2. Subject objective: Each student should be able
to
– What is the important and how Denitrification take
places in the nature?
– Obtaining an evolution of (Denitrification) in different
soil sources.
– Practical detection of nitrogen gas from reducing
nitrate compounds, through using broth medium
inoculated with standard bacteria or different
garden soil samples.

3. What is:
Denitrification

4. Nitrogen Cycle!
(1) Nitrogen Fixation
(4) Denitrification
Nitrogen
Cycle
(3) Nitrification (2) Ammonification

6. (a)
N2 (b)
N2 O
(4) Denitrification (1) Nitrogen Fixation
(c)
Nitrogen
Cycle
(3) Nitrification (2) Ammonification
NO3
NH3
Ammonia is converted Organic nitrogen is
to nitrites and nitrates. Nitrates in Soil converted to ammonium.

7. How does
nitrogen
reenter the
atmosphere in
the nitrogen
cycle?

8. Through the fourth
process called
denitrification!
(1) Nitrogen Fixation
(4) Denitrification
(3) Ammonification (2) Nitrification

9. What does
denitrification
do?

10. DENITRIFICATION:
Denitrification is defined as the reduction of nitrates to nitrites an eventually to nitrogen
gas. (NO-3) to gaseous dinitrogen (N2O, NO, and N2) these gases escape
(volatilize) into Earth's atmosphere and are not available for plant use. Because
oxygen is not necessary for denitrification to occur, this is a form of anaerobic
respiration in which the nitrates serve as electron acceptors for the denitrifying
bacteria in their energy metabolism. Denitrification takes place most rapidly in
waterlogged anaerobic soil. The four steps in the denitrification process are as
follows:
Bacteria that makes this process like:
• Pseudomonas denitrificans
• Paracoccus denitrificans
• Thiobacillus denitrificans
• Micrococcus denitrificans
• Serratia sp.
• Achromobacter sp.
• (Thermophilic denitrifier) has even been isolated from a hot spring.
The most favorable environments for these organisms are:
1. Heavily fertilized agricultural soils.
2. Sewage where nitrogenous compounds abound in considerable quantity.

12. Denitrification converts nitrates (NO3) in the soil
to atmospheric nitrogen (N2) replenishing the
atmosphere.
Nitrogen in atmosphere (N2)
Nitrates (NO3)
in Soil

13. How does the
denitrification
process work?
Nitrates in soil

14. Denitrifying bacteria live deep in soil and in aquatic
sediments where conditions make it difficult for them
to get oxygen. The denitrifying bacteria use nitrates
as an alternative to oxygen, leaving free nitrogen
gas as a byproduct. They close the nitrogen cycle!
Nitrogen in atmosphere
closes the nitrogen cycle!
(N2)
(NO3)
Denitrifying bacteria live
deep in soil and use
nitrates as an alternative
to oxygen making a
byproduct of nitrogen gas.

15. Other ways that nitrogen
returns to the atmosphere…
Emissions from industrial combustion and Volcano eruptions
gasoline engines create nitrous oxides emit nitrous oxides
gas (N2O). gas (N2O).

17. Denitrifying microorganism
• Anaerobic to reduce the nitrate to gaseous form of nitrogen.
• Room tmperature
• The predominant saturated and unsaturated fatty acids in all
denitrifying isolates are generally n-hexadecanoic acid (16:0) and
cis-11- octadecenoic acid (18:1 ω7c).
• Microscopically :Grame negative whit rod shape.
• Biochemical test and API test are probably used for identification
• 16S RNA sequencing is more reliable for characterization.
• Medium for nitrification should contain nitrate and incubated in
mesophile temperature range

18. Materials for denitrification:
• Garden soil
• Broth culture of Pseudomonas
• 2 nitrate broth tubes containing Durham tubes
• 2 nitrate free broth tubes containing Durham tubes
• -napthylamine reagent
• sulfanilic acid
• powdered zinc
• Blenders, fresh soil sample, 90 ml distilled water
• Graduate 1 ml pipette, 1 Petri plate of nitrate agar,
GasPak anaerobic jar, generator envelopes

19. Method: To isolate denitrifiers from a soil sample, the following conditions must be met
in the growth medium:
1. In this exercise a medium containing a nitrate substrate is used for gas formation and
a Durham tube is used to detect gas (N) production.
2. Some nitrate must be available, which will provide the only terminal electron acceptor
for the generation of ATP.
3. Some peptone must be present to provide essential amino acids needed by some
denitrifiers. The next step is to demonstrate the ability of the organism to generate
visible nitrogen gas. An isolate that grows on nitrate media and generates gas can be
presumed to be a denitrifier.
Procedure:
• First Period: The nitrate agar used in the Petri plate is essentially nutrient agar to
which 0.5% KNO3 is added.
• Procedure:
1. Add 10 grams of soil to 90 ml of water.
2. Blend for 2 minutes.
3. Label the bottom of a nitrate agar plate with your name and date of inoculation.
4. Pipette 1.0 ml of the blended mix onto the surface of a plate of nitrate agar.
5. Spread the inoculum over the surface of the agar with a bent glass rod.
6. Incubate the plate, inverted, at 30° C for 3 to 5 days in a GasPak anaerobic jar.

20. Second Period
• During this period, nitrate agar plates will be examined to select colonies that have developed
during the incubation period. Since the presence of growth doesn’t necessarily mean that the
organism is a denitrifier, it will be necessary to see if any of the isolates are nitrogen gas
producers; thus, Durham tube nitrate broths must be inoculated and incubated anaerobically.
Nitrate broth consists of nutrient broth plus (0.5% KNO3).
1. Inoculate one tube of nitrate broth containing a Durham tube with 1g of soil. DO NOT SHAKE
THE CULTURE TUBES DURING INCUBATION.
2. Inoculate the second tube with a loopful of Pseudomonas.
3. Repeat steps 1 and 2 with the nitrate free broth tubes.
4. Label all tubes and incubate at room temperature for 7 days.
5. Observe the tubes for gas formation.
6. Add 1 ml  - naphthalene reagent and 1 ml sulfanilic acid reagent to each of the culture tubes
and mix. The development of a red color within 30 seconds indicates that nitrites are present.
7. After carrying out step 6, any tube that fails to develop a red color could still have its full supply
of nitrate (i.e. lacks bacteria to reduce it) or it could have undergone denitrification without nitrite
being further converted to nitrogen. To distinguish between the two possibilities, a pinch of zinc
must be added to any tube that did not turn red. The zinc catalyses the reduction of nitrate to
nitrite and produces a red color within minutes if nitrate was present. Lack of a red color
indicates the absence of nitrate (and possibly the presence of nitrite).
8. Record your results.
Third Period
This period of inoculations is in preparation of trying to do a definitive identification of a denitrifier.
From an isolated colony a nutrient broth is inoculated and a gram-stained slide is made. After
incubation, the broth culture can be used as a stock culture for doing further tests to identify
your isolate. The slide will reveal the morphological nature of your organism.

22. Final Lab.
About
Nitrogen cycle