NITROGEN

• Essential dietary element- derived from animal & plant protein

• Role of N

• Nitrogen fixation, natural and synthetic, is essential for all forms of life because nitrogen is required to biosynthesize basic building blocks of plants, animals and other life forms, e.g., nucleotides for DNA and RNA and amino acids for proteins

Role of nitrogen in plants

» Major substance in plants next to water

» Building blocks

» Constituent element of» Chlorophyll

» Cytochromes

»Alkaloids

»Many vitamins

» Plays important role in metabolism, growth, reproduction and heredity

Sources of nitrogen• Atmospheric Nitrogen

–78% of atmosphere

–Plants cannot utilize this form

–Some Bacteria, Blue Green Algae, leguminous plants

• Nitrates, Nitrites and Ammonia

• Amino acids in the soil

• Organic Nitrogenous compounds in insects

Nitrogen Fixation

• Is the removal and reduction of atm. Molecular N2 & its subsequent incorporation with other elements to form nitrogenous compounds.

• “Nitrogen Fixation” is the process that causes the strong two-atom nitrogen molecules found in the atmosphere to break apart so they can combine with other atoms.

Nitrogen gets “fixed” when it is combined with oxygen or hydrogen.

Factors affecting N2 fixation

1. Presence of nitrate or ammonium :

2. Presence of certain inorganic substances

Ca, Co, Mo – influence N2 fixation along with P

3. Availability of energy source – addn. of C source increase N2 fixation

4. pH : Neutral – favours Azotobacter – Acidic-Beijerinkia

5. Soil moisture : Adequate is good for fixation

6. Temperature: Mesophilic – 30°C.

There are three ways that nitrogengets “fixed”

• Industrial N2 fixation

• Non biological N2 fixation

• biological N2 fixation

Industrial N2 fixation• Accomplished by Haber -Bosch process• Developed in Germany 1914 by Fritz haber & Karl

bosch• Process- N2 and H2 react with each other in presence

of Industrial catalyst( nickel / iron) High temperature about 500 ͦ c High pressure – 200 atmTo form NH3• Source of H2 - is methane (natural gas)• Industrial production of fertilizers and explosives

N2 + 3 H2 2NH3500C,Ni

200atm

Non biological N₂ fixation

• Non biological/ physico chemical N2 fixation involves the photochemical & electro chemical conversion of atm.N₂ to soil NO2,NO3, NH3.

• It is brought about by ionizing phenomena such as cosmic radiations,meteor trails ,lightning, thunderstorms, volcanic eruptions etc.

• These provides high energy for breaking N≡N & also for the formation of free N₂ with oxygen or hydrogen of atm H₂0

• combination of N₂ with o₂ forms nitrous and nitric oxides.

• Combination of N₂ with hydrogen forms NH3

• Nitrous and nitric oxides get hydrated with atm. Water vapour and forms nitrous and nitric acids

• Rain water bring these acids and NH3 to soil surface

• There ,the acids react with metallic ions and form metallic nitrates.

• These nitrates and NH3 enrich the surface soil

• Non biological N₂ fixation amounts to only ˂ 10% of the natural N₂ fixation

• It is common in some tropical regions, where thunder bolt are frequent.

• 1. N2 + O2 lightning 2 NO(Nitric oxide)

2. 2NO + O2 oxidation 2NO2

( Nitrogen per oxide)3. 2NO2 + H2O HNO2 + HNO3

4. 4NO2 + 2H2O + O2 4HNO3

(Nitric acid)5. CaO + 2HNO3 Ca (NO3)2 + H2O

(Calcium nitrate)6. HNO3 + NH3 NH4NO3

(Ammonium nitrate)7. HNO2 + NH3 NH4NO2

(Ammonium nitrite)

Biological Nitrogen Fixation

• Biological nitrogen fixation was discovered by the German agronomist Hermann Hellriegel and Dutch microbiologist Martinus Beijerinck.

• Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase

• The reaction for BNF is:

• N2+16ATP+8H++8 e− → 2 NH3+H2+16ADP+16Pi

• BNF is mostly accomplished by microorganisms called diazotrophs or N₂ fixers

• They include some species of bacteria, fungi, blue green algae,lichens etc.

Special features of diazotrophs

• Free living –bacteria like azotobacter, rhodopseudomonas fix atm.N₂ & also protects nitrogenase enzyme – sensitive to 0₂

• They produce exopolysaccharide (slime) which retains water and prevents diffusion of 0₂ inside cell during N₂ fixation

• Azotobacter have exceedingly high rate of respiratory metabolism thus preventing 0₂ retension inside the cell

• Blue green algae –are of 2 kinds

• One that possess heterocyst

• Other that devoid of it ( non heterocystous)

• Filamentous cyanobacteria contain pale , thick walled , hollow cells called heterocyst

• Heterocyst are the site of N₂ fixation

• They lack PSII and photosynthetic bile proteins

• non heterocystous N₂ fixing BGA like (oscillatoria) - the filaments are arranged clumps and N₂ fixation takes place in internaly organisedcell having reduced conditions

• Azospirillum paspalum survive in microaerophilicconditions associated with the rhizosphere ( area surrounding the roots ) of paddy plants - fix atm. N₂ in the rhizosphere.

Anabaena

• Soil N₂ fixers are of two categories

Non symbiotic N₂ fixers

symbiotic N₂ fixers

Non symbiotic or free living nitrogen fixers

• They inhabit both terrestrial & aquatic habitats

o Fixation carried out by free living micro-organisms

o Aerobic, anaerobic and blue green algae

Free living nitrogen fixerso Free living aerobic : Azotobacter, Beijerenckia

o Free living anaerobic : Clostridium

o Free living photosynthetic : Chlorobium, Rhodopseudomonas

o Free living chemosynthetic :Desulfovibro,Thiobacillus

o Free living fungi: yeasts and Pillularia

o Blue green algae:

o unicellular – Gloeothece , Synechococcus

o Filamentous (non heterocystous) – Oscillatoria

o Filamentous ( heterocystous) – Tolypothrix, Nostoc , Anabaena

• Ability to fix N by MO was confirmed by-technique -acetylene reduction to ethylene - diazotropic MO

• Conversion is controlled by an enzymatic complex (or nitrogenous enzyme)-reduce gaseous N - ammonia.

• Nitrogenase – sensitive to O.

• Several groups of MOs fix N in presence of minute quantities of O - Microaerobic fixers - Spirilla(Aquaspirillum & Azospirillum).

• O-not only inhibits the activity of nitrogenase-but also regulate biosynthesis.

SYMBIOTIC N₂ FIXERS

• Fixation of free nitrogen by micro-organisms living in soil symbiotically inside the plants

• ‘Symbiosis’ – coined by DeBary

• Three categories–Nodule formation in leguminous plants

–Nodule formation in non-leguminous plants

–Non nodulation

Nodule formation in leguminous plants• 2500 sp. Of family leguminosae ( Cicer arientium,

Pisum, Cajanus, Arachis) produce root nodules with Rhizobium spp.

• They fix Nitrogen only inside the root nodules

• Association provides-food and shelter to bacteria

-bacteria supply fixed nitrogen to plant

• Nodules may buried in soil even after harvesting –continue nitrogen fixation

Nodule formation in non-leguminous plants

• Some other plants also produces root nodules– Causuarina equisetifolia – Frankia

– Alnus – Frankia

–Myrica gale – Frankia

– Parasponia – Rhizobium

• Leaf nodules are also noted– Dioscorea, Psychotria

• Gymnosperms – root – Podocarpus,

- leaves – Pavetta zinumermanniana, Chomelia

Non-nodulation

• Lichens - cyanobacteria

• Anthoceros - Nostoc

• Azolla – Anabaena azollae

• Cycas – Nostoc and anabaene

• Gunnera macrophylla - Nostoc

• Digitaria, Maize and Sorghum – Spirillumnotatum

• Paspalum notatum – Azotobacter paspali

Nodule formation

• Root nodules formed due to infection of Rhizobium

• Free living bacteria growing , near the root of legumes unable to fix nitrogen in free condition

• Roots of the legumes secrete some growth factors helps in fast multiplication of bacteria

• (E.g.) Pisum sativum secretes homo serine & also carbohydrate containing protein Lectins over their surface

• Rhizobia are chemotactically attracted to root hair

• Medited by lectins, some attach to root hair cell wall

• Tryptophan is a component of the root hair exudate

• Trytophan is transformed by rhizobia to indoleacetic acid (IAA)

• This plant hormone causes the root hair to curl or branch around the attached rhizobia

• Polygalacturonase , secreted by rhizobia or possibly by the plant, depolymerizes & softens root hair cellwall

• Rhizobia gain entry into the root hair cell

• The root hair cell nucleus directs the development of infection thread

• Infection thread ,a tube consisting of cell membrane & surrounding cellulosic wall,

• Grows into root cortex & infects some tetraploidcells that proliferate & form nodule tissue

• Rhizobia are released from infection thread ,lose their rod shape

• Infected root cells swell and cease dividing.

• Bacteria within the swollen cells change form to become endosymbiotic bacteroids,which begin to fix nitrogen

• The nodule provides an oxygen-controlledenvironment

• (leghemoglobin = pink nodule interior) structured to facilitate transport of reduced nitrogen metabolites from the bacteroids to the plant vascular system, and of

• photosynthate from the host plant to the bacteroids.

Nitrogen fixing organisms found in nodules

• Rhizobium Rod shaped,motile.etc. Non sporing, non-acid fast. Rhizobia are susceptible to antibiotics, bacteriophages,

fungicides, herbicides etc.• Named after the host plant

– Pea – Rhizobium leguminosarum– Beans – R. phaseoli– Soyabeans – R. japonicum– Lupins – R. lupini

• Two types of Rhizobium-– Bradyrhizobium – slow growing spp.– Rhizobium - fast growing spp.

Structure and function of nodule

• Outermost layer of nodule-bacteriod zone-enclosed by cortical cells.

• Rate of N- fixation of nodule directly proportional to the volume of the nodule.

• Nodules are small-contain leghaemoglobin• Leghaemoglobin• Effective nodules are larger- pink in colour -due to the

presence of red coloured leghaemoglobin.• This pigment is similar to haemoglobin of blood.• Found in nodules between bacteriods & membrane

envelops, enclosing them.

• It is heme protein.• Contain heme moiety attached to a peptide chain-

represent globin part.• The amount of leghaemoglobin in nodules has direct

relationship between amount of atm N fixed by legumes.

• MW-16,000-17,000 daltons.

• Functions:

-Represents an active site of N absorption and reduction.-Acts as a specific electron carrier.-Regulate the O –supply in the nodule.-O-carrier.

Site and mechanism of nitrogen fixation in nodules

Site• Bacteroids-site of N-fixation.Mechanism• 1) Theory of Virtanen N fixation in roots appear immediately after nodule

formation. -young plants fix N than the old plants. A great part of N- converted to-L-aspartic acid and L-

glutamic acid. Apart from this alpha-alanine present in nodule-produced

from L-aspartic acid by decarboxylation. small amount of Oxime-N and nitrite-N are also present.

• 2) Theory of Burris and Wilson –hydroxylamine is the central compound of N fixation from which ammonia is formed-through reduction.

Biochemistry of nitrogen fixation

• N₂ fixers utilize atm. N₂ to synthesize NH3

• In this process , N₂ is first split up into free N₂ atoms by breaking the triple bond , with help of enzyme nitrogenase.

• This reaction is endergonic (energy consuming), it requires an input of nearly 160kcal energy.

• Free nitrogen combines with hydrogen forming NH3

• This reaction is exergonic (energy releasing)

• Mediated by enzyme hydrogenase & it releases nearly 13 kcal energy.

• BNF requires a net input of 147 kcal energy & an expenditure of nearly 16 mols of ATP per each molecule of nitrogen.

N₂ + 160Kcal 2N

2N + 3H₂ 2NH3 + 13 Kcal

NITROGENASE

Nitrogenase Complex

Two protein components: nitrogenasereductase and nitrogenase

• Nitrogenase reductase is a 60 kD homodimerwith a single 4Fe-4S cluster

• Very oxygen-sensitive • Binds Mg ATP • 4 ATP required per pair of electrons

transferred • Reduction of N2 to 2NH3 + H2 requires 4 pairs

of electrons, so 16 ATP are consumed per N2

Nitrogenase

A 220 kD heterotetramer

• Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide

(Fe8S 7-8clusters, etc)

• Four 4Fe-4S clusters plus two Fe, Mo, Co, an iron-molybdenum cofactor (fe 7 S9 Mohomocitrate)

• Nitrogenase is slow - 12 e- pairs per second, i.e., only three molecules of N2 per second

Pathway of nitrogen fixation in root nodules

• Glucose-6-phosphate acts as a electron donor

• Glucose-6-phosphate is converted to phosphogluconic acid

Glucose-6-phosphate + NADP+ + H2O 6-phosphogluconic acid + NADPH + H+

• NADPH donates electrons to ferrodoxin. Protons released and ferrodoxin is reduced

• Reduced ferrodoxin acts as electron carrier. Donate electron to Fe-protein to reduce it. Electrons released from ferrodoxin thus oxidized

• Reduced Fe-protein combines with ATP in the presence of Mg +2

• Second sub unit is activated and reduced

• It donates electrons to N2 to NH3

• Enzyme set free after complete reduction of N2 to NH3

Mo – N=NH

Mo=N-NH2

MoΞN+NH3

Mo + NH3

NΞN

Mo-NΞN

N2+16ATP+8H++8 e− → 2 NH3+H2+16ADP+16Pi

4 ATP

4 ATP

4 ATP

4 ATP

• Nitrogenase protection mechansims

• 1. Leghaemoglobin scavenges O2 to protect nitrogenase in legume rhizobium symbiosis

• 2. Confirmatory protection in Azotobacter as well as the higher respiratory rate.

• 3. Thick walls of Heterocyst protect O2 in BGA, since Nitrogenase are present in the heterocyst.

• 4. Microaerophilic nature in Azospirillum

Genes involved in Nitrogen fixation

• Genes involved in root nodule formation - called nodulin genes ( nod genes)

• Nodulin genes essential for infection of plant root and nodule formation by symbiotic N₂ fixing bacteria -divided into 2 classes

• 1) include genes that specify biochemical composition of bacterial cell surface.

• such as gene determining the synthesis of exopolysaccharides ( exo genes)

• Lipopolysaccharides (lps gene)• Capsular polysaccharides or K antigen & β 1,2 glucans

(ndu genes)

• exo & lps genes - play a role in determining host specificity

• 2) consist of nodulation genes (nod or nol)

• nod genes are involved in nodulation of particular host -called host specific nod(hsn)genes.

• Fast growing rhizobium sps - nod genes are located on large sym plasmids

• Slow growing bradyrhizobium sps – carry late nod gene on the bacterial chromosome.

• nif and fix genes - structural genes for nitrogenase enzyme

• Sym plasmid carry hup genes coding for hydrogenase activity

• Nif genes - 22 genes are involved , arranged in 7/8clusters

• Nif Q,B,A,L,F,M,Z,W,V,S,U,X,N,E,Y,T,K,D,H,J

REFERENCES

• Microbial ecology-fundamentals & application

- Atlas & Bartha. pp- 108-115

• Ramesh ,vijaya. ENVIRONMENTAL MICROBIOLOGY .pp -309-315,291-295

• Bhaskaran ,kk ENVIRONMENTAL BIOLOGY WILDLIFE CONSERVATION .3rd ed.(2013) . pp -76-78