Lab.10 methods for estimating nitrification process
Subject objective: Each student should be able to – What are the important and how Nitrification take place in the nature? – Obtaining an evolution of (Nitrification) in different soil sources. – Practical Detection of nitrite and nitrate compounds, through using broth medium inoculated by standard bacteria or different garden soil samples. – Identification of nitrifier bacteria
(a) N2 (b) N2 O (4) Denitrification (1) Nitrogen Fixation (c) Nitrogen Cycle(3) Nitrification (2) Ammonification NO3 NH3Ammonia is converted Organic nitrogen isto nitrites and nitrates. Nitrates in Soil converted to ammonium.
Now we will take a “closerlook” at theNitrification Process
• NITRIFICATION:In an aerobic environment, ammonia is liberated into the soil by the ammonification which is a part of the cycle. It does not accumulate there. If it is not used as a nitrogen source by plants or microorganisms, it is oxidized to nitrates by a two-step process called nitrification. Nitrification is the conversion of NH+4 to NO-3, this process carried out through two-step process in which ammonia (NH+3) is first oxidized to nitrite (NO-2) by chemoautotrophs: Nitrosomonas, and the nitrite (NO-2) is subsequently oxidized to nitrate (NO-3) by chemoautotrophs: Nitrobacter. The nitrate released into the soil is available to plants and microorganisms for protein synthesis.This process like nitrogen fixation, this process is uniquely associated with bacteria. Nitrate is much more readily leached from soils than is ammonia. If excessive amounts of nitrate are leached from soils, reducing soil fertility and it can accumulate in runoff water and in wells. Ammonium sulfate broth and nitrite broth are used in this part of the exercise to demonstrate the oxidation of ammonia to nitrate. Ammonia and nitrite serve as energy sources in the respective broths.• Maximum nitrification rates occur at:1. Neutral pH2. High temperatures• NH+ 4 + 1,1/2 O2 ………………. NO-2 + 2H+ + H2O + 66 Kcal. (Four genera make it)• NO-2 + 1/2 O2 …………… NO-3 + 17.5 Kcal (Nitrobacter sp., Nitrosospira sp., Nitrosococcus sp.)(Factors that favor the bacteria involved in this process belong to family Nitrobacetriaceae:• Nitrosomonas sp.• Nitrosococcus sp.• Nitrosolobus sp.• Nitrosospira sp.
Characters of nitrifying bacteria ammonia-oxidizing (AOB) bacteria• Aerobic• Alkaline pH• Temperature 20-30 °C• Motile (Flagella)• Grame negative• Different cell shape such as spindly and bacilliform
Materials for Nitrification:• Garden soil• 1 x ammonium sulfate broth (20ml)• 1 x nitrite broth (20ml)• Nesslers reagent• Trommdorfs reagent• Diphenylamine• Spot plate• Sulfuric acid (1 part conc. Sulfuric acid to 3 parts water)
• Method:1. Inoculate the ammonium sulfate and nitrite broth bottles with pinches of soil (1g). Label the bottles and shake vigorously for 5 minutes.2. Shake the bottles for 7 days at room temperature.3. Place a drop of sulfuric acid and 3 drops of Trommsdorf’s reagent in a well on a spot plate. Add a drop of culture from the ammonium broth and mix. Use a Pasteur pipette and not an inoculating loop. A blue – black color indicates the presence of nitrite.4. Test the ammonium broth for ammonia with Nessler’s reagent (see ammonification).5. Test the nitrite broth for residual nitrite.6. If no blue black color was present, test for nitrate. Add 1 drop of diphenylamine, 2 drops of sulfuric acid and 1 drop of nitrite broth culture in a well on the spot plate and mix. A blue black color indicates the presence of nitrate.7. Grams stain the organisms in the broth cultures. Record your results.