BI 304 Environmental Microbiology W15

Lab #3 The Nitrogen Cycle

(modified from Microbiology: a laboratory manual 3rd edition, Cappuccino & Sherman)

OBJECTIVE:

To demonstrate the role of soil microorganisms in the biotransformation of nitrogenous

compounds via the nitrogen cycle

THEORY:

Soil serves as a repository for many life forms including a huge and diverse microbial
population.

Life on earth could not be sustained in the absence of soil microorganisms: they degrade
dead plant and animal material to replenish the soil with basic elemental nutrients. Plants
are then able to assimilate these nutrients into organic compounds essential for their own
growth and reproduction. Thus, many soil microorganisms play a vital role in a number
of elemental cycles, including the nitrogen cycle.

The nitrogen cycle is concerned with the enzymatic conversion of nitrogenous

compounds in the soil and gaseous nitrogen from the atmosphere into inorganic nitrogen
compounds that are used by plants for synthesis of essential macromolecules like nucleic
acids and proteins. A diagram of the cycle is shown below:

http://www.windows.ucar.edu/earth/climate/images/nitrogencycle.jpg

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The cycle can be divided into four main phases:

1. Ammonification: Sequential degradation of nitrogenous organic compounds with
the release of ammonia.

2. Nitrification: Step-by-step oxidation of ammonia to nitrite (NO2-) and then to

nitrate (NO3-), a nutritional form of nitrogen that is assimilated by plants.

3. Denitrification: Reduction of nitrates not used by plants to gaseous (free)

nitrogen (N2).

4. Nitrogen fixation: Chemical combination of free nitrogen gas (N2) with other
elements to form fixed nitrogen (nitrogen-containing compounds).

In this lab we will study the processes of Ammonification, Nitrification and

Denitrification by microorganisms.

Part A: Ammonifcation

Purpose: To demonstrate the liberation of ammonia from nitrogenous organic

compounds.

Principle: Ammonification involves degradation of nitrogenous biopolymers and

subsequent release of ammonia. This process is initiated by excretion of extracellular
proteolytic enzymes that are commonly produced by such soil organisms as members of
the genera Bacillus, Clostridium and Streptomyces. These enzymes act sequentially to
hydrolyze the proteins of plant and animal origins into their constituent amino acids. The
amino acids are subsequently enzymatically deaminated, with the release of ammonia.

proteins from proteolysis by soil MOs amino acids

dead plant and polypeptides NH2-RCH-COOH
animal tissues

deamination

ammonia organic acids

NH3 + RCH - COOH

In this experiment, peptone broth, which contains an organic nitrogen substrate, is used to
demonstrate the ability of some microorganisms to degrade proteins, with the resultant
formation of ammonia. Following incubation, the presence of ammonia is detectable by
formation of a yellow colour when Nesslers reagent is added to samples of the test
cultures. The relative amount of ammonia can be determined by differences in the degree
of yellow colouration.

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Materials (per pair)

Week 1
24-hour nutrient broth cultures of Bacillus cereus, Pseudomonas fluorescens and Proteus
vulgaris.
rich garden soil
4 x 4% peptone broth tubes

Week 2
Nesslers reagent
bromothymol blue and indicator chart
spot plate

Procedure:
Week 1 (inoculation)
1. Label 3 peptone broth tubes with the names of the organisms to be inoculated. Label
the fourth tube as soil.
2. Inoculate the first three tubes with 0.1 ml of the appropriate microorganism.
3. Aseptically add 0.1 gram of garden soil to fourth experimental tube.
4. Monitors will setup a control tube without any inoculation.
5. Incubate the tubes at room temperature (RT) for one week.

Week 2 (ammonia detection)

1. Deposit a drop of Nesslers reagent into four separate depressions of a spot plate.
2. Add a loopful of each inoculated peptone broth and a loopful from the negative control
(sterile, uninoculated tube) in to the depressions. Interpretation of ammonia presence is as
follows:

Faint yellow: small amount of ammonia

Deep yellow: more ammonia
Brown precipitate: large amount of ammonia

3. Check the pH of the tubes by placing several loopfuls of each culture in separate
depressions on the spot plate and checking the pH with a pH paper strip. Compare the
colour with a colour chart to determine the pH.

4. Record all results.

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Part B. Nitrification

Purpose: To demonstrate the enzymatic conversion of ammonia to nitrates by soil

microorganisms.

Principle: Ammonia that is liberated during the ammonification process seldom

accumulates to any appreciable extent in the soil. In an aerobic environment it undergoes
bacterial oxidation to nitrates by a two-step process primarily involving the
chemoautotrophic soil organisms Nitrosomonas and Nitrobacter. This process is called
nitrification, and the chemical reactions are shown below.

Step 1. Nitrite formation

Nitrosomonas
NH4+ + O2 NO2- + H 2O
Ammonium Nitrite
ion ion

Step 2. Nitrate formation

Nitrobacter
NO2- + O2 NO3-
Nitrite Nitrate
ion ion

The nitrates released in the soil are highly soluble and are assimilated by terrestrial plants
and some microorganisms for the biosynthesis of cellular proteins.

In this procedure ammonium sulfate broth is used to demonstrate the ability of some
microorganisms to oxidize ammonia to nitrite. The nitrite broth illustrates the further
oxidation of nitrate to nitrite. Following incubation, the presence of nitrite and nitrate is
determined as follows:

Determination of nitrite production in ammonium sulfate broth cultures:

1. Test for the presence of ammonia by use of Nesslers reagent. A yellow colour
indicates that ammonia was not oxidized to nitrate. No colour change indicated the
absence of ammonia, and suggests that nitrate should be present.

2. Test for the presence of nitrite by use of Trommsdorfs reagent and sulfuric acid. The
presence of a blue-black colour is indicative of the presence of nitrite; no colour change
indicates the absence of nitrite.

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Determination of nitrate production in nitrite broth soil cultures:

1. Test for the presence of nitrite by use of Trommsdorfs reagent and sulfuric acid as
described above.
2. Test for the presence of nitrate by use of diphenylamine reagent and sulfuric acid if the
absence of nitrites (no colour change with Trommsdorfs reagent) was ascertained. The
appearance of a deep-blue colour indicates that nitrates are present.

Materials (per pair)

Week 1
rich garden soil
2 test tubes with 10 ml ammonium sulfate broth
2 test tubes with 10 mL nitrite broth

Week 2
Nesslers reagent
diphenylamine reagent
Trommsdorfs reagent

dilute sulfuric acid (1:3 in H20)

concentrated sulfuric acid
glass rod
spot plate

Procedure:
Week 1 (inoculation)
1. Inoculate one tube of ammonium sulfate broth with 0.1 gram of soil and shake
vigorously; inoculate the other tube of ammonium sulfate broth with 0.1 ml of pure
culture of Nitrosomonas and mix well. Monitors will setup a control tube with no
inoculation.
3. Inoculate one tube of nitrite broth with 0.1 gram of soil and shake vigorously;
inoculate the other tube of nitrite broth with 0.1 ml of pure culture of Nitrobacter and mix
well. Monitors will setup a control tube with no inoculation
4. Incubate all tubes at RT for one week.

Week 2: Tests for nitrites and nitrates

After 7 days, test for nitrites and nitrates as follows:
1. Nitrite Production
i) mix 3 drops of Trommsdorfs reagent with 1 drop of dilute sulfuric acid (1:3
conc.) on a spot plate

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ii) with a glass rod, transfer a drop of the culture from the ammonium sulfate
broth to this reagent mixture and stir. To avoid false results, do not use a wire loop. If
nitrites are present, an intense blue-black colour will appear.
iii) Do a test for ammonia with Nesslers reagent. When the medium becomes
negative for ammonia, all ammonium ions have been oxidized to nitrite (or may be
nitrate).
iv) Make a Gram-stained slide from each tube, and record your results. Use a
heavy inoculum for soil inoculated tubs in order to be able to see the cells.

2. Nitrate Production
i) First, test your nitrite medium culture with Trommsdorfs reagent to establish
the absence of nitrites (see above).
ii) If nitrites are lacking, test for nitrates with diphenylamine by mixing 1 drop of
diphenylamine, 2 drops of concentrated sulfuric acid, and 1 drop of the culture on the
spot plate. A blue-black colour is evidence of nitrate production.

Part C. Denitrification

Purpose: To demonstrate the reduction of nitrates to nitrogen gas.

Principle: Nitrates produced in the soil by microbial nitrification or deposited by the use
of nitrogen fertilizers must be used rapidly or they are lost by leaching off by microbial
reduction. In the latter, nitrates are reduced to nitrites, ammonia, nitrous oxide, and
finally to elemental nitrogen in the form of nitrogen gas. This process is called
denitrification.

Some of the soil microorganisms involved in denitrification are members of the genera
Pseudomonas, Bacillus and Micrococcus. Under anaerobic conditions, these organisms
have the enzyme profile enabling them to use nitrate rather than gaseous oxygen as a
final electron acceptor in their energy metabolism, thereby reducing the nitrate to gaseous
nitrogen:

NO3- NO2- NO N2
Anaerobiosis

The following experiment uses a medium containing nitrate, the substrate for nitrogen
gas formation, and a Durham tube for the detection of the evolution of nitrogen gas.

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Following incubation, the evolution of gaseous nitrogen, the end product of

denitrification, can be detected by the presence of an air bubble in the Durham tube.

Materials (per pair)

24-hour nutrient broth cultures of Pseudomonas aeruginosa and Proteus vulgaris
rich garden soil
3 x nitrate broth tubes containing Durham tubes

Procedure (in pairs):

Week 1 (inoculation)
1. Label two nitrate broth tubes with the appropriate microorganism. Label the third tube
as soil. Monitors will setup a negative control without any inoculation.
2. Aseptically add 0.1 ml of appropriate inoculum into its labeled tube; add 0.1g garden
soil to the third tube labeled soil.
3. Do not shake the tubes during inoculation.
4. Incubate the culture tubes at RT for 1 week.

Weeks 2: (observation of denitrification

1. After 7 days, examine all test cultures for the presence or absence of an air bubble in
the Durham tube. Record your observations and determine if denitrification has taken
place.

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Nitrogen Cycle Labs: Media and Reagents

Inorganic Ammonium Nitrite Medium Nitrate Medium

Compound Medium (g/L) (g/L) (g/L)
(NH4)2SO4 2.0 ----- -----
NaNO3 ---- ---- 1.0
NaNO2 ---- 1.0 ----
MgSO4.H2O 0.5 0.5 0.5
.
FeSO4 7H2O 0.03 0.03 0.03
NaCl 0.3 0.3 0.3
MgCO3 10.0 ---- ----
Na2CO3 ---- 1.0 1.0
K2HPO4 1.0 1.0 1.0
sterile dH2O 1000 mL 1000 mL 1000 mL

Nesslers Reagent (ammonia test)

DANGER! CAUTION! POISION!
Dissolve 50g of potassium iodide in 35 mL of cold distilled water. Add a saturated
solution of mercuric chloride until a slight precipitate persists. Add 400 mL of a 50%
solution of potassium hydroxide. Dilute to 1L, allow to settle and decant the supernatant
for use.

Diphenylamine Reagent (nitrate test)

CAUTION! VERY CORROSIVE!
Dissolve 0.7g of diphenylamine in a mixture of 60 mL concentrated sulfuric acid and
28.8 mL of distilled water. Cool and add slowly 11.3 mL of concentrated hydrochloric
acid. Let solution sit for 12 hours; some of the base should separate, showing that the
reagent is saturated.

Trommsdorfs Reagent (nitrite test)

Add slowly, with constant stirring, 100 mL of a 20% aqueous zinc chloride solution to a
mixture of 4.0 g of starch in water. Continue heating until the starch is dissolved as much
as possible and the solution is nearly clear. Dilute with water and add 2 g of potassium
iodide. Dilute to 1L, filter, and store in an amber bottle.