HOW DO MICROBES "FIX" NITROGEN FROM THE AIR? — NICHOLAS 457 



a mechanism for preventing free oxygen from coming in direct contact 

 with the nitrogen-fixing system and competing with nitrogen for the 

 reducing power generated by the bacteroids. When the rhizobia are 

 cultivated outside the nodules they require nitrogen compounds for 

 growth, such as nitrate, ammonia, or amino acids, as they are unable 

 to fix nitrogen without the host plant. Nodules cut out or sliced soon 

 lose their capacity to utilize nitrogen gas. 



There are two current theories of the mechanism of nitrogen fixation 

 in the nodules. The first is that fixation occurs in the membrane en- 

 velopes where the gas is activated and reduced to ammonia. Nitrogen 

 is envisaged as the ultimate acceptor of the reducing power which is 

 generated in the bacteroids and involves hemoglobin as a carrier. The 

 host plant supplies the carbon compounds which are partially oxidized 

 by the bacteroids and which then serve as a source of electrons for 

 the reduction of the activated nitrogen. The products of the incom- 

 plete oxidation of the substrates serve as acceptors of ammonia from 

 the fixation process which is needed for amino acid production in the 

 bacteroids. The acids then become available to the host plant. This 

 overall scheme is presented in figure 6. The second theory suggests 

 that hemoglobin itself is the site of nitrogen fixation in the nodule. 

 Future work will decide between these and other theories put forward 

 to explain the symbiotic system. 



The products of fixation appear to be similar in the nodules of 

 legimiinous plants as those already described for Azotohacter and 

 Clostridium^ that is, ammonia is heavily labeled with nitrogen-15 

 followed by glutamic acid. An interesting difference, however, has 

 been found in alder nodules where the amino acid citrulline contained 

 more nitrogen-15 than did glutamic acid. 



Nodulated plants of soybean were first shown by Evans and his 

 collaborators at Corvallis in Oregon to require minute amounts of 

 cobalt (0.1 microgram per liter of culture solution) when relying 

 solely on atmospheric nitrogen. Similar results were obtained subse- 

 quently with alder, Gasuarina^ and Myrica. A. vinelandii also requires 

 0.1 microgram of cobalt per liter of culture solution for nitrogen 

 fixation. Since the amount is so small it is unlikely that it functions 

 directly in nitrogen fixation but is probably required for the biosyn- 

 thesis of enzymes involved in the fixation process. Cobalt is incorpo- 

 rated into vitamin B12 coenzymes in Azotobacter and in the root nod- 

 ules of some legumes and alder. In our laboratory at Long Ashton 

 we have foimd that C. pasteuricbnum also requires cobalt or vitamin 

 B12 for nitrogen fixation. 



What of the future? 



Over 70 percent of industrial ammonia is used in the fertilizer in- 

 dustry and at present production exceeds demand, not because there 

 is no pressing need for it but because the product is expensive. Al- 



