122 PHOTO- AND CHEMOSYNTHESIS OF BACTERIA CHAP. 5 



of one molecule of carbon dioxide for each fermented molecule of butanol : 



(5.39) CiHsOH + H2O > 3 CH4 + CO2 + 7 kcal 



It seems plausible to assume, in analogy to steps (5.37) and (5.38), 

 that the last step in butanol fermentation, the decomposition of acetic 

 acid, also involves the participation of carbon dioxide, i. e., proceeds not 

 according to the "abbreviated" equation (5.34), but by a true oxidation- 

 reduction: 



(5.40) CH3COOH + CO2 > 2 CO2 + CH4 - 6 kcal 



A direct proof of the participation of free carbon dioxide in the for- 

 mation of methane in reactions which proceed with the net liberation of 

 carbon dioxide was achieved by means of radioactive carbon, C*. 

 Barker, Ruben and Kamen (1940) showed that the methane fermentation 

 of inactive ethanol by Methanosarcina methanica, in the presence of 

 radioactive carbon dioxide, gives active methane, thus estabhshing the 

 correctness of the equation: 



(5.41) 4 CH3OH + 3 C*02 > 3 C*H4 + 2 H2O + 4 CO2 + 51 kcal 



and precluding the "cancelling out" of three carbon dioxide molecules 

 on each side of this equation. A similar interpretation of acetate 

 fermentation, assumed in (5.40), thus becomes increasingly plausible. 



At first sight, the reduction of carbon dioxide to methane by the 

 methane bacteria appears as a biochemical "art for art's sake," since the 

 product escapes as a gas, carrying with it the accumulated energy. 

 However, Barker, Ruben and Kamen noticed that about 10% of radio- 

 activity supplied in the form of C*02 is found afterwards in the cell 

 material. This shows that, while a large part of reduced carbon dioxide 

 is wasted, a small proportion is utilized for the synthesis of the cell 

 material. This reminds one of the autotrophic bacteria which dissipate 

 most of the available oxidation energy, in order to reduce a small quantity 

 of carbon dioxide to carbohydrate. It seems possible that the methane 

 bacteria have solved a similar problem in a different way (the usual 

 solution being precluded by their anaerobic mode of life). Deprived of 

 oxygen, they cannot derive energy from the autoxidation of the available 

 substrate. Their solution is to use carbon dioxide as an oxidant. We 

 know that none of the available oxidation substrates — not acetate, or 

 methanol, or even hydrogen — has sufficient reducing power to bring 

 about the stoichiometric reduction of carbon dioxide to carbohydrate. 

 However, the reduction of carbon dioxide to methane requires less energy 

 per transferred hydrogen atom than the "halfway" reduction to carbo- 

 hydrate. This is why the reactions (5.35) - (5.41) are exothermal — 

 with the exception of reaction (5.40), which, however, has a positive free 

 energy of about 10 kcal. 



