‫ ادهام علي عبد جامعة االنبار كلية الزراعة‬.‫د‬.

‫ا‬
Nitrogen
• Nitrogen dynamics depend on plant
chemistry (C:N ratio, pH, decomposability),
carbon dynamics, microbial community
• Short-cuts for plants: N-fixing organisms,
mycorrhizae, direct uptake of organic N (?)
• Human nitrogen loading: fertilizer runoff
(overflows from previously almost-closed
cycles)
What’s so important about Nitrogen cycling?

essential nutrient (fertilizers, growing legumes as crops) –

changes in native species composition of ecosystem

atmospheric pollutant (burning fuels)

groundwater pollutant
 Nitrogen Cycle

N2
NONSYMBIOTIC
N2 FIXATION SYMBIOTIC DENITRIFICATION
N2 FIXATION

DECOMPOSITION PLANT UPTAKE NO3-

NITRIFICATION

AMMONIFICATION ASSIMILATORY or
Organic N NH4+ DISSIMILATORY
IMMOBILIZATION
NO3- REDUCTION
 Forms of Organic N

Amino Nucleic
sugar N acid N

Acid
Insoluble N Protein &
peptide N

Hydrolyzable
Unknown N

Labile N
 Major Inorganic N Compounds

Compound Formula Oxidation Form in soil

state
Ammonium NH4+ -3 Fixed in clay lattice, dissolved,
as gaseous ammonia (NH3)
Hydroxylamine NH2OH -1 Not detected
Dinitrogen N2 0 Gas
Nitrous oxide N2O +1 Gas, dissolved
Nitric oxide NO +2 Gas, dissolved
Nitrite NO2- +3 Dissolved
Nitrate NO3- +5 Dissolved
Nitrogen Fixation
The nodules on the roots
of this bean plant contain
bacteria called
Rhizobium that help
convert nitrogen in the
soil to a form the plant
can utilize.
 Dinitrogen Fixation
The alder, whose fat shadow nourisheth–
Each plant set neere him long flourisheth.
–William Browne (1613), Brittania’s Pastorals, Book I, Song 2

Treatment Yield (g)

Oats Peas
No N added
Non-inoculated 0.6 0.8
Inoculated with legume soil 0.7 16.4
Inoculated with sterile soil — 0.9
112 mg NO3-–N per pot added
Non-inoculated 12.0 12.9
Inoculated with legume soil 11.6 15.3
Hellriegel and Wilfarth (1888)
 Types of Biological Nitrogen Fixation
Free-living (asymbiotic)
• Cyanobacteria
• Azotobacter
Associative
• Rhizosphere–Azospirillum
• Lichens–cyanobacteria
• Leaf nodules
Symbiotic
• Legume-rhizobia
• Actinorhizal-Frankia
 Free-living N2 Fixation
Energy
• 20-120 g C used to fix 1 g N
Combined Nitrogen
• nif genes tightly regulated
• Inhibited at low NH4+ and NO3- (1 μg g-1 soil, 300 μM)
Oxygen
• Avoidance (anaerobes)
• Microaerophilly
• Respiratory protection
• Specialized cells (heterocysts, vesicles)
• Spatial/temporal separation
• Conformational protection
 Associative N2 Fixation

• Phyllosphere or rhizosphere (tropical grasses)

• Azosprillum, Acetobacter
• 1 to 10% of rhizosphere population
• Some establish within root
• Same energy and oxygen limitations as free-living
• Acetobacter diazotrophicus lives in internal tissue of sugar
cane, grows in 30% sucrose, can reach populations of 106
to 107 cells g-1 tissue, and fix 100 to 150 kg N ha-1 y-1
Estimated Average Rates of Biological N2 Fixation
Organism or system N2 fixed (kg ha-1 y-1)
Free-living microorganisms
Cyanobacteria 25
Azotobacter 0.3
Clostridium pasteurianum 0.1-0.5
Grass-Bacteria associative symbioses
Azospirillum 5-25

Cyanobacterial associations
Gunnera 10-20
Azolla 300
Lichens 40-80
Leguminous plant symbioses with rhizobia
Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus) 50-100
Pasture legumes (Trifolium, Medicago, Lupinus) 100-600
Actinorhizal plant symbioses with Frankia
Alnus 40-300
Hippophaë 1-150
Ceanothus 1-50
Coriaria 50-150
Casuarina 50
• Nitrogen Fixation

• Almost all N is in the atmosphere

• 90-190 Tg N fixed by terrestrial systems
• 40-200 Tg N fixed by aquatic systems
• 3-10 Tg N fixed by lightning
• 32-53 Tg N fixed by crops
Some biogeochemical cycling key points:

• cycling occurs at local to global scales

• biogeochemical cycles have 2 basic parts: pools and

fluxes

• elements are recycled among the biosphere, atmosphere,

lithosphere and hydrosphere

• cycles of each element differ (chemistry, rates, pools, fluxes,

interactions)

• cycling is important because it can affect many other

aspects of the environment and the quality of our lives
Nitrogen Metabolism
 Plant Nutrition

• Plant metabolism is based on

sunlight and inorganic
elements present in water,
air, and soil.
• C, H, and O and energy are
used to generate organic
molecules via
photosynthesis.
• Other chemical elements,
such as mineral nutrients,
are also absorbed from soil.
 Plant Nutrients

• Plants absorb many elements, some of which they

do not need.
• An element is considered an essential nutrient if it
meets three criteria:
• It is necessary for complete, normal plant
development through a full life cycle.
• It itself is necessary; no substitute can be
effective.
• It must be acting within the plant, not outside it.
• Many roles in plant metabolism.
 Types of Essential Nutrients

• Nine essential nutrients, called macronutrients, are

needed in very large amounts
• Eight other essential nutrients, called
micronutrients, are needed only in small amounts.
 Essential Nutrients to Most Plants
% Dry
Macronutrient Weight Component/Function
Carbon (C ) 45.0 Organic compounds

Oxygen (O) 45.0 Organic compounds

Hydrogen (H) 6.0 Organic compounds

Nitrogen (N) 1.0-4.0 Amino acids; nucleic acids, chlorophyll

Potassium (K) 1.0 Amino acids; regulates stomata
opening/closing
Calcium (Ca) 0.5 Enzyme cofactor; influences cell
permeability
Phosphorus (P) 0.2 ATP; proteins; nucleic acids;
phosphoplipids
Magnesium (Mg) 0.2 Chlorophyll; enzyme activator

Sulfur (S) 0.1 CoA; amino acids

 Essential Nutrients to Most Plants

Micronutrient Component/Function
Iron (Fe) Cytochromes; chlorophyll synthesis

Chlorine (Cl) Osmosis; water-splitting in photosynthesis

Copper (Cu) Plastocyanin; enzyme activator

Manganese (Mn) Enzyme activator; component of chlorophyll

Zinc (Zn) Enzyme activator

Molybdenum (Mo) Nitrogen fixation

Boron (B) Cofactor in chlorophyll synthesis

Nickel (Ni) Cofactor for enzyme functioning in nitrogen

metabolism
 Nitrogen: An Essential
Macronutrient
• N is not present in rock, but is abundant in the atmosphere as a
gas, N2.
• The process of converting N2 to chemically active forms of N is
nitrogen metabolism.
• Nitrogen metabolism consists of 3 stages:
• Nitrogen Fixation (N2 -> NO3-)
• Nitrogen Reduction (NO3- -> NO2- -> NH3 -> NH4+)
• Nitrogen Assimilation (transfer of NH2 groups)
• Runoff, leaching, denitrification, and harvested crops reduce soil
nitrogen.
 Nitrogen Cycling Processes
Nitrogen Fixation – bacteria convert nitrogen gas (N2)
to ammonia (NH3).
Decomposition – dead nitrogen fixers release N-
containing compounds.
Ammonification – bacteria and fungi decompose dead
plants and animals and release excess NH3 and
ammonium ions (NH4+).
Nitrification – type of chemosynthesis where NH3 or
NH4+ is converted to nitrite (NO2-); other bacteria
convert NO2- to nitrate (NO3-).
Denitrification – bacteria convert NO2- and NO3- to N2.
 Means of Nitrogen Fixation

1) Human manufacturing of synthetic fertilizers

2) Lightning

3) Nitrogen-fixing bacteria and cyanobacteria

 Nitrogen Fixing Bacteria and
Cyanobacteria
• Some are free-living in soil (E.g., Nostoc,
Azotobacter); others live symbiotically with plants
(E.g., Frankia, Rhizobium).
• These organisms have nitrogenase, an enzyme
that uses N2 as a substrate.
• N2 + 8e- + 8H+ + 16ATP -> 2NH3 + H2 + 16ADP + 16Pi
• NH3 is immediately converted to NH4+.
• Bacterial enzymes sensitive to O2.
• Leghemoglobin binds to O2 and protects enzymes.
• Symbiotic fixation rate depends on plant stage.
Natural Sources of Organic N
Source %N
Dried blood 12
Peruvian guano 12
Dried fish meal 10
Peanut meal 7
Cottonseed meal 7
Sludge from sewer treatment plant 6
Poultry manure 5
Bone meal 4
Cattle manure 2
 Symbiotic Nitrogen
Fixation
• Nitrogen-fixing bacteria fix N
(E.g., Rhizobium)
• Plants fix sugars (E.g.,legumes).
• Plants form swellings that house
N-fixing bacteria, called root
nodules.
• Mutualistic association.
• Excess NH3 is released into soil.
• Crop rotation maintains soil
fertility.
Development of a Root Nodule
• Bacteria enter the
root through an
infection thread.
• Bacteria are then
released into cell
and assume form
called bacteroids,
contained within
vesicles.
Symbiotic Nitrogen
Fixation
The Rhizobium-legume
association

Bacterial associations with certain

plant families, primarily legume
species, make the largest single
contribution to biological nitrogen
fixation in the biosphere
When this association is not present or functional, we
apply nitrogen-containing fertilizers to replace reduced
nitrogen removed from the soil during repeated cycles
of crop production.

This practice consumes fossil fuels, both in fertilizer

production and application.
Biological nitrogen fixation is the reduction of
atmospheric nitrogen gas (N2) to ammonium ions (NH4+)
by the oxygen-sensitive enzyme, nitrogenase. Reducing
power is provided by NAPH/ferredoxin, via an Fe/Mo
centre.

Plant genomes lack any genes encoding this enzyme,

which occurs only in prokaryotes (bacteria).

Even within the bacteria, only certain free-living

bacteria (Klebsiella, Azospirillum, Azotobacter),
blue-green bacteria (Anabaena) and a few symbiotic
Rhizobial species are known nitrogen-fixers.

Another nitrogen-fixing association exists between

an Actinomycete (Frankia spp.) and alder (Alnus spp.)
The enzyme nitrogenase catalyses the conversion of
atmospheric, gaseous dinitrogen (N2) and dihydrogen (H2)
to ammonia (NH3), as shown in the chemical equation below:

N2 + 3 H2  2 NH3

The above reaction seems simple enough and the

atmosphere is 78% N2, so why is this enzyme so important?

The incredibly strong (triple) bond in N2 makes this

reaction very difficult to carry out efficiently.
In fact, nitrogenase consumes ~16 moles of ATP for
every molecule of N2 it reduces to NH3, which makes it
one of the most energy-expensive processes known
in Nature.
 S

Fe

Mo

homocitrate

Fe - S - Mo electron transfer cofactor

in nitrogenase
Biological NH3 creation (nitrogen fixation) accounts for
an estimated 170 x 109 kg of ammonia every year.
Human industrial production amounts to some 80 x 109 kg
of ammonia yearly.

The industrial process (Haber-Bosh process) uses an Fe

catalyst to dissociate molecules of N2 to atomic nitrogen
on the catalyst surface, followed by reaction with H2
to form ammonia. This reaction typically runs at ~450º C
and 500 atmospheres pressure.

These extreme reaction conditions consume a huge

amount of energy each year, considering the scale at which
NH3 is produced industrially.
 The Dream…..

If a way could be found to mimic nitrogenase catalysis

(a reaction conducted at 0.78 atmospheres N2 pressure
and ambient temperatures), huge amounts of energy
(and money) could be saved in industrial ammonia production.

If a way could be found to

transfer the capacity to form N-fixing symbioses
from a typical legume host
to an important non-host crop species such as corn or wheat,
far less fertilizer
would be needed to be produced and applied
in order to sustain crop yields
Because of its current and potential economic
importance,
the interaction between Rhizobia and leguminous plants
has been intensively studied.

Our understanding of the process by which these two

symbionts establish a functional association is still not
complete, but it has provided a paradigm for many
aspects of cell-to-cell communication between microbes
and plants (e.g. during pathogen attack), and even
between cells within plants (e.g. developmental signals;
fertilization by pollen).
Symbiotic Rhizobia are classified in two groups:

Fast-growing Rhizobium spp. whose nodulation functions

(nif, fix) are encoded on their symbiotic
megaplasmids (pSym)

Slow-growing Bradyrhizobium spp. whose N-fixation and

nodulation functions are encoded on their chromosome.

There are also two types of nodule that can be formed:

determinate
and
indeterminate
This outcome is controlled by the plant host
 Determinate nodules
Formed on tropical legumes by
Rhizobium and Bradyrhizobium

Meristematic activity not persistent - present

only
during early stage of nodule formation;
after that, cells simply expand rather than
divide, to
form globose nodules.

Nodules arise just below epidermis; largely

internal
vascular system
Uninfected cells dispersed throughout nodule;
equipped to assimilate NH4+ as ureides
(allantoin and allantoic acid)

allantoin allantoic acid

 Indeterminate nodules

Formed on temperate legumes

(pea, clover, alfalfa);
typically by Rhizobium spp.

Cylindrical nodules with a persistent meristem;

nodule growth creates zones of different developmental
stages

Nodule arises near endodermis, and nodule vasculature

clearly connected with root vascular system
Uninfected cells of indeterminate nodules
assimilate NH4+ as amides (asparagine, glutamine)
 Genetics of Nitrogenase
Gene Properties and function
nifH Dinitrogenase reductase
nifDK Dinitrogenase
nifA Regulatory, activator of most nif and fix genes
nifB FeMo cofactor biosynthesis
nifEN FeMo cofactor biosynthesis
nifS Unknown
fixABCX Electron transfer
fixK Regulatory
fixLJ Regulatory, two-component sensor/effector
fixNOQP Electron transfer
fixGHIS Transmembrane complex
 Nitrogenase
Fd(ox) FeMo Cofactor

Fd(red) N2 + 8H+
8e-

nMgATP 2NH3 + H2

nMgADP + nPi 4C2H2 + 8H+ 4C2H2

Dinitrogenase
Dinitrogenase
reductase

N2 + 8H+ + 8e- + 16 MgATP → 2NH3 + H2 + 16MgADP

 Taxonomy of Rhizobia
Genus Species Host plant
Rhizobium leguminosarum bv. trifolii Trifolium (clovers)
“ bv. viciae Pisum (peas), Vicia (field
“ bv. phaseoli beans), Lens (lentils),
tropici Lathyrus
etli Phaseolus (bean)
Phaseolus (bean), Leucaena
Phaseolus (bean)
Sinorhizobium meliloti Melilotus (sweetclover),
fredii Medicago (alfalfa), Trigonella
saheli Glycine (soybean)
teranga Sesbania
Sesbania, Acacia
Bradyrhizobium japonicum Glycine (soybean)
elkanii Glycine (soybean)
liaoningense Glycine (soybean)
Azorhizobium caulinodans Sesbania (stem nodule)
‘Meso rhizobium’ loti Lotus (trefoil)
huakuii Astragalus (milkvetch)
ciceri Cicer (chickpea)
tianshanense
mediterraneum Cicer (chickpea)
[Rhizobium] galegae Galega (goat’s rue), Leucaena
Photorhizobium spp. Aeschynomene (stem nodule)
 Nitrogen Fixation

• Energy intensive process :

• N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP

+ 16 Pi

• Performed only by selected bacteria and

actinomycetes
• Performed in nitrogen fixing crops
(ex: soybeans)
Nodulation in Legumes
 Sources
• Lightning
• Inorganic fertilizers
• Nitrogen Fixation
• Animal Residues
• Crop residues
• Organic fertilizers
 Forms of Nitrogen
• Urea → CO(NH2)2
• Ammonia → NH3 (gaseous)
• Ammonium → NH4
• Nitrate → NO3
• Nitrite → NO2
• Atmospheric Dinitrogen →N2
• Organic N
 Global Nitrogen Reservoirs
Nitrogen Metric tons Actively cycled
Reservoir nitrogen
Atmosphere 3.9*1015 No
Ocean → soluble
salts 6.9*1011 Yes
Biomass 5.2*108 Yes

Land → organic
matter 1.1*1011 Slow
→ Biota 2.5*1010 Yes
 Roles of Nitrogen
• Plants and bacteria use nitrogen in the
form of NH4+ or NO3-
• It serves as an electron acceptor in
anaerobic environment
• Nitrogen is often the most limiting
nutrient in soil and water.
 Nitrogen is a key element for

• amino acids
• nucleic acids (purine, pyrimidine)
• cell wall components of bacteria (NAM).
 Nitrogen Cycles
• Ammonification/mineralization
• Immobilization
• Nitrogen Fixation
• Nitrification
• Denitrification
Mineralization or Ammonification
• Decomposers: earthworms, termites, slugs,
snails, bacteria, and fungi
• Uses extracellular enzymes → initiate
degradation of plant polymers
• Microorganisms uses:
• Proteases, lysozymes, nucleases to degrade
nitrogen containing molecules
• Plants die or bacterial cells lyse → release of organic
nitrogen
• Organic nitrogen is converted to inorganic nitrogen
(NH3)

• When pH<7.5, converted rapidly to NH4

• Example:

Urea NH3 + 2 CO2

 Immobilization
• The opposite of mineralization
• Happens when nitrogen is limiting in the
environment
• Nitrogen limitation is governed by C/N ratio
• C/N typical for soil microbial biomass is 20
• C/N < 20 →Mineralization
• C/N > 20 →Immobilization
 Microorganisms fixing
• Azobacter • Require the enzyme
• Beijerinckia nitrogenase
• Azospirillum • Inhibited by oxygen
• Clostridium • Inhibited by
• Cyanobacteria ammonia (end
product)
 Rates of Nitrogen Fixation
N2 fixing system Nitrogen Fixation (kg
N/hect/year)
Rhizobium-legume 200-300

Cyanobacteria- moss 30-40

Rhizosphere 2-25
associations
Free- living 1-2
 Bacterial Fixation
• Occurs mostly in salt marshes
• Is absent from low pH peat of northern
bogs
• Cyanobacteria found in waterlogged
soils
 Denitrification
• Removes a limiting nutrient from the
environment
-
• 4NO3 + C6H12O6→ 2N2 + 6 H20
• Inhibited by O2
• Not inhibited by ammonia
• Microbial reaction
• Nitrate is the terminal electron acceptor