37 
Exercise 18. Denitrification 
MATERIALS: 
5 tubes of Giltay’s solution 
1 fermentation tube of nitrate broth 
Soil samples 
Horse manure 
A culture Ps. pyocyaneus 
If there is an accumulation of nitrates in the soil and anaerobic conditions 
are established by the soil becoming saturated with water, in the presence of 
large amounts of organic matter loss of nitrogen may result. This loss of 
nitrogen is due to the action of anaerobic organisms which reduce nitrates. 
The nitrates may be reduced to nitrites only, or completely to free gaseous 
nitrogen. In the latter case there is an actual loss of soil fertility. 
1. Inoculate 5 tubes of Giltay’s solution as follows: 
1 tube with soil A 
1 tube with soil B 
1 tube with horse manure 
1 tube with a pure culture of Pseudomonas pyocyaneus 
1 tube leave sterile and uninoculated and label “sterile control.” 
giltay’s solution 
Water__1000.00 grams 
Potassium nitrate (KNO a )_ 2.00 “ 
Asparagin (C 4 H 8 N 8 0 3 )_ 1.00 “ 
Magnesium sulphate (MgS0 4 )_ 2.00 “ 
Calcium citrate (Ca 3 (C 6 H 5 0 7 ) 2 -f4H 8 0)_ 8.00 
Monobasic potassium phosphate (KH 2 P0 4 ) 2.00 
Calcium chloride (CaCl 8 )_ 0.20 
Iron chloride (FeCl a )_ 0.20 
2. Inoculate the fermentation tube of nitrate broth with horse manure. 
Use about one gram. 
3. Place all the cultures in the incubator for two days or one week. 
4. Test the test tube cultures first for nitrites, and if nitrites are absent, 
then test them for nitrates. 
5. Test the fermentation tube culture in the same way and if gas is present 
determine its composition as was done in Exercise 12. 
6. Record the results on the regular note paper as shown in table V. 
(a) What changes have taken place in the fermentation tube 
culture? 
( b) What has happened? 
(c) How are the changes to be explained? 
(d) What changes do the tests indicate? 
(e) Under what kind of conditions did the changes come about? 
(f) In Giltay’s solution, what substances are food for energy and 
what food for growth? 
(g) In the main, what are the changes produced in Giltay’s 
solution by the bacteria? 
( h) Where do the organisms get their oxygen? 
