Non Symbiotic Nitrogen Fixation

Non symbiotic Nitrogen Fixation

Non symbiotic (Asymbiotic) Nitrogen fixation refers to all Biological Nitrogen Fixation (BNF)
undertaken by microbial cells growing independently in soil, terrestial, and aquatic wetland
environments as saprophytes
• It also includes BNF occurring in loose or close association with the plant rhizosphere, and
endophyticall, but not requiring morphologically defined nodules
• Species of Azospirillum are able to form close associations with several members of the Poaceae
(grasses), including agronomically important cereal crops, such as rice, wheat, corn, oats, and
barley
• These bacteria fix appreciable amounts of nitrogen within the rhizosphere of the host plants
Some common examples include
• Azospirillum brasilense in association with cereal roots.
• Beijerinckia in association with the roots of Sugarcane.
• Azotobacter paspali in association with roots of tropical grass- Paspalum notatum.
Non symbiotic Nitrogen Fixation
• Around 80% of biological nitrogen fixation (BNF) is carried out by diazotrophs in symbiosis
with legumes; the rest is carried out by free living diazotrophs
• This amounts to upto 60 kg N ha-1 year-1

The level of nitrogen fixation is determined by several factors:

1. Soil temperature: Azospirillum species thrive in more temperate and/or tropical environments)
2. Ability of the host plant to provide a rhizosphere environment low in oxygen pressure
3. Availability of host photosynthates for the bacteria
4. Competitiveness of bacteria
5. Efficiency of nitrogenase
Associative nitrogen fixing bacteria in rice root systems
Key Non-symbiotic Nitrogen Fixers
Azospirillum
• Gram negative, rod-shaped bacteria
• 1 mm in diameter, highly motile
• Utilize glucose, lactate, succinate, fructose, malate, pyruvate, fumarate as
carbon source
List of N sources utilized by Azospirillum:
 Ammonium
 Nitrate
 Amino acids
 Elemental N (N2)
Azospirillum are highly adaptible and can grow under a wide range of
circusmtances that include:
 Anaerobic conditions (nitrate as electron acceptor)
 Microaerobic (elemental or ammonia used as N source)
 Fully aerobic conditions (ammonia, nitrate, amino acid or combined N only)
Azospirillum – Nitrogen Fixation
• Azospirillum can convert atmospheric nitrogen into ammonium under microaerobic conditions at
low nitrogen levels, through the action of the nitrogenase complex.
• This enzyme is built from two components: the dinitrogenase protein (MoFe protein, NifDK),
which contains a molybdenum-iron cofactor, is the site of N2 reduction; the dinitrogenase
reductase protein (Fe protein, NifH) transfers electrons from an electron donor to the nitrogenase
protein
• Most of the genetic work on nitrogen fixation by Azospirillum has been carried out with A.
brasilense
• Azospirillum only fixes nitrogen in microaerobic N-limiting conditions
• Ammonium, glutamine, nitrate and nitrite have all been shown to repress N2 fixation in A.
brasilense
• Since the reduction of N2 into ammonium is a highly energy-demanding process and the
nitrogenase enzyme complex is very sensitive to oxygen, biological N2 fixation is tightly
regulated
Azospirillum – relevance in crop cultivation

• Azospirillum species have shown to enhance crop

productivity when utilized as crop inoculants
• Field experiments in wheat inoculated with Azospirillum
species have shown a decrease in N fertilizer requirements
of upto 60%
• In addition to nitrogen fixation, many Azospirillum species
have shown various plant growth promoting benefits
Some benefits of using Azospirillum inoculants in fields
• Promotion of root hair development and branching
• Increased uptake of N, P, K and microelements
• Improved water status of plants
• Increased dry mater accumulation and grain yield
• Improves plant’s tolerance to biotic and abiotic stresses
(mediated by phytohormones acting as signalling
molecules)
Functions of Associative N2 fixing bacteria
Fix nitrogen, hence provide plants with rich N sources in the form of ammonia

Enhance the growth of plant roots (esp. rice, as interaction in rice fields is widely studied)
by exudation of growth regulators such as GA3 and IAA

Most associative N2 fixing bacteria have nitratase, which goes into plant roots after
inoculation and assist in nitrate reduction in plant hence increasing N levels, which
improves the rate of N2 fixation

Such bacteria are able to enhance plant mineral uptake

Rhizosphere colonization by associative N2 fixing bacteria enhance the growth of lateral

roots in plants
Azotobacter
• Gram-negative, oval or spherical bacteria
• Aerobic and free-living soil bacteria
• Form thick-walled cysts, multiple species produce large amounts of capsular slime
• Around six species present, some of which are motile by means of peritrichous flagella
• Usually found in neutral to alkaline soils, water, and in close association with other plants in their
rhizosphere
• Sensitive to acidic pH, high salts, and temperature
• Model organism for the study of diazotrophs (esp. asymbiotic nitrogen fixers)
• First species of Azotobacter (Azotobacter chroococcum) discovered by the Dutch scientist
Martinus Beijerinchk
• Azotobacter species are mostly free-living nitrogen fixers (some live in close association with
plants)
Azotobacter – Nitrogen Fixation
• Azotobacter species have a full range of enzymes needed to perform the nitrogen fixation:
ferredoxin, hydrogenase, and an important enzyme nitrogenase
• The process of nitrogen fixation requires an influx of energy in the form of adenosine
triphosphate
• Nitrogen fixation is highly sensitive to the presence of oxygen, so Azotobacter developed a
special defensive mechanism against oxygen, namely a significant intensification of metabolism
(high respiration rate) that reduces the concentration of oxygen in the cells
• However, in A. vinelandii , formation of alginate has been reported to play a major role in the
protection of its nitrogenase
• Formation of an alginate capsule in the cell surface forms an effective barrier for O2 transfer
into the cell
• Observations show that an increase in O2 tension induces the formation of alginate with a high
molecular weight and a greater L-guluronic acid content
Azotobacter – Nitrogenase
• Nitrogenase is the most important enzyme involved in nitrogen fixation
• Azotobacter species have several types of nitrogenase. The basic one is molybdenum-iron
nitrogenase
• An alternative type contains vanadium; it is independent of molybdenum ions and is more active
than the Mo-Fe nitrogenase at low temperatures
• Vanadium is present in V-nitrogenase in a vanadium and iron-containing protein (the Vfe protein)
• VFe proteins contains vanadium in an environment with iron, suphur and oxygen (or nitrogen or
carbon) as nearest neighbour atoms, very similar to the environment of Molybdenum in the iron
and molybdenum cofactor (FeMoco) centre of the MoFe proteins
• Key characteristic of V-nitrogenase – At low temperatures, N2 is a more effective substrate for V-
nitrogenase when compared with Mo-nitrogenase
• It can fix nitrogen at temperatures as low as 5 °C, and its low-temperature activity is 10 times
higher than that of Mo-Fe nitrogenase
• Synthesis of nitrogenase is controlled by the nif genes
Nitrogenase activity (Azotobacter) – V-nitrogenase vs. Mo-nitrogenase
Azotobacter – relevance in crop cultivation

• Besides nitrogen fixation, Azotobacter species are able to confer various plant growth promoting
benefits to the crop they are applied to
• Azotobacter species produce Thiamine, Riboflavin, Nicotin, IAA and Gibberellin
• Seed inoculation with Azotobacter have shown to improve seed germination considerably
• Several species have been shown to be effective phosphate solubilizers
• Inoculation with Azotobacter have also resulted in increased root and shoot biomass, total
chlorophyll content and carotenoids production
• Several species have been used as biopesticides due to their ability to produce siderophores and
antibiotics
Cyanobacteria
• Often called ‘blue-green algae’
• Some are aquatic and photosynthetic
• Small, usually unicellular, but often grow in large
colonies
• Majority of them are aerobic photoautotrophs
• Some show a distinct ability for heterotrophic
nutrition
• Many species are capable of living in soil and
other terrestrial habitats
• Gram-negative
• Have chlorophyll a, and photosystems I and II
that allow them to perform oxygenic
photosynthesis
• Use the pentose phosphate pathway for
carbohydrate metabolism

N2 fixing cyanobacteria
Cyanobacteria classification
Cyanobacteria

Non- heterocystous Heterocystous

Chroococcales Nostocales

Pleurocapsales Stigonematales

Oscillatoriales
Nitrogen Fixation via formation of Heterocysts
Heterocysts
• Specialized nitrogen-fixing cells formed during nitrogen
starvation
• Heterocyst protects the nitrogenase enzyme from high
oxygen concentrations
• Hence, heterocyst are responsible for creating a
microanaerobic environment
Some physiological and structural changes associated with the
formation of heterocysts include:
 Produce three additional cell walls (peptidoglycan,
glycolipid, and polysaccharide) - glycolipid forms a
hydrophobic barrier to oxygen
 Produce nitrogenase and other proteins involved in
nitrogen fixation
 Degrade photosystem II, that produces oxygen
 Up-regulate glycolytic enzymes
 Produce oxygen scavenging proteins
 Contain polar plugs composed of cyanophycin that slows
down cell-to-cell diffusion

h-heterocysts
Heterocysts

Heterocyst development in Anabaena PCC 7120

A. Anabaena PCC 7120 grown in medium containing a source of combined nitrogen grows as filaments of
photosynthetic vegetative cells
B. In the absence of combined nitrogen, heterocysts differentiate at semiregular intervals, forming a
developmental pattern of single heterocysts every 10 to 20 vegetative cells along filaments. Heterocysts
are often larger than vegetative cells, have a thicker multilayered envelope (prevent entry of oxygen), high
rate of respiration which utilizes defused oxygen, lack photosystem II (no evolution of oxygen)
Heterocysts – Nitrogen fixation

• Most heterocystous
strain fix nitrogen in the
light phase
• Import carbohydrates
from vegetative cells
• In dark phase, reductants
for nitrogen and oxygen
is generated by the
activity of the Pentose
Phosphate Cycle (PPC)
• Respiratory Electron
System (RET) generate
ATP required for the
reactions

Heterocyst metabolism and nitrogen fixation. Abbreviations: AcCoA, acetyl coenzyme A; Arg,
arginine; Asp, aspartate; b/f, cytochrome b6f complex; F6P, fructose 6-phosphate; PetF, vegetative cell
type ferredoxin; Glu, glutamate; Gln, glutamine; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; 6PG, 6-
phosphogluconate; PGA, 3-phosphoglycerate; Pi, inorganic phosphate; R5P, ribose 5-phosphate.
Nitrogenase efficiency of different species