─────────────────┬────────────────────────────┬───────────────────── 20 c.c. Extract │ CO{2} in grams in │ Duration Total + 0·2 gram. │ successive periods of │ of fermen- CO{2}. │6 hours.│18 hours.│24 hours.│ tation. ─────────────────┼────────┴─────────┴─────────┼─────────────────── Cane sugar │ 0·050 0·000 0·000 │ 6 0·05 Dihydroxyacetone │ 0·042 0·000 0·000 │ 6 0·042 Glyceraldehyde │ 0·008 0·022 0·005 │ 48 0·035 ─────────────────┴────────────────────────────┴─────────────────── Further, during an experiment in which 0·129 gram of CO{2} was evolved in 22·5 hours from 0·9 gram of glyceraldehyde in presence of phosphate, no change in free phosphate was observed, whereas in a similar experiment with glucose a loss of about 0·2 gram of P{2}O{5} would have occurred. Hence the fermentation takes place without formation of hexosediphosphate. This was confirmed by the fact that the osazone of hexosephosphoric acid was readily isolated from the products of fermentation of dihydroxyacetone (0·259 gram of CO{2} having been evolved in twenty hours) but could not be obtained from those of glyceraldehyde (0·138 gram CO{2} in twenty hours). [p108] This result is extremely interesting, although it is not impossible that the rate of fermentation of the glyceraldehyde is so slow that any phosphoric ester produced is hydrolysed as rapidly as it is formed. Lebedeff regards the experiments as proof that phosphate takes no part in the fermentation of glyceraldehyde and bases on this conclusion and his other work the following theory of alcoholic fermentation. 1. The sugar is split up into equimolecular proportions of glyceraldehyde and dihydroxyacetone:-- (a) C{6}H{12}O{6} = C{3}H{6}O{3} + C{3}H{6}O{3}. 2. The dihydroxyacetone then passes through the stages previously postulated (p. 106). (b) 4 C{3}H{6}O{3} + 4 R{2}HPO{4} = 4 C{3}H{5}O{2}PO{4}R{2} + 4 H{2}O. (c) 4 C{3}H{5}O{2}PO{4}R{2} = 2 C{6}H{10}O{4}(R{2}PO{4}){2}. (d) 2 C{6}H{10}O{4}(R{2}PO{4}){2} + 4 H{2}O = 2 C{6}H{12}O{6} + 4 R{2}HPO{4}. After which the hexose, C{6}H{12}O{6} re-enters the cycle at (a). 3. The fermentation of the glyceraldehyde occurs according to the scheme developed by Kostytscheff (p. 109), pyruvic acid being formed along with hydrogen and then decomposed into carbon dioxide and acetaldehyde, which is reduced by the hydrogen. Lebedeff, however, suggests [1914, 1, 2] that glyceric acid is first formed (1) and then converted by an enzyme, which he terms /dehydratase/ into pyruvic acid (2):-- (1) CH{2}(OH)·CH(OH)·CHO + H{2}O → CH{2}(OH)·CH(OH)·CH(OH){2} CH{2}(OH)·CH(OH)·CH(OH){2} → CH{2}(OH)·CH(OH)·COOH + 2H (2) CH{2}(OH)·CH(OH)·COOH = CH{3}·CO·COOH + H{2}O. The experimental basis for this idea is the fact that glyceric acid is fermented by dried yeast and maceration juice [compare Neuberg and Tir, 1911].