BACTERIA REDUCING NITRATES AND SULFATES 185 



manure. Wagner 32 then attempted to draw, on insufficient ground, 

 broad generalizations concerning the reduction of nitrates to gaseous 

 nitrogen by denitrifying bacteria in manure, even when added to the 

 soil. 



Gayon and Dupetit 33 isolated from the soil, in 1886, two anaerobic 

 bacteria (B. denitrificans a and jS) capable of reducing nitrates to gaseous 

 nitrogen. The two organisms were cultivated upon a medium having 

 the following composition: 34 



1. Distilled water 250 cc. 2. Distilled water 500 cc. 



KN0 3 2 grams Citric acid 5 grams 



Asparagine 1 gram KH 2 PO 4 2 grams 



MgS0 4 2 grams 



CaCl 2 0.2 gram 



FeCl 3 Trace 



Solution 2 is neutralized with a 10 per cent solution of NaOH or KOH, with 

 phenolphthalein as an indicator. The two solutions are mixed and made up to 

 1000 cc. with distilled water. 



For the isolation of denitrifying organisms, various other media can be used: 

 (1) 1000 cc. water, 10 grams glucose, 6 grams NaN0 3 , 6 grams NaCl, 0.02 gram 

 Ca 3 (P0 4 ) 2 . 35 (2) 100 cc. water, 0.5 to 1.5 grams NaN0 3 , 20 to 50 grams glycerol, 

 7 grams malic acid (neutralized with sodium carbonate), 0.5 gram sodium phos- 

 phate, 0.5 gram NaCl, 0.5 gram Na 2 C0 3 , 0.1 gram MgS0 4 . 36 (3) 1000 cc. water, 

 20 grams of calcium tartrate, citrate or malate, 10 to 20 grams KN0 3 , 0.5 gram 

 K 2 HP0 4 . 



Under anaerobic conditions, practically all the nitrate nitrogen can 

 be transformed into gaseous nitrogen. When asparagine is replaced by 

 sugar, the ammonia otherwise produced from the asparagine is not 

 formed. In the reduction of nitrate to gaseous nitrogen (so-called "ni- 

 trate fermentation"), there is an abundant accumulation of alkali, till 

 the process is stopped when the alkali concentration is equivalent to 

 1 per cent sodium carbonate. 37 When the alkali is neutralized by means 



32 Aeby, J., Dorsch, R., and Matz, Fr., and Wagner, P. Forschungen iiber 

 den relativen Dungewart und die Konservierung des Stallmistsickstoffs. Landw. 

 Vers. Sta. 48: 247-360. 1897. 



33 Gayon and Dupetit, 1886 (p. 181). 



34 Giltay, E., and Aberson, G. Denitrifizierende Organismen im Boden. 

 Arch. Neerland. 25: 341. 1892. 



36 Ampola and Ulpiani. Gazz. chim. ital. 1898, 410. 

 36 Maassen, 1901 (p. 181). 



37 Burri, R., and Stutzer, A. Uber Nitrat zerstorende Bakterien und den durch 

 dieselben bedingten Stickstoffverlust. Centrbl. Bakt. II, 1: 257-265, 350-364, 

 392-398, 422-432. 1895; 2: 473-474. 1896. 



