46 NITROGEN METABOLISM 



NHt, etc.) provided they were able to develop these struc- 

 tures. By this time nodule formation was known to be the 

 outcome of bacterial invasion of the root tissues [cf. 41, 35] 

 and the organisms living in them were described by Frank 

 under the name oi Bacterium radicicola [18]. Contrary to the 

 ideas of previous workers, Hellriegel and Wilfarth suggested 

 that the bacteria were not parasites but lived in symbiotic 

 association with the plant and endowed it with the ability 

 to grow at the expense of atmospheric Ng and thus be inde- 

 pendent of an exogenous source of fixed nitrogen. Pure 

 cultures of the nodular organisms, now placed in the genus 

 Rhizobium, were first isolated by Beijerinck, who also found 

 them free-living in the soil [3]. Like later workers, he was 

 unable to demonstrate that these organisms fixed Ng in the 

 absence of the host plant, and the mechanism of Na-fixation 

 by the symbiotic system still awaits elucidation. All the 

 strains of a given species of Rhizobium induce nodule forma- 

 tion in a restricted number of leguminous plants, termed a 

 cross-inoculation group, and it is on this basis that the 

 Rhizobium are classified into species, each species being 

 specific for one cross-inoculation group [41, i]. Although 

 nodules may be formed, they are not always effective, i.e. 

 capable of fixing nitrogen. 



Isolation of free-living N ^-fixing organisms 



During the latter half of the nineteenth century, Jodin 

 and Berthelot provided evidence that certain free-Hving 

 micro-organisms fixed atmospheric Ng , and pure cultures 

 of bacteria exhibiting this property were eventually isolated 

 by Winogradsky and Beijerinck. Each of these eminent 

 bacteriologists used the enrichment culture technique with 

 media which, apart from inorganic salts, contained only a 

 substance such as glucose or mannitol as a source of carbon 

 and energy: no nitrogenous compound was added. After 

 being inoculated with soil the cultures were incubated in an 

 atmosphere of air or nitrogen. Winogradsky thus isolated 

 the anaerobe Clostridium pasteurianum which fermented 

 glucose to acetic and butyric acids together with Hg and 

 CO 2 [49]. A few years later Beijerinck, using media 



