Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat

Abstract

The fermentation of glycerol to 1,3-propanediol, acetate, and butyrate by Clostridium butyricum was studied with respect to growth inhibition by the accumulating products. The clostridia were grown in a pHauxostat culture at low cell density and product concentration and near maximum growth rate. The products were then added individually to the medium in increasing concentrations and the resulting depression of growth rate was used as a quantitative estimate of product inhibition. Under these conditions growth was totally inhibited at concentrations of 60 g/l for 1,3-propanediol, 27 g/1 for acetic acid and 19 g/1 for butyric acid at pH 6.5. Appreciable inhibition by glycerol was found only above a concentration of 80 g/l. In a pH-auxostat without added products but with high cell density as well as in batch cultures the product proportions were different. The 1,3-propanediol concentration may approach the value of complete inhibition while the concentrations of acetic and butyric acids remained below these values by at least one order of magnitude. It was therefore concluded that 1,3-propanediol is the first range inhibitor in this fermentation.

1,3-Propanediol Production by Clostridium Butyricum in Various Fed- Batch Feeding Strategies

“…The determination of the cellular concentration (biomass) is esteemed by measure of optical density (OD) by spectrophotometer in 660 nm [12].…”

Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol

“…All controlled batch bioreactor studies were performed with defined minimal medium containing 3.5 g/liter of KH 2 PO 4 , 5.0 g/liter of K 2 HPO 4 , 3.5 g/liter of (NH 4 ) 2 HPO 4 , 0.25 g/liter of MgSO 4 ⅐ 7H 2 O, 15 mg/ liter of CaCl 2 ⅐ 2H 2 O, 0.5 mg/liter of thiamine, 0.12 g/liter of betaine, 1 ml/liter of a stock trace metal solution, 40 g/liter of glycerol (unless otherwise specified), and 10 g/liter of tetracycline (adapted from media described previously [6,21,26] 4 ) citrate. The first three salt components of the defined minimal medium were autoclaved, while the other components were sterile filtered and added to bioreactors.…”

“…For instance, chemical conversion based on the etherification of glycerol with either alcohols (methanol or ethanol) or alkenes (isobutene or 2-methylpropene) can produce useful fuels or solvents, or steam reforming of glycerol can result in methanol and hydrogen production (7). Biological conversion utilizes species belonging to the Enterobacteriaceae family, such as Klebsiella pneumoniae (11), Citrobacter freundii (5), Clostridium butyricum (4), and Pantoea agglomerans (3), to convert glycerol to 1,3-propanediol by fermentation.…”

Use of Glycerol in Biotechnological Applications

“…In C. butyricum, growth is completely inhibited at 1,3-PDO concentrations higher than 60 g l -1 . Also the byproducts acetic acid (27 g l -1 ) and butyric acid (19 g l -1 ) abolished growth of this bacterium as did glycerol concentrations of 80 g l -1 or more (Biebl, 1991, Colin et al, 2000. Growth of K. pneumoniae is inhibited at glycerol concentrations above 110 g l -1 under aerobic and above 133 g l -1 under anaerobic conditions.…”

“…Production of 1,3-PDO has been shown e.g. for strains of Klebsiella pneumoniae, Clostridium butyricum, Clostridium acetobutylicum, Clostridium pasteurianum, Clostridium butylicum, Clostridium beijerinckii, Clostridium kainantoi, Lactobacillus brevis and Lactobacillus buchneri, and Enterobacter agglomerans (Forage & Foster, 1982, Barbirato et al, 1996, Nakas et al, 1983, Schutz & Radler, 1984, Homann et al, 1990, Biebl, 1991, Biebl et al, 1992, Daniel et al, 1995. Biosynthesis of 1,3-PDO from glycerol is catalyzed in a reducing pathway involving a cytosolic two-step process.…”