38 BACTERIAL FERMENTATIONS 



reactive in the CI. kluyveri system was vinylacetic acid 

 (3-butenoic acid) . This compound at first showed the 

 reactions expected of an intermediate. Like butyrate, it 

 was oxidized to acetyl phosphate and acetate (reaction 11) 

 or, in the absence of orthophosphate, to acetoacetate (reac- 

 tion 12). 



CH 2 ==CHCH 2 COOH + H 3 P0 4 + \0 2 — > 



CH3COOPO3H2 + CH3COOH (11) 



CH 2 =CHCH 2 COOH + J0 2 -^ CH 3 COCH 2 COOH (12) 



CH 2 =CHCH 2 COOH + H 2 — > CH 3 CH 2 CH 2 COOH (13) 



2CH 2 =CHCH 2 COOH + H3PO4 + H 2 — > 

 CH 3 CH 2 CH 2 COOH + 



CH3COOH + CH 3 COOP0 3 H 2 (14) 



In the presence of hydrogen, vinylacetate was reduced to 

 butyrate (reaction 13) and in a nitrogen atmosphere it 

 underwent a typical dismutation (reaction 14) . Further- 

 more, all of these reactions occurred at rates equal to or 

 greater than the over-all rates of butyrate synthesis and 

 oxidation. However, all attempts to demonstrate the actual 

 formation of vinylacetate during the synthesis or oxidation 

 of butyrate were decisively negative. For example, when 

 C 14 -butyrate was oxidized in the presence of a pool of 

 unlabeled vinylacetate, essentially no C 14 was incorporated 

 into the vinylacetate. This and other experiments excluded 

 vinylacetate as a normal intermediate in butyrate metab- 

 olism. 



The negative results obtained with vinylacetate, aceto- 

 acetate, and other acids at the same oxidation levels, ap- 

 peared to exclude all C 4 acids as intermediates in butyrate 

 metabolism. This led inevitably to the hypothesis that 

 the actual intermediates are compounds in which the 



