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MICROBIAL FERMENTATION
AND ENZYME TECHNOLOGY
Edited by
Hrudayanath Thatoi, Pradeep K. Das Mohapatra,
Sonali Mohapatra and Keshab C. Mondal
Microbial Fermentation
and Enzyme Technology
Microbial Fermentation
and Enzyme Technology

Edited by
Hrudayanath Thatoi
(Department of Biotechnology, North Orissa University)
Pradeep K. Das Mohapatra
(Department of Microbiology, Raiganj University)
Sonali Mohapatra
(Department of Biotechnology, College of Engineering
and Technology)
Keshab C. Mondal
(Department of Microbiology, Vidyasagar University)
CRC Press
Taylor & Francis Group
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© 2020 by Taylor & Francis Group, LLC

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Library of Congress Cataloging‑in‑Publication Data

Names: Thatoi, H. N., editor.

Title: Microbial fermentation and enzyme technology / [edited by]
Hrudayanath Thatoi (Department of Biotechnology, North Orissa
University), Pradeep K. Das Mohapatra (Department of Microbiology,
Raiganj University), Sonali Mohapatra (Department of Biotechnology,
College of Engineering and technology) and Keshab C. Mondal (Department
of Microbiology, Vidyasagar University).
Description: Boca Raton : CRC Press, [2020] | Includes bibliographical
references and index.
Identifiers: LCCN 2019059892 | ISBN 9780367183844 (hardback) | ISBN
9780429061257 (ebook)
Subjects: LCSH: Enzymes--Industrial applications. | Fermentation. |
Fermentation products industry.
Classification: LCC TP248.E5 M53 2020 | DDC 660/.28449--dc23
LC record available at https://lccn.loc.gov/2019059892

Visit the Taylor & Francis Web site at

http://www.taylorandfrancis.com

and the CRC Press Web site at

http://www.crcpress.com
Contents

Preface........................................................................................................................vii
Editors..........................................................................................................................ix
Contributors.............................................................................................................. xiii
List of Abbreviations................................................................................................xvii

1. Introduction of Fermentation and Enzyme Science........................................ 1

Monika Choudhary, Sunanda Joshi and Nidhi Srivastava

2. α-Amylases: An Overview on Molecular Structure

and Biotechnological Perspectives................................................................... 13
Saptadip Samanta

3. Study of α-Amylase Based on their Compositional Parameters of Its

Gene Along with Its Protein Structure........................................................... 41
Sunil Kanti Mondal, Satadruta Das and Madhab Kumar Sen

4. Microbial Enzymes in Food Industry: Types and Applications................... 61

G. Singhal, S.S. Bhagyawant and Nidhi Srivastava

5. Fermented Foods for Health: Processes and Prospects................................ 73

Sudip Some and Amit Kumar Mandal

6. Advances in Enzymatic Applications in Food Industry................................ 85

Kashif Ameer, Protiva Rani Das and Jong-Bang Eun

7. Role of Enzymes in Development of Functional Foods

and Food Products............................................................................................99
Protiva Rani Das, Kashif Ameer and Jong-Bang Eun

8. Application of Immobilized Cells and Enzymes in the Food Industry...... 115

Judit Krisch, Erika Beáta Kerekes, Miklós Takó and Csaba Vágvölgyi

9. Dextransucrase: A Microbial Enzyme with Wide Industrial

Applications..................................................................................................... 129
Jagan Mohan Rao Tingirikari and Shadab Ahmed

10. Trends in Biosensors and Role of Enzymes as Their Sensing Element

for Healthcare Applications........................................................................... 147
Tathagata Adhikary, Amalesh Nanda, K. Thangapandi, Shantonu Roy
and Saikat Kumar Jana

v
vi Contents

11. Application of Bile Salt Hydrolase Enzyme in Cholesterol Lowering....... 165

Kriti Ghatani

12. Fermentative and Enzyme-Assisted Production of Phenolic

Antioxidants from Plant Residues................................................................. 175
Miklós Takó, Carolina Zambrano, Alexandra Kotogán,
Erika Beáta Kerekes, Tamás Papp, Judit Krisch and Csaba Vágvölgyi

13. Relevance of Microbial Enzymes in Textile Industries Emphasizing

Metabolic Engineering Panorama................................................................. 195
Dipankar Ghosh and Priyanka Talukdar

14. Microbial Degradation of Organophosphate Pesticides: A Review...........207

Sangeeta Raut

15. Microbial Production of Xylitol: A Cost-Effective Approach..................... 227

Yogita Lugani, Balwinder Singh Sooch, Vinita Dheeran
and Sachin Kumar

16. Enzymes: Key Role in the Conversion of Waste to Bioethanol................... 257

Abhijeet Thakur, Kedar Sharma, Kaustubh C. Khaire, Vijay S. Moholkar
and Arun Goyal

17. Microbial Laccase: A Vanguard Biocatalyst and Its Potentiality

towards Industrial Applications.................................................................... 269
Priyanka Ghosh and Uma Ghosh

18. Biofuel Cellulases: Diversity, Distribution and Industrial Outlook........... 283

Lavika Jain and Deepti Agrawal

19. Enzymatic Hydrolysis of Lignocellulosic Biomass Using Engineered

Microorganisms and In Silico Approaches for Enhanced Enzyme
Production: A Review..................................................................................... 299
Manish Paul, Sonali Mohapatra and Hrudayanath Thatoi

20. Agrowaste to Ethanol: Orchestrated by Enzymes from Microbes............ 319

Modhurima Chakraborti and Krishnendu Acharya

Index......................................................................................................................... 335
Preface

Microbes are the key players in converting the natural renewable substrates to value-
added products such as enzymes, organic acids, alcohols, polymers and more through
fermentation. It is a process of microbial biotechnology. Today, enhanced production
of economically important fermentation products has benefited from targeted genetic
engineering techniques to established industrial microbial strains. Enzymes are
products of living organisms and have been used in the industry for many years
due to their catalytic activities, repeated usability, etc. Enzyme has a broad range of
applications from food to detergents. Most enzymes are commercially available and
used to enhance the product quality in food, detergents, leather, paper, cosmetics, and
pharmaceuticals. The present book is dealing with 20 nos of articles covering with
many frontier areas of microbial fermentation and enzyme technology. To start with
a lucid introduction on fermentation and enzyme science, adding with an overview
on molecular structure, genetic architecture and biotechnological perspectives
of α-amylase. A well narration is presented on the role of microbial enzymes in
food industry, the importance of fermented food in healthcare sectors and also the
enzymatic contribution in functional foods and food products. The applications
of immobilized cells and enzymes have also been well documented in the present
book. Production of antioxidants and cholesterol lowering process were discussed.
Relevance of microbial enzymes in textile industries was also presented. Applications
of microbes in organophosphate pesticide degradation and the importance of enzymes
in fuel biotechnology is also noteworthy. The book also illuminates about the
enzymatic hydrolysis of lignocellulosic biomass using engineered microorganisms
and concomitant production of ethanol.
The book is written in a simple and clear text with a number of figures, tables, and
illustrations that will help the reader to better understand the subject matter. This vol-
ume is the collective efforts of several researchers, scientists, graduate students, and
postdoctoral fellows across the world who are well known in their respective areas of
specialization. This book will be a useful reference resource on fermentation, enzyme
technology, food microbiology, and biochemistry, biotechnology for researchers,
teachers and students. We express our gratitude to all of the contributing authors who
helped us enormously with their timely contributions in this volume. We are also
grateful to the reviewers who have voluntarily agreed and spared their precious time
in critically reviewing the manuscript.

vii
Editors

Dr Hrudayanath Thatoi is serving as a professor

in the Department of Biotechnology of North Orissa
University. He obtained his MSc, MPhil and PhD in
Botany from Utkal University. He is also working
as the Director, Centre for Similipal Studies, North
Orissa University. The teaching and research interests
of Dr Thatoi include Microbiology, Molecular biol-
ogy, and Industrial and Pharmaceutical Biotechnology.
Dr Thatoi has implemented several research projects
funded by University Grants Commission-Department
of Atomic Energy (UGC-DAE) Government of
India, Department of Science and Technology (DST)
Government of Odisha and Forest Department,
Government of Odisha. Till date, Dr Thatoi is credited with publication of 15 books
including two textbooks and more than 250 research papers in national and international
journals, proceedings of conferences and book chapters. He serves as member of several
scientific societies and editorial boards of the national and international journals. He is a
recipient of Fellow of Society of Applied Biotechnology. So far, he has guided 15 PhD
scholars and a number of MTech, BTech and MPhil students for their thesis/disserta-
tion works. He has visited Bangkok and Nepal to attend International Conferences on
Biodiversity and Biotechnology. Dr Thatoi is a Functional Area Expert in Ecology and
Biodiversity, being accredited by Quality Council of India (QCI), Government of India.

Dr Pradeep K. Das Mohapatra obtained his

PhD in 2008 from Vidyasagar University. He is
now working as an associate professor and head
in the Department of Microbiology, Raiganj
University, Uttar Dinajpur, West Bengal, India. He
is also acting as a Director of Professor A. K.
Bothra Environment Conservation Centre, Raiganj
University. Dr Mohapatra has significantly con-
tributed in the fields of enzyme biotechnology,
environmental and industrial microbiology. He has
worked on microbial nutrition and diversity, fer-
mentation, probiotics, immobilization, bioinfor-
matics and enzymes like tannase, xylanase, amylase, cellulase, keratinase, etc. He is
presently working on Metabiotics. Dr Mohapatra has published more than 118 origi-
nal research papers in reputed National and International Journals, 5 book chap-
ters, 5 review papers, 6 popular articles and filed 2 patents with citation of 2186,
h-index 27 and i10 index 58. He also attended 8 training programmes on microbial

ix
x Editors

research, covered 40 national and international conferences and delivered 16 invited

lectures. Dr Mohapatra has more than 11 years of research and teaching experience
and received research funds from UGC, Government of India, Biostadt India Ltd.,
Sanzyme Private Limited, etc. Seven students have been awarded a PhD under his
supervision. Dr Mohapatra received the Jr. Scientists of the Year Award 2008 and the
Environmentalist of the Year Award 2009 from the National Environmental Science
Academy, New Delhi. He was nominated as a Fellow of the Society for Applied
Biotechnology in the year 2011. He is an editorial board member of two journals and
a reviewer of many journals published by Elsevier, Blackwell, Springer and Taylor &
Francis Group. Dr Mohapatra is also associated with many academic and research
societies including AFB (France), NESA (New Delhi), BRSI (Trivandrum), AMI
(New Delhi), PBVM (Kolkata), The Indian Science Congress Association (Kolkata),
SAB (Dharward), etc.

Mrs Sonali Mohapatra pursued her BTech and

MTech in Industrial Biotechnology from Dr M.G.R
University, Chennai. Mrs Mohapatra is pursuing
her PhD in College of Engineering and Technology,
Ghatika, which is one of the premiere engineer-
ing colleges in the state of Odisha. She is currently
working as an assistant professor in the Department
of Biotechnology, College of Engineering and
technology, Bhubaneswar. Mrs Mohapatra has sig-
nificantly contributed in the fields of biofuel tech-
nology and environmental microbiology. She has
worked on fermentation, immobilization and
enzymes like xylanase, amylase, cellulase, hemicellulase, etc. Her research also deals
with the utilization of effluent produced during bioethanol production. Mrs Mohapatra
has been working on biofertilizer production using industrial waste biomass. She has
been also working on a metabolomics approach for improving the ethanol tolerance
capacity of ethalogenic microorganisms. She received international award for excel-
lence in teaching from South Asian Association, Bangkok, Thailand, in the year 2017
and young scientist award for best paper presentation in the year 2018. Mrs Mohapatra
has published more than 15 original research papers in reputed international journals,
8 international book chapters and filed 1 patent. She has covered 25 national and inter-
national conferences and has more than 6 years of research and teaching experience.
She has guided more than 14 B.tech and 5 M.tech students for their dissertation works.
Editors xi

Dr Keshab C. Mondal completed M Sc and Ph D in

Physiology from Vidyasagar University and joined
as Lecturer in the Department of Physiology, Raja
N L Khan Women’s College, in 1997 and in 2004,
in the Department of Microbiology, Vidyasagar
University, where he become Professor in 2014. His
research efforts have been focused on understand-
ing the diversity of intestinal bacteria particularly
during environmental stress. The nutritional com-
ponents, bacterial diversity and bioactive enzymes
in traditional fermented foods is also his research
field. He is developing functional foods having antiobesity and antitoxic potentiali-
ties by using probiotic bacteria and yeasts from traditional fermented foods. He has
published over 130 scientific papers, 4 reviews and 6 book chapters with citation of
3120, h-index 31 and i10 index 97. He handled 10 sponsored research projects. He was
the recipient of Indo-Hungarian fellowship in 2014 and Tempus Public Foundation
(TPF) fellowship, Hungary, for the year 2015–2016. He is a member of the Asian
Federation of Biotechnology (AFB), Indian Science Congress Association (ISCA),
Biotech Research Society of India (BRSI), Indian National Academy of Stress
Sciences (INASS), Association of Microbiologists of India (AMI) and Physiological
Society of India (PSI). He has been conferred with a Gold Medal for scoring first rank
in the MSc exam. He is a reviewer for manuscripts of several Indian and international
journals. He has over 20 years of research and teaching experience. He has produced
16 PhD students, and presently 5 students are pursuing PhD under his guidance.
Contributors

Krishnendu Acharya S.S. Bhagyawant

Molecular and Applied Mycology and School of Studies in Biotechnology
Plant Pathology Laboratory Jiwaji University
University of Calcutta Gwalior, India
Department of Botany
Kolkata, India Modhurima Chakraborti
Molecular and Applied Mycology and
Tathagata Adhikary
Plant Pathology Laboratory
National Institute of Technology
Department of Botany
Yupia, India
University of Calcutta
Deepti Agrawal Kolkata, India
Material Resource Efficiency Division
CSIR-Indian Institute of Petroleum Monika Choudhary
Dehradun, India Banasthali University
and
Academy of Scientific and Innovative Satadruta Das
Research (AcSIR) Department of Biotechnology
CSIR-HRDC Campus Visva-Bharati University
Ghaziabad, India Bolpur, India

Shadab Ahmed Protiva Rani Das

Institute of Bioinformatics and Department of Food Science and
Biotechnology Technology
Savitribai Phule Pune University Graduate School of Chonnam National
Pune, India University
Gwangju, South Korea
Kashif Ameer
Department of Food Science and
Technology Vinita Dheeran
Graduate School of Chonnam National Department of Botany
University and PMAS-Arid M.M.H. College
Agriculture University Ghaziabad, India
Gwangju, South Korea
Jong-Bang Eun
and
Graduate School of Chonnam National
Institute of Food and Nutritional University
Sciences Department of Food Science and
PMAS-Arid Agriculture University Technology
Rawalpindi, Pakistan Gwangju, South Korea

xiii
xiv Contributors

Kriti Ghatani Sunanda Joshi

Department of Food Technology Department of Bioscience and
University of North Bengal Biotechnology
Siliguri, India Banasthali University
Rajasthan, India
Dipankar Ghosh
Department of Biotechnology Kaustubh C. Khaire
(Microbial Engineering & Algal Centre for Energy
Biotechnology Laboratory) Indian Institute of Technology Guwahati
JIS University Guwahati, India
Kolkata, India
Erika Beáta Kerekes
Priyanka Ghosh Department of Microbiology
Food Technology and Biochemical Faculty of Science and Informatics
Engineering University of Szeged
Jadavpur University Szeged, Hungary
Kolkata, India
Alexandra Kotogán
Uma Ghosh Department of Microbiology
Jadavpur University Faculty of Science and Informatics
University of Szeged
Arun Goyal Szeged, Hungary
Department of Biosciences and
Bioengineering and Centre for Judit Krisch
Energy Institute of Food Engineering
Indian Institute of Technology Faculty of Engineering
Guwahati University of Szeged
Guwahati, India Szeged, Hungary

Lavika Jain Sachin Kumar

Material Resource Efficiency Biochemical Conversion Division
Division Sardar Swaran Singh National Institute
CSIR-Indian Institute of of Bio-Energy
Petroleum and Academy of Wadala Kalan, Kapurthala
Scientific and Innovative
Research (AcSIR) Yogita Lugani
Dehradun, India Enzyme Biotechnology Laboratory
Department of Biotechnology
and
Punjabi University
Academy of Scientific and Innovative Patiala, India
Research (AcSIR)
Ghaziabad, India Amit Kumar Mandal
Chemical Biology Laboratory
Saikat Kumar Jana Department of Sericulture
National Institute of Technology Raiganj University
Yupia, India Raiganj, India
Contributors xv

Sonali Mohapatra Saptadip Samanta

Department of Biotechnology Department of Physiology, Midnapore
College of Engineering and Technology Paschim Medinipur
Bhubaneswar, India Midnapore College
Midnapore, India
Vijay S. Moholkar
Centre for Energy, India, and
Madhab Kumar Sen
Department of Chemical
Department of Agroecology and Crop
Engineering
Production
Indian Institute of Technology Guwahati
Faculty of Agrobiology, Food and
Guwahati, India
Natural Resources
Sunil Kanti Mondal Czech University of Life Sciences
Department of Biotechnology Prague, Czech Republic
The University of Burdwan
Bardhaman, India Kedar Sharma
Department of Biosciences and
Amalesh Nanda Bioengineering
National Institute of Technology Indian Institute of Technology
Yupia, India Guwahati
Guwahati, India
Tamás Papp
Department of Microbiology
Faculty of Science and Informatics G. Singhal
University of Szeged Department of Bioscience and
Szeged, Hungary Biotechnology
Banasthali Vidyapith
and
Rajasthan, India
MTA-SZTE “Lendület” Fungal
Pathogenicity Mechanisms Research
Group Sudip Some
University of Szeged Chemical Biology Laboratory
Szeged, Hungary Department of Sericulture
Raiganj University
Manish Paul Raiganj, India
Department of Biotechnology
North Orissa University Balwinder Singh Sooch
Baripada, India Enzyme Biotechnology Laboratory
Department of Biotechnology
Shantonu Roy Punjabi University
National Institute of Technology Patiala, India
Yupia, India

Sangeeta Raut Nidhi Srivastava

School of Pharmaceutical Sciences Department of Bioscience and
Siksha ‘O’ Anusandhan Biotechnology
Deemed to be University Banasthali University
Bhubaneswar, India Rajasthan, India
xvi Contributors

Miklós Takó Hrudayanath Thatoi

Department of Microbiology Department of Biotechnology
Faculty of Science and Informatics North Orissa University
University of Szeged Baripada, India
Szeged, Hungary
Jagan Mohan Rao Tingirikari
Priyanka Talukdar Department of Biotechnology
Microbial Engineering Group National Institute of Technology
Department of Biotechnology Tadepalligudem, India
JIS University
Kolkata, India
Csaba Vágvölgyi
Abhijeet Thakur Department of Microbiology
Carbohydrate Enzyme Biotechnology Faculty of Science and
Laboratory Informatics
Department of Biosciences and University of Szeged
Bioengineering Szeged, Hungary
Indian Institute of Technology
Guwahati, India Carolina Zambrano
Department of Microbiology
K. Thangapandi Faculty of Science and Informatics
National Institute of Technology University of Szeged
Yupia, India Szeged, Hungary
List of Abbreviations

2G second generation
ABTS 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
AE auxiliary enzymes
Ala alanine
Arg arginine
Asn asparagine
BG β–glucosidases
BP British Petroleum
BSH bile salt hydrolase
BTU British thermal units
CAGR compound annual growth rate
CastP computed atlas of surface topography of proteins
CAZy carbohydrate-active enzymes
CAZy carbohydrate-active enzymes database
CBH cellobiohydrolase
CBM calcium binding module
CBiM carbohydrate binding module
CBP consolidated bioprocessing
CCD central composite design
CD catalytic domain
CDH cellobiose dehydrogenase
CE carbohydrate esterase
CBH cellobiohydrolase
Cel5A Cellulase5A
Cel7A Cellulase7A
Cel7B Cellulase7B
CFU colony forming unit
CHD coronary heart disease
CMC carboxy methyl cellulose
CO2 carbon dioxide
Coh cohesin
CpHMD constant pH molecular dynamics
CVD cardiovascular diseases
Cys cystine
DAD diode array detector
DDT dichlorodiphenyltrichloroethane
DM dry matter
Doc dockerin
DP degree of polymerization
DPPH 1,1-diphenyl-2-picrylhydrazyl
EC Enzyme Commission

xvii
xviii List of Abbreviations

ECD electron capture detector

ED Entner-Doudoroff pathway
EG endoglucanase
EIA Energy Information Administration
EM Embden-Meyerhof pathway
ENc expected effective number of codons
ESBRIE valuating the salt BRIdges in proteins
EU European Union
FAO Food and Agriculture Organization
FDA Food and Drug Administration
FPase filter paper degrading activity
GAE gallic acid equivalent
GBD global burden of disease
GH glycoside hydrolase
GHG green house gas
Gly glycine
GOR IV Garnier-Osguthorpe-Robson IV
GPD glucose-6-phosphate dehydrogenase
GRAS Generally Recognized as Safe
GSA generalized simulated annealing
HCA hydrophobic cluster analysis
HDL high density lipoprotein
His histidine
HPP high hydrostatic pressure
HRMS high resolution MS
Ile isoleucine
IT ion trap
IU International Unit
LAB lactic acid bacteria
LDL low density lipoprotein
Leu leucine
LiP lignin peroxidase
LPMOs lytic polysaccharide monooxygenases
LMS laccase-mediator system
Lys lysine
MALDI-TOF matrix-assisted laser desorption/ionization – time-of-flight
MANBA mannosidase beta
MD molecular dynamics
Met methionine
MnP manganese peroxidase
MS mass spectroscopy
MSW municipal solid wastes
MTMS methyltrimethoxysilane
MWCO molecular weight cut off
NAD+ nicotinamide adenine dinucleotide
NADP+ nicotinamide adenine dinucleotide phosphate
NAMD nanoscale molecular dynamics
NCBI National Center for Biotechnology Information
List of Abbreviations xix

NP nanoparticles
NPD nitrogen and phosphorus detector
Ntn N-terminal nucleophilic hydrolases
NVC non-volatile compounds
OP organophosphate
OPH organophosphorus hydrolase
PA polyamine
PAH polyaromatic hydrocarbon
PAN polyacrylonitrile
PCB polychlorinated biphenyls
PDB Protein Data Bank
PET polyethylene terephthalate
PG polygalacturonan
PHI phosphohexoisomerase
PL polysaccharide lyase
PPE phosphopentose epimerase
PPI phosphopentose isomerase
Q quadrupole
QqQ triplequadrupole
RSCU relative synonymous codon usage
SSF simultaneous saccharification and fermentation
SAXS small-angle X-ray scattering
Ser serine
SHF separate hydrolysis and fermentation
SiNP silver nanoparticles
SLH surface layer homology
SmF submerged fermentation
SOPMA self-optimized prediction method with alignment
SSCF simultaneous saccharification and co-fermentation
SSF simultaneous saccharification and fermentation
SoSF solid-state fermentation
SSFF simultaneous saccharification, filtration and fermentation
TCP 3, 5, 6-trichloro-2-pyridinol
TGase transglutaminase
Thr threonine
TIMT triosephosphate isomerase
TOF time of flight
Trp trypsin
Tyr tyrosine
US United States
Val valine
XDH xylitol dehydrogenase
XK xylulokinase
Xmod X-module
XR xylose reductase
1
Introduction of Fermentation
and Enzyme Science

Monika Choudhary, Sunanda Joshi and Nidhi Srivastava

CONTENTS
1.1 Introduction.......................................................................................................... 1
1.2 Basic Principle of Fermentation.......................................................................... 3
1.3 Biochemical Process of Fermentation................................................................. 4
1.4 Fermentation Methodology in Terms of Enzyme Production............................. 6
1.5 Factors Influencing Fermentation and Enzymes................................................. 7
1.6 Fermentation and Its Industrial Applications in Enzyme Science...................... 8
1.7 Concluding Remarks and Future Trends............................................................. 9
1.8 Conclusion......................................................................................................... 10
References................................................................................................................... 10

1.1 Introduction
Today, fermentation products profit from industrial microbial strains which are estab-
lished by targeted genetic engineering techniques. For many years, the process of
fermentation of foods was done for the production of alcoholic beverages through
barley and grapes (Campbell-Platt 1994). The environmental conditions and micro-
bial strain are employed in the development of by-product or end products. Due to
their catalytic activities, enzymes have been used in the industry for many years, and
their actions are dependent on substrate, pH, inhibitors and temperature. Each process
should be optimized. In the fabrication of over 500 profitable products, enzymes are
used (William and Akiko 2004). They have an extensive variety of applications in
diverse industrial fields.
Many commercial products are produced by a process in which enzymes are used.
There are 500 products in this category (Johannes and Zhao 2006). For thousands of
years, enzymes have been used to produce cheese, yoghurt, beer and wine (food and
beverages). Production of alcohol (ethanol) and carbon dioxide gas is done by break-
down of sugar by the enzymes in yeast:

Glucose → Ethanol + Carbon dioxide

C6H12O6 ( aq ) → 2 C2H 5OH ( aq ) + 2 CO2 ( g )

1
2 Microbial Fermentation and Enzyme Technology

The present reaction of fermentation is getting in the anaerobic condition.

The term “biotechnology” is known from the mid-1970s, and it has been described
in an incredibly uncomplicated type as applied science to the composite type of
industrial arts or scientific learning of the practical. This technology is used in mainly
fermenter set up, control, purification as well as improvement of product to character-
ize processes of chemical engineering by microorganisms and their products (Blieck
et al. 2007). In biotechnology, fermentation is the area which is incredibly important
and is moreover growing rapidly. It is an increasingly rising process. History of this
technology is longer and its future is brighter then biological sciences because of its
covering significant areas like the mankind service in medicine and food (Pátková
et al. 2000).
On a huge scale, the industries of pharmaceuticals, food and alcoholic beverages
have wide use of fermentation technology. The term “fermentation” is from the
Latin and its denoting boiling, for the reason that beverages formed by fermenta-
tion appear to be boiling. The science of fermentation is “zymology.” The produc-
tivity is affected by fermentation modes like types of strains, media and growth
conditions.
Development in production of fermented products is possible for producers
when they have knowledge about the biochemical changes in fermented foods. The
changes in production are possible by manipulation of conditions as well as strains
(John and Sons 2014). High productivity can be achieved by selection of modes of
fermentation like continuous, batch and fed-batch. Because of cell immobilization
in bioreactor, the concentration of biomass increases and therefore the concentra-
tion of biocatalyst also increases, so from it higher productivity can be attained.
There is need of evaluation and optimization of the recovery method of product from
fermentation, and its economic limitation factor also should be optimized. There are
some examples of recovery method such as filtration, homogenization and extrac-
tion (liquid and solid).
The end-product purification is also a step of this process which has high cost.
Extracellular products, biomass itself or intracellular products moreover can be the
microbial end products. Due to their catalytic activities, enzymes have been used in the
industry for many years, and these are products of living organisms (Kameswara Rao
2009). Figure 1.1 discusses the process of fermentation. Activity of enzymes should
be optimized for each process, and it is dependent on pH, temperature, inhibitors,
substrate, etc. as recovery of enzyme is difficult, the process cost reduces by appli-
cation of enzyme immobilization. Microorganisms, plants and mammalian tissues
also can be used for isolation of enzymes. However, due to their specificity as well
as accessibility, microbial enzymes are preferred. In pharmaceuticals, food, leather,
detergents, paper and cosmetics, a variety of enzymes have been used. Commercial
enzymes include proteases, lipase, pectin enzymes, milk clotting enzymes (rennet)
and amylases.
The phrase “fermentation” is used by various microbiologists to depict the produc-
tion method of the mass culture by the microorganisms.
Introduction of Fermentation and Enzyme Science 3

• Compoon of the growth medium

• Toxic by-products
• Dissolved oxygen
Fermentaon
• Final cell mass

• Mechanical stress
• Extreme temperature condion
Downstream • Composon of freezing and dry media
process • Oxygen stress

• Acidity of carrier food

• Oxygen stress
• Moisture
Storage
• Temperature

FIGURE 1.1 Fermentation process.

1.2 Basic Principle of Fermentation

For the preferred yield it is needed that the environmental conditions should be according
to the desired microorganism with the inoculation of a substrate. The crude product is used
directly for the further processing to separate definite individual molecules on or after
it. Monoseptic fermentations necessitate merely a particular microbial variety in order to
achieve the preferred biochemical modification and by the sterilization kill the microor-
ganisms which are redundant, and then a sterilized substrate is used. It is used to generate
insulin that is pharmaceutical products. In the treatment of biological waste and in many
food fermentations, there are numerous microbial species. The participation of mixed
cultures is required. It is based on the microbial compatibility of the medium constitu-
ents. For the growth maxima (Log phage), production of primary metabolite is started by
microbes as they uptake the medium nutrients; these primary metabolites then promote the
production of their secondary metabolites (stationary phase). Therefore, microbial growth
kinetics is able to return to the process of fermentation (Allen et al. 1995).
It is a technique of producing pharmaceutical proteins are produced by provid-
ing economical systems for production of therapeutic proteins by using transgenic
microbes or mammalian cell culture systems. These include vaccines, antibodies,
blood proteins, etc. As shown by Figure 1.2, the end products of fermentation are
proton sinks such as lactic acid and ethanol, whereby NADH, which permits the cell
to carry on the production of energy via glycolysis by substrate-level phosphorylation,
is recycled to NAD+. Consequently, microorganisms are helpful in turning out several
end products or by-products to retain energy balance.
4 Microbial Fermentation and Enzyme Technology

Glucose
ATP

ADP
GLUCOSE 6-
PHOSPHATE

FRUCTOSE 6-PHOSPHATE

ATP

ADP

FRUCTOSE 1:6 DIPHOSPHATE

DIHYOROXYACETONE GLYCERALDEHYDE 3-PHOSPHATE

PHOSPHATE
2 NAD+

2 NADH + H+
3 DIPHOSPHOGLYCERIC ACID
4 ADP

4 ATP

2 PYRUVIC ACID

2 CO2 2 ACETYALDEHYDE

2 NADH + H+

2 NAD+
2 ETHANOL

FIGURE 1.2 Mechanism of fermentation.

1.3 Biochemical Process of Fermentation

In diverse species of organisms with different chemical sequences, fermentation is
achieved. For glucose, two closely related fermentation pathways are used. Two mol-
ecules of three-carbon sugar are formed by partial breakdown of six-carbon glu-
cose molecule; this progression is named lactic acid fermentation. It needs anaerobic
Introduction of Fermentation and Enzyme Science 5

condition and it is found in the higher animal’s cells and a number of microorganisms.
Glycolysis is enzymes that are used in lactic acid and alcoholic fermentation. In alco-
holic fermentation, such as occurs in brewer’s yeast and some bacteria, the production
of lactic acid is bypassed, and the glucose molecule is degraded to two molecules of
the two-carbon alcohol, ethanol, and to two molecules of carbon dioxide. Interest
in the process of fermentation has continued through the ages, and much of modern
biochemistry, especially enzyme studies, has emerged directly from early studies on
the fermentation process.
Selected fungi and bacteria are used for the production of enzymes and grown
under well defined fermentation conditions. At some stage in which microbial growth
occurs, the by-products are produced (metabolites).
Still, biochemical changes as shown by Figure 1.3 (cocoaphilippinesblog.blogspot​.​
com/2016/10/cacao-beans-fermentation-process.html) and the environmental condi-
tions such as pH, occurrence of oxygen, and nutrients also affect the by-product for-
mation (Shuler and Kargi 2008).

“Microbial fermentation has been used for such purposes for many com-
mercial products and enables novel techniques to support sustainable manu-
facturing and screening for microorganisms which produce novel enzymes,
monitoring enzyme production in fermenters and following enzyme recov-
ery during purification processes, requires the use of rapid and specific assay
procedures and well defined substrates.”

Figure 1.4 discusses the extensive variety of microorganisms accessible commer-

cially, and in the starch-processing industry they have entirely replaced chemical
hydrolysis of starch. Even though a lot of microorganisms fabricate this enzyme, usu-
ally microorganisms used for their industrial application are Bacillus licheniformis,
Bacillus amyloliquifaciens and Aspergillus Niger.

Carbon
Compost

Separator Methane

Anaerobic
Feedstock Digester Gas Digester Gas
digester

Water
Process Input or Output

Process Step

Process end product

FIGURE 1.3 Detailed biochemical process of fermentation.

6 Microbial Fermentation and Enzyme Technology

Microorganism
Lactic acid bacteria
Filamentous fungi
and Yeast
Coliforms air
(Contaminated)

Fermentation Substrate
Temperature, Time Carbohydrates,
Vitamins, Proteins,
Feed water ratio Fibers

FIGURE 1.4 Substrate production using microorganisms technology.

1.4 Fermentation Methodology in Terms of Enzyme Production

At the time of 1930s, the food industry used enzyme technology for the first time to
elucidate fruit juice (Adler 1987). Microbial enzymes have many applications and
because of this the food industries have used microbial enzymes for many years.
These enzymes are also used in the leather industry (Underkofler et al. 1958).
Fermentation takes place in a fermenter, which is a huge container with volumes of
up to 1,000 cubic metres. “Nutrients based on renewable raw materials like maize,
sugars and soya these are the fermentation media and it should be sterilized and into
the fermentation medium the majority industrial enzymes are secreted by microor-
ganisms in regulate to break down the carbon and nitrogen sources.” Parameters like
consumption of oxygen, carbon dioxide formation temperature and pH are deliberate
and proscribed to optimize the fermentation progression.
In the laboratory procedure of fermentation as shown in Figure 1.5, pure and clean
nutrients are supplement to the fermenter for the enlargement of the biomass (Yamane
and Shimizu 1984). Industrial enzymes are concealed via cells into the external envi-
ronment called extracellular enzyme. For the recycling process, biomass treated with
lime to inactivate the microorganisms then can be recycled as a fertilizer and stabilize
it during storage. This technology improves the capacity and efficiency of the plant
and, with the per unit of fermentable extracts, also increases the yields of ethanol and
reduces labour, energy and capital costs (Blieck et al. 2007). Furthermore, it is help-
ful in providing greater flexibility to the final product and produces smoother taste
by improving the stability of beer (Pátková et al. 2000; Almeida et al. 2001; Reilly
et al. 2004).
Introduction of Fermentation and Enzyme Science 7

FIGURE 1.5 Process of fermentation in laboratory.

1.5 Factors Influencing Fermentation and Enzymes

There are lots of factors which have an effect on fermentation and enzyme as shown
in Figure 1.6 i.e. pH, character and composition of the medium, temperature, feeding
with precursors, integration (cycling throughout changeable environments) as well as

High Enzyme High Inhibitory

Pretreatment Severity
Demand Concentration

Mixing Issues Limited

Low Ethanol Solid Loading
Mass Transfer
Titer

Slow/Incomplete Reduced Cell Viability

Fermentation Temperature
Scarification and Ethanol Tolerant

Carbon Starvation High Cost

Enzyme Loading

FIGURE 1.6 Factors influencing fermentation and enzymes.

8 Microbial Fermentation and Enzyme Technology

shave rates in the fermenter, dissolved O2, dissolved CO2, and equipped system (e.g.
batch, fed-batch, continuous). “Variation in these factors may affect: the organoleptic
assets of the product (appearances, taste, smell, and texture), the generation of toxins;
nutritional quality; and other physico-chemical properties, the rate of fermentation;
the product spectrum and yield.”
Processing along with cereals improvement in addition to other plant products
require the use of enzymes that is originated from microbes and plants. “In the former
case, enzymes are simply extracted from the plant material and subjected to purifica-
tion and stabilization processes, or are used in the plant material as part of the pro-
cessing conditions (e.g. malt).” The applications of fermentation have been rapidly
branching in different food and pharmaceuticals industries and in the field of biofuels
energy has gained more considerable awareness (Johannes and Zhao 2006).

1.6 Fermentation and Its Industrial Applications

in Enzyme Science
For research and development food manufacturing, animal nutrition, cosmetics and
medicine are the diverse fields where enzymes are functional. “At present, almost
4000 enzymes are identified in which 200 microbial origin types are used com-
mercially, only about 20 enzymes are produced on truly industrial scale with the
improved understanding of the enzyme production biochemistry, fermentation pro-
cesses, and recovery methods, an increasing number of industrial enzymes can be
foreseeable” (Li et al. 2012).
Plentiful and renewable sources of fermentable carbohydrate are molasses from
sugar, plant cellulose, starch, from agricultural wastes and whey from cheese manu-
facturing industries.
Fermentation has various applications such as easiness of reaction, low costs,
the high specificity, and adaptability, and due to these applications present time
industries have enhancement among a more advantaged biological development.
Industrial enzymes have diverse applications, as discussed in Figure 1.7, in different
fields, i.e. agricultural field and industrial field.

“Developing countries have been relying on the biological system enriched

with vitamins and essential amino acids while the developed countries have
used the synthetic food enriched with vitamins and in the food industries
fermented enzymes have been used in food processing (Saifuddin 2013) to
produce vinegar, alcoholic foods, beverages, sausages, pickled vegetables,
cheeses, yogurts, sauces and pastes with meat-like flavours, leavened, in
current farming systems biocatalyst in organic synthesis biofuels production
and selected aspects of bioprocesses (Pátková et al. 2000) and sour-dough
breads and goes far with us in the near future” (Reilly et al. 2004).

The basic fermentation principle has been relevant to the manufacture of biofuels,
biochemicals and biomolecules through the highly developed procedure of genetic
engineering (Choct 2006). According to a research report from Austrian Federal
Environment Agency (Clouthier and Pelletier 2012), about 158 enzymes were used
in food industry, 64 enzymes in technical application.
Another random document with
no related content on Scribd:
{314}

MODUS VIVENDI, Alaskan Boundary.

See (in this volume)

ALASKA BOUNDARY QUESTION.

MODUS VIVENDI, The Newfoundland.

See (in this volume)

NEWFOUNDLAND: A. D. 1899-1901.

MOMBASA-VICTORIA RAILWAY, The.

See (in this volume)

UGANDA RAILWAY.

----------MONETARY QUESTIONS AND MEASURES: Start--------

MONETARY QUESTIONS AND MEASURES: A. D. 1895.

The situation of the Treasury of the United States.
Contract for replenishing its gold reserve.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1895 (JANUARY-
FEBRUARY).

MONETARY QUESTIONS AND MEASURES: A. D. 1895-1896.

The gold reserve in the U. S. Treasury again imperilled.
Refusal of relief by the Senate.

See (in this volume)

MONETARY QUESTIONS AND MEASURES:
UNITED STATES OF AMERICA:
A. D. 1895-1896 (DECEMBER-FEBRUARY).
MONETARY QUESTIONS AND MEASURES: A. D. 1896-1900.
The Silver Question in the United States
Presidential elections.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1896 (JUNE-NOVEMBER);
and
1900 (MAY-NOVEMBER).

MONETARY QUESTIONS AND MEASURES: A. D. 1896-1898.

Movements for monetary reforms in the United States.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1896-1898.

MONETARY QUESTIONS AND MEASURES: A. D. 1897.

Renewal of the privileges of the Bank of France.

The privileges of the Bank of France, as the fiscal agent of

the French Government, expired with the close of the year
1897, and renewal of them was opposed by the Radicals and
Socialists, who demanded the creation of a State Bank. The
government succeeded, however (June, 1897), in carrying the
measure necessary for continuing the existing system, on terms
somewhat more favorable to the state than before.

MONETARY QUESTIONS AND MEASURES: A. D. 1897 (March).

Adoption of the gold standard in Japan.

By a law of the Japanese Parliament, passed in March, 1897, to

come into force October 1, a gold monetary standard was
adopted, at the ratio of 32½ to 1, the silver dollar to be
legal tender until six months after notice given of its
withdrawal.

MONETARY QUESTIONS AND MEASURES: A. D. 1897 (April-

October).
Negotiation by American Commissioners in Europe for an
international bi-metallic agreement.

In fulfilment of the pledge given by the Republican party, at

its national convention, in 1896, that it would use efforts to
bring about an international agreement for free coinage of
gold and silver at some common ratio, the following Act was
passed by the two Houses of Congress in January and February,
1897: "That whenever after March 4, 1897, the president of the
United States shall determine that the United States should be
represented at any international conference called by the
United States or any other country with a view to securing by
international agreement a fixity of relative value between
gold and silver as money by means of a common ratio between
these metals, with free mintage at such ratio, he is hereby
authorized to appoint five or more commissioners to such
international conference; and for compensation of said
commissioners, and for all reasonable expenses connected
therewith, to be approved by the secretary of state, including
the proportion to be paid by the United States of the joint
expenses of any such conference, the sum of $100,000, or so
much thereof as may be necessary, is hereby appropriated. That
the president of the United States is hereby authorized, in
the name of the government of the United States, to call, in
his discretion, such international conference, to assemble at
such point as may be agreed upon. And he is further
authorized, if in his judgment the purpose specified in the
first section hereof can thus be better attained, to appoint
one or more special commissioners or envoys to such of the
nations of Europe as he may designate, to seek by diplomatic
negotiations an international agreement for the purpose
specified in the first section hereof. And in case of such
appointment so much of the appropriation herein made as shall
be necessary shall be available for the proper expenses and
compensation of such commissioners or envoys." Pursuant to
this Act, President McKinley, on the 12th of April, appointed
Senator Edward O. Wolcott; of Colorado, ex-Vice-President
Adlai E. Stevenson, of Illinois, and General Charles J. Paine,
of Massachusetts, to be commissioners for the purpose which
the bill describes. The commissioners first visited Paris, and
there obtained assurances from the French government of
cordial cooperation and support. They then proceeded to
London, for negotiation with the British authorities, on whose
attitude towards the movement for international action in the
matter its success was well known to depend. Some members of
the British government, conspicuously Mr. Balfour, were
outspoken advocates of bi-metallism, and much was hoped from
the discussion to be opened with them. The American
commissioners were cordially received, and they were invited
to a formal meeting, on the 12th of July, with Lord Salisbury,
the Prime Minister, Mr. Balfour, First Lord of the Treasury, Sir
Michael Hicks-Beach, Chancellor of the Exchequer, and Lord
George Hamilton, Secretary of State for India. The American
Ambassador, Mr. Hay, accompanied them to the conference, which
was held at the Foreign Office. A memorandum of the
conversation at this meeting, and at a second one held on the
15th, together with a correspondence which followed, were
published later in a parliamentary "blue book," from which the
following account is drawn. Mr. Wolcott explained the wish of
the American envoys to obtain the views of several
governments, preliminary to the inviting of an international
bi-metallic conference. He added that they expected to have,
in England, the full cooperation of the Ambassador of the
French Republic, who happened, for the moment, to be absent
from the country, but who had requested them to proceed with
the meeting in his absence. "Mr. Wolcott then presented some
reasons which, in the opinion of the Special Envoys, rendered
it desirable that some international agreement for the
restoration of bimetallism should be reached, and explained
why, in their opinion, the success of this effort depended
upon the attitude which England would take regarding the
question. He then stated that the Special Envoys requested
that England should agree to open English mints as its
 contribution to an attempt to restore bimetallism by
international agreement, and dwelt upon the importance of the
fact that France and the United States were together engaged
in an attempt to bring about such an agreement, and were
cooperating together to that end. Lord Salisbury desired to
know if the French Government would co-operate upon the basis
of opening their mints to the free and unlimited coinage of
silver.
{315}
Mr. Wolcott answered in the affirmative. Lord Salisbury then
asked at what ratio, and was informed by Mr. Wolcott that the
French Government preferred the ratio of 15½ to 1, and that
the United States were inclined to yield this point and accept
this as a proper ratio. Considerable discussion on the
question of the ratio and the method by which it should be
settled then took place. … It was then suggested that further
proceedings should be deferred until the French Ambassador
might be also present. The Chancellor of the Exchequer, in
further conversation, said that if the suggestion of opening
the English mints was to be made, he thought an answer in the
negative would undoubtedly be given. The First Lord of the
Treasury asked whether, assuming this request for opening
English mints to be refused, it was desired that the subject
be discussed upon the basis of something different and less
than the opening of English mints. Upon a mutual understanding
that in the absence of the French Ambassador, anything said
should be considered as said informally, a discussion then
took place as to the concessions that England might make
towards an international solution of the question, if it
should refuse to open English mints. Mr. Wolcott, for the
Special Envoys, presented the following as a list of
contributions which, among others, England might make towards
bimetallism if an international agreement could be effected:

1. Opening of the Indian mints. Repeal of the order making the

sovereign legal tender in India.
2. Placing one-fifth of the bullion in the Issue Department of
the Bank of England in silver.

3.
(a.) Raising the legal tender limit of silver to, say, £10.
(b.) Issuing the 20s. notes based on silver, which shall be
legal tender.
(c.) Retirement, gradual or otherwise, of the 10s. gold
pieces, and substitution of paper based on silver.

4. Agreement to coin annually £ … of silver [present silver

coinage average for five years about £1,000,000, less annual
withdrawal of worn and defaced coin for recoinage, £350,000].

5. Opening of English mints to coinage of rupees, and for

coinage of British dollar, which shall be full tender in
Straits Settlements and other silver standard Colonies, and
tender in the United Kingdom to the limit of silver legal
tender.

6. Colonial action, and coinage of silver in Egypt.

7. Something having the general scope of the Huskisson plan.

Some general conversation followed in regard to the preceding

suggestions, and the interview terminated, to be resumed on
the 15th July, 1897, when it was understood that the French
Ambassador would also be present."

At the second meeting, July 15, Baron de Courcol, Ambassador

of the French Republic, and M. L. Geoffray, French Minister
Plenipotentiary, were present, and the former spoke at length,
stating the position of the French government on the question
of the free coinage of silver. He said: "'Our population,
notably the agricultural population, finds that it has not at
its disposition sufficient resources in currency, in metallic
money. On the other hand, if the Government in the actual
state of affairs reopens the mints to the free coinage of
silver, we would be flooded by the abundance of this metal
coming from all other countries of the world, and we could not
resist the even greater evil of the inevitable depreciation of
one of our precious metals, that is to say, of the effective
destruction of the legal ratio upon which our monetary system
is based. … In other words, we think that the production of
silver, more active in certain quarters of the globe in the
last quarter of a century, is not of itself considerable
enough to change in an enduring manner the normal ratio
between gold and silver after these two metals will have been
scattered over the entire surface of the world among all
nations who are called upon to absorb them. There is, then, in
our eyes, a need which is perhaps transitory, but which is
actually common to all the commercial nations, of taking
measures adequate for assuring, by a common understanding, the
re-establishment of the normal ratio of 15½ between silver and
gold. If measures of this kind should be adopted by all the
commercial nations, we would be able to reopen our mints to
the free coinage of silver without fear of being submerged by
an excessive influx of this metal. The reopening of the mints
of all the commercial countries to the free coinage of silver
in the ratio of 15½ with gold would be the most natural and
the most efficacious means of arriving at the result sought
for. This is the desideratum which I am instructed to bring
forward here, and which I am particularly to urge upon the
English Government as a primordial condition of the success of
the common understanding. If the Government of the Queen, even
in consenting to reopen the mints in India, should refuse to
adopt the same measure for England, at least would they not be
able to take certain measures which would be, up to a certain
point, equivalent, in order to maintain the full value of
silver, and to prevent India from being the victim of a
depreciation of this metal in consequence of an unlimited
coinage? … By way of suggestion, I would indicate, as one of
the measures which the English Government might usefully
adopt, the annual purchase of a certain quantity of silver
 metal, which might afterwards be disposed of as seemed
best-either it might be preserved in ingots, or it might be
used for regular consumption, or it might be sent to India.
This quantity might be fixed approximately, at least, for a
number of years, at a sum of £10,000,000 in nominal value.
This is, perhaps, only a palliative; it is, in any event, only
one of the expedients which would be deemed necessary. But I
am to urge strongly that the English Government determine to
take measures of this kind, or other equivalent measures, if,
as I believe, it recognizes with us the necessity of improving
the monetary situation in a great part of its Empire—I may
say, in a great part of the entire world.' Lord Salisbury then
asked whether the French Government would decline to open its
mints unless England would also open her mints. The French
Ambassador replied that he preferred to discuss the subject
upon the basis that France would go to open mints if England
would consent to open her mints, but that he would not exclude
from his view the question of contributions by England towards
maintaining the value of silver, short of open mints. The
Chancellor of the Exchequer, in response to this, stated
definitely that the English Government would not agree to open
English mints to the unlimited coinage of silver, and that,
whatever views he and his colleagues might separately hold on
the question of bimetallism, he thought he could say they were
united upon this point. …
{316}
The suggestions made by the Special Envoys at the interview on
the 12th July were again read, and the Special Envoys accepted
also as important and desirable the proposal that the English
Government should purchase annually, say, £10,000,000 of
silver, with proper safeguards and provisions as to the place
and manner of its use. The French Ambassador expressed his
approval generally of the suggestions of the Special Envoys,
as being serviceable in the consideration of the question. It
was then understood that the proposals submitted by the French
Ambassador and by the Special Envoys of the United States
should be considered, and due notice given when a reply could
be made."

The proposal for the reopening of the Indian mints to silver

was submitted at once by the home authorities of the India
Office, at London, to the government of India, and the reply
of the latter was not received until the following October.
When the reply came, it extinguished hopes of the arrangement
which the American and French governments desired. In a long
despatch, the Indian government explained with clearness the
monetary situation in that country, and discussed the effect
which a return to the unrestricted mintage of silver would
have upon it, under the circumstances and prospects of the
time. "The currency system of India," said the despatch, "is
in a transition state; the Government of India in 1893 decided
to establish a gold standard, and the first step towards that
object was the closing of the mints to silver by Act VIII of
1893. The silver rupee is still the sole legal tender coin,
though the Government has by Executive orders undertaken to
receive gold and sovereigns under certain restrictions, … the
rate of exchange adopted being 16d. the rupee or 15 rupees=£1.
The measures to be taken when the transition period has passed
have not been laid down, but it is probable that the Indian mints
will be opened to gold, and gold coins will be made legal
tender to an unlimited amount; silver rupees would also
continue to be legal tender to an unlimited amount, and the
ratio between the rupee and the gold coins as legal tender
would at the same time be finally settled. The system towards
which India is moving is thus a gold standard of the same kind
as that which now exists in France and the United States, but
with a different ratio for legal tender; but for the present
the mints are closed both to gold and silver. The transition
period has lasted for more than four years, but there is
ground for hope that it is now drawing to a close. The changes
which are involved in the arrangements proposed to Her
Majesty's Government are the following. France and the United
States are to open their mints to the free coinage of silver,
continuing the free coinage of gold and the unlimited legal
tender of coins of both metals, the ratio remaining unchanged
in France and being altered to the French ratio of 15½ to 1 in
the United States. India is to open her mints to silver, to
keep them closed to gold, and to undertake not to make gold
legal tender. France and the United States would thus be
bimetallic: India would be monometallic (silver); while most
of the other important countries of the world would be
monometallic (gold). The object which the proposers have in
view is the establishment of a stable relation between the
values of gold and of silver. This would include the
establishment of a stable exchange between the rupee and
sterling currency, which was the object of the Government of
India in the proposals made in our financial despatch of the
21st June, 1892, which proposals ultimately resulted in the
adoption, in view to the attainment of that object, of the
policy of a gold standard, and in the closing of the mints to
the free coinage of silver. If, then, it were certain that the
suggested measures would result in the establishment of a
stable ratio, the Government of India might well consider
whether their adoption would not be preferable to the policy
to which they committed themselves in 1893 in the hope of
attaining the same result by isolated action on the part of
India alone. The principal questions therefore for us to
consider are whether the measures are more likely to succeed
than the policy of 1893, and what consequences to India may be
apprehended if the measures should fail of success after being
brought into operation. … The first result of the suggested
measures, if they even temporarily succeed in their object,
would be an intense disturbance of Indian trade and industry
by the sudden rise in the rate of exchange, which, if the
ratio adopted were 15½ to 1, would be a rise from about 16d.
to about 23d. the rupee. Such a rise is enough to kill our
export trade, for the time at least. If the public were not
convinced that the arrangement would have the effect intended,
or believed that it would not be permanent, the paralysis of
trade and industry would be prolonged and accompanied by acute
individual suffering, none of the advantages expected would be
 attained, and the country would pass through a critical period
which would retard its progress for years. How long the crisis
would last before normal or stable conditions were restored it
is not possible to conjecture. It would be long even if the
mercantile and banking community saw that silver was being
steadily maintained at the prescribed ratio, while any
indication of unsteadiness would greatly prolong the period by
giving foundation for doubt. If the doubt should happen to be
justified by the results, the position would be disastrous
alike to the State, to individuals, and to trade generally. …
We cannot help seeing that if the policy of 1893 is now
abandoned, and if the triple union now proposed as a
substitute should fail in its operation or should terminate,
and in its failure subject Indian trade to the violent shocks
we have described, the Government of India could not, as a
responsible Government, call upon the commercial public to
face another prolonged period of doubt, suspense, agitation,
and difficulties. For it must be clearly and fully recognized
that if India joins in the proposed measures, we shall be left
dependent, as the sole means of attaining stability in
exchange, on the success of those measures, and that if they
should fail, India must be content to remain permanently under
the silver standard with all its admitted disadvantages. … We
have given very careful consideration to the question whether
France and the United States are likely, with the help of
India, to be able to maintain the relative value of gold and
silver permanently at the ratio they intend to adopt, and have
come to the conclusion that while we admit a possibility of
the arrangements proposed resulting in the permanent
maintenance of the value of gold and silver at the ratio of
15½ to 1, the probability is that they will fail to secure
that result, and that it is quite impossible to hold that
there is anything approaching a practical certainty of their
doing so.
{317}
One reason for this conclusion is, that the arrangement would
rest on too narrow a basis. A union consisting of two
countries, with a third lending assistance, is a very
different thing from the general international union of all or
most of the important countries of the world, which was
advocated by the Government of India in the despatches of
March and June 1892 and of February and September 1886. To
afford a hope that a monetary union will succeed in
establishing stability in the relative value of gold and
silver, it is essential that the nations adhering to it should
be of such number and importance that the metallic currency of
the whole body shall be of sufficient extent to allow of the
exercise of adequate influence on the value of the two metals.
We doubt whether any two, or even three, nations in the world,
unless, indeed, one of them was Great Britain, could comply
with this condition, and we have no hesitation in saying that
France and the United States and India certainly could not. …
We have no hesitation in recommending your Lordship to refuse
to give the undertaking desired by the Governments of France
and the United States. We are quite clearly of opinion that
the interests of India demand that her mints shall not be
opened as part of an arrangement to which two or three
countries only are parties, and which does not include Great
Britain." Immediately on receiving this reply, Lord Salisbury
informed Ambassador Hay that "Her Majesty's Government feel it
their duty to state that the first proposal of the United States
Representatives is one which they are unable to accept," and
expressing a wish to know "how far the views of the American
and French Governments are modified by the decision now
arrived at, and whether they desire to proceed further with
the negotiations at the present moment."

Great Britain, Parliamentary Publications

(Papers by Command: Commercial, Number 8, 1897).

In the view of the American envoys, it seemed useless to

proceed, with no hope of cooperation from Great Britain, and
they returned to America with a discouraging report.
MONETARY QUESTIONS AND MEASURES: A. D. 1897 (December).
Adoption of the gold standard in Russia.

An imperial ukase declared the adoption of the gold standard

in Russia, authorizing the issue of a new five-rouble gold
piece.

MONETARY QUESTIONS AND MEASURES: A. D. 1900.

Settlement of the monetary system in the United States.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1900 (MARCH-
DECEMBER).

MONETARY QUESTIONS AND MEASURES: A. D. 1900 (November).

Withdrawal of legal tender silver coins in Germany.

See (in this volume)

GERMANY: A. D. 1900 (NOVEMBER).

----------MONETARY QUESTIONS AND MEASURES: End--------

MONGOLIA.

See (in this volume)

MANCHURIA AND MONGOLIA.

MONOPOLIES.

See (in this volume)

TRUSTS: UNITED STATES.

MONROE DOCTRINE, The:

As emphasized in the Treaty of International Arbitration.

See (in this volume)

PEACE CONFERENCE.
MONROE DOCTRINE, The:
Its discussion as involved in the Venezuela Boundary Question.

See (in this volume)

VENEZUELA; A. D. 1895 (JULY) and (NOVEMBER).

MONTAUK POINT, Removal of troops from Santiago de Cuba to.

See (in this volume)

UNITED STATES OF AMERICA:
A. D. 1898 (JULY-AUGUST: CUBA).

MONTENEGRO.

See (in this volume)

BALKAN AND DANUBIAN STATES.

MOROS, The.

See (in this volume)

PHILIPPINE ISLANDS; THE NATIVE INHABITANTS;
and A. D. 1899 (MAY-AuGUST).

MORTMAIN, Proposed restrictions on, in France.

See (in this volume)

FRANCE: A. D. 1901 (JANUARY).

MOSLEMS AND CHRISTIANS:

Conflicts in Armenia.

See (in this volume)

TURKEY: A. D. 1895.

MOSLEMS AND CHRISTIANS:

Conflicts in Crete.
 See (in this volume)
TURKEY: A. D. 1897 (FEBRUARY-MARCH).

MOSQUITO, The, as a carrier of disease.

See (in this volume)

SCIENCE, RECENT: MEDICAL, AND SURGICAL.

MUKDEN.

See (in this volume)

MANCHURIA AND MONGOLIA; also,
RUSSIA IN ASIA: A. D. 1891-1900.

MUNICIPAL EVENTS, Notable.

See (in this volume)

BOSTON, CHICAGO, NEW YORK, TOLEDO, LONDON.

MUNICIPAL GOVERNMENTS:
Institution in Cuba.

See (in this volume)

CUBA: A. D. 1900 (JUNE-NOVEMBER).

MUNICIPAL GOVERNMENTS:
Institution in the Philippines.

See (in this volume)

PHILIPPINE ISLANDS: A. D. 1900 (MARCH).

MUSIC: In the Nineteenth Century.

See (in this volume)

NINETEENTH CENTURY: THE MUSICAL CENTURY.
N.

NABONIDOS, Discovery of an inscription of.

See (in this volume)

ARCHÆOLOGICAL RESEARCH: BABYLONIA:
DISCOVERY OF AN INSCRIPTION.

NANSEN'S EXPEDITION, Return of.

See (in this volume)

POLAR EXPLORATION, 1896.

NASHVILLE EXPOSITION.

See (in this volume)

TENNESSEE: A. D. 1897.

NATAL.

See (in this volume)

SOUTH AFRICA.

NATIONAL DEMOCRATIC PARTY OF 1896.

See (in this volume)

UNITED STATES OF AMERICA: A. D. 1896 (JUNE-NOVEMBER.)

NATIONAL PARTY, The.

See (in this volume)

UNITED STATES OF AMERICA:
A. D. 1896 (JUNE-NOVEMBER); and
1900 (MAY-NOVEMBER).

NATIONAL SILVER PARTY.

 See (in this volume)
UNITED STATES OF AMERICA: A. D. 1896 (JUNE-NOVEMBER).

{318}

NATIONAL STEEL COMPANY, The.

See (in this volume)

TRUSTS: UNITED STATES.

NATIONALISTS, FRENCH, Revolutionary conspiracy of.

See (in this volume)

FRANCE: A. D. 1899-1900 (AUGUST-JANUARY).

NAVAL POLICY, German.

See (in this volume)

GERMANY: A. D. 1900 (FEBRUARY-JUNE).

NAVIES OF THE SEA POWERS: A. D. 1900.

The following tables are compiled from a return issued by the

British Admiralty in the spring of 1900, showing the state of
the fleets of Great Britain, France, Russia, Germany, Italy,
the United States, and Japan, including vessels then built and
in progress of construction.

Of battle-ships, there were built and building in the several

navies:

Count with a displacement

of
Great Britain. 70 821,605
France. 35 339,599
Russia. 24 262,912
 Germany. 25 191,259
Italy. 19 193,001
United States. 16 184,144
Japan. 7 92,420

Of armored and protected cruisers, there were built and

building:

Count with a displacement

of
Great Britain. 147 827,430
France. 60 297,486
Russia. 23 144,673
Germany. 22 107,844
Italy. 25 93,673
United States. 26 140,274
Japan. 23 114,479

Of unprotected cruisers, armored coast-defence vessels,

and special vessels there were built and building:

Count with a displacement

of
Great Britain. 31 104,250
France. 29 93,385
Russia. 26 66,886
Germany. 35 59,617
Italy. 3 13,821
United States. 30 77,150
Japan. 13 24,065

Of torpedo vessels and torpedo-boat destroyers

the number built and building was:
 Count with a displacement
of
Great Britain. 238 70,311
France. 305 34,002
Russia. 233 37,735
Germany. 130 20,094
Italy. 180 24,863
United States. 50 12,121
Japan. 71 9,537

A consolidation of the above figures shows a

total of ships in the principal navies as follows:

Great Britain, 486;

France, 429;
Russia, 306;
Germany, 212;
Italy, 227;
United States, 122;
Japan, 114.

A writer in the "Fortnightly Review," discussing the above

returns, points out the imperfectness of the representation
which such gross figures give of the actual naval strength of
the several Powers, and he has undertaken to correct them by a
calculation of what he calls the "fighting weight" of the
ships, based on the age of each and its displacement in tons.
He says: "The scale of depreciation for age that I have used
is as follows: Ships, built and now building, that were
launched, or which will be launched, during 1895-1899 (and
later), are reckoned at their full value of fighting weight;
i. e., at 100 per cent. Ships launched during 1890 1894 are
reckoned as now worth only 80 per cent. of their fighting
weight. The other depreciations being: Ships launched
1885-1889 are valued at 60 per cent. of their nominal fighting