CHAPTER 01

1.0 INTRODUCTION

1.1 IMPORTANCE OF NITROGEN IN RICE CULTIVATION

Plants are surrounded by the nitrogen (N) in our atmosphere. Because atmospheric

gaseous nitrogen is present as almost inert nitrogen (N2) molecules, this nitrogen is not

directly available to the plants that need it to grow, develop and reproduce. Nitrogen

deficiency is probably the most common nutritional problem affecting plants worldwide.

Healthy plants often contain 3-4% nitrogen their aboveground tissues. Nitrogen

is an important component of many important structural, genetic and metabolic

compounds in plant cells. It is a major component of chlorophyll, the compound by

which plants use sunlight energy to produce sugars from water and carbon dioxide (i.e.

photosynthesis). It is also a major component of amino acids, the building blocks of

proteins. Some proteins act as structural units in plant cells while others act as enzymes,

making possible many of the biochemical reactions on which life is based. Nitrogen is a

component of energy-transfer compounds,

Such as ATP which allow cells to conserve and use the energy released in

metabolism. Finally, nitrogen is a significant component of nucleic acids such as DNA,

the genetic material that allows cells (and eventually whole plants) to grow and

reproduce.

1.2 BIO-FERTILIZER USE IN AGRICULTURE

“Bio-fertilizer” is a substance which contains living microorganisms which, when

applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the

1

plant and promotes growth by increasing the supply or availability of primary nutrients

to the host plant.

Bio-fertilizers add nutrients through the natural processes of Nitrogen fixation ,

solubilizing phosphorus, and stimulating plant growth through the synthesis of growth

promoting substances.

Bio-fertilizers like Rhizobium, Azotobacter, Azospirillum and blue green algae

(BGA) are in use since long time ago. Rhizobium inoculants are used for leguminous

crops. Azotobacter can be used with crops like wheat, maize, mustard, cotton, potato and

other vegetable crops.

Azospirillum inoculants are recommended mainly for sorghum, millets, maize,

sugarcane and wheat. Blue green algae belonging to genera Nostoc, Anabaena, fix

atmospheric nitrogen and are used as inoculants for paddy crop grown both under upland

and low land conditions.

Biologically active products or microbial inoculants of bacteria, algae and fungi

either separately or in combination, which may enhance the availability of nutrients by

nitrogen fixation and by solubilizing soil phosphorus for the benefit of plants are called

bio-fertilizers.

1.2.1 CLASSIFICATION OF BIO-FERTILIZERS

Bio-fertilizers may be broadly classified into three groups;

Nitrogenous bio-fertilizers

Phosphatic bio-fertilizers.

Organic matter decomposers

2

1.3 BENEFITS AND ADVANTAGES OF USE OF BIO-FERTILIZER

Bio-fertilizer it is living thing, it can symbiotically associate with plant root. Involved

microorganisms could readily and safely convert complex organic material in simple

compound, so that plant easily taken up. Microorganism function is in long duration

causing improvement of the soil fertility. It increases crop yield by 20-30%. Finally it

can provide protection against drought and some soil borne diseases. Bio-fertilizer is

environmentally friendly fertilizer that not only prevents damaging the natural source but

helps to some extend clean the nature from precipitated chemical fertilizer.

1.4 OBJECTIVES

Find out the efficacy of Azotobacter nitrogen fixation in Rice rhizosphere

Evaluation of potential use of Azotobacter microbe as Bio-fertilizer in Rice cultivation.

3

CHAPTER 02

LITERATURE REVIEW

2.1 BIOLOGYCAL NITROGEN FIXATION IN RHIZOSPHERE

Biological Nitrogen Fixation (BNF) is known to occur to a varying degree in many

different environments, including soils; fresh and salt waters and sediments; on or within

the roots, stems, and leaves of certain higher plants; and within the digestive tracts of

some animals. The potential for nitrogen fixation exists for any environment capable of

supporting growth of microorganisms.

Atmospheric nitrogen (N) is a molecule composed of two atoms of nitrogen

linked by a very strong triple bond. Large amounts of energy are required to break this

bond and the molecule is therefore quite chemically non reactive.

The general chemical reaction for the fixation of nitrogen (N + 3H2 + Energy =

2NH3) is identical for both the chemical and the biological processes. The triple bond of

N must be broken and three atoms of hydrogen must be added to each of the nitrogen

atoms.

Living organisms use energy derived from the oxidation ("burning") of

carbohydrates to reduce molecular nitrogen (N2) to ammonia (NH3). The chemical

process of nitrogen fixation involves "Burning" of fossil fuels to obtain the electrons,

hydrogen atoms and energy needed to reduce molecular nitrogen. (Hubbell and Kidder.

2003)

2.1.1 SYMBIOTIC NITROGEN FIXATION

The most important contribution to Biological Nitrogen Fixation comes from the

symbiotic association of certain micro-organisms with the roots of higher plants. A

4

classic example is that of the bacteria (Rhizobium) which characteristically infect the

roots of leguminous plants (e.g., bean, soybean, clover, and peanut) with a high degree of

host specificity.

Small nodules are formed on the roots and these become filled with an altered

form of the bacteria which fix appreciable amounts of nitrogen. This symbiosis alone

accounts for 20% of global biological nitrogen fixed annually. The legumes represent a

major direct source of food for man and forage for livestock and therefore represent a

critical contribution to world food production.

2.1.2 NON - SYMBIOTIC NITROGEN FIXATION

There is great diversity in the metabolic types of free-living microorganisms which are

capable of biological nitrogen fixation. This includes about 20 genera of non-

photosynthetic aerobic (Azotobacter, Beijerinckia) and anaerobic (Clostridium) bacteria

and about 15 genera of photosynthetic cyanobacteria (blue-green algae) such as

Anabaena and Nostoc.

The significant contribution of photosynthetic (cyanobacteria) and non-

photosynthetic (Azotobacter, Clostridium) microorganisms to nitrogen fixation in the

rhizosphere of rice is well recognized.

2.1.3 ESTIMATION OF BIOLOGICAL NITROGEN FIXATION

There are common techniques can be used for estimating biological nitrogen fixation in

soil.

Acetylene (C2H2) reduction – Nitrogenase enzyme is the major enzyme in

biological nitrogen fixation which is responsible for the nitrogen fixation can

reduce acetylene (C2H2) to ethylene (C2H4) as well as N2 to NH3. Acetylene

5

and ethylene can be easily and rapidly analyzed by gas chromatography and

hence amount of n-fixed can be estimated. Number of strains can be

evaluated by this method (Singh et al., 1991).

15N gas incorporation – Nitrogen gas that is labeled with the stable isotope

15N is placed in the atmosphere of a closed 15N chamber in which the test

plants are growing. The amount of fixed nitrogen is calculated by the amount

of 15N incorporated in to the plants.

15N isotope dilution – The principle of this method is that an N2 fixing plant

growing in a medium enriched by 15N in the plant contains 15N in the plant

than does a non fixing plant growing in to the medium.

Nitrogen differences method – This classical method has generally been

used by agronomist in the field where the N2 fixing plants and non- fixing

plants are simultaneously grown.

2.2 POACEAE ASSOCIATIVE MAJOR BIOFERTILIZE MICROORGANISMS

2.2.1 Azospirillum

This is a free living or non -symbiotic bacteria (does not form nodules but makes

association by living in the rhizosphere). Azospirillum species establish an association

with many plants particularly with C4, plants such as maize, sorghum, sugarcane, etc. It

is the most common organism and can form associative symbiosis on a large variety of

plants.

2.2.2 Azotobacter

Azotobacter is a heterotrophic free living nitrogen fixing bacteria present in alkaline and

neutral soils. Apart from its ability to fix the nitrogen in soils, it can also synthesize

6

growth promoting substances which auxins, and gibberellins and also to some extent the

vitamins.

2.2.3 Acetobacter

Acetobacter diazotrophicus is a newly discovered nitrogen fixing bacteria associated

with sugarcane crop. This bacterium belongs to the alpha group of proteobacteria. It was

isolated from leaf, root, bud and stem samples of sugarcane.

2.3 Azotobacter GRASS ASSOCIATIVE BACTERIA

2.3.1 Family Azotobacteraceae

The family Azotobacteraceae contains aerobic diazotrophs with four Genera,

Azotobacter Azomonas, Beijerinckia and Derxia. The taxonomically key characters are

cell form, motility, cyst formation, G+C ratio, the formation of characteristic lipid bodies

and catalyze reaction. Members of this family are given economic importance by their

ability to fix molecular nitrogen thus to contribute to the nitrogen status of soil

(Mortimer et.al., 1981).

2.3.2 Genus: Azotobacter

Azotobacter is found on neutral to alkaline soils, in aquatic environments, in the plant

rhizosphere and philosopher. A.chroococcum is the most common species of Azotobacter

present in the soil. Most of the studies on Azotobacter have been to compare its role as a

nitrogen fixer to that of C. pasteurianum and Rhizobium. There is also interest in

Azotobacter because of it has the highest metabolic rate of any living organism and

because of its cyst formation.

7

Azotobacter species are Gram-negative, aerobic soil-dwelling bacteria. There are around

four species in the genus, some of which are motile by means of peritrichous flagella,

others are not. They are typically polymorphic, i.e. of different sizes and shapes. Their

size of the cells ranges from 2-10 µm long and 1-2 µm wide.

2.3.3 Scientific classification of Azotobacter

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Pseudomonadales

Family: Azotobacteraceae

Genus: Azotobacter

Species: Azotobacter chroococum Azotobacter vinelandii Azotobacter agilis Azotobacter paspali

2.3.4 Isolation and identification of Azotobacter

There is different culture mediums can be used for the isolation of Azotobacter which

is Ashby’s medium, Jensen’s medium, Waksman’s medium and Burk’s medium.

(Subba Rao et al., 1993).

2.3.5 Soil Dilution Plating Method

A 10g soil sample is mixed with 100ml of sterile distilled water

thoroughly.

Serial dilutions of the suspension are made using sterile distilled water.

A suitable nitrogen free agar medium specific for Azotobacter is prepared

and poured in to sterile petriplates and cooled.

The plates are incubated at 30oC for 3-4 days, after which soft milky,

mucoid colonies of Azotobacter can be seen. The young cells are not

pigmented while the older cells are pigmented. The pigment of

Azotobacter chroococum is black to brown.

8

2.3.6. Direct method:

Soil is directly spread on nitrogen free agar medium and the plates incubated at

28oC for 3 days after which the colonies develop (Subba Rao et al., 1993).

2.4 RICE CULTIVATION

Rice is the seed of the monocot plant Oryza sativa. As a cereal grain, it is the most

important staple food for a large part of the world's human population, especially in East,

South, Southeast Asia, the Middle East, Latin America, and the West Indies. It is the

grain with the second highest worldwide production, after maize.

Rice cultivation is considered to have begun simultaneously in many countries

over 6500 years ago. Two species of rice were domesticated, Asian rice (Oryza sativa)

and African rice (Oryza glaberrima).

Rice is normally grown as an annual plant, although in tropical areas it can

survive as a perennial and can produce a ratoon crop for up to 30 years. The rice plant

can grow to 1–1.8 m tall, occasionally more depending on the variety and soil fertility.

The grass has long, slender leaves 50–100 cm long and 2–2.5 cm broad. The small wind-

pollinated flowers are produced in a branched arching to pendulous inflorescence 30–

50 cm long. The edible seed is a grain 5–12 mm long and 2–3 mm thick.

World production of rice has risen steadily from about 200 million tones of paddy

rice in 1960 to over 600 million tons in 2004. Milled rice should be about 68% of paddy

by weight, although use of antiquated milling equipment in many countries means this

conversion factor can sometimes be much lower. In 2004, the top four producers were

China (26% of world production), India (20%), Indonesia (9%) and Bangladesh (5%).

9

2.4.1 Scientific Classification of Rice Plant

Kingdom: - PlantaeDivision: - Magnoliophyta

Class: - Liliopsida Order: - Poales Family: - Poaceae Genus: -Oryza

Species: -Oryza glaberrima Oryza sativa

2.4.2 RICE CULTIVATION ON LOWLAND SOIL

Most rice lands are Entisols or Inceptisols with little genetic horizon differentiation.

Other rice lands are hydromorphic associates of other soil orders. Moreover, most rice

lands are alluvial with low permeability a high water table during the rice growing

season. Both internal and external drainage are therefore poor. Because of the physical

properties of such soils are unimportant for wetland rice (Kawaguchi and Kyuma 1977).

2.4.2.1 Chemical changes in flooded soils

The main chemical changes brought about by flooding a soil (Ponnamperuma, 1972) that

have implications for land evaluation for rice are

Depletion of Oxygen

Increase in pH of acid soils and decrease in pH, of calcareous and zodiacs soils.

Decrease in redox potential,

Changes in electrical conductivity,

Reduction of Fe (II1) to Fe (I1) and Mn (1V) to Mn (I1).

Increase in total and available Nitrogen

Increase in availability of P, Si, and Mo,

Decrease in availability of S, Zn, and Cu,

Generation of organic and inorganic toxins, and sorption and desorption

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2.4.2.2. Gas transport through Rice roots

Gas transport through flood soil is very low, and therefore rice roots must supply O2 to

respiring tissues via internal gas channels, which are called parenchyma. Some of this

internally transported O2 is lost to the surrounding soil. There is disagreement about the

extent of this loss and about whether or not it is beneficial to the plants. The range is in

part due to differences in leakiness between different part of the root system and in part

due to differences in the external O2 sink. In the soil, O2 sink is enhanced by diffusion of

Fe2+ towards the root and its reaction with O2. The net O2 loss depends on the rates of

diffusion and reaction (Kirk et al., 1994).

2.5 PRODUCTION OF BIO-FERTILIZER

Bio-fertilizers are defined as biologically active products or microbial inoculants of

bacteria, algae and fungi (separately or in combination), which may help biological

nitrogen fixation for the benefit of plants.

Bio-fertilizers also include organic fertilizers (manure, etc.), which are rendered

in an available form due to the interaction of micro-organisms or due to their association

with plants. Bio-fertilizers thus include the following:

(i) Symbiotic nitrogen fixers Rhizobium spp.

(ii) Non-symbiotic free nitrogen fixers (Azotobacter, Azospirillum, etc.)

(iii) Algae Bio-fertilizers (blue green algae or BGA in association with Azolla)

(iv) Phosphate solubilizing bacteria

(v) Mycorrhizae

11

Figure 2.1 Classification of Bio-fertilizer

2.6 PRODUCTION PROCESS OF BIOFERTILIZER

2.6.1 GROWTH OF AZOTOBACTER:

Usually Azotobacter is grown on a solid medium free of nitrogen. After some times (6

months) old growth of Azotobacter is transferred to a fresh solid medium to renew the

12

growth. This procedure is repeated periodically so that the culture can be maintained in

good condition.

2.6.2 MOTHER CULTURE:

A pure growth of any organism on a small scale is called as a mother culture. Mother

culture is always prepared in a conical flask of 500 or 1000 ml. Capacity and then this

mother culture is used for further production.

For this purpose, one liter conical flasks are taken to which 500 ml of broth of

nitrogen free medium is added and these flasks are then plugged with non-absorbent

cotton, sterilized in an auto slave for 15-20 minutes at 75 lbs pressure for 15 minutes.

Flasks are then inoculated with mother culture with the help of inoculating needle

aseptically.

The flasks are transferred to shaker and shaking is done for 72-90 hours so as to

get optimum growth of bacteria in broth. Bacteria are multiplied by binary method i.e.

cell division. After about 90 days, the number of per milliliters comes to about 100

crores.

Total growth of bacteria in this broth means starter culture or mother culture,

which should carefully be done, since further purity of bio-fertilizer or quality of bio-

fertilizer depends upon how mother culture is prepared.

2.6.3 PRODUCTION ON A LARGE SCALE:

Azotobacter is multiplied on a large scale by two ways viz. Fermenter and Shaker. The

fermenter is most automatic and accurate method of multiplication of any micro-

organism. In this method, the medium is taken in a fermenter and then sterilized. After

this pH of the medium is adjusted and 1% mother culture is added.

13

In order to get an optimum growth of the Azotobacter required temperature and

oxygen supply is adjusted so that concentrated broth is made.

This concentrated broth of the culture is then mixed with a carrier previously sterilized

and bio-fertilizers are prepared. Depending upon the demand and supply suitable

fermenter is selected.

2.6.4 SELECTION OF CARRIER:

A carrier is nothing but a substance which has high organic matter, higher water holding

capacity and supports the growth of organism. In order to transport the bio-fertilizer and

becomes easy to use the suitable carrier is selected (Subba Rao et al., 1993). Generally

Lignite cool, compost and peat soil are suitable carriers for Azotobacter. Out of these

carriers lignite is most suitable for this organism, since it is cheaper, keeps organism

living for longer period and does not lower the quality of bio-fertilizers.

The lignite comes in clouds and hence it is ground in fine powder by grinding

machine. Its finesses should be 250-300 mesh. The pH of the carrier is adjusted to

neutral by adding CaCO3. The lignite naturally has a variety of micro-organism and

hence it is sterilized in autoclave at 30 lbs. Pressure for 30 minutes. After this the broth is

mixed with lignite 1:2 proportion by following method.

Galvanized trays are sterilized and used. To these trays, previously sterilized

lignite is transferred and broth is then added (lignite2: broth 1) and mixed properly.

Trays are then kept one above the other for 10-12 hours for allowing the organism to

multiply in the carrier. This mixture is then filled in plastic bags of 250 g or 500 g

capacity. Plastic bags are properly.

14

As per ISI standards, one gram of bio-fertilizer immediately after it is prepared should

have one crore cells of bacteria and 15 days before expiry date one gram of bio-fertilizer

should have 10 lakh bacteria. If bio-fertilizer is stored at 15-20 0C then it will remain

effective for 6 months. However, at 0 to 4 0C (cold storage) the bacteria will remain

active for 2 years. The storage periods are decided after testing the bio-fertilizer for that

particular storage conditions, such temperature and humidity.

2.7 APPLICATION OF BIO-FERTILIZER

Plants need nitrogen for its growth and Azotobacter fixes atmospheric nitrogen non-

symbiotically. Therefore, all plants, trees, vegetables, get benefited. However, especially

cereals, vegetables, fruits, trees, sugarcane, cotton; grapes, banana, etc. are known to get

addition nitrogen requirements from Azotobacter. Azotobacter also increases germination

of seeds. Seeds having less germinating percent if inoculated can increase germination

by 20-30%.

2.7.1 APPLICATION METHODS OF BIO-FERTILIZER

Seed inoculation: On the basis of efficiency of Azotobacter, other micro-

organisms present in the soil, benefits obtained from bio-fertilizer and

expenditure it has been fixed to use Azotobacter - bio-fertilizer at the rate of 250

g bio-fertilizer for 10-15 kg. If one knows this proportion then take a definite

quantity of seed to be inoculated. The required quantity of fresh bio-fertilizer is

secured and slurry is made by adding adequate, quantity of water. This slurry is

uniformly applied to seed; seed is then dried in shed and sown. Some stickers are

used in order to adhere of bio-fertilizer to seeds such as gum Arabia.

15

Seedling inoculation: :

This method of inoculation is used where seedlings are used to grow the crop. In

this method, seedlings required for one acre are inoculated using 4-5 packets (2-

2.5 kg). For this, in a bucket adequate quantity of water is taken and bio-fertilizer

from these packets is added to bucket and mixed properly. Roots or seedlings are

then dipped in this mixture so as to enable roots to get inoculums. These

seedlings are then transplanted e.g. Tomato, Rice, Onion, flowers.

Self inoculation or tuber inoculation:

In this method 50 liters of water is taken in a drum and 4-5 kg of Azotobacter

bio-fertilizer is added and mixed properly. Sets are required for one acre of land

is dipped in this mixture. Potato tubers are dipped in the mixture of bio-fertilizer

and planting is done.

Soil application:

This method is mostly used for fruit crops, sugarcane, and trees. At the time of

planting fruit tree 20 g of bio-fertilizer mixed with compost is to be added per

sapling, when trees became matured the same quantity of bio-fertilizer is applied.

16

CHAPTER 03

3.0 MATERIALS AND METHODS

3.1 EXPERIMENTAL SITE

The experiments were carried out in the Microbiology laboratory and pot experiments

done at Agricultural Biology Department green house in Department of Agricultural

Biology, Faculty of Agriculture, University of Peradeniya.

3.2 ISOLATION AND IDENTIFICATION OF Azotobacter

The genus Azotobacter is well established associative bacterial group which can fix the

atmospheric nitrogen by converting it to nitrate or ammonia. This group mainly associate

with C-4 plants, Hence the isolation was done from the rhizosphere of well grown guinea

grass (Panicum spp.) and also from the rice (Oryza sativa) plant. Root wash of the

above mention plants were subjected to the serial dilution techniques in order to isolate

the bacteria. The specific enrichment medium that is Ashby’s medium was used for the

isolation.

After proper inoculation period of 3-4 days with temperature of 37o C the

Azotobacter colonies were observed. On the media plates as slimy, milky, translucent

with the undulated edges.

Proper staining techniques were used for the identification of bacterium which

appears as short Cyst forming gram negative rods. Culture maintains was carried out

using sub culture techniques.

Experimental Plant

17

Rice (Oryza sativa) was used as the experimental plant. Rice seed were soaked in water

for germination before using in the experiments.

3.3 HYDROPONIC EXPERIMENT WITH Azotobacter

Hydroponic is the soil free culture for plants in agriculture. All plant nutrients will be

provided by hydroponic solutions. Some commercial hydroponic solutions are available

in markets. Hoagland’s solution is the most popular hydroponic solution used in

commercially as well as by researches. Hoagland’s solution was prepared as two

nutrients solution, with and without nitrogen solutions separately.

Regiform tanks (60×45×10 cm) were used for planting rice seedlings according to the

following treatment.

T1 – Treatment 1 T3 - Treatment 3

T2 – Treatment 2 T4 - Treatment 4

There are four treatment were prepared in this experiment.

T1 – Hoagland solution with all nutrients

T2 – Hoagland solution (without nitrogen)

T3 - Hoagland solution (without nitrogen) + Azotobacter (isolated from guinea grass) T4

- Hoagland solution (without nitrogen) + Azotobacter (isolated from Rice plant)

18

Figure: 3.1 hydroponic experiments with Azotobacter in Green house.

Each tank container, 40 plants of 5 days old rice seedlings (BG-250) were planted on

hydroponic systems with cotton plugs.

Measuring of growth rate started, 2 days after planting the seedlings. Growth parameters

used were plant height, number of leaves, number of tillers, length of mid leaf and leaf

color. Hoagland solutions and bacterial inoculums were weekly replaced to avoid the

accumulations of toxic compounds and nutrients depletion.

Finally, I was measured the dry weight in each treated plant samples.

3.4 POT EXPERIMENT with Azotobacter

3.4.1. COMPARISON BETWEEN AZOTOBACTER INOCULUMS TYPRS

Pot experiment was carried out to compare Azotobacter inoculums isolated from guinea grass

root wash, pure culture isolated from grass rhizosphere and direct inoculation to rice seeds.

Twenty (20) pots filled by ordinary soils that suitable to Rice cultivation.

There are five (05) replicates and 20 plants to each treatment

T1 – Treatment 1 T3 - Treatment 3

T2 – Treatment 2 T4 - Treatment 4

There are four (4) treatments in this experiment.

T1 - Control (without fertilizer)

T2 –Treated with guinea grass root washed inoculums

T3 - Treated with pure Azotobacter (isolated from guinea grass)

T4 – Seeds treated by pure Azotobacter (isolated from guinea grass)

19

Figure: 3.2 Pot experiment to select the best inoculation type of Azotobacter

10 days old rice seedlings that not inoculated with Azotobacter was planted on T1, T2 and

T3 treatment pots. Azotobacter inoculated seedlings were planted on T4 treatment pots.

Ashby’s broth culture medium was prepared to inoculate the Azotobacter which isolated

from guinea grass rhizosphere in to T3 treatment and T4 treatment only the seedling

inoculation by Azotobacter. T2 and T3 treatments re-inoculated again two weeks.

After 3 days of planting growth rate measurement were taken from seedlings. Growth

parameters used were plant height, number of leaves, number of tillers, length of mid

leaf and leaf color.

3.4.2 POTS EXPERIMENT TO SELECT THE BEST CARRIER MATERIAL TO

Azotobacter BIO-FERTILIZER

20

A carrier is nothing but a substance which has high organic matter, higher water holding

capacity and supports the growth of organism. In order to transport the bio-fertilizer

commonly available, easy to use suitable carrier should be selected. Generally Lignite,

compost and peat soil are consider as suitable carriers for Azotobacter in bio-fertilizer

production.

35 cement pots filled with ordinary soil suitable for rice cultivation was used for these

experiments with following treatments.

Seven (7) treatments were in this experiment.

T1 - Control Treatment (add chemical fertilizer)

T2 - Soil + Compost + Azotobacter (isolated from Rice Plant)

T3 - Soil + Charcoal + Azotobacter (isolated from Rice Plant)

T4 - Soil + Coir dust + Azotobacter (isolated from Rice Plant)

T5 - Soil + Compost + Azotobacter (isolated from Guinea grass)

T6 - Soil + Charcoal + Azotobacter (isolated from Guinea grass)

T7 - Soil + Coir dust + Azotobacter (isolated from Guinea grass)

Soil and carrier materials mixed with 1:1 ratio. All soil mixed carriers were sterilized by

autoclave under the temperature 121o C for 30 minutes in order to destroy the all of

microbes including plant pathogens.

Cell concentration of the both inoculums was measured at 660 nm with help of

spectrophotometer before incorporating with the carrier materials.

21

Comparison study was carried out with compost, charcoal and coir dust to select

the best carrier material for Azotobacter bio-fertilizers.

Figure: 3.3 Pot experiment to select the best carrier material of Azotobacter

bio-fertilizer

250ml of Azotobacter broth inoculums was added 1 kg of each carrier materials.

They were mixed properly and sealed separately in the polythene (8”×6”). These packets

were kept 24 hours under room temperature for multification of Azotobacter.

200g of each bio-fertilizer treatment was used as bio-fertilizer for the above

treatment according to the experimental design.

12 days old rice seedlings were planted on experimental pots that four (4)

seedlings in each pots. Measure of growth rate was started at 3 days after planting of

seedlings. Growth parameters used were plant height, number of leaves, number of

tillers, length of mid leaf and leaf color.2nd inoculation was done to each treatment except

control after two weeks from 1st inoculation.

CHAPTER FOUR

RESULT AND DISCUSSION

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4.1 ISOLATION AND IDENTIFICATION OF Azotobacter

Ashby’s medium was used to isolate the Azotobacter from grass rhizosphere. Bacterial

colonies were appearing as large whit, slimy colonies after 3-5 days of inoculation.

Figure 4.1 Isolated Azotobacter culture plate

When isolated Azotobacter colonies are were kept few days it’s turned to brown color

due to the production of pigments (Mortimer et al., 1981).

Figure 4.2 Ashby’s Broth medium

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Ashby’s broth medium was used to culture the isolated bacteria for the preparation of

Bio-fertilizer.

4.3 ANALYSIS OF NITROGEN FIXATION EFFICIENCY OF Azotobacter

Four treatment were used in hydroponic system,

T1 – Hoagland solution with all nutrients

T2 – Hoagland solution (without nitrogen)

T3 - Hoagland solution (without nitrogen) + Azotobacter (isolated from guinea grass)

T4 - Hoagland solution (without nitrogen) + Azotobacter (isolated from Rice plant)

Dry weight of each Plant samples are shown in table 4.1

Table 4.1 Dry weight of 20 plants in each treatment

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Treatment Dry Weight (g) ( 20

plants)

T 1 0.4730 T 2 0.3660 T 3 0.4562 T 4 0.4312

Relationships between dry weight and each treatment are shown in below figure 4.3

T 1 T 2 T 3 T 40

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5 0.473

0.366

0.45620.4312

Treatment

Dry

wei

ght(

g)

Figure 4.3 Relationships between Dry weight and Treatments

According to figure 4.3, maximum dry weight was seen in the T1 which include the all

of plant nutrients in Hydroponic solution. Minimum dry weight was in the Nitrogen

free solution. Azotobacter inoculated treatments show an increase in dry weight more

than t Nitrogen free treatment.

Moreover, shown that Azotobacter inoculation have caused to increase of dry matter

production in the inoculated solutions. Then, inoculated Azotobacter strains also

difference in Nitrogen fixation ability. Azotobacter strains which isolated from guinea

grass (T3) and rice plant (T4) show that high dry mass production in the Azotobacter

inoculated from guinea grass root zone than the rice root zone inoculation.

Other growth parameters used were as plant height, number of leaves and

length of the mid leaf of the plant. These parameters are shown in figure 4.4, 4.5 and

4.6 respectively.

25

1 st 2nd 3rd 4th 5th 6th 7th0

2

4

6

8

10

12

14

Av. plant height(cm) vs time

T-1T-2T-3T-4

No. of Readings

Plan

t hei

ght (

cm)

Figure 4.4 Relationships between average plants height and times

1 st 2nd 3rd 4th 5th 6th 7th0

0.51

1.52

2.53

3.54

4.5

No. of leaves vs time

T-1T-2T-3T-4

No. of Readings

Av. n

umbe

r of l

eave

s

Figure 4.5 Relationships between average numbers of leaves

26

1 st 2nd 3rd 4th 5th 6th 7th0123456789

10

Av. length of mid leaf (cm) time

T-1T-2T-3T-4

Number of Readings

Av. l

engt

h of

mid

leaf

(cm

)

Figure 4.6 Relationships between average length of leaf and time

Above mentioned figures show that relationship between plant heights, number of leaves

and length of the mid leaf separately. Figure 4.4 shows that average plant height and time

of the research period.

The highest value of plant height was shown in the solution which contained all nutrients

plants. Stunted growth was seen in control treatment which is without nitrogen solution.

Hence Azotobacter inoculated treatment had shown than the control. Then, Azotobacter

isolated from guinea grass, inoculated treatment has shown the plant height more than

Azotobacter isolated from rice plant.

According to figure 4.5 has shown that maximum number of leaves in all nutrient

solution treatment as well as minimum value in the control solution. Azotobacter

inoculated plants have more number of leaves than the control one. Then, figure 4.6 also

shows the same result to the above 4.4 and 4.5figures.

Overall hydroponic experiment shown that Azotobacter inoculated treatments has a

significant growth incensement due to fixation of nitrogen by Azotobacter

27

microorganisms as well as significant effect has been provided by the Azotobacter for the

rice cultivation in hydroponics systems.

4.4 ANALYSIS OF BEST INOCULATION TYPE OF Azotobacter

There are four (4) treatments used in this experiment.

T1 - Control (without fertilizer)

T2 –Treated with guinea grass root washed inoculums

T3 - Treated with pure Azotobacter (isolated from guinea grass)

T4 – Seeds treated by pure Azotobacter (isolated from guinea grass)

According to experimental design, growth parameters measured.

Table: 4.2 variation of treatment effect with plant height

a, b, Means with the same letter are not significantly different (P>0.0009

Relationships between growth parameters and time have shown in the figure 4.7, 4.8, 4.9

and 4.10

28

Treatment

Mean

T 4 37.590a T 1 36.516a T 2 35.868a,b

T 3 33.834b

.

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th05

101520253035404550

Av. plant height (cm) vs time

T 1T 2T 3T 4

No. of Readings

plan

t hei

ght (

cm)

Figure 4.7 Relationships between average plant height and time

According to the ANOVA procedure, different treatments are shown significantly

different that is due to (P>0.0009). However there is no significant different among

replicates (P>5.34). According to the Duncan's Multiple Range Test in table 4.2,

treatment means of the T4 and T1 are not significantly different that is T4 (seed inoculated)

and T1 (control) has grouped in to a one group.

But T2 treatment is in a and b group. Also T3 has significant different from T1and T4.

According to figure 4.7 graph, show the average plant height and time of the

growth phase in plant. Above graph and table shown the T4 treatment which inoculated

the Azotobacter at the seed germination stage has significant effected than the other

treatment.

Table: 4.3 variation of treatment effect with no. of leaves

29

Treatment

Mean

T 4 7.670a

T 1 7.350a

T 2 7.100a

T 3 6.050b

a, b, Means with the same letter are not significantly different (P>0.0185).

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th0123456789

10Av. number of leaves vs time

T 1T 2T 3T 4

No. of Reading

No.

of l

eave

s

Figure 4.8 Relationships between average no. of leaves and timeAccording to the ANOVA procedure, different treatments had shown significantly

different in number of leaves (P>0.0185). However there is not significant different

among replicates (P>4.46). T4 (seed inoculated), T1 (control) and T2 (guinea grass root

washed inoculums) has grouped in to a one group. T3 (pure Azotobacter culture

inoculums) grouped in to separate one.

Table: 4.4 variation of treatment effect with number of tillers

a, b, Means with the same letter are no significantly different (P>0.0173)

30

Treatment

Mean

T 2 3.950a

T 4 3.850a

T 1 3.100a,b

T 3 2.340b

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th0

0.51

1.52

2.53

3.54

4.55 Number of tillers vs time

T 1T 2T 3T 4

No. of Readings

No.

of ti

llres

Figure 4.9 Relationships between averages number of tillers and time

According to the ANOVA procedure, different treatments had shown significantly

different in number of tillers. (P>0.0173). However there is no significant different

among replicates (P>4.55). According to the Duncan's Multiple Range Test, treatment

means of the T4 and T2 are no significantly different that they are grouped in to a one

group.

Table: 4.5 variation of treatment effect with averages length of mid leaf

a, b, Means with the same letter are not significantly different (P>0.0081).

Figure 4.10 Relationships between averages length of mid leaf and time of the growth phase of rice plant.

31

Treatment

Mean

T 4 26.442a

T 1 26.128a

T 2 25.824a

T 3 23.178b

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th0

5

10

15

20

25

30

35

Length of mid leaf (cm) vs time

T 1T 2T 3T 4

No. of Reading

leng

th o

f mid

leaf

(cm

)

According to figure 4.10 show that T4 treatment has high average length of mid leaf in

the duration of vegetative period. Other treatments also are showing the relation to the T 4

treatment. According to the ANOVA procedure, different treatments had shown

significantly different in length of mid leaf. (P>0.0081). However there is not significant

different among replicates (P>5.58).

According to the Duncan's Multiple Range Test, treatment means of the T4, T1and

T2 are not significantly different that is grouped in to a one group. T3 treatment grouped

in separate one.

In this experiment was found out most suitable and efficacy Azotobacter

inoculums type to the rice cultivation. According to results of graphs and statistically

analysis was identified the relationship between among treatments and growth

parameters that used in this experiment.

T4 and T1 treatment has grouped in to a one group of average plant height and

time period of growth phase. T1 is the control one and T4 is the Azotobacter inoculated in

to seeds. Then, average number of plant leaves during the growth phase of each

treatments show the T1, T4, and T2 has grouped in to a one group according to Duncan’s

mean separation procedure. Plant height and numbers of leaves of the plant are not

32

affected by Azotobacter inoculation types. Result of average length of mid leaf is also

show the T1, T4 and T2 grouped together by analysis of Duncan’s mean separation

procedure.

According to number of tillers result shows that T2 and T4 treatment has grouped

in to one group and T1 is in another group. Significant effect has in the T2 and T4

treatments related to number of tillers in the plants.

There is no significant effect by used inoculation types of Azotobacter in rice

cultivation as overall result of this experiment according to statistical analysis.

4.5 ANALYSIS OF POTS EXPERIMENT TO SELECT THE BEST CARRIER

MATERIAL TO Azotobacter BIO-FERTILIZER.

Seven treatments were used in this pot experiment.

(T1) Treatment-1 - Control Treatment (add chemical fertilizer)

(T2) Treatment-2 - Soil + Compost + Azotobacter (isolated from Rice Plant)

(T3) Treatment-3 - Soil + Charcoal + Azotobacter (isolated from Rice Plant)

(T4) Treatment-4 - Soil + Coir dust + Azotobacter (isolated from Rice Plant)

(T5) Treatment-5 - Soil + Compost + Azotobacter (isolated from Guinea grass)

(T6) Treatment-6 - Soil + Charcoal + Azotobacter (isolated from Guinea grass)

(T7) Treatment-7 - Soil + Coir dust + Azotobacter (isolated from Guinea grass)

According to experimental design, the growth parameters were measured (plant height,

no. of plant leaves, no. of tillers and length of mid leaf in the plants)

Relationships between growth parameters and time have shown in the figure 4.11, 4.12,

4.13 and 4.14

Table: 4.6 variation of treatment effect with plant height

Treatments Mean

33

T1 63.823a

T5 60.174a, b

T3 58.792b, c

T2 57.678b, c

T4 56.172b, c

T6 54.302c, d

T7 50.712c, d

a, b, c,d Means with the same letter are no significantly different (P>0.0001).

1 st 2nd 3 rd 4 th 5 th 6 th 7 th 8 th 9 th0

1020304050607080

Av. plant height(cm) vs time

T 1T 2T 3T 4T 5T 6T 7

No. of Reading

Plan

t hei

ght(

cm)

Figure 4.11 Relationships between average plant height and time

According to figure 4.11 graphs is shown that there are no significant differences

between each treatment.

According to the ANOVA procedure, different treatments are shown significantly

different that is (P>0.0001). However there is no significant different among replicates (P>7.62).

Also the Duncan's Multiple Range Test, treatment means of the T1 and T5 are no

significantly different.

34

However means of the T1 and T3 are significantly different. According to

average plant height T1 which chemical fertilizer is added and T3 which Azotobacter

treatment (isolated from guinea grass) mixed with compost is no significant different.

Table: 4.7 variation of treatment effect with number of leaves

Treatment Mean

T1 11.40a

T5 10.40a

T3 8.16b

T2 7.85b

T4 7.15b

T6 7.13b

T7 6.15b

a, b Means with the same letter are not significantly different (P>0.0001).

35

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th 8 th

9 th 0

2

4

6

8

10

12

14Number of leaves vs time

T 1T 2T 3T 4T 5T 6T 7

Number of Readings

Av. n

o. o

f lea

ves

Figure 4.12 Relationships between average number of plant leaves and time

Above figure 4.12 shown that relation between number of leaves and time of the plant

growth phase.T1and T3 has shown high numbers of leaves than the other treatments.

According to the ANOVA procedure, different treatments had shown significantly

different in number of leaves (P>0.0001). However there is no significant different

among replicates (P>7.31). Also the Duncan's Multiple Range Test, treatment means of

the T1 and T5 are no significantly different that is T1 and T5 has grouped in to one group.

According to average number of leaves, T1 is added chemical fertilizer and T3

is that Azotobacter treatment (isolated from guinea grass) mixed with compost is not

significant different.

Figure 4.13 shows relationship between number of tillers and time of the plant growth

phase.

36

Table: 4.8 variation of treatment effect with number of tillers

Treatment Mean

T1 5.020a

T5 5.000a

T3 2.756b

T2 2.350b

T4 2.250b

T6 1.703b

T7 1.450b

a, b Means with the same letter are no significantly different (P>0.0001).

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th 8 th 9 th0

1

2

3

4

5

6

No .of tillers vs time

Series1

Series2

Series3

Series4

Series5

Series6

Series7

No. of reading

No.

of ti

llers

Figure 4.13 Relationships between averages number of tillers and time

Figure 4.13 graphs shows the maximum value of average numbers of tillers in T1 and T5

treatments as well as the other treatment shows lower value than the T1 and T5.

37

According to the ANOVA procedure, different treatments had shown significantly

different in number of tillers (P>0.0001). However there is no significant different

among replicates (P>7.31). According to the Duncan's Multiple Range Test, treatment

means of the T1 and T5 are no significantly different that is T1 and T5 has grouped in to

one group. Also other treatments which are T2, T3, T4, T5, T6 and T7 treatments grouped

in to another group.

Table: 4.9 variation of treatment effect with averages length of mid leaf

Treatment Mean

T1 44.368a

T5 41.306a, b

T3 40.674a, b

T2 40.333b

T4 38.354b, c

T6 37.373b, c

T7 35.754c

a, b,c, Means with the same letter are not significantly different (P>0.0001).

Figure 4.14 show the relationship between averages length of mid leaf and time of

growth phase of rice plant. According to Figure 4.14 graph, not show the significant

38

difference between the each treatment. All treatments show the somewhat same plant

leaf length.

1 st 2 nd 3 rd 4 th 5 th 6 th 7 th 8 th 9 th0

10

20

30

40

50

60Lengh of mid leaf(cm) vs time

T 1T 2T 3T 4T 5T 6T 7

No. of vReadings

leng

th o

f mid

leaf

(cm

)

Figure 4.14 Relationships between averages length of mid leaf and time of the growth phase of rice plant.

According to the ANOVA procedure, different treatments are shown significantly

different in length of mid leaf (P>0.0009). However there is no significant different

among replicates (P>5.34). According to the Duncan's Multiple Range Test for the

treatment means of the T1 and T5 are significantly different that is T1 and T5 has grouped

in to tow groups. And also T5 and T3 grouped together. Then T5 and T3 group has related

with T1 treatment.

According to panicles formation, T1 and T 5 treated plants has got the priority of

the panicles formation than another treatments. Then other visual observations which

color of plants, plant vigor as well as the panicles formation of plants show that T1 and T5

treatment has closed relationship other than another treatments.

39

Finally shows that seven treatments were used to this experiment and according to

results, T1 and T5 treatments closed relation performance rather than other treatments.

Therefore, assume that T1 and T5 treatments have good plant nutrients sources.

T1 was the chemical fertilizer which Urea, murate of potash (MOP) and triple

supper phosphate (TSP) in recommended ratios. T5 was compost sample inoculated by

Azotobacter which isolated from guinea grasses. According to this experiment can be

recommended that compost is the good carrier material to Azotobacter bio-fertilizer.

Then compost has good water holding capacity, high organic matters, availability as well

as the all trace nutrients for the plant growth and reproduction.

Although used the compost as carrier material in T2 treatment that is no shown

performances same to T5 treatment. Azotobacter inoculation was effected to increase the

plant growth parameters in T5 treatment than T2 treatment. Therefore Azotobacter is

more effective type was isolated from guinea grasses than the rice plant.

5.0 CONCLUSION AND RECOMMENDATIONS

Azotobacter is an associative microorganism in guinea grass (Panicum spp) and Rice

plant. Isolated Azotobacter bacterial colonies were appearing as large whit, slimy

colonies after 3-5 days of inoculation.

According to hydroponic experiment has shown that Azotobacter inoculated

treatments has a significant growth incensement due to fixation of nitrogen by

Azotobacter microorganisms as well as significant effect by the Azotobacter for the rice

cultivation in hydroponics systems.

In this experiment was found out most suitable and efficacy Azotobacter

inoculums type to the rice cultivation. According to results of graphs and statistically

analysis data was identified the relationship between among treatments and growth

40

parameters that used in this experiment. According to CRD method, there is no

significant difference among inoculation types of Azotobacter.

Compost is the good carrier material to Azotobacter bio-fertilizer than the used

other materials. Then, compost has good water holding capacity, high organic matters,

availability as well as the all trace nutrients for the plant growth and reproduction.

Azotobacter was affected to increase the plant growth parameters in Rice

cultivation. Hence, Azotobacter is more effective type was isolated from guinea grasses

than the Rice plant.

6.0 REFERENCES

Buchanan R.E and N.E.Gibbsons (1974) Bergey’s Manual of Determinative

Bacteriology, 8 th edition pp, (253-256).

Deacon, J. (2000) The Microbial World: The Nitrogen cycle and Nitrogen fixation,

Available at: http://web.reed.edu/academic/departments/biology/nitrogen, Accessed

06 Aug 2010, Institute of Cell and Molecular Biology, The University of Edinburgh.

Hubbell D.H. and Gerald Kidder (2003) Biological Nitrogen Fixation Available at:

http://edis.ifas.ufl.edu/pdffiles/SS/SS18000.pdf : Accessed 20 July 2010.

Kirk G.J.D, J.L. Solivas and C.B.M.Begg (1994) Rice root; nutrient and water use,

published by IRRI, Philliphene.

41

Kumarasinghe, K.S and D.L Eskew (1993) Isotopes studies of Azolla and nitrogen fixation

of Rice. 3300AA, Dordrecht, the Netherland.

Mostara, M.R (1995) Biofertilizer technology, marketing and usage: (6-13).

Motimer P.S , Heinz Stolp, Hans G Truper, Albert Balows and Hans G Schlegel ( 1981) A

hand Book on habitats, Isolation and Identification of Bacteria. chap.66, pp (795-801)

Vincent J.M (1982) Nitrogen fixation in legumes, Dept of Microbiology, University of Sydney.

Wilson C.E., Nathan A. Slaton and Richard J. Norman (1995) Nitrogen Fertilization of Rice in Arkansas

42