentration of glycerol can be described with a saturation function similar to that of Michaelis-Menten kinetics.

The maximum achievable concentration of 1,3-PD in the continuous fermenta­tion of glycerol can be predicted from considerations of growth and product formation stoichiometry and product inhibition (Eq.13). As discussed above, the maximum 1,3- PD production would be obtained in both strains if only 1,3-PD and acetic acid are for­med as fermentation products and at the same time there is no formation of hydrogen. Under these conditions the mole ratio of acetic acid to 1,3-PD should be 0.31 mol/mol. At pH = 7 and a residual glycerol concentration of 2-100 mmol/1 (C*GIy » CGiy » Ks) eq. (13) reduces to

^maxd-^Xl-S5) (И)

CHAc CPD

Substituting Сндс by 0.31 CPD Eq. (14) can be used to predict the maximum achievalbe

1,3- PD concentration and productivity under different dilution rate. Since all the para­meters of Eq. 14 are the same for C. butyricum and K. pneumoniae at pH 7.0 (pmax =

0. 67 h’1) the predicted theoretical maximum product concentration and productivity are applied to both strains. Fig. 4 shows the theoretical maximum propanediol concentra­tion and experimental maximum values achieved so far at different dilution rate. At high dilution rate (D > ca. 0.35 h1) nearly the same level of 1,3-PD has been obtained for C. butyricum and K. pneumoniae, being about 70 — 80% of the theoretical maxi­mum. Whereas at low dilution rate (< 0.35 h’1) K. pneumoniae reached 80 — 96% of the theoretical maximum C. butyricum reached only about 40 — 60%. At the lowest dilution rate (0.1 h’1) where experimental data are available for both strains C. butyricum rea­ched less than half of the propanediol concentration and productiviy of K. pneumoniae. Obviously, the maximum values measured for C. butyricum do not necessarily repre­sent the real maximum values achievable at the respective dilution rate. As shown for

K. pneumoniae (Menzel, K.; Zeng, A.-P.; Deckwer, W.-D. Enzyme Microbiol Technol. in press) there exists an optimum residual glycerol concentration for propanediol forma­tion at each dilution rate. So far, no systematical work has been carried out to find the optimum propanediol production at each dilution rate for C. butyricum. Another reason for the lower propanediol production by C. butyricum seems to be due to the less favor­able distribution of the by-products. At lower dilution rates the acetic acid production of C butyricum is far below the acetate level for optimal 13-PD formation. As mentioned above, for an optimal production of 1,3-PD the formation of butyric acid in C. butyri­cum and ethanol in K. pneumoniae should be as low as possible. It can be shown that the butyric acid concentration in C. butyricum culture is often higher than the ethanol concentration in the K. pneumoniae culture. However, butyric acid is nearly twice as toxic as ethanol (34). Thus, 1,3-PD concentration is limited in the C butyricum culture.

In addition, the hydrogen production in C. butyricum appeared to be higher than in К pneumoniae under substrate excess conditions. This also impedes the production of 1,3- PD in C. butyricum. Recently, Barbirato et al. (3) found that the accumulation of 3- hydroxypropionalde-hyde strongly inhibits glycerol fermentation of three enterobacteri­al species including К pneumoniae. Cameron et al. (10) showed that glycerol metabo­lism in E. coli is inhibited by the intracellular accumulation of glycerol-3-phosphate. The identification of possible inhibitory effects of intermediate metabolite(s) in C. buty­ricum is desirable.