2/o FIXATION OF FREE NITROGEN BY BACTERIA 



difficult accessibility of the interior of the nodule (the chief seat of this fission 

 fungus) to the gas to be fixed. A. B. FRANK (VIII.) has ascertained that the 

 air passages (intercellular spaces) of the nodules lead only as far as the cambium 

 layer, but not into the bacteroidal tissue; consequently atmospheric nitrogen 

 cannot penetrate directly to this tissue. On this point R. BOUQUET (I.) has 

 expressed an opinion worthy of further investigation. According to him, it 

 is the water absorbed by the roots and exhaled through the leaves which 

 conducts and gives up free nitrogen in solution to the root cells, where it is then 

 combined by the plasma. 



If, then, we must consider the question, whether the fixation of free 

 atmospheric nitrogen is effected within the nodules, to be still imperfectly solved, 

 it is, on the other hand, clearly proved that such an operation goes on in 

 (uncultivated) soil. This was first observed by M. BERTHELOT (I.) in 1885, and 

 he subsequently proved that this phenomenon is not occasioned by exclusively 

 chemical affinity, but is due to the activity of micro-organisms. Opinions on the 

 nature of the organisms were at first divided. TH; SCHLOESING, jun., and 

 EM. LAURENT (I.) ascribed the fixation of nitrogen to certain lower algse 

 (Conferva, Oscillaria, Nitzschia), and mosses (Bryum, Leptobryum). Doubt was 

 cast on this hypothesis by A. GAUTIER and R. DROUIN (I.), and P. KOSSOWITSCH 

 (II.) disproved it so far as the algae are concerned by the aid of pure 

 cultures. BERTHELOT (II.) then showed that the activity of fungi both 

 Eumycetes (such as Aspergillus niger, Alternaria tenuis, <fec.) and Schizomycetes 

 is in question. 



For more precise investigations on this matter we have to thank S. Wixo- 

 GRADSKY (II.), who described a fission fungus belonging to the group of butyric 

 acid bacteria, and bearing the name of Clostridium Pasteurianum. This occurs 

 in the form of rods, 1.2 p. broad and 5 /j. long, each producing an endospore, 

 the cells thereby swelling up to the clostridium form, and storing up in their 

 interior (though not at both poles) substances that are stained a deep blue-blade 

 by iodine. The ripe spore escapes through the wall of the mother-cell in a 

 longitudinal direction. The great resemblance between this clostridium and 

 the butyric acid bacteria described in a previous section is not only morphological, 

 but also extends to the fermentative capacity. For example, Clostridium 

 Pasteurianum acts on sugar in such a manner that both volatile acids : butyric 

 acid and acetic acid (4 : i), and gases: carbon dioxide and hydrogen (60-75 

 per cent, of H. by volume) are formed. The importance to us of this fission 

 fungus on the present occasion consists in its behaviour towards nitrogen, 

 which gas it absorbs from the atmosphere, fixes it and employs it in the elaboration 

 of organic substances. The energy necessary thereto is supplied and liberated 

 by the decomposition of sugar ; consequently it is easy to understand that a 

 definite relation exists between the amounts of sugar fermented and of nitrogen 

 combined. This ratio was determined by Winogradsky as 2.5-3 mgrms. of 

 nitrogen to 1000 mgrms. of dextrose. This element (N.) when in a state of 

 combination is not only valueless to Clostridium Pasteurianum, but when 

 present in large quantity even injurious thereto. For the cultivation of the 

 microbe which cannot be carried out on the ordinary nutrient media (gelatin, 

 bouillon) in use, though it grows on sliced potatoes use is made, preferably, 

 of an aqueous solution containing i gram K 3 P0 4 , 0.5 gram MgS0 4 , 0.01-0.02 gram 

 NaCl,FeS0 4 ,MnSO 4 , a little CaCO 3 (for fixing the acids), and 20-40 grams of 

 dextrose per litre. Clostridium Pasteurianum is strictly anaerobic, and in the 

 soil is therefore obliged to rely on the co-operation of aerobic fission fungi, 

 which remove the injurious oxygen from its sphere of influence, surround it 

 with an atmosphere of nitrogen, and as a reward for this service have the 

 opportunity of consuming the nitrogenous substances elaborated and excreted 



