WOLLO UNIVERSITY

COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES

SCHOOL OF BIO SCIENCES AND TECHNOLOGY

DEPARTMENT OF BIOTECHNOLOGY

ISOLATION AND CHARACTERIZATION OF NITROGEN FIXING BACTERIA FROM

RHIZOSPHER OF BEAN ROOT NODULES AROUND TITA AREA, SOUTH WOLLO ETHIOPIA
A RESEARCH PROPOSAL SUBMITTED TO: THE DEPARTEMENT OF
BIOTECNOLOGY, IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
DEGREE BACHCHELOR OF SCIENCE IN BIOTECHNOLOGY

BY

NAME ID
1, WONDIYE DESALEW……………………………………………………………….. 5751/11
2, MELESE AMNU…………………………………………………………………………2718/11
3, OSSIMAN …………………………………………………………………
4, FUAD AHAMMED ………………………………………………………………………………………..
5, TEJIYE WALE …………………………………………………………………………………………………..

ADVISOR: MR. CHANIE DERSO (Asst.Prof

SEPTEMBER, 2022

DESSIE, ETHIOPIA
DECULARATION
We honestly declare that this proposal title ‘‘Isolation and characterization of nitrogen fixing
bacteria from Rhizosphere of bean root nodules around Tita, Dessie’’ has been carried out
by the group members under the guidance and supervision of our advisors. Our proposal is
original and has not been submitted for the award of any degree and other university or
organization.

Advisors name Signature Date

1. Mr. Chanie Derso(Asst.Proff ___________ __________

Examiner Name Signature Date

1. _____________ ___________ ____________
2. _____________ ___________ ____________

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ACKNOWLEDGEMENT
First, and for most our heartfelt special thank for our almighty God for according us the
opportunity to carry out and complete this study. Next our special gratitude belongs to our
adviser Mr.Chanie Derso (Asst.proff) for their support in guiding and sacrifices their time to
correct this research proposal. We would like to sincerely thank the whole lectures at the Wollo
University school of bio science and technology, Department of Biotechnology.

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Table of Contents
DECULARATION...........................................................................................................................i

ACKNOWLEDGEMENT...............................................................................................................ii

LIST OF TABLES.........................................................................................................................iii

LIST OF FIGURES........................................................................................................................iv

LIST OF ABBREVIATIONS..........................................................................................................v

ABSTRACT...................................................................................................................................vi

1. INTRODUCTION.......................................................................................................................1

1.1. Background of the study.......................................................................................................1

1.2. Statement of the problem......................................................................................................3

1.3. Objectives..............................................................................................................................4

1.3.1 General objective.............................................................................................................4

1.3.2. Specific objectives..........................................................................................................4

1.4. Significance of the study.......................................................................................................4

2. LITERATURE REVIEW............................................................................................................4

2.1. Evolution of Nitrogen Fixation.............................................................................................4

2.2. Ways of Nitrogen Fixation....................................................................................................5

2.2.1. A biotic Fixation.............................................................................................................5

2.2.2. Biological fixation..........................................................................................................5

2.3. Biological nitrogen fixation..................................................................................................6

2.4. Nitrogen Fixation cycle.........................................................................................................8

2.4.1. Nitrification....................................................................................................................8

2.4.2. Assimilation....................................................................................................................8
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 2.4.3. Ammonification..............................................................................................................8

2.4.4. Denitrification................................................................................................................8

2.5. Diversity of nitrogen fixing bacteria.....................................................................................9

2.6. Stress factors affecting symbiosis and nitrogen fixation....................................................10

2.6.1. Salt and osmotic stress.................................................................................................10

2.6.2. Temperature stress........................................................................................................10

2.6.3. PH Stress......................................................................................................................10

2.6.4. Drought stress...............................................................................................................11

2.6.5. Soil fertility...................................................................................................................11

2.6.6. Heavy metals................................................................................................................11

3. MATERIALS AND METHODS..............................................................................................12

3.1. Study Area...........................................................................................................................12

3.2. Sample collection................................................................................................................12

3.3 Serial dilutions of the extracted root nodules.......................................................................12

3.4. Characterization of nitrogen fixing bacteria.......................................................................13

3.4. 1 Morphological characterization....................................................................................13

3.4.2. Biochemical Characterization......................................................................................13

3.4.3. Molecular characterization...........................................................................................15

3.6 Data Analysis.......................................................................................................................15

4. Expected result...........................................................................................................................16

5. Work Plan.................................................................................................................................17

6. Budget Summary......................................................................................................................18

7. REFERENCES..........................................................................................................................19

iv
LIST OF TABLES
Table 1: Work Plan........................................................................................................................17

Table 2: Budget Summary.............................................................................................................18

v
List of figures
Figure 1: Patterns of Nitrogen Fixation...........................................................................................7

Figure 2: Nitrogen Fixation cycle....................................................................................................9

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LIST OF ABBREVIATIONS
BNF: ----------------------------------------------------Biological Nitrogen Fixation

H2O2: -------------------------------------------------Hydrogen per oxide

KJ: ------------------------------------------------------Kilo Joules

MR: -----------------------------------------------------Methyl Red

MSA: ---------------------------------------------------Mannitol Salt Agar

YEMA: -------------------------------------------------Yeast Extract Mannitol Agar

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ABSTRACT
Background: One of the ways to increase the competitive survivability of rhizobia biofertilizers
and thus achieve better plant growth under such conditions is by modifying the rhizosphere
environment or community by addition of non-rhizobia nodule-associated bacteria (NAB) that
cause better nodulation and plant growth when co-inoculate with rhizobia. Rhizobium is the root
of legumes host nitrogen fixing bacteria which can invade root and get sugars from the plant.

Objectives: The aim of the study is to investigate the most commonly associate nodule-associated
bacteria and the rhizosphere microorganisms associate with the legume plant. Besides, the
study aims to characterize the morphological, biochemical and molecular of Nitrogen Fixing
Bacteria from root nodule of chickpea.

Methods: The characterization of isolate pure cultures through colony morphology analysis,
cellular morphology and biochemical properties may include gram staining, catalase test,
Methyl red test, Voges Proskauer test, citrate utilization test, and starch hydrolysis test then
molecular characterization will be done for isolates having potential nitrogen fixing ability.

Work plan and Budget: The study will be conducted from April 2022 to July 2022. The task
requires a total of 13530 Ethiopian Birr.

Keywords: Isolation, Characterization, Rhizosphere, Cicer Arietinum,

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1. INTRODUCTION
1.1. Background of the study
Nitrogen (N2) is an element essential for the support of all forms of life. It is found in amino
acids and proteins and many other organic compounds are derived from the nitrogen fixation
process (Abdalla et al., 2014). N2 is the most abundant gas in earths atmosphere, it is extremely
unreactive (Egamberdieva and Kucharova, 2008.). Nitrogen is the most limiting nutrient for
growth of leguminous plants like Common beans, Soya beans, chickpeas and Garden peas
because that present in the soil cannot support growth (Howieson et al., 2007). Biological
nitrogen fixation is an important part of the microbial processes (Matiru and Dakora, 2004).
Nitrogen is a major limiting factor in agricultural production even if it represents almost 80% of
the atmosphere (Abdalla et al., 2014).
Nitrification is the process by which ammonia is oxidized to nitrite ions (NO 2-) and then to
nitrate ions (NO3-), which is the form most usable by plants. Nitrates are the form of nitrogen
most commonly assimilated by plants through root hairs. In ammonification, a host of
decomposing microorganisms, such as bacteria and fungi, break down nitrogenous wastes and
organic matter found in animal waste and dead plants and animals and convert it to inorganic
ammonia (NH3) for absorption by plants as ammonium ions. Denitrification is the process by
which nitrates are reduced to gaseous nitrogen (N 2) and lost to the atmosphere. Various factors
such as the soil physico-chemical composition can interfere with the infection process and
nodulation, or can influence the activity of nitrogen-fixation during the symbiosis (Egamberdieva
and Kucharova, 2008).
What nowadays encourages the developed countries to produce and use bio-fertilizers is their
serious attention to environmental side effects resulting from the unbalanced and extreme use of
chemical fertilizers. One of the most important ways to take advantage of useful interaction of
microorganisms and to maintain diversity in agricultural ecosystems is to use useful soil
microorganisms which are living in rhizosphere environment and are capable of improving plant
nutrition and soil fertility through biological fixation of nitrogen, phosphate solubilization and
generally enhancement of plant growth. At present, bacteria around the roots are used as bio-
fertilizers (Barron, et al., 2000). Today, the use of bio-fertilizers containing crop growth

1
enhancer bacteria is increasing. These bacteria are able to improve crop growth directly or
indirectly through different mechanisms such as producing growth regulators, vitamins, amino
acids, antibiotics, and siderophores (Asgharzadehh, 2000).

The symbiotic interaction between two symbiotic components, legume and rhizobium is highly
specific and is determined via signal changes between the crop and bacterium at different stages.
At first, the host legume releases signal compounds (mainly flavonoids) within the rhizosphere
and rhizobium reacts to these signals by producing combinations which are called nodulation
factors. Then, nodulation factors are responsible for the growth of nodules in crops (Allister et
al., 2012). Nitrogen is one of the most important elements which limit crops production.
Members of the Leguminosae form the largest plant family on Earth, with around 18,000
species. The success of legumes can largely be attributed to their ability to form a nitrogen-fixing
symbiosis with specific bacteria known as rhizobia, manifested by the development of nodules
on the plant roots in which the bacteria fix atmospheric nitrogen, a major contributor to the
global nitrogen cycle.

Nutrient enrichment of soils by nitrogen fixing symbiotic bacteria present in legumes has been
known for centuries. Rhizobium spp. are well known group of bacteria that acts as the primary
symbiotic fixer of nitrogen. These bacteria infect the roots of leguminous plants, leading to the
formation of lumps or nodules where the nitrogen fixation takes place. The bacterium enzyme
system supplies a constant source of reduced nitrogen to the host plant and the plant furnishes
nutrients and energy for the activities of the bacterium. This symbiosis reduces the requirements
for nitrogenous fertilizers during the growth of leguminous crops. The isolates were grown on
yeast extract mannitol agar (YMA), and the morphologic characterization was realized by Gram
stain (Tamás et al., 2010). Rhizobium is the root of legumes host nitrogen fixing bacteria which
can invade root and get sugars from the plant. In return, they convert large amounts of dinitrogen
(N2) from the atmosphere into forms that the plants can use. We use specific primer for Rhizobia
to recognize the genetic variation between the isolates which is deferens in their efficiency of
nitrogen fixation and tolerance of biopesticide (Suliasih, 2005).

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1.2. Statement of the problem

Small-scale farmers who are the major legume producers in Ethiopia rarely apply fertilizers
during legume production. Hence, the crop is largely dependent on fixed nitrogen from native
nitrogen fixers. Isolation of rhizobia for legume production has been given a little attention in
Ethiopia due to inadequate research or negligence of researchers and unawareness of its potential
in legume production as well as lack of an intention from skilled personnel to popularize the
technology.
At present days, what encourages the developed countries to produce and use bio-fertilizers is
their serious attention to environmental side effects resulting from the unbalanced and extreme
use of chemical fertilizers. One of the most important ways to take advantage of useful
interaction of microorganisms and to maintain diversity in agricultural ecosystems is to use
useful soil microorganisms which are living in rhizosphere environment and are capable of
improving plant nutrition and soil fertility through biological fixation of nitrogen. Chemical
(Synthetic fertilizers) gives short sustain high yield product, but it causes a long-term negative
impact on the farm lands (soils) like increasing its acidity, erosion etc. So, it can be eradicate by
obtaining microbial products (bio fertilizer) as an alternative.

3
1.3. Objectives
1.3.1 General objective
 To isolate and characterize nitrogen fixing rhizobia from rhizosphere of Bean root
nodules.
1.3.2. Specific objectives
 To isolate nitrogen fixing bacteria from chickpea nodules.
 To characterize nitrogen fixing bacteria via biochemical and morphological
methods.
 To characterize nitrogen fixing bacteria at molecular level

1.4. Significance of the study

This study will contribute the community by obtaining microbial products as alternative to
chemical fertilizer or chemical nitrogen fertilizer. This eradicating the environmental side effects
resulting from the unbalanced and extreme use of chemical fertilizers. The isolation and
characterization of rhizobia is a valuable biological resource for finding bacterial strains with
effective rhizobium legume association to maximize the agricultural production. The use of bio-
fertilizers containing crop growth enhancer bacteria will increase. For the significant reduction of
the use of nitrogen fertilizer that reduce the yield of crops by replacing biofertilizer.

2. LITERATURE REVIEW
2.1. Evolution of Nitrogen Fixation
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During the evolution of earth there is aggregation of active atoms to form various molecules,
nitrogen is one of them. After continuous evolution, organisms were developed and in this
process the nitrogen form complexes (Montero et al., 2012). Stellar data suggest a primary origin
for N, but there is probably substantial secondary N at the center of galaxy. So, we know that
chemical evolution leads to the first organism formation (Berrada and Benbrahim,2014).
Nitrogen is an important atom which forms many complexes which helps in biomolecule
formation. Now the di-nitrogen is available in plenty of in the environment. At that time,
evolution occurs and the first microorganism evolved. They require nitrogen for their general
metabolism and thus they fix dinitrogen in to simple form. The evolution of nitrogen fixation
required microaerophilic condition because; nitrogenase play key role in this event and it is
inhibited by oxygen. As we know that nitrogenase is such enzyme which is blocked by oxygen
allosteric effect. So, the evolution of nitrogen fixation occurred earlier to the evolution of
cyanobacteria. However, evolutions of cyanobacteria require the oxygenic environment or it
change anoxygenic environment to oxygenic environment. There was a common ancestor which
was first evolved (progenote) which first perform the nitrogen fixation later on change in to
cyanobacteria. However, some branches of that progenote remain same as Azotobacter which
fixes nitrogen but it is not a cyanobacteria (Asgharzadehh, 2000).

2.2. Ways of Nitrogen Fixation

Nitrogen Fixation is the conversion of atmospheric nitrogen (N 2) into reactive compounds such
as ammonia (NH3) and nitrate (NO3-). The breaking of the bonds between the nitrogen atoms
requires a great deal of energy and occurs naturally in two primary ways (Reghuvaran et al.,
2014).
2.2.1. A biotic Fixation
Nitrate is the result of high energy fixation in the atmosphere from lightning and cosmic
radiation. In this process, N2 is combined with oxygen to form nitrogen oxides such as NO and
NO2, which are carried to the earth surface in rainfall as nitric acid (HNO 3). This high energy
fixation accounts for approximately 10% of the nitrate entering the nitrogen cycle (Rakash and
Rana., 2013).

5
2.2.2. Biological fixation
Biological fixation is accomplished by a series of soil micro-organisms such as aerobic and
anaerobic bacteria. Often, symbiotic bacteria such as Rhizobium are found in the roots of
legumes and provide a direct source of ammonia to the plants. In root nodules of these legumes,
the bacteria split molecular nitrogen into two free nitrogen atoms, which combine with hydrogen
to form ammonia (NH3). The following plants are common examples of legumes: clover, alfalfa,
soy beans, and chick peas. The breakdown of these legumes by bacteria during ammonification
actually returns excess nitrogen not utilized by the plant to the surrounding soil. Therefore, to
promote sustainable soil fertility, it is beneficial to use these agricultural crops in rotation with
other plants, such as corn, that are more profitable but deplete the available nitrogen in the soil-
Some free-living aerobic bacteria, such as Azotobacter, and anaerobic bacteria, like Clostridium,
freely fix nitrogen in the soil and in aquatic environments. Some members of the photosynthetic
Cyanobacteria phylum fix nitrogen in aquatic environments as well (Mohammadi et.al,. 2012).

2.3. Biological nitrogen fixation

Biological nitrogen fixation is an important part of the microbial processes (Matthew, 2008).
Nitrogen is a major limiting factor in agricultural production even if it represents almost 80% of
the atmosphere (Abdalla et al., 2014). The biological nitrogen fixation (BNF) is a natural
phenomenon consisting on the conversion of atmospheric nitrogen into ammonia by the
nitrogenase enzyme complex. This biological reduction of N 2 to NH3 is a highly endergonic
process with a minimum energy requirement of Ca.960 KJ mol -1 N-fixed (Egamberdieva and
Kucharova, 2008). Biological nitrogen fixation (BNF) is the distinguishing feature of a legume
in a cropping system. Most legume species are able to form a symbiosis with alpha- or beta-
proteo bacteria, collectively called rhizobia that use solar energy captured by the plant to break
the bond in inert atmospheric dinitrogen and form reactive N species, initially ammonium
(NH4+). As a result of this symbiosis, the legume crop requires little or no input of N fertilizer
and makes little demand on soil N reserves. Since the manufacture of synthetic fertilizer
consumes fossil fuel, thereby releasing CO 2, and the transport and spreading of organic and
synthetic N fertilizers consumes further fuel, the use of legumes in cropping systems has
immediate environmental benefits arising from reduced fossil fuel use. Nitrates from fertilizers
and soil N reserves may also leach through the soil column into ground water, and the
6
denitrification of nitrates from synthetic or organic sources is the primary source of nitrous oxide
(N2O), a powerful greenhouse gas, from agricultural soils (Philippot and Hallin, 2011).

Figure 1: Patterns of Nitrogen Fixation

7
2.4. Nitrogen Fixation cycle
2.4.1. Nitrification
Nitrification is the process by which ammonia is oxidized to nitrite ions (NO 2-) and then to
nitrate ions (NO3-), which is the form most usable by plants. The two groups of micro-organisms
involved in the process are Nitrosomas and Nitrobacter. Nitrosomas oxidize ammonia to nitrite
and Nitrobacter oxidize nitrite to nitrate.
2.4.2. Assimilation
Nitrates are the form of nitrogen most commonly assimilated by plants through root hairs. Since
heterotrophic organisms cannot readily absorb nitrogen as plants do, they rely on acquiring
nitrogen-based compounds through the food they eat. Since plants are the base of the food chain,
the nitrogen-based compounds they have assimilated into their tissue will continue to pass from
one organism to another (through consumption) as matter and energy transfers through the
ecosystem food web (Gonzalez et al., 2005)
2.4.3. Ammonification
In ammonification, a host of decomposing microorganisms, such as bacteria and fungi, break
down nitrogenous wastes and organic matter found in animal waste and dead plants and animals
and convert it to inorganic ammonia (NH 3) for absorption by plants as ammonium ions.
Therefore, decomposition rates affect the level of nutrients available to primary producers
(Gusmão et al., 2006).
2.4.4. Denitrification
Denitrification is the process by which nitrates are reduced to gaseous nitrogen (N 2) and lost to
the atmosphere. This process occurs by facultative anaerobes in anaerobic environments.
Farmers with water logged fields and soils that have high clay content are especially vulnerable
to nitrogen losses due to denitrification (shahzad et al., 2012).

8
Figure 2: Nitrogen Fixation cycle

2.5. Diversity of nitrogen fixing bacteria

Bacteria are differentiated into endosymbionts namely bacteroid forms which can reduce
dinitrogen into ammonia and this can be assimilated directly by host plants. The plant canopy
hosts a wide array of microorganisms having beneficial, harmful and neutralistic effects. It is
well-established that many soil- and plant-associated bacterial groups are able to synthesize
phytohormones. The genera under this list are steadily growing and presently include Gram-
negative and Gram-positive, symbiotic and nitrogen-fixing bacteria (Peter et al., 2002). N 2
Fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Meso Rhizobium and Sino
Rhizobium are well known for their ability to improve plant development. Many of these bacteria
can produce and excrete in their cultures more than one hormone type: Rhizobium isolates
synthesize gibberellins (GA) and auxin; Azotobacter spp., GA, auxin and cytokinin and
Acetobacter and Herb spirillum isolates, indole-3-aceticacid (IAA) and GA (Jaleel et al., 2009)

9
2.6. Stress factors affecting symbiosis and nitrogen fixation
Various factors such as the soil physico-chemical com-position can interfere with the infection
process and nodulation, or can influence the activity of nitrogen-fixation during the symbiosis
(Kinkema et al., 2006).
2.6.1. Salt and osmotic stress
Salinity is one of the major factors threatening agriculture in arid and semi-arid areas. Nearly
40% of the worlds land surface can be categorized as having a potential salinity problem
(Zahran, 2001; Niste et al., 2013). The main cause of salinity is the nutrient imbalance in the
soil, which is considered as a constraint influencing the N 2 fixing symbiosis and the survival of
both partners (Mohammadi et al., 2012; Niste et al., 2013). Salinity is concentration of dissolved
mineral salts comprising cations and anions present in the soil (soil solution) and in water. The
principal cations in solution consist of Na +, Ca2+, Mg 2+
and K+ and the major anions are Cl -,
SO42-, HCO3-, CO32- and NO3- (Hussain et al., 2014).
Rhizobia strains differ in their ability to tolerate osmotic stress and can use different adaptation
mechanisms such as intracellular accumulation of low-molecular-weight organic solutes
including amino acids such as glutamate, N- acetyl glutaminyl - glutamine, sugar and
polyamines or the accumulation of ions such as K +. Rhizobia subject to salt stress may undergo
morphological alterations, leading to changes in cell morphology and size or modifications in the
pattern of extracellular polysaccharides and lipopolysaccharides (Ventorino et al., 2012).
2.6.2. Temperature stress
High soil temperature is one of critical factors which can prevent the development of a nitrogen-
fixing association between the two symbiotic partners especially in arid and semi-arid regions.
The survival of rhizobia in soil is more affected by high temperatures than by low temperatures
because it can be deleterious (Niste et al., 2013). In arid regions, high soil temperatures affect
lives of both free and symbiotic rhizobia. Most rhizobia have an optimum growth temperature at
28-31°C and many of them are unable to grow at 38°C (ventorino et al., 2012).
2.6.3. PH Stress
Either alkaline or acidic agricultural soil has a great influence on the survival or multiplication of
rhizobia and can affect both the symbiosis partners. Most leguminous plants require a neutral or
slightly acidic soil for growth, especially when they depend on symbiotic N 2 fixation (Ventorino
10
et al., 2012). The optimum pH for rhizobia growth is considered to be between 6.0 and
7.0(Hungria and Vargas, 2000). In fact, at pH of 5.0-5.5, the nodulation in Acacia trees was
absent (Brockwell et al., 2005). The rhizobia strains vary widely in their acidity tolerance.
Rhizobium tropici and Meso rhizobium loti are considered as highly acid tolerant strains. Some
rhizobial strains can with stand and survive even in a very low pH (about 3.5). Alkalinity is less
harmful to the survival of rhizobia. And showed that the majority of these bacteria can tolerate
up to pH 9. The same result was found among strains modulating Acacia, which showed
remarkable and sometimes quite extraordinary tolerance to alkaline conditions (Brockwell et al.,
2005). For example, rhizobia strains isolated from A. farnesiana have shown an ability to adapt
and grow at pH 12.0 (Brockwell et al., 2005).
2.6.4. Drought stress
Drought stress can present a major agricultural problem which occurs when the available soil
water is reduced and the atmospheric conditions induce continuous loss of water by transpiration
or high evaporation (Brockwell et al., 2005).
2.6.5. Soil fertility
Soil fertility can also affect the biological nitrogen fixation in Rhizobium-legume symbiosis. In
fact, an excess of nitrates may cause an inhibitory action on nodulation and N2 fixation activity
(Luciñski et al., 2002).
2.6.6. Heavy metals
Heavy metals are known as the most important inorganic pollutants which persist in the soil over
long periods and have ecotoxicological effects on plants and soil micro-organisms. Some metals
such as Zn, Cu, Ni and Cr are essential for growth of both rhizobia and their host plants, whereas
others such as Cd, Hg and Pb seems to be not beneficial and could be toxic even at relatively low
concentrations (Zahran et al., 2012).

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3. MATERIALS AND METHODS

3.1. Study Area

This study will be conducted in Wollo University, Biotechnology Department Laboratory.
Dessie is located on North east of our country Ethiopia and 400 km distant from Addis Ababa.
Besides, Dessie lie on between 2470 and 2550 meters above sea level and average rainfall is
621mm.

3.2. Sample collection

The experimental material for the present study will be collected from around Tita, Dessie
farmland. Crops possessing healthy nodules with pink colour will be selected and transported to
the laboratory. Root nodules of Bean plant will be used as study material for isolation and further
morphological, molecular and biochemical characterization of Rhizobium strain. The roots will
be first washed thoroughly with sterile distilled water and nodules will be surface sterilized by
washing with 95% ethanol for 10 seconds and again will be washed in sterile distilled water for
about 5 times. Roots will be mashed with pestle mortar to obtained nodules and milky white
substances of bacteroid by dipping in phosphate buffer solution.

3.3 Serial dilutions of the extracted root nodules

After the extraction of bacteroid solution from the bean root nodules, serial dilution will be
made. 1ml of sterilized root nodule bacteroid solution will be taken in 9ml sterilized distilled
water and serially diluted up to 10-6 dilution. For identification of the colonies, 10 -4 to 10-6
dilution of nodule extract will be plated on YEMA Congo red agar media plates. The petridsh
plates will be then kept in the incubator at 37 0C for 3 days. Bacterial culture will repeat for three
times by single colony streaking on YEMA medium. Identification of the isolates will be done
via morphological, biochemical, and molecular methods.

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3.4. Characterization of nitrogen fixing bacteria
3.4. 1 Morphological characterization
Isolates will be characterized by nature of cell (gram stain) and colonies (shape, colour, margin,
nature of colony, texture)
3.4.1.1 Gram stain
The pure cultures of bacterial strains will be put for gram staining for more specific identification
of the colonies. The gram staining will be done in laminar air flow hood. The slides will be
firstly washed with ethanol and colonies will be marked on the slides with the help of inoculating
needle and will be heat fixed. Then smears will be stained in following steps a) First applied
crystal violet on each slide and kept for 1 min. b) wash via distilled water. c) Iodine added on the
slides as mordant (1 min) then 95% alcohol wash (30 sec.) and then washed with distilled water.
d) Safranin was applied on the slides and then washed with distilled water and f) air dried the
slides. Gram positive bacteria show violet color and gram-negative bacteria show pink color. The colony
feature (i.e., form, size, color, opacity, motility, and margin of the bacterial colony) will be
destined by observing the colonies on YEMA plates of the overnight grown microorganisms at
32 0C.

3.4.2. Biochemical Characterization

Isolates will be characterized by different biochemical methods like methyl red test, VP test,
catalase test (cover slip method), starch hydrolysis test, citrate utilization test, and indole test.

3.4.2.1 Methyl red test and VP test

Rhizobium will be inoculated into sterile test tubes containing methyl red-Voges Proskauer broth
(Manasa et al., 2017) at 5 mL/test tube, incubate at 30±2°C for two days, after which 5 mL of
methyl red will be added to each tube. Red broth indicates a positive result but yellow indicates a
negative result (Dinesh et al., 2015).

3.4.2.2 Voges-Proskauer test

The Rhizobium isolates will be inoculated separately into test tubes containing methyl red-Voges
Proskauer broth at 5 mL/sterile test tube and will be incubated at 30±2°C for two days. After

13
incubation, Barrit reagents A and B (5 mL each) will be added. The development of a red color
indicates a negative result (Devitt, 2009).

3.4.2.2 Indole Test

The indole test will follow the procedure of (Fadden, 2000) and Hemraj et al., 2013). Tryptone
broth medium will be prepared and poured into 10 mL test tubes (4 mL/test tube; Garg Process
Glass India Pvt. Ltd., Mumbai, India) then will be autoclaved (121°C; 15 psi; 15 min).
Rhizobium will be inoculated in broth and will incubate at 30±2°C for two days. Uninoculated
broth will serve as the control. After incubation, 1 mL of Kovac reagent (isoamyl alcohol, para-
dimethylaminobenzaldehyde and concentrated hydrochloric acid) will be added to each test tube,
including the control. Tubes will be shaken gently every 10-15 min and will be allowed to stand
until the reagent surfaced. The formation of a red ring indicates a positive result, but a yellow
ring indicates a negative result.

3.4.2.3 Citrate Utilization test

The ability to use citrate will be determined by replacing mannitol in YEM agar with an equal
amount of sodium citrate and bromothymol blue ((25 mg L-1). Fresh cultures on modified media
will be incubated for 48 h. After incubation, a positive result was indicated when green turned to
blue.

3.4.2.4 Catalase test

Catalase (CAT) activity of Rhizobium will be determined by the CAT test by placing 24 h
colonies on a glass slide and adding one drop of 30% H 2O2. The appearance of gas bubbles
indicates the presence of CAT.

3.4.2.5 Starch hydrolysis test

Starch agar medium will be prepared (Hemraj et al., 2013). Medium will poured into sterile Petri
dishes and allowed to solidify. Rhizobium will be inoculate into Petri dishes and incubated at
30±2°C for 4 days. After incubation, 5 mL of iodine solution (0.340 g iodine and 0.660 g
potassium iodide in 100 mL distilled water) (Küpper et al., 2011) was added. Formation of a

14
clear zone around colonies indicated a positive result, i.e., the ability to hydrolyze starch (Hemraj
et al., 2013).
3.4.3. Molecular characterization
3.4.3.1 Extraction: Alkaline lysis method will be used to isolate genomic DNA with minor
changes (Maniatis et al., 1982). Bacteria will be grown at 28±2°C for 4-5 days in YEM broth
with constant shaking. Centrifuged for 10 minutes at 5,000×g in Eppendorf tube, 500 µL TE
buffer containing 5µLRNAase (10 mg mL -1) and 100 µL lysozyme (15 mg mL -1), will be added
to the pellet. The suspension of cell will be incubated at 37°C for 30 min and 10%SDS (30µL)
will be added and again incubated at 70°C for 20 min. ProteinaseK10µL (20 mg mL -1) will be
added to this suspension and incubated at 45°C for 2 h. Centrifuged at 13,000 ×g for 10min and
extracted twice with (25:24) phenol/ chloroform followed by twice extractions with (24:1)
chloroform/ isoamyl alcohol. Na-acetate (3M, pH5.2) 1/10(v/v) isopropanol of 0.6 (v/v) will be
added and incubated for 1hat -20°C. It will be centrifuged at 13,000 × g for 10 min, washed
pellet with 70% ethanol, dried and suspended in 50µL TE. Quantity and quality of isolated DNA
will be determined by spectrophotometer and gel electrophoresis.
3.4.3.2 Electrophoresis: Amplified PCR products will be resolved in 0.9% agarose gel
containing 2µL of ethidium bromide (20mg L -1) in 0.5 X TBE (Trisborate-EDTA) buffer. DNA
ladders 1kb (GeneRuler, Fermentas) will be used as size marker. Gels will be visualized under
UV light and photographed.

3.6 Data Analysis

Data will be analyzed using the analysis of variance (ANOVA) procedure by SPSS and the
means will compared by Turkeys’ multiple range test (P<0.05).

4. Expected result
The isolated organism will greatly enhance agricultural production, if they are often used to
inoculate legume plants, thereby reducing the environmental threat of synthetic nitrogen

15
fertilizers. The findings will allow us a new scope for extensive research in Agricultural
Biotechnology.

16
5. Work Plan
Table 1: Work Plan

Activity July Septemper Octover

24-27 27-30 1-15 16-30 1-15 16-30 days

days days days days days
Title selection 

Write up of 
proposal

Proposal 
submission
Proposal 
presentation
Field of material 
preparation

Field of data 
collection
Lab work 
Research writing 
Research
presentation

17
6. Budget Summary
Table 2: Budget Summary

Material Unit Quantity Cost in ETB Total cost

YEMA media Kg 1 3000 3000
Iodine Kg 1 2000 2000
Ethanol-Aceton Kg 1 800 800
Safranin Kg 1 1000 1000
Pen Number 6 10 60
Flash In GB 1 350 350
A4 Paper Package 1 300 300
Print Per Paper 80 2 160
Mobil Card Number 4 15 60
Transport 2 100 200
SDS Kg 1 1000 1000
Phenol /chloroform Kg 1 1500 1500
EDTA Kg 1 1200 1200
Ethidium bromide Kg 1 1000 1000
Agarose Kg 1 900 900
Total 13, 530

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