SEMESTER IV

PAPER: B-403 (A)

(ELECTIVE)
MICROBIAL TECHNOLOGY

UNIT II
MICROBES AS BIOFERTILISERS AND BIOCONTROL AGENTS
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 Biofertilizers and their application:
Rhizobium, Azotobactor, Azospirilllum,
Cyanobacteria (BGA) and Azolla.
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Lecture delivered by: Dr. Mrutyunjay Jena

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• Imbalanced and indiscriminate use of chemical fertilisers
• Occurrence of multi-nutrients deficiency such as Zinc, boron,
sulphur etc. besides NPK
• Rain dependent agriculture - About 2/3 area
• Inadequate irrigation facilities
• Continuous fragmentation of land, unfavourable for adoption
of technology
• Land holding pattern and Predominance of marginal and
small farmers
• Causes Of Declining Crop Productivity
 Major Cause: Nutrient Deficiency In Soil
Photosynthesis: CO2 + H2O  carbohydrates (CHO) + O2

• Besides NPK, Sulphur, Zinc

And Calcium are also required
in good quantity.
• Other nutrients such as Iron,
Boron etc. though required in
small quantities, but their
deficiency significantly impacts
plant growth & life.
• Micronutrient deficiency (Zn,
Boron, Iron & Sulphur) in
Nutrients are taken up primarily by the roots in the Indian Soil is increasing
form of an aqueous solution in the soil
Innumerable experiments prove there is Significant increase in Yields by
application of secondary & micronutrients along with NPK nutrients
 The materials of biological origin commonly used to
maintain and improve soil fertility are grouped into two
main categories:

 Green manures
 Biofertilizers

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 Biofertilizers or Microbial fertilizers
or
Microbial Inoculants:
 The term biofertilizer refers to preparation containing live microorganisms, which
helps in enhancing the soil fertility either by fixing atmospheric nitrogen,
solubilization / mineralization of phosphorus and potassium or decomposing
organic wastes or by producing plant growth substances.

 Nutrient inputs of biological origin for enhanced of plant growth

 Formulations that contain living microorganisms and when applied to seed or

soil or plant surfaces colonizes the rhizosphere or interior of the plant and
promotes growth by increasing the availability of primary nutrients

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 Why Biofertilizer in Indian agriculture?
• Rice is a major crop in our country
• Cultivated in about 43mha and consume 40% of total
fertilizers
• Constitutes 42% of the total food grain production
• Primary means of livelihood for about 120-150 million homes
• More than 40% are small and marginal farmers who cannot
afford chemical fertilizers
• Alternate ecofreindly and cheap nutrient sources are BGA
and Azolla biofertilizers
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 Advantages Biofertilser
 Renewable source of nutrients
 Sustain soil health
 Supplement chemical fertilizers.
 Replace 25-30% chemical fertilizers
 Decompose plant residues, and stabilize C:N ratio of soil
 Improve texture, structure and water holding capacity of soil
 Improves soil health: Improve physico-chemical and biological properties of the soil e.g. increases water
holding capacity, soil aeration, soil aggregation and increasers soil microbial population.
 Nitrogen contribution: Increases nitrogen fixation and Nutrient Availability e.g. Contributes 20-30 kg
N.ha-1.season-1
 Increases grain yield by 10-40%.
 Stimulates plant growth by secreting growth hormones
 Eco-friendly: Non Polluting and Non-toxic Inland Production and No contamination of ground water, No
adverse effect on plant growth and soil fertility.
 Soil conditioning and pH buffering.
 Secrete fungistatic and antibiotic like substances
 Increases P-availability: Solubilize insoluble phosphorous
 Inhibits sulphide injury
 Decreases Iron toxicity
 Technologically feasible
 Low cost: Cheaper and economical, cost effective
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BIOFERTILIZER ORGANISMS

RHIZOBIUM

AZOTOBACTER

PSB

BLUE GREEN ALGAE

AZOSPIRILLUM

VA-MYCORRHIZA
 CLASSIFICATION OF BIOFERTILIZERS

BIOINOCULANTS

NITROGEN FIXERS NUTRIENT MOBILIZERS

P-Solubilizers (P. putida)
OM decomposing Fungi
(Trichoderma)
Root Colonizers (VAM) (Glomus)

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 NITROGEN FIXERS

AUTOTROPHIC HETEROTROPHIC

SYMBIOTIC ASYMBIOTIC (Free Living) SYMBIOTIC ASYMBIOTIC

A. azollae AEROBIC ANAEROBIC OBLIGATE FACULTATIVE AEROBIC ANAEROBIC

Bacillus sp. Clostridium
Azorhizobium Azospirullum
A. cycadae Ananbaena Rhodospirillum Rhizobium Klebsiella sp. Azotobacter Methanococcus
Phormidium Photorhizobium
A. anthocerotae Nostoc
Calothrix
Rhodopseudomonas
Scytonema Sinorhizobium

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 Biofertilizers are of three categories:
1. Nitrogen fixing biofertilizers:
• For legumes: Rhizobium
• For cereal: Azotobacter, Azospirillum, BGA, Azolla
2. Phosphate mobilizing biofertilizer:
• Phosphate solubilizers (PSB)
• Phosphate absorbers: Mycorrhiza
3. Cellulolyte or organic matter Decomposer:
• Cellulotytic: Cellulomonas, Trichoderma
• Lygnolytic: Arthrobacter
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 Properties of different Biofertilizers:

 Rhizobium can fix 50 to 200 kg nitrogen per hectare
per year. It increases yield by 25 to 30% and 40 to 80
kg nitrogen is leftover in the field for Subsequent
crop.

 BGA (Cyanobacteria) add up to 20 to 25 kg nitrogen Per
hectare to rice field.


 BGA, Azotobacter and Azospirillum also supply Growth
regulators such as IAA, IBA, GA & vitamins


 Azolla supplied nitrogen and also increases organic
matter in form of biomass and increases fertility
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Biofertilizer Mode of application Crop Benefit

Rhizobium Seed dressing Pulses (moong, groundnut: 50-70 KgN/ha

Lentil,Arhar,Pea,Gram) Yield:10-60%

Azotobacter Seed dressing Wheat, Maize, Barley

Vegetable: Tomato, Potato 15-20 Kg N/ha
Cash crop: Mustard, Cotton Yield:15-20%

Azospirillum Seed treatment Jwar,Bajra,Ragi, Barley 15-20 KgN/ha

and Fodder crops Yield: 10-15%

Cyanobacteria Broadcast after Rice 20-25 KgN/ha

transplantation Yield: 10-15% 14
 Advantages Cyanobacterial Biofertilser
(BENEFITS OF BGA/ CYANOBACTERIA INOCULATION)
 Rice field conditions are conducive for growth and metabolic activity of BGA
 Free living
 Nitrogen contribution: Increases nitrogen fixation and Nutrient Availability e.g.
Contributes 20-30 kg N.ha-1.season-1
 Increases grain yield
 Checks weed growth
 Addition of organic matter content.
 Releases growth promoting substances
 Improves soil health: Improve physico-chemical and biological properties of the soil
e.g. increases water holding capacity, soil aeration, soil aggregation and increasers
soil microbial population.

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 Soil conditioning and pH buffering.

 Increases P-availability: Solubilize insoluble phosphorous

 Inhibits sulphide injury

 Decreases Iron toxicity

 Technologically feasible

 Low cost: Cheaper and economical

Indirect benefits;
 Eco-friendly: Non Polluting and Non-toxic Inland Production and No contamination
of ground water,
 Residual Effect
 Renewable
 Multifaceted Action
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 Brief Introduction of Blue Green
Algae(BGA)/Cyanobacteria

 Cyanobacteria/Blue green algae constitute one of the largest groups

of Oxygenic photosynthetic prokaryotes

 Evolved 3.5 billion years ago (Proterozoic era)

 Wide range of distribution in many environments

 Some strains are capable of fixing atmospheric nitrogen and hence

can be exploited as biofertilizers

 Wide range of morphological variations observed

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 Heterocyst is the nitrogen fixing factory

Examples of heterocystous forms

Anabaena
Nostoc
Cylindrospermum

Anabena Nostoc
Cylindrospermum 18
 Non-heterocystous forms

 Phormidium
 Anacystis
 Scytonema

Phormidium Anacystis Scytonema

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 Cynobacteria/BGA fertilzer in Soil Fertility and Crop Production
 Diazotrophic cyanobacteria maintain and increase the fertility of rice fields. These algae as
bioinoculants have been found to reduce the nitrogenous consumption by about 30
percent , provide biologically fixed nitrogen in a linear fashion.
 Benefit soil health by favourably modifying the physical and chemical nature.
 The presence of heterocysts helps the algae like Anabaena, Aulosira, Calothrix,
Cylindrospermum, Hapalosiphon, Nostoc , Scytonema, Tolypothrix and Westiellopsis in
utilizing molecular nitrogen and fixing this up to ammonia under aerophillic conditions.
N2+3H2 2NH3
 These also may increase the utilization of fertlizer nitrogen by partially reducing the losses
through run-off, leaching and denitrification
 Substantial quantities of amino acids like alanin, aspartic acid, glutamic acid, vitamin B12
and auxin like substances are liberated by algae which can also be beneficial for crop plants
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 Contd.

 Mucilaginous envelope binds the soil particles together that increases

the size of soil aggregates. This reduces soil compaction, increases pore
size, aeration and water holding capacity
 Oxygen produced during algal photosynthesis reduces the oxidizable
matter in the soil, this converts the Fe++ to Fe+++, making it insoluble,
thus, reducing iron toxicity.
 Growth of microalgae has a buffering effect on soil pH bringing it to
almost neutrality from pH of 6.5-8.5.
 These have been accredited as tools for reducing the salinity in the soil
leading to better crop response.
 Some cyanobacteria are found to solubilize the insoluble phosphates in
the soil, which is supported by their profuse growth in presence of MRP.21
 Contd. •Based on their
ecology, BGA
 These can be effectively utilized as soil inoculants. biofertilizer was
There is a need to identify “Super strains” , developed especially
performing the desired functions, grow them on a for rice
large scale and make available quality inocula on
demand. •A composite culture
consisting of Nostoc,
Anabaena, Aulosira and
 Commercially viable method of producing clay Tolypothrix sp.
based l inoculum has been proposed.
 Growing the algae in separate raceways as unialgal
cultures and mixing the fresh biomass with equal
quantity of dried and finely powered clay has been
advocated.
 After mixing, the paste is sun dried, powered and
stored in polythene bags at ambient temperature.
 The method ensures high titre value and longer
shelf life. Such formulations perform additional
functions 22
 FLOWCHART OF
BGA BIOFERTILIZER PRODUCTION

Test Tube Liquid Flask Culture

Culture
Production in Indoor tanks
Quality Fresh biomass
Assessment
Mixing with carrier

Packaging and sealing Storage

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 METHODS OF PRODUCTION

 Trough method
 Pit / tank method
 Field scale production
 Nursery-cum-algal biofertilizer
production

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 Carrier materials
Availability of suitable carrier material is the biggest challenge
in biofertilizer production.
 Soil based
 Carrier based
 Paddy-Straw

Our product consists of four strains of

cyanobacteria as a consortium.
 Anabaena variabilis
 Nostoc muscorum
 Aulosira fertilissima
 Tolypothrix tenuis

 Selected on the basis on niitrogen fixing

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potential
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Colonisation of BOF Slag by Microalgae

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Commercial Production of BGA Biofertilizer

Perfection of a protocol for the large scale multiplication of algae has made
viable commercial production of the algal inoculum.
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(a) (b)

(c) (d)

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 SOME GENERAL FACTS ON AZOLLA

 Azolla is an aquatic heterosporous pteridophyte

 Cosmoploitan in distribution

 Grows well in pH range of 4-10 but best growth reported between 7 and 7.5

 Optimum temperature for growth is 23-30°C

 The size of the plant is 1-3 cm and doubling time is 2-3 days

 Somatic chromosome number (2n) is between 40-60

 Reproduces vegetatvely as well as sexually

 Able to fix atmospheric nitrogen (40-60 Kg N ha-1 crop)

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 The nitrogen fixing capacity is due to the symbiont Anabaena azollae
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 BENEFITS OF APPLICATION OF AZOLLA

 High nitrogen fixing ability

 High biomass production due to rapid growth

 Increases the availability of Zn, Fe and Mn

 Improves soil physico-chemical properties

 Improves fertilizer use efficiency

 Controls weed growth

 Release plant hormones and vitamins

 Checks loss of water 44

WHO IS DOING THE JOB OF NITROGEN
FIXATION IN AZOLLA?

THE SYMBIOTIC NITROGEN FIXING BLUE GREEN ALGAE

ANBAENA AZOLLAE

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ANBAENA AZOLLAE
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 NITROGEN CONTRIBUTION BY AZOLLA

 Green manuring (GM) provides : 20-40 kg N/ha

 Dual cropping once provides : 30 kg N/ha

 Dual cropping twice provides : 60 kg N/ha

 GM + dual cropping once provides : 50-60 kg N/ha

 GM + dual cropping twice provides : 80-90 kg N/ha

 Incorporated Azolla decomposes within 8-10 days and releases 67%

of its nitrogen within 35 days

 N from Azolla releases slowly as compared to chemical – N but faster

than N from BGA
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 MULTIFACETED USES OF AZOLLA

N2 FIXER
H2 GAS PRODUCER
SOIL CONDITIONER

WATER PURIFIER BIOREMEDIATOR

AZOLLA

HUMAN AND CATTLE FEED

K+SCAVENGER GREEN MANURE
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 MAINTENANCE OF AZOLLA

 Azolla can be maintained out door in:

 Nursery plots
 Ponds/ditches/canals
 Concrete tanks
 Polythene sheet lined pits or simple earthen ditches
 Outdoor maintenance is generally soil based
 It is inexpensive
 Azolla can be maintained indoor in:
 Trays/pots/dishes etc.
 Indoor maintenance is generally medium based
 It is expensive
 Some common growth media used for maintenance are:
 IRRI medium
 Espianase and Watanabe medium
 Hoagland medium (1/2 or 2/5 strength)
 Medium consists of both macro and macronutrients without combined nitrogen49
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MAJOR FACTORS INFLUENCING
SUCCESS

LIGHT
WATER
pH
TEMPERATURE
NUTRIENTS
INSECTS AND PESTS 56
Rhizobium, Azotobacter, Azospririllum and PSB

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 RHIZOBIUM INOCULANT
 Rhizobium is a gram negative (-ve) bacilliform bacterium.
 It grows in the Rhizosphire of the legumes.
 The fixed atmospheric nitrogen is then made available to the
legumes.
 Neither the legume nor the bacterium can fix nitrogen alone.
 Different species of Rhizobium shows specific host-range.
Ex: R. trifoli infects-Clover
R. phaseoli infects-Bean
R. leguminosarum infects-Pea
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 Preparation of Rhizobium Inoculant:-
 Selection of suitable bacterial strain by selective healthy plants.
 Isolation of Rhizobium from nodules.
 Identification of the strain.
 Preparation of inoculums culture.
 Large scale production.

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 Isolation of Rhizobium

Procedure:
 Uproot healthy leguminous plants.
 Wash the root system tap water.
 Wash with 0.1% acidified H2Cl2 for 4-5 minutes.
 Wash with sterile water.
 Dipped with 70% ethanol and again wash with water.
 The nodules are then washed in a small volume of sterile water.
 The washed liquid taking the Rhizobia cells. It is then spread on the surface of
YEMA culture plates.
 Culture plates are incubated at 260C-280C for 10 days (4-5 days large colonies
will appear also small colonies appear).
 Round, colourless or white with central colour develops red rot and entire
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margin.
 Mass Culture
1. By Fermentation – starter culture
o YEMA Medium.
o 28oC-30oC
o 108 to 109 cells/ml.
o not stored more than 24 hours.
2. Carriers of Rhizobium Inoculant.
o Charcoal
o Farm yield manure (FYM)
o Paddy husk
o Coconut cell powder
o Coffee husk
3. Processing
o Drying: the carrier material is sun dried up to a moisture of 5%
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o Grinding:-Powder form 100-200 mesh
 Application of Rhizobium inoculants
o Carrier based cultures are suspended in 10% sugar or solution in water.
o Or sometimes 10% solution of gum-arabic is used as sticker.
o Seeds are then added to the slurry and thoroughly mixed for uniform seed
coating.
o Spread the seeds and dried in shade and are sown immediately.
Pelleting:-
o Acidic or acid soil. Use seeds as pelleted.
o 40% gum anaemic or 5% carboxy methyl cellulose is added to the slurry
before it is applied to the seeds.
o CaCO3 powder can pass through 300 mesh sieve is added to wet inoculated
seeds and mixed thoroughly for two minute until the seeds are evenly coated..
o The lime coated seeds looks like white tablets then hard and for an hour by
spreading on a cleaning surface. Then sown immediately.
o Other pelleting agents used are gypsum, rock phosphate, super phosphate,
and charcoal.

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 Quality control
o type of carrier material
o Expiry date and temperature
o pH
o Method of use
o Packet inforamtion
o Packet should be ISO mark and number

Crop Response
Rhizobium Seed dressing Pulses (moong, groundnut: 50-70 KgN/ha
Lentil, Arhar, Pea,Gram) Yield:10-60%

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 AZOTOBACTER INOCULANT

o Azotobacter is an non-symbiotic nitrogen fixing bacterium. It is a soil

borne heterotrophic bacterium.
o It is grows in the rhizosphere of plants. Beijerinck first isoated and
described two species Azotobacter chrococcum (soil borne) and A. agalis
(water borne)
o The bacterium morphologicaly pleomorphic ranging from rod shape to
coccoids cells.
o Gram – negative, motile by peritrichous flagella or some time flagella not
found.
o pH optimum of growth and nitrogen fixation: 7 -7.5
o Never produce endopsores but forming cysts.

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 ISOLATION

o Azotobacter can be isolated from the soil by culturing soil sample on a nitrogen
free agar medium.
o Plates are incubated at 300C for 3 to 4 days.
o Azotobacter colonies appear as flat, soft, milky and mucoid on the agar plates.

MANUFACTURE

 The Jensen’s liquid culture medium is used for large scale preparation of
Azotobactor inoculants.
For mass production
 Rotary shockers
 Bioreactors
 large flask culture

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 Application: procedure for use
 For transplanted crops application of the inoculants is done by dropping the
seedlings in the slurry of the carrier based Azotobacter for 10 to 30 minutes
and planted immediately.
 For sugarcane, many packets of which are recommended at regular intervals
in the early stage of crop growth.
 The second and subsequent inoculations are done by pouring the slurry near
the root zone.
 The inoculant is also mixed with farm land manure and broad cost near the
root zone.

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 Role and benefit
 Fixed atmospheric nitrogen and increase soil fertility.

 Produce biologically active substances some of them are vitamin-B,

Nicotinic acid and panthothenic acid, biotin, heteroauxin and gibberllins.

 Such compounds increase germination and vigour of young plants

 Also produces antibiotics and fungistatic compounds against soil borne

pathogens like Fusarium, Altermaria and Trichodema

CROP RESPONSE
Crop % increasing
Onion ...................................22
Rice ......................................23
Binjal ...................................42
Cabbage................................50
Wheat...................................30
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Maize....................................71
 Azospirillum
 Non-symbiotic nitrogen fixer
 Beijernick in 1925, isolated a nitrogen fixing bacterium named it
Spirillium lipoferum. Later , in 1978 , J.J. Tarrand et al renamed this as
Azospirillum lipoferum.
 Garm negetaive bacteria. Grow well aerobically also in presence of
combined nitrogen such as ammonium salt.
 It is lives in rizosphere soils and occurs in association outside the roots
also insides the roots of c3 and c4 plants.
 Most isolates have been found from cereal plants.
 They more prominent in tropical soils than the temperate soils.
 They never form nodules like symboitic bacteria .
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 Isolation
 It is widely distributed in tropical soils and is usually found in association with
graminaceous roots, the native isolates from rhizospheric soils or from root cortex.
 Prepare NfB semi-solid media in 15 ml cotton serum vials
 Collect rhizopshere soil from the roots of cereal or grasses and suspend 10 g soil in 90
ml of sterile water..
 do serial dilution for 10 times
 Finally add 1 ml of final serial dilution tube to the medium and incubate for 40 hrs at
350 C.

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 Mass culture
For mass production
 Rotary shockers
 Bioreactors
 large flask culture

Carrier Materials

 Same as used for Rhizobiium and for Azotobacter.

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 Application: procedure for use

Application of Azospirillum in paddy fields improve nitrogen efficiency is common now a

days.
Application of Azospirillum is done in three steps.
1. Seed dressing
2. during seedling transplantation
3. as spray on the leaves on the thirtieth day.
Five packets of (1000 g) Azospirillum is applied at the time sowing as seed dressing
Ten packet s are mixed with 2 kilos soil and 2 kilos dried manure and broadcast in the
entire nursery and a day before seedlings are transplanted.
On 30th days 10 packets mixed with 400 litres of water . This permits the bacteria to fix
atmospheric nitrogen on the phylospere also. 71
 Role and benefit

 Treatment or use of this fertilisers reduces 25% of chmeical fertilisers to get

the same or more yield of paddy.
 Fixes atmospheric nitrogen to the soil . It can fic 15 to 25 kg of atmospheric
nitrogen per hectare.
 Promotes crop growth by producing ecenssial growth hormones gibberlic
acui (GA) and indole acetic acid (IAA)
CROP RESPONSE
 In sugarcane application could reduce the nitrogen chemical fertilsers
by about 25 %
 Increase Sucrose yield
 Seed inoculation of rice, barley and maize , wheat increased both
straw yield and grain yield
 Maintain the nitrogen and phosphorous ratio and increased mineral
nutriet uptake ability
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 Effectively controlled the pest of sorghum shootfly by increasing total
Phospahte –Solubilising Microbes (PSB)
 Phosphorous is an important nutrient for plants and microbes.
 Most of the soil phosporous in in no-available form
 Several microbes have the ability to solubilse phosporous.
 They can convert insoluble inorganic phosphate into simple and soluble
forms.
 Soil borne bacteria of the Genera Pseudomonas, Bacillus and many fungi
have capacity to solubilise chemically –fixed soil phosphrous and rock
Bacillus megatherium
phosphate. Aspergillus niger
B. Subtilis A. flavus
B. Mycoides A. Fumigatus
B. Fluorescence Penicelium digitatum
Pseudomonas putida Fusarium oxysporum
P. liquifacians Candida sp.
P. calcis Pythium sp.
Xanthomonas sp.
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Aerobactor aerogenes
Bacteria that can solubilse phosphate are colletively called phosphobacteria.
These microbes can also mineralise organic phosphorous to solubile forms by enzymatic
activity.

Isolation
 1 g soil from rhizospere soil suspended sterile wter and go for serial
dilution.

 Serial dilutions of the suspensions and plate on phosphate containing

media.
 Incubate for 4 to 5 days.
 Bacterial colonies will be appera on theplate.
 Clear zone around the colonies indicate phosphate solubilisation.
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 Mass culture
For mass production
 Pikovskaya liquid medium
 Rotary shockers
 Bioreactors
 large flask culture
Carrier Materials

 Same as used for sterilized peat soil and lignite)

 The mixture is cured for a week in large trays at 280 C
 Packed in plastic bags and stored at 15 to 200 C until use

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 Role and benefit

 Treatment or use of this fertilisers r25-30 phosphate upta ke capacity

 Promotes crop growth by producing important essential growth hormones
 Also PSB increased the crop productivity capacity about 20- 30%.

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 AGRICULTURE

Important input for better crop nutrient management in soil

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