Syntheses of Glucose, Cellobiose and Other Saccharides by Acetobacter acetigenum in a Lactate-buffered Glycerol Medium

Abstract

WE have cultivated Acetobacter acetigenum N.C.I.B. 8132, a cellulose-forming micro-organism, in 10 litres of medium in 40 ‘Glaxo’ flasks, which were shaken during incubation at 30° for twenty-one days. The medium contained (gm./l.): potassium dihydrogen phosphate, 2.0; crystalline magnesium sulphate, 1.0; crystalline ferrous sulphate, 0.01; ammonium hydrogen phosphate, 2.5; calcium D-pantothenate, 0.002 ; riboflavin, 0.002; biotin, 0.0001; glycerol, 30; lactic acid, 10.2; the pH value was adjusted with potassium hydroxide to 4.6. The metabolism solution was Seitz-filtered to remove cells and cellulose and concentrated to 500 ml. over barium carbonate, in vacuo at 35°. Activated charcoal, ‘Ultrasorb S.C. 120/240’ (British Carbo Norit Union) as used by Hughes and Whelan¹, was sieved through a British Standards 170 mesh and the material retained on the sieve was used to pack two ‘Pyrex’ glass columns (138 × 7.8 cm.) to a depth of 105 cm. In each column 1.9 kgm. of this charcoal rested on a pad of glass-wool overlayed with 100 gm. of ‘Celite’ added as a slurry in water. The columns were washed each with 5 litres of 50 per cent (v/v) aqueous ethanol and then each with 20 litres of distilled water. After removal of insoluble barium salts the metabolism concentrate was rediluted to 10 litres with distilled water and passed through the columns during 14 hr. Each column was eluted with 80 litres of distilled water followed by aqueous ethanol solutions (each of 50 litres) in which the respective ethanol percentages (v/v) were 0.5, 5, 10, 20, 35, and 50. Six aqueous ethanolic fractions, A ₁–A ₆, were collected and concentrated at 35°. Portions of each were de-ionized with ‘Zeo-Karb 225’ and ‘Deacidite E’ and chromatographed on paper against authentic sugars using (a) n-propanol/water/ethylacetate, 7:2:1 by volume; (b) pyridine/water/ethylacetate, 1:2:2 by volume; (c) ethyl acetate/acetic acid/water, 3:1:3 by volume. The chromatograms gave evidence of the presence of the following sugars: A ₂, glucose, fructose, xylose, and ribulose; A ₃ similar to A ₂ together with a trace of cellobiose; A ₄, mainly cellobiose ; A ₅, cellotriose and cellotetraose. Fraction A ₃ was then de-ionized and submitted to column partition chromatography according to the procedure of Lemieux, Bishop and Pelletier², which involves the use of ‘Celite No. 535’ to hold the stationary phase. After irrigation with water-saturated n-butanol, the column was extruded and cut into zones as determined by an alkaline permanganate spray. A fraction was separated which, after several further passages through the ‘Celite’ and a final purification by band paper chromatography, yielded 45 mgm. of a substance which had the mobility of glucose when chromatographed in two different solvent systems. An acetate was prepared by heating with anhydrous sodium acetate and acetic anhydride, and after purification this had m.p. 131°, alone or mixed with authentic β-penta-acetyl glucose of m.p. 131°. The infra-red spectra of both acetates were compared and no significant differences could be observed. (Found: C, 49.3; H, 5.8 per cent. Calc. for C₁₆H₂₂O₁₁: C, 49.2; H, 5.7 per cent.)