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 Microbial
Biotechnology
An Interdisciplinary Approach
 Microbial
Biotechnology
An Interdisciplinary Approach

Pratyoosh Shukla
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742
© 2017 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works

Printed on acid-free paper

Version Date: 20161019

International Standard Book Number-13: 978-1-4987-5677-8 (Hardback)

This book contains information obtained from authentic and highly regarded sources. Reasonable
efforts have been made to publish reliable data and information, but the author and publisher cannot
assume responsibility for the validity of all materials or the consequences of their use. The authors and
publishers have attempted to trace the copyright holders of all material reproduced in this publication
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Contents

Foreword, vii
Preface, ix
Contributors, xi

Chapter 1   ◾   Bacterial Exopolysaccharides: Major Types

and Future Prospects 1
Aparna Banerjee and R ajib Bandopadhyay

Chapter 2   ◾   Bioremediation: A Novel Green Technology

to Clean Up the Highly Contaminated
Chromites Mining Sites of Odisha 21
Swati Sucharita Panda and Nabin Kumar Dhal

Chapter 3   ◾   Recent Developments in Food Biotechnology

to Improve Human Health with Probiotics
with Special Emphasis on Lowering Cholesterol 35
Renu Agrawal

Chapter 4   ◾   Molecular Characterization and

Quantification of Microbial Communities in
Wastewater Treatment Systems 59
Jashan Gokal, Oluyemi Olatunji Awolusi, Abimbola
Motunrayo Enitan, Sheena Kumari, and Faizal Bux

Chapter 5   ◾   Thermostable Enzymes and Their Industrial

Applications 115
Santhosh Kumar, Nanthakumar Arumugam,
Kugenthiren Permaul, and Suren Singh

v
vi   ◾    Contents

Chapter 6   ◾   Microbial Enzymes for Pulp and Paper

Industry: Prospects and Developments 163
Puneet Pathak, Prabhjot K aur, and Nishi K. Bhardwaj

Chapter 7   ◾   Rhizobacteria: Tools for the Management

of Plant Abiotic Stresses 241
Anjali Singh, Ajay Shankar, Vijai Kumar Gupta, and Vishal Prasad

Chapter 8   ◾   Betulin Biotransformation toward Its

Antitumor Activities: A Brief Overview 263
Dhirendra Kumar and K ashyap Kumar Dubey

Chapter 9   ◾   Optimizing the Performance of Wastewater

Treatment Plants and Effluent Quality Using
Evolutionary Algorithms 287
Abimbola Motunrayo Enitan, Josiah Adeyemo, Gulshan Singh,
and Folasade Adeyemo

Chapter 10   ◾   Production of Fructooligosaccharides as

Ingredients of Probiotic Applications: Future
Scope and Trends 311
Ruby Yadav, Puneet Kumar Singh, and Pratyoosh Shukla

Chapter 11   ◾   Avenues in Ophthalmic Optical Coherence

Tomography in Medical Biotechnology:
Prospects and Future Trends 325
R aju Poddar, Vinod Aggarwal, Varun Gogia, Mayank Bansal,
Shika Gupta, Rohan Chawla, and Pradeep Venkatesh

INDEX349
Foreword

T he book Microbial Biotechnology: An Interdisciplinary Approach,

edited by Dr. Pratyoosh Shukla, covers some of the latest applications
of microorganisms from a practical point of view. The field of microbial
biotechnology, in the context of the so-called cell factories, is of great
interest and the number of groups involved in such projects is growing
exponentially.
Although the book chapters cover different aspects of microbial bio-
technology, I want to highlight two of them. The first refers to the field
of functional foods; several chapters deal with the production of probiot-
ics and prebiotics, and their effect on gastrointestinal health. The second
topic is microbial bioremediation, which is exemplified in this book by
the use of microbes to clean up mining sites and by the optimization of
wastewater treatments. Other issues having a significant impact are also
addressed in the book: for example, the use of microbial enzymes in pulp
and paper industries, the different applications of exopolysaccharides, or
the latest developments in medical biotechnology, among others.
In summary, there is no doubt about the interest of the contents dis-
played in this book. I am sure that the book Microbial Biotechnology: An
Interdisciplinary Approach will provide the scientific community with
great benefits for the coming years.

Francisco Plou
Research Scientist at Spanish CSIC
Honorary Professor at Autonomous University of Madrid
Madrid, April 4, 2016

vii
Preface

T he book describes the interdisciplinary scope of biotechnology

and discoveries thereof. This book briefs the reader on various novel and
innovative ideas of emerging biotechnology. The key features are described
below to highlight the important contents of the book:

1. The book envisages the recent ideas of novel findings in microbiology.

2. It also provides insights into various interdisciplinary research
avenues.
3. There are very few books available covering the diversity of topics
described in this book.
4. Some key areas of modern biotechnology are also covered in this
book, which are not available in any such books in the market.
5. Enhanced and simplified descriptions are the key components of
this book, which provide unique benefits to its readers.

This book will also act as an important means of information on research-

ers working in interdisciplinary areas of research. The chapters outlined
in this book cater to the needs of researchers working in the areas of bac-
terial exopolysaccharides, microalgal proteomics, applications of micro-
bial L-asparaginases, novel aspects of bioremediation, probiotics and their
impact on society, microbial community analysis in wastewater treatment
techniques, etc. The book focuses on describing the above-mentioned
aspects and on diversifying the understanding of microbial biotechnology
to an expanded level.

ix
x   ◾    Preface

This book will be a valuable resource to senior undergraduate and grad-

uate students, researchers, professionals, and other interested individuals
or groups working in the areas mentioned in the book.

Pratyoosh Shukla, PhD

December 2016
Rohtak, India
Contributors
Folasade Adeyemo
Institute for Water and Wastewater
Technology (IWWT)
Durban University of
Technology
Durban, South Africa
Josiah Adeyemo
Department of Civil and
Structural Engineering
Masinde Muliro University of
Science and Technology
Kakamega, Kenya
Vinod Aggarwal
Dr. Rajendra Prasad Centre for
Ophthalmic Sciences
A.I.I.M.S.
New Delhi, India
Renu Agrawal
CSIR-CFTRI and Rural
Development Programme
Mysore, India
Nanthakumar Arumugam
Department of Biotechnology
and Food Technology
Durban University of Technology
Durban, South Africa
Oluyemi Olatunji Awolusi
Institute for Water and Wastewater
Technology
Durban University of Technology
Durban, South Africa
Rajib Bandopadhyay
Department of Botany
The University of Burdwan
Burdwan, West Bengal, India
Aparna Banerjee
Department of Botany
The University of Burdwan
Burdwan, West Bengal, India
Mayank Bansal
Dr. Rajendra Prasad Centre for
Ophthalmic Sciences
A.I.I.M.S.
New Delhi, India
Nishi K. Bhardwaj
Avantha Centre for Industrial
Research & Development
Yamuna Nagar, Haryana, India
Faizal Bux
Institute for Water and Wastewater
Technology (IWWT)
Durban University of Technology
Durban, South Africa
xi
xii   ◾    Contributors
Rohan Chawla
Dr. Rajendra Prasad Centre for
Ophthalmic Sciences
A.I.I.M.S.
New Delhi, India
Nabin Kumar Dhal
Environment and Sustainability
Department
CSIR-IMMT
Bhubaneswar, India
Kashyap Kumar Dubey
University Institute of
Engineering and Technology
(UIET)
Maharshi Dayanand University
Rohtak, Haryana, India
Abimbola Motunrayo Enitan
Institute for Water and Wastewater
Technology (IWWT)
Durban University of Technology
Durban, South Africa
Varun Gogia
Dr. Rajendra Prasad Centre for
Ophthalmic Sciences
A.I.I.M.S.
New Delhi, India
Jashan Gokal
Institute for Water and Wastewater
Technology
Durban University of Technology
Durban, South Africa
Shika Gupta
Dr. Rajendra Prasad Centre for
Ophthalmic Sciences
A.I.I.M.S.
New Delhi, India
Vijai Kumar Gupta
School of Natural Sciences
NUI Galway
Galway, Ireland
Prabhjot Kaur
Avantha Centre for
Industrial Research &
Development
Yamuna Nagar, Haryana, India
Dhirendra Kumar
Department of Biotechnology
University Institute of
Engineering and Technology
(UIET)
Maharshi Dayanand University
Rohtak, Haryana, India
Santhosh Kumar
Department of Biotechnology and
Food Technology
Durban University of Technology
Durban, South Africa
Sheena Kumari
Institute for Water and Wastewater
Technology (IWWT)
Durban University of Technology
Durban, South Africa

Swati Sucharita Panda
Environment and Sustainability
Department
CSIR-IMMT
Bhubaneswar, India
Puneet Pathak
Avantha Centre for Industrial
Research & Development
Yamuna Nagar, Haryana, India
Kugenthiren Permaul
Department of Biotechnology and
Food Technology
Durban University of Technology
Durban, South Africa
Raju Poddar
Department of Bioengineering
Birla Institute of Technology
Ranchi, India
Vishal Prasad
Institute of Environment and
Sustainable Development
Banaras Hindu University
Varanasi, India
Ajay Shankar
Institute of Environment and
Sustainable Development
Banaras Hindu University
Varanasi, India
Pratyoosh Shukla
Department of Microbiology
Maharshi Dayanand University
Rohtak, Haryana, India
Contributors   ◾   xiii
Anjali Singh
Institute of Environment and
Sustainable Development
Banaras Hindu University
Varanasi, India
Gulshan Singh
Institute for Water and Wastewater
Technology (IWWT)
Durban University of
Technology
Durban, South Africa
Puneet Kumar Singh
Department of Microbiology
Maharshi Dayanand University
Rohtak, Haryana, India
Suren Singh
Department of Biotechnology and
Food Technology
Durban University of
Technology
Durban, South Africa
Pradeep Venkatesh
Dr. Rajendra Prasad Centre for
Ophthalmic Sciences
A.I.I.M.S.
New Delhi, India
Ruby Yadav
Department of Microbiology
Maharshi Dayanand University
Rohtak, Haryana, India
 Chapter 1

Bacterial
Exopolysaccharides
Major Types and Future Prospects

Aparna Banerjee and Rajib Bandopadhyay

CONTENTS
Abstract.................................................................................................................2
Introduction.........................................................................................................2
EPS Producing Bacteria: Major Types..............................................................4
Soil Inhabitants...............................................................................................4
Lactic Acid Bacteria.......................................................................................4
Halophiles........................................................................................................4
Thermophiles..................................................................................................8
Psychrophiles..................................................................................................8
EPS from Pathogenic Bacteria.................................................................8
Regulation of EPS Production...........................................................................9
Present Studies on Bacterial EPS.....................................................................10
Heteropolysaccharides.................................................................................10
Homopolysaccharides..................................................................................12
Future Prospects of Bacterial EPSs.................................................................13
Food Industry................................................................................................13
Pharmaceutical Industry.............................................................................13
Biomedical Application...............................................................................14
Bioremediation and Wastewater Treatment..............................................14
Patenting in the Field of Bacterial EPS...........................................................14
Conclusion.........................................................................................................16
Acknowledgment...............................................................................................17
References...........................................................................................................17
1
2   ◾    Microbial Biotechnology

ABSTRACT
Exopolysaccharide (EPS) is secreted by bacteria for their survival in harsh
environmental conditions as a protective mechanism. Repeating sugar
units, attached with proteins, lipids, organic and inorganic compounds,
metal ions, and DNA are found in EPS. Bacterial EPSs have possible com-
mercial applications in pharmaceutical industry, food processing, drug
detoxification, bioremediation, and in many more. The most used and
patented bacterial EPSs are xanthan, cellulose, gellan, alginate, etc. Varied
applications of microbial EPSs are somewhat unexplored and their study
is persistently enhancing toward isolation and characterization of novel
EPSs as renewable capital. Downstream processing for purification and
genetic engineering for increased EPS biosynthesis require more attention.

INTRODUCTION
Polysaccharides is an important content of microbial cell walls, either as
storage capsular polysaccharides or as biofilm called as exopolysaccharides
(EPSs) secreted by microbes in its surrounding. Presently, isolation and
characterization (Figure 1.1) of new microbial EPS is of key scientific inter-
est, because EPS has shown promising application as texture enhancers,
gelling agents, emulsifiers, viscosifiers, and also as the newest nanovector
for drug delivery, resulting in sustained release of drugs. Bacterial EPS own
a varied range of property which is not found in traditional plant polymers.
Although it competes for algal (alginates, carrageenans, and ulvan), crus-
tacean (chitin) or plant polysaccharides, its production level is less due to
green house effect, global warming, marine pollution, sea level increment,
loss of key stone species, crop failure, and overall climate change impacts.
Microorganisms provide a controlled production in bioreactors, with-
out any variation due to the physiological states encountered for higher
organisms (1). But downstream processing of bacterial polysaccharides has
cost-intensive steps, as the expenses needed for substrates requirement for
microbial growth and bioreactors are too high (2). Moreover, cultivation
of microorganisms in a fermenter allows growth optimization and pro-
duction yield either by physiological study or by genetic modification. For
high-value pharmaceutical industry, bacterial polysaccharides can be pro-
duced at a feasible economic cost. Research in the field of bacterial EPS pro-
duction is till now done on most available EPSs such as cellulose, xanthan
gum, levan, glucan, cellulose, sphigan, hyaluronan, and succinoglycan, of
which xanthan gum from Xanthomonas sp., gellan from Sphingomonas
 Bacterial Exopolysaccharides   ◾   3

Centrifugation of microbial culture

Isolation of supernatant

Precipitation of EPS using

methanol, ethanol, or iso-propanol

Isolation of EPS from

centrifugated pellet and vacuum drying

Morphological Physical characterization Chemical

characterization characterization
Molecular weight
Fluorescence microscopy determination GC-MS, HPLC
Total carbohydrate FTIR
Scanning electron
content
microscopy 1
H NMR and 13C NMR
CHNS analysis
Thermo gravimetric
analysis

FIGURE 1.1 Flowchart elucidating the main steps of isolation and characteriza-
tion of bacterial EPS.

elodea and Sphingomonas paucimobilis, cellulose from Gluconacetobacter

xylinus, alginate from Pseudomonas aeruginosa and Pseudomonas putida,
levan from Zymomonas mobilis and Halomonas eurihalina, succinoglycan
from Agrobacterium radiobacter, Rhizobium meliloti, and Agrobacterium
tumefaciens, Glucan from Leuconostoc dextranicum show strong potential
commercial uses. All of these are very closely related structures and share
high level of homology in many biosynthetic pathways.
The word EPS is first termed by Sutherland (3) for high molecular
weight marine bacterial carbohydrate polymers. EPSs are produced in
large amounts surrounding the microbial cells in extreme environments
of Antarctic ecosystems, hypersaline lakes, hot water springs, or in deep-
sea hydrothermal vents. Among all other adaptations for survival in
extreme conditions, such as high temperatures, extreme salt levels, low
pH, temperature variation, and high radiation zone, EPS biosynthesis is
the most common protective mechanism by extremophilic microorgan-
isms. EPS producing bacteria can be screened by the presence of glossy
4   ◾    Microbial Biotechnology

and slimy colony appearance, so that they can be further chosen for mass
production (4).

EPS PRODUCING BACTERIA: MAJOR TYPES

Several groups of bacteria are capable of EPS production and they can be
broadly categorized into five groups (Table 1.1): soil inhabitants, lactic acid
bacteria, halophiles, thermophiles, and psychrophiles.

Soil Inhabitants
The most famous EPS producers are soil inhabiting rhizobia. It forms
large amounts of polysaccharides when grown in pure cultures and also
into the rhizosphere. Rhizobium meliloti, Rhizobium leguminosarum, and
Rhizobium tropici are the three most studied EPS producing soil inhab-
iting bacteria (5,6). Regulation of motility related genes and presence of
quorum sensing proteins in rhizobia resulted into a complex EPS bio-
synthesis pathway. EPS production by bacteria and biofilm formation
enhances soil fertility and improved plant growth (7). Pantoea (formerly
Enterobacter) agglomerans isolated from mangrove forest had very high
ultraviolet radiation tolerance. The water-soluble EPS was extracted and
was further tested for its ultraviolet radiation protection and free radical
scavenging activities (8). Micrococcus luteus isolated from Egyptian soil,
produced a maximum of 13 g/L EPS and the EPS showed high antioxidant
activity (9).

Lactic Acid Bacteria

Lactic acid bacteria (LAB) improve the flavor, aroma, and texture of
milk, meat, and vegetables and therefore they are used in food fermenta-
tions. LAB usually produce small amounts of EPS (100–200 mg/L) (10)
but Lactobacillus sakei produced up to 4 g/L EPS (11). In fermented milk
(12) and during cheese production (13), small amounts of EPS produc-
tion change the texture and properties of the products. Several strains
of Streptococcus thermophilus produce EPS in milk or in same type of
media. The thermophilic LAB are also capable of producing EPS, such as
Streptococcus thermophilus and Lactobacillus delbrueckii (12).

Halophiles
The diversity of halophilic bacteria so far isolated and characterized can be
categorized into four different classes according to its salt requirement for
their growth, which include slight halophiles, moderate halophiles, extreme
TABLE 1.1 Recent Studies on Bacterial Exopolysaccharides, Its Chemical Composition, and Probable Function
Bacterial Type Bacterial Strain Maximum EPS Constituents of EPS Function of EPS References
Production
Soil inhabitant Pseudomonas 4.5 g/L Fructose:glucose:mannose = 4:1:0.6. NMR indicated Phosphate solubilizing Taguett et al.
fluorescens EPS produced was levan with β-(2 → 6)-linked activity (32)
fructose units
Rhizobium tropici 4.08 g/L Mannose (0.86%, 1.49%, and 2.68%), rhamnose Shear-resistant nature, Lemos et al.
(2.58%, 2.49%, and 0.60%), glucuronic acid (8.6%, soil stabilizing agent (5)
5.97%, and 3.57%) and trace of galacturonic acid
Micrococcus luteus 13 g/L Mannose:arabinose:glucose:glucuronic In vitro DPPH Asker et al.
acid = 3.6:2.7:2.1:1.0 radical-scavenging (33)
Main backbone consists of mannose units linked activity, with an EC50
with (1 → 6) glycosidic bonds and arabinose units value of 180 µg/mL
linked with (1 → 5) glycosidic bonds. There is a side
chain consisting of mannose units linked with
(1 → 6) glycosidic bonds at C3, when all glucose and

Bacterial Exopolysaccharides   ◾   5

most of glucuronic acid are found in the side chain.
Lactic acid Lactobacillus sakei 2 g/L and IR spectra of both EPS were same as commercial Antiviral and Vázquez et al.
bacteria and Leuconostoc 1.4 g/L dextran; specifically α-(1 → 6) glucan with immunomodulatory (34)
mesenteroides approximately 6% substituted side chain glucose activity
Lactobacillus – Mannose:fructose:galactose:glucose = 8.2:1:4.1:4.2 Antibacterial, Wang et al.
plantarum antioxidant, and (35)
anticancerous activity
Streptococcus 12.14 g/L – Antibacterial, Kanmani
phocae antioxidant, and et al.(36)
flocculating activity
(Continued)
 6   ◾    Microbial Biotechnology
TABLE 1.1 (Continued) Recent Studies on Bacterial Exopolysaccharides, Its Chemical Composition and Probable Function
Maximum EPS
Bacterial Type Bacterial Strain Production Constituents of EPS Function of EPS References
Streptococcus Tetrasaccharide of glucose: galactose = 1:1 and – Marshall
thermophilus and heptasaccharide composed of et al. (37)
Lactobacillus galactose:glucose:rhammose = 5:1:1
Delbrueckii
Halophile Halomonas 3.89 g/L Monosaccharide (%): glucose (24 ± 1.73), glucuronic Flocculating and Amjres et al.
stenophila acid (7.5 ± 0.37), mannose (5.5 ± 0.17), fucose emulsifying activities (38)
(4.5 ± 0.36), galactose (1.2 ± 0.17), and rhamnose
(1 ± 0.05)
Halomonas 1.7 g/L Monosaccharide: 72% mannose, 27.5% glucose, and Emulsifying Llamas et al.
almeriensis 0.5% rhamnose. hydrophobic (39)
Low-molecular-weight EPS: 1.1% protein, 70% substrates and
mannose, and 30% glucose pseudoplasticity
Thermophile Brevibacillus 2.08 g/L Glucose:galactose:mannose: Nonpathogenic Yildiz et al.
thermoruber galactosamine:mannosamine =57.7:16.3:9.2:14.2:2.4 promising EPS (25)
Geobacillus 1.12 g/L Carbohydrate content = 98% Anti-cytotoxic activity Kambourova
tepidamans Protein content = 1.8% against Avarol et al. (24)
Uronic acids = 0.2%
(Continued)
TABLE 1.1 (Continued) Recent Studies on Bacterial Exopolysaccharides, Its Chemical Composition and Probable Function
Maximum EPS
Bacterial Type Bacterial Strain Production Constituents of EPS Function of EPS References
Psychrophile Pseudoalteromonas – – Inhibitory activity Chen et al.
sp. S-5 against human (40)
leukemia K562 cells
Pseudomonas sp. – Carbohydrate content = 33.81% ± 2.59, composed of Emulsifier and Mercade
ID1 glucose (17.04% ± 0.32), galactose (8.57% ± 1.15), cryoprotector et al. (41)
and fucose (8.21% ± 1.12). Total uronic acid
content = 2.40% ± 0.33%.

Bacterial Exopolysaccharides   ◾   7

Pseudoalteromonas 8.61 g/L – Cryoprotector Liu et al. (42)
sp. strain
SM20310
Zunongwangia Highly complex α-mannan polymer of 2-α-, Cryoprotector Liu et al. (28)
profunda 6-α-mannosyl residues, where 6-α-mannosyl
residues are branched at 2nd position with 1–2
t-mannosyl residues
8   ◾    Microbial Biotechnology

halophiles, and borderline halophiles. The halotolerant bacteria in con-

trast show tolerance to a wide range of salinity stress, from high salt con-
centration to zero salt requirement (14). Many halophilic Archaea such as
Haloferax, Haloarcula, Halococcus, Natronococcus, and Halobacterium are
also good EPS producers (15–17). H. maura, H. eurihalina, H. ventosae,
and H. Anticariensis of Halomonas genus are the most dominant halo-
philic EPS producing group. The characteristic features of EPS synthesized
by Halomonas strain are unusually high sulfate content and uronic acids in
large amounts which result in good jellifying properties (18). Most halophiles
show high levels of heavy metal resistance as it is found in coastal areas.

Thermophiles
Thermophiles are currently classified as: moderate thermophiles (50–
70°C) and extremothermophiles (>70°C) based on its optimal growth
temperatures; extremothermophiles grow optimally above 80°C and are
also termed as “hyperthermophiles.” They inhabit a wide range of habitats
from geothermal springs and solfataric (sulfur) fields, shallow submarine
hydrothermal systems, geothermally heated oil reservoirs to abyssal hot-
vent environments or hot coal-refuse piles (19).
Different hyperthermophilic microorganisms such as Thermotoga
maritima, Archaeoglobus fulgidus, and Thermococcus litoralis produce EPS
significantly (20–22). Works has also been done on EPS producing Bacillus
licheniformis from marine hot springs (23), glucan producing Geobacillus
tepidamans from Bulgarian hot springs (24), Brevibacillus thermoruber
from geothermal springs of Turkey and Bulgaria (25).

Psychrophiles
A large portion of reduced carbon reserve of the ocean is EPS and it
enhances the survival rate of psychrophilic marine bacteria by modify-
ing the physicochemical environment around the bacterial cell. Antarctic
marine environments are rich in bacterial EPSs which help the micro-
bial communities to survive under extreme cold temperature and salin-
ity with least nutrient availability. EPS produced by a new genus of
Pseudoalteromonas, isolated from Antarctic sea ice at −2°C and 10°C
showed higher uronic acid content than EPS produced at 20°C (26).

EPS from Pathogenic Bacteria

Surface attaching bacteria come together in a hydrated polymeric matrix
for biofilm formation. EPS biosynthesis by these virulent communities
 Bacterial Exopolysaccharides   ◾   9

results in resistance to antimicrobial drugs, which is the root to many

chronic and persistent bacterial infections. Bacterial EPS is also the basis
of growth of pathogenic microorganism in biofilms as it provides the sub-
stratum for microbial growth in mats; for example, dental caries by acido-
genic Gram-positive cocci, cystic fibrosis pneumonia by Pseudomonas
aeruginosa and Burkholderia cepacia, nosocomial infection by different
Gram-negative bacteria are all biofilm-mediated infection. Nitric-oxide-
mediated signaling results in EPS production in Shewanella oneidensis.
LasI-dependent QS provides maturation signal and results in the forma-
tion of a thick and differentiated biofilm of Pseudomonas aeruginosa.
Presently, diguanylate cyclase and its product cyclic di-guanosine mono-
phosphate (c-di-GMP) are key biomedical targets for the inhibition of
biofilm development.

REGULATION OF EPS PRODUCTION

A complex process involving the transport of organic and inorganic mol-
ecules to the outer surface, its adsorption to that surface and finally the
formation of an unalterable attachment result in the growth of EPS as bio-
film. The most studied regulatory mechanism controlling EPS production
is QS. QS maintains bacterial intercellular communication and regulate
gene-specific expression to increase cell density.
Two QS processes described in bacteria are AI-1 and AI-2. Autoinducer-1
(AI-1) regulates intraspecies communication and AI-2 regulates interspe-
cies communication. Gram-negative bacteria secrete AI molecule: N-acyl
homoserine lactone (AHL) that control cell density. The detection of accu-
mulated AHL signal by bacteria switches on transcriptional effectors to
activate silent genes above a certain threshold concentration. This results
into cell density dependent gene expression and behavioral change. Gram-
positive bacteria communicate with modified oligopeptides and mem-
brane-bound histidine kinase sensor as receptors. Signaling is controlled
by multiple phosphorylations which modify the activity of the regulator
(27). The omnipresent bacterial c-di-GMP is an important messenger in
controlling bacterial biofilm formation. Cyclic nucleotides are synthe-
sized by external stimuli on various signaling domains within N-terminal
region of dimeric diguanylate cyclase. It initiates condensation of two
molecules of guanosine triphosphate opposite to each other within the
C-terminal region of the enzyme. Additionally, the formation of a long-
term stable aerobic granule, a superior biofilm for biological wastewater
treatment, can be controlled by stimulating c-di-GMP.
10   ◾    Microbial Biotechnology

Substrate (a) is first catabolized by glycolytic pathways into pyruvate

while entering the cell. Under aerobic conditions, it is converted to acetyl-
CoA and enters TCA cycle (b). UDP-Glc, UDP-Gal, and GDP-Man are
interconverted by epimerization, oxidation, decarboxylation, reduction,
and rearrangement (c) reactions to form energy-rich monosaccharide tri-
phosphates. EPS biosynthesis and polymerization occurs through any of
the following mechanisms (d).
Wzx–Wzy system (left) synthesize EPS by the sequentially transferring
monosaccharides from NDP-sugars to a polyprenylphosphate lipid carrier.
For polymerization by polymerase enzyme Wzy, modified repeating units
are transported across the inner membrane by a flippase enzyme Wzx to
the periplasmic place. Polysaccharide copolymerase (PCP) determines the
polymer length, while an outer membrane polysaccharide export protein
OPX forms the intermembrane channel.
The ABC-transporter system (right) results in EPS formation at the
cytoplasmic side of the inner membrane by adding sugar residues to the
nonreducing end of the polymer and export it across the inner membrane,
followed by its translocation across the outer membrane by PCP and OPX
(Figure 1.2).

PRESENT STUDIES ON BACTERIAL EPS

Microbial EPSs are usually linear molecules with different side chains.
Association of high molecular weight polymer chains results in complex
entanglement. These high molecular weight polymers show a tendency
to form double-stranded helices (kappa carrageenans, xanthan, succino-
glycan, and gellan) and sometimes triple stranded (curdlan, schizophyl-
lan) (28,29) also. Commonly found monosaccharides in bacterial EPSs
are d-glucose, d-galactose, and d-mannose; l-fucose and l-rhamnose;
and N-acetylhexosamines, N-acetyl-d-glucosamine, and N-acetyl-d-
galactosamine. Uronic acids such as d-glucuronic and d-galacturonic acid
are also present in few microbial EPSs. Common and commercially most
exploited types of bacterial EPSs (Figure 1.2) are xanthan, gellan, cellu-
lose, sphingan, hyaluronan, alginate, etc.

Heteropolysaccharides
1. Xanthan gum: It is the first industrially produced biopolymer,
which is extensively studied and widely accepted commercially.
Xanthomonas genus of bacteria secretes this heteropolysaccharide
 Bacterial Exopolysaccharides   ◾   11

(a)

ADP + Pi Substrate
ATP Glycerol
Glucose
Fructose

Man-6-P Glucose-6-P

Fructose-6-P
(c) ADP + Pi NAD+

Glycolysis
(b)
Man-1-P Glc-1-P ATP NADH + H+
Acetyl CoA

TDP-Glc Pyruvate

UDP-Glc CO2
GDP-Man
FADH2 TCA NAD+
cycle
FAD NADH + H+
GDP-Fuc TDP-Rha UDP-Gal UDP-GlcA
ATP ADP + Pi
NDP-sugars

NDPs
NMP
NDPs ADP + Pi
(d) NMP
NDP-sugars
Cytoplasm

NDP-sugars ATP ABC

P
Wzy Wzx P
PCP

Inner membrane
PCP

P P
P P

Peptidoglycan
OPX
OPX

Outer membrane

EPS EPS Extracellular

environment

FIGURE 1.2 Schematic diagram showing biosynthetic pathways of EPS syn-

thesis by Gram-negative bacteria. (a) Substrate entry to the cell, (b) entry
of phosphorylated sugars in TCA cycle, (c) interconversion of sugars, and
(d) EPS polymerization. Fuc, fucose; Gal, galactose; Glc, glucose; GlcA, gluc-
uronic acid; Man, mannose; Rha, rhamnose; GDP, guanosine diphosphate;
TDP, tyrosine diphosphate; NMP, nucleoside monophosphate. (Adapted
from Freitas, F., Alves, D. V., and Reis, M. A. M. 2011. Trends in Biotechnology
29:388–398.)
12   ◾    Microbial Biotechnology

and it has a glucose backbone with trisaccharide side chain of gluc-

uronic acid, mannose, pyruvil, and acetyl residues.
2. Sphingans: These are heteropolysaccharides produced by members
of the genus Sphingomonas having a characteristic tetrasaccharide
backbone of rhamnose, glucose, and glucuronic acid. Different vari-
ations of sphingans are gellan, welan, rhamsan, and diutan. They
have difference in composition and linkage of the side chains, for
example, gellan contains acetyl and glyceryl residues in side chain,
whereas welan has a rhamnose or mannose containing a side chain
branch.
3. Hyaluronan: Repeating disaccharide units of glucuronic acid and
N-acetylglucosamine form a linear structured polymer named
hyaluronan. Bacterial strains, for example, Pseudomonas aeru-
ginosa and group A and C Streptococci attenuated strains are
observed to produce this high market value EPS.
4. Succinoglycan: This is a branched bacterial EPS with glucose and
galactose backbone and tetrasaccharide side chains of glucose resi-
dues. Succinate, pyruvate, and acetate are also commonly found in
it. Many soil inhabiting bacteria such as Rhizobium sp., Alcaligenes
sp., Pseudomonas sp., and Agrobacterium sp. are good producers of
succinoglycan.
5. Alginate: This is a linear copolymer of block-structured poly-
mannuronic acid and poly-guluronic acids. Alginate is secreted by
Pseudomonas aeruginosa and Azotobacter vinelandii. The main dif-
ference between algal and bacterial alginate is the algal one is an
acetylated polysaccharide.

Homopolysaccharides
1. Glucans: Glucans are glucose homopolysaccharides differing
in glycosidic bond, degree and type of branching, chain length,
molecular mass, and polymer conformations. Glucans are of two
types—α-glucans (reuteran, dextran, mutan, and alternan) and
β-glucans (e.g., cellulose and curdlan). Bacterial genera, such as
Gluconacetobacter, Agrobacterium, Aerobacter, Achromobacter,
Azotobacter, Rhizobium, Sarcina, and Salmonella are able to pro-
duce cellulose. Extracellular enzyme dextransucrase that form
α-glucans are produced from sucrose in several bacterial genera
 Bacterial Exopolysaccharides   ◾   13

of Lactobacillus Leuconostoc and Streptococcus. Substrate synthe-

sis for cellulose production starts from the glycolytic intermedi-
ate glucose-6-phosphate. Curdlan is glucans homopolysaccharide
of β-(1 → 3)-linked glucose residues produced by bacteria such as
Agrobacterium biobar and A. tumefaciens, and it forms characteris-
tic elastic gels after heating in aqueous suspension. Curdlan produc-
tion by Alcaligenes faecalis is developed for commercial use in gel
production.
2. Levan: It is synthesized from sucrose by the extracellular enzyme
levansucrase (EC 2.4.1.10), also known as sucrose 6-fructosyltrans-
ferase by several bacterial genera of Bacillus, Rahnella, Aerobacter,
Erwinia, Streptococcus, Pseudomonas, and Zymomonas (30). Levan
is highly branched water-soluble fructose homopolysaccharide.
Due to the presence of β-(2 → 6) linkage, levan is soluble in oil.

FUTURE PROSPECTS OF BACTERIAL EPSs

The future of bacterial EPS production will be glorious, as immense poten-
tial applications of EPS are already established in vitro. The most accepted
bacterial EPS as drug delivery systems are xanthan and cellulose. There
are many others and the most promising are the ones mentioned below.

Food Industry
First industrially marketed EPS dextran is produced by LAB and is used
in confectionary to improve moisture retention, maintenance of viscos-
ity, and to inhibit sugar crystallization. It acts as gelling agents in jelly
and gum. It inhibits water crystal formation in ice cream and also gives
the desired body and mouth feel in pudding. Due to the growing demand
in natural and minimally processed foods, the use of antimicrobial com-
pounds produced by LAB is of huge scientific and commercial interest as
a safe and natural food preservative. Nisin produced by Lactococcus lactis
and Reuterin produced by Lactococcus reuteri are widely used as natural
antibacterial food preservatives.

Pharmaceutical Industry
Recently there is a huge growing demand for LAB as probiotics. The
characteristic features of LAB strains as probiotics are its acid and bile
tolerance, producing antimicrobial compounds against pathogens and
adherence, and colonization in human intestinal mucosa.
14   ◾    Microbial Biotechnology

Bacterial EPS xanthan gum synthesized by Xanthomonas campestris is

a sustained drug delivery system and increase drug effectivity. Bacterial
cellulose nanocrystals produced by Gluconacetobacter xylinus showed
superior properties than plant-derived cellulose and are recently patented
by FDA for using as transdermal drug delivery system, tablet excipient,
and aerogel–hydrogels.

Biomedical Application
Polysaccharides of marine Vibrio, Pseudomonas, and Bacillus lichenifor-
mis showed to have antitumor, antiviral, and immune stimulant activity.
Alteromonas infernus isolated from deep-sea hydrothermal vent, pro-
duced a low-molecular weight heparin-like EPS with good anticoagulant
property. An l-fucose containing polysaccharide Clavan showed prom-
ising roles in tumor cell colonization prevention in lung, regulation of
white blood-cell formation, rheumatoid arthritis treatment, antigen syn-
thesis for antibody production, and in cosmeceuticals as a skin mois-
turizing agent. Water-soluble EPS from Pantoea agglomerans showed
protective activity against UV radiation by its free radical scavenging
activity (8).

Bioremediation and Wastewater Treatment

Bacterial EPS can effectively perform bioremediation, as bacteria grow-
ing within the biofilm have higher adaptation to different extreme envi-
ronments and increased survival rate. Biofilm reactors are mainly used to
treat municipal and industrial wastewater. As EPSs have good floccula-
tion activity and are able to bind metal ions in solutions, it is used in the
removal of heavy metals from the environment. Sulfate reducing bacte-
ria, Enterobacter and Pseudomonas species are the major group of bacte-
ria commonly found in metal contaminated wastewaters and are highly
efficient in anaerobic degradation of organic pollutants and heavy metal
precipitation from wastewater. Recently Acidithiobacillus thiooxidans
and Acidithiobacillus ferroxidans are proved to be potent accumulators of
Fe3+ (4).

PATENTING IN THE FIELD OF BACTERIAL EPS

Bioactive microbial EPSs have shown enormous growth in patent-
ing from last 30 years as the first patent in “recombinant DNA plasmid
for xanthan gum synthesis” was published in 1987 by Merck. Till then
 Bacterial Exopolysaccharides   ◾   15

TABLE 1.2 Important Patents in the Field of Bacterial Exopolysaccharide in the

Last Two Years
Patent Filing Date Applicants Title
20150150959 06.04.2015 Children’s Medical Bacterial biofilm matrix as a
Center Corporation platform for protein delivery
20150079137 19.03.2015 Polymaris Exopolysaccharide for the
Biotechnology treatment and/or care of the skin,
mucous membranes, and/or nails
20140348878 27.11.2014 Ai et al. Strain of exopolysaccharide-
secreting Lactobacillus brevis and
application thereof
20140322273 30.10.2014 Ai et al. Strain of exopolysaccharide
secreting Lactobacillus plantarum
and application thereof
20140171386 19.06.2014 Cheuk et al. Method and composition to
reduce diarrhea in a companion
animal
20140057018 27.02.2014 Ashraf Hassan Processed cheese with cultured
dairy components and
manufacturing
20140037687 06.02.2014 Apariin et al. Exopolysaccharide of Shigella
sonnei bacteria, method for
producing same, vaccine and
pharmaceutical composition
containing same
20140037597 06.02.2014 Senni et al. Sulfated depolymerized derivatives
of exopolysaccharides (eps) from
mesophilic marine bacteria,
preparing same, and uses thereof
in tissue regeneration
Source: http://tgs.freshpatents.com/Exopolysaccharide-bx1.php

bacterial xanthan gum from Xanthomonas campestris have different pat-

ents with varying applications. Some of the recent patenting trends in last
2 years have excelled with lactic acid bacteria, soil inhabitants, and halo-
philes (Table 1.2). More research is needed in the field of EPS producing
thermophiles and psychrophiles, as EPS is produced in large quantities to
survive in extreme harsh environment. Hyaluronan because of its highly
hydrophilic nature is currently used in pharmaceutical industry and has
the highest market value of 1 billion US$ among all other bacterial EPSs,
which indicates a strong market demand for it. But the main hindrance in
using hyaluronan commercially is its high cost, which is around 100,000
US$. Other important bacterial exopolysaccharides like xanthan, gellan,
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As to the great oak flaring to the wind—
To the grave’s low hill as to the Matterhorn
That shoulders out the sky.

And so he came.
From prairie cabin up to Capitol,
One fair Ideal led our chieftain on.
Forevermore he burned to do his deed
With the fine stroke and gesture of a king.
He built the rail-pile as he built the State,
Pouring his splendid strength through every blow,
The conscience of him testing every stroke,
To make his deed the measure of a man.
So came the Captain with the mighty heart:
And when the step of Earthquake shook the house,
Wrenching the rafters from their ancient hold,
He held the ridgepole up, and spiked again
The rafters of the Home. He held his place—
Held the long purpose like a growing tree—
Held on through blame and faltered not at praise.
And when he fell in whirlwind, he went down
As when a kingly cedar green with boughs
Goes down with a great shout upon the hills,
And leaves a lonesome place against the sky.

—Copyright by Doubleday, Page & Co., New York, and used by

kind permission of author and publisher.

HONEST POVERTY
By Robert Burns

Is there for honest poverty

That hings his head, an’ a’ that?
The coward slave, we pass him by—
We dare be poor for a’ that,
The rank is but the guinea’s stamp,
 The man’s the gowd for a’ that.

What though on hamely fare we dine,

Wear hoodin’ gray, an’ a’ that?
Gie fools their silks, and knaves their wine—
A man’s a man for a’ that.
For a’ that, an’ a’ that,
Their tinsel show, an’ a’ that,
The honest man, tho’ e’er sae poor,
Is king o’ men for a’ that.

Ye see yon birkie ca’d a lord,

Wha’ struts, an’ stares, an’ a’ that?
Tho’ hundreds worship at his word,
He’s but a cuif for a’ that.
For a’ that, an’ a’ that,
His riband, star an’ a’ that,
The man of independent mind,
He looks an’ laughs at a’ that.

A prince can mak a belted knight,

A marquis, duke, an’ a’ that!
But an honest man’s aboon his might—
Guid faith, he mauna fa’ that!
For a’ that, an’ a’ that,
Their dignities, an’ a’ that,
The pith o’ sense an’ pride o’ worth
Are higher rank than a’ that.

Then let us pray that come it may

(As come it will for a’ that)
That sense and worth o’er a’ the earth
Shall bear the gree an’ a’ that!
For a’ that, an’ a’ that,
It’s comin’ yet for a’ that,
That man to man the world o’er
Shall brithers be for a’ that.
 AN INCIDENT OF THE FRENCH CAMP
By Robert Browning

You know, we French stormed Ratisbon:

A mile or so away,
On a little mound, Napoleon
Stood on our storming-day;
With neck outthrust, you fancy how,
Legs wide, arms locked behind,
As if to balance the prone brow,
Oppressive with its mind.

Just as perhaps he mused, “My plans

That soar, to earth may fall,
Let once my army-leader Lannes
Waver at yonder wall,”—
Out ’twixt the battery-smokes there flew
A rider, bound on bound
Full-galloping; nor bridle drew
Until he reached the mound.

Then off there flung in smiling joy,

And held himself erect
By just his horse’s mane, a boy:
You hardly could suspect—
(So tight he kept his lips compressed,
Scarce any blood came through)
You looked twice ere you saw his breast
Was all but shot in two.

“Well,” cried he, “Emperor, by God’s grace

We’ve got you Ratisbon!
The marshal’s in the market-place,
And you’ll be there anon
To see your flag-bird flap his vans
Where I, to heart’s desire,
Perched him!” The chief’s eye flashed; his plans
 Soared up again like fire.

The chief’s eye flashed; but presently

Softened itself, as sheathes
A film the mother-eagle’s eye
When her bruised eaglet breathes;
“You’re wounded!” “Nay,” his soldier’s pride
Touched to the quick, he said:
“I’m killed, sire!” And his chief beside,
Smiling the boy fell dead.

THE FOOL’S PRAYER

By Edward Roland Sill

The royal feast was done. The King

Sought some new sport to banish care,
And to his jester cried: “Sir Fool,
Kneel now, and make for us a prayer!”

He bowed his head, and bent his knee

Upon the monarch’s silken stool;
His pleading voice arose: “O Lord,
Be merciful to me, a fool!

“No pity, Lord, could change the heart

From red with wrong to white as wool;
The rod must heal the sin; but, Lord,
Be merciful to me, a fool!

“’Tis not by guilt the onward sweep

Of truth and right, O Lord, we stay;
’Tis by our follies that so long
We hold the earth from heaven away.

“These clumsy feet, still in the mire,

Go crushing blossoms without end;
These hard, well-meaning hands are thrust
 Among the heart-strings of a friend.

“The ill-timed truth we might have kept—

Who knows how sharp it pierced and stung?
The word we had not sense to say—
Who knows how grandly it had rung?

“Our faults no tenderness should ask,

The chastening stripes must cleanse them all;
But for our blunders—oh, in shame
Before the eyes of heaven we fall.

“Earth bears no balsam for mistakes;

Men crown the knave and scourge the tool
That did his will; but Thou, O Lord,
Be merciful to me, a fool!”

The room was hushed; in silence rose

The King, and sought his gardens cool,
And walked apart, and murmured low,
“Be merciful to me, a fool!”

IKE WALTON’S PRAYER

By James Whitcomb Riley

I crave, dear Lord,

No boundless hoard
Of gold and gear,
Nor jewels fine,
No lands, nor kine,
Nor treasure-heaps of anything.—
Let but a little hut be mine
Where at the hearthstone I may hear
The cricket sing,
And have the shine
Of one glad woman’s eyes to make,
For my poor sake,
 Our simple home a place divine;—
Just the wee cot—the cricket’s chirr—
Love, and the smiling face of her.

I pray not for

Great riches, nor
For vast estates, and castle-halls,—
Give me to hear the bare foot-falls
Of children o’er
An oaken floor,
New-rinsed with sunshine, or bespread
With but the tiny coverlet
And pillow for the baby’s head;
And pray Thou, may
The door stand open and the day
Send ever in a gentle breeze,
With fragrance from the locust-trees,
And drowsy moan of doves, and blur
Of robin-chirps, and drone of bees,
With after hushes of the stir
Of intermingling sounds, and then
The good-wife and the smile of her
Filling the silences again—
The cricket’s call,
And the wee cot,
Dear Lord of all,
Deny me not!

I pray not that

Men tremble at
My power of place,
And lordly sway,—
I only pray for simple grace
To look my neighbor in the face
Full honestly from day to day—
Yield me his horny palm to hold,
And I’ll not pray
For gold;—
The tanned face, garlanded with mirth,
It hath the kingliest smile on earth—
The swart brow, diamonded with sweat,
Hath never need of coronet.
And so I reach,
Dear Lord, to Thee,
And do beseech
Thou givest me
The wee cot, and the cricket’s chirr,
Love, and the glad sweet face of her.

THE LAST TATTOO

(DEDICATED TO THE REMAINING MEMBERS OF THE G. A. R.)
By John Milton Scott

Blow soft and low, O fife, to-day,

For thinner grow our ranks of blue;
The years our priceless heroes slay
Until the fewer grow more few
And dear familiar voices still
As patriot graves with patriots fill.

Beat soft and low, O drum, to-day

As tho’ you were a trembling sigh;
Dear, paling lips their last prayer say
While more and more dear comrades die,
Their feet across the dark door’s sill
As patriot graves with patriots fill.

Float gently, flag, and droop to-day

As droop the grasses o’er the brook;
They few and fewer grow each May;
For those we love we vainly look,
So many sunny smiles grow chill
As patriot graves with patriots fill.
O hush, exultant sounds, to-day!
For they are gone, these ranks on ranks
Who loved to hear the shrill fife play
And with their comrades render thanks,—
O Time, how many brave you kill
And patriot graves with patriots fill!

O, silken every sound to-day

And soften every bugle brave!
We can not bid our vision stay
From seeing our last comrade’s grave,—
O dear, last-billowed comrade hill!
Lone, last of graves our patriots fill!

O angel choir, wing low that day

And silken sing a Bethlehem strain
And all your pipes of welcome play!
Altho’ their brothers they have slain,
In brother love their hands grow white,
For what they did they thought was right.

Not into graves, but into skies,

Where love and life eternal are!
God’s reveille has bid them rise
Beyond earth’s sun and morning star
Where all men just love-brothers be
As One once said in Galilee.
 LYRIC SELECTIONS IN POETRY

L’ENVOI
By Rudyard Kipling

When Earth’s last picture is painted, and the tubes are twisted and
dry,
When the oldest colors have faded, and the youngest critic has died,
We shall rest, and, faith, we shall need it—lie down for an æon or
two,
Till the Master of All Good Workmen shall put us to work anew!

And those that were good shall be happy: they shall sit in a golden
chair;
They shall splash at a ten-league canvas with brushes of comet’s
hair;
They shall find real saints to draw from—Magdalene, Peter, and
Paul;
They shall work for an age at a sitting and never be tired at all!

And only the Master shall praise us, and only the Master shall blame;
And no one shall work for money, and no one shall work for fame,
But each for the joy of working, and each, in his separate star,
Shall draw the thing as he sees it for the God of Things as They Are!

OUR FLAG
By John Milton Scott
(Written expressly for this work)

’Tis homes make a country and children make homes

Where the heart is held true and the truth never roams,
Where joy is abounding and life overflows,
And love is the rapture which every one knows,
Where the pride of all hearts is the boy at his play,
His eyes like the sun overshining the day,
His cheeks like the roses his grandmother grew,
Shot through with a dimple the size of a dew
Which gives to his smile irresistible grace
As his sister looks down in his uplifted face;
In such bright-shining faces our true eyes may see
The love which shall honor our Flag of the Free.

Whatever they say, however they brag,

’Tis these put the red in our flag,
Not our patriot deaths,
Not our gold nor our lands,
Not our fifes nor our drums,
Not our captains’ commands;
But the homes and the children,
Our country’s true worth,
The grace and the greatness,
The glory of earth,—
The children, the children,
Light-hearted and free
Who play in the sunshine
And pray at our knee,—
O ’tis homes and the children
Where joys never lag,—
’Tis these keep the red in our flag,
Our flag of the red, white and blue
To which home-hearts and child-hearts are true.

’Tis the mothers of men who give us our lives,

Who give us our children, who give us our wives,—
O ’tis woman’s great heart which hallows and trues
And makes the straight line to which our ax hews;
’Tis our wives and our daughters who keep our feet straight
In the paths where God’s honor and man’s honor mate;
Where woman is honored as mother and wife
The war drum throbs never nor screams the shrill fife;
There freedom and justice with honor and truth
Keep our nation alive in the vigor of youth,—
There smile the bright heavens which never wax old,
And there a free flag will forever unfold.

Whatever they say, however they brag,

’Tis these put the white in our flag.
Not our patriot deaths,
Not our gold nor our lands,
Not our fifes nor our drums,
Not our captains’ commands;
But the women, the women,
Our country’s true worth,
The grace and the greatness,
The glory of earth,—
The women, the women
With hands free to do
Will build a great state
As tender, as true,—
O the free hearts of women,
Unchoked by hate’s slag,
’Tis these keep the white in our flag,
Our flag of the red, white and blue
To which our love-honor is true.

’Tis city and country where good neighbors live

And their love and their labors so joyously give,
That bodies be clothed and hungers be fed,
That with love in our heart and truth in our head,
Great thoughts and great dreams together we share
As we meet at the market or kneel at our prayer;
When our feet at one fireside make mellow our speech
As together we plan our ideals to reach,
What visions together we wisely may find
To make our earth friendlier, truer, more kind,
That our flag tell the beauty of man to the world
Wherever in freedom and justice unfurled.
 Whatever they say, however they brag,
’Tis these put the blue in our flag,—
Not our patriot deaths,
Not our gold nor our lands,
Not our fifes nor our drums,
Not our captains’ commands;
But our neighbors, good neighbors,
Our country’s true worth,
The grace and the greatness,
The glory of earth,—
Our neighbors, good neighbors,
Without hurt or hate,
Whose love and whose labors
Have builded our state,—
O, ’tis neighbors, good neighbors
Whose hearts never sag,—
’Tis these keep the blue in our flag,
Our flag of the red, white and blue
Which good neighbors ever renew.

“Where the vision is not the people will die,”

Said the word of truth sounding God’s voice from the sky;
As trees draw their vigor from the sun-quickened air,
That they grow globing fruits which make the year fair,
So the dreams and the visions of young men and maids
Show heavens of glory through which our flag wades
Where dreams come awake and visions fulfill
In a world that’s so human no hatreds can kill,—
There the noble ideal forever leads on;
There are stars for our nights and suns for our dawn,
The dreamers and lovers by God justified
In a love-world Christ visioned and for which He died.

Whatever they say, however they brag,

’Tis these put the stars in our flag;
Not our patriot deaths,
Not our gold nor our lands,
 Not our fifes nor our drums,
Not our captains’ commands;
But our visions and dreams,
Our country’s true worth,
The grace and the greatness,
The glory of earth,—
The dreamers, the dreamers,
Love-visioned by God,
Bring the stars to our earth,
To the stars lift our sod;
’Tis the visions and dreams,
Scaling mountain and crag,—
’Tis these keep the stars in our flag,
Our flag of the red, white and blue
Which from dream hearts unfolded and flew.

Without a free earth there’s no sky for our flag,

And vainly of rights and of freedom we brag;
There Tyranny still is exploiting its slaves,
And we buy rights to live, and, then, buy our graves;
Such flag of the free our poverty mocks
As the ways of progression the privileged Greed blocks;
But free land and free men make our flag’s holy sky,
And our winds never weary with Poverty’s cry.
Here man to his fellow is never for sale,
And free men to free men give good neighbor hail,
In whose cheery words we ever shall hear
The flap of our flag and our patriots’ cheer.

Whatever they say, however they brag,

’Tis these make a sky for our flag.
Not our patriot deaths,
Not our gold nor our lands,
Not our fifes nor our drums,
Not our captains’ commands;
But free land and free men,
Our country’s true worth,
 The grace and the greatness,
The glory of earth,—
The freemen, the freemen
With brothering palms
Who love one another
And praise God in psalms,—
O ’tis free land and free men,
And no poverty’s rag,—
’Tis these make a sky for our flag,
Our flag of the red, white and blue
To which freemen forever are true.

THANKS FOR AMERICA AND ITS FLAG!

By John Milton Scott

Dear God, whose Heart is Freedom’s home,

Whose joy is that Thine earth be free,
We thank Thee for our native land
And for its growing liberty;
We praise Thee for its holy Flag
By precious blood so consecrate,
A banner born of patriot love
And weaving in its folds no hate.

Its glory shines anear, afar

On murk and midnight tyranny,
A streak of Freedom’s blessed dawn
Which tyrant-hating eyes do see,
And, taking heart, they braver toil
Their country’s liberties to gain,
Till some bright day no land is found
But sings great Freedom’s glad refrain.

Our banner’s red speaks patriots’ blood,

Its white a noble faithfulness,
Its blue of truth, and all its stars
 Are hopes for grander days to bless;
For it, for all who made it great,
The living ones or sacred dead,
We thank Thee through our smiles and tears
Who love its white and blue and red.

We’ll take it as their sacred trust,

And, as they, keep it true and tried,
To pass it stainless when we die,
That all its love and truth abide.
O may it deeper meanings gain
Through all the changing, growing years,
Fulfilling every liberty,
The rainbow of each captive’s tears.

And may it brother other flags,

Behold in each some human worth,
Till peace divine whites each and all,
A fellowship that fills our earth;
O then no enemy is found
Upon the wide world’s mother-breast,
In every heart Christ-gentleness,
And every flag with Christ-love blest.

THERE WAS A MAN

By David Starr Jordan

There was a man who saw God face to face;

His countenance and vestments evermore
Glowed with a light that never shone before,
Saving from him who saw God face to face.
And men, anear him for a little space,
Were sorely vexed at the unwonted light.
Those whom the light did blind rose angrily;
They bore his body to a mountain height
And nailed it to a tree; then went their way,
 And he resisted not nor said them nay,
Because that he had seen God face to face.

There was a man who saw Life face to face;

And ever as he walked from day to day,
The deathless mystery of being lay
Plain as the path he trod in loneliness;
And each deep-hid inscription could he trace;
How men have fought and loved and fought again;
How in lone darkness souls cried out for pain;
How each green foot of sod from sea to sea
Was red with blood of men slain wantonly;
How tears of pity warm as summer rain
Again and ever washed the stains away,
Leaving to Love, at last, the victory.
Above the strife and hate and fever pain,
The squalid talk and walk of sordid men,
He saw the vision changeless as the stars
That shone through temple gates or prison bars,
Or to the body nailed upon the tree,
Through each mean action of the life that is,
The marvel of the Life that yet shall be.

TO A MOCKING-BIRD IN CALIFORNIA
By John Milton Scott
(Written expressly for this Reader)

“Gertie! Gertie! Gertie!” “Peter! Peter! Peter!”

In the morn when wings are fleeter,
In the noon when skies are bright
You call these names in wild delight.

Who is this “Gertie,” who this “Peter”

Who go rapturing through your meter?

Did you hear beneath your tree

These names called in ecstasy,
When your heart caught fire, and flames
In love—calling these dear names?

Did Gertie’s heart go twitter, tweeter

When she heard the call of Peter?
Did Peter’s heart beat wild and hurty
When he heard the call of Gertie?
And who this “Gertie,” who this “Peter”
Teaching you such silk-toned meter?

Mocking-birds have thuswise sang

Since Time’s song of joy upsprang,
And to each your lyric brought
Something that his spirit sought;
Some perfect which the heart still dreams,
Though Sorrow’s sands fill all the streams,
No waters in their olden place,
Nor in your eyes the olden face;
Nor in your ears that olden voice;
Yet something makes us still rejoice
And rapture dreams with mating birds
As if our hearts filled with their words.
“Gertie! Gertie! Gertie!” “Peter! Peter! Peter!”
Who set the mock-bird’s throat to meter?

Maybe Eve called Adam so

In dark days when shadowed woe;
Thus called Adam in the dark
When Eve’s heart in fear called “Hark!”

Or might it be in Abram’s time

Love taught you this sweetheart rhyme,—
Some trembling tones in Haran’s tongue
Ere the world-famed march begun?

Or when Ruth gleaned th’ alien corn,

Maybe, then, your song was born,
’Neath the whisp’ring palms one hour
Where you refuged from a shower?

Or some youth in David’s band

Taught your throat in Israel’s land,—
Maybe David’s self, before
His song-heart the king-cares wore,
When his boy-heart whistled true
As wildly free as now are you?
His psalm of joy you often heard
Which now you sing without his word?
With his maiden, were you there
When his first kiss was like a prayer?

You heard his son, the song-wise king,

In heart-beat, song-beat rapturing
So fine, his songs are scriptures now
In which true lover hearts may bow,
Learning how to rapture speech,
That heart to heart through words may reach?

Maybe Greek, when Helen’s charm

Made old Homer’s heroes arm?
Or some dark-eyed odalisque
When Egyptian lips were kist?
Or did some Roman maiden sigh
When Cæsar’s soldier said Good-by?

Spake some shepherd on that night

Just before the Christmas light
Burst upon the flocks so still,
And the winds with angels fill,—
Spake some shepherd in a tryst
Just before he saw Babe-Christ?

Maybe she, the Magdalene,

Ere the ways of shame were seen,
Heard and said ’neath purpling vine
 These sweet, holy words of thine?
Did she find, Christ-cleansed and pure,
Him whose words were thy throat’s lure,
And did they both together then
Tell the Christ’s love for all men?

Or some Christian’s true heart-call

Ere the martyr’s cup of gall
Pressed the lips by love caressed
Which unto death the Christ confessed?

Or later, with the centuries gone,

Your song, in a Castilian dawn,
Raptures to a red, red rose,
And Columbus stronger grows
For his journey far away
Within his heart your brave, bright lay?

Wept black eyes in sun-bright Spain

When dared his crew the unknown main?
Those sorrow-tones you’re calling now,
Your rippling wave-sounds from his prow;
We almost hear the whistling sails
In your wild song which never fails
Of courage which can travel far
To bring a joy back from a star,
Or bring the moon’s remotest beam
To build in joy a Jacob’s dream

O’er which the song-glad angels go

To bring the smiles of heaven below,
That hearts which pillow on hard stone
May have a song for every moan.

Did they hear you in that breeze

Blowing o’er uncharted seas,
Remembering, then, the night-eyed maid
In whose smile all fears were laid?
Perhaps your lure was on the wave,
The first call that the New World gave,
As Fate urged him on and on,
Into that splendid glory drawn
Wherein a New World was his gift
In which our starry flag can lift,
Proclaiming all men equal, free,
A world of brothers,—yet to be?

O ’twere fine, if we but knew

’Twas your song hailed that brave crew,—
Columbus’ ears enraptured by
Your song-flights in this new sky,
By your welcome to this shore
Which welcomes exiles ever more,—
All song-tongues your singings span,
You a true American
With welcome for all alien feet
Who with Freedom here would meet.

Did doe-eyes in joyous France

To such words in rapture dance,
Giving that charmèd land its grace
Where each face, a lover’s face,
Sets the heart to music’s notes
As they thrill from bird-sweet throats?

Did Lafayette from your free wings

Catch the song which Freedom sings;
As he hearkened to your cheer,
Growing dearer and more dear,
Till upon our country’s soil
He nobly wrought in battle-toil,
That our flag might float as free
As your song-flights in his tree?

But my questions lose their way,—

You sing what tender lovers say,—