Isolation and Identification

of bacteria in
Biofertilised soil
The Thesis is submitted in the partial fulfillment

Of the requirements for the degree of

BACHELOR OF TECHNOLOGY
In
BIOTECHNOLOGY
By

KALYAN KUMAR CHOUBEY

20BT8045.
Under the guidance of

Dr. KAUSTAV AIKAT

PROFESSOR
Department of Biotechnology

National Institute of Technology

Durgapur, India
MAY 2024

i
 DEPARTMENT OF Biotechnology
NATIONAL INSTITUTE OF TECHNOLOGY
DURGAPUR, INDIA

DECLARATION
I the undersigned declare that the thesis work entitled “Isolation and Identification of
Bacteria from Biofertilised Soil “, submitted towards partial fulfillment of requirements for
the award of the degree in Bachelor of Technology in Biotechnology, is my original work
and this declaration does not form the basis for award of any degree or any similar title to
the best of my knowledge.

-----------------------------------------------
Durgapur Kalyan Kumar Choubey
May, 2024 Roll No. -20BT8045

ii
 DEPARTMENT OF BIOTECHNOLOGY
NATIONAL INSTITUTE OF TECHNOLOGY
DURGAPUR, INDIA

CERTIFICATE OF RECOMMENDATION
This is to certify that the thesis entitled “Isolation and Identification of Bacteria in
Biofertilised Soil “, submitted by Name of the Student of Department of
BIOTECHNOLOGY, National Institute of Technology, Durgapur, in partial fulfillment of
the requirements for the award of the degree in Bachelor of Technology in
BIOTECHNOLOGY is a bonafide record of work carried out by him/her under my/our
guidance during the academic year 2023 – 2024.

Head Dr. Kaustav Aikat

Department of BIOTECHNOLOGY Professor
National Institute of Technology Department of BIOTECHNOLOGY
Durgapur National Institute of Technology
Durgapur

iii
 DEPARTMENT OF BIOTECHNOLOGY
NATIONAL INSTITUTE OF TECHNOLOGY
DURGAPUR, INDIA

CERTIFICATE OF APPROVAL
This is to certify that we have examined the thesis entitled “Isolation and Identification

Of bacteria in Biofertilised Soil”, submitted by Kalyan Kumar Choubey and hereby

accord our approval of it as a study carried out and presented in a manner required for its
acceptance in partial fulfillment of the requirements for the award of the degree in
Bachelor of Technology in BIOTECHNOLOGY for which it has been submitted. It is to
be understood that by this approval the undersigned do not necessarily endorse or approve
any statement made, opinion expressed or conclusion drawn therein but approve the thesis
only for the purpose for which it is submitted.

Examiners:

Name Signature

iv
 ACKNOWLEDGEMENTS
Foremost, I would like to express my sincere gratitude to my supervisor Dr. Kaustav
Aikat, Professor, Department of BIOTECHNOLOGY, National Institute of Technology
Durgapur for enlightening me with the first glance of research, and for his patience,
motivation, enthusiasm, and immense knowledge. His inspiring guidance, systematic
approach, sensible criticisms, and close support throughout the course of this project work
helped me overcome the problems at many critical stages of the assignment leading and
enabling towards the accomplishment of this project.

It gives me great pleasure to acknowledge the support and help of Reshmi Verma (my
PhD senior) to clarify my long technical queries. I am also thankful to my classmates and
friends for their love and support.

Finally, I feel great reverence for all my family members and the Almighty, for their
blessings and for being a constant source of encouragement.

Durgapur Kalyan Kumar Choubey

May, 2024 Roll No. (s)-20BT8045

B.tech, Biotechnology

Department of BIOTECHNOLOGY

NIT Durgapur

v
 ABSTRACT
Biofertilizers are increasingly recognized as a sustainable substitute for chemical
fertilizers in modern agriculture. Nonetheless, their adoption faces obstacles such
as inconsistent effectiveness, limited understanding and awareness, high
production expenses, competition from chemical alternatives, and potential
environmental implications. This study focuses on isolating bacteria from soil
samples previously treated with biofertilizers. The conducted tests aim to
comprehensively explore the properties of these bacteria, laying the groundwork
for potential replacements for chemical fertilizers. This research addresses a
significant contemporary challenge in agricultural sustainability.

vi
 CONTENTS
Content Page No.
1. Chapter 1: Introduction
1-4
1.1 Fertilizer 1
1.2 Eco-Friendly Soil Enhancers: Exploring Biofertilizers as Green 1-2
Alternatives
1.2 Biofertilizers and their types
2-4
2. Chapter 2: Literature Review
5-8
2.1 Cyanobacteria
5-6
2.2 Potassium solubilizing bacteria
7-8
3. Chapter 3: Materials and Methods
9-11
3.1 Solid and Liquid Sample 9

3.2 Physical and Chemical Properties of Sample 9

3.3 Culture Medium 9

3.4 Isolation and Determination of Bacteria present in sample 10

3.5 Morphological identification 10

3.6 Biochemical identification 10-11

4. Chapter 4: Results and Discussions

12-16
4.1 Colony morphology
12
4.2 Catalase Test
13
4.3 Citrate utilization Test
13
4.4 Indole acetic Acid Test
13
4.5 Results
14
4.6 Discussion
15-16
5. Chapter 5: Concluding Remarks and Scope for Future Work
17-18
5.1 Concluding Remarks
17
5.2 Scope for Future work
18
6. References
19

vii
 LIST OF FIGURES
Fig. No. Title Page No.

CHAPTER 4: RESULTS AND DISCUSSION

1.1 The BG-11 agar plate showing the multitude of cyanobacterial colonies

1.2 Shows the pink-colored solution indicating a positive result for the indole
Acetic acid test
1.3 Negative result of oxidase test

1.4 Positive result of Citrate test

viii
 LIST OF TABLES
Table No. Name Page No.
1.1 Study of the colonial morphology of the bacterial samples
1.2 Values of spring stiffness and damping coefficient

ix
 Chapter 1
INTRODUCTION
1.1 INTRODUCTION

Agriculture serves as the cornerstone of human civilization, catering to our needs for
sustenance, economic progress, and essential resources like food, fiber, and fuel. Over the
ages, farming practices have evolved from traditional subsistence methods to the modern,
industrialized approaches we see today. Amidst these changes, one constant requirement has
persisted: the necessity to replenish soil nutrients to ensure fertility and sustain crop yields.

The soil, acting as a fundamental medium for plant growth, comprises a dynamic ecosystem
teeming with diverse microorganisms, including bacteria, fungi, protozoa, and nematodes,
collectively referred to as soil microbiota. These microorganisms play pivotal roles in
nutrient cycling, organic matter decomposition, and overall soil vitality. However, the
extensive use of intensive agricultural techniques like tillage, monoculture, and heavy
reliance on chemical fertilizers can disrupt this delicate balance within the soil microbiota,
leading to soil degradation and diminished fertility.

Chemical fertilizers, ubiquitous in modern agriculture, are engineered to provide crops with
essential nutrients like nitrogen (N), phosphorus (P), and potassium (K). They dissolve
readily in water and are readily absorbed by plant roots, offering an immediate nutrient
source for plant growth. However, the excessive dependency on chemical fertilizers has
raised numerous environmental concerns, including contamination of groundwater,
eutrophication of water bodies, and depletion of soil biodiversity.

1.2 Eco-Friendly Soil Enhancers: Exploring Biofertilizers as Green Alternatives

To overcome the limitations associated with chemical fertilizers and encourage sustainable
agricultural methods, biofertilizers have emerged as a promising solution. These fertilizers
harness living microorganisms, primarily bacteria, capable of nitrogen fixation, phosphorus
solubilization, and production of growth-promoting substances like phytohormones and

Isolation and Identification 1

enzymes, all of which facilitate plant growth. Establishing symbiotic or associative
relationships with plant roots, these microorganisms enhance nutrient absorption, soil
structure, and overall plant health.

A notable advantage of biofertilizers lies in their environmentally friendly and sustainable

characteristics. Unlike chemical fertilizers, which are synthetic and often derived from non-
renewable sources, biofertilizers are sourced from renewable materials, are biodegradable,
and leave no harmful residues in soil or water bodies. Moreover, biofertilizers have the
potential to mitigate environmental pollution caused by chemical fertilizers, such as nitrogen
leaching into groundwater or greenhouse gas emissions during production.

Beyond their environmental benefits, biofertilizers offer economic advantages. By potentially

reducing the overall cost of fertilization, as they can supplement or replace chemical
fertilizers, which can be costly, biofertilizers contribute to economic savings for farmers.
Additionally, they can enhance crop quality, leading to increased yields and improved market
prices. Furthermore, the adoption of biofertilizers supports sustainable agricultural practices,
reduces reliance on external inputs, and fosters self-sufficiency in nutrient management.

1.3 Biofertilizers and their types

1. Nitrogen-fixing biofertilizers: These biofertilizers contain nitrogen-fixing bacteria, which

convert atmospheric nitrogen into a form that can be taken up by plants. Examples of
nitrogen-fixing biofertilizers include Rhizobium, Azotobacter, and Azospirillum.

2. Phosphate-solubilizing biofertilizers: These biofertilizers contain bacteria that can

solubilize insoluble forms of phosphorus in the soil, making them available for plant uptake.
Examples of phosphate-solubilizing bacteria include Bacillus, Pseudomonas, and Aspergillus.

3. Potassium-releasing biofertilizers: These biofertilizers contain bacteria that can release

potassium from minerals in the soil, making them accessible to plants. Examples of
potassium-releasing bacteria include Bacillus and Paenibacillus.

Isolation and Identification 2

4. Plant growth-promoting biofertilizers: These biofertilizers contain bacteria that produce
growth-promoting substances, such as phytohormones, enzymes, and siderophores, which
stimulate plant growth and development. Examples of plant growth-promoting bacteria
include Azospirillum, Bacillus, and Pseudomonas.

5. Mycorrhizal biofertilizers: These biofertilizers contain mycorrhizal fungi, which form

mutualistic associations with plant roots and help in nutrient uptake, especially phosphorus.
Examples of mycorrhizal fungi include Glomus and Rhizophagus.

6. Vermicompost: This is organic matter that has been decomposed by earthworms,

resulting in a nutrient-rich fertilizer that improves soil structure, water retention, and nutrient
availability.

7. Algal Biofertilizers: These are derived from various types of algae, such as seaweeds
and cyanobacteria, which are rich in nutrients like nitrogen, phosphorus, potassium, and
micronutrients essential for plant growth.

8. Compost-Based Biofertilizers: These are produced from composted organic materials,

such as kitchen scraps, yard waste, and manure, providing a balanced mix of nutrients and
improving soil health.

9. Fish Amino Acids (FAAs): These are derived from fish waste or byproducts,
containing amino acids, proteins, and other nutrients beneficial for plant growth.

Biofertilizers are increasingly recognized as a sustainable substitute for chemical fertilizers in

modern agriculture, offering numerous advantages such as enhancing soil fertility, improving
nutrient availability, fostering plant growth and vitality, and mitigating environmental
impacts. Nevertheless, their adoption faces challenges, including efficacy variability, limited
awareness, high production costs, competition with chemical counterparts, and environmental
concerns. Overcoming these hurdles necessitates ongoing research, innovation, standardized
production methods, supportive policies, farmer involvement, and integration with other
sustainable agricultural approaches.

By bolstering soil health, curbing chemical inputs, conserving resources, and championing
environmental stewardship, biofertilizers hold promise for sustainable agriculture. However,
optimizing their formulations, bolstering efficacy and stability, ensuring safety, and fostering

Isolation and Identification 3

environmental sustainability require further research and development. Farmer education,
extension services, and participatory research can bolster knowledge, awareness, and
adoption of biofertilizers. Enabling policies and regulations can create conducive
environments for their use, fostering collaboration among academia, industry, and farmers.

With concerted efforts in research, innovation, education, and policy backing, biofertilizers
can integrate seamlessly into mainstream agricultural practices, paving the way for more
sustainable and eco-friendly agricultural systems.

Isolation and Identification 4

 Chapter 2
LITERATURE REVIEW
2.1 Cyanobacteria

Cyanobacteria, a diverse group of Gram-negative photosynthetic prokaryotes, inhabit

virtually every corner of the Earth, thriving in a wide range of environments, from rocky
shores to hot springs, and even in extreme conditions like drought, desiccation, and high
salinity. They withstand various stresses including UV radiation, temperature fluctuations,
anaerobic conditions, and nitrogen depletion. Integral to global nutrient cycling,
cyanobacteria possess the remarkable ability to convert atmospheric CO2 and N2 into organic
compounds using the enzymes Rubisco and nitrogenase, respectively. Some species can fix
nitrogen via specialized cells called heterocysts.

Once disregarded or viewed as nuisances, cyanobacteria are now acknowledged for their
potential in biotechnological applications. Establishing pure cultures of cyanobacteria, known
as axenic cultures, is crucial for studying their physiology, genetics, and taxonomy. However,
this process involves labor-intensive techniques like single-cell isolation, UV irradiation,
filtration, antibiotic treatment, and density gradient centrifugation, with success rates varying
by strain. To grasp their ecological roles and exploit their biotechnological potential, it's vital
to estimate and conserve cyanobacterial diversity in unexplored habitats through systematic
surveys, collection, and characterization of pure cultures.

Identifying cyanobacteria in nature is often feasible based on their distinctive colors, such as
green, blue-green, or olive green. Nevertheless, microscopic examination and pigment
analysis are commonly necessary for precise identification. Tropical conditions, such as those
in India, provide favorable environments for the luxuriant growth of cyanobacteria in various
ecosystems, including soil, freshwater bodies, oceans, saline backwaters, estuaries, and
saltpans. Although studies on cyanobacteria have been carried out in the Western Ghats
region of Maharashtra in India, they have mostly been restricted to freshwater and paddy
fields, with limited studies on cyanobacteria from soil samples. Pune, located in Maharashtra,
India, has favorable climatic conditions for the growth of cyanobacteria, with three distinct
seasons (summer, monsoon, and winter) and an average temperature ranging from 20°C to

Isolation and Identification 5

28°C. The present study aims to investigate the diversity and distribution of cyanobacteria in
Pune, Maharashtra, India, with a focus on soil samples. This study will contribute to the
understanding of cyanobacterial biodiversity in this region and their potential
biotechnological applications. Soil samples were collected from 5-10 cm deep soil layers
using scalpels by inserting them into the soil, making circles, and lifting them up. Random
collections were made from different spots in a locality to maximize diversity. Soil samples
were collected in labelled polythene zip pouches and information such as location, habitat,
date of collection, temperature, and soil type was recorded. Fresh biomass attached to rocky
substratum was also collected and stored in labelled family plastic bottles. The collected soil
samples were shade dried and stored in wide-mouth screw-cap glass bottles for further
isolation and identification in the laboratory. The isolation of individual species of
cyanobacteria from crude samples is crucial for establishing pure cultures, as field samples
may contain multiple cyanobacterial species as well as other bacterial and fungal spores.
Mechanical separation is considered the most effective method for obtaining pure cultures of
cyanobacteria by isolating them from other organisms.

A total of 227 cyanobacterial samples were collected from 45 localities in Ahmednagar,

Pune, and Satara districts of Maharashtra state. Among the collected samples, 20
cyanobacterial species belonging to 13 genera, 8 families, and 3 orders were identified.
Nostocales was the dominant order, occurring in 85% of the samples, with the family
Nostocaceae being the most frequently observed family, occurring in 40% of the samples.
Among the 13 genera, Nostoc was the most dominant, occurring in about 38.46% of the
samples, followed by Anabaena (23.08%). The highest relative abundance was observed in
Nostoc calcicola, which was found in 131 samples with a relative abundance of 57.71%.
Nostoc punctiforme was found in 119 samples with a relative abundance of 52.42%, followed
by Nostoc entophytum in 102 samples with a relative abundance of 44.93%. The least
relative abundance (4.41%) was observed in Hapalosiphon welwitschii, which was found in
only 10 soil samples out of the collected samples. The study conducted in the Ahmednagar,
Pune, and Satara districts of Maharashtra state in India collected a total of 227 cyanobacterial
samples from 45 localities. Among these samples, 20 cyanobacterial species were identified,
belonging to 13 genera, 8 families, and 3 orders. The dominant order was Nostocales,
accounting for 85% of the frequency, with the family Nostocaceae being the most dominant
family at 40% frequency. Stigonematales and Chroococcales challenge and obtain pure
cultures of cyanobacteria for research and other applications.

Isolation and Identification 6

2.2 Potassium solubilizing bacteria

Potassium plays a vital role in facilitating the metabolic and physiological functions of plants,
while also bolstering their resilience against both biotic and abiotic stressors. In soil
environments, the majority of potassium (approximately 90-98%) exists in non-exchangeable
mineral forms. However, certain rhizobacteria possess the capability to break down these
minerals, converting them into soluble potassium forms that are readily accessible to plants.
This transformative process, referred to as solubilization, is orchestrated by potassium
solubilizing bacteria (KSB). These organisms are increasingly being commercialized as
biofertilizers, offering an eco-friendly alternative to synthetic fertilizers. Similarly, nitrogen-
fixing rhizobacteria (NFR) are also gaining prominence for their contributions to sustainable
food production systems worldwide.

Apart from solubilizing potassium, these bacteria also possess plant growth promoting traits.
They produce plant growth hormones, siderophores, antibiotics, and can solubilize phosphate,
zinc, and potassium in soil, thereby accelerating plant growth and development. This makes
them promising candidates for sustainable farming practices as they can replace synthetic
insecticides and promote protection against phytopathogens. The diversity of plant growth
promoting rhizobacteria (PGPR) strains is vast, with various genera such as Aeromonas,
Agrobacterium, Bacillus, Pseudomonas, Rhizobium, and Streptomyces, among others,
reported to exhibit PGP traits.
The present study aims to isolate and characterize bacterial isolates from rhizospheric soil for
their potassium solubilization efficiency and PGP traits. This research has the potential to
contribute to the development of eco-friendly and sustainable strategies for improving crop
yields, promoting plant growth, and enhancing plant resistance against pathogens, ultimately
supporting the sustainable production of food. The spread plate method was used to isolate
bacteria from the rhizospheric soil of Solan, Himachal Pradesh using NA plates. The colonies
were selected, purified, and stored for further study. The best potassium solubilizing bacteria
were identified through 16S rRNA sequencing. In this study, 30 different bacterial strains
were isolated from rhizospheric soil, and two of them, AKY2 and HPY10, showed potassium
solubilization ability on Aleksandrow agar plates with solubilization zones of 10 mm and 22
mm, respectively, after 5 days of incubation

Isolation and Identification 7

The potassium solubilization efficiency (KE) of AKY2 and HPY10 was measured as 2.6 and
23.2, respectively. The quantitative estimation of potassium released by these isolates using a
flame photometer showed that AKY2 released 7.29 mg/L and HPY10 released 8.66 mg/L of
potassium after 10 days of incubation. The 16S rRNA sequencing of HPY10 identified it as
Serratia marcescens. Previous studies have shown that other bacterial isolates could solubilize
potassium minerals in liquid Aleksandrov broth medium, releasing 13.71 to 23.88 mg/L of
potassium. The potassium solubilization zones on silicate culture media ranged from 0.65 cm
to 1.50 cm depending on the isolate. This study provides evidence of the potassium
solubilization ability of bacterial isolates AKY2 and HPY10 and their quantitative estimation
of potassium release. The identification of HPY10 as Serratia marcescens through 16S rRNA
sequencing adds to the understanding of the bacterial species involved in potassium
solubilization. Further research on the characterization of these bacterial isolates for their
plant growth promoting (PGP) traits, such as the production of plant growth hormones,
siderophores, and antibiotics, can provide insights into their potential as biofertilizers for
sustainable agriculture. In this study, bacterial strain AKY2 was found to exhibit multiple
plant growth promoting traits, including ammonia production, antagonistic activity against
Fusarium oxysporum, zinc solubilization, hydrogen cyanide (HCN) production, indole-3-
acetic acid (IAA) production, and a phosphate solubilization index of 3.4. AKY2 was also
capable of growing at 2% and 4% NaCl concentrations. Both AKY2 and HPY10 showed
excellent potassium solubilization ability at pH 7.

Chapter 3

Isolation and Identification 8

 Materials and methods:

3.1 Soil sample

Approximately 100 g of two soil samples were taken from the rhizosphere of various plants
present in the same stretch of land. The sealed soil sample and the liquid sample were
immediately placed in a refrigerated container. Each sample was used to isolate the Bacteria
after bringing it back to the laboratory. The samples were serially diluted in Phosphate Buffer
Saline (PBS) in dilutions of 10-1, 10-2, 10-3, 10-4 and 10-5 to make the samples workable
and get distinct colonies of the microorganisms.

3.2 Physical and Chemical properties of the samples

The soil sample collected had a gravel-like coarse texture, with low organic matter content.
The soil can be classified as lateritic soil which is generally high in iron content. The soil was
very porous making it less resistant to drought. The soil sample had a pH of 6.

3.3 Culture medium

Aleksandrow Agar consisted of Magnesium sulphate 0.500 g/L, Calcium carbonate 0.100
g/L, Potassium aluminosilicate 2.000 g/L, Glucose 5.000 g/L, Ferric chloride 0.005 g/L
Calcium phosphate 2.000 g/L, Agar 20.000 g/L, Final pH ( at 25°C) - 7.2±0.2

BG-11 agar media consisted of Sodium nitrate (NaNO3) 1.500 g/L, Dipotassium hydrogen
phosphate (K2HPO4) 0.040 g/L, Magnesium sulfate, heptahydrate (MgSO4.7H20) 0.075 g/L,
Calcium chloride dihydrate 0.036 g/L, Citric acid 0.006 g/L, Ferric ammonium citrate 0.006
g/L, EDTA, disodium salt 0.001 g/L, Sodium carbonate 0.020 g/L, Trace metal mix 1.000 ml.
Trace metal mix has the following composition of Boric acid 2.860 g/L, Manganese chloride,
tetrahydrate 1.810g/L, Zinc sulfate, heptahydrate 0.222 g/L, Sodium molybdate, dihydrate
0.390 g/L, Copper sulfate, pentahydrate 0.079 g/L, Cobalt nitrate, hexahydrate 0.0494 g/L,
Final pH ( at 25°C) - 7.10

3.5 Isolating and determination of Bacteria present in soil.

Isolation and Identification 9

For the isolation of cyanobacteria, the soil sample and the liquid sample were homogenized
in sterile distilled water and serially diluted. Aliquots of each dilution were spread on BG-11
agar medium and incubated at 30◦C for 24 - 48 hours. Colonies were selected carefully from
the plates; the colonies were further purified by streaking each colony-forming unit separately
on another BG-11 media.

3.6 Morphological identification

The morphology of the colony on culture medium plates was studied.
The shape, margin, color, surface, etc., were observed and noted down respectively.

3.7 Biochemical identification

The following tests were performed for the identification of bacteria
1. Catalase test - The catalase test is a laboratory test used to determine the presence of
catalase enzyme in a bacterial or biological sample. Catalase is an enzyme that helps to break
down hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). This enzyme is
produced by many aerobic and facultatively anaerobic bacteria as a defense mechanism
against the toxic effects of hydrogen peroxide.

2. Oxidase test - The oxidase test is used as a presumptive test to help identify bacteria based
on their ability to produce cytochrome c oxidase enzyme. This enzyme is found in the
electron transport chain of aerobic and facultative anaerobic bacteria, and its presence or
absence can be used as a characteristic to differentiate between different bacterial species or
groups. For example, Gram-negative bacteria such as Pseudomonas, Neisseria, and Moraxella
are known to be oxidase-positive, while many Enterobacteriaceae, which are also Gram-
negative bacteria, are oxidase-negative.

3. Indole test - is used to determine the ability of bacteria to produce indole, a metabolic by-
product of tryptophan metabolism. Tryptophan is an amino acid that can be found in many
proteins and is an essential component of the bacterial growth medium. The indole test is
commonly used as a part of the identification scheme for enteric bacteria, which are a group
of Gram-negative bacteria that commonly inhabit the intestines of humans and animals.

4. Citrate utilization test - is used as a presumptive test to help identify bacteria based on their
ability to utilize citrate as the sole carbon source for growth. Some bacteria can transport and

Isolation and Identification 10

metabolize citrate, while others do not. This test is commonly used as a part of the
identification scheme for enteric bacteria, which are a group of Gram-negative bacteria that
commonly inhabit the intestines of humans and animals.

Chapter
4

Results & discussions

Isolation and Identification 11

The observations and results from the various tests performed were noted.

Fig 1.1: – The BG-11 agar plate showing the bacterial colonies

Some of the colonies were obtained for the BG-11 agar plate. Individual colonies were then
again inoculated on separate agar plates to get a colony of single strain of cyanobacteria.

4.1 Colony morphology

A brief study on the characteristics of the morphology of the various bacterial colonies was
carried out.

Attributes Observations
Bg11 Aleksandrow
Whole Colony Circular Circular
Colony edge Entire Entire
Colony surface Smooth Glistening Smooth Glistening
Colony elevation Raised flat Convex

Table 1.1: study of the colonial morphology of the bacterial samples

4.2 Catalase Test

Isolation and Identification 12

The catalase test was conducted using freshly prepared 3% hydrogen peroxide solution to
ensure accuracy. Bacterial cultures were inoculated onto clean glass slides or into test tubes
containing sterile water using sterile loops or swabs, ensuring the cultures were pure and
well-isolated. Hydrogen peroxide solution was then added directly onto the bacterial cultures
using droppers, and observations were made immediately for bubbling. Positive catalase
activity was indicated by the rapid formation of bubbles within a few seconds of hydrogen
peroxide addition

4.3 Citrate utilisation Test

In conducting the citrate utilization test, we first prepared Simmons Citrate Agar as per the
manufacturer's instructions. This medium, containing citrate as the sole carbon source and
bromothymol blue as a pH indicator, was crucial for our investigation. Using a sterile loop,
we inoculated the surface of the agar plate with a well-isolated bacterial culture, ensuring
even distribution across a portion of the plate. After proper inoculation, we incubated the
plate aerobically at 37°C for 24-48 hours to provide optimal conditions for bacterial growth
and citrate utilization. Following the incubation period, we carefully examined the plate for
signs of bacterial growth and any color changes in the medium. A color change from the
initial green to blue indicated citrate utilization by the bacteria.

4.4 Indole Acetic Acid test

The Indole acetic acid test was performed to show that the bacteria, which is being isolated
from the soil sample can produce the plant hormones IAA (Indole acetic acid) and hence may
serve as a potent biofertilizer. For the test, we are going to use bacteria isolated and grown
the Aleksandrow’s agar media plates since the potassium solubilizing bacteria has been
previously shown to possess the property of producing indole acetic acid as an extracellular
product ( S.C.Kammar and R.C.Gundappagol, 2016 ).

For performing the test, the Salkowski reagent was used which is a mixture of a mixture of
0.5 M ferric chloride (FeCl3 ) and 35% perchloric acid (HClO4), which upon reaction with
IAA yields a pink colored solution due to the formation of a complex with and reduction of
ferric ion ( Kamnev et al., 2001).

The cells were allowed to grow in a suspension media of Luria Bertani broth and incubated at
36 degrees Celsius for 72 hours. The cell-containing media was then centrifuged at 10000rpm
for 10 mins and the supernatant was used as the substrate for the identification reaction. The
supernatant and Salkowski reagent were mixed and kept in the dark for an hour to let the

Isolation and Identification 13

reaction take place. After completion the characteristic pink color of the IAA and ferric
complex was observed, concluding that the result was positive.

4.5 Results of the Biochemical Test

Several tests were performed on the isolated bacteria in BG11 to gain a better insight into the
various metabolic processes and characteristics of the bacteria. Catalase test was important
because nitrogen-fixing bacteria require that enzyme to protect the ROS (reactive oxygen
species) sensitive enzymes necessary for nitrogen fixation. The Oxidase test showed that the
bacteria isolated were anaerobic in nature. A positive result in the citrate utilization test for
bacteria can be conclusive that the bacteria may be able to respond to stress when
conventional sources of carbon are scarce for consumption. Positive indole test showed the
ability of bacteria to produce indole.

Biochemical Tests Results

Catalase Test +Ve
Oxidase Test -Ve
Citrate utilisation +Ve
Indole Test +Ve
Table 1.2: Shows the result of the various biochemical tests performed on bacteria

Fig 1.2: shows the pink-colored solution indicating a positive result for the indole acetic acid test

Fig 1.3: Negative result of oxidase test

Isolation and Identification 14

 Fig 1.4: Positive result of Citrate test

4.5 Discussions
A brief study on the characteristics of the colony morphology of the various bacterial
colonies was also carried out. Based on the results of the various tests conducted, including
catalase, oxygenase, citrate utilization, and indole tests, it can be inferred that the isolated
bacteria exhibit characteristics consistent with certain types of bacteria other than
cyanobacteria. The presence of catalase activity suggests their potential to protect reactive
oxygen species (ROS)-sensitive enzymes required for various physiological functions.
Additionally, the absence of oxidase activity indicates an anaerobic nature, which may be
characteristic of certain bacterial groups. The positive indole test confirms the bacteria's
capability to produce indole, further supporting their identification as a specific type of
bacteria. However, the positive result in the citrate utilization test suggests the bacteria may
belong to a group capable of utilizing citrate as a carbon source, which is not typical of
cyanobacteria. Therefore, while these results provide valuable insights into the properties of
the isolated bacteria, further analysis is needed to definitively identify their taxonomic
classification.

Isolation and Identification 15

 Chapter
5

CONCLUSION AND SCOPE OF

FUTURE WORK
5.1 CONCLUSION
In this work, the various methods and techniques for the isolation of bacteria from soil
samples which has been fertilized using biofertilizers were performed. The importance of
agriculture and bringing about a change in the fertilizer scenario needs to change, due to the

Isolation and Identification 16

increasing amount of stress chemical fertilizers induce in the soil biome. Various studies have
shown that chemical fertilizers tend to bioaccumulate which causes some serious health
issues in the human body.

The study involved a comprehensive examination of the colony morphology of various

bacterial colonies, alongside conducting tests including catalase, oxygenase, citrate
utilization, and indole tests. The results suggest that the isolated bacteria exhibit
characteristics consistent with certain bacterial groups other than cyanobacteria. Catalase
activity indicates their potential to protect enzymes sensitive to reactive oxygen species,
while the absence of oxidase activity suggests an anaerobic nature, characteristic of specific
bacterial groups. Positive indole test results confirm the bacteria's ability to produce indole,
supporting their identification. However, the positive outcome in the citrate utilization test
implies potential membership in a group capable of citrate utilization, contrary to
cyanobacteria traits. Further analysis is warranted to definitively classify these bacteria.

Cyanobacteria also help in enriching the soil around it by the formation of various
compounds which promote plant growth. As they were able to grow in BG 11 plates it can be
inferred that they can grow in soils where soluble forms of nitrogen are below optimum and
hence help in enriching the said soil. The Indole Acetic Acid test was performed to show that
the bacteria, which is being isolated from the soil sample, can produce the plant hormones
IAA (indole acetic acid) and hence may serve as a potent biofertilizer. The test came out
positive showing that the bacteria isolated by us can indeed prove to be very useful as a
biofertilizer.

5.2 SCOPE OF FUTURE WORK

In future studies, an important avenue for exploration lies in conducting confirmatory tests to
definitively identify the bacterial culture as cyanobacteria. While the results of the catalase,
oxygenase, citrate utilization, and indole tests provided valuable insights into the properties
of the isolated bacteria, further confirmation is necessary to ascertain their taxonomic
classification. One potential confirmatory test could involve analyzing the presence of
characteristic pigments, such as phycocyanin and chlorophyll, which are unique to
cyanobacteria. Spectrophotometric analysis or chromatography techniques could be

Isolation and Identification 17

employed to detect and quantify these pigments in the bacterial culture. Additionally,
molecular methods, such as polymerase chain reaction (PCR) targeting specific
cyanobacterial genes or 16S rRNA sequencing, could be utilized to identify genetic markers
unique to cyanobacteria. By integrating these confirmatory tests into future research
endeavors, we can enhance the accuracy and reliability of bacterial identification, paving the
way for a more comprehensive understanding of microbial diversity and ecological roles in
various environments.

We can always expand the scope of this project and further our research in the future to better
help the farmers with a cheap and ecologically favorable alternative and assist our society
combat hunger.

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Isolation and Identification 18

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