Nitrification of organic compounds in Soil

 

Aim

To demonstrate nitrification in the given soil sample

Principle

Nitrification is the process by which ammonia (NH₃) or ammonium (NH4⁺) is converted to nitrate (NO₃⁻). Nitrification is the  result of oxidation of ammonium to nitrite (NO₂⁻) by nitrosifying or ammonia-oxidizing bacteria and oxidation of nitrite (NO₂⁻) to nitrate (NO₃⁻) by the nitrite-oxidizing bacteria. Nitrification is an important step in the nitrogen cycle in soil. The nitrate released into the soil is highly soluble and easily assimilated by photoautotrophic bacteria, algae, and plants that convert it into the amino acids needed for their  enzyme and protoplasm construction.

The chemoautotrophic bacteria such as Nitrobacter, Nitrococcus, Nitrosococcus, and Nitrosomonas  use the energy of reduced nitrogen compounds, such as ammonium and nitrite, as an energy source for the autotrophic production of organic compounds. Russian soil microbiologist Winogradsky showed that Nitrosomonas species and Nitrobacter species take part in nitrification, which is one of the essential phases of nitrogen cycle.

There are several factors influencing the growth of nitrifying bacteria in soil. Presence of good amount of organic matter in soil is important for the growth and proliferation of the nitrifying bacteria. In acid soils, nitrification is poor due to decrease in the population of nitrifying bacteria. Water logged soil deficient in oxygen are not congenial for nitrification. Similarly either too low (below 5⁰C) and too high soil temperature (above 40⁰C) is not conducive for the optimum functioning of nitrifying microorganisms. 

Materials required

Ammonium Sulfate Broth (Nitrite Forming Broth), Nitrite Broth (Nitrate Forming Broth),Trommsdorf's reagent, diphenyl amine reagent, soil sample.

Methodology

1. Tubes containing ammonium medium and nitrite medium were prepared and sterilized.

2. A loopful of soil was inoculated into the tubes. The tubes were then incubated at room temperature for 3-7 days.

3. Following incubation, test for nitrite was carried out by adding 2-3 drops of Trommsdorf's reagent to the incubated tubes and observed for colour change.

4. Similarly, the test for nitrate was carried out by adding 2-3 drops of diphenyl amine to incubated tubes and observing the colour change.

5. The results were compared with the uninoculated control and recorded.

Observation  

Ammonium sulfate broth (nitrite forming broth) was observed to see if ammonium has been converted to nitrite (NH₄⁺  --->     NO₂^-). Nitrate forming broth will be observed to see if nitrite has been converted to nitrate (NO₂^-  ---> NO₃^-).

Result

When Trommsdorf’s reagent was added to the tubes containing the ammonium medium, a blue black coloration was obtained. Similarly, when diphenyl amine reagent was added to the tubes containing nitrite medium, a blue black coloration was obtained which showed the presence of nitrifiers in the soil sample.

Detection of nitrification in soil sample   (left hand side)

Colour development	Intensity	Inference
No color	-	No nitrite/nitrate
Pale blue	+	Small amount of nitrite/nitrate
Blue	++	More nitrite/nitrate
Blue/black	+++	Large amount of nitrite/nitrate