Advances in Plant & Microbial Biotechnology: Rita Kundu Rajiv Narula Editors

Rita Kundu · Rajiv Narula Editors
Advances
in Plant &
Microbial
Biotechnology
Advances in Plant & Microbial
Biotechnology
Rita Kundu • Rajiv Narula
Editors
Advances in Plant
& Microbial Biotechnology
Editors
Rita Kundu Rajiv Narula
Department of Botany State University of New York
University of Calcutta Albany, NY, USA
Kolkata, West Bengal, India
ISBN 978-981-13-6320-7 ISBN 978-981-13-6321-4 (eBook)

https://doi.org/10.1007/978-981-13-6321-4
Library of Congress Control Number: 2019934372
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Foreword
The field of Plant and Microbial Biotechnology is a broad domain which encom-
passes both the macroscopic and the microscopic members of the world. The utility
of plant systems for the development of human welfare is realized from time imme-
morial. The application of plant biotechnology encompasses the fields for the effec-
tive production of value-added materials like food and biochemical and
pharmaceutical products; it also emphasizes the control of plant growth and devel-
opment and plant protection against several biotic and abiotic factors.
In the last century, there had been a drastic change in the concept of microbes as
pathogens only to beneficial role as it leads to the generation of thousands aspects
of research with microbes used in bioremediation of pollutants; utilization of wastes;
amelioration of the nutritional conditions of soil; production of variety of fermented,
probiotic food; and so on. There was a drastic paradigm shift in the traditional use
of inorganic substances to the unique application of plant and microbial technology
for the eco-friendly way of industrial production of materials, which gave rise to a
volley of research areas.
The topic of the current issue of Advances in Plant & Microbial Biotechnology
is a compilation of the contributions from leading workers and researchers from
different arenas of plant and microbial biotechnology and is a successful outcome
of the 1st International Conference on Biotechnology and Biological Sciences,
BIOSPECTRUM 2017, organized by the Department of Biotechnology, University
of Engineering & Management, Kolkata. This is an admirable issue comprising of
17 different research topics which would definitely inculcate the scientific knowl-
edge of researchers and scientists working in the field of Plant and Microbial
Biotechnology and aims to give an idea on the ongoing cutting edge research areas
on these domains.
The book focuses on the background research for the development of technology
to be handed over from “lab to land” for the improvement in the production of crops
like rice, wheat, and sugarcane. Biogenic production of silver nanoparticles and its
use in waste removal are also emphasized. The book contains the improved tech-
niques for the production of various enzymes of commercial interests, plant hor-
mones, sugar alcohols, hydroxy fatty acids, phytochemicals, and biofertilizers. The
cytotoxic potentials and genetic toxicity potentials of various plant parts are meticu-
lously analyzed. The book also focuses on the recent context of interspecies
v
vi Foreword
interaction of the biofilm formed by the extracellular products of Pseudomonas sp.

isolated from ocular infection. The role of siderophores for sustained maintenance
of soil health is also discussed in the book.
This book is a nice blend of plant and microbial biotechnology focusing on vari-
ous techniques and methodologies being used in the recent field of research. The
authors used various analytical instrumentations accompanied by biochemical assay
techniques providing results which can have a far-sighted impact on the societal
benefit.
I am delighted to go through the chapters and hope the readers at large will be
benefited from the technologies described to face various challenges in the field of
industrial production of commodities of plant and microbial origin.
Associate Professor and Head Rina Rani Ray

Department of Biotechnology
Maulana Abul Kalam Azad University of Technology
Preface
BIOSPECTRUM 2017 was organized by the Department of Biotechnology,

University of Engineering & Management, Kolkata. This conference brought forth
a unique bridging forum for the interaction among eminent academicians, scientists,
researchers, and professionals from various branches of biological sciences, a plat-
form to exchange knowledge and expertise enriching researchers with opportunities
of networking and collaboration across the globe. This conference promoted an
intense dialogue between academia and industry to bridge the gap between aca-
demic research, industry initiatives, and governmental policies. This was fostered
through panel discussions, keynote lectures, invited talks, and industry exhibits
where academia was exposed to state of practice and results from trials and current
research trends. There was participation from different domains of biotechnology
with plant biotechnology assuming a pivotal role.
This book is an assimilation of some of the newest and upcoming research topics
in the field of plant biotechnology. Readers of this book will be immensely bene-
fited in obtaining novel ideas which will open up newer directions in the field of
research in plant biotechnology. This book offers original, peer-reviewed articles
dealing with all aspects of fundamental and applied research in the field of plant
biotechnology. The coverage extends to other current applied areas of molecular
biology, genetics, biochemistry, and cell and tissue culture. This book provides a
single platform for articles that discusses and describes the scope of modern tech-
nologies to address the increasing demands for high yielding crop production,
encompassing plant tissue culture, role of genetic engineering, and extending the
exploitability of plants to incorporate other sustainable uses.
Finally, the editors are indebted to the International Advisory Committee and the
Technical Program Committee for their valuable guidance and support. We express
our sincere thanks to the students for their consistent support and spirited participa-
tion; we express our heartfelt gratitude to the management, staff, and faculty mem-
bers of the University of Engineering & Management, Kolkata, for making the
conference a success.
Kolkata, West Bengal, India Rita Kundu

Albany, NY, USA Rajiv Narula
vii
Acknowledgment
The book is the outcome of the International Conference on Biotechnology and

Biological Sciences, BIOSPECTRUM 2017. The editors of the book would like to
acknowledge the help of all the people involved in making this book, Advances in
Plant and Microbial Biotechnology, more specifically to the authors and reviewers
who helped in the whole process. Without their support, this book would not have
become a reality.
First, the editors would like to thank each one of the authors for their research
contribution. Our sincere gratitude goes to the chapters’ authors who contributed
their time and expertise to make this book.
Second, the editors are extremely thankful to the organizers of the international
conference BIOSPECTRUM and the management, faculty, and staff members of
the University of Engineering & Management (UEM), Kolkata, India. The editors
are grateful to the chancellor, Prof. Dr. Satyajit Chakrabarti, for his motivation and
encouragement.
Third, the editors wish to acknowledge the valuable contribution of the reviewers
regarding the improvement of the quality, coherence, and content presentation of
the chapters.
ix
Introduction
Biotechnology is the use or manipulation of an organism or parts of an organism.

By this definition, the origin of biotechnology dates back to the early era of civiliza-
tion, when people first begin to cultivate food crops. While the early applications are
certainly still employed today, modern biotechnology is primarily associated with
molecular biology, cloning, and genetic engineering to increase the yield as well as
to improve the quality of the crop. Within the last 50 years, several key discoveries
revolutionized the biological sciences that enabled the rapid evolution of the biosci-
ences. These discoveries enabled scientists to isolate and manipulate genes, which
has facilitated the growth of the biotechnology industry.
Life on earth would not have flourished without plants. Plants are the source of
oxygen which is required by all living beings for respiration. Plants absorb carbon
dioxide and produce oxygen by photosynthesis. This not only maintains oxygen on
earth but also decreases carbon dioxide content of the atmosphere which in turn
reduces global warming which is an important environmental problem.
Plants are valued greatly for their therapeutic properties and considered as excel-
lent sources of medicines. Plants have been used for medicinal purposes from pre-
historic times. Among ancient civilizations, India has been known to be a rich
repository of medicinal plants. The forest in India is the principal source of a large
number of medicinal and aromatic plants, which are largely used as raw materials
for the manufacture of drugs and perfumery products. About 8,000 herbal remedies
have been codified in AYUSH systems in India. Ayurveda, Unani, Siddha, and folk
(tribal) medicines are the major systems of indigenous medicines. Treatment with
medicinal plants is considered safe as there are no or minimal side effects. These
remedies are in sync with nature, which is the biggest advantage. The golden fact is
that the use of herbal treatments is independent of any age groups and sexes.
Over the past few years, a number of techniques have been developed which
have advanced basic research in plant sciences and their application in agriculture.
The very first of these studies deals with modifications to the growth of plant cells,
tissues, organs, and protoplasts in tissue culture. The second area which is fast gain-
ing worldwide attention is genetic engineering. This emerging technology has led to
the manipulation of plant genetic material which will facilitate breeding better qual-
ity crop varieties.
xi
xii Introduction
Plant biotechnology delivers significant and tangible benefits to farmers, con-

sumers, and the environment around the globe. It has improved farm incomes
through increased crop yields and reduced use of agrochemicals, protected natural
habitats by increasing the production on existing cropland, and allowed for improv-
ing waterways and reducing soil erosion. Biotech crop varieties have significantly
increased plant productivity along with protecting the environment from an increas-
ing chemical burden.
The 11 research papers in the book comprise of different facets of advancements
in research in plant biotechnology.
Ayurveda, the oldest Indian indigenous medicine system of plant drugs, is known
from very early times for preventing or suppressing various tumors using natural
drugs. A chapter illustrates the cytotoxic potential of Moringa oleifera Lam.
Entomopathogenic fungi Cordyceps sp. are one of the unique and valuable sources
of bioactive compounds which help to treat various diseases. An experimental work
explains the strain improvement strategies of Cordyceps sp.
Another very important work emphasized on rice crop improvement by develop-
ment of low-cost blue-green algal biofertilizer comprising of consortium of four
blue-green algal strains, namely, Anabaena variabilis, Nostoc muscorum,
Tolypothrix tenuis, and Aulosira fertilissima, using different carrier materials, i.e.,
fly ash (100%), soil (100%), montmorillonite (100%), fly ash + soil (1:1), and fly
ash + montmorillonite (1:1).
Apart from medicines, plants have industrial application. In this respect, a chap-
ter elaborates studying the modification introduced into the fatty acid molecule of
Camelina sativa to enable it to become a potential resource to synthesis biolubri-
cants. A chapter on production of bioethanol describes pretreatment procedures of
rice straw for improving enzymatic hydrolysis which facilitates the conversion into
ethanol. Lignocellulosic materials are the potential renewable energy resources for
the production of transportation fuels. In one study, sorghum biomass was used as a
model lignocellulosic substrate for the production of xylulosic ethanol.
This book can be used by students, academicians, and researchers working on
the domain of plant biotechnology as a reference book.
Contents
1 Dilute Acid Pretreatment Efficiency on Various Solid

Loadings and Effect of Different Neutralizing Agents
on Xylulosic Ethanol Production�� 1
Narendra Naik Deshavath, Bijayeeni Singh Deo, Jyothika Boddu,
Komali Vykuntam, Vaibhav V. Goud, and Venkata Dasu Veeranki
2 Effect of BGA Biofertilizers Using Different Carrier
Materials on Rice Crop�� 9
Rajinder Kaur and Dinesh Goyal
3 Identification of Differentially Expressed Terminal Heat
Stress-Associated Proteins in Developing Grains
in Wheat (Triticum aestivum L.)�� 13
Davinder Sharma, Ratan Tiwari, Vijay Kumar Gupta,
Jagadish Rane, and Rajender Singh
4 The Genetic Toxicity Potential of Heavy Metals (Zn, Cu)
on Vigna radiata, Triticum aestivum, and Cicer arietinum�� 19
Aparajita Shilpie and Kamal Nayan Mishra
5 Production, Optimization, and Characterization
of Siderophore by Pseudomonas aeruginosa (C3)
Isolated from Rhizospheric Soil�� 27
Chhaya Verma, Apoorva Dixit, and Rajesh Kumar
6 Development of Marker in the Soft Gold
Mushroom Cordyceps spp. for Strain Improvement�� 33
Loknath Deshmukh, Diva Gupta, and Sardul Singh Sandhu
7 Optimization of Microwave-Assisted Pretreatment
of Rice Straw with FeCl3 in Combination with H3PO4
for Improving Enzymatic Hydrolysis�� 41
Bikash Kumar and Pradeep Verma
xiii
xiv Contents
8 Profiling Indolic Auxins Produced by the Strains

of Aspergillus Using Novel HPTLC Technique�� 49
Dhavalkumar Patel, Anoshi Patel, Disha Vora, Kinjal Desai,
Sudeshna Menon, Sebastian Vadakan, and Dweipayan Goswami
9 Comparative Analysis of Cytotoxic Potential of Crude
Extracts and Fractionated Isolates from Moringa oleifera Lam.�� 59
Kinjal Desai and Vincent Braganza
10 Hydroxy Fatty Acid from Camelina sativa Seed Oil
for Industrial Application�� 69
Neha Sharma, Lekha Charan Meher, Mitesh Mittal,
and Sanjai Kumar Dwivedi
11 Comparison of Different Planting Methods to Determine
the Precision of Phenotyping Wheat in Field Experiments�� 77
Davinder Sharma, Jagadish Rane, Rajender Singh,
Vijay Kumar Gupta, and Ratan Tiwari
12 Effect of Extraction Temperature and Different Carrier
Agents on Physicochemical and Antioxidant Properties
of Spray-Dried Murraya koenigii (Linn.) Leaf Extract�� 85
Vandana Sablania, Sowriappan John Don Bosco,
and Shubham Rohilla
13 Isolation and Characterization of Microbial Asparaginase
to Mitigate Acrylamide Formation in Food �� 95
Mausumi Ray, Sunita Adhikari (Nee Pramanik), and Pradyut Kundu
14 Small Colony Variant Selection, Biofilm Induction,
and Interspecies Interactions of Ocular Clinical
Pseudomonas aeruginosa �� 101
Sadhana Sagar and Shilpa Deshpande Kaistha
15 Comparative Analysis of Phytochemicals of Healthy
and Symptomatic Clerodendrum inerme�� 115
Sonal, Sharmita Gupta, Yati Prabha, and S. K. Soni
16 Synthesis of Silver Nanoparticle of Aqueous Extract
of Allium Fistulosum and Its Efficiency Against Bacterial
Contaminants from Industrial Waste Water
and Its Photocatalytic Potential�� 121
Uma Ramaswamy and Vicky Mani
17 Exploration of Biocontrol and Growth-Promoting Activity
of Bacterial Strains Isolated from the Sugarcane Crop �� 129
Beenu Shastri, Anil Kumar, and Rajesh Kumar
About the Editors
Dr. Rita Kundu is a professor at the University of Calcutta’s Department of

Botany. With a specialization in Cell Biology and Genetics, she has been engaged
in cancer biology research. She has studied cell death through apoptosis, autophagy,
or any other way in cervical cancer cell lines using traditional medicinal plants and
other organic sources (marine/freshwater algal compounds, microbial compounds,
synthetic organic compounds) to regulate cell proliferation. Currently, her main
interest is in identifying metal-resistant crop (rice) varieties in southern West Bengal
and studying their stress responses. She is the author of numerous papers.
Dr. Rajiv Narula received his Bachelor’s Degree in Botany from the esteemed
Presidency College (now Presidency University), Kolkata. He went on to complete
a Master’s in Biotechnology at GGD University, Chhattisgarh, and a PhD in
Environmental Engineering at Clarkson University, Potsdam, NY. After completing
his doctoral thesis on Pathogen Reduction and Recycling of Bedding Materials on
Dairy Farms (May 2011), he joined the State University of New York, Canton, in
2011, and is currently a member of its Environmental Science and Chemistry
Department.
xv
Dilute Acid Pretreatment Efficiency
on Various Solid Loadings and Effect 1
of Different Neutralizing Agents
on Xylulosic Ethanol Production
Narendra Naik Deshavath, Bijayeeni Singh Deo,

Jyothika Boddu, Komali Vykuntam, Vaibhav V. Goud,
and Venkata Dasu Veeranki
Abstract
Lignocellulosic materials are the potential renewable energy resources for the
production of transportation fuels. In the present study, sorghum biomass is used
as a model lignocellulosic substrate for the production of xylulosic ethanol.
Dilute sulfuric acid pretreatment was performed at different solid loadings (2.5%
to 15% w/v) to hydrolyze the maximum hemicellulosic content of sorghum bio-
mass. As a result, significant xylan conversion efficiency was achieved and
xylose found to be a predominant sugar present in the acid hydrolyzate. The
pretreatment-derived acid hydrolyzate was divided into five fractions, and each
fraction was neutralized with different alkaline agents such as calcium hydroxide
(Ca(OH)2), potassium hydroxide (KOH), magnesium hydroxide (Mg(OH)2),
sodium hydroxide (NaOH), and ammonia (NH3) to determine the effect of alka-
line agents on Pichia stipitis growth and xylulosic ethanol production. Among all
the alkaline agents, Ca(OH)2 was found to be a significant neutralizing agent
which showed comparatively higher ethanol yield and productivity during the
fermentation.
Keywords
Pretreatment · Sorghum biomass · Solid loadings · Ethanol
N. N. Deshavath · V. V. Goud · V. D. Veeranki (*)

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati,
Guwahati, Assam, India
e-mail: [email protected]; [email protected]; [email protected]
B. S. Deo
Utkal University, Bhubaneswar, Odisha, India
J. Boddu · K. Vykuntam
National Institute of Technology Durgapur, Durgapur, West Bengal, India
© Springer Nature Singapore Pte Ltd. 2019 1

R. Kundu, R. Narula (eds.), Advances in Plant & Microbial Biotechnology,
https://doi.org/10.1007/978-981-13-6321-4_1
2 N. N. Deshavath et al.
1.1 Introduction
Bioethanol is a substitute to petroleum-based fuels which can also conquer the fos-
sil fuel depletion. Lignocellulosic biomass is the most abundant, economically via-
ble renewable energy resource that can be utilized for the production of bioethanol
[9]. Generally, agricultural residues such as corn stover, sorghum stalks, sugarcane
bagasse, and wheat straw are the major sources of lignocellulosic biomass for bio-
ethanol production. Cellulose, hemicellulose, and lignin are the major constituents
of lignocellulosic biomass. Cellulose is a homopolymer which is made up of glu-
cose units, whereas heteropolymeric structure of hemicellulose is made up of
xylose, arabinose, and other organic acids [11].
In a traditional way, conversion of lignocellulosic biomass into bioethanol can be
performed in three steps such as pretreatment, enzymatic hydrolysis, and fermenta-
tion. Pretreatment is an essential process which mostly hydrolyzes the hemicellu-
losic fraction, and the resulting residual solid biomass constitutes mostly cellulose
which can be further hydrolyzed by cellulase (biocatalyst) for the production of
glucose monomers. Xylose and glucose are found to be principal sugars formed
during the pretreatment and enzymatic hydrolysis, respectively. As compared to
enzymatic hydrolyzate, pretreatment-derived hydrolyzate has less significance in
the production of bioethanol. Efficiency of prehydrolyzate fermentation was
deterred due to the presence of sulfate and fermentative inhibitors such as furans,
organic acids, and other phenolic compounds [6].
However, several processes were well established to decrease the fermentative
inhibitor effect such as ion exchange, overliming, and treatment with activated car-
bon [1, 5, 10]. Among them, overliming is a well-known process which effectively
decreases the fermentative inhibitor concentration. Overliming is a process where
the prehydrolyzate pH increases up to 9, 10, and 11 by the addition of alkali
(Ca(OH)2) and then adjusted to cultivation pH of microorganisms. While increasing
the pH of prehydrolyzate at elevated levels sugar loss can occur, it is a profound
drawback of overliming process [8].
Therefore, in the present study, dilute sulfuric acid pretreatment of sorghum bio-
mass was performed at different solid loadings. The pretreatment-derived acid
hydrolyzate was neutralized with different alkaline agents and then subjected to
fermentation by Pichia stipitis 3498.
1.2 Materials and Methods
1.2.1 Dilute Sulfuric Acid Pretreatment
Sorghum biomass pretreatment was carried out with 0.2 M sulfuric acid at 121 °C
for 2 h reaction time with 2.5–15% (w/v) solid loading. After pretreatment, solid
and liquid fractions were separated through 0.2 μm nylon membrane filter. The solid
1 Dilute Acid Pretreatment Efficiency on Various Solid Loadings and Effect… 3
portion was washed with distilled water to attain neutral pH and then dried at
45 ± 3 °C. The liquid portion was divided into five fractions and heated to 50 °C for
30 min. Among the five fractions, two fractions were neutralized by slow addition
of Ca(OH)2 and Mg(OH)2, respectively. The remaining three fractions were neutral-
ized with liquid form of 10N KOH, 10N NaOH, and 25% NH3, respectively. These
hydrolyzates were stored at −20 °C until fermentation. Prior to fermentation, cen-
trifuge the hydrolyzates to remove the precipitate, and then supernatant was adjusted
to cultivation pH (i.e., 6).
1.2.2 Microorganism and Seed Culture Preparation
Pichia stipitis 3948 strain was procured from the National Collection of Industrial
Microorganisms (NCIM), Pune, India. P. stipitis was subcultured on modified
YPDX agar plates which contains (g/L) yeast extract, 10; peptone, 20; glucose, 5;
xylose, 15; and agar, 20, and incubated at 30 °C for 48 h. A colony from the plate
was inoculated into 100 ml of autoclave sterilized YPDX liquid growth medium and
incubated at 30 °C with 120 rpm for 18 h. Initial pH of the YPDX medium was
adjusted to 6. The cells were harvested by centrifugation at 8000 rpm for 10 min and
resuspended in sterile distilled water to adjust the final concentration of 40 g/L
which served as inocula for bioethanol production. Cell growth was observed by
measuring the absorbance at 600 nm (OD600) using Agilent Cary 100 UV-visible
spectrophotometer.
1.2.3 Fermentation
Fermentation was carried out in sterile 25 mL Erlenmeyer flasks containing 10 mL

of fermentation medium which include 0.2 mL of 50X concentrated nutrient solu-
tion (1.7 g of yeast nitrogen base, 1 g of urea, and 6.56 g of peptone in 20 mL of
water), 0.5 mL of inocula gives an initial cell concentration of 2 g/L, and an appro-
priate quantity of hydrolyzate was added to reach the desired volume. Initial pH of
the media was adjusted to 6 and incubated at 30 °C with 120 rpm.
1.2.4 Analytical Method
Quantification of pretreatment-derived carbohydrates, fermentative inhibitors, and

fermentation-produced ethanol was carried out by high-performance liquid chroma-
tography (HPLC) [3].
1.3 Result and Discussion
1.3.1 Pretreatment of Sorghum Biomass
According to our previous studies, dilute acid pretreatment was conducted with 5%
solid loading, and the maximum hemicellulose conversion was attained at 121 °C
with 0.2 M sulfuric acid for 120 min [2–4]. Therefore, in the present study, we made
an attempt to increase solid loading of pretreatment process to meet the desired
levels of sugar concentration for successful xylulosic ethanol production. According
to the HPLC analysis, different types of carbohydrates were formed from their
respective polymeric carbohydrates such as xylobiose, xylose, and arabinose
derived from hemicellulose, cellobiose, and glucose derived from cellulose. Among
all the carbohydrates, xylose is found to be the principal carbohydrate formed dur-
ing the pretreatment (Fig. 1.1a). It is a fact that pretreatment is an essential step
which significantly hydrolyzes the hemicellulose content and the percentage of
xylan present in hemicellulose is comparatively higher than that of arabinose [3].
Hence, higher concentration of xylose is found (Fig. 1.1a).
From Fig. 1.1a, linear increase of carbohydrates concentration was observed by
increasing the sorghum biomass solid loading (2.5–15%), with a R2 value of 0.98
and above. Besides carbohydrates, fermentative inhibitors like furfural (a degrada-
tion product of pentose sugars), 5-hydroxymethyl furfural (a degradation product of
glucose), and acetic acid (derived from acetylated xylan) were also formed (data not
shown).
1.3.2 eutralization of Acid Hydrolyzates for Xylulosic Ethanol

N
Production
During the neutralization process, sugar loss was not found, and the possible stoi-
chiometric chemical reactions are shown in Fig. 1.2. The resulting MgSO4, Na2SO4,
K2SO4, and (NH4)2SO4 are solubilized in their respective hydrolyzates; however,
CaSO4 is a solid which could be removed through centrifugation. Apart from these,
at industrial-scale production, moisture heat-sterilized medium is used for fermen-
tation. Therefore, in the present study, instead of using filter sterilization method,
autoclave sterilization of fermentation medium was performed. Generally, xylose
degradation can be initiated when the temperature reaches above 120 °C [7].
Therefore, minute amounts of xylose (4–5 mg) loss could be possible during the
autoclave sterilization of fermentation medium.
Since glucose is the preferred substrate, P. stipitis starts to utilize xylose after
glucose consumption. Due to the presence of K2SO4, Na2SO4, and (NH4)2SO4 in the
fermentation medium, sugar consumption and ethanol production were delayed
(Fig. 1.1b–d). It took 144 h to reach the maximum ethanol concentration (Table 1.1).
In addition to this, 15%, 18%, and 27% of unconverted xylose were still present in
the fermentation media of KOH-, NaOH-, and NH3-treated hydrolyzate at 144 h.
The presence of sulfate ions decreases the microbial metabolic growth which
35 2
Linear fit of Xylose (R =0.994) Glucose
2 25
30 Linear fit of Xylobiose (R =0.982)
Xylose
Concentration (g L-1)
2
Linear fit of Cellobiose (R =0.982)
25 2 20 Ethanol
Linear fit of Arabinose (R =0.997)
2 Xylose
Linear fit of Gluocse (R =0.994)
20 Cellobiose 15
15 Xylobiose
Gluocse 10
10 Arabinose
5
5
0 0
-5
(a) -5
(b)
2.5 5.0 7.5 10.0 12.5 15.0 0 24 48 72 96 120 144
Solid Loading (%) Time (h)
Glucose Glucose
25 25
Xylose Xylose
20 Ethanol 20 Ethanol
15 15
10 10
5 5
0 0
-5
(c) -5
(d)
0 24 48 72 96 120 144 0 24 48 72 96 120 144
Time (h) Time (h)
25 Glucose 25 Glucose
Xylose Xylose
20 Ethanol 20 Ethanol
15 15
10 10
5 5
0 0
(e) (f)
-5 -5
0 24 48 72 96 120 144 0 24 48 72 96 120 144
Time (h) Time (h)
Fig. 1.1 (a) Pretreatment efficiency on different solid loadings for carbohydrates yield, (b) fer-
mentation profiles of (b) KOH-, (c) NaOH-, (d) NH3-, (e) Ca(OH)2-, and (f) Mg(OH)2-neutralized
hydrolyzates: sugar utilization and ethanol production
Fig. 1.2 Possible

stoichiometric chemical
reactions of different
alkaline agents when
reacted with H2SO4
Table 1.1 Summary of fermentation results from sorghum biomass pretreatment-derived

hydrolyzate
Different alkaline agents used for neutralization of
acid hydrolyzates
Parameters Ca(OH)2 Mg(OH)2 KOH NaOH NH3
Maximum ethanol concentration (g/L) 11.3 10.13 9.4 9.12 8.1
Fermentation time (h) 96 96 144 144 144
Ethanol yield on substrate (gp/gs) 0.37 0.33 0.30 0.30 0.26
Theoretical ethanol yield (%) 73.2 65.7 59.7 59 52.5
Ethanol productivity (g/L/h) 0.11 0.1 0.065 0.063 0.056
Total sugars utilized (%) 97 98 85 82 73
eventually affects the ethanol yield. Neutralization with Ca(OH)2 and Mg(OH)2
showed better results as compared to other neutralizing agents. Ca(OH)2 forms cal-
cium sulfate (CaSO4) when reacted with H2SO4. Therefore, removal of sulfate in the
form of CaSO4 (prior to the fermentation) enhanced ethanol production (Fig. 1.1e).
Highest ethanol concentration (11.3 g/L) and yield (0.37 gp/gs) are evidences of the
Ca(OH)2-treated hydrolyzate fermentation. To the best of our knowledge, this is the
first report in which Mg(OH)2 has been used for neutralization of acid hydrolyzate,
and the results are found to be significant with 10.3 g/L of ethanol concentration
(Figure 1.1f) along with 0.33 gp/gs ethanol yield. According to the literature, MgSO4
has superior advantage in the yeast-based fermentation processes. Availability of
Mg2+ ions in the fermentation medium significantly influenced the metabolic growth
of microbes [12]. Previous studies also suggest that magnesium ions are important
in stimulating the central pathway of carbohydrates catabolism, especially ethanol
production process [12]. However, detailed summary of fermentation results are
shown in Table 1.1 for better understanding the effect of different alkaline agents on
xylulosic ethanol production.
1.4 Conclusions
During the pretreatment process, with increase in solid loading of sorghum bio-
mass, the amount of carbohydrates concentration increased with a R2 value of 0.98
and above. During fermentation, 11.3 g/L and 10.13 g/L ethanol production were
observed in both Ca(OH)2- and Mg(OH)2-neutralized hydrolyzates, respectively, at
96 h fermentation period, whereas 9.4 g/L, 9.12 g/L, and 8.1 g/L ethanol concentra-
tions were observed in KOH-, NaOH-, and NH3-treated hydrolyzate fermentation,
respectively, at 144 h. Prolonged fermentation is not feasible for the commercializa-
tion of ethanol production process; therefore, fermentation was stopped at 144 h.
Acknowledgment Authors would like to thank Miss Sushmita Mahanta, a summer trainee at
Centre for the Environment, IIT Guwahati, for her help in proofreading.
References
1. Chandel AAK et al (2011) Detoxification of lignocellulosic hydrolysates for improved
bioethanol production. Biofuel Production-Recent …, 2012, p. 989572. https://doi.
org/10.1155/2012/989572
2. Deshavath NN, Dasu VV et al (2017a) Development of dilute sulfuric acid pretreatment method
for the enhancement of xylose fermentability. Biocatal Agric Biotechnol 11(March):224–230.
https://doi.org/10.1016/j.bcab.2017.07.012
3. Deshavath NN, Mohan M et al (2017b) Dilute acid pretreatment of sorghum biomass to maxi-
mize the hemicellulose hydrolysis with minimized levels of fermentative inhibitors for bio-
ethanol production. 3 Biotech. Springer Berlin Heidelberg 7(2):1–12. https://doi.org/10.1007/
s13205-017-0752-3
4. Deshavath NN et al (2018) Chemical composition analysis of various genetically modified
sorghum traits: pretreatment process optimization and bioethanol production from hemicel-
lulosic hydrolyzates without detoxification. Biochem Pharmacol Elsevier B.V. https://doi.
org/10.1016/j.jece.2018.08.002
5. Gírio FM et al (2010) Hemicelluloses for fuel ethanol: a review. Bioresour Technol
101(13):4775–4800. https://doi.org/10.1016/j.biortech.2010.01.088
6. Huang CF et al (2009) Enhanced ethanol production by fermentation of rice straw hydroly-
sate without detoxification using a newly adapted strain of Pichia stipitis. Bioresour Technol
100(17):3914–3920. https://doi.org/10.1016/j.biortech.2009.02.064
7. Liu X et al (2012) Quantification of glucose, xylose, arabinose, furfural, and HMF in corn-
cob hydrolysate by HPLC-PDA-ELSD. Carbohydr Res Elsevier Ltd 353:111–114. https://doi.
org/10.1016/j.carres.2012.03.029
8. Mohagheghi A, Ruth M, Schell DJ (2006) Conditioning hemicellulose hydrolysates for fer-
mentation: effects of overliming pH on sugar and ethanol yields. Process Biochem 41(8):1806–
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9. Naik SN et al (2010) Production of first and second generation biofuels: a comprehensive
review. Renew Sust Energ Rev 14(2):578–597. https://doi.org/10.1016/j.rser.2009.10.003
10. Palmqvist E, Hahn-Hägerdal B (2000) Fermentation of lignocellulosic hydrolysates I: inhibi-
tors and mechanism of inhibition. Bioresour Technol 74(1):25–33
11. Rowell R (2012) Handbook of wood chemistry and wood composites, 2nd edn. CRC Press,
Boca Raton. https://doi.org/10.1201/b12487
12. Walker GM (1994) The roles of magnesium in biotechnology. Crit Rev Biotechnol
14(4):311–354
Effect of BGA Biofertilizers Using
Different Carrier Materials on Rice Crop 2
Rajinder Kaur and Dinesh Goyal
Abstract
The present study laid emphasis on rice crop improvement by development of
low-cost blue-green algal biofertilizer comprising of consortium of four ARM
blue-green algal strains, viz. Anabaena variabilis (ARM 441), Nostoc muscorum
(ARM 442), Tolypothrix tenuis (ARM 443) and Aulosira fertilissima (ARM 444)
using different carrier materials, i.e. fly ash (100%), soil (100%), montmorillonite
(100%), fly ash + soil (1:1) and fly ash + montmorillonite (1:1). Pot trial was
conducted to study their effect on rice cultivar PUSA 1121 using nonsterile soil
with a control without inoculation of blue-green algal consortium. At the time of
harvest (after 90 days of inoculation), consortium of ARM culture showed
highest nitrogen content (0.149%) and carbon (0.39%), respectively, at treatment
T3 involving fly ash + soil (1:1) followed by highest grain yield (g per pot) of
14.3 and 12.75 which was recorded in treatment T3, fly ash + soil (1:1) as
compared to control. Therefore in the present study, fly ash with combination of
soil (1:1) was observed as a good carrier material in place of soil or MMT alone
for showing highest nitrogen, carbon and phosphorus content promoting cheap
and adaptable method by farmers for organic farming.
Keywords
Blue-green algae · Biofertilizers · Soil · Fly ash · Soil properties · Rice
R. Kaur (*)
Department of Biotechnology, Beant College of Engineering and Technology,
Gurdaspur, Punjab, India
D. Goyal
Department of Biotechnology, Thapar University, Patiala, Punjab, India

https://doi.org/10.1007/978-981-13-6321-4_2
10 R. Kaur and D. Goyal
2.1 Introduction
Indian agriculture was mostly organic before the advent of the Green Revolution;
however, widespread adoption of nutrient-responsive and high-yielding varieties
and use of inorganic fertilizers, weedicides and insecticides only resulted in high
crop yield [1]. It has been reported that excessive utilization of chemical fertilizers
has made nitrogen as the second limiting factor after water, for which the use of
biofertilizers becomes necessary to prevent soil deterioration and maintain soil
fertility. Biofertilizers are best developed substitute for chemical fertilizers such as
cyanobacteria which are capable of fixing nitrogen [2]. Amongst the array of
biofertilizers developed for different crops, cyanobacteria, popularly known as
blue-green algae, constitute the most important inputs in rice cultivation [4, 10]. In
addition, the use of cyanobacteria as biofertilizers can improve plant growth and
crop yield as they add organic matter to soil, thus improving soil texture [6].
Application of such algal biofertilizers has proved to be sustainable, eco-friendly,
cheap and easily manageable and improves the nutrient status as well as soil health
[3]. Carrier-based algal biofertilizers can make the rice production system more
viable and reduce the ecological hazards caused due to synthetic fertilizers and can
serve as one of the components of integrated plant nutrient supply system [7]. In the
present study, consortium of four ARM blue-green algal strains Anabaena variabilis
(ARM 441), Nostoc muscorum (ARM 442), Tolypothrix tenuis (ARM 443) and
Aulosira fertilissima (ARM 444) procured from the Indian Agricultural Research
Institute, New Delhi, were prepared using different carrier materials (fly ash (100%),
soil (100%), montmorillonite (100%), fly ash + soil (1:1) and fly ash + montmorillonite
(1:1)). Their effect on rice cultivar PUSA 1121 was examined in pot experiment
under glasshouse conditions, using nonsterile soil with a control without inoculation
of blue-green algal consortium.
2.2 Methodology
For development of low-cost BGA biofertilizers using different carrier materials,

electrostatic precipitator (ESP) coal fly ash was collected from GGSS Thermal Power
Plant, Ropar, Punjab. Wood charcoal and montmorillonite were purchased from local
market located in Patiala, Punjab, as per required quantity. Garden soil was collected
from experimental plot Science & Technology Entrepreneurs’ Park (STEP), Thapar
University, Patiala. Soil, charcoal and montmorillonite were crushed into fine powder
and air-dried for their physiochemical properties. Comparative study of
physicochemical and mineralogical content was done before the pot trial of each
carrier material [9]. Different treatments of fly ash (100%), soil (100%), montmorillonite
(100%), fly ash + soil (50:50%) and fly ash + montmorillonite (50:50) on rice cultivar
PUSA 1121 were examined by pot experiment using nonsterile soil with a control
without inoculation of blue-green algal consortium. At the time of harvest (after 90
days of inoculation), soil total nitrogen (%), organic carbon (%) and phosphorus
content (mg kg−1) and grain yield were examined with respective to control.
Table 2.1 Influence of consortium of blue-green algal inoculants on soil physicochemical proper-
ties in pot experiment with rice crop after 90 DAT (days after transplanting)
BGA consortium of ARM cultures
Organic Carbon Total Nitrogen Ava. P
Treatments pH (%) (%) (mgkg−1)
T1 Soil 100% 8.44 ± 0.5b 0.29 ± 0.04 bc 0.075 ± 0.005 d 12.95 ± 1.3 c
T2 MMT 100% 8.33 ± 0.24 c 0.31 ± 0.01 b 0.095 ± 0.012 c 11.69 ± 1.10 d
T3 Fly ash + soil 8.21 ± 0.58e 0.39 ± 0.11 a 0.149 ± 0.034 a 14.98 ± 0.96 b
(50:50)
T4 Fly 8.29 ± 0.17d 0.33 ± 0.02 b 0.137 ± 0.001 b 15.74 ± 0.52 a
ash + MMT
(50:50)
T5 Fly 8.13 ± 0.22f 0.30 ± 0.001 bc 0.120 ± 0.011 b 11. 25 ± 0.95 d
ash + MMT
(10:50)
T6 Fly ash 7.68 ± 0.51g 0.24 ± 0.07 ab 0.092 ± 0.047 b 13.01 ± 0.62 c
100%
Control Soil without 8.44 ± 0.6a 0.21 ± 0.001 c 0.007 ± 0.001 e 4.54 ± 0.32 e
inoculum
L.S.D 0.036 0.054 0.003 0.379
(P<0.05)
Fig. 2.1 Effect of

inoculation of consortium
of BGA (ARM cultures)
on grain yield of rice crop
(g per pot)
Pot trials on PUSA 1121 examined that consortium of ARM-procured cultures with
the combination of fly ash and soil (1:1) T3 showed nitrogen content of 0.149% and
carbon of 0.39%, respectively (Table 2.1), followed by highest grain yield (g per pot)
of 14.3 and 12.75 that was recorded in treatment T3, fly ash +soil (1:1) as compared
to control (Fig. 2.1). Physicochemical properties of soil before the inoculation of
BGA consortium have been shown in Table 2.2. Significant enhancement of organic
C and total N and P content over control could be contributed by growth-promoting
12 R. Kaur and D. Goyal
Table 2.2 Physicochemical Parameters Mean ± SE

analysis of soil used in pot pH 8.45 ± 0.07
culture experiment before the
Organic Carbon (%) 0.20 ± 0.01
transplantation of rice and
inoculation of BGA Total Nitrogen (%) 0.011 ± 0.004
consortium Ava. Phosphorus (mgkg−1) 5.41 ± 0.6
Water Holding capacity (%) 33 ± 0.1
role of cyanobacterial inoculants in the rhizospheric zone of rice fields [8] and fly ash
which itself contained some available P [5].
2.4 Conclusion
The present investigation concluded that fly ash with combination of soil (1:1) was
observed as a good carrier material in place of soil or MMT alone for showing
highest nitrogen, carbon and phosphorus content promoting cheap and adaptable
method by farmers for organic farming. Incorporation of fly ash-based algal
biofertilizers to the soil can enhance more crop productivity with reduced dosage of
urea for long-term sustainability of soil nutrients.
References
1. Pabbi S (2008) Cyanobacterial biofertilizers (review). J Eco-friendly Agric 3(2):95–111
2. Elanwar M, Osman H, Mostafa M, El-Sheekh M, El-Naggar AH, Gheda SF (2010) Effect of
two species of cyanobacteria as biofertilizers on some metabolic activities, growth, and yield
of pea plant. Biol Fertil Soils 46:861–875
3. Pereira I, Ortega R, Barrientos L, Moya M, Reyes G, Kramm V (2009) Development of a
biofertilizer based on filamentous nitrogen –fixing cynobacteria for rice crops in Chile. J Appl
Phycol 21:135–144
4. Dhar DW, Prasanna R, Singh BV (2007) Comparative performance of three carrier based blue
green algal biofertilizers for sustainable rice cultivation. J Sustain Agric 30:41–50
5. Gaind and Gaur (2002) Impact of fly ash and phosphate solubilising bacteria on soybean pro-
ductivity. Bioresour Technol 85:313–315
6. Maqubela P, Pearson NSM, Muchaonyerwa P, Acqui LPD, Pardo MT (2010) Effects of cya-
nobacteria strains selected for their bioconditioning and biofertilization potential on maize dry
matter and soil nitrogen status in a South African soil. Jpn Soc Soil Sci Plant Nutr 56:552–559
7. Mittra BN, Karmakar S, Swain KD, Ghosh CB (2005) Fly ash – a potential source of soil
amendment and a component of integrated plant nutrient supply system. International Ash
Utilization Centre for Applied Energy Research, University of Kentucky, Paper#28. http://
www.flyash.info
8. Prasanna R, Jaiswal P, Nayak S, Sood A, Kaushik BD (2009) Cyanobacterial diversity in the
rhizosphere of rice and its ecological significance. Indian J Microbiol 49:89–97
9. Kaur R, Goyal D (2014) Mineralogical comparison of coal fly ash with soil for use in agricul-
ture. J Mater Cycles Waste Mater 18(1):186–200
10. Valiente EF, Ucha A, Quesada A, Leganes F, Carreres R (2000) Contribution of N2 fixing
cyanobacteria to rice production: availability of nitrogen from 15N-labelled cyanobacteria and
ammonium sulphate to rice. Plant Soil 221:107–112
Identification of Differentially Expressed
Terminal Heat Stress-Associated 3
Proteins in Developing Grains in Wheat
(Triticum aestivum L.)
Davinder Sharma, Ratan Tiwari, Vijay Kumar Gupta,

Jagadish Rane, and Rajender Singh
Abstract
Terminal heat stress (THS) causes abrupt modulation in the expression of stress-
related proteins in developing seeds. These differential expressions are thought
to enhance thermotolerance. Hence, a field experiment was conducted with three
spring wheat genotypes, Raj 4014 (heat sensitive), K 7903 (heat escaper) and
WH 730 (heat tolerant), for the identification of THS-associated proteins. To
expose the plants to different levels of temperatures at the time of grain filling,
the crop was sown during the second week of November and during the first
week of January. The average ambient temperatures during the grain growth
phase between anthesis and physiological maturity were 25.5 °C and 24.9 °C
when sown in November and 31.3 °C and 32.0 °C when sown in January in
2012–2013 and 2013–2014, respectively. SDS-PAGE revealed considerable dif-
ferences in grain proteome at different stages of grain filling in response to
THS. Results showed that RAJ 4014 and K 7903 had very high homology in
terms of qualitative pattern of protein bands as compared to WH 730. RAJ 4014
and K 7903 showed the expression of two new THS-responsive proteins (~ 40
and 105 kDa) at 7 days post anthesis (DPA) under THS. These protein bands
appeared in WH 730 subsequently at 14 DPA but with low intensity. While pro-
tein bands of ~ 90 and 42 kDa appeared in K 7903 at 7 DPA, other two genotypes
D. Sharma
Presenting author, ICAR-Indian Institute of Wheat & Barley Research,
Karnal, Haryana, India
R. Tiwari · R. Singh (*)
ICAR-Indian Institute of Wheat & Barley Research, Karnal, Haryana, India
V. K. Gupta
Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India
J. Rane
School of Drought Stress, ICAR-National Institute of Abiotic Stress Management,
Baramati, Maharashtra, India

https://doi.org/10.1007/978-981-13-6321-4_3
14 D. Sharma et al.
showed these bands at 14 DPA under both normal and THS conditions. These
information could help in designing a strategy for developing heat-tolerant culti-
vars in molecular breeding programmes.
Keywords
Terminal heat stress · Differentially expressed proteins · SDS-PAGE ·
Thermotolerance
3.1 Introduction
As a cool season crop, wheat is sensitive to high temperature, but with the avail-
ability of extensively adapted semidwarf cultivars, wheat cultivation has spread into
temperate regions nearness to the equator especially in Southeast Asia. Some culti-
vars of wheat have versatile characters to survive above threshold level tempera-
tures. Wheat evolved appropriate mechanisms such as escape, avoidance and/or
staying green to cope with terminal heat stress (THS) by abrupt modulation in the
expression of stress-related proteins in developing seeds. These differential expres-
sions are thought to enhance thermotolerance [1]. Several studies have provided
evidence that THS provokes cessation of conventional protein synthesis, accompa-
nied by an increased synthesis of stress-related proteins, mainly, such as heat-shock
proteins (HSPs) [2].
HSPs act as network of chaperone machinery, in which many chaperones act in
concert or interact with other stress response mechanisms for facilitating protein
refolding and stabilizing the polypeptides which improve and establish the normal
physiological processes under high temperature stress to ensure normal growth and
development [3]. HSPs can be broadly grouped as high molecular weight (HMW)
HSPs and low molecular weight (LMW) HSPs. HMW-HSPs have essential func-
tions in providing protection against aggregation and misfolding of non-native poly-
peptides under heat stress conditions. However, LMW-HSPs constitute complexes
with unfolded proteins and other HSPs for stabilization of unfolded proteins [4].
Expression of specific stress-related proteins is genotypic dependent, and SDS-
PAGE protein banding patterns could be important to investigate different thermo-
tolerance mechanisms mentioned above. The study was undertaken to compare the
SDS-PAGE protein profile in three wheat genotypes having different thermotoler-
ance mechanism with a view to generate information which could provide a novel
strategy for improving heat tolerance in molecular breeding programmes.
The experiment was conducted at Indian Institute of Wheat and Barley Research
(IIWBR), Karnal (29° 42′ N, 77° 2′ E), in the Indo-Gangetic Plain in Northwestern
India, with mildly alkaline soil (Typic ustochrept) over a period of 2 years
3 Identification of Differentially Expressed Terminal Heat Stress-Associated Proteins… 15
(2012–2013 and 2013–2014). Experiments were laid out in a randomized complete

block design with ten replications. To expose the plants to different levels of tem-
peratures at the time of grain filling, the crop was sown under optimal irrigated
conditions during the second week of November as control and under very late
sown (LS) irrigated conditions during the second week of January. The average
ambient temperatures during the grain growth phase between anthesis and physio-
logical maturity ranged from 25.1 to 25.4 °C when sown in November and 32.0 to
32.6 °C when sown in January in 2012–2013 and 2013–2014, respectively. Three
spring wheat genotypes, viz. Raj 4014 (heat sensitive) [5, 6], K 7903 (heat escaper)
[5, 6] and WH 730 (heat tolerant) [5–7], with a wide range of genetic background
were chosen. As days to flowering in selected genotypes ranged from 7 to 12 days
in each experiment, previous observations were used for staggered sowing to ensure
synchrony in flowering and thus nearly the same level and magnitude of exposures
to ambient temperatures during grain filling across the genotypes. Sowing was done
with the IIWBR dibbler to ensure precision in planting [5, 6]. Dibbling tool was
used to place 3 seeds at 1 locus without any overlap at 6.5 cm depth within a small
experimental unit of 4 rows of 50 cm length with 20 cm space between the rows and
10 cm between the plants within the rows. Seeds were hand-planted in uniformly
created cavities at the rate of single seed/cavity at 72 seeds/plot. Two weeks after
sowing, 2 of 3 plants were uprooted to finally keep 1 plant/locus and 24 plants/plot.
Developing spikes were sampled in the morning hours, at 5 days gap from anthesis
to 35 DPA. All grains from each spikelet were removed, frozen and stored at
−80 °C. To extract crude protein, 12 grains were homogenized with a pestle in a
precooled mortar that contained 2.5 mL frozen extraction medium consisting of
100 mM HEPES-NaOH (pH 7.5), 2 mM EDTA, 50 mM 2-mercaptoenthanol and
12.5% (v/v) glycerol. The homogenate was filtered through multilayered cheese
cloth and followed by centrifugation at 12,000 g for 15 min at 4 °C. The supernatant
obtained was used for the SDS-PAGE protein profiling. Soluble protein from devel-
oping seed samples of each treatment determined by Lowry method [8] and the
samples of each corresponding to 12 μg protein were mixed with sample buffer and
resolved on 10% SDS-PAGE. The protein bands were resolved using the Hoefer
SE600 electrophoresis unit.
One-dimensional SDS-PAGE revealed considerable differences in grain proteome

at different stages of grain filling in response to terminal heat stress. Heat stress
induced both qualitative (presence/absence) and quantitative (differential expres-
sion) alterations in the protein profile (Fig. 3.1). More than 30 protein bands were
detected which included both common and specific bands among the investigated
genotypes. An examination of the protein profile of genotypes grown under both the
sowing conditions revealed that RAJ 4014 and K 7903 had very high homology in
terms of qualitative pattern of protein bands as compared to WH 730.
16 D. Sharma et al.
Fig. 3.1 SDS-PAGE protein profiling of three different wheat genotypes (1- K 7903, 2- RAJ 4014
and 3- WH730) at 7, 14, 21 and 28 days post anthesis of grain filling under timely and late sown
conditions. A mixture of markers (M) was used for SDS-PAGE. Arrows show the change in
expression of existing/new proteins. 12 μg of proteins was loaded in each well of the polyacryl-
amide gel (10%) for SDS-PAGE
There was much quantitative variation in protein bands among the genotypes. At
7 and 14 DPA, almost all protein bands showed upregulation in both tolerant (K
7903 and WH 730) and sensitive (RAJ 4014) genotypes under late sown as com-
pared to the timely sown condition (Fig. 3.1). RAJ 4014 and K 7903 showed the
expression of two new heat stress-responsive proteins (~ 40 and 105 kDa) at 7 DPA
under late sown condition. The expression of these proteins was high in RAJ 4014
as compared to K 7903. These protein bands appeared in WH 730 subsequently at
14 DPA but with less expression. On the contrary, protein bands (~ 90 and 42 kDa)
3 Identification of Differentially Expressed Terminal Heat Stress-Associated Proteins… 17
were present in K 7903 only under the timely and the late sown condition, respec-
tively, at 7 DPA, while these were highly expressed in all the three genotypes at
14 DPA under both the sowing conditions. A protein band (~ 30 kDa) showed down-
regulation under late sown condition in all the three genotypes. Few proteins (~ 68,
70 and 60 kDa), which were upregulated during early grain filling under late sown
condition, showed their downregulation at 21 DPA (Fig. 3.1). On the other hand,
protein bands (~ 40 and 90 kDa) were downregulated under timely sown condition.
In addition, a protein of ~ 40 kDa disappeared from all genotypes under both the
sowing conditions. As the grain filling progresses, some new protein bands (~ 68
and 72 kDa) appeared, while some previously existed protein bands (~ 40 and
44 kDa) disappeared under late sown condition at 28 DPA (Fig. 3.1).
Besides these, few protein bands which were upregulated previously showed
their downregulation under both sowing conditions. Protein profiling showed both
qualitative and quantitative changes under heat stress (Fig. 3.1). These heat stress-
responsive changes were predicted to be related to starch biosynthesis enzymes,
heat-shock proteins or anti-oxidant enzymes. The differences in SDS-PAGE protein
profile under heat stress are in agreement with the earlier results reported in wheat
[9–12]. Wheat and barley proteome analysis under heat stress also showed the simi-
lar pattern of expression of heat stress-related proteins [12]. A positive association
of HSP with different intensity of temperature stress (from 25 to 46 °C) has been
reported in wheat [13, 14]. Similarly, [9] reported that several HMW-HSPs (83–
94 KDa) and LMW-HSPs (16–42 KDa) appeared in wheat developing seeds after
heat shock in Mustang and Sturdy varieties of wheat with distinct levels of acquired
thermotolerance. A growth stage-specific expression profile revealed the role of Hsp
70 gene in THS tolerance [15]. SDS-PAGE profile study revealed that differential
SDS-PAGE protein profile exhibited new set of proteins during late sown in thermo-
tolerant cultivars while few proteins were observed constantly in all wheat cultivars
in both timely and late sown conditions. These unique proteins could adjust the
tolerance mechanism of wheat under high temperature at different stages of grain
filling and could be used as a stage-specific screening marker to identify high-
temperature-tolerant wheat genotypes.
Acknowledgements We gratefully acknowledge the financial support from ICAR for Network
Project on Transgenic in Crop: Functional Genomics in Wheat.
References
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tive and grain-filling phases. Crit Rev Plant Sci 30(6):491–507
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biochemical and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci
14(5):9643–9684
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4. Wahid A, Gelani S, Ashraf M, Foolad MR (2007) Heat tolerance in plants: an overview.

Environ Exp Bot 61(3):199–223
5. Sharma D, Singh R, Rane J, Gupta VK, Mamrutha HM, Tiwari R (2016) Mapping quantitative
trait loci associated with grain filling duration and grain number under terminal heat stress in
bread wheat (Triticum aestivum L.). Plant Breed 135(5):538–545
6. Sharma D, Tiwari R, Gupta VK, Rane J, Singh R (2018) Genotype and ambient temperature
during growth can determine the quality of starch from wheat. J Cereal Sci 79:240–246
7. Pandey GC, Mamrutha HM, Tiwari R, Sareen S, Bhatia S, Siwach P, Tiwari V, Sharma I (2015)
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Physiol Mol Biol Plants 21(1):93–99
8. Lowry OH, Rosebrough NJ, Farrand AL, Randall RJ (1951) Protein measurement with the
Folin Phenol reagent. J Biol Chem 193:265–270
9. Krishnan M, Nguyen HT, Burke JJ (1989) Heat shock protein synthesis and thermal tolerance
in wheat. Plant Physiol 90(1):140–145
10. Sharma NPM, Babu SS, Udayakumar M (2007) Heat shock response of wheat cultivars differ-
ing in thermotolerance. Indian J Plant Physiol 12(4):327–336
11. Chauhan S, Srivalli S, Nautiyal AR, Khanna-Chopra R (2009) Wheat cultivars differing in heat
tolerance show a differential response to monocarpic senescence under high temperature stress
and involvement of serine proteases. Photosynthetica 47(4):536–547
12. Kumar RR, Goswami S, Sharma SK, Singh K (2012) Protection against heat stress in wheat
involves change in cell membrane stability, antioxidant enzymes, osmolytes, H2O2 and tran-
script of heat shock protein. Int J Plant Physiol Biochem 4(4):83–91
13. Zivey M (1987) Genetic variability for heat shock protein in common wheat. Theor Appl
Genet 74(2):209–213
14. Satbhai R, Kale A, Naik R (2016) Study on effect of high temperature stress in wheat geno-
types using SDS protein profile. J Wheat Res 8(1):51–58
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New Series 35(3):233–243
The Genetic Toxicity Potential of Heavy
Metals (Zn, Cu) on Vigna radiata, Triticum 4
aestivum, and Cicer arietinum
Aparajita Shilpie and Kamal Nayan Mishra
Abstract
Optimum amount of heavy metal in soil is essential for proper growth of plants.
However, presence of these metals in higher concentration is detrimental and
harmful for plant kingdom. Accumulation of heavy metals in soil can inhibit the
growth as well as ability of absorbance of nutrients from soil in several plants.
Toxic tolerance and response toward these heavy metals vary among plant spe-
cies, and this variation is directly related to genetic constitution of plant genome.
The present study is a comparative account on the effect of ZnSO4 and CuSO4 on
Vigna radiata, Triticum aestivum, and Cicer arietinum plants exposed to heavy
metals. The plants were treated with different concentrations of ZnSO4, CuSO4,
and combined ZnSO4 and CuSO4. Different concentrations of Zn and Cu inde-
pendently showed significant effect on different parts of plants. A significant
synergetic effect was also observed in treated groups when compared with con-
trolled plants. This study contributes valuable information on effect of heavy
metals present in excess amount which lead to the changes in genotypic and the
phenotypic characteristics of plants.
Keywords
Genetic toxicity · Toxic tolerance · Heavy metals · Plant growth
A. Shilpie (*)
IIT Roorkee, Roorkee, Uttarakhand, India
K. N. Mishra
Pandit Ravishankar Shukla University, Raipur, Chhattisgarh, India

https://doi.org/10.1007/978-981-13-6321-4_4
20 A. Shilpie and K. N. Mishra
4.1 Introduction
Heavy metals are highly toxic environmental pollutants having harsh effects on
plants, animals, and human being. The factors which are generally responsible for
heavy metal contamination in environment are anthropogenic (viz., mining, smelt-
ing operation, and agriculture) as well as natural activities (viz., earthquake, volca-
nic activity), and these are taken by human being and other animals through the
food chain, thus affecting them [20]. Optimum ranges of trace metals are necessary
for growth, but below and above the optimum level, they cause deficiencies and
toxicity, respectively. They also affect enzymatic activities in plants and animals.
Al, B, Cu, Ni, Zn, As, Ba, Cd, Cr, Hg, Mo, Ni, Pb, and Se are the metals which
generally cause harsh effects in plants as well as in animals including human [13,
14]. Plants are good specimens to judge and estimate the environmental pollution
and toxicity caused by heavy metals. Phytotoxicity of metals is well proved [10, 19,
24]. Toxic heavy metal toxicity affects the plant growth; reduces the yielding capa-
bility, growth of root and shoot, homeostasis, and rate of nutrient absorption; and
also gets accumulated in plants [18]. Plants give some indication of heavy metal
toxicity by producing some biomarkers or endpoints which can be detected and
examined at different levels of molecular and cellular organizations to check and
control the heavy metal toxicity in an environment [5, 6].
Heavy metal gives rise to some genotoxic effects; they can be classified into four
types: mutagenesis, clastogenesis, aneugenesis, and recombinogenesis [17].
Response against heavy metals in a plant can be checked by various ways like
observing morphological and molecular changes [18]. Among the heavy metals,
excess amount of zinc in soil affects the metabolic activities of plants; it reduces the
growth of both root and shoot and causes senescence and gives rise to chlorosis in
the younger leaves; it reaches to older leaves after prolonged exposure [7–9]. Copper
(Cu) is a metal which is considered as a micronutrient for plants [22] and helps in
CO2 assimilation and ATP synthesis. But high concentration of Cu in soil has some
cytotoxic effect; it produces stress and damage to plants, inhibits the growth of
plant, and causes leaf chlorosis [12].
The experiment was performed on three plants, mung bean (Vigna radiata), wheat
(Triticum aestivum), and gram (Cicer arietinum), which were grown in pot, in con-
trolled conditions. The concentrations of copper and zinc which were tested in soil
4 The Genetic Toxicity Potential of Heavy Metals (Zn, Cu) on Vigna radiata, Triticum 21
Table 4.1 The soil concentration of the Cu/Zn pre- and posttreatment
Soil concentration Pretreatment (mg/kg) Posttreatment (mg/kg)
Copper 58.75 58.38
Zinc 354.62 352.44
Table 4.2 The concentration of ZnSO4, CuSO4, and combined ZnSO4 and CuSO4
Heavy metals Concentration 1 Concentration 2 Concentration 3
Control Distilled water Distilled water Distilled water
CuSO4 50 μM 175 μM 200 μM
ZnSO4 0.25 mM 0.625 mM 1 mM
ZnSO4:CuSO4 0.75 mM:150 μM 0.75 mM:300 μM 1.5 mM:150 μM
(1:1) (1:2) (2:1)
before sowing the seed were shown in Table 4.1. And they are treated regularly with
different concentrations of ZnSO4, CuSO4, and combined ZnSO4 and CuSO4 [1, 11,
23] given in Table 4.2. Not much changes in concentration of Cu/Zn in soil profile
(before and after treatment) had been observed in concentration due to the reason
that treatments were given to the plants by spraying method on their leaves; there
were no contact of soil with heavy metal solutions. The percentage of seed germina-
tion was calculated after 24 h in room temperature (Table 4.3). After 10 days, plants
were taken out from pot, washed with distilled water and nonionic detergent Tween
20. Plants were cut into three parts, viz., leaves, stems, and roots, and DNA was
isolated from each part separately by modified CTAB method [2, 16]. DNA of con-
trolled and treated samples was compared by the process of agarose gel electropho-
resis. Due to treatment of heavy metals, growth and development of plants
deteriorated; changes in protein function must be a reason behind this. That is why
protein estimation was performed by the Bradford method [4] (Fig. 4.1).
22
Table 4.3 Seed germination percentage was calculated on the basis of the number of seed germinated
Seed germination Seed germination Seed germination
Heavy metals Concentration 1 percentage (%) Concentration 2 percentage (%) Concentration 3 percentage (%)
Control Distilled water Vigna radiata: 92 Distilled water Vigna radiata: 96 Distilled water Vigna radiata: 92
Triticum aestivum:100 Triticum aestivum:96 Triticum aestivum: 96
Cicer arietinum: 96 Cicer arietinum:92 Cicer arietinum: 96
CuSO4 50 μM Vigna radiata:72 175 μM Vigna radiata: 68 200 μM Vigna radiata: 60
Triticum aestivum:88 Triticum aestivum:72 Triticum aestivum:68
Cicer arietinum:80 Cicer arietinum:72 Cicer arietinum:64
ZnSO4 0.25 mM Vigna radiata: 76 0.625 mM Vigna radiata: 72 1 mM Vigna radiata: 68
ZnSO4:CuSO4 0.75 mM:150 μM Vigna radiata: 80 0.75 mM:300 μM Vigna radiata: 76 1.5 mM:150 μM Vigna radiata: 60
A. Shilpie and K. N. Mishra
Fig. 4.1 Variation in seed germination rate during treatment of heavy metals (3 days) along with
control samples
4.3 Results (Figs. 4.2, 4.3, and 4.4; Graphs 4.1, 4.2, and 4.3)
Fig. 4.2 Leaf samples: comparison between absorbance of leaf samples (gram, wheat, moong)
treated with different concentrations of Zn and Cu
Fig. 4.3 Root samples: comparison between absorbance of root samples (gram, wheat, moong)
Fig. 4.4 Stem samples: comparison between absorbance of stem samples (gram, wheat, moong)
Graph 4.1 DNA

electrophoretic gel picture
of leaf samples from
treated plants
Graph 4.2 DNA

electrophoretic gel pictures
of stem samples from
treated plants
Graph 4.3 DNA

electrophoretic gel picture
of root samples from
treated plants
4.4 Discussion
The same result was observed by Verma et al. [23]. They used various concentra-
tions of CuSO4 solution, viz., 50, 200, 500, and 1000 μM, and found that less con-
centration (<50 μM) of it is necessary for seedling growth in mung bean. Higher
concentration affects adversely. And he also found that there was high protein con-
tent in root than shoot as compared to control plants.
Ashagre et al. [3] performed the same experiment on tomato’s growing seed to
check the effect of copper and zinc. And he found high concentration (p < 0.05)
decreases the rate and amount of germination, length of root and shoot, as well as
capacity to endure the stress conditions. Minimum growths were observed at
600 ppm and maximum in controlled samples.
Most of the experimental reports [1, 15, 21] explained the same result in different
plants.
4.5 Conclusion
Heavy metals affect the plant cells adversely. The total protein content was found to
be varying as compared to control samples, along with varying concentrations of
heavy metals. The DNA was also affected by heavy metals. This may be due to the
adverse effects of heavy metal on the metabolic activities. Senescence was found in
younger leaves.
References
1. Ahmad N, Alatar AA, Faisal M, Khan MI, Fatima N, Anis M, Hegazy AK (2015) Effect
of metals (Cu and Zn) on the development of sarpagandha (Rauvolfia serpentina) cultured
in vitro. Biol Plant 59(1):11–17
2. Allen GC, Flores-Vergara MA, Krasnyanksi S, Kumar S, Thompson WF (2006) A modified

protocol for rapid DNA isolation form plant tissues using cetyltrimethylammonium bromide.
Nat Protoc 1(5):2320–2325
3. Ashagre H, Almaw D, Feyisa T (2013) Effect of copper and zinc on seed germination, phyto-
toxicity, tolerance and seedling vigor of tomato (Lycopersicon esculentum L. cultivar Roma
VF). Int J Agric Sci Res 2(11):312–317
4. Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
5. Briat JF, Lebrun M (1999) Plant responses to metal toxicity. Comptes Rendus de l’Academie
des Sciences/Life Sci 322:43–54
6. Burton MAS (1986) Biological monitoring of environmental contaminants, a technical report.
MARC, London
7. Choi JM, Pak CH, Lee CW (1996) Micronutrient toxicity in French marigold. J Plant Nutr
19:901–916
8. Ebbs SD, Kochian LV (1997) Toxicity of zinc and copper to Brassica species: implications for
phytoremediation. J Environ Qual 26:776–781
9. Fontes RLS, Cox FR (1998) Zinc toxicity in soybean grown at high iron concentration in nutri-
ent solution. J Plant Nutr 21:1723–1730
10. Foy CD, Chaney RL, White MC (1978) The physiology of metal toxicity in plants. Annu Rev
Plant Physiol 29:511–566
11. Johnson SE, Lauren JG, Welch RM, Duxbury JM (2005) A comparison of the effects of micro-
nutrient of seed priming and soil fertilization on the mineral nutrition of chickpea (Cicer ari-
etinum), Lentil (Lens culinaris), Rice (Oryza sativa) and Wheat (Triticum aestivum) in Nepal.
Exp Eng 41:427–448
12. Lewis S, Donkin ME, Depledge MH (2001) Hsp70 expression in Enteromorpha intestinalis
(Chlorophyta) exposed to environmental stressors. Aquat Toxicol 51:277–291
13. Logan TJ, Traina SJ (1993) Trace metals in agricultural soils. In: Allen HE, Perdue EM, Brown
DS (eds) Metals in groundwater. Lewis Publishers, Boca Raton, pp 309–347
14. Mehera A, Farago ME (1994) Metal ions and plant nutrition. In: Farago ME (ed) Plants and the
chemical elements: biochemistry, uptake tolerance and toxicity. VCH, Weinheim, pp 32–66
15. Mittal N, Vaid P, Kaur A (2015) Effect on amylase activity and growth parameters due to metal
toxicity of iron, copper and zinc. Indian J Appl Res 5(4):662–664
16. Murray HG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic
Acids Res 8:4321–4325
17. Panda BB, Panda KK (2000) Genotoxicity and mutagenicity of metals in plants. In: Prasad
MNV, Strzalka K (eds) Physiology and biochemistry of metal toxicity and tolerance in plants.
Springer, The Netherlands, pp 3–172
18. Pirselova B (2011) Monitoring the sensitivity of selected crop to lead, cadmium and arsenic.
J Stress Physiol Biochem 7(4):31–38
19. Prasad MNV (1997) Trace metals. In: Plant ecophysiology. Wiley, New York, pp 263–273
20. Rajeshwari TR, Sailaja N (2014) Impacts of heavy metals on environmental pollution. J Chem
Pharm Sci 3(0974-2115):175–181
21. Singh D, Shakya N, Katiyar DK, Verma A, Narayan R, Niyazi R (2010) Impact of copper tox-
icity on black gram and its remedial approach for minimization of metal toxicity. Res Environ
Life Sci 3(3):133–138
22. Thomas F, Malick C, Endreszl EC, Davies KS (1998) Distinct responses to copper stress in the
halophyte, Mesembryanthemum crystallium. Physiol Plant 102:360–368
23. Verma JP, Singh V, Yadav J (2011) Effect of copper sulphate on seed germination, plant growth
and peroxidase activity of Mung bean (Vigna radiata). Int J Bot 7(2):200–204
24. Woolhouse HW (1983) Toxicity and tolerance in the response of plants to metals. In: Lang O,
Nobel PS, Osmond CB, Zeigler H (eds) Encyclopedia of plant physiology, Physiological plant
ecology III, vol 12. Springer-Verlag, Berlin, pp 245–300
Production, Optimization,
and Characterization of Siderophore 5
by Pseudomonas aeruginosa (C3) Isolated
from Rhizospheric Soil
Chhaya Verma, Apoorva Dixit, and Rajesh Kumar
Abstract
In this study production of siderophore was analyzed in fluorescent pseudomo-
nad named as C3 (Pseudomonas aeruginosa). Out of ten isolates, this strain was
selected for optimization of siderophore production; it produced 51.45 SU (sid-
erophore unit). Findings of this experiment showed that siderophore production
was increased when growth medium was added with potassium nitrate, 0.073 mg/
ml FeCl3, 200 ppm cadmium, sucrose (C source), and 0.5% NaCl in shaking
condition at pH 6 after 5 days of incubation. Siderophore production was
increased with incubation time and cadmium concentration, but after 6 days,
siderophore production was reduced. Further, siderophore extraction was done
by crystallization process, formed by adding methanol after maintaining pH 3 by
mixing of sulfuric acid, ferrous sulfate, and 50% ammonium sulfate. Produced
crystal was purified and characterized TLC (thin layer chromatography) and
FTIR (Fourier-transform infrared spectroscopy) analysis. In TLC analysis the
specific spot from the extracted siderophore was found to correspond with a spot
of standard siderophore with the same Rf value. In FTIR analysis similar peak
was observed as reported in earlier studies. This study was based on siderophore
production, optimization, and characterization of extracted siderophore.
Keywords
Siderophore · Pseudomonas · Optimization · FTIR
C. Verma · A. Dixit · R. Kumar (*)

Rhizosphere Biology Laboratory, Department of Environmental Microbiology, Babasaheb
Bhimrao Ambedkar University (A Central University), Lucknow, Uttar Pradesh, India

https://doi.org/10.1007/978-981-13-6321-4_5
28 C. Verma et al.
5.1 Introduction
Iron is a transition metal and essential micronutrient for almost all organisms which
consist of bacteria and fungi and plants in the various metabolic and informational
cellular pathways. In plant the iron is essential for many processes such as respira-
tion, photosynthesis, and nitrogen fixation. However, microorganisms are unable to
obtain enough iron present in soil as immobilized form and cannot be transported in
the cells. For utilization of unavailable iron, the plant and microbes produced low
molecular weight (<10 KD) iron-chelating secondary metabolites termed as “sid-
erophore” [1]. Siderophore has various applications in the field of agriculture, medi-
cine, etc. In present time the siderophore production in microbes is analyzed by
using the CAS agar assay [2]. In this assay competition was developed between the
ferric complex of an indicator dye chrome azurol S (CAS) and a chelator or sidero-
phore for iron. Iron is removed from CAS by siderophore because of higher affinity
of siderophore for iron.
Ionic forms of iron are insoluble under physiological conditions and hence are
difficult to assimilate for microorganisms, although iron is abundantly present in the
environment. Three to four gram of iron is generally present in human body, out of
which hemoglobin and myoglobin acquire the major share of 70%. To make avail-
able siderophore for biotechnological and medicinal application is important to
increase siderophore production by evaluating process parameter. Many different
environmental factors affect the synthesis of siderophore, notably the chemical
nature of the organic carbon and energy source, metals, Fe3+, amino acids, and
organic nitrogen sources [3, 4]. Any factor influencing siderophore production
influences the performance of PGPR in plant growth promotion and phytopathogen
suppression [5]. Therefore, present study was aimed to regulate the cultural param-
eter for improvement of siderophore production and characterization.
All the ingredients and media used in these experiments were purchased from
Himedia Laboratories Pvt Ltd (India) and Rankem Pvt Ltd (India). The strain C3
used in this experiment was collected from Rhizosphere Biology Laboratory and
characterized by morphological and biochemical methods.
5.2.1 Siderophore Production
Siderophore production by different strains of Pseudomonas aeruginosa was tested

by chrome azurol S (CAS) agar assay. Formation of yellow-orange zone around the
colonies indicates the siderophore production. CAS-shuttle assay was used for the
quantitative estimation of siderophore [2]. In this method, 3–4-day grown culture
broths were centrifuged at 10,000 rpm in cooling centrifuge at 4 °C for 10 min.
After this obtained cell-free supernatant was mixed with 0.5 ml CAS solution in
5 Production, Optimization, and Characterization of Siderophore… 29
1 ml of culture filtrate. The developing blue color was determined after 20 min of
incubation by using spectrophotometer at absorbance 630 nm. The percentage of
siderophore units was calculated by standard formula.
5.2.2 Optimization of Siderophore
There are several parameters affecting production of siderophore used for the opti-
mization studies; such factors are different nitrogen and carbon source, different
NaCl and FeCl3 concentration, pH, and days, etc. For nitrogen source potassium
nitrate, ammonium sulfate, ferrous ammonium sulfate, and glycine were added in
CAS media. In analysis of effects of carbon source, dextrose, glucose, sucrose, lac-
tose were added in CAS media. Three parameters of FeCl3 0.135 mg/ml, 0.270 mg/
ml, and 0.067 mM and for NaCl effects 0.5, 1, 3, 5% NaCl were used in the media.
Different concentrations of cadmium 0, 50, 100, and 200 ppm were used. Siderophore
production was also checked in shaking and static condition. The effect of days on
siderophore production was determined by incubating the flask at different days
such as 3, 6, and 9 days.
5.2.3 Crystallization and Characterization of Siderophore
For study of chemical structure, siderophore was extracted from culture supernatant
of C3 strain as crystal form by modified method of Tank et al. [6]. Produced crystal
was purified and characterized by TLC and FTIR analysis. In TLC analysis the
specific spot from the extracted siderophore was found to correspond with a spot of
standard siderophore with the same Rf value. In FTIR analysis similar peak was
observed as reported in earlier studies.
Siderophore production of isolate C3 was done on solid CAS blue agar plates.
Production of siderophore was observed with the formation of clear orange color
zone around the growth. In quantitative analysis the result showed that C3 strain
produces high amount of siderophore approximately 51.45 SU. In presence of FeCl3
the growth of Pseudomonas increases, and it is vital for its growth. However, De
Villegas [7] stated that above 10 μM concentration of FeCl3 has a negative effect on
siderophore production, and other study favors that highest siderophore production
occurs at iron concentration above 50 μg/mL [8]. Our results show maximum sid-
erophore production at 0.027 M concentration, and at 0.27 no production was
reported (Fig. 5.1d). Sodium chloride is also an important growth factor like other
supplement to the medium. The concentration of siderophore was maximum at 3%
NaCl than other concentrations. The 1% and 8% also showed positive result, but no
production was found at 5% (Fig. 5.1c).
30 C. Verma et al.
A B C D
80 40 60
40
60 30 30 40
40
SU
SU
SU
20
SU
20 10 20
20
0 0 10
0
0 0.27 0.027 0.013
1% 3% 5% 8%
Nitrogen source Carbon source FeCl3
NaCl
E F G
100 52 60
50 40
SU
SU
SU
50 20
48
0
0 46 0 ppm 50 ppm 100 200
3rd 6th 9th Shaking Static ppm ppm
days Growth condition Cd
Fig. 5.1 Siderophore production at different parameters including (a) nitrogen source, (b) carbon
source, (c) NaCl, (d) FeCl3, (e) days, (f) growth condition (shaking and static), (g) cadmium
concentration
Efficiency of biosynthesis of siderophore in strains was also affected by the car-

bon sources. Four different sugars (sucrose, dextrose, glucose, and lactose) were
used for optimization of siderophore production. In Fig. 5.1b, it was found that
dextrose, sucrose, and lactose were good for C3, but lactose was useless for sidero-
phore production. However, Sharma et al. [9] reported that glucose stimulated sid-
erophore production in Pseudomonas. Impact of nitrogen sources on siderophore
production was studied by addition of various nitrogenous compounds like urea,
potassium nitrate, ammonium sulfate, glycine, and FAS (ferrous ammonium sulfate)
supplemented to the medium in which FAS show negative siderophore production,
while remaining source had good response (Fig. 5.1a).
Different cadmium concentrations are used to check the production of sidero-
phore. Cadmium concentrations 0, 50, 100, and 200 ppm were used for the opti-
mization of siderophore. In this test at low concentration (50 ppm) of cadmium,
very little amount was produced, but at high concentration (200 ppm), sidero-
phore was produced positively as shown in Fig. 5.1g. At different incubation con-
ditions, we found that siderophore production was higher at shanking condition
than static because of oxygen availability (Fig. 5.1f).The inoculated culture media
was incubated for various days such as 3, 6, and 9 days at 30 °C (Figure 5.1e).
Siderophore production was enhanced day by day and reduced after 9th day of
incubation. As time spends, iron is depleted from the media. Therefore, increase
in pH may be coincidental to the increase in siderophore concentration. Along
with siderophore production, the pH of media was increased from 6.8 to 10.
Sayyed et al. [3] reported that siderophore production was declined if more iron
content was found in CAS medium.
5 Production, Optimization, and Characterization of Siderophore… 31
S1
1087.4
3.2
1142.7
3.0
2.8
3211.2
2.6
2.4
2.2
1433.5
Absorbance
2.0
622.1
1468.6
1.8
1.6
1.4
723.4
1677.8
979.7
1.2
1.0
0.8
0.6
0.4
0.2
0.0
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers (cm-1)
Fig. 5.2 FTIR characterization of siderophore crystal
5.3.1 Crystallization and Characterization of Siderophore
Obtained crystal was transparent, bright fine needle shaped, and white in appear-
ance. The crystal of extracted siderophore was analyzed by using FTIR analysis on
KBr pellets with the 4000–400 cm−1 range of wave numbers. Results of FTIR show
that crystals obtained had hydroxamate functional group which correlated with the
peaks obtained from FTIR analysis of PBHA crystals and pyoverdine (Fig. 5.2).
Finding showed that the standard crystals are hydroxamate crystal.
5.4 Conclusion
The present study depends on siderophore production and optimization in florescent

pseudomonad isolated from rhizosphere. The isolate C3 was selected for study
because it produced maximum amount of siderophore. Optimization of growth con-
dition and media composition was done for maximum siderophore production.
Maximum production was observed at different conditions, but in respect of incuba-
tion time, maximum production was reported at 6th day of incubation. Extracted
siderophore was characterized by FTIR and TLC analysis. The use of siderophore
as biocontrol agent against selected plant pathogens is an emerging idea, and such
field of study is an environment-friendly alternative for chemical pesticides.
References
1. Saharan BS, Nehra V (2011) Plant growth promoting rhizobacteria: a critical review. Life Sci
Med Res 21:1–30
2. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination
of siderophores. Anal Biochem 160:47–56
32 C. Verma et al.
3. Sayyed RZ, Chincholkar SB (2010) Growth & siderophore production Alcaligenes faecalis is
influenced by heavy metals. Indian J Microbiol 50(1):179–182
4. Sayyed RZ, Naphade BS, Chincholkar SB (2005) Ecologically competent rhizobacteria
for plant growth promotion & disease management. In: Rai MK, Chikhale NJ, Thakare PV,
Wadegaonkar PA, Ramteke AP (eds) Recent trends in biotechnology. Scientific Publisher,
Jodhpur, pp 1–16
5. Sharma S, Kaur M (2010) Antimicrobial activities of rhizobacterial strains of Pseudomonas
and Bacillus strains isolated from rhizosphere soil of carnation. Indian J Microbiol 50:229–232
6. Tank N, Rajendran N, Patel B, Saraf M (2012) Evaluation and biochemical characterization
of a distinctive pyoverdin from a Pseudomonas isolated from chickpea rhizosphere. Braz
J Microbiol 43:639–664
7. Díaz de Villegas ME, Villa P, Frías A (2002) Evaluation of the siderophores production by
Pseudomonas aeruginosa PSS. Rev Latinoam Microbiol 44(3–4):112–117
8. Manninen M, Sandholm TM (1993) Methods for the detection of Pseudomonas siderophores.
J Microbiol Methods 19:223–234
9. Sharma T, Kumar N, Rai N (2016) Production and optimization of siderophore produc-
ing Pseudomonas species isolated from Tarai region of Uttarakhand. Int J Pharm Biol Sci
7(1):306–314
Development of Marker in the Soft Gold
Mushroom Cordyceps spp. for Strain 6
Improvement
Loknath Deshmukh, Diva Gupta, and Sardul Singh Sandhu
Abstract
Natural drugs play extensive role and are the basis of traditional systems for cure
and treatment of diseases. Entomopathogenic fungi Cordyceps Spp. are one of
the unique and valuable sources of bioactive compounds which help in treatment
of various diseases like nervous disorders, cardiovascular diseases, tumors, age-
ing, hypo-sexuality, etc. A significant decrease in natural production of Cordyceps
Spp. has been observed in the last few decades from protected biosphere reserves,
due to thorough and illegal harvesting. This compels the necessity of artificial
cultivation and strain improvement strategies. For identifying improved strains
of the fungi, different methods using antifungal agents, UV irradiation and anti-
biotic compound have been carried out for effective selection systems. The two
fungi did not show any marker when treated with UV-irradiated conidia for
ammonia assimilation. Further when these isolates were treated with some anti-
fungal agents, again no remarkable marker was found against any antifungal
agents. Consecutively, an antibiotic hygromycin was also tested against the iso-
lates. The gradient decrease in radial growth was observed on increasing the
concentration of antibiotic from 200 to 900 μg/ml showing a radial zone of
15mm to 4mm in Cordyceps sinensis. While the isolate Cordyceps militaris is
found to be highly susceptible showing no growth in any of the concentrations of
hygromycin. This may be the evidence for the presence of selectable marker
gene hph in the isolates and can be used as a selectable marker tool for selection
in the strain improvement of Cordyceps Spp.
Keywords
Cordyceps Spp. · Strain improvement · Marker · Hygromycin
L. Deshmukh (*) · D. Gupta · S. S. Sandhu

Fungal Biotechnology and Invertebrate Pathology Laboratory, Department of Biological
Science, R.D. University, Jabalpur, Madhya Pradesh, India

https://doi.org/10.1007/978-981-13-6321-4_6
34 L. Deshmukh et al.
6.1 Introduction
Today’s research is paying attention towards strain improvement strategies for

increasing productivity to broaden the application potential. Fungi are second larg-
est and diverse group of organisms existing in nature after insects [1]. Medicinal
mushrooms are thought to produce bio-metabolites seizing various medicinal func-
tions including antitumor, antioxidant, radical scavenging, immunomodulating, car-
diovascular, anti-hypercholesterolemic, detoxification, antiviral, antibacterial,
antiparasitic, antifungal, hepatoprotective and antidiabetic effects [2]. The aware-
ness on development of genomic assets of entomopathogenic fungi are emerging
day by day [3, 4]. Genus Cordyceps is an entomopathogenic higher fungus parasit-
izes on larva of Lepidoptera and has been used as traditional medicine since ancient
times [5, 6]. Hike in price of wild Cordyceps mushroom and capsule, i.e., $32,000/
kg and $5.8/g, respectively, justified the name “soft gold mushroom”. This hike is
due to imbalance in supply and demand in the marketplace [7, 8]. The collection
difficulties and insufficient data availability regarding its production compel the
necessity of artificial cultivation and strain improvement strategies for this mush-
room so that large amount of biomass for functional foods and medicines becomes
easy to harvest [9].
The genetic makeup and molecular biology of these fungi are indispensable to
understand the in vitro production of fruiting body and bio-metabolites like cordy-
cepin and polysaccharides. In favour of enhancing the metabolic capacities of
Cordyceps Spp., genetic improvement and manipulation are mandatory. The selec-
tion and identification of these genetic changes can be established inside the organ-
isms through some selectable and non-selectable markers [10]. In the present work,
the methods used for detecting the presence of markers in the genome of C. militaris
and C. sinensis are UV irradiation, antifungal drugs and antibiotics.
6.2.1 Sample Collection and Maintenance
Cordyceps sinensis was collected from Pithoragarh District of Uttarakhand, and

lyophilized culture of Cordyceps militaris (3936) was procured from MTCC
Chandigarh, India. Both fungal isolates were grown on different media to optimize
their higher growth rate at incubation of 20°C for 10 days. Pure cultures were main-
tained on optimized media PPDA (Potato dextrose agar supplemented with pep-
tone) for C. sinensis and SMYEA (Sabouraud maltose agar supplemented with
yeast extract) for C. militaris at 4°C for further experimental purpose.
6 Development of Marker in the Soft Gold Mushroom Cordyceps spp… 35
6.2.2 Marker Development
Different methods were used to develop an efficient marker to achieve targeted

function and their responsible altered gene performance.
6.2.2.1 UV Irradiation

Fungal suspensions of both the cultures were made in 0.05% Tween-80 solution
(1 × 105 conidia ml−1) and were spread on sterilized media plates. All plates were
exposed under UV radiation (15 cm apart) for different time intervals ranging from
15 to 120 min in protected UV chamber. All UV-irradiated plates were incubated
under dark condition at 20 °C. Further, UV-irradiated grown colonies were tested
for nitrogen assimilation under different nitrogen sources (sodium nitrate, sodium
nitrite, ammonium chloride, glutamate).
6.2.2.2 Antifungal Sensitivity Test

Antifungal agents like griseofulvin, ketoconazole and clotrimazole were selected.
Fungal cultures were grown on media supplemented with antifungal agents in dif-
ferent concentration range from 0.5 to 2.5 mg/ml. Growth patterns of inoculated
cultures were measured by means of colonial diameter with zone measuring scale
(Himedia Laboratories, India).
6.2.2.3 Antibiotic Sensitivity Test

To test the sensitivity of C. militaris and C. sinensis against selected antibiotic, cul-
tures were inoculated on different concentrations of hygromycin B antibiotic (200–
900 μg/ml) supplemented in media plates. Growth pattern of fungus determines the
sensitivity of antibiotic.
6.3 Results
6.3.1 Development of Marker
The effect of UV irradiation on both the cultures was determined. 27 and 31 colo-
nies of C. militaris and C. sinensis, respectively, were procured from UV-irradiated
culture. A total of 58 colonies were tested and observed for the screening of nitrogen
assimilation, but none of colonies showed any marker when treated with
UV-irradiated conidia. All colonies were grown normally when growth media was
supplemented with different nitrogen sources. This result indicated that no nitrogen
assimilation gene is defected by UV irradiation (Fig. 6.1).
In this sequence, when these isolates (C. militaris and C. sinensis) were treated
with three antifungal agents like griseofulvin, ketoconazole and clotrimazole, no
remarkable marker was found against any of the tested antifungal agents. Both cul-
tures were showing moderate resistance against the used three antifungal agents; no
sensitivity was shown by C. militaris and C. sinensis (Fig. 6.2).
Fig. 6.1 UV-irradiated colonies tested under nitrogen sources. (a) C. militaris, (b) C. sinensis
Fig. 6.2 Antifungal sensitivity test of C. militaris and C. sinensis (a) Test plate, (b) Control
The antibiotic hygromycin B was used to test sensitivity against the isolates (C.
militaris and C. sinensis). The minimum inhibitory concentration of hygromycin B
on the growth of C. militaris and C. sinensis was successfully determined. 200 μg/
ml concentration of hygromycin B was completely able to inhibit the growth of C.
militaris which suggested that C. militaris was completely sensitive on this con-
centration. It is apparent that Cordyceps militaris is highly susceptible and shows
no growth in any of the concentrations of hygromycin (Fig. 6.3). However, a ramp-
ing decrease in radial growth was observed on increasing the concentration of anti-
biotic from 200 μg/ml to 900 μg/ml showing a radial zone of 15 mm to 4 mm in
Cordyceps sinensis as depicted in Table 6.1 and Graph 6.1 This might be the evi-
dence of the presence of selectable marker gene hph in the isolates and can be used
as a selectable marker tool for selection in the strain improvement of aforesaid
Cordyceps Spp.
Fig. 6.3 Sensitivity test of antibiotic hygromycin B on C. militaris and C. sinensis

(a) Negative control, (b) positive control, (c, d, e, f and g) growth of C. militaris and C. sinensis on
growth media containing increasing order of hygromycin concentration
Table 6.1 Antibiotic sensitivity test of C. militaris and C. sinensis by hygromycin B

Hygromycin C. C. Hygromycin C. C.
concentration militaris sinensis concentration militaris sinensis
S.No (μg/ml) (mm) (mm) S.No (μg/ml) (mm) (mm)
1. Positive control 26 40 6. 600 00 4
2. 200 00 15 7. 700 00 4
3. 300 00 14 8. 800 00 5
4. 400 00 10 9. 900 00 3
5. 500 00 6 –
6.4 Discussion
The market demand of Cordyceps Spp. regarding its medicinal value is increasing
sharply [11]. Strain improvement techniques for increased production of this spe-
cies are very crucial. For generating fungal mutants, inactivation of gene is found
to be a very feasible one [12]. Strain improvement techniques like transformation
and protoplast fusion require markers for selection of transformants and fusants.
The provision for genetic engineering of strain requires homologous integration of
gene, a recyclable marker which is a bidirectional positive selection system of
transformants [13]. Most of the cultures in laboratories tolerate ampicillin, chlor-
amphenicol, erythromycin and tetracycline like antibiotics at their much high dose
Graph 6.1 Graphical representation of effect of hygromycin B on C. militaris and C. sinensis

growth on solid media
for resistance cassettes; hence aminoglycoside resistance marker like hygromycin

B from Streptomyces hygroscopicus is used for positive selection [14]. In a study
by Zheng et al. [15], growth of Cordyceps militaris JM4 was found to be inhibited
by hygromycin B at the concentration of 400 μg/ml, and transformants of C. mili-
taris were detected in selective PPDA plates by the presence of hygromycin B
resistance gene in its integration DNA. The development of marker inside an
organism can be versatile in function to understand the genetic diversity and struc-
ture. The mating system and ecology in Cordyceps Spp. can also be used in the
study by these markers [10].
6.5 Conclusion
In the present work, an attempt to develop markers for C. militaris and C. sinensis
was done. While looking for nitrogen assimilation defect, the UV-irradiated colo-
nies of cultures did not show any of the defects. The cultures were also found resis-
tant to antifungal agents like griseofulvin, ketoconazole and clotrimazole. The
antibiotic sensitivity test of C. militaris and C. sinensis with hygromycin B showed
positive selection system for improved strains. C. militaris was found highly sus-
ceptible to hygromycin, whereas C. sinensis shows a gradient decrease in radial
growth from 15 mm to 4 mm on increasing concentration of 200 μg/ml to 900 μg/
ml. Therefore, these can serve as valuable tool for genetic analysis of strains and
also for selection of transformants. This will help for desirable strain development
with higher production rate as well as resource conservation of this important
medicinal mushroom Cordyceps.
Acknowledgements The authors owe huge gratitude to the Vice Chancellor, R.D. University
Jabalpur, (M.P.). An eternal gratefulness to the Head, Department of Biological Science,
R.D. University, Jabalpur, (M.P.), for allowing the authors to complete this research. We thank
Bio-Design Innovation Centre, Department of Biological Science, R.D. University, Jabalpur, (M.P.),
India, for providing facilities and funding to complete this research and bringing it to the world.
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Fungal Biol 115(3):265–274
Optimization of Microwave-Assisted
Pretreatment of Rice Straw with FeCl3 7
in Combination with H3PO4
for Improving Enzymatic Hydrolysis
Bikash Kumar and Pradeep Verma
Abstract
Pretreatment is a key step to alter the recalcitrance structure of lignocellulosic
biomass for enhancing enzymatic hydrolysis. Rice straw is an agricultural residue
which is one of the potential substrate for ethanol production. In the present work,
optimization of microwave-assisted pretreatment of rice straw in FeCl3 solution
with H3PO4 was performed. The effect of concentration of FeCl3 and H3PO4 along
with pretreatment time was evaluated. The optimal pretreatment condition was
found as follows: 250mM FeCl3, 3%H3PO4, 155°C, and 20 minutes. The pre-
treated pulp was subjected to enzymatic hydrolysis using commercial cellulase
for assessing effectiveness of pretreatment system. The maximum saccharifica-
tion per pulp and per biomass was observed as 98.9% and 66.4%, respectively,
under enzyme load of 3 FPU/g of substrate after incubation for 48 h.
Keywords
Microwave · Enzymatic hydrolysis · Lignocellulosic biomass · Delignification
7.1 Introduction
The rapid increase in energy demand, fast depletion of fossil fuel reservoirs, and
environment pollution caused by use of fossil fuels have forced the government
and scientific community to search for alternative sources of energy generation
that are inexpensive, eco-friendly, and renewable and can efficiently replace con-
ventional fossil fuels [1]. Lignocellulosic biomass is regarded as one of the most
promising alternative to fossil fuel. It can be transformed into biofuel and various
B. Kumar · P. Verma (*)

Bioprocess and Bioenergy Laboratory, Department of Microbiology,
Central University of Rajasthan, Ajmer, Rajasthan, India

https://doi.org/10.1007/978-981-13-6321-4_7
42 B. Kumar and P. Verma
value-added products which can offer sustainable system to help meet up this
global necessity. Rice is one of staple crop of the world with annual production of
481.54 million metric tons of which India contributes 106.50 million metric tons
(2016–2017) [2]. Each kilogram of rice obtained after harvesting generates
1–1.5 kg of the straw [3]. Based on this it can be estimated that about 106.5–159.8
million metric tons of rice straw was produced in India (2016–2017) of which a
part goes as cattle feed. However, the remaining part is waste and disposal of
which is a major concern. Most often these residues are burnt resulting in pollu-
tion. Thus, utilization of rice straw for its conversion to biofuel is one approach
that can help in lignocellulosic waste management. The bioconversion of ligno-
cellulosic biomass to ethanol involved three major steps: first pretreatment for the
opening of the crystalline structure of cellulose by breaking down of lignin, sec-
ond conversion of cellulose to glucose by hydrolysis with combination of enzymes,
and third fermentation of sugars to ethanol [4]. Pretreatment is one of the most
essential steps in the cost-effective conversion of lignocellulosic biomass to bio-
ethanol or other bio-based products. The major goal of the pretreatment is to
improve the accessibility of cellulose to the hydrolytic enzymes. Pretreatment
breaks the physical barrier by disrupting the cell wall, removing hemicellulose or
lignin fractions, reducing the cellulose crystallinity, improving porosity in the
biomass structure, and increasing the accessible surface area. As a consequence,
hydrolytic enzymes can easily access the cellulose fibers and act with higher effi-
ciency. Previously various physical methods such as ball milling [5], irradiation
[6], and chemical pretreatment methods such as ammonia [7], alkali [8], dilute
acids [9], and organosolv process [10] have been utilized frequently which can
help to modify the structural framework of lignocellulosic biomass and improve
the saccharification of the cell wall carbohydrates [11]. However, the existing
pretreatment technologies have certain limitations such as formation of inhibitory
compounds which hinder fermentation, inadequate separation of cellulose and
lignin, and considerable production of wastes [12].
In order to disrupt the recalcitrance structures of lignocelluloses, microwave
heating has been used [13]. Unlike conventional heating microwave irradiation has
offered advantage over conventional heating methods due to nonthermal or thermal
effects [14]. Microwave irradiation has increased reaction rate and reduced reaction
time. Verma and Chaturvedi [15] have carried out studies using microwave pretreat-
ment of lignocellulosic biomass especially for the efficient enzymatic hydrolysis of
woody biomass. Similar approach has also been used by Sindhu et al. [16] where
they used microwave-assisted surfactant pretreatment of chilli postharvest residue
for the production of bioethanol and biopolymer. On the other hand, some micro-
wave sensitizer chemicals can enhance the effect of irradiation, which will result in
improved delignification with negligible or no carbohydrate degradation [17].
However only few studies have been carried out especially for the enzymatic hydro-
lysis of rice straw using microwave system and microwave pretreatment. Liu et al.
[18] demonstrated effect of FeCl3 pretreatment on corn stover. In the present study,
7 Optimization of Microwave-Assisted Pretreatment of Rice Straw with FeCl3… 43
optimization of microwave pretreatment of rice straw with FeCl3 in combination

with H3PO4 at different pretreatment time was evaluated. Enzymatic hydrolysis of
the pretreated pulp was performed to assess the impact of the microwave-assisted
FeCl3 pretreatment on rice straw.
7.2.1 Materials
The chemicals used were purchased from HiMedia, India, and Merck, India. The
chemicals are of analytical grades. Rice straw was collected from local farms of
Rajasthan, India. They were washed with water in order to remove dirt and mud,
followed by oven drying at 50 °C. They were grounded in an electric grinder, sieved
to 2–5 mM mesh size, and stored in airtight polythene bags until use.
7.2.2 Microwave Pretreatment of Rice Straw Using FeCl3
Rice straw (2g) was soaked in aqueous solution of FeCl3 of different concentrations
50–400 mM for 24 h. The solid to liquid ratio was maintained at 20:1 (w/w) in all
the experiment .The treatment was performed in a microwave reactor with a 700W
magnetron “Microwave Reaction system SOLV, Multiwave Pro” (Make: Anton
Paar, Austria). The program was set up as heating as fast as possible with high stir-
ring during attaining of the desired temperature and holding temp for desired time
with fast stirring, then followed by cooling the system to 70 °C with slow stirring
before opening the system. After the reaction, the pulp fraction was separated by
vacuum filtration and washed three times with 150 ml of distilled water. The pulp
fraction or pulp yield was calculated by using equation
Wi − Wf
Pulp Yield ( % ) = ∗ 100
Wi
where Wi and Wf are weight of substrate before and after pretreatment,
respectively.
7.2.3 Effect of H3PO4 Concentrations
Rice straw (2g) was treated with different concentrations of H3PO4 (0.5–5%) at
155 °C for 30 min to study the effect of H3PO4 concentration on saccharification
obtained from pulp fractions.
7.2.4 Effect of Pretreatment Time
Rice straw (2g) samples soaked in 250 mM FeCl3 for 24 h and 3%H3PO4 were
added just before start of reaction which were subjected to microwave treatment at
155 °C for 10, 20, and 30 min in order to study effect of pretreatment time. The
pretreated pulp was subjected to enzymatic hydrolysis using commercial cellulase.
7.2.5 Enzymatic Saccharification of Pulp
The enzymatic saccharification of pulp was performed as method suggested by

Verma et al. [17]. The wet pulp fraction was hydrolyzed with a commercial cellu-
lase preparation, “ONOZUKA R-10” from Trichoderma viride (HiMedia). The cel-
lulase enzyme loading was 1FPU/g substrate. Enzymatic hydrolysis was performed
at a substrate concentration of 2% in 0.05 M sodium acetate buffer (pH 4.5) contain-
ing 0.02% sodium azide at 50 °C in rotary shaker water bath (Tempo, India) at
140 rpm for 48 h. The saccharification ratio per pulp was calculated according to the
NREL LAP-009 procedure[19]. After enzymatic hydrolysis, 1ml sample was col-
lected from each tube; the samples were placed in 1.5 ml eppendorf tubes, and then
the solutions were centrifuged at 5000 rpm for 5 min. The appropriate dilutions
were made to estimate sugar yield using dinitrosalicylic assay [20].
The saccharification ratio per pulp was evaluated based on how much pulp
fraction was susceptible to the enzymatic hydrolysis. The saccharification per
biomass was based on the weight percentage of reducing sugar to the original
biomass. All enzymatic hydrolysis experiments were performed in duplicate.
7.2.6 ffect of Enzyme Load on Saccharification Yield

E
on Pretreated Pulp
Rice straw (2g) was pretreated with 250 mM FeCl3 in combination with 3% H3PO4
at 155 °C for 20 min, and then the obtained pretreated pulp fractions were hydro-
lyzed with different concentrations i.e. 1, 3, and 5 FPU/g (filter paper unit per gram
of substrate) of commercial cellulase “ONOZUKA R-10” in order to examine max-
imum saccharification yield for evaluating the optimum enzyme dose. The control
set was incubated without addition of enzyme.
7.3 Results and Discussion
7.3.1 Microwave Pretreatment of Rice Straw Using FeCl3
In the present work, we compared the effect of different concentrations of FeCl3

(50–400 mM) on microwave treatment of rice straw at 155 °C for 30 min. The maxi-
mum pulp yield of 90.2% was observed for control (soaked in distilled water) which
may be because of minimum breakdown of lignocellulosic constituents of rice

straw. With increase in concentration of FeCl3, pulp yield gradually decreased which
may be due to loss of lignin (data not shown) and breakdown of carbohydrate poly-
mer. The obtained pulp was subjected to enzymatic hydrolysis using commercial
cellulase. The maximum saccharification per pulp and per biomass of 43.1% and
25.3%, respectively, (Fig. 7.1a) was obtained with 250 mM FeCl3.
The results clearly show that with an increase in FeCl3 concentration from 50 to
250 mM, the saccharification yield gradually increased; however above 250 mM
decrease in the saccharification yield was observed which may be due to loss of cel-
lulosic part. The saccharification yield per biomass was 10.1-fold higher than the
control, which was better than the results obtained by Lü and Zhou [21]. However,
they pretreated rice straw in microwave at 140 mM FeCl3, 160 °C for 19 min fol-
lowed by utilization of Trichoderma viride and Bacillus pumilus for the production
of reducing sugars.
Fig. 7.1 (a) Pulp yield and saccharification yields of microwave pretreated rice straw in different
concentrations (50–400 mM) of FeCl3 at 155 °C for 30 min. (b) Pulp yield and saccharification
yields of microwave pretreated rice straw in different H3PO4 concentrations (0.5–4%) at 155 °C for
30 min. (c) Pulp yield and saccharification yields of microwave pretreated rice straw in 250 mM
FeCl3 with 3% H3PO4 at 155°C for different time intervals. (d) Effect of enzyme dose on sacchari-
fication yield of the pretreated pulp obtained after microwave pretreatment of rice straw with
250 mM FeCl3 and 3% H3PO4 at 155 °C for 20 min
7.3.2 Effect of H3PO4 Concentrations
The rice straw was subjected to addition of different concentrations (0.5–4%) of

H3PO4 for microwave treatment at 155 °C for 30 min. The maximum pulp yield of
63.7% was obtained by addition of 2% H3PO4. The maximum saccharification per
pulp and per biomass obtained was 39.01% and 24.03%, respectively, (Fig.7.1b) for
the pretreatment under 3% acid concentration.
7.3.3 Effect of Pretreatment Time
The rice straw was soaked in 250 mM FeCl3 for 24 h; 3% H3PO4 was added with just
before start of pretreatment. The pretreatment was performed at 155 °C for different
pretreatment time, i.e., 10, 20, and 30 min. The maximum pulp yield of 82.97% was
obtained after pretreatment of 10 min, whereas pulp yield of 67.13% and 61.43%
for 20 and 30 min, respectively. Maximum saccharification per pulp and per bio-
mass of 77.03% and 57.09%, respectively (Fig. 7.1c), was obtained for pretreatment
for 20 min which was much higher as compared to saccharification yields for pre-
treatment for 10 and 30 min. This can be explained as low pretreatment time
(10 min) caused low delignification of lignocellulosic biomass due to inefficient
breakdown of lignin carbohydrate complexes that resulted in less accessibility of
cellulase enzyme to cellulosic part. The pulp yields for 20 and 30 min of pretreat-
ment are in comparable range, but the saccharification yield for 30-min pretreat-
ment is relatively low. It can be due to loss of cellulosic component with long
pretreatment time (30 min) which finally affects the overall saccharification yield.
The optimum pretreatment time obtained was 20 min, which is comparable to the
results obtained by Lü and Zhou [21] where the optimum irradiation time was
obtained as 19 min.
7.3.4 Effect of Enzyme Dose on Saccharification Yields
The enzymatic hydrolysis was performed for pulp obtained by microwave

pretreatment of rice straw in 250 mM FeCl3 and 3% H3PO4 at 155 °C for 20 min
with different cellulase enzyme load, i.e., 1, 3, and 5 FPU/g of substrate. The
maximum saccharification per pulp and per biomass of 98.9% and 66.4%,
respectively, was obtained at enzyme load of 3 FPU/g after 48 h of incubation
(Fig. 7.1d). The saccharification per pulp and per biomass by enzymatic hydrolysis
of pretreated rice straw with enzyme load of 5 FPU/g was obtained as 97.8 and 62.3,
respectively, which are comparable to the saccharification yields obtained for
enzyme load of 3 FPU/g. The enzyme load higher than 3 FPU/g did not result in any
increase in saccharification yield; it may be due to its adsorption onto the substrate
that restricted the diffusion process through the structure as explained by Martín
et al. [22] where enzyme loading higher than 15 FPU/g did not result in any increase
in initial rate.
7.4 Conclusion
The conditions for microwave-assisted FeCl3 in combination with H3PO4

pretreatment of rice straw were optimized. The optimal conditions were found as
follows: 250 mM FeCl3 concentration, 3% H3PO4 concentration, 155 °C pretreatment
temperature, and 20-min pretreatment time. Enzymatic hydrolysis of pretreated
pulp with enzyme load (3 FPU/g) for 48 h resulted in maximum saccharification per
pulp and per biomass as 98.9% and 66.4%, respectively. The pretreatment step and
cost of enzyme are rate-limiting step of the commercial biofuel production. The
present work gives an efficient pretreatment strategy resulting in high amount of
reducing sugars at low enzyme load. It will lead to develop cost-effective
lignocellulosic biorefinery concept.
Acknowledgment The authors acknowledge the financial support provided by Department of

Biotechnology, Government of India through project sanction wide no: BT/PR7333/
PBD/26/373/2012.
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Profiling Indolic Auxins Produced
by the Strains of Aspergillus Using Novel 8
HPTLC Technique
Dhavalkumar Patel, Anoshi Patel, Disha Vora, Kinjal Desai,

Sudeshna Menon, Sebastian Vadakan,
and Dweipayan Goswami
Abstract
Fungi are recognized to have dealings with plants by several known mechanisms,
one of which is through the production of phytohormones, auxins. Indole-3-
acetate (IAA), indole-3-butyrate (IBA), and indole-3-pyruvate (IPyA), i.e.,
indolic auxins, are most widely yield by the strains of fungi through which they
interact with plants. Fungal strains produce these indolic auxins by tryptophan
(Trp)-dependent and tryptophan-independent pathways. Under present study, we
found Aspergillus flavus strain PGFW, Aspergillus niger strain BFW, and
Aspergillus caespitosus strain DGFW as the utmost effective IAA-producing
strains from the rhizosphere of well-growing wheat plant which was determined
by spectrophotometric method that uses Salkowski reagent. This method though
has a flaw that it is not specific to IAA but develops color by reacting with all the
other indolic derivatives. We found that for the spectrophotometric method, the
absorption maxima (λmax) of the mixture containing indolic compounds tend to
shift when compared to pure standard. To overcome this limitation, high-
performance thin-layer chromatography (HPTLC)-based etiquette is technologi-
cally advanced for the first time to precisely detect and calculate the amount of
IAA and IBA in the assortment of 100 to 1000 ng per spot ignoring other Trp
derivatives. HPTLC analysis showed that all the three strains under current study
could produce indolic auxins by Trp-dependent and Trp-independent pathways,
but the amount of indolic auxins produced was enhanced in presence of Trp.
These strains may act as phytoaugmentor or phytopathogen, but for their mode
of action, they produce various indolic auxins which can be profiled by the novel
method described in this paper.
D. Patel · A. Patel · D. Vora · K. Desai · S. Menon · S. Vadakan · D. Goswami (*)

Department of Biochemistry and Biotechnology, St. Xavier’s College (Autonomous),
Ahmedabad, Gujarat, India
e-mail: [email protected]

https://doi.org/10.1007/978-981-13-6321-4_8
50 D. Patel et al.
Keywords
Aspergillus spp. · Indolic compounds · HPTLC
8.1 Introduction
Fungi residing in the rhizosphere are known to yield auxins like indole-3-acetic acid
(IAA) and benefit plant growth [3]. Such fungi live symbiotically with plants and
supports plant growth in return plant provide sugars and amino acids to the fungi for
their survival. Such fungi are named plant growth-promoting fungi (PGPF) [10].
The microbial biosynthesis of auxins has undertaken penetrating examination which
has reasoned that diverse strains of fungi produce IAA from tryptophan (Trp) while
other fungi produced IAA even in the absence of Trp [7].
Spectrophotometric technique is the most generally used to distinguish indolic
subordinates delivered by microorganisms and parasites. The spectrophotometric
strategy utilizes the old-fashioned Salkowski reagent (FeCl3 broken down in per-
chloric corrosive/sulfuric corrosive) which reacts to the indolic subordinates to cre-
ate color complexes [11]. This procedure is basic yet profoundly uncertain as it
gives a non-particular color complex response with all the indolic subordinates
fashioned by the growth and gives detection of aggregate indolic content rather than
exact amount of IAA. High-performance thin-layer chromatography (HPTLC) is
recently reported to be very truthful in distinguishing indolic units created by
microbes [7].
Here for the first time, we are reporting the method development to analyze
indolic molecules from fungi using HPTLC. For the study we chose three efficient
IAA-producing strains from the rhizospheric soil of wheat plant. We also compared
the senility of HPTLC method with routinely spectrophotometric.
8.2 Experimental
8.2.1 Materials and Reagents
Indolic auxins, standard IAA (99.0%) and standard IBA (99.0%), were procured
from Hi-Media. Solvents isopropanol, n-butanol, methanol, and ammonia solution
(25% v/v) were procured from Merck Ltd. Aluminum backed silica gel 60F254
TLC foils with 0.25-mm thickness were purchased from Merck (Darmstadt,
Germany).
8.2.2 Instrumentation
The HPTLC apparatus used in the experimentations was obtained from CAMAG
(Muttenz, Germany) which essentially contained of two parts: Linomat 5 which is a
8 Profiling Indolic Auxins Produced by the Strains of Aspergillus Using Novel HPTLC… 51
programmed applicator snug with a Hamilton syringe (100 μl) for precise stacking
of samples onto TLC foils; second is Scanner 3 for the scanning of HPTLC foils
after drying.
8.2.3 Standard Solutions
Standard solutions of both the auxins (IAA and IBA) were prepared in pure methanol
in a range of 100 μg/ml.
8.2.4 Strains of PGPF Used for Study
Three strains, Aspergillus flavus strain PGFW (KY964054), Aspergillus niger strain
BFW (KY964055), and Aspergillus caespitosus strain DGFW (KY964056), capa-
ble to produce IAA-producing strains from the rhizospheric soil of Triticum aesti-
vum (wheat) plant (22°61′N, 72°93′E).
8.2.5 ssessment of the IAA Produced by PGPF Strains

A
by Means of the Traditional Spectrophotometric Method
The preliminary IAA-producing capability of these strains was estimated using

conventional spectrophotometric method which involves the use of Salkowski
reagent [8]. Fungal isolates were cultivated in the potato dextrose broth enhanced
with Trp (1 mg/ml of broth) at 25 ± 2 °C for 168 h. From each of these isolates, their
culture supernatants were assorted with Salkowski reagent (50 ml, 35% of perchloric
acid and 1 ml 0.5 M FeCl3 solution) in the proportion of 1:1. Pink color develops
indicating the production of IAA, its optical density was taken at 530 nm.
Concentration of the IAA bent was assessed against the standard curve of IAA in
the array of 10–100 μg/ml.
8.2.6 Extraction of Indolic Auxins
Indolic auxins were haul out from culture supernatants as described by [6]. Briefly,
fungal strains were grown for 168 h in the potato dextrose broth enhanced with Trp
(1 mg/ml of broth) at 25 ± 2 °C. Culture supernatants were brought to pH 2.5 by
means of 1 N HCl and mined thrice using equivalent volume of ethyl acetate. A por-
tion of ethyl acetate was air desiccated and redissolved in one tenth volume of meth-
anol [8]. These methanolic extracts were further used for HPTLC study.
52 D. Patel et al.
8.2.7 HPTLC Procedure
8.2.7.1 Sample Application

Sample loading on TLC foils (sized 10 × 15 cm) was completed using a Linomat 5
applicator (CAMAG, Muttenz, Germany) which confined Hamilton 100 μl syringe.
8.2.7.2 Calibration Curves

Calibration curves of IAA and IBA (100 μg/ml) were set as per the technique
previously developed by Goswami et al. [7] within the range of limit of detection
(LOD) and limit of quantitation (LOQ). Each standard, i.e., IAA and IBA were
loaded from 4 to 20 μl, was distinctly on TLC in the form of bands (size 10 × 10 cm).
After proper loading of the standard, TLC plate was air dehydrated for 600 s, and
the developed band were envisioned under UV with a wavelength of 254 nm. Pre-
saturated twin chambers with the solvent system were used for development of TLC
(containing standard). Solvent system used as mobile phase was isopropanol/n--
butanol/ammonia/water [10:6:3:1 (v/v)]. TLC plates were air desiccated, and the
developed band was scanned with Scanner 3 (Camag) in absorbance-reflectance
mode at 254 nm.
8.2.7.3 Densitometric Analysis of Chromatogram

For quantitative densitometric examination, apex region of developed spots for
distinct sample was measured by direct scanning at 254 nm utilizing the Camag
TLC Scanner 3 with deuterium source at an inspection rate of 20 mm/s. The slit
measurement setting was 6 mm in length into 0.45 mm in width, and data resolve
was 100 μm per step utilizing filter factor Savitzky-Golay 7, pattern refinement was
set to the least slant, and show scaling was set to programmed.
8.2.7.4 D etermination of IAA from Extracted Indolic Auxins

from Fungal Supernatant
Fungal methanolic extract (containing indolic auxins formed by fungal strains) and
indolic standards (IAA and IBA) were laden on single TLC plate. On TLC plate
(sized 15 × 10 cm), 16 bands were laden on entire TLC, where 2 bands laden with
individual standards, 2 bands laden with combination of standards auxins (IAA and
IBA), and 2 bands laden with each fungal methanolic extract in different volumes.
For Aspergillus flavus strain PGFW, Aspergillus niger strain BFW, and Aspergillus
caespitosus strain DGFW, the methanolic extracts laden per band were 20 μl and
30 μl correspondingly. TLC plate was developed in pre-saturated twin rack chamber
as mentioned above densitometric analysis was completed to perceive and calculate
IAA and IBA in the methanolic fungal extracts; measured values were equated with
the values attained by means of the spectrophotometric method.
8.3 Results
8.3.1 I AA Production by PGPF Strains Examined by Means

of Spectrophotometric Method
Spectrophotometric analysis recommended that all the three strains under current
study could produce indolic auxins under both the conditions (culture medium with
and without supplementation of Trp). Maximum values of IAA produced by these
strains are represented in Table 8.1.
8.3.2 HPTLC
8.3.2.1 Calibration Curves

TLC plate laden with standard indoles were allowed to advance in twin rack chamber
pre-saturated with solvent system isopropanol/n-butanol/ammonia/water [10:6:3:1
(v/v/v)] for 75 min. On development, the TLC plate was air desiccated and studied
for retention factor (Rf) values underneath short UV (254-nm wavelength) where Rf
value of IAA was 0.63 and Rf value of IBA was 0.70. For concocting calibration
curve, each standard was laden independently extending from 400 to 2000 ng per
band. Densitometric study presumed that Trp possessed linear correlation with
concentration of standard laden and peak zone of the developed band in the range of
100–1000 ng per spot. Standard auxins exhibited parallel linearity in the array of
100–1000 ng per band and 100–500 ng per band of IAA and IBA correspondingly.
Qualified visualization of standard IAA and IBA standards on solitary TLC is
revealed in Fig. 8.1. Further, LOD and LOQ were determined for each standard
auxin (IAA and IBA) as of the calibration curve where which were 96.1 ng and
291.2 ng per spot correspondingly for IAA and 83.4 ng and 252.7 ng per spot cor-
respondingly for IBA.
8.3.2.2 D etermination of IAA and IBA from Fungal Supernatant

Containing Indolic Auxins
On precise standardization, this method was rummage sale to perceive and quantify
IAA and IBA from fungal extracts. TLC after development is shown in Fig. 8.2.
Bands 1 and 2 are laden with standard IAA (20 μl and 30 μl individually), bands 3
and 4 are laden with standard IBA (20 μl and 30 μl individually), bands 5 and 6 are
laden with extracted indolic auxin from A. flavus without Trp in media (20 μl and
30 μl individually), bands 7 and 8 are laden with extracted indolic auxin from A.
flavus strain PGFW in media supplemented with Trp (20 μl and 30 μl respectively),
bands 9 and 10 are laden with extracted indolic auxins from A. niger strain BFW
without Trp in media (20 μl and 30 μl individually), bands 11 and 12 are laden with
extracted indolic auxin from A. niger strain BFW in the media supplemented with
Trp (20 μl and 30 μl individually), bands 13 and 14 are laden with extracted indolic
auxins from A. caespitosus strain DGFW without Trp in media (20 μl and 30 μl
individually), and bands 15 and 16 are laden with extracted indolic auxin from A.
54
Table 8.1 HPTLC analysis of IAA from three Aspergillus spp.

μg of
Area ng of IAA μg of IAA per Spectrophotometric results
Culture Sample under ng of IAA per ml IAA per ml of μg of IAA per showing production (μg of
Strains media loaded curve per band sample sample broth ml of broth IAA per ml of broth)
A. flavus without 20 10173.00 3681.30 184065.01 184.07 18.41 17.58 ± 1.17 85.67 ± 4.33
strain PGFW Trp 30 13519.10 5024.04 167467.90 167.47 16.75
with Trp 20 16628.90 6271.95 313597.51 313.60 31.36 30.10 ± 1.78 112.29 ± 8.34
30 22566.50 8654.61 288487.16 288.49 28.85
A. niger strain without 20 7551.90 2629.49 131474.72 131.47 13.15 12.26 ± 1.26 82.67 ± 3.22
BFW Trp 30 9498.60 3410.67 113689.14 113.69 11.37
with Trp 20 12703.60 4696.79 234839.49 234.84 23.48 23.25 ± 0.33 139.72 ± 7.44
30 18205.00 6904.41 230147.14 230.15 23.01
A. caespitosus without 20 8534.50 3023.80 151189.81 151.19 15.12 14.55 ± 0.81 106.62 ± 6.44
strain DGFW Trp 30 11445.90 4192.09 139736.49 139.74 13.97
with Trp 20 13552.10 5037.28 251863.96 251.86 25.19 26.54 ± 1.92 156.08 ± 5.44
30 21855.60 8369.34 278978.06 278.98 27.90
D. Patel et al.
All tracks @ 254 nm
700.0
700.0
[AU]
[AU]
500.0
500.0
400.0
400.0
300.0
300.0
200.0
200.0
100.0
100.0 0.0
100.0
[mm]
80 0
70 .0
.0
60
0.0
.
50 0
40 0
-0.20 -0.10
.0
30 0
0.00 0.10 IAA
.
0.20 0.30 0.40 0.50 IBA 20 0
.
10
0.60 [Rf] 0.80 0.0

.
.
Fig. 8.1 Displays the 3D densitogram of the TLC foil (same) where concentration of peak defines
the area underneath the curve. It can be understood that as the concentration of standard auxin
(IAA and IBA) increases as the zone underneath the curve for an equivalent band also rises
Fig. 8.2 Display the image of TLC foil under 254-nm UV light
56 D. Patel et al.
Fig. 8.3 Displays the 2D densitogram of fungal extracts containing indolic auxin compounds (a)
band laden with extract of A. flavus strain PGFW without Trp (track 5 of this figure), (b) track
laden with extract of A. flavus strain
caespitosus strain DGFW in media supplemented with Trp (20 μl and 30 μl indi-
vidually). Area under the curves of IAA and IBA yield by fungal isolates are shown
in Fig. 8.3. These values are used to calculate IAA produced by fungal isolates
which is represented in Table 8.1.
It was found strains produced IAA or IBA or both. Their actual production
detected by spectrophotometric method is much greater than the HPTLC-derived
values. This is because fungal isolates produce several other molecules other than
IAA and IBA (Fig. 8.2) which may be falsely detected as IAA by the spectrophoto-
metric method.
8.4 Discussion
Spectrophotometric technique is the most extensively used to perceive indolic

derivatives from Trp among the existing approaches. The spectrophotometric tech-
nique uses the outmoded Salkowski reagent (FeCl3 dissolved in perchloric acid/
sulfuric acid) which responds to the indolic auxins to develop color [1, 6, 10]. This
technique is easy and simple but extremely imprecise as it gives a non-precise color
response with all the indolic auxin produced by fungi and make available detection
of entire indole content relatively than precise detection of both IAA and
IBA. Therefore, the spectrophotometric technique provides inaccurate amounts of
IAA/IBA produced by PGPFs [13]. Excluding the spectrophotometric technique,
thin-layer chromatographic (TLC) technique [8, 9, 12] and high-performance
liquid chromatographic (HPLC) approaches are also castoff [12]. TLC affords
merely qualitative detection of indolic auxins, while HPLC is very delicate [2], but
needs extremely purified samples which make procedure of sample preparation
monotonous; HPLC study also necessitates longer time intervals for detection and
standardization [4, 5].
8.5 Conclusion
Hence, in this paper we account the use of high-performance thin-layer

chromatography (HPTLC) for the immediate detection and quantification of IAA
produced by quite a few PGPFs. We are also providing comparison of its sensitivity
with outmoded spectrophotometric technique. To the best of our data, this is the
foremost report which describes quantification of IAA and IBA from fungal isolates
by means of HPTLC.
Acknowledgments Authors are thankful to the Gujarat State Biotechnology Mission (GSBTM)
for providing the funding under FAP 2016 GSBTM/MD/PROJECTS/SSA/5041/2016-17 project
and St. Xaveir’s College (Autonomous), Ahmedabad-380009 for providing necessary facilities.
References
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61(2):793–796
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of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) produced by rhizofungi from
l-tryptophan (Trp) using HPTLC. J Microbiol Methods 110:7–14
8. Goswami D, Vaghela H, Parmar S, Dhandhukia P, Thakker JN (2013) Plant growth promoting
potentials of Pseudomonas spp. strain OG isolated from marine water. J Plant Interact
8(4):281–290
9. Hartmann A, Singh M, Klingmüller W (1983) Isolation and characterization of Azospirillum
mutants excreting high amounts of indoleacetic acid. Can J Microbiol 29(8):916–923
10. Karnwal A (2009) Production of indole acetic acid by fluorescent Pseudomonas in the presence
of L-tryptophan and rice root exudates. J Plant Pathol Microbiol 91:61–63
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of yam (Dioscorea rotundata L.) minisetts by Bacillus subtilis isolated from culturable
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Comparative Analysis of Cytotoxic
Potential of Crude Extracts 9
and Fractionated Isolates from Moringa
oleifera Lam.
Kinjal Desai and Vincent Braganza
Abstract
According to World Health Organization, most of the world’s population depends
upon plants as an important element in primary healthcare systems. Ayurveda is
India’s oldest indigenous medicine system of plant drugs. It is known from for
preventing or suppressing various tumors using natural drugs, one such being
Moringa oleifera Lam. This plant has reported antioxidant properties for both
fruits and leaves (Luqman S, Srivastava S, Kumar R, Maurya AK, Chanda D,
Evid Based Complement Alternat Med 2012(December):e519084. https://doi.
org/10.1155/2012/519084, 2011); moreover, its extracts have exhibited antican-
cer properties in vitro in case of hepatocarcinoma as well as antitumor-promoting
activities for skin cancer in rat models (Guevara AP, Vargas C, Sakurai H,
Fujiwara Y, Hashimoto K, Maoka T, Kozuka M, Ito Y, Tokuda H, Nishino H,
Mutat Res/Genet Toxicol Environ Mutagen 440(2):181–188. https://doi.
org/10.1016/S1383-5718(99)00025-X, 1999).
The aim of this study is to investigate the antiproliferative activity of various
extracts from Moringa oleifera Lam. and isolate the active compounds. Fifteen
extracts were prepared form dried leaves of Moringa oleifera Lam. using five
solvent systems and three methods of extraction. In vitro screening was done
using Schizosaccharomyces pombe and MCF-7 cell line. The fractionation of
active crude extracts was performed by silica gel column chromatography and
fractions evaluated for cytotoxicity. The aqueous, methanolic and hydrometha-
nolic extracts exhibited cytotoxicity against MCF-7 cell line at lower concentra-
tions compared to lymphocytes. Our findings showed the following: (a) the crude
extracts of Moringa oleifera Lam. exhibited cytotoxic potential; (b) the extracts
K. Desai
Department of Biochemistry, St Xavier’s College, Ahmedabad, Gujarat, India
V. Braganza (*)
Loyola Centre for Research and Development, Ahmedabad, Gujarat, India

https://doi.org/10.1007/978-981-13-6321-4_9
60 K. Desai and V. Braganza
were selectively more toxic to tumor cell line compared to normal lymphocytes.
(c) The crude extract showed better anti-proliferative activity compared to frac-
tion separated. The reason could be that the compounds in crude extract have a
synergistic effect resulting in better activity.
Keywords
Antiproliferative · Schizosaccharomyces pombe MCF-7 · Cytotoxic potential
9.1 Introduction
Plants have formed the basis of sophisticated traditional medicine systems that have
been in existence for thousands of years [3]. Several groundbreaking and important
drugs have been isolated from plants. Some significant examples are quinine (anti-
malaria), morphine (pain reducer), Taxol and camptothecin (anticancer). Screening
over 110,000 compounds from 35,000 plants by the National Cancer Institute for
about 25 years led to the isolation from the bark of the Pacific yew tree, Taxus brevi-
folia, Taxol® (paclitaxel), a cytotoxic diterpene alkaloid [4]. Studies of Taxol
against several cancer cells established its excellent anticancer potential, especially
against leukemia cells [5].
Moringa oleifera Lam., belonging to family Moringaceae, is one of the most
commonly cultivated species. The common name for this plant is drumstick tree,
and it is widely cultivated in tropical and subtropical regions. It is a drought-resistant
tree whose fruits and leaves are used in culinary preparations. It has also been used
in herbal medicine [6].
All the plant parts have been used for various ailments, like the seeds are consid-
ered to be antipyretic and are reported to have antimicrobial activity. The leaves and
its extract are believed to reduce blood glucose levels and are used to treat sores and
burns. The roots of the plant have anti-inflammatory activities [7]. Reported phyto-
chemical evaluation of Moringa oleifera Lam. has indicated the presence of various
phytosterols, glucosinolates, and isothiocyanates [8]. The leaves of this plant exhibit
antioxidant properties due to the presence of phenolics [9].
Biological activity and cytotoxicity of drugs, chemicals, and extracts are very
commonly assessed using various model systems. Yeast has been one of the earliest
species to be used as a model organism. Due to its sequence homology and con-
served gene sequences, it serves as a good model for correlation with mammalian
systems [10] so can be used for screening of new therapeutics. Another commonly
used system is cell lines. This is generally a preliminary screening before clinical
trials and also a methodology to narrow down to a specific fraction or chemical or
drug that is effective. Thus, in our study, extracts that showed cytotoxicity in the
prescreening study using mutant yeasts were selected for further screening using
cell lines to confirm their therapeutic potential.
The focus of modern science is to identify and isolate active principles from
plants. Their isolation can be achieved using bioassay-guided fractionation. The
9 Comparative Analysis of Cytotoxic Potential of Crude Extracts and Fractionated… 61
process involved would be preparing crude extracts and fractionating the com-
pounds and assessing each fraction for desired biological activity. Once such active
fraction is obtained, further characterization would enable identification of the com-
pounds. Hence, leading to discovery of new therapeutics form plants. Many such
examples are reported of bioassay-guided fractionation where active principles have
been successfully identified and isolated. For example, 2-methoxy-1,
4-naphthoquinone (MNQ), an antimicrobial compound, was identified and isolated
from Impatiens balsamina L. using bioassay-guided fractionation [11]. Hence, frac-
tionation, isolation, and identification are crucial steps for drug development from
natural products coupled with efficacy and toxicity studies.
9.2 Materials and Method
9.2.1 Plant Material and Preparation of Crude Extracts
Plant materials originally bought from government nursery, Gandhinagar, and

planted at the greenhouse of the Xavier Research Foundation were used for the
study. The leaves were air dried under shade and crushed finely. This powder was
then used for all extraction purposes. Three different experimental conditions [12–
14], (i) lower temperatures (25 °C) (ii) elevated temperature and (iii) sonication,
have been used for extraction. Moreover, five solvent systems were used which
were (i) water, (ii) methanol, (iii) ethyl acetate, (iv) chloroform/methanol (1:1), and
(v) water/methanol (1:1). The solvents were selected for the whole azeotropic range
varying from polar to nonpolar solvents.
The entire range of solvents was used for each of the experimental conditions.
For each of the conditions, 1gram of powder was weighed and suspended in 20 ml
of selected solvent [15, 16].
The extraction procedure involved continuous agitation and changing of solvent
until solvents were rendered colorless. The extracts obtained were pooled, and the sol-
vent was allowed to evaporate in a pre-weighed crucible. The residue was resuspended
in tissue culture grade DMSO to make a stock of 10mg/ml and stored at 4 °C [13].
9.2.2 Culturing Yeast and Cytotoxicity Assay
The study involved use of genetically modified yeast strain as a prescreening tool to
check the effectiveness of the crude extracts. The yeast used for this study was
Schizosaccharomyces pombe purchased from National Collection for Yeast Culture
(NCYC) with accession number 1683. This yeast that has its cdc 2 gene modified
has an altered cell cycle. This model has been used to screen for antiproliferative
activity of various crude extracts using methyl thiazole tetrazolium (MTT) assay
[17, 18]. Moreover, MTT assay was performed for wild-type yeast as well as to
ensure selective toxicity of extracts against mutant yeast. The media used for cultur-
ing yeast comprised of yeast extract, peptone, dextrose (YEPD) broth. For each
assay, a fresh inoculum was introduced into YEPD broth and allowed to grow until
it gave an absorbance of 0.9 at 660 nm. After the incubation, 100μl of broth with
cells (~106) were transferred to 96-well microtiter plate. The crude extracts (in trip-
licates) at concentrations of 50, 100, 150, and 200 μg per well were added. Blank
wells contained YEPD without cells. Yeast cells without any extract were kept as
control. DMSO was used as vehicle control. Vinblastine at concentration of 0.16 μM
(IC50 of vinblastine) was used as positive control. After exposure of 24 h, 20 μl of
MTT dye (2.5 mg/ml) was added to each well. The plate was then incubated in dark
for 4 h. In order to dissolve the formazan crystals formed post the incubation, 100 μl
of DMSO was added, and the plate agitated for 5 min. The absorbance was read at
570 nm using a microplate reader. Based on the absorbance, cell viability was cal-
culated as shown below:
ODof sample – OD of blank
Cell viability =
OD of yeast cells – OD of blank
Results of MTT assay for yeast were used to select extracts to be screened in the
cell lines.
9.2.3 Culturing MCF-7 Cell Line and Screening Assay
The study was done on MCF-7 cell line purchased from National Cell Culture
Collection (NCCS), Pune. The cell line was cultured using Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10% fetal bovine serum in T-75 flasks.
The flasks were maintained at 37 °C, 5% CO2 in completely humidified atmosphere.
The cells were used for cytotoxicity assay when they reached a confluence of 90%.
These cells were resuspended at a concentration of 4 × 105 cells/ml and aliquoted
into 96-well plate (100 μl/well). After 24 h, the cultivated cells were treated with
crude extracts at concentrations of 0.1, 1.0, 10, and 100 μg/ml. Cells were exposed
to vinblastine 0.16 μM (positive control) and DMSO 1% (vehicle control). Phosphate
buffered saline (PBS) was added to three wells serving as control. MTT assay was
performed 48 h after exposure [19]. The toxicity assessment of crude extracts was
done using peripheral blood lymphocyte culture (PBLC) [20]. 1.0 ml of heparinized
blood was withdrawn from a healthy donor and transferred to microfuge tube con-
taining 0.1 ml of phytohemagglutinin (PHA). The tube was then incubated at 4 °C
for 45 min and centrifuged at 350×g for 10 min. The erythrocytes settled at the bot-
tom of the tube, and colorless fluid supernatant was transferred to T-75 flask con-
taining RPMI-1640 supplemented with 20 mM glutamine and 1.5% sodium
bicarbonate. The flask was incubated at 37 °C in a 5% CO2 and humidified incubator
for 48 h. After incubation, plated in a 96-well plate at density of 106 cells/ml (100 μl/
well). The crude extracts were then introduced at concentrations of 0.1, 1.0, 10, and
100 μg/ml and incubated for 24 h. MTT assay was performed as mentioned earlier
to assess cytotoxicity. The cell viability was calculated. Therefore, our study focuses
on identifying cytotoxicity of crude extracts on breast cancer cell line and toxicity
assessment on lymphocytes as well. Based on the results, the crude extracts that
exhibited toxicity against the tumor cell line and non-cytotoxic to lymphocytes were
used for further experiments.
9.2.4 olumn Chromatography and Fractionation of Crude

C
Extracts
The crude extracts exhibiting cytotoxicity selectively against the cell lines were
fractionated using silica gel column chromatography. Figure 9.1 depicts the scheme
for fractionation of selected extract.
9.2.5 Statistical Analysis
The cytotoxicity assay was performed using MTT assay. The cytotoxic potential
was correlated to decrease in viable number of cells. A dose-response curve was
generated, and the different doses were compared using one-way ANOVA. Moreover,
the various extracts were also compared to find significant difference in their effi-
cacy. Analysis for the same was done using one-way ANOVA.
The inhibitory concentration (IC50) is essential to compare efficacy of a com-
pound to standards or other similar active principles. IC50 was calculated for all the
extracts using regression analysis on a percent inhibition versus dose plot.
Fig. 9.1 Fractionation

scheme for crude extract of Water: methanol extract at
Moringa oleifera Lam. elevated temperature of Moringa
oleifera Lam.
Fractionation with solvent system

(ethyl acetate:methanol:water)
(2:1.5:3)
8 fractions processed for second

fractionation.
2 fractions obtained labelled as

MO1 and MO2
9.3 Results
9.3.1 Cytotoxicity Assay Using Yeast
The cell viability was calculated after performing MTT assay of yeast cells both
wild and mutant exposed to various extracts at dose of 50–200 μg/ml. A dose-
response curve was plotted, and linear regression analysis was performed to obtain
IC50 values.
This study evaluates cytotoxicity potential of the extracts for both wild-type and
mutant yeast cells to assess selective cytotoxicity. Table 9.1 compares IC50 of crude
extracts of Moringa oleifera Lam. for mutant and wild-type yeast. The water, meth-
anol, and water/methanol extracts had a significant difference (p value < 0.01)
between IC50 values for wild-type and mutant yeast in all three methods employed
for extract preparation.
9.3.2 MTT Assay of MCF-7 Cell Line
Based on the results obtained, the water, water/methanol, and methanol extracts for
all three plants were chosen for study in cell line MCF-7.
The IC50 values for crude extracts of Moringa oleifera Lam. were calculated
from dose-response curves and compared between MCF-7 cells and lymphocytes.
The water/methanol extract at elevated temperature had an IC50 of 244.6 μg/ml and
was least toxic to lymphocytes compared to other extracts. The methanol extract by
sonication and at 25 °Cexhibited greater cytotoxicity at lower dose and were less
Table 9.1 IC50 values of crude extracts of Moringa oleifera Lam. for wild-type and mutant yeast
IC50 (μg/ml)
Method employed Solvent system used Mutant Wild type Significance
25 °C Water 204.087 650.862 *
Methanol 302.664 534.948 **
Ethyl acetate 315.964 241.945 ns
Chloroform/methanol 275.792 273.321 *
Water/methanol 234.449 1157.89 **
Elevated temperature Water 296.761 475.309 ***
Methanol 196.724 442.826 **
Chloroform/methanol 186.799 221.464 ns
Water/methanol 144.225 479.292 **
Sonication Water 287.270 407.896 **
Methanol 192.607 391.567 ***
Chloroform/methanol 243.971 237.273 ns
Water/methanol 218.541 482.635 ***
p values, ns = nonsignificant: p value > 0.05, *p value < 0.05, **p value < 0.01, ***p value < 0.001
Table 9.2 IC50 values of crude extracts of Moringa oleifera Lam. for MCF-7 and lymphocytes
IC50 (μg/ml)
Method employed Solvent system used MCF-7 PBMC Significance
25 °C Water 9744.8 1556.1 **
Methanol 6502.4 1929.1 **
Water/methanol 2213.3 14650.7 **
Elevated temperature Water 2202.6 8082.1 **
Methanol 7516.3 1418.5 **
Sonication Water 10881.1 3524.2 **
Methanol 1130.0 170.71 **
p values, ns = nonsignificant: p value > 0.05, *p value < 0.05, **p value < 0.01, ***p value < 0.001
toxic to lymphocytes. The sonication extract was toxic to lymphocytes as seen in

Table 9.2. Based on these results, the water/methanol extract (elevated temperature)
was selected for fractionation and further study.
9.3.3 ytotoxicity Assessment of Fractions and Comparison

C
with Crude Extract
The cytotoxicity of fraction MO1 and MO2 was expressed as percent cell viability
and compared to the cytotoxicity of water/methanol extract that was subjected to
fractionation. As seen in Fig. 9.2, the fractions MO1 and MO2 were not able to
reduce the viability even up to 70% at dose of 100 μg/ml, whereas an equivalent
reduction in cell viability a dose of 100 μg/ml was seen in case of crude extract.
9.4 Discussion
Moringa oleifera Lam. has been used traditionally for treatment of various ailments
including cancer. Anticancerous activity of leaves of this plant and their extracts
against a variety of cell lines have been reported [21–23]. However, our report on
breast cancer cell line with a huge range of extracts and simultaneous toxicity
assessment with lymphocytes is first of its kind. The present study evaluated the
cytotoxic effects of crude extracts prepared by various methods and solvent systems
for three plants using fission yeast. The cdc2 gene in fission yeast is highly con-
served to the human cdc2 and plays a central role in regulating the onset of S-phase
of cell cycle as well as for initiation of mitosis. Hence, it works as a key checkpoint
regulator in yeast cell cycle. The alteration in this gene would lead to loss of regula-
tion of cell cycle, a situation similar to cancer cells, and therefore such mutant yeast
was used for screening plant extracts in our study. Similar studies using yeast have
been reported for various chemical agents [24]. Moreover, the ability of plumbagin
100
% Cell viability 80
60
MO1 fraction
40 MO2 fraction
20 Crude
0
0.1 1 10 100
Concentration (µg/ml)
Fig. 9.2 Cell viability of crude extract and fraction MO1 and MO2 of Moringa oleifera Lam.,
after 48 h exposure at a dose of 0.1–100 μg/ml
(plant based compound) as an anticancer agent and the pathway it affects was stud-
ied using a yeast system [25].
Moreover, the study also focused on assessing the cytotoxicity of crude extracts
of this plant not only against MCF-7 cell line but also toxicity assessment for nor-
mal peripheral blood monocytes (PBMCs). This will ensure the selectivity of extract
to affect cancerous cell rather than normal cells. This finding is essential in process
of drug development.
9.5 Conclusion
The cytotoxicity analysis has revealed the ability of water methanolic extract (ele-
vated temperature) to be a better cytotoxic agent than the fractions. The probable
reasons could be that in the crude extract compounds are exerting a synergistic
effect and hence more effective. Even traditional systems of medicine like Ayurveda,
traditional Chinese medicine, and European phytotherapy generally believe that a
synergy of all ingredients of plants will bring about the maximum of the therapeutic
efficacy. Moreover, the water methanolic extract (elevated temperature) was more
toxic to MCF-7 compared to lymphocytes indicating selective action against can-
cerous cells, hence can be used a therapeutic agent. The mode of action of the
extract can be further explored using specific metabolite assays and in vivo studies
can be performed as well.
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H, Nishino H (1999) An antitumor promoter from Moringa oleifera Lam. Mutat Res/Genet
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3. Zhao T, Sun Q, Marques M, Witcher M, Zhao T, Sun Q, Marques M, Witcher M (2015)
Anticancer properties of Phyllanthus emblica (Indian Gooseberry), anticancer properties of
Phyllanthus emblica (Indian Gooseberry). Oxidative Med Cell Longev 2015(June):e950890.
https://doi.org/10.1155/2015/950890
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medicinal uses. Phytother Res 21(1):17–25. https://doi.org/10.1002/ptr.2023
7. Kumar VV, Hussain Z, Verma M (2014) Antiinflammatory and antinociceptive activity of
Moringa oleifera. J Biomed Pharm Res 3(1):14–21
8. Ndhlala AR, Mulaudzi R, Ncube B, Abdelgadir HA, du Plooy CP, Van Staden J (2014)
Antioxidant, antimicrobial and phytochemical variations in thirteen Moringa oleifera Lam.
cultivars. Molecules 19(7):10480–10494. https://doi.org/10.3390/molecules190710480
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jbeb.2014.1101
10. Botstein D, Chervitz SA, Michael Cherry J (1997) Yeast as a model organism. Science (New
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11. Yang X, Summerhurst DK, Koval SF, Ficker C, Smith ML, Bernards MA (2001) Isolation of
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pone.0045023
Hydroxy Fatty Acid from Camelina sativa
Seed Oil for Industrial Application 10
Neha Sharma, Lekha Charan Meher, Mitesh Mittal,
and Sanjai Kumar Dwivedi
Abstract
Renewable bio-based feedstocks are viable alternative to crude petroleum for
fuel and chemical demands. Moreover, mineral oil-based lubricants used in
industries and transport sector are associated with environmental concerns.
Camelina sativa is an oilseed crop, and the oil may be a potential feedstock for
fuel and lubricant base stocks. The present study revealed that the Camelina oil
contains 82.3% of unsaturated fatty acids (monounsaturated fatty acid 33.6% and
polyunsaturated fatty acid 48.7%). The physicochemical properties of Camelina
oil reveal the presence of long chain fatty acid and high unsaturation. The oil
may be chemically modified for lubricant applications and eco-friendly. The
article describes the modification of Camelina oil to hydroxy fatty acid by the
process of splitting, separation of saturated fatty acid so that the unsaturated fatty
acid having iodine value 160gI2/100 g is further epoxidized to obtain hydroxy
fatty acids. The hydroxy functionality introduced into the Camelina fatty acid
serves as a potential renewable material for synthesis of bio-lubricant base
stocks.
Keywords
Bio-lubricant · Camelina sativa seed oil · Hydroxy fatty acid · Iodine value
N. Sharma (*) · L. C. Meher · M. Mittal

Defence Institute of Bio-energy Research, Project Site Secunderabad,
Secunderabad, Telangana, India
S. K. Dwivedi
Defence Institute of Bio-Energy Research, Haldwani, Uttarakhand, India

https://doi.org/10.1007/978-981-13-6321-4_10
70 N. Sharma et al.
10.1 Introduction
In the past three decades, world energy demand is increasing continuously with
increase in industrialization. The total world energy consumption in 2012 was
1.384 × 1019 million tons of oil equivalent (Mtoe) and is estimated to reach
1.586 × 1019 Mtoe and 2.055 × 1019 Mtoe in 2020 and 2040, respectively, i.e. almost
a 48% increase from 2012 to 2040 [1].
The crude petroleum serves as raw materials for lubricants, polymers and
personal care products. The lubricants from petroleum are being widely used in
machineries and industries. The conventional mineral lubricants are usually envi-
ronmentally unacceptable because of their low biodegradability and toxicity and
lead to contaminate the whole environment [2]. The bio-based products are environ-
ment friendly to replace petrochemicals, and it is a strategic approach to national
security [3]. Therefore, more attention has been focused to reduce the dependency
from fossils source by synthesis bio-based chemicals from renewable and sustain-
able resources that should reduce the environmental impact.
Vegetable oils are considered to potential bio-lubricant-based stock to replace
conventional mineral oil-based lubricating oil and synthetic esters. Vegetable oil-
based lubricant is used due to its enhanced biodegradability; superior, low toxicity;
and low temperature properties. It has been studied that castor and lesquerella fatty
acid estolide esters were formed by reacting with different types of saturated, unsat-
urated and branched fatty acids with low temperature properties [4]. Castor oil con-
sists 12 hydroxy fatty acids; these properties of castor fatty acid have application as
lubricant base stocks such as estolide esters and their derivatives [5]. In this context,
various studies have been carried out for synthesis of precursor for lubricant base
stock.
Camelina sativa is an oilseed crop, and the oil may be a potential feedstock for
lubricant base stocks. The physicochemical properties of Camelina oil reveal the
presence of long chain fatty acid and 82.3% of high unsaturation. The present study
describes the modification of Camelina oil to hydroxy fatty acid by the process of
splitting followed by isolation of unsaturated fatty acid which is further epoxidized
to obtain hydroxy fatty acids with multiple –OH groups that may serve as a potential
renewable material for synthesis of bio-lubricant base stocks.
10.2.1 Extraction and Analysis of Camelina Oil
The dried seeds were powdered, and the oil was isolated by Soxhlet extraction
method using petroleum ether (60–80 °C) as solvent. In each extraction experiment,
50–60 g of crushed Camelina sativa seed was taken in a thimble. The extract was
collected and the oil was obtained after removal of solvent under vacuum. The oil
was characterized for its physicochemical properties as per IS-548:1976, Part-1
10 Hydroxy Fatty Acid from Camelina sativa Seed Oil for Industrial Application 71
method of Bureau of Indian Standards, and the fatty acid composition was carried
out by gas chromatographic analysis as per method IS-548:1976, Part-3.
10.2.2 Splitting of Fatty Acid from Camelina Oil
Saponification of Camelina oil with potassium hydroxide was followed by

phosphoric acid treatment to obtain free fatty acids. The organic layer was washed
with water till the wash water was neutral, followed by drying with anhydrous
sodium sulphate. The acid value and iodine value were estimated. During typical
experiments, 100 g Camelina oil was mixed with 80 ml water, 60 ml ethanol and
30 g KOH, followed by addition of phosphoric acid to lower pH up to 4.0 and stirred
for a few minutes, then washed with water to remove phosphoric acid and the
remaining reaction mixture. The fatty acids were dried under vacuum.
10.2.3 I solation of Unsaturated Fatty Acid by Urea Inclusion

Complex
During the urea inclusion complex method [6], 40 g of free fatty acid was added to
400 ml methanol in 1 l conical flask boiled at 65 °C under water bath. 30 g of urea
was added. It was allowed to cool in freeze at 7–8 °C overnight. The urea complexes
were separated by filtration and the unsaturated fatty acids were isolated.
10.2.4 Epoxidation of Unsaturated Fatty Acid
The epoxidation was carried out in the laboratory with 30 g unsaturated free fatty
acid under ice bath taken in a 150 ml round-bottom flask, and 5.8 ml of formic acid
was added to it followed by gradual addition of 30.9 ml hydrogen peroxide (30%
solution) with stirring and cooling. The flask containing the reactants was heated up
to 40–45 °C in a water bath with stirring at 600 rpm for 6 h. After the reaction was
complete, the mixture was cooled down to room temperature, washed with distilled
water. The epoxidized fatty acid was dried under vacuum to obtain colourless trans-
parent, viscous liquid. The oxirane oxygen content (OOC) of epoxidized Camelina
oil was determined as per AOCS Official Method Cd 9-57, 1993, iodine value of
epoxidized Camelina oil was estimated according to IS :548 1964 (Part I) using
Wijis solution.
10.2.5 R
ing Opening of Epoxidized Fatty Acid to Hydroxy Fatty
Acid
The acid-catalysed ring opening was studied in the laboratory. In typical experiment
50 g of epoxidized fatty acid was taken in a 250 ml flat-bottom flask with addition
72 N. Sharma et al.
of 2% H2SO4 with stirring. The reaction was conducted at below 100 °C using an oil
bath and stirring at 600 rpm for 4 h. The hydroxyl value of hydroxy fatty acid physi-
cochemical properties was measured according to using IS-548:1976 Part-1 method
of Bureau of Indian Standards.
10.3.1 Extraction and Analysis of Camelina Oil
Camelina seed contained 28–31 wt% of oil. The iodine value of Camelina oil is
140gI2/100 g. The physicochemical properties of Camelina oil are listed in
Table 10.1. The oil was found to be yellowish clear liquid. The saponification value
is 180 mg KOH/g which indicated long chain of fatty acids in it. The iodine value
of the Camelina oil is high, i.e. 140 g gI2/100 g, which indicates that the oil is rich
in unsaturated fatty acid. The fatty acid distribution in Camelina oil is listed in
Table 10.2. The fatty acid composition of Camelina oil contains 82.3% of unsatu-
rated fatty acids (monounsaturated fatty acid 33.6% and polyunsaturated fatty acid
48.7%) which indicates oil rich in unsaturation.
10.3.2 Splitting of Fatty Acid of Camelina Oil
The aim of splitting the triglycerides is to obtain the free fatty acid. Free fatty acids
were obtained by saponification of Camelina oil with potassium hydroxide followed
by acidification with phosphoric acid. The iodine value of free fatty acid of Camelina
oil was 143 gI2/100g, and its acid value was 212 mg KOH/g.
10.3.3 U
rea Inclusion Complex Formation for Isolation
of Unsaturated Fatty Acid
Saturated fatty acid was precipitated out by urea inclusion complex method and
separated from the oil, obtaining unsaturation fatty acids. The iodine value of unsat-
urated fatty acid was 162 gI2/100g which revealed the presence of high unsaturation
in the oil, and its acid value was 212 mgKOH/g.
Table 10.1 Physicochemical properties of Camelina oil

S.No Properties Results
1. Appearance Yellowish colour, clear liquid
2. Acid value (mg KOH/g) 2.7
3. Saponification value (mg KOH/g) 180
4. Iodine value (gI2/100g) 140
Table 10.2 Fatty acid S.No. Fatty acid Weight%

composition of Camelina oil 1. Myristic acid (C14:0) 0.2
2. Palmitic acid (C16:0) 7.3
3. Stearic acid (C18:0) 3.1
4. Oleic acid (C18:1) 14.0
5. Linoleic acid (C18:2) 22.2
6. Linolenic acid (C18:3) 23.9
7. Arachidic acid (C20:0) 3.2
8. Gadoleic acid (C20:1) 14.8
9. Ecosadienoic acid (C20:2) 1.8
10. Ecosatrienoic acid 0.8
(C20:3)
11. Behenic acid (C22:0) 0.8
12. Eruic acid (C22:1) 4.8
13. Unidentified 3.1
Fatty Acid
Mixture
O Urea/ MeOH
Cooling
C
HO + Urea+ SFA
inclusion Complex
Unsaturated Fatty Acid
Epoxidation
O O O
C
HO
Epoxidized Fatty Acid
Ring Opening of Oxiranes
O OH
OH
C
HO HO HO
Hydroxy Fatty Acids
Fig. 10.1 The process of preparation of hydroxy fatty acid from Camelina oil fatty acid
10.3.4 E
poxidation of Unsaturated Fatty Acid and Ring Opening
of Oxirane
Epoxidation is an important step where the fatty acid rich in C=C unsaturation
forms oxirane ring which was followed by ring opening reaction to form hydroxy
fatty acid. The conversion of C=C bond to oxirane and further hydroxy groups was
introduced to form hydroxy fatty acid as shown in Fig. 10.1.
74 N. Sharma et al.
Table 10.3 Physicochemical properties of free fatty acid, unsaturated fatty acid, epoxidized fatty
acid and hydroxy fatty acid
Properties
Iodine value Acid value Oxirane oxygen Hydroxyl value
S.No Product (gI2/100g) (mgKOH/g) content (wt. %) (mgKOH/g)
1.
2. Free fatty acid 143 198 – –
3. Unsaturated 162 198 – –
fatty acid
4. Epoxidized 9.49 178 8.11 –
fatty acid
5. Hydroxy fatty 08 162.48 0.28 289
acid
In typical experiment the epoxidation is carried out with double bond to formic
acid molar ratio 1:0.75 and double bond to hydrogen peroxide ratio 1:1.5 at 46.7 °C
for 6 h. The percentage of oxirane oxygen content is 8.1 which shows the extent of
epoxidation is >88% conversion of unsaturated moiety. The iodine value decreases
from 162 to 9.49 gI2/100g which further revealed >94% conversion during epoxida-
tion. The ring opening of epoxidized fatty acids was carried out with sulfuric acid at
below 100 °C for 3 h. The obtained hydroxy fatty acid having iodine value 8gI2/100g
and oxirane oxygen content 0.28% indicated the presence of low unsaturated in it.
The physicochemical properties of free fatty acid, unsaturated fatty acid, epoxidized
fatty acid and hydroxy fatty acids are listed in Table 10.3.
The hydroxyl value of the hydroxy fatty acid is 289 mg KOH/g which is higher
than the hydroxyl value of castor oil (160–168 mg KOH/g) indicating that the
Camelina oil is converted to hydroxy fatty acid with multiple –OH functionality
groups.
10.4 Conclusion
The Camelina oil may be chemically modified to derive industrially important

oleochemicals. The present article described the method to derive hydroxy fatty
acid which is having –OH functional group. The castor oil with its ricinoleic acid
(12-hydroxy fatty acid) finds various industrial applications, i.e. for lubricant base
stock. The camelina-based hydroxy fatty acid with higher hydroxyl value is best
suited as precursor to derive estolide with free –OH group to impart better
lubricity.
Acknowledgements The authors express their gratitude to DRDO, Ministry of Defence,

Government of India, for providing financial assistance to Ms. Neha Sharma in the form of Senior
Research Fellowship. The authors are also grateful to Dr. J.Vollmann, BOKU-University of Natural
Resources and Applied Life Sciences, Vienna, Austria, for providing the nucleus seed of Camelina
cv. Calena.
References
1. World energy demand and economic outlook Date of access: 28/05/2017, International Energy
Outlook 2016 (IEO2016) Chapter 1. https://www.eia.gov/outlooks/ieo/pdf/world.pdf
2. Soni S, Agarwal M (2014) Lubricants from renewable energy sources–a review. Green Chem
Lett Rev 7(4):359–382
3. Rani S, Joy ML, Nair KP (2015) Evaluation of physiochemical and tribological properties of
rice bran oil–biodegradable and potential base stoke for industrial lubricants. Ind Crops Prod
65:328–333
4. Cermak SC, Brandon KB, Isbell TA (2006) Synthesis and physical properties of estolides from
lesquerella and castor fatty acid esters. Ind Crops Prod 23:54–64
5. Potula SB, Korlipara VP, Bhamidipati VSKR, Krishnasamy S, Rachapudi BNP (2014) Castor
oil fatty acid based estolide esters and their derivatives as potential lubricant base stocks USP
patent no US8,742,150 B2
6. Hayes DG, Bengtsson YC, Van Alstine JM, Setterwall F (1998) Urea complexation for the
rapid, ecologically responsible fractionation of fatty acids from seed oil. J Am Oil Chem Soc
75(10):1403–1409
Comparison of Different Planting
Methods to Determine the Precision 11
of Phenotyping Wheat in Field
Experiments
Davinder Sharma, Jagadish Rane, Rajender Singh,

Vijay Kumar Gupta, and Ratan Tiwari
Abstract
Lack of uniformity of plant stand while conducting a field experiment can sub-
stantially contribute to errors in the prediction of association between plant phe-
notype and genotype. Among the several factors that can contribute to
experimental errors, inconsistent seed depth and plant spacing often occur due to
lack of precision when seeds are sown by hand or seed drills. Hence, we compare
three planting methods, novel dibbling, seed drill, and hand sowing, to determine
the most efficient method for precision phenotyping in field. We showed the
advantage of the new methods over conventional methods of sowing, viz., seed
drill and by hand. Compared with conventional methods, the new method
improved the consistency in plant spacing substantially as indicated by reduction
in standard deviation at least by three times. The desired seed depth (6.5 cm) and
plant spacing (10 cm intra- and 20 cm inter-row spacing) could be maintained
with greater precision in dibbling method than in seed drill or hand sowing
method. The reduction in error and the least coefficient of variation (CV%) for
the plant traits measured in the new method relative to other methods indicated
possibility of enhancing precision in phenotyping responses of wheat plants
under field condition.
D. Sharma
Presenting author, ICAR-Indian Institute of Wheat & Barley Research,
Karnal, Haryana, India
J. Rane
School of Drought Stress, ICAR-National Institute of Abiotic Stress Management,
Baramati, Maharashtra, India
R. Singh · R. Tiwari (*)
ICAR-Indian Institute of Wheat & Barley Research, Karnal, Haryana, India
V. K. Gupta
Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India

https://doi.org/10.1007/978-981-13-6321-4_11
78 D. Sharma et al.
Keywords
Precision phenotyping · Planting methods · Plant stand · Coefficient of variation
11.1 Introduction
Reducing experimental error is one of the major challenges while phenotyping a

large number of genotypes for genetic variations under field conditions. Nongenetic
variations in field crop experiments result from extraneous variations. Lack of uni-
formity of plant stand while conducting a field experiment can substantially contrib-
ute to errors in the prediction of association between plant phenotype and genotype.
Among the several factors that can contribute to plant stand, inconsistent seed depth
and plant spacing often occur due to lack of precision while planting. Any error dur-
ing the planting can leave a permanent effect on the plant performance throughout
their life cycle [1]. The microclimate may be altered considerably in the field by
uneven space and depth of planting which can contribute to nongenetic variations
and hence affect the precision of phenotyping. Individual development was influ-
enced directly by the level of crowding [2]. Further, variability in plant space and
emergence can cause crowding stress, which can result in a reduction in the capture
and utilization of resources by plants [3]. The lack of precision in phenotyping lim-
its our ability to dissect the genetics of quantitative traits. Since conventional meth-
ods, including hand sowing and seed drills, often fail to place the seeds at consistent
depth or space, we compared these planting methods with dibbling method to deter-
mine the most efficient method for precision phenotyping in field experiments.
11.2 Material and Methods
The experiments were conducted at the Indian Institute of Wheat and Barley
Research (IIWBR) (29°42′ N latitude and 77°2′E longitudes at an altitude of 250 m
above the mean sea level) in the Indo-Gangetic Plain in northwestern India, with
mildly alkaline soil (typic ustochrept) during the crop seasons 2012–2013 and
2013–2014. Experiments were laid out in a randomized complete block design with
four replications using a split plot arrangement where the planting methods com-
posed the main plots and genotypes of the subplots. The experiment was conducted
with four wheat (Triticum aestivum L.) genotypes, viz., DBW 88, DBW 96, DBW
97, and DBW 123. In both the crop seasons, experiments were planted in the third
week of November. Agronomic practices were followed as recommended for irri-
gated timely sown conditions. Three planting methods used:
1. Seed drill method (SDM): Seeds were sown at 96 seeds/plot in four lanes with
the help of seed drill (precision plot drill; equipment code, ER-03; CIAE, Bhopal,
Hyderabad). Drill was set to drop seeds at the interval of 10 cm and at the depth
of 6.5 cm.
11 Comparison of Different Planting Methods to Determine the Precision… 79
2. Hand sowing method (HSM): Furrows in the soil were made with a marked hoe,
to the predetermined depth, 6.5 cm. Seeds in each row were dropped by hand and
subsequently covered with surrounding soil. Seeds were sown at 96 seeds/sub-
plot in four lanes.
3. Dibbling tool method (DBM) [4, 5]: Dibbling area (11.7 m × 6.5 m) was divided
into 16 subplots (2.3 m × 1 m) with path (0.5 m) on either side with the help of
threads. Dibbler was set to create cavities of 6.5 cm deep with inter- and intra-
row spacing of 10 and 20 cm, respectively. With one seed placed in each cavity,
each subplot initially had 288 seeds. Two weeks after sowing, two of three plants
were uprooted to finally keep one plant/spot and 96 plants of a genotype in each
subplot.
11.2.1 Data Measurements and Analysis
Depth of seeding was measured during the morning hours of the next day of sowing
by removing the soil very carefully with the help of a narrow strip of metal to locate
the seed, and then scale was used to measure the depth from the soil surface. Three
weeks after sowing, the variability in plant spacing was measured by using a scale.
Leaf chlorophyll status (SPAD) was measured (GS 73; [6]) with a chlorophyll meter
(Minolta SPAD-502, Osaka, Japan). Productive tillers (PT) as tillers bearing spike
were recorded at the time of harvest. The postharvest measurements included thou-
sand grain weight (TGW) and yield per plant (GY). During the collection of data,
border effects were eliminated by sampling plants at the center of each subplot. All
the data were analyzed using the SAS statistical software program, PROC MIXED
(SAS version 9.3, SAS Institute Inc., Cary, NC, USA) with a MODEL statement
containing the ALPHA = 0.05 and DDFM = Satterthwaite option. Replication was
treated as a RANDOM effect, but planting method (PM) and genotype (GEN) were
treated as fixed effects. An estimate statement was used to derive means for the main
effects and their interaction. The LSMEANS statement included the
ADJUST = TUKEY option for using the Tukey range test (adjusted for multiple
comparisons) to evaluate a ranked (high to low) ordering of main effects means for
significant differences. Combined analysis was performed due to homogeneous
experimental error and non-significant interaction in year × planting method ×
genotypes.
The standard deviation (SD) of means was used to determine the variability in seed
depth and space between the plants for each of the planting methods, and results
revealed that the SD was much higher for seed drill method (SDM) and hand sowing
method (HSM) as compared to dibbling method (DBM) (Fig. 11.1). Thus, the
desired seed depth (6.5 cm) and plant spacing (10 cm intra- and 20 cm inter-row
spacing) could be maintained with greater precision in DBM than in SDM or
80 D. Sharma et al.
Fig. 11.1 Plot depicting deviations in seed depth and plant spacing obtained with three different
planting methods and graph showing the coefficient of variation (CV%) for the investigated traits
under different planting methods. SDM seed drill method, HSM hand sowing method, and DBM
dibbling method
HSM. Combined variance analysis of all the traits over the years showed that differ-
ences in effects of planting methods were significant for SPAD, PT, and GY except
TGW. However, planting method × genotype interaction was significant for SPAD
and GY (Table 11.1). Compared with conventional methods, the new method
improved the precision in phenotyping as indicated by reduction in percent of error
variance (Table 11.2) and coefficient of variation (CV) at least by one-half and two
times, respectively (Fig. 11.1). Further, the differential responses of genotypes rep-
resented by measured traits were more conspicuous and consistent across the years
in those plants which were sown by DBM than by SDM or HSM. High-quality
genotypic and phenotypic data are essential to dissect the genetic architecture of
complex traits. While the power of current genotyping approaches enables high-
density genotyping of entire genomes, field phenotyping is the bottleneck.
Hence, the present study was aimed to find an efficient method that can enhance
our precision to differentiate genotypic responses by minimizing experimental
errors arising from the inconsistent crop establishment. The results obtained from
this experiment clearly revealed that factors contributing to the initial crop estab-
lishment were effectively addressed by the novel dibbling method for planting that
takes into consideration the variation in plant responses due to aboveground factors
such as solar radiation [7] and belowground factors such as nutrient and soil mois-
ture [8] which influence plant growth and development. Although there was no sig-
nificant response in plant growth under different planting methods used, variation
within the genotype was much greater in SDM or HSM than in DBM. We attribute
it to the consistent plant spacing in DBM that resulted in uniformity in the intercep-
tion of available solar radiation, water use, and nutrient uptake which collectively
contributed to the homogenous crop stand as revealed by different parameters rela-
tive to the same observed in other methods. In DBM, the observed seed depth and
plant spacing closely matched the desired values (Fig. 11.1) because of equidistant
cavities of same depth created by the dibbler.
Additionally, three seeds sown at one locus but in separate cavities completely
eliminated the possibility of gaps that might have been formed due to lack of germi-
nation at the place. Seed drill might get out of level due to lack of perfection in the
leveling of the field, inconsistent speed, or jerks due to which seed placement at
variable depth could occur. Uneven seed placement by hand or seed drill often cre-
ate bunching and gaps in the field. In addition, lack of germination at some locus
added further variation in plant spacing. Moreover, hand sowing method was least
precise as there was no perfect control over placement of seeds. Further, the lack of
homogeneity among neighboring plants could be due to seedling emergence which
Table 11.1 Source of variation, degree of freedom for the F-tests, and probability values obtained
in a PROC MIXED analysis of variance of agronomic traits for planting method (PM) and geno-
type (GEN) main effects and their interaction for combined 2 years
Source of variation num df den df SPAD PT TGW GY
PM 2 6 0.0108 <0.0015 0.2038 <0.0081
GEN 3 75 <0.0001 <0.0001 <0.0001 <0.0001
PM*GEN 6 75 0.0092 0.5602 0.1196 0.0002
82 D. Sharma et al.
Table 11.2 Error as percent of total sum of squares in different planting methods followed in the
experiment for various traits studied
Traits SPAD PT TGW GY
SDM 31.3 55.7 38.3 54.8
HSM 23.2 53.5 19.7 20.8
DBM 9.6 29.6 14.2 13.2
Error% = (error sum of squares/total sum of squares) × 100
SDM seed drill method, HSM hand sowing method, DBM dibbling method, SPAD leaf chlorophyll
status, PT productive tillers, TGW thousand grain weight, GY grain yield
occurs over a period of several days due to variability in soil resistance and soil
moisture or due to uneven compaction of soil by human feet or tractor wheels [1]
which could be avoided in DBM. The precision in sowing by DBM was reflected in
higher efficiency in differentiating the genotypes based on their phenotypes. DBM
could help in identifying the differences in SPAD among all the genotypes, while
the other two methods were not efficient. Canopy structure is the major determinant
of the quantity and the distribution of radiation and consequently of SPAD. Continuous
and spatially homogeneous canopy establishment in DBM favors precision in mea-
surement, while discontinuous and spatially heterogeneous canopy in HSM or SDM
made it difficult to distinguish genotypes on the basis of this trait. [9] reported that
the uniform plant spacing could greatly enhance precision in radiation-based pheno-
typing. The differential responses of genotypes could also be seen for traits other
than SPAD in those plants which were sown with DBM.
In addition, varietal responses are more conspicuous and consistent across the
years in DBM while highly inconsistent in HSM and SDM. This was mainly due to
reduction in error (Table 11.1) as indicated by the substantially less coefficient of
variation (CV%) for each trait in the DBM as compared to other methods (Fig. 11.1).
Experimental error is the difference between a genotype treated alike in replicated
experiment and is the primary basis for making a decision whether an observed dif-
ference is real or just due to chance. The CV can be used to indicate the stability of
phenotypes in field experiments. Experiments with low CV and with reduced extra-
neous variations improve the ability to detect differences. The ability to detect
genetic differences increases as the experimental error decreases. DBM minimizes
the extraneous variations which ultimately contribute to greater precision in pheno-
typing. It is suggested that this method can enhance our capacity to identify genes
and gene marker for crop improvement through phenotyping under field
conditions.
Acknowledgments We gratefully acknowledge the financial support from ICAR for the Network
Project on Transgenic in Crop: Functional Genomics in Wheat.
References
1. Nielsen RL (1991) Stand establishment variability in Corn. Purdue University AGRY-91-01
2. Lauer J (1994) Should I be planting my corn at a 30-inch row spacing? Wisconsin crop man-
ager. Crop Agron 1(6):311–314
3. Tollenaar M, Deen W, Echarte L, Liu W (2006) Effect of crowding stress on dry matter accu-
mulation and harvest index in maize. Agron J 98(4):930–937
4. Sharma D, Singh R, Rane J, Gupta VK, Mamrutha HM, Tiwari R (2016) Mapping quantitative
trait loci associated with grain filling duration and grain number under terminal heat stress in
bread wheat (Triticum aestivum L.). Plant Breed 135(5):538–545
5. Sharma D, Tiwari R, Gupta VK, Rane J, Singh R (2018) Genotype and ambient temperature
during growth can determine the quality of starch from wheat. J Cereal Sci 79:240–246
6. Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for growth stages of cereals. Weed
Res 14(6):415–421
7. Sinclair TR, Muchow RC (1999) Radiation use efficiency. Adv Agron 65:125–265
8. Casper BB, Jackson RB (1997) Plant competition underground. Annu Rev Ecol Evol Syst
28:545–570
9. Blackmer TM, Schepers JS, Vigil MF (1993) Chlorophyll meter readings in corn as affected by
plant spacing. Commun Soil Sci Plant Anal 24(17–18):2507–2516
Effect of Extraction Temperature
and Different Carrier Agents 12
on Physicochemical and Antioxidant
Properties of Spray-Dried Murraya
koenigii (Linn.) Leaf Extract
Vandana Sablania, Sowriappan John Don Bosco,

and Shubham Rohilla
Abstract
The work evaluated the effect of extraction temperature on Murraya koenigii L.
extract, prepared at an extraction temperature of 80 °C and 90 °C. The prepared
Murraya koenigii L. leaf extract was encapsulated with different carrier agents
such as guar gum, pectin, and whey protein isolates at a concentration of 1%,
5%, and 10% respectively. Spray drying was done at an inlet temperature of
130 °C with constant feed flow rate (8 rpm) at a pressure of 0.4 kg/cm2 and outlet
temperature of 80 °C. The encapsulated powders were analyzed for their physi-
cochemical properties such as moisture content, water activity, bulk density,
tapped density, flow properties, antioxidant activity, and total phenolic content.
The extract encapsulated with pectin and whey protein isolates did not show
significant difference on DPPH activity at varied extraction temperature, whereas
the extract encapsulated with guar gum showed more activity at an extraction
temperature of 80 °C. The extract encapsulated with whey protein isolates
showed more total phenolic content at an extraction temperature 90 °C. Therefore,
it can be considered as a good source of active phytochemicals. Thus from this
study, encapsulated Murraya koenigii L. extract could be considered as an impor-
tant source of antioxidant and anti-inflammatory in human cells.
Keywords
Murraya koenigii L. · Spray drying · Antioxidant activity · Total phenolic
content
V. Sablania · S. J. D. Bosco (*) · S. Rohilla

Department of Food Science and Technology, Pondicherry University, Puducherry, India

https://doi.org/10.1007/978-981-13-6321-4_12
86 V. Sablania et al.
12.1 Introduction
In current status, there is an increase in demand of herbal products due to their

higher antioxidant activities, which have the potential to protect the body against
free radical damage and degenerative diseases. Several studies were done to identify
new compounds capable to inactivate free radicals generated by metabolic path-
ways in human tissues and cells. Murraya koenigii L. leaf is a much demanded spice
commodity in South India and all over the world. It belongs to the Rutaceae family,
used for its characteristic color and flavor [1]. Around 14 species of Murraya are
found all over the world in which Murraya koenigii and Murraya paniculata are the
most popularly used variety in India. Murraya koenigii leaf is known for its aro-
matic, spicy, bitter taste and acrid, cooling, and strong fragrance. It is described to
have antidiabetic, antioxidant, antidysenteric, anticarcinogenic, stimulant, hypogly-
cemic, and antimicrobial activities due to the presence of tocopherol, β-carotene,
and lutein [2]. There is an increase in demand of naturally present antioxidants for
the treatment of oxidative stress-related diseases. Plants extract are usually available
in the form of liquid and viscous solution which cannot be stored for a longer period
of time in liquid form. Hence the drying of these extracts could be a better alterna-
tive to increase the shelf life of the liquid feed without much altering in its chemical
composition. Therefore, the drying techniques such as spay drying and freezed dry-
ing can be used for the drying of extracts. Any product in dried form shows signifi-
cant merits over conventional liquid extract, including chemical, physicochemical,
and microbiological stability, easy to transport, less space for storage, and the use of
powders for its pharmaceutical applications [3]. Several researchers use spray-
drying technique for encapsulation of any herbal extract with their efficacy and
better stability in powder form. With this scenario Murraya koenigii L. leaf extract
was prepared at varied extraction temperature and encapsulated with carrier agents
such as whey protein isolates, pectin, and guar gum in order to produce dried pow-
der with maximum retention of antioxidant activity and total phenolic content.
12.2.1 Extract preparation
Fresh curry leaves were purchased from the local market of Pondicherry, washed
under running tap water, and then leaves were taken out from the stalks and kept for
drying in tray dryer at 60 °C for 4 h (Ezidri Ultra FD1000). The tray-dried leaves
were ground into a grinder (Kenstar Karishma), and then 100 g of dried powder was
mixed in 1000 mL of distilled water and kept in water bath at a temperature of 80 °C
and 90 °C for 1 h for the extraction of bioactive extract. After 1 h it was filtered
through muslin cloth, and again 500 mL of distilled water was added into the
remaining residues and kept in water bath at same temperature for another 30 min.
After 30 min the extract was filtered through muslin cloth and mixed with the previ-
ously prepared extract.
12 Effect of Extraction Temperature and Different Carrier Agents… 87
12.2.2 E
ncapsulation of Curry Leaf Extract with Different Carrier
Agents
The prepared curry leaf extract was blended with different carrier agents such as
whey protein isolates (10%), guar gum (1%), and pectin (5%) and then kept over-
night at 4 °C for complete hydration of carrier agents into the extract. Many studies
have been conducted to optimize the operational conditions for the spray-dried juice
and extract powders. High inlet temperature leads to degradation of phenolic com-
pounds; therefore, the inlet and outlet temperature were kept constant at 130 °C and
80 °C, respectively. The feed solution was fed into pilot plant spray dryer with a
co-current flow rate of 8 rpm and pressure 0.4 kg/cm2 for atomization. The process
conditions were optimized by taking into account the experimental studies and pre-
viously conducted studies to enable the maximum powder yield with more antioxi-
dant activity. The powder was collected in pre-weighed glass bottles and refrigerated
until the analysis to be done.
12.3 Physicochemical Analysis of Spray-Dried Powder
12.3.1 Powder Yield
The spray-dried powder yield was calculated by using the formula given by
Santhalakshmy et al. [4].
12.3.2 Moisture Content
Moisture content was determined by the method given in AOAC manuals [5].
12.3.3 Water Activity
The water activity of the powder was measured by using the electronic instrument
(AquaLab Series 4TE, Decagon Devices, Inc., Pullman, Washington, USA).
12.3.4 Bulk and Tapped density
Bulk density and tapped density of spray-dried powders were measured by using the
method given by Tonon et al. [6] and Goula et al. [7].
12.3.5 Total Phenolic Content
Total phenolic content in spray-dried samples were determined by Folin-Ciocalteu

assay given by Shah et al. [8] with slight modifications.
12.3.6 DPPH Radical Scavenging Activity
The free radical scavenging activity was determined by the method given by Shah
et al. [8] with slight modification.
12.4 Statistical Analysis
The data interpretation was accomplished by SPSS software SPSS 20.0 (IBM
Corporation, Armonk, New York). The one-way analysis of variance (ANOVA) was
done by using duplicate values and mean values which were separated by using
Duncan’s multiple range test (p ≤ 0.05). All the data were expressed as the
mean ± standard deviation.
12.5.1 Powder Yield
The powder yield of encapsulated extract with different carrier agents is shown in
Table 12.1. The powder yield was observed to be increased with increase in extraction
temperature. The maximum yield of the powder is obtained with whey protein isolates
(6.70–9.15%) followed by pectin (2.90–5.25%) at an extraction temperature of 80 °C
and 90 °C, whereas guar gum showed a decrease in yield of powder with increased
extraction temperature (0.70–0.50%). Whey protein isolates are considered to be a
strong surfactant which specially migrates to the droplet air interface. Immediate reac-
tion of hot air with extract in the presence of high concentration of protein content
developed a film on particle and resulted in higher powder yield as compared to other
carrier agents. Whereas in the case of guar gum, increase in extraction temperature
leads to decrease in powder yield which could be due to the sticky behavior of guar
gum inside the spray dryer chamber and resulted in reduced powder yield [9].
12.5.2 Moisture Content
The moisture content of the spray-dried powder is found to be in the range between
3.57% and 5.84% (Table 12.1). Significant difference was observed with increase in
extraction temperature of the curry leaf powder. There was an increase in moisture
content with increased extraction temperature from 80 to 90 °C. Spray-dried
12
Table 12.1 Physicochemical analysis of encapsulated curry leaf extracts using different carrier agents
Whey protein isolate Pectin Guar gum
Parameters 80 °C 90 °C 80 °C 90 °C 80 °C 90 °C
Yield (%) 6.70 9.15 2.90 5.25 0.70 0.50
Moisture (%) 3.57 ± 0.16c 4.18 ± 0.28b 3.80 ± 0.21c 4.60 ± 0.50a 4.82 ± 0.11b 5.84 ± 0.56a
Water activity 0.33 ± 0.01ab 0.35 ± 0.01a 0.28 ± 0.02c 0.34 ± 0.06b 0.31 ± 0.02b 0.34 ± 0.04a
Bulk density (g/mL) 0.18 ± 0.07 d 0.14 ± 0.03e 0.30 ± 0.00a 0.25 ± 0.09 b 0.20 ± 0.00 c 0.13 ± 0.02 e
Tapped density (g/mL) 0.22 ± 0.12c 0.24 ± 0.04c 0.38 ± 0.05a 0.41 ± 0.00a 0.30 ± 0.00b 0.19 ± 0.05d
Carr index 36.27±0.07 a,b 35.81 ± 0.31a,b 25.47 ± 5.52d 38.43 ± 2.20a 33.99 ± 0.00b,c 32.13 ± 0.51c
Hausner ratio 1.56 ± 0.07b 1.55 ± 0.07b 1.37 ± 0.00d 1.62 ± 0.05a 1.51 ± 0.00 b,c 1.47 ± 0.01c
TPC (mgGAE/g) 46.62 ± 4.11b 71.16 ± 8.25a 19.01 ± 1.95c 47.23 ± 4.43b 51.14 ± 1.40b 52.91 ± 0.43b
All these values are mean ± standard deviation of duplicate readings. In each row superscripts show significant difference at P ≤ 0.05
Effect of Extraction Temperature and Different Carrier Agents…
89
powder obtained with whey protein isolates showed less moisture content in com-
parison to other carrier agents which could be due to its high concentration. Increase
in concentration of carrier agent results in decrease in moisture content reported by
Abadio et al. [10]. Increase in extraction temperature leads to release more sub-
stance from the dried powder. Constant carrier agent spontaneously increases the
moisture content with increase in extraction temperature. In general, less concentra-
tion of carrier agents and low inlet temperature could also be a reason for increase
in moisture content of the obtained spray-dried powder. The same results were also
observed by Goula et al. [7].
12.5.3 Water Activity
Water activity is the free water availability in food, which is considered to be a better
medium for the growth of microorganism and make the food unstable which leads to
the deterioration of the quality of food. Water activity of spray-dried powder was
found to be in the range between 0.28 and 0.35 (Table 12.1). Obtained spray-dried
powders showed water activity below 0.6 is considered to be appropriate for powder’s
stability. Hence, water activity between 0.2 and 0.3 minimizes the microbial propaga-
tion and oxidative and enzymatic activity [11]. Water activity was found to be increased
with increase in extraction temperature due to increase in moisture content of spray-
dried powder, in which the intermolecular bonds stabilize the solution by increasing its
viscosity and leads to retention of more water molecules in its matrix [12].
12.5.4 Bulk and Tapped Density
Encapsulated curry leaf extract showed significant effect on bulk density at different
extraction temperature (Table 12.1). It was found to be in the range between 0.13
and 0.30 g/mL. Bulk density was found to be decreased significantly with increase
in moisture content. Goula et al. [7] suggested that high bulk density is linked with
low moisture content which shows that there is an inverse relationship between the
residual moisture and bulk density. Powder encapsulated with pectin showed higher
value of bulk density, whereas powder obtained with whey protein isolates showed
lowest bulk density. Ferrari et al. [13] reported that high molecular weight of carrier
agent leads to less entrapment of particles and ultimately increases the bulk density
of the powder. It was observed that, stickier the particles of an agglomerate, the more
interspaces will be formed between them, and bulk density of powder decreases.
Tapped density was found to be higher in comparison to bulk density which could
be due to change in the volume, compaction of the particles, and transitions from
loosely packed particles to a more compact form, which increases the cohesive
forces among particles. There was no significant difference found on tapped density
of spray-dried powder at varied extraction temperature with different carrier agents.
Lower bulk density results in more accumulation of air into the powder and leads to
increase the possibility of the powder to oxidize and reduce the storage stability [4].
12.5.5 Carr Index and Hausner Ratio
Flowability is an important parameter for any of the powder material, and it was
expressed as the Carr index, whereas cohesiveness was expressed as a Hausner
Ratio. The higher the value of Carr index, the lower will be the flowability [14].
Hausner ratio shows an interparticle friction between the particles and indicates the
compaction properties of materials. From Table 12.1, the Hausner value of the
spray-dried powder found to be more than 1.25 resulted in the formation of agglom-
erates. Increased extraction temperature significantly leads to decrease in Hausner
ratio. Cohesiveness determines the flow properties and consistency of powders.
Low value of cohesiveness results in better flowability of powder. Chemical compo-
sition of powder such as protein, fat, moisture, and dietary fiber content also affects
the flowability of powders. The Carr index of the obtained spray-dried powder
found to be in the range between 25.47 and 38.43 resulted in reduced flowability
which could be due to the particle morphology and its interaction. The flowability
of powder was found to be increased with increase in extraction temperature, but
there was no significant effect of carrier agents reported on flow properties.
12.5.6 Total Phenolic Content
Total phenolic content (TPC) of encapsulated Murraya koenigii leaf extract is

shown in Table 12.1. It showed a significant effect of extraction temperature and
different carrier agents on phenolic content of encapsulated powder. Total phenolic
content is found to be increased with increase in extraction temperature. This indi-
cates that the extraction temperature is the key factor for the extraction of bioactive
compounds and leads to increase in total phenolic content. Total phenolic content of
powder obtained with whey protein isolates ranged from 46.62 to 71.16 mg gallic
acid equivalent (GAE)/g followed by guar gum 51.14–52.91 mg GAE/g and then
pectin 19.01–47.23 mg GAE/g. Powder obtained with WPI showed highest total
phenolic content which is due to its high emulsification property. Suhag and Nanda
[15] reported that retention of phenolic compounds was higher in honey powder
encapsulated with WPC, whereas Flores et al. [16] also observed that blueberry
pomace extract showed more anthocyanin stability when encapsulated with whey
protein isolate.
12.5.7 DPPH Scavenging Radical Activity
A DPPH assay was conducted to assess the scavenging effect of encapsulated pow-
der. While interacting with the DPPH free radical, the antioxidants transfer an elec-
tron to DPPH and neutralize the free radicals [17], due to which the extract color
changed from purple to golden yellow. Figure 12.1 shows the scavenging effect of
Murraya koenigii L. leaf extract encapsulated with different carrier agents at varied
extraction temperature. The scavenging activity of encapsulated powder obtained
Fig. 12.1 Antioxidant activity of encapsulated powder extracted at an extraction temperature of

(a) 80 °C and (b) 90 °C
with pectin is found to be more in comparison to other carrier agents which may be
due to the formation of gel networks which stabilizes the aqueous phase, by increas-
ing the viscosity [12].
Therefore, pectin is considered to anticipate the wall matrix component in a
homogenous liquid mixture. With increase in extraction temperature from 80 °C to
90 °C leads to decrease in scavenging activity. Extraction temperature of Murraya
koenigii leaves showed significant difference on scavenging activity of encapsulated
powders which means temperature is the key factor leading to a higher extraction of
bioactive compounds. The chemical structure of antioxidants decides their integral
reactivity toward free radicals. Scavenging activity of pectin is found to be 71.77%,
whereas guar gum and WPI showed 58.59% and 47.60%, respectively, at concentra-
tion of 500 μg/mL. The scavenging activity was not found to be correlated with the
total phenolic content. Cho et al. [18] also observed that the antioxidant activity of
the extracts from Enteromorpha prolifera is not directly correlated with their TPC
(r2 = 0.12).
12.6 Conclusion
The study was done to produce spray-dried powder of Murraya koenigii leaf extract
which was extracted at varied extraction temperature. The extract was encapsulated
with different carrier agents. The physicochemical parameters were found to be
desirable at an extraction temperature of 80 °C, whereas the antioxidant properties
were found to be more effective at an extraction temperature of 90 °C. Extract
encapsulated with pectin showed more antioxidant activity, whereas extract encap-
sulated with whey protein isolates showed more total phenolic content. Therefore,
the retention of bioactive compounds would be more effective if blends of carrier
agents will be used for the encapsulation of Murraya koenigii leaf extract.
Acknowledgment We acknowledge the Department of Food Science and Technology,

Pondicherry University, Pondicherry, for providing facilities, and are also grateful to UGC for
providing Junior Research Fellowship.
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Isolation and Characterization of Microbial
Asparaginase to Mitigate Acrylamide 13
Formation in Food
Mausumi Ray, Sunita Adhikari (Nee Pramanik),

and Pradyut Kundu
Abstract
Asparaginase is an enzyme which is used in food processing industry and also
used as a medicine. It is used to treat acute lymphoblastic leukemia, acute
myeloid leukemia and non-Hodgkin’s lymphoma. In food manufacturing it is
used to decrease the acrylamide which is occurring in some starch-based foods
during baking, frying and roasting. Acrylamide has carcinogenic effect on ani-
mals and human. The free amino acid asparagine reacts with sugars like glucose
and fructose during Maillard reaction under high temperature and low moisture
condition. To reduce acrylamide in food products, bacterial LA (L-asparaginase)
is used. LA catalyzes the conversion reaction of L-asparagine to L-aspartic acid
and ammonia. In present investigation, characterization of an extracellular LA
from an isolated Bacillus sp. strain M6 was carried out in batch scale fermenta-
tion process. The effect of pH, temperature and incubation time was measured
and the highest asparaginase activity (47 IU/ml) was achieved at pH 7.0, tem-
perature 30 °C.
Keywords
Acrylamide · L-asparaginase · Bacillus sp
M. Ray (*) · S. Adhikari (Nee Pramanik)

Department of Food Technology & Biochemical Engineering, Jadavpur University,
P. Kundu
Department of Food Processing Technology, A. P. C Ray Polytechnic,

https://doi.org/10.1007/978-981-13-6321-4_13
96 M. Ray et al.
13.1 Introduction
Acrylamide is a chemical compound which has carcinogenic effects on human as

well as on animals [1]. In 2008 a research work was conducted on acrylamide and
it was found that different types of human cancer such as ovarian, endometrial,
breast and kidney cancer are linked with high exposure to acrylamide [2, 3].
Acrylamide is linked with asparagine and Maillard reaction in foods [4–8]. Many
processes are used to reduce the formation of acrylamide in foodstuffs. Acrylamide
formation during baking and frying depends on moisture content and baking tem-
perature/time. Longer baking times are required to minimize the formation of acryl-
amide if final products are prepared to the same final moisture but with less color
development [9, 10]. The acrylamide load can be reduced by substituting ammo-
nium bicarbonate with inverted sugar and raising agents or by spraying with glycine
or by adding inorganic salts and organic acids [11, 12]. Alteration of the sensorial
properties like flavor, texture, browning [13] and formation of other undesirable
compounds [14] may lead to less consumer acceptance. The changes may even
affect the technology of the process like reduction of yeast fermentation properties
in bread [15].
It has been also found that asparagine is the major ingredient for the formation of
acrylamide in cereal food products. Increased amount of asparagine in various cere-
als can enhance the production of acrylamide. To reduce acrylamide in different
baked and fried products, the free asparagine content needs to be reduced by the
enzyme asparaginase that hydrolyzes asparagine to aspartic acid and ammonia.
Over 90% of acrylamide content is reduced when asparaginase-treated mashed
potato, potato flakes, rye flour, and wheat flour are incubated [8, 11, 16].
The objective of the present work is to isolate a potent bacterial strain capable of
producing asparaginase enzyme and optimize various process parameters for maxi-
mum production of the desired enzyme.
13.2.1 Isolation of a Potent Bacterial Strain
For the isolation of a potent bacterial strain, 10 g of soil was transferred to conical
flask containing 100 ml of sterile modified M9 medium having the following com-
position (g/L): KH2PO4, 3.0; Na2HPO4, 6.0; NaCl, 0.5; MgSO4,7H2O, 0.12; and
CaCl2, 2H2O, 0.001. The medium was enriched with 1% asparagine. Then the coni-
cal flask was kept in incubator for 24 h. The diluted soil sample was added to the
modified M9 agar medium enriched with 1% asparagine and 0.005% phenol red.
Plates were incubated at 37 °C for 24 h. The asparaginase enzyme production was
accompanied by an increase in pH of the medium; as a result there will be forma-
tion of pink zone around the colonies that help in identification of asparaginase
production. The selected colonies were isolated and maintained on nutrient agar
slant at 4 °C.
13 Isolation and Characterization of Microbial Asparaginase to Mitigate Acrylamide… 97
13.2.2 Enzyme Assay
Asparaginase enzyme assay was performed for each isolated colonies to select the
most potent strain using UV–visible spectrophotometer. During estimation ammo-
nia was produced due to the action of asparaginase enzyme on the substrate aspara-
gine. The ammonia produced was proportional to the amount of the enzyme present
and it was detected using Nessler’s reagent. The reaction mixture contained 0.5 ml
of 0.08 mM l-asparagine, 1.0 ml of 0.1 M acetate buffer (pH 5.0) and 0.5 ml of
enzyme solution. With the addition of 0.5 ml of 15 % trichloroacetic acid solution
after 30 min of incubation, the reaction was terminated. One unit (IU) of LA is the
amount of enzyme that produces 1 μM of NH3 per minute under the optimum assay
conditions.
13.2.3 Optimization of Process Parameters
13.2.3.1 Optimization of pH
The selected strain of the organism was grown at different pH i.e., 4, 5, 6, 7 and 8 at
30 °C for 24 h. After completion of 24 h the fermented broth was centrifuged and
the clear supernatant was taken for the enzymatic assay.
13.2.3.2 Optimization of Temperature

The selected strain of the organism was grown at different temperatures, i.e., 25, 30,
35 and 40 °C for 24 h. After completion of 24 h, the fermented broth was centri-
fuged, and the clear supernatant was taken for the enzymatic assay.
13.2.4 Morphological Study of the Isolated Strain
The morphological, cultural and biochemical characteristics of the isolated strain

were studied according to Bergey’s Manual of Determinative Bacteriology [17].
13.3.1 Screening of Bacterial Strains for LA Activity
Fifteen different bacterial strains were isolated from soil samples. After screening of
LA production capability one particular strain (M6) was selected as it was the most
potent strain.
98 M. Ray et al.
Fig. 13.1 Optimization of 50
Enzyme Concentration
pH for maximum enzyme
production by the isolated
40
strain 30
(IU/ml)
20
10
0
2 7 12
pH
Fig. 13.2 Optimization of 60
Enzyme Concentration
temperature for maximum 50
enzyme production by the
40
isolated strain
(IU/ml)
30
20
10
0
20 30 40 50
Temperature ( °C)
13.3.2 Morphological Study of the Isolated Strain
The isolate was aerobic, gram positive and rod shaped and showed sub-terminal
endospore on spore staining. Positive growth of the strain was observed in the
medium that contained glucose, arabinose, galactose, lactose and sucrose. The
strain had the capability to hydrolyze starch, casein and can use citrate but could not
produce hydrogen sulfide (H2S). Positive urease and catalase activity had been
shown by the isolated strain. According to the morphological and biochemical char-
acteristics of the strain M6 was identified as Bacillus sp. M6.
13.3.3 Optimization of Process Parameters
The production of LA by the isolated Bacillus sp. M6 was found maximum at pH

7.0. The asparaginase activity at this optimum pH was 40 IU/ml. To study the
effect of temperature on the enzyme production by the isolated Bacillus sp. M6
pH was fixed to 7.0 but the incubation temperature was varied. It was found that
at 35 °C maximum enzyme production was obtained and it was 47 IU/ml
(Figs. 13.1 and 13.2).
13 Isolation and Characterization of Microbial Asparaginase to Mitigate Acrylamide… 99
13.4 Conclusion
In the present study 15 organisms were isolated from soil sample capable of produc-
ing L-asparaginase enzyme. After screening the strain marked as M6 was selected
as the most potent one. The morphological and biochemical test of the strain was
performed and it was identified as Bacillus sp. M6. The strain was capable to pro-
duce 47 IU/ml of the desired enzyme at pH 7.0 and temperature 35 °C. The other
environmental conditions for maximum enzyme production by the isolated strain
can be further optimized. The enzyme can be produced in large quantity, purified
and applied to raw materials before application of high-temperature treatment like
baking or deep-oil frying to reduce its asparagine content. As a result lesser quantity
of acrylamide will be formed.
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Small Colony Variant Selection, Biofilm
Induction, and Interspecies Interactions 14
of Ocular Clinical Pseudomonas
aeruginosa
Sadhana Sagar and Shilpa Deshpande Kaistha
Abstract
Pseudomonas aeruginosa, an opportunistic pathogen, is frequently isolated from
ocular infections. In this study, antibiotic resistance and biofilm formation ability
of ocular isolate, Ps. aeruginosa AV2 is characterized. Ps. aeruginosa AV2 is
shown to be a potent multiple drug resistant, biofilm former using the adherence,
Congo red binding, and acyl homoserine lactone production assays. The extra-
cellular products of the isolate show strong antimicrobial activity against inter-
species ocular isolates Staphylococcus aureus, Micrococcus luteus, Bacillus
cereus, and Enterobacter aerogenes. Antimicrobial activity was also found
against reference strains S. aureus ATCC 25923 and S. aureus ATCC 33529. The
active compounds in the cell-free extract (CFE) were characterized to be stable
at 55 °C and pH 7.0 and non-proteinaceous in nature. ATR-FTIR spectroscopy
peaks show the presence of amines, phenolic, and lactone groups. In the presence
of the CFE, S. aureus, E. coli, and Ps. aeruginosa exhibit approximately twofold
induction in biofilm type of growth. While the CFE has no effect on interspecies
mature biofilms, it showed induction of intraspecies mature biofilm. The results
suggest that although Ps. aeruginosa secondary metabolites may be character-
ized as possessing antimicrobial activity, they may in fact increase intra- and
interspecies cells to adhere to substrates and form biofilms. Inter-/intraspecies
S. Sagar
Agriculture Microbiology, Rani Lakshmi Bai Central Agricultural University,
Jhansi, Uttar Pradesh, India
S. D. Kaistha (*)
Department of Microbiology, Institute of Biosciences & Biotechnology, Chhatrapati Shahu Ji
Maharaj University, Kanpur, Uttar Pradesh, India

https://doi.org/10.1007/978-981-13-6321-4_14
102 S. Sagar and S. D. Kaistha
microbial infections are common in the eye. Hence, polymicrobial interactions at

infectious site may lead to development of microbial populations such as bio-
films that are refractory to antimicrobial treatments.
Keywords
Biofilm · Antimicrobial activity · Pseudomonas aeruginosa cell-free extract
14.1 Introduction
Ps. aeruginosa is a gram-negative ubiquitous opportunistic pathogen isolated typi-

cally from the eye, skin, wound, burn, catheter, and cystic fibrosis infections. The
inhibitory activity of Ps. aeruginosa through its extracellular products on co-species
has been the subject of several reports [1–3]. A plethora of primary and secondary
metabolites, antibiotics, signaling molecules, nutrients, and enzymes regulate the
interactions and growth of surrounding communities. Interactions between intra- and
interspecies organisms occur with the help of diffusible products which are produced
at different stages of microbial growth. The responses to such signals can be varied
including synergism and antagonism. Ps. aeruginosa is a producer of several antimi-
crobial compounds such as hydrogen cyanide, pyocyanin, phenazines, quinolones,
etc. [3, 4]. There are, however, also reports of the persistence of multispecies with
Pseudomonas. Hence, the isolation, for example, of S. aureus, a gram-positive cocci,
frequently co-isolated with Ps. aeruginosa despite its sensitivity to Pseudomonas
exotoxins [5, 6]. Interspecies interactions of Ps. aeruginosa have severe clinical
implications as microbial consortia are being frequently isolated from infections
which were considered to be primarily Ps. aeruginosa infections [7]. Clinical infec-
tions due to microbial consortia are likely to be develop increasing resistance to high
generation antimicrobial agents by horizontal gene transfer [8].
Microorganisms may exist as free living or in the form of communities called
biofilms [8, 9]. Biofilm form of growth is a survival strategy for several microorgan-
isms under conditions of stress [8]. Antimicrobial settings often induce transcrip-
tional changes in growth forms leading to the development of biofilm communities
which insulate the encapsulated organisms. In many infectious and ecological set-
tings, microbial consortia compete with each other in an exopolymeric matrix of the
biofilms [8, 9]. Within biofilms, cell-cell communication occurs using diffusible
chemical signaling messengers or quorum sensing signals that can affect gene
expression profiles as well as metabolic changes within the biofilm [9]. Biofilm
formation as a mechanism responsible for the persistence of interspecies with Ps.
aeruginosa within clinical settings is therefore explored in this study.
Interspecies interactions have been studied previously with Ps. aeruginosa
NCNC6750 and Ps. aeruginosa PA01 [3]. There are few reports of the multispecies
interactions with freshly isolated clinical Pseudomonas [2]. The nature of such
interactions within clinical isolates remains poorly reported. In the present study,
the interspecies interactions with clinical isolate from ocular infection have been
14 Small Colony Variant Selection, Biofilm Induction, and Interspecies Interactions… 103
explored. A clinical isolate Ps. aeruginosa AV2 was isolated from an ocular infec-
tion and its virulence traits characterized. Further, the effect of Ps. aeruginosa AV2
and its extracellular product on microbial growth, both as planktonic and biofilms,
was studied on multiple cultures such as S. aureus and E. coli.
14.2.1 Bacteria
Ps. aeruginosa AV2, B. cereus, M.luteus, E.aerogenes, E. coli, and S. aureus are
clinical isolates from ocular infections obtained from Department of Microbiology,
CSJM University, Kanpur. The cultures were maintained on Tryptone Soya Media
at 37 °C. Ps. aeruginosa PA01 and Chromobacterium violaceum ATCC 12472 are a
kind gift from Prof I. Ahmad, AMU, Aligarh, and Prof R McLean, Texas State
University, TX, USA. Ps. aeruginosa ATCC 15442, S. aureus ATCC 25893 (MSSA),
and S. aureus ATCC 33592 (MRSA) were purchased from American Type Culture
Collection (Hi Media, India) and all the cultures were maintained on Tryptone
Soya (TS) Media at 37 °C.
14.2.2 Characterization of Cell-Free Extract
Log phase culture of Ps. aeruginosa AV2 was extracted at different time points post
infection, centrifuged, filtered, and supernatant used as cell-free extract (CFE). The
composition of the CFE was determined by measurement of exopolysaccharide and
protein content using phenol-sulfuric acid method [10] and Folin-Lowry method,
respectively [10]. Varying treatments of pH (3.0, 5.0, 7.0, and 9.0) and temperature
(37 °C, 55 °C, 70 °C, and 95 °C for 30 min) and proteinase K treatment (0.1 mg/ml
for 20 min) of the cell-free extract were adjusted prior to setting up antimicrobial
zone of inhibition assay. Spectra for ATR-FTIR spectroscopy were obtained with a
Bruker Vertex 70 ATR-FTIR spectrometer. For each spectrum, eight interferograms
were collected at 4 cm−1 resolution and 4000–600 cm−1 wave-number range.
14.2.3 C. violaceum Bioassay
The bioassay was performed as per the modified protocol of McLean et al. [11]. Log
phase culture of C. violaceum ATCC12472 was spread on TS agar. 10 μl of Ps.
aeruginosa PA01 and Ps. aeruginosa AV2 was added to the bored wells in the agar.
Zone of inhibition of growth of C. violaceum and zone of no pigmentation were
recorded.
14.2.4 Antimicrobial activity
Antimicrobial activity was determined using the colony biofilm, spot culture method
as well as agar well diffusion in a zone of inhibition assay. Overnight grown test
cultures were spread on tryptone soya peptone agar and dried for 10 min.
Subsequently log phase culture of Ps. aeruginosa AV2 was spot inoculated on the
agar, air-dried and incubated for 24 h at 37 °C. Cell-free extract was used in agar
well diffusion assay for determination of antimicrobial activity against different
cultures. Zone of inhibition was measured in mm.
Antimicrobial activity was measured in arbitrary units (AU). It was defined as
the maximum dilution which produced a clearly visible antibacterial zone. The
reciprocal of the dilution gave the titer of antibacterial activity in AU per ml. It is
defined as reciprocal of the highest dilution resulting in a clear zone of growth
inhibition.
14.2.5 Biofilm Formation and Eradication Assay
Static biofilm formation assay was used as per modified protocol O’Toole and
Kotler [12]. Isolates were grown in 1.5 ml polyethylene tubes containing 500 μl of
TS broth (TSB) and 96-well microtiter plates containing 200 μl of TSB for different
time point at 37 °C for 24 h. For biofilm eradication assays, 24 h mature biofilm was
washed prior to treatment with the cell-free extract (1:1). After 24 h of incubation,
biofilms were washed with sterile saline. Cultures were removed and planktonic
growth measured spectrophotometrically at A630. Static surface with biofilms was
washed with sterile saline. Adherent bacteria were stained with 1%w/v crystal vio-
let for 20 min. Stained adherent bacteria were washed and detached using 200 μl of
dimethyl sulfoxide and solubilized biofilms measured using microtiter plate reader
(Thermo Scientific, USA) at A630. Results are mean of three experiments done in
triplicates.
14.2.6 Motility
Motility was determined by swimming plate, swarming plate, and twitch plate assay
as described previously [13].
14.2.7 Exopolysaccharide Determination
Exopolysaccharide measurement for biofilms was performed by phenol-sulfuric

method [14] as well as Congo red binding assay [15].
14.2.8 Hydrophobicity
Microbial hydrophobicity assay was performed as described by Rosenberg et al. [16].
14.2.9 Acyl Homoserine Lactone
The production of acyl homoserine lactone was checked as per modified protocol [17].
14.2.10 Antibiotic Sensitivity
Antibiotic susceptibility tests for Ps. aeruginosa AV2 and Ps. aeruginosa PA01
isolates were performed by disk diffusion method (Hi Media) on Mueller Hinton
Agar (HiMedia, India) as per CLSI nomenclature [18].The antibiotics tested include
ampicillin (Ap, 10 μg), cephalothin (Ch, 30 μg), colistin sulfate (Cl, 10 μg), genta-
micin (G, 10 μg), streptomycin (S, 10 μg), and tetracycline (T, 30 μg).
14.2.11 SCV induction
Double agar overlay method was used wherein upper layer of TSA containing 160
AU/ml of CFE was prepared. S. aureus ATCC 25983 was spread on the plate and
growth measured up to 48 h of incubation at 37 °C for the emergence of SCV. In
additionally, 24 h S. aureus mature biofilm was treated with 160 AU/ml of CFE for
24 h with incubation at 37 °C. Titers of S. aureus were determined by dilution plat-
ing of biofilm cells on TSA in the absence or presence of CFE as per double agar
overlay method assay. Frequency of SCV generation was calculated by determining
the ratio of SCV to normal phenotype cells.
Statistics Statistical analysis was done using student’s t test. One way ANOVA
was used for comparison of means. All experiments were repeated at least thrice in
triplicates. p ≤ 0.05 was considered as biologically significant.
14.3 Results
14.3.1 Bacterial Characterization
Ps. aeruginosa AV2 was isolated from intraocular lens-associated microbial kerati-
tis infection and characterized using standard microbiological and biochemical tests
as per Bergey’s determinative bacteriology [19]. The isolate was also characterized
for its ability to form biofilm in static biofilm assay [12] and antibiotic susceptibility
using disk diffusion method as per CLSI nomenclature [18]. Table 14.1 shows bio-
film characterization of the isolate on the basis of static biofilm assay; swarming,
twitching, and swimming motility; production of acyl homoserine lactone; and EPS
production. Ps. aeruginosa PA01 was used as standard reference strain for biofilm
formation. Ps. aeruginosa AV2 is a moderate biofilm former and shows resistance
to ampicillin, cephalothin, colistin sulfate, co-trimoxazole, gentamicin, streptomy-
cin, and tetracycline. Isolate shows strong swimming, swarming, and twitching
motility. Exopolysaccharide production and acyl homoserine lactone production
were comparable to standard reference strain for biofilm studies, Ps. aeruginosa
PA01.
14.3.2 Antimicrobial Activity and CFE Characterization
The antimicrobial activity of the isolate was serendipitously discovered during a

motility assay; large zones of inhibition were seen to a contaminating clinical iso-
late B. cereus. Cell-free extracts (CFE) were obtained at different times post incuba-
tion (6–72 h) to determine the kinetics of the production of the antimicrobial
metabolite using zone of inhibition assay. Figure 14.1 shows the kinetics for growth
of Ps. aeruginosa AV2 and its antimicrobial metabolite production against B.
cereus. The antimicrobial product was a secondary metabolite which shows maxi-
mum activity at 48 hrs post inoculation. Further, spot culture assay was carried
out with Ps. aeruginosa AV2 against B. cereus, M.luteus, S. aureus ATCC 25983
(MSSA), S. aureus ATCC 33529 (MRSA), E. aerogenes, E. coli, and Ps. aerugi-
nosa ATCC 15442 (Fig. 14.2). 48 h cell-free extract was obtained and used in agar
Table 14.1 Biofilm characteristics and antibiogram of Ps. aeruginosa AV2 and Ps. aeruginosa
PA01
Characteristics Ps. aeruginosa AV2 Ps. aeruginosa PA01
Antibiograma ApRChRClRCoROfR GRSRTR ApRChRClRCoROfR GRSRTR
Biofilm formation assayb
Polyethylene 1.305 ± 0.54 1.576 ± 0.275
Polystyrene 0.296 ± 0.091 0.188 ± 0.011
Hydrophobicityc 11.94 ± 5.25% 39.4 ± 5.2%
Exopolysaccharide productiond(A540) 0.5 ± 0.177 0.37 ± 0.2
Congo red binding assaye(A630) 0.392 ± 0.005 0.54 ± 0.8
Acyl homoserine lactonef 0.412 ± 0.055 0.491 ± 0.085
production
Motility (Zone in mm)
Swimming 31.0 ± 12 34.33 ± 10.27
Swarming 25.25 ± 0.6 47 ± 6.68
Twitching 12.25 ± 1.2 25.66 ± 14.05
a
Ap, ampicillin (10 μg); Ch, cephalothin (10 μg); Cl, colistin sulfate (10 μg); Co, co-trimoxazole
(25 μg); G, gentamicin (10 μg); S, streptomycin (10 μg); T, tetracycline (30 μg), bBiofilm forma-
tion assay [18], chydrophobicity assay [23], dexopolysaccharide production [4], eCongo red bind-
ing assay [9], facylhomoserine lactone assay [25],e motility[22] were performed by using equal
cells of OD = 0.5 from both the isolates.
Results are mean of triplicates and represent one of three experiments
14 1
Inhibition (mm)
Zone of inhibition (mm)

0.9
12
Growth curve 0.8
Growth OD 630nm
10 0.7
8 0.6
0.5
6 0.4
4 0.3
0.2
2
0.1
0 0
0 6 12 18 24 48 72
Time (hrs)
Fig. 14.1 Growth kinetics of Ps. aeruginosa AV2 and CFE antimicrobial activity, shown as zone
of inhibition in mm (mean ±SD), against Bacillus cereus isolate at different time points
Fig. 14.2 Antimicrobial

35.00 25.3
activity of Ps. aeruginosa 30.00 22.2
23
AV2 in spot culture assay 25.00 20
using cells and zone of 11.6
20.00 12.1 15
inhibition assay using
15.00 10
cell-free extract (CFE). 7 7
10.00
Data shown as zone of 3
5.00 0 0 0
inhibition in mm
(mean ± SD) represent 0.00
Bacillus cereus
Micrococcus luteus
Staphylococcus
aureus (ATCC 25893)
Staphylococcus
aureus ATCC 33529
Pseudomonas
aeruginosa ATCC
15542
Escherichia coli
Enterobacter
aerogenes
means of three experiments
diffusion assay to determine the range of activity against gram-positive and gram-
negative bacteria (Fig. 14.2). Ps. aeruginosa AV2 and its CFE were found to inhibit
growth of S. aureus ATCC 25893 (MSSA), S. aureus ATCC 33592 (MRSA), B.
cereus, and E.aerogenes but had no effect on gram-negative E. coli and Ps. aerugi-
nosa ATCC 15442. Maximal antimicrobial activity of the extracellular product was
targeted at gram-positive bacteria such as S. aureus and B. cereus at 160 AU/ml.
The physicochemical characteristics of the cell-free extract were determined by
various treatments followed by checking its activity against quality control refer-
ence organism S. aureus ATCC 25893 in a zone of inhibition assay. Antimicrobial
activity of the cell-free extract was found to be acid stable from pH (5.0–7.0) but
alkali unstable (pH 9.0) and temperature stable at 55 °C for 30 min but inactivated
beyond 75 °C for 10 min. Activity was unaffected by proteinase K treatment (1 mg/
ml), suggesting primarily non-proteinaceous nature (Fig. 14.3). Exopolysaccharide

35.00
27.30 27 28
30.00
25.00 20.60
20.00 14.60
15.00 12
10.00
5.00 0.00 0 0
0.00
Fig. 14.3 Physicochemical characteristics of the Ps. aeruginosa AV2 CFE following treatments
at various temperatures (37 °C, 55 °C,70 °C, 90 °C), pH (pH 3.0, 5.0, 7.0, 9.0), and proteinase K
(0.1 mg/ml) treatment. Data shown as zone of inhibition in mm (mean ±SD) represent means of
three experiments
Fig. 14.4 Effect of Ps.

aeruginosa AV2 (PAV2)
and Ps. aeruginosa PA01
(PA01) in violaceum
bioassay using
Chromobacterium
violaceum ATCC 12472
content of the CFE was determined by the phenol-sulfuric method [14] and found
to be 1.5 mg/ml, whereas protein content was found to be 6.4 mg/ml [10]. The effect
of the CFE on quorum sensing was tested using C. violaceum ATCC 12472 in agar
well diffusion bioassay. However, CFE was found to inhibit growth of C. violaceum
ATCC 12472 and not affect pigment formation (Fig. 14.4). Ps. aeruginosa PA01
was used as positive control as a producer of C-4 and 3 oxo-C12 AHL [20]. Hence,
activity is primarily antimicrobial and does not affect production of dodecanoyl-
homoserine lactone, quorum sensing molecule for which violaceum production is
bioassayed.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy
was used to determine the molecular structure of the cell-free extract. Figure 14.5
shows the characteristic absorption spectra of the peaks obtained for the CFE. The
three main peaks at 1737 cm−1, 1366 cm−1, and 1217 cm−1 correspond to lactones or
primary amines, carbohydrate groups inclusive of OH/CH groups, and C–O stretch-
ing and O–H deformation of carboxyl, phenols, and aromatic ethers [20].
100
99.8
Transmittance [%]
99.6
99.2
99.0
98.8
3853.41
3770.20
3018.14
2357.82
1737.68
1635.41
1217.85
1134.06
1059.52
1445.01
1366.86
686.46
3500 3000 2500 2000 1500 1000
Wavelength (cm-1)
Peaks (cm-1) Characteristics

3550-3853 (-OH) hydroxyl stretch intermolecular stretch for water
2357 NH or CO stretching vibrations
1737 Primary amines and associated amides, lactones
1445 CH2 bending vibration involving methyl, methylene and methoxy groups
1217 C-O stretching and O-H deformation of carboxyls, phenols, and aromatic ethers
CO Amide III
1366 CH and OH deformational vibrations (carbohydrates)
983-1060 C=CH2
686-771 -CH=CH2 (trans)
Fig. 14.5 ATR-FTIR spectroscopy of Ps. aeruginosa AV2 CFE. Table lineate the characteristics
of the various peaks found in the CFE spectra
14.3.3 Effect on Biofilm Formation and Biofilm Eradication
The 48 h CFE was used to study its effect on biofilm formation of S.aureus (MSSA
and MRSA), Ps. aeruginosa, and E. coli strains. Figure 14.4 shows that while no
effect was observed on planktonic growth of both the Staphylococcal isolates, bio-
film formation was induced at biologically significant levels. In Ps. aeruginosa,
biofilm induction was found to be over 50% with a decrease in planktonic cells,
while in E.coli cells significant induction was observed in growth of planktonic as
well as biofilm growth (Fig. 14.6).
The effect of the supernatant was also studied on preformed biofilms. No effect
was observed in the planktonic and biofilm forming cells for the Staphylococcal
cells. E. coli-associated planktonic cells were mitigated with no effect on biofilm
formation (Fig. 14.7). Presence of the CFE induced biofilm formation in preformed
Ps. aeruginosa, biofilm likely due to presence of quorum sensing lactones. Acyl
homoserine lactone assay for the CFE extract suggests that the biological active
component responsible for biofilm induction is likely to be homoserine lactone
(Table 14.1). Although the CFE showed antimicrobial properties in agar diffusion
assay as well as mitigation of planktonic cells in liquid culture, it was shown to
induce biofilm formation particularly for all gram-positive and gram-negative iso-
lates tested with no effect on preformed biofilm cells.
Control -Planktonic CFE-Planktonic

Control- Biofilm CFE-biofilm
1.2
Biofilm formation (A 630) 1
0.8
0.6
0.4
0.2
0
S. aureus E. coli Ps. aeruginosa S. aureus
(MSSA) (MRSA)
Fig. 14.6 Effect of Ps. aeruginosa AV2 CFE on planktonic growth and biofilm in a biofilm forma-
tion assay
1.2
Control-Planktonic CFE- Planktonic
1 Control- Biofilm CFE- Biofilm

Biofilm eradication (A 630nm)
0.8
0.6
0.4
0.2
0
S. aureus E. coli Ps. aeruginosa S. aureus
(MSSA) MRSA
Fig. 14.7 Effect of Ps. aeruginosa AV2 CFE on 24hr mature biofilm of different cultures
14.3.4 Selection of S. aureus single colony variants (SCV)
In order to determine the cause of enhanced biofilm following treatment with CFE,
Ps. aeruginosa AV2 CFE were used in SCV assay for S. aureus ATCC 25983 as
described in material and method. Interestingly, small colony variants (SCV) for S.
aureus appeared following 48 h of incubation (Fig. 14.8). When the SCV were sub-
sequently plated on fresh TSA, they regained their morphological size suggesting
reversible phenotype switch. The S. aureus SCVs were characterized by small col-
ony size, reduced growth rate, and pigmentation. S. aureus biofilm cells treated with
CFE for 24 h were plated on TSA in the presence and absence of CFE.
Fig. 14.8 Induction of S.

aureus small colony
variants by Ps. aeruginosa
AV2 CFE
Figure 14.8 shows the SCV in the presence of CFE in comparison to control
untreated cells. The frequency of the SCV generation in biofilms was found to be
1.26 × 10−4. Hence, CFE can select S. aureus SCV in biofilm mode as well double
agar plate assay.
14.4 Discussion
In this study we isolated a biofilm forming clinical isolate of Ps. aeruginosa AV2
and compared the effect of its extracellular supernatant on planktonic and biofilm
modes of growth of co-cultures. Antimicrobial activity at 48 h post-logarithmic
growth was detected against predominantly gram-positive clinical ocular isolates
such as S. aureus, M. luteus, B. cereus, and E. aerogenes but not to E. coli and Ps.
aeruginosa ATCC 15542. The cell-free extract responsible for the antimicrobial
activity was found to be non-proteinaceous and heat stable up to 55 °C. ATR-FTIR
spectroscopy analysis revealed strong peak at 1737 cm−1which correlate to structure
containing lactones group [20].
Multispecies biofilms comprising of Ps. aeruginosa and S. aureus or E. coli have
been reported from clinical samples [4–7, 10, 21]. Ps. aeruginosa secretes a number
of extracellular products such as pyocyanin, hydrogen cyanide, or quinolone N
oxides that can act against normal microflora as well as host cells [4]. Toxicity
against S. aureus by Ps. aeruginosa has been reported to target specifically the elec-
tron transport chain [1, 22]. Whereas in a similar co-culture experiment Ps. aerugi-
nosa conditioned supernatant gave rise to synergistic effect on E coli growth [23].
In this study, we report that cell-free supernatant of Ps. aeruginosa is capable of
inhibiting growth of co-cultures in zone of inhibition assay but inducing biofilm
formation without affecting mature biofilm. More than 50 quinolone products from
Ps. aeruginosa are produced having significant antibiotic activity against gram-
positive organisms [6, 23]. Las A protease from Pseudomonas also shows notable
antistaphylococcal activity [6]. Utilization of Staphylococcus as source of iron has
also been reported in isolates from cystic fibrosis [24].
Stress environments can induce cells to form biofilms [9]. Low concentrations of
antibiotics can induce biofilm formation in many pathogenic settings [25, 26]. The
mechanisms for such activity are not very well understood. Ps. aeruginosa AV2
supernatant which showed antimicrobial activity in a zone of inhibition assay showed
significant induced biofilm formation in all isolates studied. Hence, secondary

metabolite such as antibiotic may work to trigger gene regulation inducing biofilm
formation, while quorum sensing lactones may induce formation of gram-negative
biofilms. The antimicrobial activity produced by the Pseudomonas herein showed no
effect or dispersal of mature biofilms. Extracellular polysaccharides produced by the
pel and psl system were found to disrupt S. epidermis biofilms independent of its
bactericidal activity evidenced with mutant studies [3]. Yet, presence of
Staphylococcus in infectious settings suggests that subpopulation of Staphylococcus
can persist and coexist. In the present study, SCV developed within the zone of inhi-
bition following exposure to Ps. aeruginosa AV2 CFE or secreted products from
colony biofilm. Small colony variants as a survival strategy by S. aureus in the pres-
ence on Ps. aeruginosa supernatant has been reported recently [21].
Once a biofilm is formed, it is extremely difficult to eradicate. The effectiveness
of Ps. aeruginosa exotoxins on neighboring species in conditions of biofilms may
hence be far more limiting compared to when it is in free living conditions. The role
of quorum sensing-dependent extracellular products in competing with other spe-
cies is likely to differ compared to metabolites produced as planktonic cells and
require further investigations. Hence the extracellular products of Ps. aeruginosa
may have differential effect on cultures depending on their growth status [2].
In light of differential behavior of antimicrobial compounds in the case of plank-
tonic as well as biofilm forming cells, it is imperative to not depend only on zone of
inhibition assay or liquid assays for antimicrobial activity but also study effects of
the compounds on biofilm forming cells. Ps. aeruginosa and S. aureus multispecies
biofilms have been identified in a number of infectious diseases such as cystic fibro-
sis, otitis media, etc. The role of Ps. aeruginosa extracellular products in enhancing
the biofilm formation in multispecies biofilms may be a responsible for the refrac-
tory nature of such microbial growths.
Acknowledgment Financial support from Department of Science and Technology and

Department of Atomic Energy, Government of India, is gratefully acknowledged. We are grateful
for ATR-FTIR facility used at Indian Institute of Technology, Kanpur.
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lular products inhibit staphylococcal growth, and disrupt established biofilms produced by
Staphylococcus epidermidis. Microbiology 7:2148–2156
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BW, Miller SI (2006) Selection for Staphylococcus aureus small-colony variants due to growth
in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 103:19890–19895
6. Kessler E, Safrin M, Olson JC, Ohman DE (1993) Secreted LasA of Pseudomonas aeruginosa
is a staphylolytic protease. J Biol Chem 10:7503–7508
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EL, Lynch SV, Bohannan BJ, Green JL, Maurer BA, Kolter R (2010) Relationship between
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Pseudomonas aeruginosa. Environ Microbiol 12(5):1293–1303
8. Anderson GG, O’Toole GA (2008) Innate and induced resistance mechanisms of bacterial
biofilms. In: Romeo T (ed) Bacterial biofilms. Springer, Heidelberg, pp 85–105
9. López D, Vlamakis H, Kolter R (2010) Biofilms. Cold Spring Harb Perspect Biol 12(7):398
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determination of sugars and related substances. Nature 28:167–174
11. McLean RJC, Pierson LS III, Fuqua C (2004) A simple screening protocol for the identifica-
tion of quorum signal antagonists. J Microbiol Meth 58(3):351–360
12. O’Toole GA, Kotler R (1998) Initiation of biofilm formation in Pseudomonas fluorescen-
sWCS365 proceed via multiple, convergent signaling pathways: a genetic analysis. Mol
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13. Rashid MH, Kornberg A (2000) Inorganic polyphosphate is needed for swimming, swarming
and twitching motilities of Pseudomonas aeruginosa. PNAS 97(9):4885–4890
14. Kay WW, Phipps BM, Ishiguro EE, Trust TJ (1985) Porphyrin binding by the surface array
virulence protein of Aeromonassalmonicida. J Bacteriol 164:1332–1336
15. Abate G, Mshana RN, Miorner H (1998) Evaluation of a colorimetric assay based on
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for rapid detection of
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16. Rosenberg M (1984a) Bacterial adherence to hydrocarbon: a useful technique for studying cell
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Comparative Analysis of Phytochemicals
of Healthy and Symptomatic 15
Clerodendrum inerme
Sonal, Sharmita Gupta, Yati Prabha, and S. K. Soni
Abstract
Clerodendrum, a flowering plant, belongs to family Lamiaceae. The aim of pres-
ent study was to analyse phytochemicals from the leaves of healthy and viral
symptomatic C. inerme plants. Leaves having chlorotic spots, yellow-green
patches, leaves curling, etc. were due to virus infection as seen in electron
microscopy. Extraction was done using solvents chloroform, acetone, methanol,
ethanol and water. For analysis of phytochemicals, each extract was subjected to
qualitative test for identification of various constituents like alkaloids, steroids,
flavonoids, coumarins, quinones, flavanones, terpenoids, tannins and phenolic
compounds and proteins. After analysing all constituents, it has been confirmed
that symptomatic leaves of C. inerme have less amount of phytochemicals as
compared to healthy leaves of C. inerme. The major purpose of this study was to
make a comparative account of the phytochemical constituents of healthy and
symptomatic Clerodendrum inerme.
Keywords
C. inerme · Healthy · Symptomatic · Phytochemicals
15.1 Introduction
The medicinal plants are beneficial for healing and curing of diseases of human,
because of phytochemical constituents present in them. Plants are found to contain
chemical constituents, which are used as natural drugs to treat common fungal and
bacterial infections. Medicinal plants are frequently used in different combinations
Sonal · S. Gupta (*) · Y. Prabha · S. K. Soni

Botany Department, D.E.I., Agra, Uttar Pradesh, India

https://doi.org/10.1007/978-981-13-6321-4_15
116 Sonal et al.
of medicines in India because of least side effect and cost which makes it suitable
for therapeutic applications in tribal medicine. About 25 percent of all new medi-
cines are indirectly or directly derived from higher monocot and dicot plants [5].
Paste and leaf extract are used in treatment of malaria, inflammation, itching skin
diseases and infected wounds. The genus Clerodendrum entails more than 450 spe-
cies of tropical and sub-tropical zones of the world, which involves herbs, shrubs
and trees. C. inerme is used for the purpose of landscaping or hedge plants in roads,
streets, parks, etc. It is an evergreen shrub of 1–1.8 m tall having smooth and woody
stems. Leaves are green, smooth and simple and have venation pinnate, entire mar-
gins and opposite leaves with shiny dorsal surface. Plants have attractive flowers in
white colour which are arranged in clusters [2]. Stamens are four, usually in pairs of
two of variable length and thrusting beyond the mouth of red to yellow petals [1]. In
the present study, viral symptomatic and healthy leaves of C. inerme were screened
for the presence of phytochemicals. Phytochemical constituents were extracted
using various solvents, like chloroform, ethanol, methanol, acetone and
water. Extracts were used for identification of various constituents like proteins,
alkaloids, flavonoids, steroids, coumarins, flavanones, quinones, terpenoids and tan-
nins and phenolic compounds.
15.2.1 Collection of Plant Material
Healthy and symptomatic leaves of C. inerme were collected from hedge grown
outside of the botany department, faculty of science, DEI, Dayalbagh, Agra (UP)
(Figs. 15.1 and 15.2). Healthy and symptomatic leaves of C. inerme were cleaned
with tap water and dried in shade. After 7 days, leaves were grounded in
Fig. 15.1 Image of C.

inerme plant showing
healthy leaves
15 Comparative Analysis of Phytochemicals of Healthy and Symptomatic… 117
Fig. 15.2 Image of C.

inerme plants showing
chlorotic spots, leaf
curling, etc
mortar-pestle. Grounded leaf material of healthy and symptomatic plants was stored
in airtight containers, separately.
15.2.2 Extraction of Phytochemicals
To 100 ml conical flasks, each containing 50 ml of solvents, namely, chloroform,

ethanol, methanol, acetone and water, 1 gm of dried plant material was added. These
flasks were stored at room temperature for 72 h. Prepared extracts were preserved
in cold at 4 °C till further application.
15.2.3 Phytochemical Evaluation
Extracts obtained from healthy and symptomatic C. inerme plants were assessed for
presence of various constituents like alkaloids, steroids, terpenoids, flavonoids, phe-
nol and tannins, quinones, flavanones, coumarins, etc.
15.2.3.1 Test for Proteins

To 1 ml of plant extract, 2–3 drops of ninhydrin reagent were appended. Purple
colour appeared which indicated the presence of proteins [4].
15.2.3.2 Test for Alkaloids

Two ml of extract was added in a test tube along with 5–6 drops of Wagner’s reagent.
Formation of reddish-brown precipitate confirmed the presence of alkaloids [4].
15.2.3.3 Test for Steroids

One ml of crude extract with 2 ml of chloroform was taken. To this 2 ml of concen-
trated sulphuric acid and acetic acid was added. The presence of steroids was con-
firmed, when greenish colour developed [6].
118 Sonal et al.
15.2.3.4 Test for Terpenoids

Two ml of extract was taken in a test tube. One ml of chloroform and sulphuric acid
each was added to it. Appearance of greyish colour ratified the presence of terpe-
noids [6].
15.2.3.5 Test for Flavonoids

Two percent of sodium hydroxide was added to 2 ml of plant extract. Visualization
of yellow colour which later becomes colourless on addition of 5–6 drops of diluted
acid showed the presence of flavonoids [6].
15.2.3.6 Test for Phenol and Tannins

Two percent of ferric chloride was added to 2 ml of plant extract. Development of
blue green or black colour confirmed the presence of tannins and phenols [6].
15.2.3.7 Test for Quinones

Take 1 ml of extract in a test tube; 1 ml of concentrated sulphuric acid was added.
Red colour indicated the presence of quinones [3].
15.2.3.8 Test for Flavanones

To the substance, concentrated sulphuric acid was added; orange to crimson red
colour confirms the presence of flavanones [3].
15.2.3.9 Test for Coumarins

To 1 ml of extract, 1 ml of ten percent sodium hydroxide was added. Visualization
of yellow colour ratified the presence of coumarins [3].
15.3 Results
Results of phytochemical analysis are summarised in Tables 15.1 and 15.2.
Table 15.1 Preliminary phytochemical analysis of healthy and symptomatic C. inerme leaf
extracts in solvents (aqueous, acetone and chloroform)
Water Acetone Chloroform
Sr. no. Phytoconstituents Healthy Symptomatic Healthy Symptomatic Healthy Symptomatic
1. Proteins +++ ++ +++ + +++ –
2. Steroids – – +++ – +++ +
3. Terpenoids – – – – +++ +
4. Flavonoids ++ + ++ ++ ++ +
5. Phenol and – – – – +++ +++
tannins
6. Quinones ++ + + + +++ ++
7. Flavanones ++ ++ ++ ++ +++ +
8. Coumarins – – – – – –
9. Alkaloids +++ ++ – – +++ –
15 Comparative Analysis of Phytochemicals of Healthy and Symptomatic… 119
Table 15.2 Phytochemical analysis of healthy and symptomatic C. inerme leaf extracts in sol-
vents (ethanol and methanol)
Ethanol Methanol
Sr. no. Phytoconstituents Healthy Symptomatic Healthy Symptomatic
1. Proteins – – – –
2. Steroids + + + –
3. Terpenoids + – – –
4. Flavonoids +++ + ++ +
5. Phenol and tannins + + – –
6. Quinones + – – –
7. Flavanones – – ++ +
8. Coumarins – – – –
9. Alkaloids + + ++ +
+ indicates average colour presence, ++ indicates good colour, +++ indicates very good colour
Visual colour observations were made during phytochemical analysis.
15.4 Conclusion
The results revealed the presence of medicinally important constituents in the

extract of healthy leaves of C. inerme. Chloroform, water and acetone were found
most suitable for the extraction of phytoconstitutents from healthy leaves of C.
inerme, whereas extracts from symptomatic leaves were showing least results.
Studies show the presence of phytochemicals, which are important physiologically
as well as medicinally in the treatment of different ailments. Therefore these extracts
from C. inerme could be seen as a good source for useful drugs.
References
1. Chakraborthy GS, Mazumdar A, Singh S, Verma P (2013) Clerodendrum inerme: a review.
Pharmacophore 4(6):230–232
2. Chethana GS, Harivenkatesh KR, Gopinath SM (2013) Preliminary phytochemical analysis of
Clerodendrum inerme. Int Res J of Pharm 4(5):208–209
3. Firdouse S, Parwez A (2011) Phytochemical investigation of extract of Amorphophallus cam-
panulatus tubers. Int J of Phytomed 3:32–35
4. Misra CS, Pratyush K, LipinDev MS, James J, ThaliyilVeettil AK, Thankamani VA (2011)
Comparative study on phytochemical screening and antibacterial activity of roots of Alstonia
scholaris with the roots, leaves and stem bark. Int J Res Phyto Pharm 2:77–82
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screening of crude leaf extract of Clerodendrum Philippinum Schauer. Int J Inst Pharm LSci
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3(12):10–14
Synthesis of Silver Nanoparticle
of Aqueous Extract of Allium Fistulosum 16
and Its Efficiency Against Bacterial
Contaminants from Industrial Waste
Water and Its Photocatalytic Potential
Uma Ramaswamy and Vicky Mani
Abstract
In this study eco-friendly synthesis and characterization of silver nanoparticle
using aqueous extract of whole plant Allium fistulosum Linn and its antibacterial
efficiency in waste water and photocatalytic potential were studied. Green syn-
thesis of silver nanoparticle (AgNP) was performed by treating the 5% aqueous
plant extract with 1 mM concentration of silver nitrate at 60 °C. AgNPs were
characterized by using UV-visible spectroscopy, scanning electron microscopy
and Fourier transform infrared spectroscopy. Silver surface plasmon resonance
(SPR) occurred at 432 nm for 1 mM AgNP. The silver nanoparticle size ranges
from 66 to 86 nm. Silver nanoparticle of Allium fistulosum treated with industrial
waste water with different time durations (12, 24 and 72 h). In 72 h AgNP exhib-
ited antibacterial efficiency against pathogenic bacteria Proteus vulgaris, Shigella
dysenteriae and Escherichia coli isolated from industrial waste water. Synthesized
silver nanoparticle has potent photocatalytic dye degradation for safranin and
methyl orange at 43.1% and 34.6%, respectively.
Keywords
Silver nanoparicle · Allium fistulosum · Dyes · Antibacterial
U. Ramaswamy (*)
Department of Biochemistry, Dwaraka Doss Goverdhan Doss Vaishnav College,
Chennai, Tamilnadu, India
V. Mani
Department of Biochemistry, University of Madras, Chennai, Tamilnadu, India

https://doi.org/10.1007/978-981-13-6321-4_16
122 U. Ramaswamy and V. Mani
16.1 Introduction
Nanomaterials are becoming a part of our daily life, but properties and behavior of
material at the nanoscale differ significantly when compared to microscale.
Nanotechnology provide the solutions to many medical, social and environment
problems. Metals such as gold, silver, copper and zinc are widely used in the syn-
thesis of nanoparticles; silver has the unique property to fight against the pathogens
and it has been used in herbal medicine Allium fistulosum L. is a member of
Amaryllidaceae family that comes under the genus Allium and it is a perennial
onion commonly called as spring onion, in Hindi as “hara pyaz,” in tamil as “thal
vangayam.” The roots and bulbs have been used for the treatment of febrile disease,
headache, abdominal pain, diarrhea, snakebite, ocular disorders and habitual abor-
tion, as well as having antifungal and antibacterial effects [4]. Recently numerous
studies have indicated that A. fistulosum has antifungal activity [3, 6]. The study
focuses on the synthesis of silver nanoparticle of aqueous extract of Allium fistulo-
sum and its efficiency against bacterial contaminants from industrial effluent and its
photocatalytic potential.
16.2.1 Collection and Authentication of Allium fistulosum
Allium fistulosum plant was collected from Avadi market, Chennai and it was
authenticated by Dr. S. Jayaraman, Director of Plant Anatomy Research Centre,
West Tambaram, Chennai (Reg. no: PARC/2017/3436).
16.2.2 Aqueous Extract of the Allium fistulosum
50 g of fresh whole plant Allium fistulosum was crushed finely and mixed in 100 ml
of deionized water mixture boiled for 8 min [1]. Cool the extract it was filtered with
Whatman number 1 filter paper and the extract was made up to 100 ml with distilled
water. The extracts were stored at 4 °C and used for silver nanosynthesis.
16.2.3 Preparation of Silver Nitrate
One millimolar (1 mM) silver nitrate solution (AgNO3) – 0.084 g of silver nitrate –
was weighed and dissolved in 500 ml of distilled water and stored in brown bottle.
Biosynthesis of Silver Nanoparticles 10 ml of A. fistulosum extract was mixed to

90 ml of prepared 1 mM AgNO3 solution with constant stirring at 60°C for 20 min.
16 Synthesis of Silver Nanoparticle of Aqueous Extract of Allium Fistulosum… 123
16.2.4 Characterization of Silver Nanoparticles
UV-Visible Spectroscopy 2 ml of the reaction mixture was taken and the optical
density was measured from a wavelength of 200–800 nm using the Shimadzu
UV-visible spectrophotometer.
16.2.5 Fourier Transform Infrared Spectroscopy (FTIR)
FTIR analysis of silver nanoparticles was carried out by potassium bromide pellet
method to identify the possible biomolecules responsible for the reduction of silver
ions using Bruker-Alpha spectrometer.
16.2.6 Scanning Electron Microscopy (SEM)
SEM analysis was carried out to determine the size and shape of the biosynthesized
nanoparticles of A. fistulosum using Hitachi gu SU 6600.
16.2.7 Photocatalytic Degradation of Dye
Photocatalytic degradation of dye was estimated by colorimetric method. Typically

10 mg of safranin and methyl orange dyes were added to 1000 mL of double-
distilled water used as stock solution. About 1 mg of biosynthesized silver nanopar-
ticles was added to 100 mL of safranin and methyl orange dye solution. The dye
solution along with silver nanoparticles was mixed in a cyclomixer for 15 min. The
solution was kept in sunlight from morning to evening. At regular intervals of time,
3 ml of the solution was withdrawn and taken optical density using UV-visible spec-
trophotometer at wavelength 450 and 520 nm for methyl orange and safranin,
respectively.
Dye degradation ( % ) = A − B / A ×100
where A is the initial concentration of dye solution and B is the concentration of dye
solution exposed to sunlight [5].
16.2.8 Antibacterial Effect of Silver Nanoparticles of A. fistulosum
To detect the presence of bacteria in industrial waste water was determined by pour
plate method [2]. 100 ml of industrial waste water (sample B) was taken in two
flasks namely, BT and BC where BC served as control for sample B and BT served
as sample B treated with 10mg of silver nanoparticles of A. fistulosum and it was
incubated for 12, 24 and 72 h. After incubation 100 μl of samples (BC and BT) were
poured in each 20 ml of nutrient agar plate kept for incubation at 37 °C for 12, 24,
and 72 h. After incubation the colonies of bacteria were analyzed. Based on the
morphology and color of the colonies in selective media (MacConkey agar and
Salmonella-Shigella agar (SS)) for specific bacterial species (Klebsiella, Escherichia
coli, Proteus, Shigella) were chosen and done pour plate method. The colonies were
counted for both control and test plate.
16.3 Results and Discussions
UV-Visible Spectrum Analysis Optimum intensity of UV-visible spectra peak or

SPR (surface plasmon resonance) band centered at 432 nm for 10 ml of the aqueous
extract of A. fistulosum treated with 1 mM silver nitrate (Fig. 16.1).
FTIR Analysis FTIR spectrum of AgNPs of A. fistulosum extract shows bend at

3318 cm−1, 1637, 1015 cm−1 corresponds to OH stretches, H-bonded alcohols and
phenol, NH bend, primary amines, N-O asymmetric stretch nitro compounds, C-O
stretch alcohols, carboxylic acids, esters and ether, respectively (Figs. 16.2 and
16.3). FTIR spectrum of AgNPs suggested that the particle was surrounded by dif-
ferent organic molecules such as alcohols, ketones, carboxylic acid and terpenoids.
SEM Analysis The AgNP formed were spherical in shape, agglomerated, and
polydispersed. The AgNP diameter varied from 66 to 86 nm (Fig. 16.4).
Photocatalytic Degradation of Dyes Absorption peaks of methyl orange and saf-

ranin dyes at 450 and 520 nm for methyl orange and safranin dyes were decreased
initially with the increase of the exposure time. This clearly indicates the photocata-
Fig. 16.1 UV-visible spectra of aqueous extract of A. fistulosum with 1 mM silver nitrate
Fig. 16.2 FTIR analysis of aqueous of Allium fistulosum
Fig. 16.3 FTIR analysis of silver nanoparticle of Allium fistulosum (1 mM)
lytic degradation of methyl orange and safranin dye. Dye degradation efficiency of
AgNP of A. fistulosum was 34.6% and 43.1% for methyl orange and safranin,
respectively (Fig. 16.5).
Effect of AgNP of A. fistulosum on Bacteria of Domestic and Industrial Waste

Water
Findings of the study revealed that in AgNP-treated industrial waste water (BT)
colonies of Shigella and Proteus were less than control plate (BC) in 24 h incuba-
tion. In 72 h there was no growth of E. coli, Shigella and Proteus in BT plate
Fig. 16.4 SEM analysis

of silver nanoparticle of
Allium fistulosum
50
Safrannin
45
Methyl orange
40
Dye degradation(%)
35
30
25
20
15
10
5
0
1 2 3 4 24 48 72
Exposure Time(hours)
Fig. 16.5 Photocatalytic degradation of methyl orange and safranin by the AgNPs of A.
fistulosum
Table 16.1 Mean values of total bacterial count of industrial waste water in (a) MacConkey and
(b) SS agar medium
Time (h) E. coli Klebsiella Time (h) Shigella Proteus
BC BT BC BT BC BT BC BT
12 h 800 650 850 700 12 h 490 300 350 200
24 h 810 150 860 600 24 h 500 150 360 150
72 h 815 NIL 879 380 72 h 500 NIL 368 NIL
compared to BC plate of both MacConkey and SS agar (Table 16.1). This result
shows that silver nanoparticle has the good ability to inhibit E. coli, Proteus and
Shigella in industrial waste water.
16.4 Conclusion
In this study a simple, eco-friendly, pollutant-free and economic biological proce-

dure has been developed to synthesize the AgNPs of Allium fistulosum. The silver
nanoparticles were characterized by UV-visible spectra, FTIR spectra and scanning
electron microscopy. Silver nanoparticles of Allium fistulosum exhibited potent pho-
tocatalytic activity against methyl orange and safranin. Silver nanoparticles of
Allium fistulosum exhibited potent antibactericidal effect on E. coli, Proteus and
Shigella on industrial waste water.
References
1. Benjamin G (2011) IPCBEE, vol 5. IACSIT Press, Singapore
2. Dosoky R, Kotb S, Farghali M (2015) Efficiency of silver nanoparticles against bacterial con-
taminants isolated from surface and ground water in Egypt. J Adv Vet Anim Res 2(2):175–184
3. Sang S, Lao A, Wang Y, Chin CK, Rosen RT, Ho CT (2002) Antifungal constituents from the
seeds of Allium Fistulosum L. J Agric Food Chem 50:6318–6321
4. Shogakukan (1985) Shanghai science and technology publisher and shougakukan, Tokyo. Dict
Chin Drugs 3:1599–1600
5. Vanaja M, Paulkumar K, Baburaja M, Rajeshkumar S, Gnanajobitha G, Malarkodi C,
Sivakavinesan M, Annadurai G (2014) Degradation of methylene blue using biologically syn-
thesized silver nanoparticles. Bioinorg Chem Appl, Article ID 742346
6. Yin MC, Tsao SM (1999) Inhibitory effect of seven Allium plants upon three Aspergillus spe-
cies. Int J Food microbial. 49:49–56
Exploration of Biocontrol and Growth-
Promoting Activity of Bacterial Strains 17
Isolated from the Sugarcane Crop
Beenu Shastri, Anil Kumar, and Rajesh Kumar
Abstract
Naturally occurring bacteria were isolated from the internal tissues of stalks as
well as from roots of sugarcane crop and from the rhizospheric soil. The highest
numbers of bacterial populations were isolated from the rhizospheric zone.
Isolated bacterial strains were subjected to antagonistic activity in vitro against
Colletotrichum falcatum fungus causing red rot disease in sugarcane crop. Most
of the isolated bacteria showed antagonistic activity against C. falcatum in vitro.
Isolated antifungal isolates were identified morphologically and biochemically.
Further, the potential strains were examined for various plant growth promoting
traits and hydrolytic enzymes production. Bacteria isolated from rhizospheric as
well as from endophytic zone of sugarcane crop showed the inhibition of red rot
pathogen as well showed the in vitro plant growth promotory traits. Thus, iso-
lates help in biocontrol of red rot as well as can be used for increment of sugar-
cane yield.
Keywords
Sugarcane · Endophytes · Rhizospheric bacteria · Colletotrichum falcatum
B. Shastri · A. Kumar · R. Kumar (*)

Rhizosphere Biology Laboratory, Department of Environmental Microbiology, School for
Environmental Sciences, Babasaheb Bhimrao Ambedkar University,
Lucknow, Uttar Pradesh, India

https://doi.org/10.1007/978-981-13-6321-4_17
130 B. Shastri et al.
17.1 Introduction
Saccharum spp. (sugarcane) is an important agro-industrial crop cultivated in vari-

ous regions (tropical and sub-tropical) of the world. Their valuable product includes
high concentrations of sucrose, or sugar, in the stem and more recently used in etha-
nol production, which is an important renewable biofuel source. Brazil is the largest
producer for sugarcane, and its area under production has expanded from 5.81 mil-
lion hectare (M ha) in 2005 to 10.4 million ha in 2014 growing at an average annual
rate of 6.79% [1]. Sugarcane is cultivated in many regions of India with total area
covering of 5.01 M ha [1]. Sugar consumption is largest in India with an annual
consumption of about 19 million MT (metric tons) [2]. Therefore, to sustain the
increasing population and maintain the productivity as well as production, this area
has become a major focus of research.
Various abiotic and biotic factors, including a wide range of fungal and bacterial
diseases, are adversely affecting the growth and performance of sugarcane in the
field. Red rot is one of the most dreadful diseases of sugarcane, caused by the fun-
gus Colletotrichum falcatum. It not only causes severe loss in yield and quality but
also reduces the quality of juice [3, 4]. It can reduce cane weight by up to 29% and
loss in sugar recovery by 31% [5].
Current control strategies for red rot disease involve the use of resistant varieties
and fungicide applications, but their efficacy is limited; therefore, novel and envi-
ronmentally sound strategies to control disease of sugarcane are needed. Biocontrol
agents provide an excellent alternative to chemical pesticides and biofertilizer.
Pseudomonas, Bacillus, Burkholderia, Agrobacterium, Streptomyces, etc. are com-
monly genera used as biocontrol agents.
17.2 Methodology
For sample collection or isolation, red rot susceptible healthy sugarcane variety
Co1148 and the rhizospheric soil were collected from the Indian Institute of
Sugarcane Research (IISR) farms, Lucknow. Endophytic bacteria and rhizospheric
bacteria were isolated from the internal tissues of roots and stalks of sugarcane
variety Co1148 and from the rhizospheric soil using the protocol of Viswanathan
et al. [6]. Isolated bacteria were then studied for dual plate assay for antagonism on
potato dextrose agar (PDA) plates against red rot fungus, i.e., Colletotrichum falca-
tum, and were incubated for 7–10 days at 28 °C. The percentage inhibition was
measured by the formula [(C−T)/C] × 100, where C is the radial colony growth of
fungal pathogen in control and T is the radial colony growth in dual plate assay [6].
Morphological (gram staining and motility) and biochemical characterization
(IMViC, catalase, oxidase, urease, H2S) of the isolated antifungal bacterial isolates
was performed as per the Bergey’s manual of Systematic Bacteriology [7].
17 Exploration of Biocontrol and Growth-Promoting Activity of Bacterial Strains… 131
17.2.1 In Vitro Evaluation of Plant Growth-Promoting Activities
Quantification of indoleacetic acid (IAA) was performed according to Gordon and

Weber [8]. Solubilization of calcium phosphate was done according to Pikovskaya
[9] on Pikovskaya agar medium. The confirmation of siderophore production was
performed according to Schwyn and Neilands [10] universal method, with chrome
azurol S (CAS) agar plate assay. Ammonia production was evaluated, after adding
Nessler’s reagent to a peptone broth containing bacterial culture after 72 h of incu-
bation, and yellowish brown color was recorded [11]. For hydrocyanic acid (HCN),
the Bakker and Schippers [12] method was followed in nutrient agar medium, sup-
plemented with 4.4 g/L of glycine by observing the color change of filter paper
placed on lid of plates.
17.2.2 Screening for Hydrolytic Activity
Chitinase activity was detected on colloidal chitin agar medium with 0.5% colloidal
chitin, according to method of Berger and Reynold [13]. Cellulase activity was per-
formed on Czapek’s mineral salt medium containing 1% CMC [14]. The amylolytic
activity was detected on starch agar medium containing 1% soluble starch [15]. The
caseinase enzyme activity of culture was examined on skimmed milk agar medium
showing halo zone around the colonies [16].
Rhizospheric soil gave the highest number of bacterial isolates (20) as compared to
endophytic bacteria (8) isolated from sugarcane variety Co1148. Thus, total 28 bac-
teria were isolated collectively. Dual plate assay showed that out of the total 28
bacterial isolates, 14 bacterial isolates showed in vitro antagonism against
Colletotrichum falcatum. Percentage inhibition showed that out of 14 bacteria, 4
bacterial isolates (So-6, So-9, So-22, and R-17) were showing inhibition more than
50% against C. falcatum in vitro (Table 17.1).
Table 17.1 Antagonism of selected isolates to Colletotrichum falcatum on PDA

S. No Isolates C. falcatum mycelia growth (mm) on tenth day % Inhibition
1. Control 74 –
2. So-6 32 57
3. So-9 28 62
4. So-22 25 66
5. R-17 30.3 59
Table 17.2 Identification and characterization of isolates

Isolates So-6 So-9 So-22 R-17
Gram nature + − − −
Motility − − − −
Catalase + + + +
Indole − − − −
Citrate − + − +
H2S production − − − −
Urease − − − −
MR − − − −
VP − − − −
Oxidase − − − −
IAA + + + +
HCN − − − −
Ammonia + + + +
PSB + − − +
Siderophore − + − −
Chitinase − − − −
Amylase − + − +
Cellulase + − − −
Caseinase − + + +
17.3.1 Identification of Antifungal Isolates
Four bacterial isolates (So-6, So-9, So-22, and R-17) were identified morphologi-
cally as well as biochemically (Table 17.2).
17.3.2 In Vitro Screening of Plant Growth Promoting Activities
All the four isolates that showed inhibition above 50% (So-6, So-9, So22, and R-17)
were showing positive results for IAA production (Table 17.2). Phosphate solubili-
zation is a very important trait of plant growth promotion as microorganisms solu-
bilize insoluble phosphate by producing phosphatase enzyme and making it
available to the plants. Isolates So-6 and R-17 were showing positive result for
phosphate solubilization. In our study, all the four isolates were able to produce
ammonia. Fravel [17] suggested that ammonia production by microorganisms is
also related for inhibitory action on fungal pathogen. None of the bacteria showed
positive result for HCN. Siderophore is an iron-chelating secondary metabolite pro-
duced by several microorganisms that bind insoluble Fe3+ and make it available for
its own growth and also for plants. Less availability of iron results in the growth
inhibition of phytopathogens which happen due to chelating iron by siderophore
and hence enhance the growth of plant. Thus, it promotes plant growth in an indirect
way. In the present study, only isolate So-9 were showing positive results for
siderophore.
17 Exploration of Biocontrol and Growth-Promoting Activity of Bacterial Strains… 133
17.3.3 Hydrolytic Enzyme Activity
There are several mechanisms by which microorganisms bring about control of

plant diseases. Lytic enzymes such as amylases, cellulases, caseinase and chitinase
have the ability to degrade structural fungal cell walls therefore they are related to
hyperparasitic activities [18, 19]. In the present study none isolates showed chitin-
ase activity. Isolates So-9 and R-17 showed positive results for amylase production
and only isolates So-6 showed positive results for cellulase. Isolates So-9, So-22
and R-17 gave positive test for caseinase production (Table 17.2).
17.4 Conclusion
The bacteria isolated in this work have the possibility to be used in the control of red
rot disease because of the presence of various lytic enzymes and plant growth pro-
motory traits as discussed above. Isolates So-6, So-9, and So-22 have been isolated
from the rhizospheric soil, and isolates R-17 have been isolated from root region of
sugarcane variety. These findings showed that endophytic as well as rhizospheric
bacteria act as a biocontrol agent which promotes the growth of plants indirectly by
protecting them from phytopathogens by means of inhibiting the growth of patho-
genic organisms.
Acknowledgments The authors acknowledge Dr. Ram Ji Lal, Prinicipal Scientist (now retired)
and Dr. Dinesh Singh (Principal Scientist), IISR, Lucknow for providing various assistance.
References
1. Food and Agricultural Organization (FAO) of United Nations: Economic and Social
Department: The Statistical Division (2014)
2. Vishwakarma SK, Kumar P, Nigam A et al (2013) Pokkah Boeng: an emerging disease of
sugarcane. J Plant Pathol Microbiol 4:170
3. Satyavir S (2003) Red rot of sugarcane – current Scenario. Indian Phytopathol 56:245–254
4. Duttamajumder SK (2008) Red rot of sugarcane. Indian Institute of Sugarcane Research,
Lucknow
5. Hussnain Z, Afghan S (2006) Impact of major cane diseases on sugarcane yield and sugar
recovery, Annual report. Shakarganj Sugar Research Institute, Jhang
6. Viswanathan R, Rajitha R, Sundar RA, Ramamoorthy V (2003) Isolation and Identification of
Endophytic Bacterial Strains from Sugarcane Stalks and Their In Vitro Antagonism against the
Red Rot Pathogen. Sugartech 5(l & 2):25–29
7. Krieg NR, Holt JG (1984) Bergey’s manual of determinative bacteriology, vol 1. The Williams
and Wilkins Co, Baltimore
8. Gordon SA, Weber RP (1951) Colorimetric estimation of indole-acetic acid. Plant Physiol
26:192–195
9. Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with vital activity of
some microbial species. Microbiol 17:362–370
10. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination
of Siderophore. Anal Biochem 160:47–56
11. Dye DW (1962) The inadequacy of the usual determinative tests for identification of
Xanthomonas sp. New Zeal J Sci 5:393–416
12. Bakker AW, Schippers B (1987) Microbial cyanide production in the rhizosphere in relation
to potato yield reduction and Pseudomonas spp-mediated plant growth-stimulation. Soil Biol
Biochem 19:451–457
13. Berger LR, Reynolds DM (1958) The chitinase system of a strain of Streptomyces griseus.
Biochim Biophys Acta 29:522–534
14. Farkaš V, Lišková M, Biely P (1985) Novel media for detection of microbial producers of cel-
lulase and xylanase. FEMS Microbiol Lett 28(2):137–140
15. Mishra S, Behera N (2008) Amylase activity of a starch degrading bacteria isolated from soil
receiving kitchen wastes. Afr J Biotechnol 7(18):3326–3331
16. Berg G, Roskot N, Steidle A et al (2002) Plant-dependent genotypic and phenotypic diversity
of antagonistic rhizobacteria isolated from different Verticillium host plants. Appl J Environ
Microbiol 68(7):3328–3338
17. Fravel DR (1988) Role of antibiosis in the biocontrol of plant diseases. Annu Rev Phytopathol.
26:75–91
18. Kim PI, Chung KC (2004) Production of an antifungal protein for control of Colletotrichum
lagenarium by Bacillus amyloliquefaciens. FEMS Microbiol Lett 234:177–183
19. Oppenheim AB, Chet I (1992) Cloned chitinase in fungal plant-pathogen control strategies.
Trends Biotechnol 10:392–394

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Rita Kundu · Rajiv Narula Editors

ISBN 978-981-13-6320-7 ISBN 978-981-13-6321-4 (eBook)

Library of Congress Control Number: 2019934372

© Springer Nature Singapore Pte Ltd. 2019

interaction of the biofilm formed by the extracellular products of Pseudomonas sp.

Associate Professor and Head Rina Rani Ray

BIOSPECTRUM 2017 was organized by the Department of Biotechnology,

Kolkata, West Bengal, India Rita Kundu

The book is the outcome of the International Conference on Biotechnology and

Biotechnology is the use or manipulation of an organism or parts of an organism.

Plant biotechnology delivers significant and tangible benefits to farmers, con-

1 Dilute Acid Pretreatment Efficiency on Various Solid

8 Profiling Indolic Auxins Produced by the Strains

Dr. Rita Kundu is a professor at the University of Calcutta’s Department of

Narendra Naik Deshavath, Bijayeeni Singh Deo,

N. N. Deshavath · V. V. Goud · V. D. Veeranki (*)

© Springer Nature Singapore Pte Ltd. 2019 1

1.2 Materials and Methods

1.2.1 Dilute Sulfuric Acid Pretreatment

1.2.2 Microorganism and Seed Culture Preparation

Fermentation was carried out in sterile 25 mL Erlenmeyer flasks containing 10 mL

1.2.4 Analytical Method

Quantification of pretreatment-derived carbohydrates, fermentative inhibitors, and

1.3 Result and Discussion

1.3.1 Pretreatment of Sorghum Biomass

1.3.2  eutralization of Acid Hydrolyzates for Xylulosic Ethanol

Fig. 1.2 Possible

Table 1.1 Summary of fermentation results from sorghum biomass pretreatment-derived

© Springer Nature Singapore Pte Ltd. 2019 9

For development of low-cost BGA biofertilizers using different carrier materials,

Fig. 2.1 Effect of

2.3 Result and Discussion

Table 2.2 Physicochemical Parameters Mean ± SE

Davinder Sharma, Ratan Tiwari, Vijay Kumar Gupta,

© Springer Nature Singapore Pte Ltd. 2019 13

3.2 Materials and Methods

(2012–2013 and 2013–2014). Experiments were laid out in a randomized complete

3.3 Result and Discussion

One-dimensional SDS-PAGE revealed considerable differences in grain proteome

4. Wahid A, Gelani S, Ashraf M, Foolad MR (2007) Heat tolerance in plants: an overview.

Aparajita Shilpie and Kamal Nayan Mishra

© Springer Nature Singapore Pte Ltd. 2019 19

4.2 Materials and Methods

Graph 4.1 DNA

Graph 4.2 DNA

Graph 4.3 DNA

2. Allen GC, Flores-Vergara MA, Krasnyanksi S, Kumar S, Thompson WF (2006) A modified

Chhaya Verma, Apoorva Dixit, and Rajesh Kumar

C. Verma · A. Dixit · R. Kumar (*)

© Springer Nature Singapore Pte Ltd. 2019 27

5.2 Materials and Methods

5.2.1 Siderophore Production

Siderophore production by different strains of Pseudomonas aeruginosa was tested

5.2.2 Optimization of Siderophore

5.2.3 Crystallization and Characterization of Siderophore

5.3 Result and Discussion

Efficiency of biosynthesis of siderophore in strains was also affected by the car-

Fig. 5.2 FTIR characterization of siderophore crystal

5.3.1 Crystallization and Characterization of Siderophore

Kolkata, West Bengal, India Rita Kundu

1 Dilute Acid Pretreatment Efficiency on Various Solid

8 Profiling Indolic Auxins Produced by the Strains

1.2 Materials and Methods

1.2.1 Dilute Sulfuric Acid Pretreatment

1.2.2 Microorganism and Seed Culture Preparation

1.2.4 Analytical Method

1.3 Result and Discussion

1.3.1 Pretreatment of Sorghum Biomass

1.3.2 eutralization of Acid Hydrolyzates for Xylulosic Ethanol

2.3 Result and Discussion

3.2 Materials and Methods

3.3 Result and Discussion

4.2 Materials and Methods

5.2 Materials and Methods

5.2.1 Siderophore Production

5.2.2 Optimization of Siderophore

5.2.3 Crystallization and Characterization of Siderophore

5.3 Result and Discussion

5.3.1 Crystallization and Characterization of Siderophore

6.2 Materials and Methods

6.2.1 Sample Collection and Maintenance

6.2.2 Marker Development

6.2.2.1 UV Irradiation

6.2.2.2 Antifungal Sensitivity Test

6.2.2.3 Antibiotic Sensitivity Test

6.3.1 Development of Marker

7.2 Materials and Methods

7.2.2 Microwave Pretreatment of Rice Straw Using FeCl3

7.2.3 Effect of H3PO4 Concentrations

7.2.4 Effect of Pretreatment Time

7.2.5 Enzymatic Saccharification of Pulp

7.2.6 ffect of Enzyme Load on Saccharification Yield

7.3 Results and Discussion

7.3.1 Microwave Pretreatment of Rice Straw Using FeCl3

7.3.2 Effect of H3PO4 Concentrations

7.3.3 Effect of Pretreatment Time

7.3.4 Effect of Enzyme Dose on Saccharification Yields

8.2.1 Materials and Reagents

8.2.3 Standard Solutions

8.2.4 Strains of PGPF Used for Study

8.2.5 ssessment of the IAA Produced by PGPF Strains

8.2.6 Extraction of Indolic Auxins

8.2.7 HPTLC Procedure

8.2.7.1 Sample Application

8.2.7.2 Calibration Curves

8.2.7.3 Densitometric Analysis of Chromatogram

8.2.7.4 D etermination of IAA from Extracted Indolic Auxins

8.3.1 I AA Production by PGPF Strains Examined by Means

8.3.2.1 Calibration Curves

8.3.2.2 D etermination of IAA and IBA from Fungal Supernatant

9.2 Materials and Method

9.2.1 Plant Material and Preparation of Crude Extracts

9.2.2 Culturing Yeast and Cytotoxicity Assay

9.2.3 Culturing MCF-7 Cell Line and Screening Assay