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.

What is:

Denitrification

Nitrogen Cycle!

(1) Nitrogen Fixation

(3) Nitrification (2) Ammonification

(4) Denitrification

Nitrogen

Cycle

(1) Nitrogen Fixation

(3) Nitrification (2) Ammonification

(4) Denitrification

Nitrogen

Cycle

Nitrates in Soil Ammonia is converted

to nitrites and nitrates.

Organic nitrogen is

converted to ammonium.

(a)

(b)

(c)

N2

NH3

NO3

N2O

How does

nitrogen

reenter the

atmosphere in

the nitrogen

cycle?

fourth Through the

called process

denitrification!

(1) Nitrogen Fixation

(2) Nitrification (3) Ammonification

(4) Denitrification

What does

denitrification

do?

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.

Denitrification converts nitrates (NO3) in the soil

to atmospheric nitrogen (N2) replenishing the

atmosphere.

Nitrates (NO3) in Soil

Nitrogen in atmosphere (N2)

How does the

denitrification

process work?

Nitrates in soil

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!

Denitrifying bacteria live deep in soil and use nitrates as an alternative to oxygen making a byproduct of nitrogen gas.

Nitrogen in atmosphere closes the nitrogen cycle!

(NO3)

(N2)

Other ways that nitrogen

returns to the atmosphere…

Emissions from industrial combustion and gasoline engines create nitrous oxides gas (N2O).

Volcano eruptions emit nitrous oxides gas (N2O).

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

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

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.

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.

Final Lab.

About

Nitrogen cycle