Dr. P.V. Madhusoodanan

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Welcome
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Biotechnology of Biological Nitrogen Fixation
Dr. P.V. Madhusoodanan Prof. Head Department of
Botany University of Calicut Email
pvmadhu_at_gmail.com web http// pvmadhu.tripod.com
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BIOFERTILIZERS

• BIOFERTILIZERS
• FOR SUSTAINABLE AGRICULTURE
• AND CLEAN ENVIRONMENT

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Biofertilizers
Fertilizers with life
Microorganisms (inoculam) applied to improve the
fertility of the soil
N P K
Nitrogen and Phosphorous Why N2 is scarce in the
soil?
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Norman E. Borlaug E. Borlaug

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Bad type of storing ! The Hindu 6th Sep 2010
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Relevance of Biofertilizers

• HYV are fertilizer responsive- Green revolution
  - Grow more food
• Susceptible to pests and pathogens
• Demand high amount of chemical fertilizers and
  pesticides
• Agroindustries are the major contributors of
  pollution in the
• world
• Farmers apply excess amount of chemicals
• Chemical fertilizers provide one nutrient but
  wipes out essential
• elements
• They alter the pH of the soil
• Affect the soil dynamics by the
  soil/microorganisms- kill them-
• earthworms
• Deteriorate agroecoystem-Chemophobia
• Biofertilizers can substitute to a great extent
  the chemical fertilizers
• Organic farming- Sustainable development
• One straw revolution- Masanobu Fukuoka

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BIOLOGICAL NITROGEN FIXATION

• N?N
• Diazo
• 79 inert vital element
• Ionization energy 336 kcal/mol
• Haber-Bosch Process, 8000C, high pressure (300
  Atm.) N2 NH3
• 1.5 kg fuel needed for 1 kg N fertilizer to
  farm.
• BNF only 1/150
• N2 deficiency no nitroplasts 200 mt/y by
  few microorganisms
• Nitrogenase enzyme BNF nif genes
• Example- Potential of enzyme catalyzed reaction

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Biogeochemical Cycle
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Nitrogen Cycle
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Nitrogen cycle
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• Drews (1928) Cyanobacteria fix N2
• De (1939) Cyanobacteria fix N2 in paddy fields-
  Indian
• Beijerlink (1888) isolated Rhizobium
• Reduction of Gaseous N2 to NH3
• N2 8H 8e- 16ATP16H2O 2NH3 H2 16 ADP
  16 Pi

• Terminus Technicus chemically impossible
• Eukaryotic cells completely lack this ability
• The intermediate steps of N2 fixation still
  unknown

N ?N HNNH H2N NH2
2NH3 Diimine Hydrazine
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The total cost of N2 reduction is 8 electron
transferred and 16 ATP hydrolyzed
(physiologically 20-30 ATPs)

• Nitrogenase also reduces H2 O H2
  

HNNH H2 N2 2H2
This happens when ATP level is low- Futile cycle
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• Nitrogenase
• 2 iron - S proteins
• Individually inactive
• 1. Mo-Fe protein 36 Fe and 36S 2 Mo atoms
• 220,000 MW component I/ Nitrogen reductase
• 2. Fe protein - 4Fe 4S cluster/ 60,000 MW
• Component II/ Nitrogenase reductase
• Both O2 sensitive
• Fe protein more sensitive
• Anaerobic envt. essential

First isolated from Clostridium pasteurianum
(1960) CRL of Dupond de Numeurs (USA)
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Chemistry of Nitrogen fixation
Nitrogenase Glutamine synthatase Glutamate
synthase (GOGAT Glutamate oxogluterate
aminotranferase)
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Genetics of N2 Fixation
Klebsiella pneumoniae facultative anaerobe- Fix
N2 only under anaerobic condition- related to
E.coli Easy to culture Nif genes are studied
only in K. pneumoniae
Genetic map
Nif operons appx gene location
Nitrogenase DG Q B A L
F M V S U X N E Y K D H J 0
8 12 16
20 28 kb
NR
Synthesis of Fe-Mo cofactor
Synthesis polypeptide for electrone transport NR
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The genes for histidine (DG) and nif genes are
linked 15 nif genes have been identified 2
unknown function x,y
Gene Coding H - Nitrogenase reductase
FJ - Synth. polypeptide DK -
Nitrogenase MSV - maturation of complete BQVNE
- Fe Mo Co factor functional nitrogenase XY
- Unknown AL - Regulate expression
of nif genes
Nif genes of other organisms are similar but
usually the genes are scattered- not clustered as
in K. When a small portion of chromosome of K.
pneumoniae was transferred to E.coli, the latter
started N2 fixation
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Major Biofertilizers
Important microorganisms as Biofertilizers
Microorganisms Nutrient fixed Actinorrhizae
(Frankia sp) 150 kg N/ha/Y Azospirillum 10-2
0 kg Azotobacter gt 20 kg Rhizobium 50-100
kg Azolla (Anabaena) 120-150 kg (gt
900kg) Cyanobacteria gt 25 kg Mycorrhizae
(VAM) Solubilize P
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N2- Fixing Organisms
Free Living N2- Fixers

• Obligate Aerobes
• Azotobacter
• Beijerenkia
• Azotcoccus
• 2. Obligate aerobes that fix N2 at low O2
• Azospirillum
• Thiobacillus
• Rhizobium
• 3. Facultative anaerobes- fix N2 under O2 free
  conditions
• Klebsiella pneumoniae
• Bacillus polymax
• Escherichia intermedia

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4. Obligate anaerobes Clostridium Desulfovibrio 5.
Phototropic bacteria Rhodospirillum Chromatium Ch
lorobium 6. BGA/ Cyanobacteria a. Unicellular
aerobic Gloethece Aphanothece b. Filamentous
heterocystous - aerobic/ anaerobic Nostoc,
Anabaena, Cylindrospermum c. Filamentous
non-heterocystous Oscillatria, Lyngbya,
Plectonema
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Symbiotic N2- Fixers
Rhizobium- Leguminosae (12,000 spp.) Ulmaceae
Parasponia
Non-Rhizobium N2 Fixers Frankia
(Actinorrhizae) Alnus (Betulaceae) Elaegnus
(Elaegnaceae) Coenothus (Rhamnaceae) Dryas
(Rosaceae) Coriaria (Coriariaceae) Casuarina
(Casuarinaceae) Lichens Collema-
Nostoc Dendriscocaulon- Scytonema Water
fern Azolla- Anabaena
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Cycads Cycas- Nostoc, Anabaena Higher
Plants Haloragaceae- Gunnera- Nostoc Associative
Symbiosis and casual Assn. Phyllophore-
Azotobacter sp. Roots of grasses- Azotobacter
Azospirillum
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Frankia nodules and Frankia cells
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Azospirillum
Azotobacter
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Root nodules Rhizobium
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Steps in the formation of root nodule infected by
Rhizobium
Root nodule bacteria and symbiosis with legumes
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Stem nodule Sesbania rostrata
Section of root nodule Coronilla varia
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Azolla
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Cyanobacteria
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Scytonema guyanense
Scytonema simplex
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Westiellopsis prolifica
Calothrix marchica
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Dichothrix sp.
Stigonema dendroideum
Calothrix brevissima
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Azolla rubra
Anabaena azollae
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Azolla filiculoides
Azolla pinnata
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VAMF
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Ectomycorrhizae
Endomycorrhizae
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Effect of biofertilizers on Casuarina
equisetifolia saplings 1. Control (sterilized
soil) 2. Plant inoculated with Rhizobium sp. 3.
Plant inoculated with Azotobacter sp. 4. Plant
inoculated with Azospirillum sp. 5. Plant
inoculated with Phosphobacteria 6. Plant
inoculated with microbial consortium
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Effect of biofertilizers on Casuarina
equisetifolia saplings 1. Control (sterilized
soil) 2. Plant grown in unsterilized soil 3.
Plant inoculated with microbial consortium and
farm yard manure (FYM)
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Advantages of Azolla application

• Contribute up to 40 N2
• Accommodate P and other nutrients and releases
  slowly
• Can be applied as a dual crop in paddy fields
• Suppresses weeds
• Use as mulch, compost, fish feed, cattle feed,
  etc.
• Edible as vegetable- Azolla thoran, Azolla vada,
  etc.- rich in protein
• Helps in detoxify Heavy metals and many other
  pollutants
• It also reduces nematode population
• Aquatic Fern
• Maintenance during summer

Disadvantages
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Economic Importance of Cyanobacteria

• N2 fixers- biofertilizers

• Chinese use Nostoc commune as a delicacy

• Japanese use Nostoc commune, N. verrucosum,
  Aphanothece sacrum and Brachytrichia quoi as
  side dishes

• 1521- Barnal Diaz accompanied Cortez recorded
• Mexico city (now) ooze from Lakes
  having a flavour like cheese Spirulina
  platensis

• Chad (Africa) people collect Spirulina cakes
  from lakes and consume.

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• Anabaena cylindrica- one of the richest known
  sources of Vit. B12

• Amino acid production- Oscillatoria,
  Westiellopsis

• H2 (fuel) production- Phormidium, Calothrix

• Antibiotics production- Phormidium, Dichothrix,
  Lyngbya

• Phormidium valderianum shows anti-hepatitis-B

• Single cell protein- Spirulina

• Cosmetics and beauty products- anti wrinkle
  creams, skin
• ointments, pimple lotion, biolipstics, etc.

• Effluent treatment- bioflocculation- Phormidium,
  Oscillatoria

• Aquaculture- fish feed supplement

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Spirulina (CFTRI)

• L.V.Venkataraman
• Tablets and powder (Ambadi Gr., Madras)
• Cattle and fish feed supplement
• ß-carotene as food colour and pro- vitamin-A
• Phycocyanin-water soluble pigment (Lina Blue)
• used in eye shadow, eye-liner biolipstic
  (Sigam Algae Co., Bangkok)

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Spirulina major constituents
Proteins 65.71 Fat 6.7 ß Carotene 320, 000
u/100g Calcium 658 mg P 977 mg Fe 47.7
mg Sodium 796 mg Potassium 1.14
mg Lysine 2.99 Cystein 0.47 Methionine 1.38
Phenyl alanine 2.87 Theonine 3.04
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Spirulina capsules
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Spirulina capsules
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Negative

• Poisonous - Microcystis spp. give a bad smell on
  decaying

• Decrease fish and other aquatic fauna

• When blooms, biofouling

Biological softwares - M.S. Swaminathan
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Cyanobacterial biotechnology
Application of scientific and engineering
principles to the processing of materials by
biological agents to produce goods and services
Mass production- Biofertilizers- flakes Azolla-
rural technology- TNAU
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Biolistics
Electroporation
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Isolation and Pure Culture
Dilution method Streak method Culture media
Sterilization - inoculation Agar plates- Agar
slope- mass culture
Composition of culture media for cyanobacteria
Chu-10 pH 6.5- 7.0 (Chu, 1942) Ca (NO3)2 0.04
g/l K2HPO4 0.01 MgSO4 7 H2O 0.025 Na2
CO3 0.02 Na2 SiO3 0.025 FeCl3 0.8 mg/l
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Nitrogen free medium (Fogg, 1949)
KH2PO4 0.2 MgSO4 7H2O 0.2 CaCl2
2H2O 0.1 Fe EDTA 1.0 ml A5 (Trace elements)
sol. 1.0 ml
Constituents of A5 solution (Arnen, 1938)
MnCl2 4 H2O 1.81 MoO3 0.0077 ZnSO4
4H2O 0.222 CuSO4 5H2O 0.079 H3BO3 2.86
Lab Manual. IARI, 1987
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BG-11 medium (/100ml) ((Rippka et al., 1979)
NaNO3 6 g KH2 PO4.3H2O 400 mg MgSO4.7H2O 750
mg CaCl2.2H2O 360 mg Citric acid 600 ml EDTA
(Na2Mg salt) 100 mg Na2NO3 200 mg Glass
distilled water 1 lit. Trace metal solution 1 ml
For preparing N2 free medium NaNO3 may be omitted
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Contd.
composition of trace metal solution A5 C
(after Rippka et al., 1979)
H3 BO3 2.86 g/l MnCl2.4H2O 1.81
g/l ZnSO4.7H2O 0.222 g/l Na2MoO4.2H2O 0.390
g/l CuSO4.5H2O 0.079 g/l Co(NO3)2.6H2O 0.0494
g/l
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Growth characteristics of certain cyanobacterial
isolates in BG-11 medium
A. Anabaena sp. B. Aulosira sp. C.
Cylindrospermum majus D. Nostoc linckia E.
Scytonematopsis sp. F. Aphanothece stagnina G.
Anabaena sp. H. Microcystis aeruginosa I.
Calothrix marchica J. Westiellopsis prolifica
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CULTURE

• Rhizobium Yeast Extract Mannitol Agar Medium
  (YEMA) Vincent, 1970
• Azotobacter Modified Burks Medium (Rennie,
  1981)
• Phosphobacteria Sundara Rao Sinha, 1963
• VAM spore isolation from soil Gerdemann
  Nicolson, 1963
• Azotobacter Azospirillum are commercially
  available.

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Protoplast fusion (Nottingham Univ., Dr. Davey)
PEG Transfer of Cyanobacteria as symbionts
Genetic Engineering
Transfer of nif genes synthesis of nitroplasts?
Ti Plasmid mediated gene transfer
Electroporation
Microinjuction
Microprojectile bombardment (biolistics)
Azolla dual cropping- Development of rural
technology
Lab. Field problems
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Protoplast fusion
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Genetically engineered Nicotiana tabacum
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Chemical fertilizers are 1. Expensive 2.
Eliminate other nutrients 3. Kill
microorganisms 4. Alter soil pH and dynamics 5.
Deteriorate agroecosystem 6. Cause Health Hazards
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Chloroplast / Bacterial uptake
Kawaguti Yamasu (1965) discovered chloroplasts
in marine gastropod Elysia atroviridis- feeds on
Codium fragile functional? Taylor (1968) in
Elysia viridis Codium tomentosum Trench (1969)
gastropod Tridachia crispata Functional
chloroplast 2-3 µm double membrane Thylakoids-
in tubules of digestive diverticulam
Giles Sarafis (1972) Caulerpa sedoides
- Chloroplasts grown in chicken egg white-
division of chloroplasts occurred.
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Plant cells Bonnet Erickson (1974) PEG
transferred chloroplasts of Vaucheria dichotoma
into protoplasts of Daucus carota not functional
Bacteria into chloroplasts Rhizobium Klebsiella
nif genes into tomato M.R. Davey- Nottingham
Univ. Cyanobacteria Endosymbiotic
cyanobacterium- Cyanelle occurs in biflagellate
protozoa Cyanophora paradoxa and acts as
chloroplasts
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Biofertilizer/ BGA Centers in India
IARI, New Delhi TNAU- Coimbatore, TN MKU-
Madurai, TN BARD- Thiruchirappally, TN CRRI-
Cuttack, Orissa CAS- Chennai, TN Calicut
University- Kerala
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t h a n k s
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