Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose

Abstract

The metabolism of CIostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (9gluc,,se = 11.1 mmol h-' g-') was around four times lower than the specific rate of glycerol catabolism (qglycero, = 57-4 mmol h-' g-'). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways. INTRODUCTIONOver recent years there has been an increase in research and development efforts involving clostridial fermentation, namely the fermentation of glucose by butyric acid bacteria (Woods, 1993). Clostridia are able to ferment the carbon source to a variety of end-products by means of branched fermentation pathways. Generally, the different branches of fermentation are not equivalent with respect to the generation of energy or reducing equivalents, andAbbreviations: DHA, dihydroxyacetone; fd, ferredoxin; 3-HPA, 3-hydroxypropionaldehyde; PD, propanediol.the amount of reduced versus neutral and oxidized products has the potential for a great deal of natural variation. It was shown (Jungermann et al., 1973; Petitdemange e t d. , 1976) that the reduced electron carrier ferredoxin (fd) plays a pivotal role in electron distribution in the cell in that it can either transfer electrons via hydrogenase to generate hydrogen or transfer electrons to the pyridine nucleotides via the appropriate fd oxidoreductase. The activities of three enzymes, NADH-fd oxidoreductase, NADPH-fd oxidoreductase and hydrogen...

Effect of glucose on glycerol metabolism by Clostridium butyricum DSM 5431

“…butyricum on glycerol is preceded by the induction of four enzymes : GDH, DHA kinase, glycerol dehydratase and 1,3-PD dehydrogenase (Dabrock et al 1992 ;Abbad-Andaloussi et al 1996). In this study, it is shown that the induced synthesis of these enzymes is repressed by the inclusion of glucose in the growth medium chiefly at the start of fermentation ; the levels of 1,3-PD dehydrogenase and GDH were then similar to those found in extracts of cells grown on glycerol.…”

“…When glycerol and glucose are compared as carbon sources for growth, they show a number of differences in catabolism (Abbad-Andaloussi et al 1996). In glucose-grown cells, the difference between the specific production rate of NADH and the specific consumption rate indicates an excess of NADH which was reoxidized by the NADH-ferredoxin reductase and the reduced ferredoxin (fd) reoxidized by hydrogenase (Abbad-Andaloussi et al 1996).…”

“…In glucose-grown cells, the difference between the specific production rate of NADH and the specific consumption rate indicates an excess of NADH which was reoxidized by the NADH-ferredoxin reductase and the reduced ferredoxin (fd) reoxidized by hydrogenase (Abbad-Andaloussi et al 1996). In contrast, growth on glycerol indicates that the NADH produced by glycerol dehydrogenase (GDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is not sufficient for 1,3-PD For inoculum preparation, spores were transferred to RCM medium, heat shocked at 80°C for 10 min and incubated at 35°C under anaerobic conditions in Hungate tubes.…”

Glycerol fermentation by (open) mixed cultures: A chemostat study

“…To investigate glycerol fermentation, most previous studies have applied pure cultures; Klebsiella aerogenes or Clostridium butyricum are the best known examples (Papanikolaou et al, 2000;Zeng et al, 1993). In a study with C. butyricum, 1,3-propanediol emerged when the culture was fed with glycerol instead of glucose, whilst the production of organic acids (butyrate and acetate) decreased (AbbadAndaloussi et al, 1996). Ethanol production from glycerol has also been investigated, because for certain bacterial strains under similar operational conditions, the ethanol yields obtained on glycerol are higher than those obtained on glucose (Dharmadi et al, 2006;Ito et al, 2005;Jarvis et al, 1997).…”

Properties and sequence of the coenzyme B12-dependent glycerol dehydratase of Clostridium pasteurianum

“…The activity of all four key enzymes known from 1,3-propanediol-forming enteric bacteria has been determined in crude extracts of C. pasteurianum [8]. This was also done for C. butyricum, except that the activity of the dihydroxyacetone kinase was not measured [9]. Recently, we have cloned the genes coding for glycerol dehydratase and 1,3-propanediol dehydrogenase of C. pasteurianum in Escherichia coli.…”