Topic 4: Biological Nitrogen Fixation

##Discussion points

o Nodulation/infection, development, host strain interaction, infectivity, effectively and

efficiency.

o Biochemistry of nitrogen fixation, role of leg-heamoglobin.

o Acetylene reduction assay.

o Effect of combined N- application, water stress and temperature.

o Nitrogen supplying capacity of nitrogen fixing systems.

# Nitrogen Fixation
o The atmosphere contains about 78% by volume of molecular nitrogen.

o However plants do not absorb nitrogen in this form, it is taken up by plants in the form
of nitrates or ammonium.

o Thus the highly stable triple bonds in the nitrogen molecule have to be broken down to
produce nitrates or ammonium, which are directly available to plants.

o This requires very high temperatures of about 600°C and atmospheric pressure of about
200 atmospheres to be achieved in the presence of a catalyst to combine the nitrogen
with hydrogen to produce ammonium.

o This reaction or rather process is referred to as nitrogen fixation.

o Biological nitrogen fixation (BNF) is then a biological process whereby atmospheric

nitrogen (N=N) is reduced to ammonia in the presence of nitrogenase enzyme complex.
o Nitrogenase is a biological catalyst found naturally only in certain microorganisms such as
the symbiotic Rhizobium and Frankia, or the free-living Azospirillum and Azotobacter.

o Leguminous plants fix atmospheric nitrogen by working symbiotically with special

bacteria, rhizobia, which live in the root nodules.

o Rhizobia infect root hairs of the leguminous plants and produce the nodules.

o The nodules become the home for bacteria where they obtain energy from the host plant
and take free nitrogen from the soil air and process it into combined nitrogen.

o In return, the plant receives the fixed N from nodules and produces food and forage protein.
#The N-cycle
#Root Nodules
#The nodulation process
#The stepwise events

1. The legume plant releases chemicals (flavanoids) which are detected by the bacteria using its sensory Nod D
gene.
2. Nod D gene produces Nod factors that switch on the other Nod genes (Nod A,B,C,.E).
3. The Nod genes (i.e., A/B/C) produce their own nod factors that cause the bacteria to move to legume root
hair.
4. Once the bacteria is in attachment, the root hairs then start to curl and the rhizobia start to multiply inside the
coils.
5. The wall of the root hair then degrades due to infection and an infection thread is formed.
6. The infection thread grows until it reaches the interior of the root and then it spreads to affect other root cells
7. The multiplied rhizobia still in the curl are released into the infection thread resulting in the other root cells
being infected.
6. All infected and affected root cells rapidly divides.
7. Under the direction of the bacteria, the dividing root cells form spherical structures that are hollow inside
called bacteriods.
8. Once bacteria move into the bacteriods, they express Nif genes that lead to the production of the nitrogenase
enzymes, these enzymes are capable of converting atmospheric N2 into ammonia (NH3)
o Due to the high energetic cost of fixing dinitrogen, a significant part of nitrogen fixation occurs near the plant roots
surface, where there is an influx of sugar to power processes such as nutrient and water uptake.
#Specificity and effectiveness
o There are roughly 1,300 leguminous plant species in the world.
o But it is important to note that not all bacteria in the genus Rhizobium will form a symbiotic
relationship with all legume crops and that once formed, not all symbioses fix N2 with
equal effectiveness.
o In fact, there is a certain specificity to the process.
o This means that a given legume cultivar nodulated by different strains of the same species
of Rhizobium would fix different amounts of nitrogen.
o Selection of elite strains of Rhizobium is based on this observation.
o Similarly, a given strain of Rhizobium will nodulate and fix different amount of N2 in
symbiosis with a range of cultivars of the same plant species.
o Also, the free-nodulating Gliricidia or promiscuous varieties of soybean can nodulate
profusely and fix a great deal of nitrogen depending on the effectiveness of the rhizobial
populations present.
o A Rhizobium that nodulates cowpea may not nodulate Leucaena and vice versa.
o RJ (1, 2, 3, 4 .....) are host/plant genes found in soyabean that reduce the effectiveness of
nodules formed by certain rhizobia ssp.
o Leguminous species mutually susceptible to nodulation by a particular group of bacteria
constitute a cross-inoculation group.
o Mechanisms of recognition between the microsymbiont and the host-plant have been
suggested to be specific.
#Biochemistry of Nitrogen fixation
#Biochemistry of nitrogen fixation details
o BNF can be represented by the following equation, in which two moles of ammonia are produced from one
mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions):
N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi
o This reaction is performed exclusively by prokaryotes (the bacteria and related organisms), using an enzyme
complex termed nitrogenase.
o This enzyme consists of two proteins (i.e., an iron protein and a molybdenum-iron protein).
o The reactions occur while N2 is bound to the nitrogenase enzyme complex.
o The Fe protein is first reduced by electrons donated by ferredoxin then the reduced Fe protein binds ATP and
reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH.
o In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to
H2N-NH2, and this in turn is reduced to 2NH3.
o Depending on the type of microorganism, the reduced ferredoxin which supplies electrons for this process is
generated by photosynthesis, respiration or fermentation.
o The nitrogenase enzyme complex is highly sensitive to oxygen.
o It is inactivated when exposed to oxygen, because oxygen reacts with the iron component of the proteins.
o Although this is not a problem for anaerobic bacteria, it could be a major problem for the aerobic species such as
cyanobacteria (which generate oxygen during photosynthesis) and the free-living aerobic bacteria of soils, such as
Azotobacter and Beijerinckia.
o In the symbiotic nitrogen-fixing organism, Rhizobium, the root nodules contain oxygen-scavenging molecules such
as leghaemoglobin, which appear as a pink colour when the active nitrogen-fixing nodules of legume roots are cut
open.
o The pigment leghaemoglobin is a unique metabolite of this type of symbiosis.
o Leghemoglobin is a macromolecule synthesized by both, symbiotic partners (i.e., the rhizobia and the host plant).
o Rhizobium synthesizes the heme portion while the plant synthesizes the globine.
o Leghaemoglobin is found only in the nodules and is not produced by either the bacterium or the plant when grown
alone.
o Leghaemoglobin may regulate the supply of oxygen to the nodule tissues in the same way as haemoglobin regulates
the supply of oxygen to mammalian tissues.
#Short-term Estimation of BNF: Acetylene Reduction Assay

o Nitrogenase not only catalyzes the reduction of atmospheric N2 to NH3, but can also reduce
acetylene (C2H2) into Ethylene (C2H4).
o The acetylene reduction assay (ARA) is carried out on detached nodules, de-topped roots,
or whole plants in a closed vessel containing 10% acetylene.
o A gas chromatograph is used to determine the amount of ethylene formed.
o Data are usually expressed as nanomoles or micromoles of ethylene produced per hour per
plant or per weight unit of nodules.
o The acetylene reduction assay provides an instant measure of nitrogenase activity (but not
necessarily of N2 fixed) under the experimental conditions.
o A problem that is inherent in ARA is the need to calibrate the rates of ethylene production
with the actual rates of N2 fixation.
o The commonly used ratio of 3:1 for acetylene reduced per N2 fixed is not always valid.
o Also, nitrogenase activity of some legumes declines considerably once nodules or roots are
detached from the rest of the plant.
o Variation in light intensity, temperature, and moisture in the field will increase the level of
variation of nitrogenase activity and will reduce the significance of integration of short-
term assays.
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