1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO2, H2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH2, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a "green" polyester is a most cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this p range 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed.

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The initial steps of glycerol dissimilation and 1,3‐propanediol (1,3‐PD) formation by Klebsiella pneumoniae anaerobically grown on glycerol were studied by quantifying the in vitro and in vivo activities of enzymes in continuous culture under conditions of steady state and oscillation and during transient phases. The enzymes studied included glycerol dehydrogenase (GDH), glycerol dehydratase (GDHt), and 1,3‐propanediol oxidoreductase (PDOR). Three conclusions can be drawn from the steady‐state results. First, glycerol concentration in the culture is a key parameter that inversely affects the in vitro activities (concentrations) of all three enzymes, but has a positive effect on their in vivo activities. Growth rate significantly affects the ratio of in vitro and in vivo enzyme activities under low glycerol concentrations, but not under glycerol excess. Second, whereas the flux through the oxidative pathway of glycerol dissimilation is governed mainly by the regulation of in vivo enzyme activity on a metabolic level, the flux through the reductive pathway is largely controlled by the synthesis of enzymes. Third, GDHt is a major rate‐liming enzyme for the consumption of glycerol and the formation of 1,3‐PD in K. pneumoniae at high glycerol concentrations. Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated. The average values of the in vitro activities during an oscillation cycle agreed well with their corresponding values for nonoscillating cultures under similar environmental conditions. Experiments with step changes in the feed concentration of glycerol demonstrated that growth and product formation are very sensitive to changes of substrate concentration in the culture. This sensitivity is due to the dynamic responses of the genetic and metabolic networks. They should be considered when modeling the dynamics of the culture and attempting to improve the formation of 1,3‐PD. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 544–552, 1998.

Oscillation and hysteresis phenomena are observed in the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae in long‐term cultivations under a variety of conditions. In this work, the conditions for the occurrence of these phenomena are reported and the patterns of cell growth and metabolism under oscillation are characterized. During an oscillation period, the formation rates of CO2, H2, and formate and the consumption rate of alkali periodically pass values of maxima and minima, the latter being close to zero. The formation of biomass and fermentation products such as 1,3‐propanediol, acetate, and ethanol also undergo periodic changes which shift maxima and minima. Sustained oscillation occurs only under conditions of substrate excess within a distinct regime. At pH 7.0, it is only found at dilution rates above 0.15 h−1 under the experimental conditions. At lower pH values, oscillations are more likely to happen, even at a relatively low dilution rate and low substrate excess. Whereas the amplitude of oscillations at pH 7.0 depends on both the dilution rate and the residual glycerol concentration (CGlyc) the interval of oscillations appears to be only a function of CGlyc. An increase of CGlyc in culture damps the oscillation and leads to its disappearance at CGlyc = 1100 to 1200 mmol/L (pH 7.0). The operation mode was also found to be an important parameter in determining the stability and actual state of the culture, resulting in hysteresis under certain conditions, particularly at low pH values. Generally, a large perturbation of cultivation conditions tends to cause oscillation and hysteresis. The results unambiguously demonstrate that the oscillation and hysteresis phenomena shown in this work are bound to genuine metabolic fluctuations of the microorganism. They reveal several differences and new features compared with those reported in the literature and cannot be readily explained by the mechanisms known so far. © 1996 John Wiley & Sons, Inc.