Subject objective: Each student should be able to:• Learn about the important of ammonification process in the nature.• Obtain an evolution of the ammonification potentials in different soil samples.• Determine protease activity through detecting ammonium using broth culture with garden soil and through using soil extract.
Ammonification: Bacteria, fungi, actinomycetesdecomposers break down amino acids from deadanimals and wastes into nitrogen ammonium. Bacteria decomposers break down amino acids into ammonium
Principle• The nitrogen in most plants and animals exists in the form of protein. When these organisms die, the protein is broken down to amino acids, which in turn are deaminated to liberate ammonia. This process of the production of ammonia from organic compounds is called ammonification. Soil bacteria (e.g. Bacillus, Proteus, and Pseudomonas) produce the proteases that accomplish ammonification. Once ammonia is released into the soil it dissolves in water to form the ammonium ion (NH4 +). Some of these ions are used by plants and microorganisms to synthesize amino acids.• In this exercise, peptone is used as an organic nitrogen substrate. The ability of different bacteria and the organisms in a soil sample to break down the organic nitrogen and release ammonia will be examined. Ammonia can be detected by adding Nessler’s reagent to samples – if ammonia is present, the samples will turn yellow – brown.
A. Mineralization (Ammonification) – the conversion of organic nitrogen (proteins, amino sugars, nucleic acids, chitin) to ammonium (NH4+), a mineral form who? heterotrophic bacteria and fungi – ‘decomposers’ generic equation:B. Immobilization – microbial uptake of inorganic nitrogen and incorporation into organic forms who? heterotrophic bacteria and fungi – ‘decomposers’1) Ammonium assimilation
The ammonium is either:1. taken up by the plants (only in a few types of plants)2. ammonium can be adsorbed and fixated (stuck) on to the negativelycharged soil particles or be taken up by plants.3.Ammonium (NH4) Stored in the soil up to later be changed intoinorganic nitrogen, the kind of nitrogen that most plants can use. Bacteria converts organic nitrogen to ammonium (NH4) Ammonium (NH4) is used by some plants Ammonium (NH4) is stored in soil.
First Procedure for Estimation of Ammonia in soil:FIRST PERIOD: (Inoculation)• Materials:1. 2 tubes of peptone broth2. Rich garden soil3. Broth cultures of Bacillus, Proteus and PseudomonasProcedure:1. Inoculate one tube of peptone broth with 1gm of soil sample, save the other tube for a control.2. Incubate the tube at room temperature for 3–4 days and 7 days.SECOND AND THIRD PERIODS: (Ammonia Detection)After 3 or 4 days, test the medium for ammonia with the following procedure. Repeat these tests again after a total of 7 days of incubation.• Materials:1. Nessler’s reagent, Spot plate2. pH-meter or pH paper.Procedure:1. Deposit a drop of Nessler’s reagent into two separate depressions of a spot plate.2. Add a loopful of the inoculated peptone broth to one depression and a loopful from the sterile uninoculated tube in the other, then add 1-2 dopes of nesslers reagent. Interpretation of ammonia presence is as follows:• Faint yellow color—small amount of ammonia• Deep yellow—more ammonia• Brown precipitate—large amount of ammonia3. Check the pH of the two tubes by pH-meter or pH paper.
Second procedure for estimation of ammonia and ammonium in soil samples:Inorganic nitrogen (NH+4) and nitrogen process rate measurements:Accumulation of inorganic nitrogen is measured by extracting each soil sample with 80 ml of 2M KCl. After adding KCl and shaking each container by hand to suspend the soil, sample containers were placed on a rotary shaker at speed (100rpm/min.) for 1 h, and then shaken again by hand to re-suspend the soil. Samples were filtered (Whatman No. 42 filter paper) All soil extracts were frozen at (-20°C) to prevent secondary formation of nitrite ions by microbial or chemical redox reactions from ammonium ions or nitrate ions. Gross N mineralization was measured on whole soil samples.• Titration method used for ammonium (NH+4) measurement:Extracted NH+4 from soil samples were determined by titration method (19) by treating (20 ml) of extracted soil with five drops of methyl red reagent and titration was done with (0.05 N) ofH2SO4 until the end point of reaction yellow to red, then sample boiled off to room temperature and same step of titration was repeated to the same color, and distilled water corresponding of the blank test serves as the control sample.• Following equations were used for determination of NH+4 in soil water extract: – N1 × V1 = N2 × V2N1: unknown N of (NH+4)?V1: 20ml water extract of soil sample.N2: 0.05N of (H2SO4)V2: (ml) of (0.05N) H2SO4 correspond to (ml) of NH+4 at the endpoint of reaction.a: consumption of 0.05N H2SO4 in ml for the water extract of soil sample.b: consumption of 0.05N H2SO4 in ml for the distillated water (blank).Then this equation is used for converting known (NH+4) N from first equation to ppm. – ppm = Known (NH+4)N × 18 × 1000Molecular weight of (NH+4) = 18
What happens toammonium (NH4) stored in the soil?