Ammonification of organic compounds in Soil

 Aim

To demonstrate ammonification in the given soil sample

Principle

      Nitrogen in most plants and animals exist in the form of proteins. When an organism dies, its proteins are attacked by the proteases of soil bacteria to produce polypeptides (peptones) and amino acids. This process is called peptonization. Then, the amino groups on the amino acids are removed by a process called deamination, producing ammonia (NH₃). In most soils, the ammonia is trapped by the moisture in soil to form ammonium ions (NH₄⁺). The release of organically bound nitrogen as inorganic ammonium ions, called ammonification is the consequence of microbial activity found in the soil. In addition to the ammonification of amino acids, other compounds such as nucleic acids, urea, and uric acid go through the ammonification process. The bacteria that accomplish it (Bacillus, Clostridium, Proteus, Pseudomonas, and Streptomyces) are called ammonifying bacteria. 

Ammonification of organic compounds is a very important step in the cycling of nitrogen in soils, since most autotrophs are unable to assimilate amino acids, nucleic acids, urea, and uric acid and use them for their own enzyme and protoplasm construction.

      There are several factors influencing ammonification of proteins in the soil. In acidic soil, fungi are more active than bacteria. If there is a ready supply of energy rich carbohydrates, the microorganisms do not attack the complex nitrogenous compounds. Thus for ammonification, rich amount of carbohydrates in soil is harmful. Besides proteins, other nitrogen containing organic substances like urea are also attacked by microorganisms to yield ammonia.

Materials required

Peptone Broth, soil sample, Nessler’s reagent.

Methodology

1.     Tubes containing peptone broth 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 ammonia was carried out by adding 2 drops of Nessler’s reagent to the incubated tubes and observed for colour change.

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

Observation

The presence of dissolved ammonia is detected with Nessler’s reagent, which contains potassium iodide, mercuric iodide and sodium hydroxide. Development of a dark yellow orange or brown colour upon the addition of Nessler’s reagent to the culture medium indicated positive result. The intensity of the colour is directly proportional to the concentration of ammonia dissolved in culture media.

Result

       When Nessler’s reagent was added to the tubes, yellow-orange precipitate was obtained, which showed the presence of ammonifiers in the soil sample.

Detection of ammonification in soil sample using Nessler’s reagent    (left hand side)

Colour development	Intensity	Inference
No color	-	no ammonia
Pale Orange color	+	small amount of ammonia
Orange	++	more ammonia
Brown precipitate	+++	large amount of ammonia