146 PBOvisiON OF energy: fermentation 



when growing on alcohol and acetate it synthesises butyric and 

 caproic acids with the Hberation of some hydrogen. If acetic 

 acid, isotopically labelled (C*) in the position CHgC^OOH, is 

 added to the culture, the resulting acids are labelled in the 

 positions CH3.C*H2.CH2.C*OOH (butyric) and CHg.C^Hg.CHa. 

 C*H2.CH2.C*OOH (caproic) indicating that they are formed by 

 condensation of acetate molecules. Cell-free enzyme prepara- 

 tions have been made which will accomplish the synthesis of 

 butyric and caproic acids from ethyl alcohol and acetate under 

 anaerobic conditions. Analysis of the reactions involved shows 

 that the alcohol is first oxidised to acetaldehyde and acetyl- 

 phosphate with the liberation of hydrogen, and this hydrogen 

 is then utilised to reduce (acetylphosphate -f acetate) to 

 butjrric acid. The over-all reactions can be represented: — 



1. 2CH3.CH2OH + HsPO^^CHg.CHO -f CH3.COOPO3H2 +3H2 



2. CH3.COOPO3H2 + CH3.COOH + 2H,->CH3.CH2.CH2.COOH -)- 



H3PO4 + H2O 



The initial step involved in the condensation of acetylphosphate 

 and acetate has not yet been clarified. The work on acetone 

 production, outlined above, suggests that acetoacetic acid 

 might be concerned but if this substance is added to the enzyme 

 preparation it is either split irreversibly to acetylphosphate and 

 acetate or, in the presence of hydrogen, reduced to ^-hydroxy- 

 butyric acid and does not give rise to butyric acid under any 

 condition tested. A number of other possible intermediate 

 substances have now been tested and the only one which will 

 give rise to butyric acid is vinyl acetate, CH2 = CH.CH2.COOH, 

 but isotope studies indicate that it is not, in fact, involved in 

 the production of butyric acid from acetylphosphate and 

 acetate. The present situation concerning the synthesis of 

 butyric acid by CI. kluyveri can be summarised diagram- 

 matically as follows: — 



CH3.CH2OH CH2 : CH.CH2.COOH 



I j 4H 



CH3.COOH -f CH3.COOPO3H2 >? >CH3.CH2.CH2.COOH -f H3PO4 



t 2H 



CH,.CO.CHo.COOH- 



