ISOLATION AND IDENTIFICATION OF MICROBES

FROM CHEMICAL FERTILIZER CONTAMINATED SOIL

COLLECTED FROM JAGATSINGHPUR DISTRICT

A Dissertation submitted to

Department of Botany

CHRIST COLLEGE, CUTTACK

Affiliated to

UTKAL UNIVERSITY

for the partial fulfilment of [Link]. Degree in Botany

by

MADHUSMITA PATRA

Roll No: -10416C224007

Under the Guidance of

[Link] NAYAK
Lecturer in Botany

CHRIST COLLEGE,
CUTTACK-753008, ODISHA

1
 SESSION: 2022-2024

CERTIFICATE

This is to certify that the dissertation entitled “Isolation and

identification of microbes from chemical fertilizer contaminated soil
from Jagatsinghpur District” submitted in partial fulfilment of the
requirement for the award of the Degree of Master of Science in Botany
to Christ College, Cuttack, Odisha, is a faithful record of Bonafede
research work carried out by Madhusmita Patra, Roll NO. 10416C224007
under my guidance and supervision. No part of this work has been
submitted for any other Degree or Diploma.

Date: Signature of guide

2
 Declaration
I, Madhusmita Patra, hereby declare that the dissertation entitled
“Isolation and identification of microbes from chemical fertilizer
contaminated soil from Jagatsinghpur District” submitted to Christ
College, Cuttack in partial fulfilment of the requirements for the award of
the Degree of Master of Science in Botany is a record of original work
done by me during January-March 2024 under the supervision and
guidance of Mrs. Nibedita Nayak, Lecturer in Botany, Christ College,
Cuttack, Odisha.

Place: Christ College, Cuttack Signature

Date:

3
 ACKNOWLEDGEMENT
I would like to express my gratitude to all the people who have helped me to
complete my project successfully.

First and foremost, I would like to express my sincere gratitude to our

Principal Mrs. MADHUSMITA PATRA for giving me the opportunity and
the resources to conduct my research.

My Special thanks to Mr. SOVAN PANDA, Head of the Department for

his constant guidance, motivation and support.

With massive gratification, I wish to express my profound gratitude and

heartfelt thanks to my research supervisor, guide Mrs. Nibedita Nayak for
his valuable guidance, keen interest, constant encouragement and
cooperation throughout the course of the dissertation work despite his busy
schedule.

I am thankful to SBIO SCIENCE Pvt. Ltd. for helping me complete my

project.

I am also thankful to my other teachers of the Department for their valuable

suggestions, encouragement and beneficial appreciation during my
dissertation work.

My sincerest thanks go to my family members and friends for their

enthusiastic support in dissertation work.

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 CONTENTS
CHAPTER PAGE NO

1. INTRODUCTION
1.1 Types of Fertilizer
1.2 Fertilizers and Soil Pollution
1.3 Impact of Fertilizers as Pollutants
1.4 What is Microbes and its Type
1.5 Soil Microbes and Their Importance
1.6 Antibiotics

2. OBJECTIVE

3. REVIEW OF LITERATURE

4. MATERIALS AND METHODS

4.1 Isolation of Microbes
4.1.1 Sample Collection
4.1.2 Serial Dilution
4.1.3 Different Cultures
4.2 Identification of Microbes
4.2.1 Colony Morphology
4.2.2 Gram Staining
4.2.3 Biochemical Tests
4.3 Antimicrobial Activity
4.4 Biodegradation of Fertilizers

5. RESULT AND DISCUSSION

6. CONCLUSION

5
7. REFERENCES

ABSTRACT

6
 1. INTRODUCTION
Fertilizers are integral part of current agricultural production as they provide essential mineral
elements for positive crop growth and blossoming harvests. Even high yielding varieties of
crop plants possibly do not reach to their full potential without getting a balanced dose of
fertilizers. Fertilizers are broadly classified grouped in to two: (i) inorganic or chemical
fertilizers which include nitrogenous, phosphatic, potassic, and complex fertilizers and (ii)
organic fertilizers which comprise farm yard manure, bone meal, compost, green manure, etc.
The increasing population forced agriculture intensification accompanied with additional use
of fertilizers that have played vital role to meet the demand of food across the globe. During
1970s and 1980s, one-third of the increase in cereal production worldwide and half of the
increase in India’s grain production have been attributed to increased fertilizer consumption
(FAO, 2013). It has been reported that to feed 6127.7 million populations in the year 2000 the
consumption of nitrogen (N), phosphorus (P), and potassium (K), the key components of
inorganic fertilizers, was 64.9, 25.9, and 18.2 kg ha_1, respectively, which increased to 85.8,
33.2, and 20.4 kg ha_1, respectively in the year 2014 when the world population reached
7243.8 million (FAO, 2013). Moreover, total fertilizer nutrient (NþP2O5þK2O) consumption
was estimated at 170.7 and 175.7 million tons in 2010 and 2011, respectively. The extent to
which the world food production relies on the application of fertilizers can easily be
understood as the estimated consumption of N, P, and K fertilizers is expected to increase
from current consumption levels by 172%, 175%, and 150%, respectively by 2050. World
fertilizer production is accompanied by an abrupt increase in the proportion of urea in world
N production that comprises roughly 40% of all N fertilizers produced. This boost in fertilizer
consumption has resulted in a shift in the nutrient composition of runoff and leaching leading
to deterioration of soil health. [1]

1.1 Types of Fertilizers:

On the basis of the nature of the elements present Fertilizers are of following
types:

[Link] Fertilizers: These types of fertilizers generally supply nitrogen to the soil.

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Examples - Ammonium sulphate {(NH4)2 SO4} Calcium ammonium nitrate (CAN), basic
Calcium ammonium nitrate (Ca (HD), CaO), Calcium cyanamide (CaNCN), Urea etc.

2. Phosphorus Fertilizers: These fertilizers provide phosphorus to the soil. Examples: Super
phosphate of Lime, triple super phosphate, phosphate slag. Ammoniated phosphates, Nitro
phosphates etc.

3. Potash Fertilizers: These fertilizers provide potassium to the plants. Examples: Potassium
chloride, Potassium sulphate, Potassium nitrate etc.

4. NP Fertilizer: These fertilizers contain two elements i.e., nitrogen and phosphorus. These
are formed by mixing together both the fertilizers.

Examples: Dihydrogen ammoniated phosphate NH HIPO and calcium Super phosphate

Ca(H2PO4)2.

5. NPK or Complete Fertilizers: These types of fertilizers provide all the three essential
elements viz Nitrogen, phosphorus and potassium to the soil. It is obtained by mixing all the
three types of fertilizers in Suitable proportions.

Fertilizer can also be classified on the action of these fertilizers on the plant. This
classification is as follows:

1. Direct Fertilizers: These include ammonium and potassium salts, nitrates,

superphosphates etc. which are directly utilized by the plants.

2. Stimulant Fertilizers: These types of fertilizers improve the condition of the soil. When
they are added to the soil, it neutralizes and makes it loose. Examples: humus, lime, gypsum
etc.

3. Mixed Fertilizers: It contains two or three primary elements. When the fertilizing
materials mixed, it is known as mixed fertilizers. For Example: expression like 4-8-2 is used
for a mixed fertilizer containing 4% Nitrogen, 8% phosphorus and 2% potash.

1.2 Fertilizers and Soil Pollution:

Indiscriminate and long-term use of fertilizers has become a significant source of soil and
water pollution [2] which put the pristine terrestrial and aquatic ecosystems downstream and

8
human health at risk. Soils naturally contain heavy metals (HMs) such as cadmium (Cd),
mercury (Hg), arsenic (As), chromium (Cr), lead (Pb), etc.
These metals are non-biodegradable and build up to dangerous quantities over time.
Excess application of fertilizers aggravates the situation by lowering the soil pH which
facilitates HMs availability. Furthermore, fertilizers also contain a wide range of HMs from
the source materials; therefore, application of these fertilizers adds more HMs to the soils.
Excessive accumulation of HMs deteriorates the physical and biological characteristics of
soil and adversely affect plant growth, and physiological and biochemical processes leading
to the degeneration of organelles and cells that may result in plant death [3]. Consumption of
such crops and crop products facilitates the entry of metals in the food chain where they
impair our ecosystem, and human and animal health [4]. Apart from agriculture, fertilizers
are also extensively used to maintain and improve the beauty of home lawns, gardens, and
parks. Thus, urban soils in park and residential areas may also have a significant impact on
human health as they are directly exposed to HMs [5]. Continuous application of N fertilizers
may result in negative impacts on agro-ecosystem such as leaching, Pollution of water
resources, gaseous emissions to atmosphere thus causing irreparable dam- age to the Overall
ecosystem and environment. Similarly, phosphorus (P) is one of the major essential
macronutrients for biological growth and application of P fertilizers is indispensable
component for crop production. However, the availability of P to plants is a serious issue
because of its fixation and precipitation behaviour in soil which lowers the efficiency of
added P. It has been reported that more than 80% of applied P as fertilizers Precipitates in the
presence of metal ion complexes in soil [6]. The synthesis of calcium phosphate from
municipal wastewater and its application for the removal of metals from acidic effluents. In
addition to these constraints, the prices of P fertilizers jumped up Several folds during recent
years making P fertilizers not-affordable to a common resource poor farmer.

1.3 Impact of Fertilizes as Pollutants:

1. Degradation of Soil Quality –

Fertilizers pollute soil due to waste material released from raw materials used in
manufacturing process. Because metals are not biodegradable, they can contaminate soil by
accumulating in it as a result of excessive Phosphate fertilizer use. Because of the overuse of
NPK fertilizers, agricultural practises have been reduced. Wheat, maize, and gram grown in

9
such soils have lower protein content. Furthermore, the carbohydrate content of such crops
degrades in quality. When there is an excess of potassium in the soil, the vitamin C and
carotene Content of vegetables and fruits decreases. Excessive fertilizer use can alter soil
fertility and acidity levels. This Is why soil should be tested at least once every three years to
determine the exact amount of fertilizer to be Used. The loss of humus reduces the soil’s
ability to store nutrients.

2. Change in Biology of Water Bodies–

Eutrophication is caused by the overuse of fertilizers. Fertilizers contain chemicals such as

NO3 and PO4, which Are washed into lakes and oceans. These substances are toxic to aquatic
life because they promote the growth of Algae in bodies of water and reduce oxygen levels.
This results in a toxic environment and the extinction of aquatic life as well as other aquatic
animals and plants. It indirectly disrupts the food chain because different types of fish in
water bodies are the primary food source for many birds and animals in the environment.

[Link] on Human Health-

Fertilizers, which contain NZ and other chemicals, have an impact on ground water systems
and are used for drinking. One of the most common outcomes is the development of blue
baby syndrome, which occurs in Infants whose skin tissues are low in oxygen, causing their
skin to appear purplish or blue in colour. Various Studies have revealed that the use of lawn
fertilizers and pesticides can endanger one’s health. Cancer and Chronic diseases in humans,
particularly in children, are examples of these risks.

4. Climate Change–

Fertilizers contain substances and chemicals such as methane, carbon dioxide, ammonia, and
nitrogen, and the emission of these gases contributes to the presence of greenhouse gases in
the environment. As a result, global warming and weather changes occur. Carbon dioxide,
methane, and nitrous oxide are the three most Significant greenhouse gases, Nitrous oxide is
a nitrogen by-product.

1.4 Microbes:

Technically Microbe is an organism of microscopic size, which may exist in its single celled
form or as a Colony of cells. The possible existence of unseen microbial life was suspected

10
from ancient times, such as in Jain scriptures from sixth century BC India. The scientific
study of microorganisms began with their Observation under the microscope in the 1670s by
Anton van Leeuwenhoek. In the 1850s, Louis Pasteur found that microorganisms caused food
spoilage, debunking the theory of spontaneous generation.

In the 1880s, Robert Koch discovered that microorganisms caused the diseases tuberculosis,
cholera, Diphtheria, and anthrax. Some are pathogenic and many are beneficial Because
microorganisms include most unicellular organisms from all three domains of life, they ca be
extremely diverse. Two of the three Domains, Archaea and Bacteria, only contain
microorganisms. The third domain Eukaryota includes all multicellular organisms as well as
many unicellular protists and protozoans that are microbes. Microorganisms can have very
different habitats and live everywhere from the poles to the equator, deserts, geysers, rocks
sand the deep sea. Some are adapted to extremes such as very het or very cold conditions [7].

1.5 Soil Microbes and Their Importance:

Soil ecosystems are highly complex, as they contain a large number of microbial species, Soil
Microorganisms are essential for plant nutrient cycling and decomposition of organic matter
[8]. However, environmental conditions and disturbances are likely to influence the microbe.
Population structure and their function in soils, which could alter the soil characteristics [9].
All these microbes are beneficial for building soil health, structure and fertility. Soil microbes
occur enormous biodiversity in the soil and are responsible for the majority of soil enzymatic
processes in soil they also store energy and nutrients in their biomass.

The soil is more than simply a source of nutrients to plants. It is a complex ecosystem of soil-
dwelling Organisms. Plants exhibit a diverse array of interactions with these microbes and
other Irving organisms thus, they improve plant nutrition and health and improve the quality
of the soil. This important activity takes place in the rhizosphere. The rhizosphere is an
intense hub of activity in the soil. In this root zone around plants, plant roots feed nearby sol
microbes in exchange for plant nutrients. In this way, the rhizosphere is the “bridge” where
unavailable minerals are turned into plant nutrients. Plants need the minerals found in the soil
to grow, photosynthesis, flower, pollinate and produce fruit or seeds. Although soils are
composed of around 45% minerals, most of those minerals are not in a form plant They can
use Soil microbes are the bridge between the soil minerals and the plant roots:

11
 Nitrogen - Although plants are surrounded by nitrogen in its gaseous form in the
atmosphere. Only microbes are able to turn it into a usable form. Microbes also transform soil
organic nitrogen into mineral nitrogen (ammonium and nitrate) that plants can take up.

 Sulphur - It takes soil microbes to change organic sulphur in the soil into plant- available
sulphate.

 Phosphorus - Organic phosphorus is mineralised to plant-available forms only through

microbial activity, many other micronutrients also benefit from the microbial bridge to make
them plant- available. With nutrient movement and nutrient holding capacity, microbes can
Change the form of a nutrient to make It plant-available; change a mineral’s acidity or
alkalinity; hold nutrients in a way that makes it easier for a Plant to take them up when
needed.

Generally, isolation and characterization of microbes from contaminated soil has been limited
depending upon the ability to culture microorganisms from environmental samples
unfortunately only a fraction of microorganisms can be cultured in the laboratory. We cannot
isolate all of them, it has been estimated that the microbial community in one gram sell, 4000
different DNA units can exist. Also, hardly 1% of microorganisms of soil can be cultivated
with classic laboratory techniques and unknown if this percentage is representative of total
microbial population [10].

1.6 Antibiotics:

Antibiotics, in one form or another, have been in use for centuries. The vast majority of novel
antibiotics have been detected by screening of “wild isolates” obtained from soil and other
natural habitats. Although a wide taxonomic range of microbes have the ability to produce
antibiotics. Thus, over 55% of the antibiotics detected between 1945 and 1978 originated
from the genus Streptomyces, representing a total of more than 5,000 compounds [11]. Drugs
that have been synthesized by chemical procedures in the laboratory are called synthetic
drugs while those produced by bacteria and fungi are called antibiotics [12]. The antibiotics
are widely distributed in the nature, where they play an important role in regulating the
microbial population of soil, water, sewage, and compost. Those that are currently of greatest
use have derived from a relatively small group of microorganisms belonging to the genera
Penicillium, Streptomyces, Cephalosporium, Micomonospora and Bacillus [13]. Antibiotics
are low molecular-weight (non-protein) molecules produced as secondary metabolites,

12
mainly by microorganisms that live in the soil. While many antibiotics are known to exist,
efforts to discover new antibiotics still continue. Therefore, many species such as
Streptomyces, Bacillus and Penicillium have been studied continuously for their ability to
produce antibiotics [14]. In addition, due to the fact that Bacillus species have produced
antibiotics in the soluble protein structure and that these antibiotics have been found to be
cheaper and more effective in studies conducted to date, these microorganisms are preferable
for commercial production.

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 2. OBJECTIVE

 To isolate a diverse range of microorganisms from soil contaminated with chemical

fertilizers, aiming to elucidate the microbial community composition and diversity
in response to anthropogenic pollution.

 To identify and characterize the isolated microorganisms using molecular and

biochemical techniques, facilitating the understanding of their metabolic pathways
and potential interactions with chemical pollutants present in the soil.

 To assess the bioremediation potential of the isolated microorganisms through

laboratory scale experiments, evaluating their ability to degrade or detoxify chemical
contaminants, commonly found in fertilizer-contaminated soil, thus contributing to
the development of sustainable remediation strategies for polluted environments.

14
 3. REVIEW OF LITERATURE
The potential of microbial isolates to biodegrade agricultural soil artificially contaminated
with Dichlorvos pesticide was studied by S. E. Agarry et al. in 2013. They isolated four
strains of bacteria i.e. proteus vulgaris, vibrio sp., serrati asp. and Acinetobacter sp. And
evaluated in order to discover their ability to biodegrade dichlorvos pesticide in medium
supplied with different nutrients (NH4NO3, KH2PO4 and NPK ([Link]) fertilizer). The
result showed that the bacteria isolates were able to grow in nutrient medium containing
Dichlorvos as the only carbon source [15].

Jiangwei et al. in 7 October 2010 isolated a strain ZHU-1 capable of utilizing chlorpyrifos as
the sole carbon sources and energy from soil. ZHU-1 was identified as Bacillus licheniformis
based on analysis of morphology, physiological and biochemical characters and 16S rRNA.
The addition of ZHU-1 to soil treated with chlorpyrifos resulted in a higher degradation rate
than non-inoculated soils, the degradation rate of chlorpyrifos (100 mg kg-1) could reach
99% or above after 14 days. The microbial manure added by strain ZHU-1 can be applied not
only as fertilizer, but also in degrading chlorpyrifos residue in soil. This study may provide
basis for prevention and control of pesticides pollution [16].

Vasvi Chaudhry et al. in 3 February 2012 studied to characterized and compare microbial
community structure, diversity and bacterial phylogeny from soils of chemically cultivated
land (CCL), organically cultivated land (OCL), and fallow grass land (FGL) for 16 years and
were under three different land use types. As a result, they saw that, Proteo bacteria,
Bacteroidetes, and Gemmatimonadetes were found to be significantly abundant in OCL soil.
On the contrary, Actinobacteria and Acidobacteria were significantly abundant in CCL and
FGL, respectively. Their findings supported the view that organic compost amendment
(OCL) activates di verse group of microorganisms as compared with convention ally used
synthetic chemical fertilizers [17].

15
Jean Louise cocsondamo et al. in march 3, 2022 collected soils planted with rise from
different regions of Japan. Soil P was sequentially fractionated using the Hedley method.
iPSB were isolated using selective media supplemented with tricalcium phosphate (Ca-P),
aluminium phosphate (Al-P), or iron phosphate (Fe-P). Representative isolates were selected
based on the P solubilization index and soil sampling site. Identification was performed using
16S rRNA and rpoB gene sequencing. Effectiveness was screened based on rice cultivar
Koshihikari growth supplemented with Ca-P, Al-P, or Fe-P as the sole P source. Despite the
relatively homogenous soil pH of paddy field sources, three sets of iPSB were isolated,
suggesting the influence of fertilizer management and soil types. Most isolates were
categorized as β-Proteobacteria (43%). To the best of our knowledge, this is the first study to
describe the genera Pleomorphomonas, Rhodanobacter, and Trinickia as iPSB. Acidovorax
sp. JC5, Pseudomonas sp. JC11, Burkholderia sp. JA6 and JA10, Sphingomonas sp. JA11,
Mycolicibacterium sp. JF5, and Variovorax sp. JF6 promoted plant growth in rice
supplemented with an insoluble P source. The iPSBs obtained may be developed as microbial
inoculants for various soil types with different P fixation capacities [18].

Sri widawati et al. in December 20,2018 isolated indigenous nitrogen fixing bacteria and
evaluated its potential. Twenty-five isolates were obtained, and eight of them (Azospirillum
sp., Azospirillum lipoferum, Azotobacter chroococcum, A. paspalii, and Rhizobium sp.) were
identified as nitrogen-fixing bacteria. A greenhouse experiment was conducted using factorial
completely randomized design with three replications. The first factors were fertilizers, i.e.,
NPK; A. lipoferum CBT4 + NPK; A. lipoferum CBT4; and without fertilizer (control). The
second factors were soil types, i.e., A (fertile soil from Cibinong), B (soil from Bangka
Botanical Garden), C (soil from post tin mines two years after mining), and D (soil from
active tin mining). Result showed that Azospirillum lipoferum CBT4 isolated from C (soil
from post tin mines two years after mining) exhibited the highest IAA, Ca-P solubilizing
ability, and PME-ase activity. This species survived up to a population of 107 CFU/gram soil
in the three types of post tin mining soils and could be a potential plant-growth promoting
rhizobacteria (PGPR) species for effectively improving the growth of S. bicolour plant on
post tin-mining soil [19].

Mingchao Ma et al. in 2018 isolated 163 effective bacterial PGPR strain from rhizospheres of
plants in four provinces of China to investigate its effects on soybean growth and soil
bacterial community composition. According to capacities for mineral potassium and
phosphate solubilization, the best strain (designated 3016) was selected and identified as

16
Paenibacillus mucilaginosus based on biochemical characterization and phylogenetic
analysis. It showed a higher capacity for nitrogen fixation and phytohormone production than
commercial strains. They use P. mucilaginosus as an inoculant for seed dressing survived in
the soybean rhizosphere as revealed by a species-specific PCR method. Inoculation
significantly improved symbiotic nodulation, soybean growth parameters, nutrient contents
and yields. At last, they concluded that inoculation with P. mucilaginosus 3016 had beneficial
effects on both soybean growth and soil quality, and is a potential candidate for developing
commercial inoculants of PGPR to be used as a bio-fertilizer [20].

Zerihun Tsegaye et al. in march 2019 collected 426 samples of tef (Eragrostis tef)
rhizosphere soils from east Shewa zone, Oromia regional state. Out of which they isolate 200
morphologically different bacterial pure colonies and screened for their PGP traits and
biocontrol properties. Among these 40.5% isolates were positive for phosphate solubilization.
36% were positive for IAA production, 4.5% were positive for ammonia production, 19 %
were positive for (EXPS), 15.5% were positive for protease production, 12.5% were positive
for HCN productions, 9.5 % were positive for cellulase production, 4% were positive for
amylase production, 3.5% were positive for chitinase production. For abiotic stress tolerance
test, all of the isolates were grown well at 20oc and 30oc and neutral pH, 27% isolates were
grown well at 4oc, 25.5% grew at 40oc, 25.5% were grown well on pH-9 and pH-11, 23.5%
were tolerated pH-5, 3.5% grew at 50oc and 60oc, 13.5% were grown well on 5% NaCl
(w/v), 3.5% were grown well on 10 and 15% NaCl (w/v), which indicated these isolates can
survive in some extreme conditions. Totally 15 bacterial species having PGP traits, biocontrol
properties, and abiotic stress tolerance ability were identified using the Biolog bacterial
identification system. Among these, the majority of the identified PGPR have utilized
carbohydrate, carboxylic acid, and amino acid, which are the main components of plant root
exudates [21].

Md. Atikur Rahman et al. in December 2018 isolated six-different bacterial strains capable of
degrading Carbofuran, Emamectin Benzoate and Thiamethoxam from eight different soil
samples. The isolates were characterized by using different conventional and molecular
methods. The strains were identified molecularly into different genotypes using amplified
ribosomal DNA restriction analysis (ARDRA) and partial sequencing of 16S rDNA. The
ARDRA pattern clustered them into 3 groups. Among the isolates three were identified as
Achromobacter spp. and one as Diaphorobacter sp. by biochemical tests. It was further
confirmed by the partial 16S rDNA sequencing. The two identified potential bacteria can be

17
used for biodegradation of different pesticides which can have a significant environmental
impact in soil farm [22].

Md. Mahinur Rahman et al. in June 19, 2017 studied soil samples from Dhaka city were
collected from fish, vegetables and fruits dump area. They sub cultured bacterial population
in trypticase soya agar (TSA) plate. Nineteen colonies were isolated, cultured and
characterized by gram staining and biochemical tests. Six isolates were found to be gram
negative while thirteen were gram positive. All isolates were positive in oxidase, catalase,
citrate, and protease tests. Eight isolates showed coagulase negative and nine were coagulase
positive. It was found that all bacterial isolates were sensitive to tetracycline,
chloramphenicol, gentamycin, ciprofloxacin, azithromycin and ceftriaxone. About 95% of the
bacterial isolates were resistant to penicillin-G and ampicillin. About 89%, 26%, 21% and
11% of the bacterial isolates were resistant to amoxicillin, co-trimoxazole, nalidixic acid and
erythromycin, respectively [23].

Alemayehu Letebo Alebejo et al. in August 2017 isolated Rhizobium from yeast extract
mannitol agar medium. A total of 120 rhizobium were isolated from four samples. One isolate
from each sample was selected for further characterization. Isolate LLsm1, CPsm1, CPnm1
and Esm1 were found to be negative for MR-VP and starch hydrolysis test. All isolates were
found to be positive and negative for catalase and citrate utilization test respectively. They are
also found to be Gram-negative, rod-shaped morphology, fast grower and indole producers.
All the isolates were found with poor absorption of Congo red dye and no growth on the
YEMA with 2% NaCl. Therefore, all isolates were confirmed as Rhizobia and plant growth
promoting bacterial strains. These properties suggest that rhizobium isolated in this study
could find potential application for development of the sustainable agriculture as to be a good
candidate of biofertilizer which help in soil fertilization without applying chemical fertilizers
[24].

Thirty bacterial and ten yeasts isolates were obtained from PAH and PCB contaminated soil
by Vera M. Karličić1*et al. in 2016 with an aim of determining the presence of PGP
mechanisms. As a result, three bacterial (Serratia liquefaciens, Micrococcus sp. and Serratia
sp.) and two yeast isolates (Candida utilis and Candida tropicalis) were recognized as PGP
strains. Among them, Serratia sp. showed the highest indole production (25.5 µg/ml).
Analyses of metal tolerance (Cu+2, Cr+6 and Ni+2) revealed that Serratia liquefaciens,
Micrococcus sp., Serratia sp. and Candida tropicalis were capable to tolerate significant

18
concentration of metals. As a result of this study several bacterial and yeast strains were
attributed as potential plant growth promoters which can be applied in future remediation
activities and environmental quality improvements [25].

Jayaraj [Link]. in 10 July 2023 study bacteria from agricultural soil systems that have been
polluted with pesticides were isolated, identified, and their ability to tolerate pesticides was
examined. Target bacterial species were isolated from Psidium guajava (L) and Abelmoschus
esculentus (L) cultivating an agriculture field. From 10 distinct soil samples collected from an
agricultural field, 27 bacterial species were extracted, and the capacity of these
microorganisms to withstand pesticides was examined. Only three bacterial species (PRB-
S1P2, PRB-S1P3, and PRB-S6P1) are capable to grow on Nutrient agar medium with
different concentration of pesticides dimethoate, Thiamethoxam and Imidacloprid. Apart
from these three, one bacterial species was highly tolerant to all test pesticides. The highest
pesticide tolerant bacteria are Pseudomonas nitroreducens was identified through 16s rRNA
sequencing and the sequences were submitted to the NCBI with the accession No:
ON624333.1. Hence, the bacteria can be subjected to further study of its use in the field of
bioremediation [26].

Y. jayasri [Link]. in 10 march 2015 isolated pesticide degrading bacteria from ground nut crop
soil. The isolate was identified as Bacillus aerius based on its morphological, biochemical
and16Sr DNA analysis Pesticide degradation capability of the bacteria was screened on MSM
containing 25, 50, 75 and 100 mg of pesticide concentrations. Optimum degradation is up to
50mg of pesticide. Further bacterial optimal conditions were calculated based on different pH
and temperature measurements. The optimum conditions of the bacterium were found to be
30 to 35ºC and pH 5 to 6, where good tolerance against chlorpyrifos was observed. The study
shows elite bacterial isolate of optimal pH and temperature growth with pesticide degradation
[27].
Salam [Link]. in 2014 was isolated, purified, and characterize of soil isolates having
antimicrobial activity against Bacillus strains. Soil samples were serially diluted and plated
on isolation agar media. Potential colonies were screened, purified, and stored in glycerol
stock. Isolates were morphologically and biochemically characterized. Genomic DNA was
extracted from the identified isolate, and analysed using 16s rRNA sequencing. The sequence
analysis revealed of the strain to be Streptomyces, Bordetella and Achromobacter. The
culture isolate was grown in the production medium and then isolated with the antibiotic

19
compound. The compound isolated was tested for the antibacterial activity using the tube
method and Well plate method. The compound showed high potential of antibacterial activity
and the activity is dose dependent [28].

S.E. Agarry [Link]. in 2013 was isolated a bacterial consortium which degraded Dichlorvos
pesticide from agricultural soil using pour plate method. This consortium was composed of
four pure strains which were characterized based on their morphological and biochemical
characteristics. The strains were presumptively identifying as Proteus vulgaris, Vibrio sp.,
Serratia sp. and Acinetobacter sp. The consortium and the four bacteria were evaluated in
order to discover their ability to biodegrade Dichlorvos pesticide in medium supplied with
different nutrients (NH4NO3, KH2PO4 and NPK ([Link]) fertilizer). The results showed that
the bacterial consortium and the four bacteria isolates were able to grow in nutrient medium
containing Dichlorvos as the only carbon source. Moreover, the bacterial consortium was
able to remove greater amount of DDP in soil amended with inorganic fertilizer (NPK) than
those amended with NH4NO3 and KH2PO4, respectively. These results indicate that the
isolated strains can be used for waste biodegradation or bioremediation of organophosphate
pesticide- contaminated soil or water [29].

20
 4. MATERIALS AND METHODS
4.1. Isolation of Microbes:

4.1.1 Sample Collection-

Soil samples were collected from different agricultural plots. The samples were collected
from 15 cm depth from each plot with a stainless-steel soil prob. The soil cores from the same
plot were placed in a clean plastic bucket and mixed thoroughly to form a composite sample.
Composite samples were transferred immediately into sterilized polythene bags. once in the
laboratory, all visible roots and plant fragments were removed manually from the soil sample.
I collected four soil samples from four different field which are in Jagatsinghpur district. Two
samples collected from vegetable field, one sample collected from Rice field and another one
sample collected from moong dal field.

4.1.2 Serial Dilution-

A serial dilution is a stepwise series of dilutions that are performed to reduce the
concentration of a substance in a solution to a more usable concentration. Serial dilutions are
made by performing the same dilution multiple times, using certain amounts of previous
solutions added to the subsequent dilution. it is often used to avoid transferring very small
volumes to make a highly diluted solution.

To perform serial dilution the following steps are:

 seven test tubes were taken and arranged in a row and added 9ml of distilled water to
each tube.

21
  Marked the tubes as 10-1 up to 10-7. Added 1ml of sample in the 1 st test tube and mix
well by vertexing.
 Then 1ml sample taken from the 1st test tube and added it to the second test tube. The
step was repeated 7 times to reach 10-7 dilution.

[Fig.1: Serial Dilution of Soil Sample]

4.1.3 Different Culture:

(A) Primary Culture:

A mixed culture or primary culture is one that contains more than one type of organism
growing in a sterile medium, such as agar. It is the initial growth of microorganisms.

To perform primary culture the steps are as follows:

 At first, we have to prepare nutrient agar media and then autoclave it.
 Then pour the media in the petri plate inside laminar Air Flow Chamber.
 Wait for solidification for 30 to 45 minutes.
 Take 100 micro litre of the sample and spread on the media by using L –
spreader.
 At last, incubate the plate in the incubator for 24 hours.

(B) Sub Culture:

Sub culturing is a procedure of transferring of microorganisms into fresh nutritive medium

from its primary culture. This process allows for the propagation and purification of specific
microbial strains for further study.

22
To perform sub culture, we have to follow the following steps:

 At first, we have to prepare nutrient agar media and then autoclave it.
 Then pour the media in the petri plate inside laminar Air Flow Chamber.
 Wait for solidification for 30 to 45 minutes.
 After that take one colony from the primary culture with the help of inoculation
loop and streak on the sub culture plate.
 At last, incubate the plate in the incubator for 24 hours.

(C) Pure Culture:

Pure culture is a laboratory culture technique containing a single species of organism. A pure
culture is usually derived from mixed culture (one containing many species) by transferring a
small sample into new.

To perform pure culture the steps are as follows:

 At first prepare the nutrient agar media and autoclave it.

 Then pour the media inside the test tubes and keep it in slanting position and
wait for solidification.
 Streak the bacteria from subculture with the help of inoculation loop.
 Incubate it for 24 hours.

(D) Broth Culture:

In this method bacteria are growing in a liquid growth medium. they are used to grow and
maintain cultures for a laboratory. Different bacteria may grow differently in broth culture.

To perform broth culture the steps are as follows:

 First prepare a nutrient agar media and autoclave it.

 Then pour the media inside the test tubes and wait to cool down.
 Take one inoculum of pure culture and mixed with the broth media.
 At last, incubate it for 24 hours.

4.2 Identification of Microbes:

4.2.1 Colony Morphology:

23
Bacteria grow on a solid media as colonies. A colony is defined as a visible mass of
microorganism all originating from a single mother call; therefore, a colony constitutes a
clone of bacterial all genetically alike. Colony morphology is a method that used to describe
the characteristics of an individual colony of bacteria. It can be used to identify them.
Different types of bacteria will produce different looking colonies are circular in shape, and
others are irregular. A specific terminology is used to describe common colony types. These
are:

 FORM- It is defined as the basic shape of the colony. For example: round, irregular,
filamentous, rhizoid, punctiform.
 SIZE- The diameter of the colony. For example: Punctiform, small, moderate, large.
 ELEVATION- This describes the side view of a colony. For example: Flat, raised,
convex, umbonate, crateriform.
 MARGIN/BORDER – The edge of a colony. For example: entire, lobate, scalloped,
filiform, undulate, curled.
 SURFACE- The smoothness of surface of the colonies. For example: smooth, viscid,
mucoid, dry.
 OPACITY- For example: transparent (clear), translucent, opaque etc.
 COLOUR/PIGMENTATION- For example: white, buff, red, purple, etc.

24
 [Fig. 2: Morphological Characteristics of Bacterial Colonies]

4.2.2 Gram Staining:

Gram staining is a common technique used in microbiology to differentiate bacteria into two
groups based on the characteristics of their cell walls: Gram-positive and Gram-negative.
This staining method involves several steps:

1. Preparation of a bacterial smear: Culture from the solid media: using a sterile pipette,
add one drop of sterile saline or sterile water to the center of the microscopic slide.
Aseptically pick a small amount of an isolated colony with a loop and gently mix into the
drop of sterile /saline water using circular motions. Mix evenly to make a thin smear.

2. Fixation: The slide is gently heated or treated with chemicals to fix the bacterial cells in
place.

3. Staining: The slide is flooded with crystal violet stain, which binds to the peptidoglycan
layer of the bacterial cell wall in all cells.

4. Iodine Treatment: A mordant, usually iodine, is applied, which forms a crystal violet-
iodine complex within the cell wall, enhancing the retention of the stain.

5. Decolorization: The slide is washed with a decolorizing agent, typically alcohol or

acetone. This step differentiates between Gram-positive and Gram-negative bacteria. Gram-
positive bacteria retain the crystal violet-iodine complex due to their thick peptidoglycan
layer, while Gram-negative bacteria lose the stain because of their thinner peptidoglycan
layer and the presence of an outer lipid membrane.

6. Counterstaining: The slide is then treated with a contrasting stain, usually safranin or
fuchsine, which stains the decolorized Gram-negative bacteria pink or red, while the Gram-
positive bacteria remain purple.

By observing the color of the bacterial cells under a microscope, microbiologists can
determine whether the bacteria present are Gram-positive or Gram-negative. This information

25
can help in identifying and classifying the types of bacteria causing the spoilage. However,
Gram staining is mainly effective for bacteria and is not typically used for identifying fungi,
which have different cell structures and staining properties.

4.2.3 Biochemical Tests:

(A) Catalase Test:

Catalase is an enzyme that breakdown hydrogen peroxide in to water and oxygen. This test is
use to identify the organism is produced the catalase enzyme or not.

The catalase test can be performed as follows:

 Obtain a glass slide and a bottle of hydrogen peroxide.

 A small amount of organism is collected from a well isolated 18–24-hour
colony with sterile inoculating loop or wooden stick smear a small number
of bacteria onto the dry slide.
 However, no agar must be picked up with the colony, especially when the
culture is picked up from the blood agar.
 A drop of 3% H2O2 on to the organism on the microscope slide by using a
dropper.
 The formation of bubbles is observed against a dark background to enhance
readability. Bubbles= catalase positive. No bubbles = catalase negative.

(B) Starch Hydrolysis Test:

The starch hydrolysis test is also known as amylase test, is a biochemical test that is used to
determine the ability of bacteria to produce amylase and utilize starch as a carbon source.
This test is used to identify bacteria that can hydrolyze starch.

Composition of starch agar per 1000 ml:

Beef extract- 5gms, Starch- 2gms, Agar- 18gms, Peptone- 5gms, NaCl- 5gms

Preparation of starch agar:

26
  Measure the appropriate amount of Starch Agar media powder (25.0 grams per 1000
mL) and dissolve it in the water of the required volume in a conical flask (or glass
bottle).
 Stir well in a magnetic stirrer or manually and heat to boil to completely dissolve the
media powder.

 Autoclave the medium at 121°C and 15 lbs. pressure for 15 minutes.

 When the medium is cooled to around 40 to 45°C, in a sterile petri dish (of 10cm
diameter) dispense about 20 to 25 mL of the medium and let it solidify.
Reagent:

Gram’s Iodine Solution is required for the detection of starch hydrolysis.

Preparation of gram’s iodine solution:

 Dissolve 25 grams of Iodine Crystal and 50 grams of potassium iodide in 500 mL of
distilled water to make Lugol’s iodine stock solution.
 Dissolve 5 grams of sodium bicarbonate (NaHCO3) in 100 mL of distilled water to
make 5% sodium bicarbonate solution.
 Mix 60 mL of Lugol’s iodine solution and 60 mL of 5% sodium bicarbonate solution
with 220 mL of distilled water to make Gram’s Iodine Solution.

Procedure:

 Using a sterile inoculating loop (or cotton swab) pick up the sample bacteria from
several fresh colonies (of about 18 to 20 hours).
 Streak the bacteria in the form of short and thick straight lines over the surface of the
bacteria. (Multiple bacteria can be tested in a single plate forming multiple lines.)
 Incubate at 35±2°C for at least 48 hours.
 Following incubation, add a few drops of iodine solution directly over the colonies
and observe for the formation of a clear halo around the colonies.

A positive result is indicated by the formation of a clear halo around the colonies and the
development of dark blue to purple-blue colour in the surrounding medium after the
addition of Gram’s Iodine.

27
 A negative result is indicated by no clear halo around the colonies and the development
of dark blue to purple-blue colour in the surrounding medium after the addition of Gram’s
Iodine.

(C) Indole Test:

This test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan
to indole, which accumulates in the medium. Tryptophanase is a bacterial enzyme that is
involved in degradation of tryptophan to indole.

Procedure:
 Prepare the media (Peptone-10gm, NaCl-5gms, Tryptophan-1gms) and autoclave.
 Pore the media in the test tube and cool down, Add Kovac’s reagent.
 Mix 100 µl of bacterial culture and incubate it at 37℃ for 24-48 hours.

(D) Methyl Red Test:

Methyl red test, commonly known as MR test is used to determine the ability of an organism
to produced and maintain stable acid end products from glucose fermentation. MR test along
with the VP test is performed simultaneously because they are physiologically related and are
performed on MRVP broth.
In the methyl red test (MR test), the test bacteria is grown in a broth medium
containing glucose. If the bacteria has the ability to utilise glucose with production of a stable
acid, the colour of the methyl red changes from yellow to red, when added into the broth
culture.

Procedure:
 Prepare the media (Peptone-7gms, Glucose-5gms, K2HPO4-5gms) and autoclave it.
 Pour the media inside the test tube and wait for 2 minutes.
 Then add few drops of methyl red indicator.
 Add 100 micro litre of bacterial culture and incubate for 24 hours at 37℃.

(D) VP Test:

28
Voges–Proskauer or VP is a test used to detect acetoin in a bacterial broth culture. The test is
performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth,
which is a glucose-phosphate broth that has been inoculated with bacteria. A cherry red
colour indicates a positive result, while a yellow-brown colour indicates a negative result.

Procedure:

 Using a sterile inoculating loop, pick up well-isolated colonies of sample bacteria

from 18 to 24 hours old culture and inoculate the broth.
 Incubate the tubes aerobically for 18 to 24 hours at 35±2°C.
 Following incubation, transfer 2 mL of broth to a clean (sterile if possible) test tube.
 Add 6 drops of Reagent A (5% α-naphthol solution) and mix properly by shaking.
 Add 3 drops of Reagent B (40% KOH solution) and mix properly by shaking.
 Observe for the formation of red-pink colour at the surface of the medium within 30
minutes. Continuously shake the tube vigorously during the 30-minute waiting
period.
 If no colour is developed (negative reaction) re-incubate the remaining broth for
additional 24 hours and test again.

(E) Citrate Utilization Test:

The citrate utilization test is a part of the IMVIC test that differentiates organisms on the
basis of their ability to use citrate as a sole source of energy.

Procedure:
The media (ammonium dihydrogen phosphate -0.04, Magnesium sulphate-0.008, sodium
citrate-0.08g, Dipotassium phosphate-0.04g, sodium chloride, -0.2g, bromothymol blue-
0.08g) was prepare and autoclaved it at 121 0C under 15 psi for 15 minutes. Then cool it in
slanted position. Bacteria cultures were inoculated in appropriate labelled test tube. Then
incubated at 370C for 24-48 hours.

(F) Carbohydrate Fermentation Test:

29
This test is used to determine whether a bacteria can utilize a certain carbohydrate or not.
While fermenting carbohydrates, organic acids are produced as end products. These produced
acids will decrease in pH of the medium. This decrease in pH causes the pH indicator present
in the medium to change colour.
 Positive fermentation is denoted by the colour change of the media from reddish-
orange to yellow.
 Negative fermentation is denoted by no colour change of the medium (remains
reddish-orange).
Procedure:
The broth medium (peptone-0.4g, sodium chloride-0.2g, beef extract-0.04g, carbohydrate-
0.4, phenol red-0.018g) was prepared and sterilised. Using sterile inoculating loop well
isolated bacterial colony was picked up and inoculated in the appropriate labelled broth. Then
the tubes are incubated at 370C for 24 hours.

4.3 Antimicrobial Activity:

Antimicrobial susceptibility tests are used to determine which specific antibiotics a particular
bacterium is sensitive to. There are two types of methods i.e. well diffusion method and disk
diffusion method.
The well diffusion method is a common procedure for evaluating the antibacterial activity of
substances, such as antibiotics or plant extract.

Procedure:
Prepare NA media and autoclave it. Pour the media in to Petri plate and wait for
solidification. Add 0.1ml of bacterial culture and spread it. Make a well. Add antibiotic to the
well. Incubate it at 370C for 24hours.
Formula: Zone of inhibition= D-d

D= D1+D2+D3
3
The disk diffusion method is a second method of determining antibiotic sensitivity. With this
technique, disks impregnated with various antibiotics are placed on the surface of an agar
plate that has been inoculated with the organism isolated. The antibiotic diffuses out word

30
from the disk over a standard incubation time, and the diameter of the zone of inhibition is
measured. The size of this zone is compared with standards to determine the sensitivity of the
organism to the drug.
The measurement of the zone of inhibition at present is carried out by using a physical ruler
like a meter scale as shown in figure 1 or using a vernier capers. The scale is placed above the
petri-dish and the value of the diameter is read using the human eye.

4.4 Biodegradability test:

Biodegradation is a natural process by which organic chemicals in the environment are
converted to simpler compounds, mineralised and redistributed through elemental cycles such
as the carbon, nitrogen and sulphur cycle. Biodegradability testing measures the complex
biochemical process that occurs when microorganisms consume a given type of material.

Procedure:
 Microorganisms capable of degrading chemical fertilizer are selected and cultured to
create an inoculum.
 The chemical fertilizer samples are prepared for testing, usually by diluting 1gm of
fertilizer in to 10ml of distilled water.
 In each test tube necessary nutrient medium was taken and 100 µl of broth was added
to it after that add 0.5ml of diluted fertilizer.
 The test tubes are placed under controlled conditions (temperature, pH, etc.) and
incubate it for a specific period of time, typically one-several weeks to allow
microbial degradation occur.
 After 7 days each sample degradation rate was observed by spectrophotometry
method.

31
 5. RESULT AND DISCUSSION
(A) Sample collection:

[Fig. 3: Collection of Samples from Different Fields]

32
 [Fig. 4: Collected Samples Were Placed in Containers]

(B) Serial dilution:

After serial dilution I got a series of solutions with decreasing concentrations of the soil
samples.

[Fig. 5: Serial Dilution of Four Different Soil Samples]

(C) Cultures:

33
 [Fig. 6: Primary Culture of Diluted Samples]

1(A) 1(B)

2(A) 2(B)

3(A) 3(B)

34
 4(A) 4(B)
[Fig. 7: Sub Culture Streaking from Primary Culture]

1(A) 1(B) 2(A) 2(B)

3(A) 3(B) 4(A) 4(B)

35
 Sample 1 sample 2 sample 3 sample 4
[Fig. 8: Pure Culture and Broth Culture of the Samples]

(D) Colony morphology:

Sample-1 sample-2

CHARACTERS S-1 S-1 S-2 S-2 S-3 S-3 S-4 S-4

Colony1 Colony2 Colony1 Colony2 Colony1 Colony2 Colony1 Colony2
Size punctiform Large moderate moderate moderate small small moderate
Moderate Moderate Small moderate

36
Colour Gray Gray White Gray Gray Off-white Yellow Off-white

Texture Smooth Dry Dry Viscoid Viscid Dry Smooth Dry

Elevation Raised Flat Flat Raised Flat Umbonate Convex Flat

Form Punctiform Filamentous Irregular Round Round Irregular Round Filamentous

Margin Scalloped Filiform Undulate Entire Entire Undulate Entire Filiform

Opacity Translucent Opaque Opaque Opaque Transparent Opaque Translucent Opaque

Sample-3 sample-4
[Fig. 9: Morphological Study of Selected Colonies Isolated from Different Samples]

[Table.1: The Table Showing the Characteristics of 8 Colonies Selected from Primary
Culture Petri Plate of Four Different Samples]
(E) Gram’s Staining:
After the bacterial strains developed in the pure culture slants loopful of the strains were
utilized for gram staining purpose. Microscopically, it was found that the strains of
1(A),1(B),2(A),2(B),3(A),3(B),4(A) and 4(B) were all purple in staining which means all are
positive and according to the shape strain 1(A) is rod shaped(bacillus) and rest of all are
sphere shaped (Cocco-bacillus).
Colonies in petri Colour of the Shape of the Bacteria
plate Colonies Colonies
S-1(A) Purple Rod shaped bacillus
S-1(B) Purple Sphere-shaped Cocco-bacillus
S-2(A) Purple Sphere-shaped Cocco-bacillus
S-2(B) Purple Sphere-shaped Cocco-bacillus
S-3(A) Purple Sphere-shaped Cocco-bacillus
S-3(B) Purple Sphere-shaped Cocco-bacillus
S-4(A) Purple Sphere-shaped Cocco-bacillus
S-4(B) Purple Sphere-shaped Cocco-bacillus
[Table.2: The Table Showing the Gram Staining Characteristics of the Bacterial Species
Strain]

37
38
 [Fig. 10: Gram Staining of Isolated Bacteria from Different Samples]

(F) Biochemical tests:

After doing several biochemical tests, I was able to know that 1(A) bacteria strain showing
positive result in all biochemical test except methyl red, VP and citrate utilization test. 1(B)
bacteria strain showing positive result in all biochemical test except CFT-3 (sucrose), VP and
CUT test.
2(A) bacteria strain showing negative results only in methyl red and CUT test and other are
showing negative result. 2(B) bacteria strain showing positive result in catalase, CFT-2, CFT-
3 and VP test.
3(A) bacteria strain showing negative result in starch hydrolysis, CFT-1, indole and methyl
red test. 3(B) showing negative result only in catalase, indole and methyl red test.

39
4(A) bacteria strain showing positive results only in CFT-3, methyl red and VP test other are
showing negative result. 4(B) bacteria showing positive result in CFT-2, CFT-3, indole and
methyl red test.

Tests 1(A) 1(B) 2(A) 2(B) 3(A) 3(B) 4(A) 4(B)

Catalase Less Less

+Ve +Ve -Ve +Ve +Ve -Ve -Ve -Ve
Starch
Hydrolysis +Ve +Ve -Ve -Ve -Ve +Ve -Ve -Ve
Indole
+Ve +Ve -Ve -Ve -Ve -Ve -Ve +Ve
Methyl
Red -Ve +Ve +Ve -Ve -Ve -Ve +Ve +Ve
VP Test
+Ve -Ve -Ve +Ve +Ve +Ve +Ve -Ve
Citrate
Utilisation -Ve -Ve +Ve -Ve +Ve +Ve -Ve -Ve
CFT-1
+Ve +Ve -Ve -Ve -Ve +Ve -Ve -Ve
CFT-2
+Ve +Ve -Ve +Ve +Ve +Ve -Ve +Ve

CFT-3 +Ve -Ve -Ve +Ve +Ve +Ve +Ve +Ve

[Table.3: Different Biochemical Tests to Identify Bacteria]

40
1(A) 1(B) 2(A) 2(B)

3(A) 3(B) 4(A) 4(B)

[Fig. 11: Catalase Test Results of Eight Bacteria]

1(A) 1(B) 2(A) 2(B)

41
 3(A) 3(B) 4(A) 4(B)

[Fig. 12: Starch Hydrolysis Test Results of Eight Bacteria]

Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)

42
Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)

[Fig. 13: Indole & Methyl Red Test Results of Eight Bacteria]

Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)

43
Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)

[Fig. 14: VP & Citrate Utilization Test Results of Eight Bacteria]

44
Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)

45
Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)

46
 Sample-1 (a, b) Sample-2 (a, b) Sample-3 (a, b) Sample-4 (a, b)
[Fig. 15: CFT-1, 2, 3 Test Results of Eight Bacteria Respectively]
(G) Antimicrobial test:

Sample Antibiotics Diameter(D) Zone of inhibition

(D-d)
(d=8mm)
1(A) A(azithromycin) D(A) =28.3mm 20.3mm

P(penicillin) D(P) =24.3mm 16.3mm

C(cefpodoxime) No zone formation No. zone formation

1(B) A(azithromycin) D(A) =19.6mm 11.6mm

47
 P(penicillin) D(P) =29.3mm 21.3mm

C(cefpodoxime) D(C) =15.5mm 7.5mm

2(A) A(azithromycin) D(A) =17.5mm 9.5mm

P(penicillin) D(P) =21mm 13mm

C(cefpodoxime) D(C) =21mm 13mm

2(B) A(azithromycin) D(A) =13.7mm 5.5mm

P(penicillin) D(P) =20.5mm 12.5mm

C(cefpodoxime) D(C) =22.5mm 14.5mm

3(A) A(azithromycin) D(A) =19mm 11mm

P(penicillin) D(P) =27.3mm 19.3mm

C(cefpodoxime) D(C) =22.5mm 14.5mm

3(B) A(azithromycin) D(A) =23mm 15mm

P(penicillin) D(P) =25mm 17mm

C(cefpodoxime) D(C) =22mm 14mm

4(A) A(azithromycin) D(A) =17mm 9mm

P(penicillin) D(P) =23.3mm 15.3mm

48
 C(cefpodoxime) D(C) =19mm 11mm

4(B) A(azithromycin) D(A) =17mm 9mm

P(penicillin) D(P) =18.5mm 10.5mm

C(cefpodoxime) D(C) =20.6mm 12.6mm

[Table.4: Antimicrobial Activity of Azithromycin, Penicillin, Cefpodoxime against

Isolated Bacteria]

1(A) 1(B)

2(A) 2(B)

49
 3(A) 3(B)

4(A) 4(B)

[Fig. 16: Antimicrobial Activity of Azithromycin, Penicillin, Cefpodoxime against

Isolated Bacteria]

(H) Biodegradation Test:

Samples NPK SSP DAP Gromor

1(A) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7
1.33 1.07 1.34 1.33 1.31 1.30 1.20 1.17
1(B) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7
1.33 1.17 1.33 1.32 1.32 1.32 1.28 1.27
2(A) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7
1.32 1.17 1.33 1.31 1.28 1.26 1.25 1.21

2(B) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7

50
 1.34 1.16 1.32 1.26 1.30 1.29 1.27 1.27

3(A) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7

1.33 1.12 1.33 1.32 1.31 1.31 1.20 1.18
3(B) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7
1.34 1.18 1.33 1.33 1.30 1.30 1.25 1.23
4(A) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7
1.34 1.25 1.33 1.31 1.32 1.33 1.28 1.28
4(B) Day-0 Day-7 Day-0 Day-7 Day-0 Day-7 Day-0 Day-7
1.33 1.37 1.32 1.30 1.33 1.33 1.29 1.32
[Table.5: The OD Value of Degradation of NPK, SSP, DAP, Gromor Fertilizer By
Different Isolated Bacteria]

Culture 1(A): Day-0 Culture 1(A): Day-7

Culture 1(B): Day-0 Culture 1(B): Day-7

51
 Culture 2(A): Day-0 Culture 2(A): Day-7

Culture 2(B): Day-0 Culture 2(B): Day-7

Culture 3(A): Day-0 Culture 3(A): Day-7

52
Culture 3(B): Day-0 Culture 3(B): Day-7

Culture 4(A): Day-0 Culture 4(A): Day-7

Culture 4(B): Day-0 Culture 4(B): Day-7

53
 [Fig. 17: Visualization of Day_0 & Day_7 of Degradation of NPK, SSP, DAP, Gromor
Fertilizer by Different Isolated Bacteria]
Microbial community structure or soil microbial ecology is an important feature of soil
quality. Although the microbial plate method is traditional, it is still being used for microbial
isolation and culture. This study encompassed assessing the ability of traditional culture
method. By using serial dilution and different culture techniques 8 bacterial species were
isolated. After isolation, colony morphology can be used to identify the bacterial shape, size,
colour, texture, elevation, opacity, form and margin etc. (Table 1)

After the bacterial strains developed in the pure culture slants loopful of the strains were
utilized for gram staining purpose. Microscopically it was found that the strains of
1(A),1(B),2(A),2(B),3(A),3(B),4(A) and 4(B) were all purple in staining which means all are
positive and according to the shape strain 1(A) is rod shaped (bacillus) and rest of all are
sphere shaped (Cocco-bacillus). (Table 2)

Out of the 8 bacterial isolates 1(A), which is +Ve bacillus rod shaped bacteria, showing
maximum inhibition zone for Azithromycin antibiotic whereas 2(b), which is coccobacillus
very short rods or oval shaped bacteria, showing minimum zone of inhibition for
Azithromycin antibiotic. For Penicillin, 2(A) bacteria showing no zone formation which
means it is negative, 1(B) bacteria (Cocco-bacillus gram positive) showing maximum zone of
inhibition and 4(B)
(Cocco-bacillus gram positive) showing minimum zone of inhibition. Similarly, for
Cefpodoxime, 1(A) bacteria showing no zone formation which means it is negative, 2(B) and
4(A) showing maximum zone of inhibition whereas 1(B) showing minimum zone of
inhibition. (Table-4)

6. CONCLUSION

54
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