Effects of hydrogen partial pressure on autotrophic growth and product formation of Acetobacterium woodii.

Low aqueous solubility of the gases for autotrophic fermentations (e.g., hydrogen gas) results in low productivities in bioreactors. A frequently suggested approach to overcome mass transfer limitation is to increase the solubility of the limiting gas in the reaction medium by increasing the partial pressure in the gas phase. An increased inlet hydrogen partial pressure of up to 2.1 bar (total pressure of 3.5 bar) was applied for the autotrophic conversion of hydrogen and carbon dioxide with Acetobacterium woodii in a batch-operated stirred-tank bioreactor with continuous gas supply. Compared to the autotrophic batch process with an inlet hydrogen partial pressure of 0.4 bar (total pressure of 1.0 bar) the final acetate concentration after 3.1 days was reduced to 50 % (29.2 g L(-1) compared to 59.3 g L(-1)), but the final formate concentration was increased by a factor of 18 (7.3 g L(-1) compared to 0.4 g L(-1)). Applying recombinant A. woodii strains overexpressing either genes for enzymes in the methyl branch of the Wood-Ljungdahl pathway or the genes phosphotransacetylase and acetate kinase at an inlet hydrogen partial pressure of 1.4 bar reduced the final formate concentration by up to 40 % and increased the final dry cell mass and acetate concentrations compared to the wild type strain. Solely the overexpression of the two genes for ATP regeneration at the end of the Wood-Ljungdahl pathway resulted in an initial switch off of formate production at increased hydrogen partial pressure until the maximum of the hydrogen uptake rate was reached.

Continuous gas fermentation by Acetobacterium woodii in a submerged membrane reactor with full cell retention.

Acetogenic bacteria like Acetobacterium woodii represent an ancient group of anaerobic microorganisms which use hydrogen and carbon dioxide to produce acetate. Cell concentrations and space-time yields are usually low in gas fermentations. A standard stirred­tank bioreactor with continuous gas supply was equipped with a customized submerged microfiltration unit. A. woodii showed similar growth behavior with an initial maximal growth rate of 1.2 d(-1) in continuous gas fermentations with full cell retention and varying dilution rates. A steady increase of cell mass concentrations was observed with the highest biomass formation at the highest dilution rate. By contrast the final acetate concentrations were lowest at the highest dilution rate. The highest final acetate space-time yield of 148 g l(-1) d(-1) was measured at the highest dilution rate (increase by factor 8 compared to a standard batch process or by factor 37 compared to published data). The highest reported cell concentration of A. woodii in gas fermentations of nearly 14 g l(-1) cell dry weight was achieved in the submerged membrane bioreactor with increased yeast extract concentrations in the feed medium. Product inhibition was observed when acetate concentrations exceeded 8-12 g l(-1) causing a steady decrease in cell mass specific acetate production rates.

Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts.

Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non-surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven-step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo-enzymatic preparation of UDCA-the two-step reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid using a mutant of 7ß-hydroxysteroid dehydrogenase (7ß-HSDH) from Collinsella aerofaciens and 3α-hydroxysteroid dehydrogenase (3α-HSDH) from Comamonas testosteroni. Three different one-pot reaction approaches were investigated using whole-cell biocatalysts in simple batch processes. We applied one-biocatalyst systems, where 3α-HSDH, 7ß-HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two-biocatalyst systems, where 3α-HSDH and 7ß-HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one-pot reaction. The best result was achieved by the one-biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100 mM DHCA to >99.5 mM 12-keto-UDCA within 4.5 h in a simple batch process on a liter scale.