Nitrogen Fixation

DEPARTMENT OF BIOTECHNOLOGY
 Why N2?
• Major requirement in plants next to water.
• Building Blocks of biomolecules
• Constituent element of
 chlorophyll
 Cytochrome
 Alkaloids
 Many vitamins

Role in Metabolism, growth, reproduction and

heredity
DEPARTMENT OF BIOTECHNOLOGY
Ammonia assimilatory cycle:
How nitrogen enters biological pathways Amino acids
proteins
Pathway 1 purines
pyrimidines
GS
NH4+ + glutamate + ATP glutamine + ADP + Pi

GOGAT
glutamate -ketoglutarate + glutamine

Pathway 2

GDH
NH4+ + -ketoglutarate glutamate

Amino acids
proteins
DEPARTMENT OF BIOTECHNOLOGY
 N2
dinitrogen gas
Denitrification Nitrogen fixation
(78% of air)
the Haber Process and
lightning

N 2O
nitrous oxide
The Nitrogen Cycle
NH4+
ammonium
Denitrification BIOSPHERE

NO3-
nitrate
nitrification
nitrification
NO2-
nitrite
DEPARTMENT OF BIOTECHNOLOGY
The Nitrogen Cycle N2
dinitrogen gas
(78% of air)

Biological nitrogen fixation

N 2O
nitrous oxide nitrification
Prokaryotes
assimilation
NH4+
Plants ammonium
consumption
Animals

uptake
NO3 -

nitrate
NO2-
nitrite
DEPARTMENT OF BIOTECHNOLOGY
 Sources of Nitrogen
 Atmospheric N2
* 78% of atmosphere
* Plants cannot utilize this form
* Some bacteria, Blue-green algae, leguminous plants
 Nitrates, Nitrites and Ammonia
* Nitrate is the chief form
 Amino acids in the soil
* some organisms make use of this form of N2
* some higher plants also uptake aa
 Organic N2 sources
* Insectivorous plants

DEPARTMENT OF BIOTECHNOLOGY
 Nitrogen Fixation

Conversion of free Nitrogen into nitrogenous

salts for uptake by plants

DEPARTMENT OF BIOTECHNOLOGY
 Types of N2 fixation

DEPARTMENT OF BIOTECHNOLOGY
 Non-biological fixation
• Found during the rainy season as a consequence of lightning

DEPARTMENT OF BIOTECHNOLOGY
 Biological Fixation

• Fixation of atmospheric nitrogen into nitrogenous

salts with the help of micro-organisms

• 2 types
* Symbiotic
* Asymbiotic

DEPARTMENT OF BIOTECHNOLOGY
 Greensulfur Bacteroides
Prokaryotes Spirochetes
Deinococci

Green, Chlamydiae
nonsulfur
Thermotoga Gram positive

Cyano
bacteria
Rhizobium
a Bradyrhizobium
Sinorhizobium
Agrobacterium
b Azospirillum
d
g Herbaspirillum
Desulfoivbrio
E. coli
Klebsiella
Azotobacter
Proteobacteria

DEPARTMENT OF BIOTECHNOLOGY
 Ecology of nitrogen-fixing bacteria

DEPARTMENT OF BIOTECHNOLOGY
 Asymbiotic Nitrogen Fixation
• Nitrogen fixed by free living microorganisms

• Bacteria
Free living aerobic: Azotobacter, Beijerinckia
Free living anaerobic: Clostridium
Free living photosynthetic: Chlorobium, Rhodopseudomonas
Free living chemosynthetic: Desulfovibrio, Thiobacillus
• Fungi : yeasts and Pillularia
• Blue Green Algae
Unicellular: Gloeothece, Synechococcus
Filamentous (non-heterocystous) – Oscillatoria
Filamentous (heterocystous) – Tolypothrix, Nostoc, Anabaena

DEPARTMENT OF BIOTECHNOLOGY
 Symbiotic Nitrogen Fixation
• Fixation of free nitrogen by micro-organisms in soil living
symbiotically inside the plants

• ‘Symbiosis’ coined by DeBary

• 3 types
* Nodule formation in leguminous plants
* Nodule formation in non-leguminous plants
* Non-nodulation

DEPARTMENT OF BIOTECHNOLOGY
 Nodule formation-Leguminous plants
• ~ 2500 sp of Leguminosae (Cicer, Cajanus, Pisum, Arachis etc)
produce root nodules with Rhizobium sps.

• Fix nitrogen only inside the root nodules

• Association provides - food and shelter to bacteria

- nitrogen to the plant

• Nodules may be buried in soil even after harvesting –

continue nitrogen fixation

DEPARTMENT OF BIOTECHNOLOGY
 Nodule formation- non-Leguminous plants
• Some other plants also produce root nodules
+ Casuarina equisetifolia- Frankia
+ Alnus – Frankia
+ Myrica gale – Frankia
+ Parasponia - Rhizobium

• Some plants produce leaf nodules

+ Diascorea, Psychotria

• Gymnosperms - root- Podocarpus

- leaves – Pavetta, Chomelia

DEPARTMENT OF BIOTECHNOLOGY
 Non-nodulation

• Lichens : Cyanobacteria
• Anthoceros : Nostoc
• Azolla : Anabaena azollae
• Cycas: Nostoc, Anabaena
• Gunnera macrophylla: Nostoc
• Digitaria, Maize, Sorghum: Spirillum notatum
• Paspalum notatum : Azotobacter paspali

DEPARTMENT OF BIOTECHNOLOGY
 Symbiotic Nitrogen Fixation
• Small, knob-like protrusions
• Size and shape varies
• Spherical, flat-finger like or elongated
• Pin-head to 1cm in size
• Various sp of Rhizobium
- Pea : Rhizobium leguminosarum
- Beans : R. phaseoli
- Soyabeans : R. japonicum
- Lupins : R. lupini
• 2 types of Rhizobium
* Bradyrhizobium – slow growing sp
* Rhizobium – fast growing sp

DEPARTMENT OF BIOTECHNOLOGY
 Rhizobium

• Gram negative
• Non-spore forming
• Micro-aerobic
• Show a degree of specificity
• The 2 partners- host and bacteria are recognized by chemical
substance
• LECTINS- Phytoagglutinins (Carbohydrate containing plant
proteins)

DEPARTMENT OF BIOTECHNOLOGY
 legume

Fixed nitrogen Fixed carbon

(ammonia) (malate, sucrose)

rhizobia

DEPARTMENT OF BIOTECHNOLOGY
 Obvious signs of nodulation by common rhizobial species

MEDICAGO LOTUS
(alfalfa) (birdsfoot trefoil)
DEPARTMENT OF BIOTECHNOLOGY
 Physiology of a legume nodule

DEPARTMENT OF BIOTECHNOLOGY
Very early events in the Rhizobium-legume symbiosis

Flavonoids rhizosphere Nod-factor

nod-gene
inducers

DEPARTMENT OF BIOTECHNOLOGY
 Sinorhizobium meliloti

chromosome
NodD
nod-gene inducers
from alfalfa roots plasmid
(specificity)

pSym
activated NodD
positively regulates
nod genes

nod genes

DEPARTMENT OF BIOTECHNOLOGY
 NodM
Nod factor
biosynthesis

NodC
Nod factor R-group
“decorations”
determine host
specificity
NodB

Nod Factor: a
lipooligosaccharide

DEPARTMENT OF BIOTECHNOLOGY
 Rhizobium Attachment and infection

Nod factor
(specificity)
Invasion through infection tube
Flavonoids
(specificity)

Bacteroid Nitrogen
differentiation fixation

Formation of
nodule primordia
From Hirsch, 1992.
New Phyto. 122, 211-237
DEPARTMENT OF BIOTECHNOLOGY
Rhizobium encoding GFP from jellyfish as a marker

Infection thread

(From Quaedvlieg et al. Plant Mol. Biol. 37: 715-727, 1998)

DEPARTMENT OF BIOTECHNOLOGY
Bacteria divide as they traverse infection thread

DEPARTMENT OF BIOTECHNOLOGY
 Nodule development

Enlargement of the nodule,

nitrogen fixation and
exchange of nutrients

DEPARTMENT OF BIOTECHNOLOGY
 The Nodulation Process

• Chemical recognition of roots and Rhizobium

• Root hair curling
• Formation of infection thread
• Invasion of roots by Rhizobia
• Cortical cell divisions and formation of nodule
tissue
• Bacteria fix nitrogen which is transferred to
plant cells in exchange for fixed carbon

DEPARTMENT OF BIOTECHNOLOGY
 Biological nitrogen fixation:

nitrogenase
N2 + 8 flavodoxin + 8H + 16 MgATP + 18 H2O
- + 2-

2NH4+ + 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4- + H2

1. Rare, extremely energy consuming conversion because of stability

of triply bonded N2
2. Produces fixed N which can be directly assimilated into N
containing biomolecules

DEPARTMENT OF BIOTECHNOLOGY
 Exchange of nutrients during Rhizobium-legume symbiosis

Malate
to bacteria

nitrogen-
fixing bacteroid
containing Rhizobium

TCA
ATP ADP+Pi
NH4+
to plant
N2 NH4+

DEPARTMENT OF BIOTECHNOLOGY
 A growing population must eat!

•Combined nitrogen is the most common limiting nutrient in agriculture

•Estimated that 90% of population will live in tropical and subtropical areas
where (protein-rich) plant sources contribute 80% of total caloric intake.
•In 1910 humans consumed 10% of total carbon fixed by photosynthesis, by
2030 it is predicted that 80% will be used by humans.
DEPARTMENT OF BIOTECHNOLOGY
 Why chemical fertilizers aren’t the answer

Consumes 1.4%
of total fossil
fuels annually

•Production of nitrogenous fertilizers has “plateaued” in recent years

because of high costs and pollution

•Estimated 90% of applied fertilizers never reach roots and contaminate

groundwater

DEPARTMENT OF BIOTECHNOLOGY
Current approaches to improving biological
nitrogen fixation

1 Enhancing survival of nodule forming bacterium by improving

competitiveness of inoculant strains

2 Extend host range of crops, which can benefit from biological

nitrogen fixation

3 Engineer microbes with high nitrogen fixing capacity

What experiments would you propose if you were to follow each of these
approaches?

DEPARTMENT OF BIOTECHNOLOGY