BUTYRIC ACID-BUTANOL FERMENTATIONS 37 



since it must involve a minimum of three reactions: a con- 

 densation and two reduction steps. The simplest possible 

 mechanism of butyrate synthesis and oxidation, used as a 

 working hypothesis in subsequent experimentation, is shown 

 in the following reaction sequence: 



CH3COOPO3H2+CH3COOH ^=± CH3COCH2COOH 



1 



±2H 

 ±2H \Y 



CH3CH2CH2COOH ^=± CH3CHOHCH2COOH 



±H 2 



In this sequence acetyl phosphate was postulated to con- 

 dense with acetate to form acetoacetate which was reduced 

 first to /?-hydroxybutyrate and then to butyrate. Both of 

 the postulated intermediates were known to accumulate 

 during the oxidation of fatty acids in animals under certain 

 circumstances. 



The above sequence could readily be tested with CI. 

 Kluyveri extracts by finding out whether acetoacetate and 

 /?-hydroxybutyrate react as the scheme predicts. Stadtman 22 

 found that they do not so react. /3-Hydroxybutyrate could 

 not be reduced to butyrate nor oxidized to acetyl phosphate. 

 Acetoacetate could undergo a phosphoroclastic conversion 

 to acetyl phosphate and acetate, but this reaction was too 

 slow and occurred under the wrong conditions to partici- 

 pate in butyrate oxidation. Furthermore, although aceto- 

 acetate could be reduced enzymatically with molecular 

 hydrogen as the scheme predicts, the product was not butyr- 

 ate but /3-hydroxybutyrate, which, could not be further 

 reduced. These observations eliminated acetoacetate and 

 /J-hydroxybutyrate as intermediates in butyrate synthesis. 



A variety of other four-carbon acids at the oxidation 

 levels of acetoacetate and /J-hydroxybutyrate were then 

 tested. 22 - 23 The only one of these acids that proved to be 



