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Nitrogen Cycle:
Nitrification and Denitrification
n
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1. Definition: The biological formation
(oxidation) of NH4+ to produce nitrite
and nitrate. 2. Significance: Beneficial or Harmful?
» a. Allow for easier uptake of N by plants.
» b. Increases loss of N to leaching.» c. Increases risks of NO3- toxicity.
Nitrification: I. Introduction
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Nitrification: I. Introduction
» d. Increases soil acidity.
» e. Increases N loss due to denitrification.
» f. It is a necessary intermediate in returning N
to the atmosphere.
3. End product of fertilizer N is nitrate whether
the fertilizer is added as urea, anhydrousammonia or ammonium nitrate.
4. Potential pollutant
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II. Microbiology: Nitrifying Bacteria
1. Obligate aerobes
2. Derive their carbon solely from CO2 or
carbonates. 3. Derive energy from oxidation of NH4+
or NO2- (Chemoautotrophs).
4. The following genera are known tooccur in soils:
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II. Microbiology: Nitrifying Bacteria
a. Oxidize ammonium to nitrate
» i. Nitrosomonas - ellipsodal or short rods
» ii. Nitrosococcus - spherical cells
» iii. Nitrosospira - spiral-shaped cells
» iv. Nitrosobolus - lobate
b. Oxidize nitrite to nitrate (NO2-
NO3-)» Nitrobacter
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III. Biochemistry of Nitrification.
NH4+ + 1/2O2 NH2OH + H+ NO2- NO3-
Nitrosomonas Nitrobacter
1. Reaction involves a hydroxylamineintermediate
NH3 +O2 + 2H+ + 2e NH2OH + H2O
2. The oxygen in the nitrate product is formed
from H2O not molecular O2. NH3 NH2 OH (HNO?) NO NO2
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Nitrification
3. NO2- + H2O H2O.NO2 HO3- + 2H
2H + 1/2O2 H2O
4. Above reaction is an example of
hydration and dehydrogenation.
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IV . Factors Affecting Nitrification
1. Acidity (pH):
Significant correlation between NO3-
production and pH Optimum pH between 6.5-7.5
low rates at pH less than 6.0 and negligible
below pH of 4.5.
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Nitrification
2. Aeration:
» O2 is an obligate requirement, making adequate
aeration essential (Nitrosomonas and Nitrobacter are strict aerobes).
3. Moisture:
» Affects indirectly by controlling O2
availability.
Proceeds readily at -0.1 to -1MPa moisture
tension.
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Nitrification
4. Temperature
» Process slow below 5oC and above 40oC.
» Optimum between 30 and 35o C. 5. Organic Matter
» Starting point.
» It's decomposition may deplete supplies of available NH4+ and O2 for the nitrifiers.
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V. Fate of Nitrates in Soils
Once NO3- is formed in soils it is subject to
the following:
» 1. It may undergo denitrification by micro-organisms to gaseous oxides of nitrogen
and N2
» 2. It may be taken up by organisms and used to
synthesize amino acids (assimilatory
reduction).
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Nitrification
» 3. In the absence of O2 it may be used by
microorganisms as an electron acceptor
and become reduced to NH4+ (dissimilatory
reduction).
» 4. It may be leached from the upper profile to
deeper soils layers or to ground water.
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VI. Control of Nitrification
1. Use of microbial inhibitors e.g. N-serve
(Nitrapyrin).
2. Use of N-compounds that release N slowly
because of limited solubility in water or slow
rate of microbial decomposition e.g. urea
formaldehyde complexes, oxamide.
3. Use nitrogen compounds with materials thatdelay the release of N.
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VII. Desirable Characteristics of
Nitrification Inhibitors
1. Specific in blocking NH4+ NO3-
2. Able to move with fertilizers through
soil 3. Non-toxic to plants
4. Non-toxic to plants plant root
microenvironment 5. Not easily bio or chemical degradable
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VIII. Nitrate Pollution
1. Nitrate can be a significant environmental
pollutant.
2. This is because of its potential role in
» a. eutrophication,
» b. infant methemoglobinemia associated with
nitrate rich waters and vegetable,
» c. animal methemoglobenimia and d. formation
of nitrosoamine.
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VIII. Nitrate Pollution
a. Eutrophication:
» This is the process whereby waters are enrichedwith nutrients.
» N03- in rivers encourage algae and rooted
plants to flourish.
b. Methemoglobinemia:
» Arises when nitrate consumed in water, foodand feed is reduced in the gastrointerstinal tractto nitrite this nitrite enters the blood stream
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VIII. Nitrate Pollution
and reacts with hemoglobin to from met-hemoglobin.
» This leads to an impairment of O2 transport.
» It is serious for infants under three months andin ruminants.
» Standards of NO3- by WHO in water is 10ppmnitrate-N.
c. Animal may die from consuming plant specieswith high levels of NO3-.
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VIII. Nitrate Pollution
d. Nitrosoamines:
» 1. These compounds are formed from thecondensation of a secondary amine and
nitrite.
R’ R
NH + NO2 N-NO + OH-
R R’
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VIII. Nitrate Pollution
2. They are carcinogenic, mutagenic and
teratogenic i.e. they can induce cancer,
mutation and cause birth defects or sometimes death in fetuses.
3. Reaction can be carried out by microbial
enzymes or non-enzymatically byorganic materials present in soils.
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Denitrification:
Definition: Denitrification is the microbial reduction of NO3-
and NO2- and then to gaseous N2O and N2. This process is different from dissimilatory nitrate
reduction and also from assimilatory nitratereduction.
Assimilatory Nitrate Reduction: NO3- to NH4+ in the course of biosynthesis of
Amino Acids and proteins.
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I. Introduction:
Dissimilatory Nitrate Reduction:
» Certain microbial species use NO3- as
electron acceptor in absence of O2 to produce NH4+, but not N2, as an end
product.
» Pathway doesn’t lead to nitrogen lossfrom the system .
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+5 +4H +3 +6H -4
2HNO3 2HNO2 ? [2NH2OH] 2NH4+-H2O -2H2O
d. Extensive microbes can reduce NO3- to NO2- in the presence of organic matter and absence of O2.
This process is called nitrate respiration. NO3- NO2-
Nitrate respiration
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II. Microbiology
Although the capacity for denitrification is
found in many bacteria active in proteolysis,
ammonification and other transformation,the capacity for true denitrification is
limited to certain bacteria.
The active species are largely limited to thegenera Pseudomonas, Bacillus and
Paracoccus.
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II. Microbiology
Fungi and actinomycetes have not beenimplicated in N2 production.
The denitrifying bacteria are aerobic, butnitrate is used as the electron source inabsence of O2.
Thus the active species grow aerobically
without nitrate and anaerobically in its presence.
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III. Biochemistry of Denitrification
Reaction
+5 +3 +2 +1
0
2NO3- ® 2NO2- ® 2NO ® N2O ® N2
nitrate nitrite nitric oxide nitrous oxide
reductase reductase reductase reductase
¬ Photoautotrophs « Chemoautotrophs ® Chemoautotrophs®
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III. Biochemistry of Denitrification
a. Under field conditions, not all the intermediategaseous products are converted to N2 by each of thespecific reductase enzymes involved.
b. Nitrate Reductase
1. Nitrate Reductase contains Mo, Fe, and labile sulfidegroups
2. Both Mo and Fe are necessary for enzyme activity.
3. Reaction:
2NO3- + 10H N2 + 4H2 +2OH-
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III. Biochemistry of Denitrification
c. Nitrite reductase (NO2-)
1. 2NO2- + 6H ® N2 + 2H2 + OH-
2. Soluble and found in soluble fraction of cytoplasm.
d. Nitric Oxide (NO) reductase :
1. Membrane Bound,
E-FeII + NO2- ® E-FeII ® E-FeII.NO ® E-FeII.(N2O3) ® E-FeII + N2O (E = enzyme, FeII=Iron Group)
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III. Biochemistry of Denitrification
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IV. Factors Affecting Denitrification
1. Soil Nitrate Content.
» At low NO3- concentration the kinetics of
reduction appear to be first order.» Nitrate concentration has been observed to
influence the N2:N2O ratio in the gaseous
product of denitrification.
» At high NO3- levels N2 is the predominant, and
at low levels it is often N2O
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IV. Factors Affecting Denitrification
2. Carbon Availability.
» Most denitrification is accomplished byheterotrophs and is therefore, the process is
strongly dependant on the availability of carbon.
» Available carbonaceous substrates supplyelectrons. Their decomposition also produces
CO2 and reduces the O2 levels, thus increasingthe demand for NO3- as an electron acceptor during microbial growth.
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IV. Factors Affecting Denitrification
» The increased amounts of easily decomposablecarbon provided by root exudates and rootexfoliates in the rhizosphere suggests that thisregion may be more conducive todenitrification than non-rhizosphere soil.
3. Soil Water Content.
» The influence of water is linked to its role ingoverning O2 diffusion to sites of microbial
activity. Increases in soil water content to levelsthat interfere with air diffusion progressivelyincrease denitrification potential.
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IV. Factors Affecting Denitrification
» Oxygen is repressive to all the nitrogen oxide
reductases, but slightly less repressive to NO3-
reductase than to the others.
» Generally, in the field, and providing there are
no abnormal high O2 consumption rate,
denitrification is lacking or insignificant at
moisture levels less than 60% of moistureholding capacity, which is approximately
equivalent to -0.01MPa moisture stress.
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IV. Factors Affecting Denitrification
3. Soil pH.
» Most denitrifying bacteria grow best near
neutrality (pH 6-8).» Denitrification becomes slow but may still
remain significant below pH 5.
» It is negligible or absent below pH 4
» The degree of soil acidity also influences the
N2O:N2 ratio in the evolved gases.
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IV. Factors Affecting Denitrification
4. Temperature.
» Minimum temperature for denitrification is
about 5oC, maximum temperature, about75oC.
» Optimum temperature is around 25oC and
above.