464 FIXATION OF FREE NITROGEN 



of the plants, at 5.2 per cent. ; at the time when the fruit begins to 

 form, at 2.6 per cent.; and after the fruit had become ripe, at 1.7 per 

 cent. The exact manner in which the nitrogen is taken up by the 

 plant from the root tubercles is not known. Frank found even higher 

 percentages namely, 6.94 and 7.44 per cent, of nitrogen and since 

 the latter is present in the form of proteids it means a percentage of 

 43.4 and 46.5 of dry proteid substance. 



Clover and other leguminosse are now frequently used as so-called 

 green manure for improving impoverished soil. These leguminosse 

 which can obtain their nitrogen supply with the assistance of the 

 nitrogen-fixing bacteria from the air are planted in the nitrogen-poor 

 oil. They are allowed to grow, and instead of being harvested are 

 plowed under. The chemical and biological facts of nitrogen absorp- 

 tion have been known for a few decades only, but the ancient Romans 

 had already noted the fact that impoverished soil could be improved 

 by the planting of clover. 



Clostridium Pasteurianum. In addition to those bacteria which have 

 the power of nitrogen fixation in symbiotic community with legumin- 

 osse, there are other bacteria which exhibit the same power in soil 

 alone and without being in symbiosis with other higher organisms. 

 The investigations of Winogradski have demonstrated this. He 

 described a nitrogen-fixing bacterium under the name of Clostridium 

 Pasteurianum. It is a rod about 5 micra long, 1.2 micra wide, which 

 produces end spores, and in doing so assumes the clostridium shape. 

 At the same time it forms in its interior (but not at the poles) sub- 

 stances which are stained deep black blue with iodin solution. The 

 mature spores escape in the longitudinal axis of the organism. The 

 organism belongs to the group of butyric-acid bacteria, to which the 

 bacilli of black-leg and malignant edema also belong. The Clos- 

 tridium Pasteurianum is a strict anaerobe like most members of this 

 group. It forms butyric and also acetic acid in the presence of 

 carbohydrates, which are used as a source of energy; it can fix free 

 nitrogen from the air. The organism does not grow on the general 

 artificial culture media, but on potatoes. As the medium best adapted 

 for its growth the following is recommended : 



Phosphate of potassium (K 3 PO 4 ) 1.0 



Sulphate of magnesium 0.5 



Chloride of sodium, 



Sulphate of iron, 



Sulphate of manganese .... 1 . ..... each 0.01 to 0.02 



Carbonate of calcium, enough to neutralize. 



Glucose 20. 00 to 40.0 



Water, enough to make 1000 . 00 c.c. 



As the Clostridium Pasteurianum is strictly anaerobic it can develop 

 in soil only in the presence of aerobic bacteria, which use up the oxygen. 



Azotobacters. A group of aerobic bacteria able to fix nitrogen has 

 been discovered by Beyerinck. He has named them Azotobacter. 

 These organisms are oval bacteria, 4 to 6 micra long; they are 



