NITROGEN METABOLISM 223 



ments of the micro-organisms, which in their turn fix 

 nitrogen. The legumes, if grown in sterile soil, require 

 combined nitrogen for their growi^h, do not produce 

 nodules and do not fix nitrogen. In non-sterile soil 

 growth can occur in the absence of combined nitrogen, 

 nodules are produced and atmospheric nitrogen is fixed. 

 Beijerinck isolated the responsible organism, now know 

 as Bhizobium leguininosarum and induced it to grow apart 

 from the plant. The process of fixation of nitrogen by 

 Bhizobium is considered by Virtanen and Laine to pro- 

 ceed through formation of hydroxylamine as a first step. 

 They observed that when peas were grown in sterile 

 sand inoculated with Bhizobium soluble nitrogen com- 

 pounds were excreted by roots carrying nodules only. 

 The nitrogen compounds were identified as the oxime 

 of oxalacetic acid (1 to 2 per cent.), Z-aspartic acid and 

 jS-alanine. Ammonia could not be detected. Oxalacetic 

 acid is also an essential intermediate and is found in 

 liighest concentration at noon on bright days and lowest 

 in the dark. The active nodules contain a red pigment 

 which is identical with, or very similar to, haemoglobin. 

 Nodules may be formed by non-nitrogen fixing strains 

 of Bh. leguminosarum but are not pigmented, nor are 

 those formed by active strains in the absence of oxygen. 

 If the plants are kept in the dark for several days the 

 pigment becomes green, due to opening of the porphyrin 

 ring, and the system can no longer fix nitrogen, even if 

 the plants are transferred to the light. Virtanen suggests 

 that the haemoglobin acts as an oxygen transfer system, 

 becoming oxidised to methaemoglobin with simultaneous 

 reduction of atmospheric nitrogen to hydroxylamine : — 

 N2 + Methaemoglobin (Fe^^^) ^=^ 



NH2OH -f haemoglobin (FeU). 

 The hydroxylamine condenses with oxalacetic acid, 

 formed by the plant in the breakdown of sugars, to give 

 the oxime : — 

 COOH.CO.CH2.COOH + KH2OH > COOH.C.(NOH).CHoCOOH + H^O 



