Glycerol can be converted to 1,3-PD by a number of bacteria including strains of the genera Klebsiella, Citrobacter, and Clostridium under anaerobic conditions. Among these, Klebsiella pneumoniae and Clostridium butyricum have been most intensively studied. The pathway of anaerobic dissimilation in these strains is depicted in Fig. l.

Fig. l Pathway of anaerobic glycerol dissimilation in К pneumoniae and C. butyri­cum.

Glycerol is fermented by a dismutation process involving two parallel pathways. Through the oxidative pathway, glycerol is dehydrogenated by an NADMinked enzy­me, glycerol dehydrogenase, to dihydroxyacetone (DHA) which is then further metabo­lized to pyruvate. Through the parallel reductive pathway, glycerol is dehydrated by a В ^-dependent glycerol dehydratase to form 3-hydroxypropionaldehyde which is then reduced to 1,3-PD by an NADH-linked oxidoreductase, 1,3-PD dehydrogenase. The physiological role of the 1,3-propanediol pathway is to regenerate the reducing equiva­lents (NADH2) which are released from the formation of DHA and during the further oxidation of dihydroxyacetonephosphate (DHAP) as well as from biosynthesis. The enzymes leading to the formation of 1,3-PD and DHAP have been studied by many re­searchers with strains of Enterobacteriaceae (9,19,20,25,29). Enzymes active in the gly­cerol metabolism of C butyricum were measured very recently (1,2).

The further metabolism of DHAP is essential to provide ATP for cell growth and for the necessary phosphorylation of dihydroxyacetone. In addition, it provides the reducing equivalents for the 1,3-PD pathway, leading to the formation of 1,3-PD. De­spite its importance for an optimum production of 1,3-PD the metabolism of DHAP and its subsequent metabolites has received little attention in the past. In fact, the pathways of DHAP (and pyruvate) oxidation have been taken from glucose metabolism without experimental evidence. There is few work concerning the enzymes which catalyze the metabolism of pyruvate in pure glycerol fermentation. Zeng et al. (33,38) applied the pathway stoichiometry to analyze the fermentation of glycerol in both K. pneumoniae and C. butyricum, with particular emphasis on the regulation of pyruvate metabolism and its influences on the 1,3-PD yield and selectivity. Fig. 2 summarizes the pathways of pyruvate metabolism in K. pneumoniae and C. butyricum grown anaerobically on glycerol, respectively.

(1) Pyruvate formate-lyase

(2) Pyruvate dehydrogenase

(3) Pyruvaterferredoxin oxidoreductase

Fig. 2. Pyruvate metabolism during anaerobic fermentation of glycerol.

The cleavage of pyruvate to acetyl-CoA and C02 is assumed to be carried out by the enzyme pyruvate: ferredoxin oxidoreductase in C. butyricum and by the enzyme pyruvate-formate lyase in K. pneumoniae in the literature. Acetic acid and butyric acid are the main fermentation products of pyruvate in C. butyricum. K. pneumoniae produ­ces no butyric acid, but ethanol as one of the main products. Minor products include 2,3-butanediol, lactic and formic acid. At low pH value a significant amount of 2,3-bu — tanediol is formed in the glycerol fermentation of K. pneumoniae (7).

Analysis of continuous culture data of C. butyricum revealed that the reduced ferredoxin (Fdred) formed during oxidation of pyruvate to acetyl-CoA is not completely cleaved into hydrogen and oxidized ferredoxin under conditions of glycerol excess (38). Instead, part of the reducing power from Fdred is transferred to NAD+ under the forma­tion of NADH2. The enzymes catalysing this reaction had been previously described for C acetobutylicum (21) and recently for C. butyricum (2). In K. pneumoniae reducing equivalents released from pyruvate cleavage by the pyruvate formate lyase are trapped in formate and cannot be transferred to NAD. It was therefore surprising to find sub­stantial deviations of the ratio of 1,3-PD to hydrogen from the calculated one based on the action of pyruvate formate lyase in this species. We could recently demonstrate by enzyme assays that pyruvate dehydrogenase, which is normally the enzyme complex for an aerobic pyruvate decarboxylation in Enterobacteriaceae, is simultaneously involved in this anaerobic fermentation process (Menzel et al., GBF, unpublished data). Factors that affect the pyruvate dehydrogenase activity in K. pneumoniae are being studied in continuous culture with the goal of further increasing the activity of this enzyme for a high yield and flux of 1,3-PD. Although the activity of this enzyme is desirable its si­multaneous involvement in addition to pyruvate formate lyase in the K. pneumoniae culture gives rise to unfavorable dynamic behavior of the pathways such as oscillation and hysteresis under a variety of conditions (37).