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Current Developments
in Biotechnology and
Bioengineering
Production, Isolation and Purification
of Industrial Products
Edited by
Ashok Pandey, Sangeeta Negi,
Carlos Ricardo Soccol
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List of Contributors
M. Adsul DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Limited
Cristóbal N. Aguilar Food Research Department, School of Chemistry,

Autonomous University of Coahuila, Saltillo, Coahuila, México
A. Angel-Cuapio Universidad Autónoma Metropolitana-Iztapalapa, Mexico City,

DF, Mexico
G.S. Anisha Government College for Women, Trivandrum, Kerala, India
P. Binod CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
J. Buenrostro-Figueroa Department of Biotechnology, Division of Health and

Biological Sciences, Metropolitan Autonomous University, Iztapalapa, México
S. Chakraborty Indian Institute of Technology Guwahati, Guwahati, Assam, India
M.L. Chávez González Food Research Department, School of Chemistry,

G.-Q. Chen Tsinghua University, Beijing, China
S. Chen Hubei University, Wuhan, PR China
Juan C. Contreras-Esquivel Food Research Department, School of Chemistry,

J.D. Coral Bioprocess Engineering and Biotechnology Department, Federal

University of Paraná (UFPR), Curitiba, PR, Brazil
J.C. de Carvalho Bioprocess Engineering and Biotechnology Department, Federal

xxi
xxii List of Contributors
J. de Oliveira Bioprocess Engineering and Biotechnology Department, Federal

A. Dhillon Indian Institute of Technology Guwahati, Guwahati, Assam, India
M.J. Fernandes Bioprocess Engineering and Biotechnology Department, Federal

R. Gaur Indian Oil Corporation Limited, R&D Centre, Faridabad, India
A. Goyal Indian Institute of Technology Guwahati, Guwahati, Assam, India
L.R.C. Guimarães Bioprocess Engineering and Biotechnology Department, Federal

M. Haridas Kannur University, Kannur, India
R. Hemamalini Indian Institute of Technology Delhi, New Delhi, India
Ayerim Hernandez-Almanza Food Research Department, School of Chemistry,

A. Illanes Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
J. Isar University of Delhi South Campus, New Delhi, India
A. Joseph Kannur University, Kannur, India
S.G. Karp Bioprocess Engineering and Biotechnology Department, Federal

N. Karthik CSIR-National Institute for Interdisciplinary Science and Technology

(NIIST), Trivandrum, India
R. Kaushik University of Delhi South Campus, New Delhi, India
S.K. Khare Indian Institute of Technology Delhi, New Delhi, India
P.C.S. Kirnev Bioprocess Engineering and Biotechnology Department, Federal

D. Kothari Indian Institute of Technology Guwahati, Guwahati, Assam, India

List of Contributors xxiii
C. Larroche Blaise Pascal University, Aubière Cedex, France
L.A.J. Letti Bioprocess Engineering and Biotechnology Department, Federal

O. Loera-Corral Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, DF,

Mexico
A.I. Magalhães, Jr. Bioprocess Engineering and Biotechnology Department,

Federal University of Paraná (UFPR), Curitiba, PR, Brazil
A.B.P. Medeiros Bioprocess Engineering and Biotechnology Department, Federal

J.D.C. Medina Bioprocess Engineering and Biotechnology Department, Federal

F. Miranda-Hernández Universidad Autónoma Metropolitana-Iztapalapa, Mexico

City, DF, Mexico
N.R. Nair CSIR-National Institute for Interdisciplinary Science and Technology

S. Nair Dow Chemicals GmBH, Dubai, UAE
K.M. Nampoothiri CSIR-National Institute for Interdisciplinary Science and

Technology (NIIST), Trivandrum, India
A. Nandan CSIR-National Institute for Interdisciplinary Science and Technology

S. Negi Motilal Nehru National Institute of Technology, Allahabad, India
M.G.B. Pagnoncelli Bioprocess Engineering and Biotechnology Department,

Federal University of Paraná (UFPR), Curitiba, PR, Brazil; Federal Technological
University of Parana, Dois Vizinhos, Brazil
A. Pandey Center of Innovative and Applied Bioprocessing, (a national institute

under Dept of Biotechnology, Ministry of S&T, Govt of India), Mohali, Punjab, India
A.K. Patel DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation
Limited
xxiv List of Contributors
V. Rajulapati Indian Institute of Technology Guwahati, Guwahati, Assam, India
S. Ramachandran Insight Professional Institute, Dubai, UAE
A. Rani Indian Institute of Technology Guwahati, Guwahati, Assam, India
C. Rodrigues Bioprocess Engineering and Biotechnology Department, Federal

Rosa M. Rodríguez-Jasso Food Research Department, School of Chemistry,

R. Rodríguez Food Research Department, School of Chemistry, Autonomous

University of Coahuila, Saltillo, Coahuila, México
L.V. Rodríguez Durán Department of Biotechnology, Division of Health and

Biological Sciences, Metropolitan Autonomous University, Iztapalapa, México
Héctor A. Ruiz Food Research Department, School of Chemistry, Autonomous

A. Sabu Kannur University, Kannur, India
R. Saini DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Limited
S. Sajitha CSIR-National Institute for Interdisciplinary Science and Technology

S. Saran University of Delhi South Campus, New Delhi, India
R.K. Saxena University of Delhi South Campus, New Delhi, India
V.C. Sekhar CSIR-National Institute for Interdisciplinary Science and Technology

K. Sharma Indian Institute of Technology Guwahati, Guwahati, Assam, India
R. Sindhu CSIR-National Institute for Interdisciplinary Science and Technology

R.P. Singh Punjabi University, Patiala, Punjab, India

List of Contributors xxv
R.S. Singh Punjabi University, Patiala, Punjab, India
Reeta R. Singhania DBT-IOC Centre for Advanced Bioenergy Research, IndianOil

Corporation Limited
C.R. Soccol Bioprocess Engineering and Biotechnology Department, Federal

T.S. Swapna Government Victoria College, Palakkad, India
D. Tan Xían Jiaotong University, Xían, China
L. Thomas CSIR-National Institute for Interdisciplinary Science and Technology

M.V. Ushasree CSIR-National Institute for Interdisciplinary Science and Technology

P. Valencia Universidad Técnica Federico Santa María, Valparaíso, Chile
L.P.S. Vandenberghe Bioprocess Engineering and Biotechnology Department,

K. Vibha Motilal Nehru National Institute of Technology, Allahabad, India
J. Vidya CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
N. Vijayan Kannur University, Kannur, India
N. Vivek CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
Q. Wang Hubei University, Wuhan, PR China
X. Wei Hubei University, Wuhan, PR China
A.L. Woiciechowski Bioprocess Engineering and Biotechnology Department,

J. Yin Tsinghua University, Beijing, China

xxvi List of Contributors
A. Zandoná Filho Bioprocess Engineering and Biotechnology Department, Federal

P.A. Zárate Food Research Department, School of Chemistry, Autonomous

S.F. Zawadzki Bioprocess Engineering and Biotechnology Department, Federal

About the Editors
Ashok Pandey
Professor Ashok Pandey is Eminent Scientist at the Center of
Innovative and Applied Bioprocessing, Mohali (a national
institute under the Department of Biotechnology, Ministry
of Science and Technology, Government of India), and
former chief scientist and head of the Biotechnology
Division at the CSIR’s National Institute for Interdisciplinary
Science and Technology at Trivandrum. He is an adjunct
professor at Mar Athanasios College for Advanced Studies
Thiruvalla, Kerala, and at Kalasalingam University, Krishnan
Koil, Tamil Nadu. His major research interests are in the
areas of microbial, enzyme, and bioprocess technology,
which span various programs, including biomass to fuels
and chemicals, probiotics and nutraceuticals, industrial
enzymes, solid-state fermentation, etc. He has more than
1100 publications and communications, which include 16
patents, 50+ books, 125 book chapters, and 425 original and review papers, with an h index
of 75 and more than 23,500 citations (Google Scholar). He has transferred several tech-
nologies to industries and has been an industrial consultant for about a dozen projects for
Indian and international industries.
Professor Pandey is the recipient of many national and international awards
and fellowships, which include Elected Member of the European Academy of Sciences
and Arts, Germany; Fellow of the International Society for Energy, Environment and
Sustainability; Fellow of the National Academy of Science (India); Fellow of the Biotech
Research Society, India; Fellow of the International Organization of Biotechnology and
Bioengineering; Fellow of the Association of Microbiologists of India; honorary doctorate
degree from the Université Blaise Pascal, France; Thomson Scientific India Citation
Laureate Award, United States; Lupin Visiting Fellowship; Visiting Professor at the
Université Blaise Pascal, France, the Federal University of Parana, Brazil, and the École
Polytechnique Fédérale de Lausanne, Switzerland; Best Scientific Work Achievement
Award, Government of Cuba; UNESCO Professor; Raman Research Fellowship Award,
CSIR; GBF, Germany, and CNRS, France fellowships; Young Scientist Award; and others.
He was chairman of the International Society of Food, Agriculture and Environment,
Finland (Food & Health) during 2003e04. He is the Founder President of the Biotech
xxvii
xxviii About the Editors
Research Society, India (www.brsi.in); International Coordinator of the International

Forum on Industrial Bioprocesses, France (www.ifibiop.org); chairman of the
International Society for Energy, Environment & Sustainability (www.isees.org); and vice
president of the All India Biotech Association (www.aibaonline.com). Professor Pandey
is editor-in-chief of Bioresource Technology, Honorary Executive Advisor of the Journal of
Water Sustainability and Journal of Energy and Environmental Sustainability, subject
editor of the Proceedings of the National Academy of Sciences (India), and editorial board
member of several international and Indian journals, and also a member of several
national and international committees.
Sangeeta Negi
Dr. Sangeeta Negi is an assistant professor in the Department
of Biotechnology at the Motilal Nehru National Institute of
Technology, India. She has a First Class Master’s degree in
biochemistry and a PhD in biotechnology from the Indian
Institute of Technology, Kharagpur. She has also worked as
an academic guest at the Biological Engineering Department,
Polytech Clermont-Ferrand; the Université Blaise Pascal,
France; and the Bioenergy and Energy Planning Research
Group, Swiss Federal Institute of Technology, Lausanne,
Switzerland. Dr. Negi’s current research interests are in the
areas of biofuels, industrial enzymes, and bioremediation. She is an editorial board
member of the Journal of Waste Conversion, Bioproducts and Biotechnology and the Journal
of Environmental Science and Sustainability. She has been awarded as Outstanding
Reviewer by Elsevier and has won the Young Scientist Award by DST at the National
Seminar on Biological and Alternative Energies Present and Future, organized by Andhra
University, Visakhapatnam, in 2009. She has also won the Best Poster Award at the
International Congress on Bioprocesses in Food Industries (2008) at Hyderabad. Dr. Negi
has contributed to nearly 70 publications, including review articles, original papers, and
conference communications.
About the Editors xxix

Professor Carlos Ricardo Soccol is the research group leader
of the Department of Bioprocesses Engineering and
Biotechnology at the Federal University of Paraná (UFPR),
Brazil, with 20 years of experience in biotechnological
research and development of bioprocesses with industrial
application. He graduated with a BSc in chemical engi-
neering (UFPR, 1979), Master’s in food technology (UFPR,
1986), and PhD in Génie Enzymatique, Microbiologie et
Bioconversion (Université de Technologie de Compiègne,
France, 1992). He did his postdoctoral work at the Institut
ORSTOM/IRD (Montpellier, 1994 and 1997) and at the
Université de Provence et de la Méditerranée (Marseille,
2000). He is an HDR Professor at the École d’Ingénieurs Supériure of Luminy,
MarseilleeFrance. He has experience in the areas of science and food technology, with
emphasis on agro-industrial and agro-alimentary biotechnology, acting in the following
areas: bioprocess engineering and solid-state fermentation, submerged fermentation,
bioseparations, industrial bioprocesses, enzyme technology, tissue culture, bio-
industrial projects, and bio-production. He is currently the Coordinator of Master
BIODEV-UNESCO, associate editor of five international journals, and editor-in-chief of
the journal Brazilian Archives of Biology and Technology. Professor Soccol has received
several national and international awards, which include the Science and Technology
Award of the Government of Paraná (1996); Scopus/Elsevier Award (2009); Dr. Honoris
Causa, Université Blaise Pascal, France (2010); Outstanding Scientist at the 5th
International Conference on Industrial Bioprocesses, Taipei, Taiwan (2012); and Elected
Titular Member of the Brazilian Academy of Sciences (2014). He is a technical and sci-
entific consultant for several companies, agencies, and scientific journals in Brazil and
abroad. He has supervised and mentored 96 Master of Science students, 48 PhD stu-
dents, and 14 postdoctoral students. He has 995 publications and communications,
which include 17 books, 107 book chapters, 270 original research papers, and 557
research communications in international and national conferences and has registered
44 patents. His research articles as of this writing have been cited (Scopus database) 5600
times with an h index of 36.
Preface
This book is a part of the comprehensive series Current Developments in Biotechnology and
Bioengineering, comprising nine volumes (Editor-in-chief: Ashok Pandey), and deals with the
production, isolation, and purification of industrial products produced by biotechnological
processes. This book covers recent technological advances of a great number of biotechno-
logical products and is divided into four different parts: Production of Industrial and
Therapeutic Enzymes, Organic Acids, Biopolymers and Other Products, and Products
Isolation and Purification.
Part 1 is devoted to the production of industrial and therapeutic enzymes. The first
chapter describes the current and future trends of production, application, and strain
improvement of a-amylases, one of the most important enzymes used in industry.
a-Amylases find application in several industrial processes, such as starch liquefaction,
desizing of textiles, detergents, baking, bioethanol production, etc. Glucoamylase is another
enzyme extensively used in the food and fermentation industries, mainly for the saccharifi-
cation of starch, brewing, and production of high-fructose syrup, which are discussed in
Chapter 2. Cellulases, b-glucosidases, and xylanases are the second most used enzymes in
industry by sales volume, with an increasing demand since 1995 in several industrial appli-
cations, comprising detergents and textiles, animal feed, food, paper, and biofuels. These
enzymes are discussed in Chapters 4, 5, and 6 of this book. Chapter 7 discusses proteolytic
enzymes, also known as “proteases,” which are used to cleave the peptide bonds connecting
two amino acids. They are produced mainly by microorganisms and have great commercial
value, being used in food, dairy, detergents, and leather processing. Lipolytic enzymes are
hydrolases comprising 15 families of lipases, as shown in Chapter 8 of this book through a
study of the industrial applications and other important aspects of these enzymes. The
purpose of Chapters 9 and 10 is to present an overview of laccases and peroxidases, covering
their production and use in the pretreatment of lignocellulosic biomass and biopulping, and
also projecting new perspectives on improving such processes and products using these
enzymes. Sources of production, strategies, characteristics, applications, and industrial
importance of therapeutic enzymes, such as L-glutaminase, L-asparaginase, and penicillin
acylase, are presented and discussed in Chapters 11, 12, and 13. Other enzymes, such as
phytases, chitinases, keratinases, tannases, aminopeptidases, nattokinases, and poly-
saccharide lyases, are reviewed in Chapters 14 to 23, covering recent advances, production
methods, potential applications, and the global market.
The second part of the book is dedicated to organic acids. In Chapters 24 and 25, lactic
acid and citric acid production, synthesis (covering factors that affect biochemical pathways),
and recovery are addressed. Chapter 26 reviews the microbial production of gluconic acid,
properties of glucose oxidase, production, recovery, and applications. Succinic acid is an
important platform molecule, used as an intermediate in the production of numerous
everyday products, among which are pharmaceuticals and adhesives, representing a total
immediate addressable market of more than $7.2 billion. Chapter 27 presents an analysis of
the current market, biological-based production processes, enzymatic regulation, and
recovery systems of succinic acid.
xxxi
xxxii Preface
Part 3 discusses polymer production and other products. Polylactide (PLA), derived
from lactic acid, a biodegradable polyester, has applications in packaging, textiles, and the
biomedical and pharmaceutical industries. Chapter 28 reviews the properties and applica-
tions of PLA, focusing on recent technologies and improvement of production techniques.
Polyhydroxyalkanoates (PHAs), a family of environmentally friendly polyesters that can be
synthesized by a wide range of microorganisms as carbon and energy reserves, have been
considered an alternative to petroleum-based chemicals. The composition and structural
diversity of PHAs have led to various properties and endless applications to form a PHA value
chain. Chapter 29 briefly introduces their production and application, highlighting the lab-
oratory production by the microbial strains developed using genetic and/or metabolic en-
gineering or synthetic biology techniques. Industrial production, recent technologies, and
improvement of PHA production are also discussed. Poly-g-glutamic acid (g-PGA) is a natural
polymer, synthesized by various strains of Bacillus spp., that is used in food, cosmetics,
agriculture, and the wastewater industry. Chapter 30 provides updated information on the
biosynthesis, fermentation, purification, and application of g-PGA. In Chapter 31, recent
developments in the biological production of 1,3-propanediol by various natural and
genetically engineered microorganisms, nonnative 1,3-propanediol producers, as well as
mixed cultures, are discussed. Important aspects of downstream processing and various
methods and steps involved in the extraction and purification of 1,3-propanediol from the
fermentation broth are also covered in this chapter. The production of petroleum-based
plastics is a challenging environmental problem, causing the production and consumption
of biodegradable plastics to receive considerable attention nowadays. Chapter 32 provides an
overview of the degradation mechanisms of biodegradable polymers, with particular
emphasis on the main parameters affecting the degradation of these polymeric biomaterials.
In Chapter 33 the potential of biological control is presented and discussed as a promising
alternative to chemical pesticides. The final two chapters of this book, Chapters 34 and 35,
present the most relevant downstream processes to extract, isolate, purify, and refine
fermentation products.
We are confident that this book will be profitable to students, professors, researchers,
and professionals interested in studying biotechnology and bioengineering. We thank
Dr. Kostas Marinakis, Book Acquisition Editor; Ms. Anneka Hess; and entire production team
at Elsevier for their help and support in bringing out this volume.
Editors
Ashok Pandey
Sangeeta Negi
1
a-Amylases
R. Sindhu1, *, P. Binod1, A. Pandey2

1
CSIR-NATIONAL INSTITUTE F OR INTERDISCIPLINARY SCIENCE AND TECHNOLOGY (NIIST),
TRIVANDRUM, INDIA; 2 CE NTER OF INNOVATIVE AND APPLIED BIOPROCESSING,
(A NATIONAL INSTITUTE UNDER DEPT OF BIOTECHNOL OGY, MINISTRY OF S&T, GOVT OF
INDIA), MOHALI, PUNJAB , INDIA
1.1 Introduction
1.1.1 Starch
Starch is the major polysaccharide food reserve in nature after cellulose. It serves as an
important source of nutrition for other living organisms [1]. It is synthesized in the
plastids present in leaves and accumulates as insoluble granules in higher and lower
plants. Starch is composed of a large number of glucose units joined by glycosidic bonds.
It consists of two types of molecules: amylose and amylopectin. Amylose is a linear,
water-insoluble polymer of glucose joined by a-1,4 bonds, whereas amylopectin is a
branched, water-soluble polysaccharide with short a-1,4-linked linear chains of 10e60
glucose units and a-1,6-linked side chains with 15e45 glucose units. The levels of
amylase and amylopectin vary among different starches. Generally, starch is composed
of amylose and amylopectin in the range 25e28% and 72e75%, respectively.
1.1.2 Amylases
Amylases are the enzymes that break down starch, or glycogen. These enzymes are
produced by a variety of living organisms, ranging from bacteria to plants to humans.
Though amylases are produced by several microorganisms, those produced by fungi and
bacteria have dominated applications in the industrial sector [2]. Bacteria and fungi
secrete amylases to the outside of their cells to carry out extracellular digestion, which
breaks down the insoluble starch, and then the soluble end products (such as glucose or
maltose) are absorbed into the cells.
Amylases constitute a class of industrial enzymes occupying about 25% of the enzyme
market. Because of the increasing demand for these enzymes in various industries, there
is enormous interest in developing them with better properties, such as raw starch-
degrading amylases suitable for industrial applications, and cost-effective production
*
Corresponding Author.
Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products
http://dx.doi.org/10.1016/B978-0-444-63662-1.00001-4 3
Copyright © 2017 Elsevier B.V. All rights reserved.
4 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING
techniques. Although amylases can be derived from several sources, including plants,
animals, and microorganisms, microbial enzymes generally meet industrial demands.
A large number of microbial amylases are available commercially and they have almost
completely replaced the chemical hydrolysis of starch in the starch processing industry
[3]. One of the most important advantages of using microbes for the production of
amylases is the bulk production capacity and the fact that microbes can be genetically
modified to produce enzymes with desired characteristics [4]. These enzymes are of great
significance in biotechnology, with various applications ranging from food, fermentation,
and textiles to the paper industry. Each application of a-amylase requires unique
properties with respect to specificity, stability, and temperature and pH dependence.
Modern technologies such as computational packages and online servers are the
current tools used in protein sequence analysis and characterization. The physico-
chemical and structural properties of these proteins are well understood with the use of
computational tools. The protein sequence profile, such as number of amino acids and
sequence length, and the physicochemical properties of the protein, such as molecular
weight, atomic composition, extinction coefficient, aliphatic index, instability index, etc.,
can be computed by ProtParam, and the secondary structure prediction, sequence
similarity, evolutionary relationships, and 3-D structure of various proteins can be
computed using the ESyPred3D server [5].
1.1.3 Classification of Amylases

Based on the mechanism of breakdown of starch, the molecules are classified into three
types: a-amylase, b-amylase, and amyloglucosidase. a-Amylase reduces the viscosity of
starch by breaking down the bonds at random, thereby producing variably sized chains
of glucose. b-Amylase enzyme breaks the glucoseeglucose bonds by removing two
glucose units at a time, thereby producing maltose. Amyloglucosidase is the enzyme that
breaks successive bonds from the nonreducing end of the straight chain, producing
glucose. Many microbial amylases usually contain a mixture of these amylases. This
chapter focuses only on a-amylases.
a-Amylases (EC 3.2.1.1) are starch-degrading enzymes that catalyze the hydrolysis of
internal a-1,4-O-glycosidic bonds in the polysaccharides with the retention of the
a-anomeric configuration in the products. Most of the a-amylases are metalloenzymes,
which require calcium ions (Ca2þ) for their activity, structural integrity, and stability.
They belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes [6,7].
Based on the end-product formation a-amylases are classified as saccharifying and
liquefying amylases. The saccharifying a-amylases are further classified as maltose
forming, maltotetraose forming, maltopentaose forming and maltohexaose forming
based on the end products formed [1].
The a-amylase family is the largest family of glycoside hydrolases, transferases, and
isomerases, comprising 30 different enzyme specificities. These enzymes are classified
into four groups: endoamylases, exoamylases, debranching enzymes, and transferases.
Endoamylases are enzymes that cleave internal a-1,4 bonds resulting in a-anomeric
Chapter 1 a-Amylases 5
products. Exoamylases are enzymes that cleave a-1,4, or a-1,6 bonds of the external
glucose residues resulting in a- or b-anomeric products. Debranching enzymes are
enzymes that hydrolyze a-1,6 bonds leaving linear polysaccharides. Transferases are
enzymes that cleave a-1,4 glycosidic bonds of the donor molecule and transfer part of
the donor molecule to a glycosidic acceptor, forming a new glycosidic bond [7].
1.2 Sources of a-Amylase

a-Amylases are universally distributed throughout the plant, animal, and microbial
kingdoms. The enzymes from microbial sources have dominated applications in in-
dustrial processes [2]. Though a-amylases have been derived from several microbial
sources, including bacteria, fungi, yeast, and actinomycetes, the enzymes produced from
bacterial and fungal sources have dominated applications in industrial sectors. Because
of their short growth period, their biochemical diversity, and the ease with which
enzyme concentrations might be increased by environmental and genetic manipulation,
the enzymes from microbial sources generally meet industrial demands.
1.2.1 Plant a-Amylases

Plants store carbon predominantly as starch and the metabolism of starch is essential to
all life. Family 1 a-amylases are characterized by having a secretary signal peptide.
This plays an important role in the degradation of extracellular starch in cereal grain
endosperms. Family 2 a-amylases are characterized by having no predicted targeted
peptide and are localized in the cytoplasm. These amylases have been identified from
monocotyledons, dicotyledons, and gymnosperms. They become most active when the
plastidial starch reserves of leaves are more depleted. They are involved in general stress
responses. Family 3 a-amylases are characterized by having a large N-terminal domain,
which contains a large predicted chloroplast transit peptide. These enzymes are
responsible for degrading plastid-bound starch in storage tissues and leaves [8].
1.2.2 Bacterial a-Amylases

a-Amylases are produced from various bacterial sources, including Bacillus, Brevi-
bacterium, Clostridium, Halomonas, Naxibacter, Nesterenkonia, Paenibacillus,
Pseudomonas, Streptomyces sp., etc. Among the bacterial sources, Bacillus sp. is widely
used, especially for the production of thermostable a-amylases. Bacillus subtilis, Bacillus
stearothermophilus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus acid-
ocaldarius, Bifidobacterium bifidum, and Bifidobacterium acerans are important sources
used for a-amylase production [9]. Alkaline and thermotolerant amylases have been
reported from Bacillus sp., B. licheniformis, and Bacillus halodurans [10]. Other bacteria
producing a-amylase include Anoxybacillus beppuensis [11], Bacillus laterosporus [12],
Bacillus acidicola [13], Chryseobacterium taeanense [14], Clostridium sp. [15],
Microbacterium foliorum [16], Nesterenkonia sp. [17], Thermococcus sp. [18], Anoxybacillus
flavithermus [19] etc.
1.2.3 Fungal a-Amylases

Several fungal species also produce a-amylases, including Acremonium, Aspergillus,
Penicillium, Mucor, Neurospora, and Thermomyces sp. Among the fungal sources, the
genus Aspergillus has been widely used for the production of a-amylases. Aspergillus
niger, Aspergillus flavus, and Aspergillus oryzae are important sources used among the
fungal sources [20,21]. Other fungal strains producing a-amylase include Thermomyces
lanuginosus [22].
1.3 Production of a-Amylase

1.3.1 Production Methods
To meet the industrial demand, it is essential to develop a low-cost medium for the
production of a-amylase. It can be produced by submerged fermentation (SmF) and
solid-state fermentation (SSF). The production is affected by a variety of physiological
factors, which include pH, temperature, aeration, inoculum concentration, inoculum
age, composition of the growth medium, surfactants, carbon source, nitrogen source,
etc. [23]. Interactions of these parameters have a significant influence on the production
of the enzyme. Generally, SmF is carried out using synthetic media, incorporating me-
dium constituents such as nutrient broth and soluble starch, as well as other compo-
nents, which are very expensive. Replacement of such constituents by cheaper carbon
and nitrogen sources as well as nutrients would benefit the process in cost reduction.
Agricultural by-products offer potential benefits in this regard [7].
SSF is defined as the process in which the growth of microorganisms is carried out on
solid substrates with negligible free water, or free-flowing water [24]. SSF plays an
important role in the production of enzymes. Agro-industrial substrates are considered
the best substrates for SSF processes. It is of special interest in those processes in which a
crude fermented product may be used directly as an enzyme source. The common
substrates used for SSF processes are wheat bran, rice bran, cassava waste, palm oil
waste, banana waste, tea waste, coconut oil cake, coir pith, corn cobs, etc. In SSF, it is
important to provide optimized water content and to control the water activity of the
fermenting substrate. At times, SSF is preferred to SmF because of its simple technique,
low capital investment, lower levels of catabolite repression and end-product inhibition,
low wastewater output, better product recovery, and high-quality production [25].
Continuous and fed-batch studies are more effective for the production of a-amylase.
The study conducted by Lee and Parulekar [26] revealed that the a-amylase production
by B. subtilis TN 106 was enhanced when batch cultivation was extended with fed-batch
cultivation, and the enzyme activity was 54% higher in a two-stage fed-batch operation
compared to a single-stage batch culture. Mishra and Maheswari [27] reported
a-amylase from a thermophilic fungus, T. lanuginosus; the enzyme was a dimeric protein
with a molecular mass of 42 kDa with optimum pH and temperature of 5.6 and 65 C,
respectively. The enzyme produced high levels of maltose from potato starch, suggesting
its usefulness in the commercial production of maltose and glucose syrups. The study
conducted by Krishna and Chandrasekharan [28] revealed that banana peel could be
utilized as a potential substrate for a-amylase production by A. niger. Saxena and Singh
[29] screened various agro-industrial residues for amylase production from Bacillus sp.
and found mustard oil cake to be the best substrate. The strain produced 5400 U/g of
amylase at 1:3 moisture content, 20% inoculum, and an incubation period of 72 h. Yang
and Wang [30] reported a-amylase production by Streptomyces rimosus TM 55 using
sweet potato residue and peanut meal residue as a substrate. The strain produced
1903 U of a-amylase after 96 h of incubation.
Ramachandran et al. [20] used coconut oil cake (COC), a by-product of oil extraction
from dried copra, as a substrate for the production of a-amylase from fungi. COC sup-
plemented with 0.5% starch and 1% peptone enhanced a-amylase production by
A. oryzae. COC serves as a source of soluble proteins and lipids thus providing essential
nutrients for the growth of and enzyme synthesis by the organism. Production of
a-amylase by B. amyloliquefaciens under SSF using corn gluten meal (CGM) was re-
ported by Saban et al. [31]. The study revealed that a-amylase production in a medium
with CGM was five times higher than that in a medium containing starch and other
components. Utilization of CGM as a substrate makes the process economically viable
because CGM is a by-product of starch-based industries.
Production and optimization of a-amylase from A. oryzae CBS 819 using a by-product
of wheat grinding (gruel) as the sole carbon source was done by Kammoun et al. [32].
Various process parameters affecting the production were optimized by adopting a
BoxeBehnken design, which increased the enzyme production from 40.1 to 151.1 U/mL.
Murthy et al. [33] reported coffee by-products as suitable substrate for the production of
a-amylase under SSF. Coffee waste was converted into value-added products by
fermentation using Neurospora crassa CFR 308. The optimum conditions for a-amylase
production were moisture content of 60%, pH 4.5, incubation temperature of 27 C,
particle size of 1 mm, and incubation time of 5 days. Under optimized conditions the
strain produced 7084 U/gds of a-amylase.
Syed et al. [34] reported extracellular amylase production by Streptomyces gulbar-
gensis DAS 131 by SmF. The highest amylase production was observed when the medium
was supplemented with 1% starch. The enzyme was thermotolerant and stable at pH 9.0.
Starch and peptone were good sources of carbon and nitrogen. Sharma and
Satyanarayana [13] reported enhanced production of acidic high-maltose-forming and
Ca2þ-independent a-amylase by B. acidicola; a maximum enzyme titer of 366 IU/L was
attained after 36 h of fermentation at pH 4.5, 33 C, with 0.5 vvm aeration. The enzyme
titer was 10,100 IU/L in fed-batch fermentation. One of the main advantages of fed-
batch fermentation over the batch fermentation is that the concentration of limiting
substrate is maintained at low levels, thus avoiding the repressing effect of high substrate
concentration and thereby minimizing the accumulation of inhibitory metabolites.
A highly thermostable and calcium-independent a-amylase from A. beppuensis

TSSC-1 was reported by Kikani and Singh [11]. This organism produced a monomeric
a-amylase with optimal pH and temperature of 7.0 and 55 C, respectively. The key
findings of this study were cost-effective purification, high thermostability, and broad
pH stability. The enzyme exhibited Ca2þ independence and resistance to chemical
denaturation, which could make it suitable for many industrial applications. Another
agro-industrial residue, date waste, has also been used as the substrate for the pro-
duction of a-amylase using yeast, Candida guilliermondii CGL-A10 [35]. Maximum
enzyme production was attained in SmF (2056 mmol/L/min). Rajagopalan et al. [15]
used sugarcane bagasse hydrolyzate for the production of a-amylase produced by a
solventogenic Clostridium sp. BOH3. The strain used starch directly without any pre-
treatment and produced extracellular amylase (7.15 U/mg protein) and butanol almost
equivalent to 90% of the yield equivalent to glucose. Sugarcane bagasse was used by
Roohi and Kuddus [16] to produce a cold-active a-amylase from M. foliorum GA2.
Maximum enzyme production (6610 U) was observed when fermentation was carried
out in a medium containing 40% bagasse, 0.0003 M lactose, at pH 8.0, with incubation
temperature of 20 C for 5 day at static conditions. This was the first report on cold-
active a-amylase production from M. foliorum GA2. Table 1.1 shows various microor-
ganisms used for the production of a-amylase.
1.3.2 Factors Influencing the Production of a-Amylase

Production of a-amylase by SSF and SmF is affected by a variety of physicochemical
factors [3]. These include media composition, incubation temperature, inoculum age,
carbon source, nitrogen source, pH, phosphate concentration, aeration, and others.
Table 1.1 Strains and Strategies Adopted for a-Amylase Production

Method of
Microorganism Production Substrate Enzyme Yield References
Bacillus subtilis TN 106 Fed batch [26]
Streptomyces rimosus TM55 SSF Sweet potato residue/ 2642.7 U/gds [30]
peanut residue
Aspergillus oryzae SSF Oil cake 9196 U/gds [20]
Aspergillus oryzae OBS819 SmF 151.1 U/mL [32]
Neurospora crassa CFR 308 SSF Coffee waste 7084 U/gds [33]
Streptomyces gulbargensis DAS 131 SmF Starch [34]
Bacillus acidicola SmF 366 IU/L [13]
Anoxybacillus beppuensis TSSC-1 [11]
Candida guilliermondii CGL-A10 SmF 2056 mmol/L/min [35]
Clostridium sp. BOH3 SmF Sugarcane bagasse 7.15 U/mg protein [15]
hydrolyzate
Microbacterium foliorum GA2 SmF Bagasse 6610 U/mL [16]
SmF, submerged fermentation; SSF, solid-state fermentation.

1.3.2.1 Incubation Temperature

The effect of temperature on a-amylase production is related to the growth of the or-
ganism. Temperature control is very important in fermentation processes because
growth and production of enzymes are sensitive to temperature. Hence, the optimum
temperature varies with the culture. a-Amylases have been produced by various mi-
crobes over a wide range of temperature. Productions in SSF as well as in SmF are
usually carried out in the range 25e37 C. However, psychrophilic and thermophilic
temperatures have also been reported for the production. For example, a-amylase
production was attained at 55 C by the thermophilic fungi Thermomonospora fusca [36]
and T. lanuginosus [27] and at 80 C by a hyperthermophilic bacterium, Thermococcus
profundus [36]. A psychrophilic bacterium, Alteromonas haloplanktis, produced
a-amylase at 4 C [37].
1.3.2.2 pH
The pH of the fermentation medium plays an important role in enzyme production. It
induces morphological changes in the organisms as they are sensitive to the concen-
tration of hydrogen ions present in the medium. A pH change in the medium affects the
growth as well as the product stability. Unlike SmF, in which pH control is almost
mandatory for a-amylase production, in SSF processes, generally there is no need to set,
or control, the pH, as the substrates (agro-industrial residues) mostly possess excellent
buffering capacity and keep the pH favorable for the growth and activity of the culture.
Most of the Bacillus strains used commercially for the production of a-amylases have an
optimum pH of 6.0 or 7.0. Some of the medium components eliminate the need for pH
control. Yabuki et al. [38] reported that A. oryzae 557 accumulated a-amylase in the
mycelia when grown in phosphate, or sulfate-deficient, medium and it was released
when the mycelia were placed in a medium with pH above 7.2. Based on the optimal pH
for activity, a-amylases are classified as acidic, neutral, and alkaline [1].
1.3.2.3 Carbon Sources

a-Amylase production could be either constitutive or inducible. Galactose, inulin, and
glycogen are suitable substrates for a-amylase production in SmF. Supplementation with
lactose; an analog of maltose, a-methyl-D-glucoside; and yeast extract induces the
production [7]. Several agro-residues such as wheat bran, rice bran, vegetable peels, fruit
peels, cassava bagasse, and vegetable-oil-extracted residues are used as substrates for
a-amylase production in SSF. Most studies on a-amylase production by A. oryzae suggest
that the general inducer molecule is maltose. Eratt et al. [39] observed a 20-fold increase
in enzyme activity when maltose and starch were used as inducers in A. oryzae NRC
401013. Xylose and fructose support good growth, but they are strongly repressive [40].
1.3.2.4 Nitrogen Sources

Organic as well inorganic nitrogen sources are used for the production of a-amylases,
although organic sources have been preferred over inorganic nitrogen sources. Commonly
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you—but it hurts me so.”
Peggy sprang up and began to dress, puffing and whistling with
desperation. As soon as she was dressed she ran to the door and
opened it. All was still, a world of green and white. “The fire is almost
out,” she reported, “and I can see Mr. Smith’s horse’s tracks.”
It was about ten o’clock when a couple of horsemen suddenly

rounded the point of the forest and rode into the clearing. One of
them, a slender, elderly man with gray, curly beard and a skin like
red leather, dismounted and came slowly to the door, and though his
eyes expressed surprise at meeting women in such a place, he was
very polite.
“Mornin’, ma’am,” he said, with suave inflection.
“Good morning,” Peggy replied.
“Fine snowy mornin’.”
“It is so.” She was a little irritated by the fixed stare of his round,
gray eyes.
He became more direct. “May I ask who you are and how you
happen to be here, ma’am?”
“You may. I’m Mrs. Adams. I came up here with my husband,
Professor Adams.”
“Where is he?”
“He has gone up the trail toward Frémont. He is a botanist.”
“Is that his horse’s tracks?”
Alice called sharply, “Peggy!”
Mrs. Adams turned abruptly and went in.
The stranger turned a slow gaze upon his companion.
“Well, this beats me. ’Pears like we’re on the wrong trail, Bob. I
reckon we’ve just naturally overhauled a bunch of tourists.”
“Better go in and see what’s inside,” suggested the other man,
slipping from his horse.
“All right. You stay where you are.”
As he stepped to the door and rapped, Peggy opened it, but Alice
took up the inquiry.
“What do you want?” she asked, imperiously.
The man, after looking keenly about, quietly replied: “I’m
wonderin’ how you women come to be here alone, but first of all I
want to know who made them tracks outside the door?”
Alice ignored the latter part of his question and set about
satisfying his wonder. “We came up here with a geological survey,
but my horse fell on my foot and I couldn’t ride, so the men had to
leave me behind—”
“Alone?” sharply interrogated the man.
“No; one man stayed.”
“What was his name?”
“I don’t know. We called him Smith.”
“Was he the man that rode away this morning?”
“What does that matter to you?” asked the girl. “Why are you so
inquisitive?”
He maintained his calm tone of mild authority. “I’m the sheriff of
Uinta County, ma’am, and I’m looking for a man who’s been hiding
out in this basin. I was trailin’ him close when the snow came on
yesterday, and I didn’t know but what these tracks was his.”
Peggy turned toward Alice with an involuntary expression of
enlightenment, and the sheriff read it quickly. Slipping between the
two women, he said:
“Jest a minute, miss. What sort of a looking man was this Smith?”
Alice took up the story. “He was rather small and dark—wasn’t he,
Peggy?”
Peggy considered. “I didn’t notice him particularly. Yes, I think he
was.”
The man outside called: “Hurry up, Cap. It’s beginning to snow
again.”
The sheriff withdrew toward the door. “You’re both lying,” he
remarked without heat, “but it don’t matter. We’ll mighty soon
overhaul this man on the horse—whoever he is. If you’ve been
harboring Hall McCord we’ll have to take you, too.” With that threat
as a farewell he mounted his horse and rode away.
Peggy turned to Alice. “Did you know that young fellow was an
outlaw?”
“Yes: I saw his picture and description on a placard in the railway
station. I recognized him at once.”
“Why didn’t you tell me?”
“Well, I liked his looks, and, besides, I wanted to find out if he
were really bad or only unfortunate.”
“What has he done?”
“They say he held up a train!”
“Merciful Heavens! a train-robber! What’s his real name?”
“The name on the placard was Hall McCord.”
“And to think he was in the same room with us last night, and you
were chumming with him! I can’t understand you. Are you sure he is
the robber?”
“Yes. He confessed to having tried to rob the express car.”
“He seemed such a nice fellow. How did he come to do it?”
Alice concluded not to honor the other girl by bringing her into the
discussion. “Oh, it is hard to say. Need of money, I suppose. Poor
boy, I pity him.”
“They’ll get him, sure. They can follow his tracks as easy as
anything. I don’t suppose I ought to say it, but I hope he’ll get away.
Don’t you?”
“Yes, I do!” was Alice’s fervent response. “But see! it’s snowing
again. It may cover his trail.”
Peggy went to the door and gazed long and keenly at the peaks.
When she turned her face was solemn. “Allie, this is getting pretty
serious for us. If the men don’t come to-day they may get snowed up
entirely.”
Alice stifled a wail. “Oh, if I were only able to walk I wouldn’t mind.
I could help gather fuel and keep the fire going.”
“There’s plenty of wood for another day, but I’m worried about the
men. Suppose they are up on that glacier?”
“I’m not worried about them, but I know they are worrying about
us. They’ll surely start back this morning; but they may not be able to
reach us till night.”
The light of the morning had turned gray and feeble. The air was
still and the forest soundless, save now and then when a snow-laden
branch creaked with its burden.
There was something majestic as well as menacing in this all-
pervading solemn hush.
Peggy went about her duties as cheerfully as she could, but with a
wider knowledge of mountaineering than Alice had. She was at heart
quite terrified. “We’re going to miss our nice outlaw,” she remarked.
“He was so effective as a purveyor of wood.” Then she went to the
door and looked out. “That sheriff will never keep his trail,” she said.
“What’s that?” suddenly asked Alice.
Both listened. “I hear it!” whispered Peggy. “It’s a horse—there!
Some one spoke.”
“It’s Freeman!” Alice joyously called out. “Coo-hoo!”
No one replied, and Peggy, rushing to the door, met the young
outlaw, who appeared on the threshold with stern, set face.
“Who’s been here since I left? Your party?”
Peggy recoiled in surprise and alarm, and Alice cried out, “Why
did you come back?”
“Two men on horseback have been here since I left. Who were
they?” His voice was full of haste.
“One of them said—he was the—the sheriff,” Alice replied, faintly.
He smiled then, a kind of terrifying humor in his eyes. “Well, the
chances are he knew. They took my trail, of course, and left in a
hurry. Expected to overhaul me on the summit. They’ve got their
work cut out for ’em.”
“How did they miss you?” the girl asked, huskily.
“Well, you see, when I got up where I could view the sky I was
dead sure we were in for a whooping big snow-storm, and I just
couldn’t leave you girls up here all alone, so I struck right down the
cañon in the bed of the creek—the short cut. I don’t like to backtrail,
anyway; it’s a bad habit to get into. I like to leave as blind a trail as I
can.” His face lightened up, grew boyish again. “They’re sure up
against a cold proposition about now. They’ll lose my track among
the rocks, but they’ll figure I’ve hustled right on over into Pine Creek,
and if they don’t freeze to death in the pass they’ll come out at
Glover’s haymeadow to-morrow night. How’s the wood-pile holding
out?”
“Please go!” cried Alice. “Take your chance now and hurry away.”
“I’m not used to leaving women in such a fix. The moment I saw
that blizzard was beginning all over again I turned back.”
“You haven’t had any breakfast?” said Peggy.
“Nothing to speak of,” he replied, dryly. “I wasn’t thinking of
breakfast when I pulled out.”
“I’ll get you some.”
Alice could not throw off the burden of his danger. “What will you
do when my people return?”
“I don’t know—trust to luck.”
“You are very foolish. They are certain to come to-day.”
“They won’t know who I am if you women don’t give me away.”
“I’m sure Freeman—Professor Ward—will know you, for he also
saw the placard.”
“That’s no sign. Suppose he does—maybe he won’t think it is his
job to interfere. Anyway”—here his voice became decisive—“I won’t
leave you in such a fix as this.” His eyes spoke to her of that which
his tongue could not utter. “I wanted an excuse to come back,
anyway,” he concluded. “No matter what comes now, my job is here
to protect you.”
She did not rebuke him, and Peggy—though she wondered at his
tone—was too grateful for his presence even to question Alice’s
motive in permitting such remarks.
As for Alice, she felt herself more and more involved in the
tangled skein of his mysterious life. His sudden and reckless
abandonment of the old love which had ruined him, and the new and
equally irrational regard which he now professed for her, filled her
with a delicious marveling.
He appealed to a woman’s imagination. He had the spice of the
unknown. In her relationship with Ward there was no danger, no
mystery—his courtship narrowly escaped being commonplace. She
had accepted his attentions and expected to marry him, and yet the
thought of the union produced, at its warmest, merely a glow of
comfort, a sense of security, whereas the hint of being loved and
protected by this Rob Roy of the hills, this reckless Rough Rider of
the wilderness, was instinct with romance. Of course his devotion
was a crazy folly, and yet, lying there in her rough bunk, with an
impenetrable wall of snow shutting out the rest of the world, it was
hard not to feel that this man and his future had become an
inescapable part of her life—a part which grew in danger and in
charm from hour to hour.
Full two miles above the level of her own home, surrounded by
peaks unscalably wild and lonely, deserted by those who should care
for her, was it strange that she should return this man’s adoring gaze
with something of the primal woman’s gratitude and submission?
The noon darkened into dusk as they talked, slowly, with long
pauses, and one by one the stirring facts of the rover’s life came out.
From his boyhood he had always done the reckless thing. He had
known no restraint till, as a member of the Rough Riders, he yielded
a partial obedience to his commanders. When the excitement of the
campaigns was over he had deserted and gone back to the round-up
wagon and the campfire.
In the midst of his confidences he maintained a reserve about his
family which showed more self-mastery than anything else about
him. That he was the black sheep of an honorable flock became
increasingly evident. He had been the kind of lad who finds in the
West a fine field for dare-devil adventure. And yet there were
unstirred depths in the man. He was curious about a small book
which Alice kept upon her bed, and which she read from time to time
with serene meditation on her face.
“What is that?” he asked.
“My Bible.”
“Can I see it?”
“Certainly.”
He took it carefully and read the title on the back, then turned a
few of the leaves. “I’m not much on reading,” he said, “but I’ve got a
sister that sends me tracts, and the like.” He returned to the fly-leaf.
“Is this your name?”
“Yes.”
“‘Alice Mansfield,’” he read; “beautiful name! ‘New York City’!
That’s pretty near the other side of the world to me.” He studied the
address with intent look. “I’d like to buy this book. How much will you
take for it?”
“I’ll trade it for your weapon,” she replied.
He looked at her narrowly. “You mean something by that. I reckon
I follow you. No, I can’t do that—not now. If I get into business over
the line I’ll disarm, but in this country a fellow needs to be protected.
I want this book!”
“For the fly-leaf?”
He smiled in return. “You’ve hit it.”
She hesitated. “I’ll give you the book if you’ll promise to read it.”
He clapped the covers together and put the volume in his pocket.
“It’s mine! I’ll read every word of it, if it takes an age, and here’s my
hand on it.”
She gave him her hand, and in this clasp something came to her
from his clutching fingers which sobered her. She drew her hand
away hastily and said: “If you read that book—and think about it—it
will change your whole world.”
He, too, lost his brightness. “Well, I’m not so anxious to keep up
this kind of life. But if anybody changes me it will be you.”
“Hush!” she warned with lifted finger.
He fell back, and after a little silence went out to wait upon the fire.
“It seems to me,” said Peggy, reprovingly, “that you’re too gracious
with this mountaineer; he’s getting presumptuous.”
“He doesn’t mean to be. It’s his unsophisticated way. Anyhow, we
can’t afford to be captious to our host.”
“That’s true,” admitted Peggy.
The night shut down with the snow still falling, but with a growing
chill in the air.
“The flakes are finer,” the outlaw announced, as he came in a little
later. “That is a good sign. It is growing colder and the wind is
changing. It will pinch hard before sun-up, and the worst of it, there’s
no way to warm this cabin. We can’t have the door open to-night. I’m
worried about you,” he said to Alice. “If only those chumps had left a
man-size ax!”
The two women understood that this night was to bring them into
closer intimacy with the stranger than before. He could not remain
outdoors, and though they now knew something of his desperate
character, they had no fear of him. He had shown his chivalry. No
one could have been more considerate of them, for he absented
himself at Peggy’s request instantly and without suggestion of
jocularity, and when he came in and found them both in bed he said:
“I reckon I’ll not make down to-night—you’ll need all your blankets
before morning”; and thereupon without weighing their protests,
proceeded to spread the extra cover over them.
Alice looked up at him in the dim light of the candle and softly
asked: “What will you do? You will suffer with cold!”
“Don’t worry about me; I’m an old campaigner. I still have a
blanket to wrap around my shoulders. I’ll snooze in a corner. If you
hear me moving around don’t be worried; I’m hired to keep the fire
going even if it doesn’t do us much good inside.”
The chill deepened. The wind began to roar, and great masses of
snow, dislodged from the tall trees above the cabin, fell upon its roof
with sounds like those of soft, slow footfalls. Strange noises of
creaking and groaning and rasping penetrated to Alice’s ears, and
she cowered half in fear, half in joy of her shelter and her male
protector. Men were fine animals for the wild.
She fell asleep at last, seeing her knight’s dim form propped
against the wall, wrapped in a blanket Indian-wise, his head bowed
over the book she had given him, a candle smoking in his hand.
She woke when he rose to feed the fire, and the current of cold air
which swept in caused her to cover her mouth with the blanket. He
turned toward her.
“It’s all over for sure, this time,” he said. “It’s cold and goin’ to be
colder. How are you standing it? If your feet are cold I can heat a
stone. How is the hurt foot?” He drew near and looked down upon
her anxiously.
“Very much easier, thank you.”
“I’m mighty glad of that. I wish I could take the pain all on myself.”
“You have troubles of your own,” she answered, as lightly as she
could.
“That’s true, too,” he agreed in the same tone. “So many that a
little one more or less wouldn’t count.”
“Do you call my wound little?”
“I meant the foot was little—”
She checked him.
“I didn’t mean to make light of it. It sure is no joke.” He added,
“I’ve made a start on the book.”
“How do you like it?”
“I don’t know yet,” he answered, and went back to his corner.
She snuggled under her warm quilts again, remorseful, yet not
daring to suggest a return of the blanket he had lent. When she
woke again he was on his feet, swinging his arms silently. His candle
had gone out, but a faint light was showing in the room.
“Is it morning?” she asked.
“Just about,” he replied, stretching like a cat.
The dawn came gloriously. The sun in far-splashing splendor
slanted from peak to peak, painting purple shadows on the snow and
warming the boles of the tall trees till they shone like fretted gold.
The jays cried out as if in exultation of the ending of the tempest, and
the small stream sang over its icy pebbles with resolute cheer. It was
a land to fill a poet with awe and ecstatic praise—a radiant, imperial,
and merciless landscape. Trackless, almost soundless, the mountain
world lay waiting for the alchemy of the sun.
VI
The morning was well advanced when a far, faint halloo broke
through the silence of the valley. The ranger stood like a statue,
while Peggy cried out:
“It’s one of our men!”
Alice turned to the outlaw with anxious face. “If it’s the sheriff stay
in here with me. Let me plead for you. I want him to know what
you’ve done for us.”
The look that came upon his face turned her cold with fear. “If it is
the sheriff—” He did not finish, but she understood.
The halloo sounded nearer and the outlaw’s face lightened. “It’s
one of your party. He is coming up from below.”
Impatiently they waited for the new-comer to appear, and though
he seemed to draw nearer at every shout, his progress was very
slow. At last the man appeared on the opposite bank of the stream.
He was covered with snow and stumbling along like a man half dead
with hunger and fatigue.
“Why, it’s Gage!” exclaimed Peggy.
It was indeed the old hunter, and as he drew near his gaunt and
bloodless face was like that of a starved and hunted animal. His first
word was an anxious inquiry, “How are ye?”
“All well,” Peggy answered.
“And the crippled girl?”
“Doing nicely. Thanks to Mr. Smith here, we did not freeze. Are
you hungry?”
The guide looked upon the outlaw with glazed, protruding eyes.
“Hungry? I’m done. I’ve been wallerin’ in the snow all night and I’m
just about all in.”
“Where are the others?” called Alice from her bed.
Gage staggered to the door. “They’re up at timber-line. I left them
day before yesterday. I tried to get here, but I lost my bearin’s and
got on the wrong side o’ the creek. ’Pears like I kept on the wrong
side o’ the hog-back. Then my horse gave out, and that set me afoot.
I was plum scared to death about you folks. I sure was.”
Peggy put some food before him and ordered him into silence.
“Talk later,” she said.
The outlaw turned to Alice. “That explains it. Your Professor Ward
trusted to this man to take care of you and stayed in camp. You can’t
blame him.”
Gage seemed to have suddenly become old, almost childish. “I
never was lost before,” he muttered, sadly. “I reckon something must
have went wrong in my head. ’Pears like I’m gettin’ old and foolish.”
Alice exchanged glances with the outlaw. It was plain that he was
in no danger from this dazed and weakened old man who could think
of nothing but the loss of his sense of direction.
As the day advanced the sun burned clear. At noon it was warm
enough to leave the door open, and Alice, catching glimpses of the
flaming world of silver and purple and gold, was filled with a desire to
quit her dark corner.
“I’m going to get up!” she exclaimed. “I won’t lie here any longer.”
“Don’t try it!” protested Peggy.
“I’m going to do it!” she insisted. “I can hobble to the door if you
help me.”
“I’ll carry you,” said the outlaw. “Wrap her up and I’ll get her a
seat.”
And so, while Mrs. Adams wrapped her patient in a blanket, the
outlaw dragged one of the rough, ax-hewn benches to the door and
covered it with blankets. He put a stone to heat and then re-entered
just as Alice, supported by Peggy, was setting foot to the floor.
Swiftly, unhesitatingly, and very tenderly he put his arms about her
and lifted her to the bench in the doorway before the fire.
It was so sweet to feel that wondrous body in his arms. His daring
to do it surprised her, but her own silent acquiescence, and the
shiver of pleasure which came with the embarrassment of it,
confused and troubled her.
“That’s better,” he said as he dropped to the ground and drew the
blankets close about her feet. “I’ll have a hot stone for you in a
minute.”
He went about these ministrations with an inward ecstasy which
shone in his eyes and trembled in his voice. But as she furtively
studied his face and observed the tremor of his hands in tender
ministration she lost all fear of him.
After three days in her dark corner of the hut the sunshine was
wondrously inspiring to the girl, although the landscape on which she
gazed was white and wild as December. It was incredible that only a
few hours lay between the flower-strewn valley of her accident and
this silent and desolate, yet beautiful, wilderness of snow. And so, as
she looked into the eyes of the outlaw, it seemed as though she had
known him from spring to winter, and her wish to help him grew with
every hour of their acquaintanceship.
She planned his defense before Ward and Adams. “When they
know how kind and helpful he has been they can but condone his
one rash deed,” she argued in conclusion.
He was sitting at her feet, careless of time, the law, content with
her nearness, and mindful only of her comfort, when a distant rifle-
shot brought him to his feet with the swiftness of the startled stag.
“That’s your expedition,” he said, “or some one who needs help.”
Again the shots rang out, one, two, three—one, two, three.
“It’s a signal! It’s your party!”
Peggy uttered a cry of joy and rushed outside, but Alice turned an
unquiet gaze on the outlaw. “You’d better fly!”
“What is the use?” he answered, bitterly. “The snow is so deep
there is no show to cross the range, and my horse is weak and
hungry.”
Gage appeared at the door. “Lemme take your gun, stranger; I
want to answer the signal.”
“Where’s your own?”
“I left it on my horse,” the old man answered, sheepishly.
The young fellow looked at Alice with a keen glitter in his eyes. “I’ll
make answer myself,” he said; “I’m very particular about my
barkers.”
Alice, as she heard his revolver’s answering word leap into the
silent air and bound and rebound along the cliffs, was filled with a
sudden fear that the sheriff might be guided back by the sound—and
this indeed the fugitive himself remarked as he came back to his
seat beside her.
“If he’s anywhere on this side of the divide he’ll sure come back.
But I’ve done my best. The Lord God Almighty has dropped the
snow down here and shut me in with you, and I’m not complaining.”
There was no answer to be made to this fatalism of utterance, and
none to the worship of his eyes.
“Lift me up!” commanded Alice; “I want to look out and see if I can
see anybody.”
The outlaw took her in his arms, supporting her in the threshold in
order that she might see over the vast sea of white. But no human
being was to be seen.
“Take me back—inside,” Alice said to the man who had her in his
arms. “I feel cold here.”
Once again, and with a feeling that it was, perhaps, for the last
time, he carried her back to her bench and re-enveloped her in her
blankets.
“Stay here with me now,” she whispered to him, as she looked up
into his face.
And the outlaw, filled with gladness and pride, threw himself on
the floor beside her.
VII
The signal pistol-shots came nearer and nearer, but very slowly;
and as the outlaw sat beside Alice’s couch he took her Bible from his
pocket and said:
“I made a stab at reading this last night.”
She smiled. “I saw you. How did you like it?”
“I didn’t exactly get aboard someway.”
“What was the trouble?”
“I guess it was because I kept thinking of you—and my own place
in the game. Three days ago I didn’t care what became of me, but
now I want a chance. I don’t see any chance coming my way, but if I
had I’d make use of it.” He looked at her a moment in silence, then
with sudden intensity broke forth. “Do you know what you mean to
me? When I look at your face and eyes I’m hungry for you.”
She shrank from him and called to Mrs. Adams.
He went on. “Oh, you needn’t be afraid. I just wanted to say it,
that’s all. If there was only some other way to straighten myself—but
I can’t go to jail. I can’t stand up to be clipped like a poodle-dog, then
put on striped clothing and walk lock-step—I can’t do it! They’ll put
me in for ten years. I’d be old when I got out.” He shuddered. “No, I
won’t do that! I’d rather die here in the hills.”
She grew white in sympathy. “It is a frightful price to pay for one
insane act, and yet—crime should be punished.”
“I’m getting my punishment now,” he replied, with darkly brooding
glance. “There’s a good old man and two women, my sisters, waiting
for me down the slope. If I could reach home I’d try to live straight,
but it’s a long and dangerous trail between here and there.”
Peggy now ran into the cabin. “It’s the expedition,” she
announced. “I can see Freeman.”
“I reckon this is where I get off,” said the outlaw in a tone of
mingled relief and dismay.
“No, no!” Alice entreated. “Stay till Freeman comes. He will help
you. Let me explain to him. I know he will not betray you.”
He looked at her again with that intent, longing worship in his
eyes, and answered, “I accept the chance for the sake of one more
hour with you.”
The outlaw stepped to the door, and he saw a man at the head of
his train mid-leg deep in snow, leading his horse, breaking the way
for his followers, who were on foot, crawling, stumbling, and twisting
among the down-timber, unmindful of the old trail.
At sight of that big and resolute leader, with flowing black beard
and ruddy face, the outlaw was filled with jealous sadness. To find
Ward a man of superb physical prowess, the kind that measures
peaks for the fun of it, was disturbing, and without defining his feeling
he was plunged into melancholy musing. And when later Ward
entered, and, stooping over the couch, kissed Alice, the end of his
idyl seemed to him announced.
In the bustle of the moment, in the interchange of anxious, hurried
inquiries, the outlaw stood aside in the corner, unnoticed, till Alice
caught Ward’s arm and said:
“Freeman, this is Mr. Smith, to whom we owe a great deal. He has
taken the utmost care of us. We would have frozen but for him.”
Ward shook hands with the outlaw, but wonderingly asked of
Alice, “But where was Gage?”
The outlaw answered, “Gage got lost and only turned up a couple
of hours ago.”
Ward turned to Alice in horror. “Good Lord! And you were here
alone—crippled—in this storm?”
“No—that’s what I’m telling you. Mr. Smith came and took care of
us. He brought our wood, he cooked for us, he kept our fire going.
He gave up his bed, even his blankets, for us. You should be very
generous to him.”
Ward again reached a hearty hand. “I’m tremendously obliged to
you.”
The outlaw quailed under all this praise. “There was mighty little to
do,” he answered. “I only shared my fire with them.”
Ward studied him closer. “Haven’t we met before?”
“No, I reckon not.”
“I’m quite sure I’ve see you somewhere. What are you doing up in
here?”
Alice interposed. “What are we going to do?”
Ward turned to the outlaw. “What would you advise? I’ve only had
one idea, and that was to reach this cabin. Now what would you do?”
The outlaw was ready. “I would send a part of the men with the
horses down the valley to grass and I’d wait here till Miss Mansfield
is able to ride.”
“Will this snow go off?”
“That’s my notion.”
“It’s certain we can’t camp here—the horses must have grass.”
“I’ll be able to ride in a day or two,” Alice said, bravely.
“We could frame up a portable bed and carry you,” suggested the
outlaw; “but it can’t be done to-night, so you’d better send your outfit
down to the marsh to camp. The horses are worn out and so are the
men.”
“Will you guide them to grass and help them find shelter?”
The outlaw hesitated for an instant, and Alice interposed: “No, no!
Let Gage do that. I want Mr. Smith to remain here.”
Ward perceived in her entreaty something of anxiety and fear, and
after the men and horses had started down the slope he turned to
the outlaw and said: “I’m mighty grateful to you, Mr. Smith. It must
have surprised you to find these women here.”
The outlaw dryly replied, “It did!”
Alice added: “It was in the middle of the night, too; but Mr. Smith
was very nice about it. He slept outdoors without a word of
complaint.”
Ward had figured the situation to conclusion: “Smith is a poacher,”
and though he had a savage dislike of these illicit game-
slaughterers, he could not but be glad of the presence of this
particular outlaw, and resolved to overlook his trade in gratitude for
his cabin and service.
The outlaw helped Adams and Ward to clear away the snow for a
tent, and Alice, seeing the three men thus amicably joined in her
defense, could not find it in her heart to condemn one of them as a
criminal. Here in the white isolation of the peaks the question of
crime and its punishment became personal. To have this man’s fate
in her hand was like grasping the executioner’s sword for herself.
“If women had to punish criminals themselves, with their own
hand,” she asked, “how many of them would do it?”
Peggy came in and whispered to her: “No one else seems to have
recognized him. He may get away safely. I hope he will. Shall we tell
the men who he is?”
“Yes, we shall have to do that soon, but I’m afraid they won’t take
the sentimental view of him that we do. I tremble to think of what
they will do when they know.”
Ward explained to Adams: “Our friend Smith here is a poacher—
but as our account stands I don’t feel it my duty to report him, do
you?”
“No; Peggy tells me he has acted like a gentleman all through.”
In this spirit they made themselves comfortable for the night.
The sun set gloriously, but the air bit ever sharper, and while
Peggy went about her cooking, assisted by her husband and the
outlaw, Alice pulled Ward down to her bedside and hurriedly began:
“You remember that placard we read in the station—the one about
the train-robber?”
“Yes!”
“Well, this is the man—our Mr. Smith.”
Ward looked at her a moment with reflective eyes, then
exclaimed: “You’re right! I thought I’d seen him somewhere.”
“And the sheriff is after him. He was here yesterday morning.”
“Here?”
“Yes. You see, Mr. Smith stayed with us till he thought the storm
was over, then rode away, intending to cross the divide, but when the
snow began again he turned back. He said he couldn’t leave us
alone. He left us just before dawn, and four or five hours afterward
the sheriff came. Of course he saw the poor fellow’s trail and
instantly set off after him.”
“But why didn’t they meet?”
“Because Mr. Smith came back a different way and then the
blizzard came on and covered up his tracks. He thinks the sheriff has
gone on over the divide. You must help him, Freeman. Help him to
get away and find some way to give him a start. Nobody could have
been more considerate, and I can’t see him taken by these cold-
blooded men who want that two thousand dollars’ reward. He really
could have escaped, only for us. He came back to protect us.”
Ward pondered. “The problem is not so easy of solution. A train
robbery is a pretty serious matter. I’m very grateful to him, but to
connive at his escape is itself a punishable act. Why did you tell me?
I could have passed it over—”
“Because I’m afraid the sheriff may come back at any moment.”
Ward’s brow was troubled. “I could ignore his deed and pretend
not to know who he is, but definitely to assist a bandit to escape is a
very serious matter.”
“I know it is; but remember he gave up his chance to cross the
divide in order to keep us from suffering.”
“I wish you hadn’t told me,” he repeated, almost in irritation. “If the
sheriff only keeps on over the range Smith can take care of himself.”
As the outlaw re-entered the cabin Alice acknowledged in him
something worth a woman to love. In the older man was power,
security, moral, mental, and physical health, the qualities her reason
demanded in a husband; but in the other was grace and charm,
something wildly admirable. He allured as the warrior, intrepid and
graceful, allured the maiden, as the forest calls the householder.
Something primordial and splendid and very sweet was in her feeling
toward him. There could be no peaceful wedlock there, no security of
home, no comfort, only the exquisite thrill of perilous union, the
madness of a few short weeks—perhaps only a few swift days of
self-surrender, and then, surely, disaster and despair. To yield to him
was impossible, and yet the thought of it was tantalizingly sweet.
When she looked toward Ward she perceived herself sitting
serenely in matronly grace behind a shining coffee-urn in a well-
ordered, highly civilized breakfast-room, facing a most considerate
husband who nevertheless was able to read the morning paper in
her presence. When she thought of life with the outlaw all was dark,
stormy, confused, and yet the way was lit by his adoring eyes. A
magical splendor lay in the impulse. His love, sudden as it seemed,
was real—she was certain of that. She felt the burning power, the
conjury of its flame, and it made her future with Ward, at the
moment, seem dull and drab.
“Why, why could not such a man and such a passion come with
the orderly and the ethical?” she asked herself.
At the best he was fitted only for the mine or the ranch, and the
thought of life in a lonely valley, even with his love to lighten it, made
her shudder. On one side she was a very practical and far-seeing
woman. The instant she brought her reason to bear on the problem
she perceived that any further acquaintance with this man was
dangerous. They must part here at this moment, and yet she could
not let him go without in some way making him feel her wish to help
him.
VIII
Ward and the outlaw were discussing plans for getting out of the
basin when Adams came in to say, “A couple of other weary
wanderers are turning up.”
“The sheriff!” instantly exclaimed Alice, her face whitening in swift
dismay.
In that moment the forester was transformed. With a weapon in
his hand he stood aside, his eyes on the door, a scowl of battle on
his face. He resembled a wolf with bared fangs ready to die
desperately.
Ward, quick to read his purpose, interposed. “Wait!” he
commanded. “Stay here; I’ll see them. Don’t be rash.”
As he passed out into the firelight the outlaw, without relaxing his
vigilance, said in a low voice, “Well, girl, I reckon here’s where I say
good night.”
“Don’t resist,” she pleaded. “Don’t fight, please! Please! What is
the use? Oh, it’s too horrible! If you resist they will kill you!”
There was no fear in his voice as he replied: “They may not; I’m
handy with my gun.”
She was breathless, chilled by the shadow of the impending
tragedy. “But that would be worse. To kill them would only stain your
soul the deeper. You must not fight!”
“It’s self-defense.”
“But they are officers of the law.”
“No matter; I will not be taken alive.”
She moaned in her distress, helplessly wringing her hands. “O
God! Why should I be witness of this?”
“You won’t be. If this is the sheriff I am going to open that door and
make a dash. What happens will happen outside. You need not see
it. I’m sorry you have to hear it. But I give you my word—if you must
hear something I will see to it that you hear as little as possible.”
The latch clicked—he stepped back, and again stood waiting,
silent, rigid, ready to act, murderous in design.
Mrs. Adams entered quickly, and, closing the door behind her,
hurriedly whispered: “It’s the sheriff. Hide! The men will hold them as
long as they can. Hide!”
The outlaw looked about and smiled. “Where?” he asked, almost
humorously. “I’m not a squirrel.”
“Under the bunk. See, there is room.”
He shook his head. “No, I refuse to crawl. I won’t sneak. I never
have. I take ’em as they come.”
“For my sake,” pleaded Alice. “I can’t bear to see you killed. Hide
yourself. Go to the door,” she said to Peggy. “Don’t let them in. Tell
Freeman—” She rose and stood unsteadily, forgetful of her own
pain.
Mrs. Adams urged her to lie down, but she would not. The
moments passed in suspense almost too great to be endured.
“Listen!” commanded the outlaw. “They’re coming in.”
As they harkened Ward’s voice rose clearly. “You can’t miss the
camp,” he was saying, as if speaking to some one at a distance.
“Just keep the trail in the snow and you’ll find them. I’m sorry we
can’t put you up—but you see how it is.”
“They’re going!” exclaimed Alice. “Thank God, they’re going!”
“It can’t be they’ll go without searching the shack,” the fugitive
muttered, in no measure relaxing his attitude of watchful menace.
“They’re playing a game on us.”
Again the latch clicked, and this time it was Ward who confronted
the outlaw’s revolver mouth.
“It’s all right,” Ward called, instantly understanding the situation.
“They’re gone. The old man was about played out, for they’ve been
fighting snow all day, but I told him we couldn’t take care of them
here and they have gone on down to the camp. He thinks you got
over the divide. You are all right for the present.”
“They’ll come back,” replied the other. “It only puts the deal off a
few hours. They’ll return, trailin’ the whole camp after them. What
can I do? My horse is down there in the herd.”
“That’s bad,” exclaimed Ward. “I wonder if I could get him for
you?”
“If I had him he’s weak and hungry, and the high places are feet
deep in drifts. It doesn’t signify. I’m corralled any way you look at it,
and the only thing left is to fight.”
“There’s our trail to the glacier,” Ward musingly suggested; “it’s a
pretty deep furrow—you might make it that way.”
A spark of light leaped into the man’s eyes. “How far up does it
run? Where does it end?”
“In Glacier Basin, just at timber-line.”
The outlaw pondered, speaking his thoughts aloud. “From there
across to the Indian reservation there isn’t a wolf track.... It’s a man’s
job crossing there, almost sure death, but it’s my only show.” He had
replaced his weapon in his belt and was weighing his chance, his
eyes fixed on Alice’s face. To leave this shelter, this warm circle of
light, this sweet girlish presence, and plunge into the dark, the cold,
and the snow, was hard. No one but a man of unconquerable
courage would have considered it. This man was both desperate and

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M. Adsul DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Cristóbal N. Aguilar Food Research Department, School of Chemistry,

A. Angel-Cuapio Universidad Autónoma Metropolitana-Iztapalapa, Mexico City,

G.S. Anisha Government College for Women, Trivandrum, Kerala, India

P. Binod CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

J. Buenrostro-Figueroa Department of Biotechnology, Division of Health and

S. Chakraborty Indian Institute of Technology Guwahati, Guwahati, Assam, India

M.L. Chávez González Food Research Department, School of Chemistry,

G.-Q. Chen Tsinghua University, Beijing, China

S. Chen Hubei University, Wuhan, PR China

Juan C. Contreras-Esquivel Food Research Department, School of Chemistry,

J.D. Coral Bioprocess Engineering and Biotechnology Department, Federal

J.C. de Carvalho Bioprocess Engineering and Biotechnology Department, Federal

J. de Oliveira Bioprocess Engineering and Biotechnology Department, Federal

A. Dhillon Indian Institute of Technology Guwahati, Guwahati, Assam, India

M.J. Fernandes Bioprocess Engineering and Biotechnology Department, Federal

R. Gaur Indian Oil Corporation Limited, R&D Centre, Faridabad, India

A. Goyal Indian Institute of Technology Guwahati, Guwahati, Assam, India

L.R.C. Guimarães Bioprocess Engineering and Biotechnology Department, Federal

M. Haridas Kannur University, Kannur, India

R. Hemamalini Indian Institute of Technology Delhi, New Delhi, India

Ayerim Hernandez-Almanza Food Research Department, School of Chemistry,

A. Illanes Pontiﬁcia Universidad Católica de Valparaíso, Valparaíso, Chile

J. Isar University of Delhi South Campus, New Delhi, India

A. Joseph Kannur University, Kannur, India

S.G. Karp Bioprocess Engineering and Biotechnology Department, Federal

N. Karthik CSIR-National Institute for Interdisciplinary Science and Technology

R. Kaushik University of Delhi South Campus, New Delhi, India

S.K. Khare Indian Institute of Technology Delhi, New Delhi, India

P.C.S. Kirnev Bioprocess Engineering and Biotechnology Department, Federal

D. Kothari Indian Institute of Technology Guwahati, Guwahati, Assam, India

C. Larroche Blaise Pascal University, Aubière Cedex, France

L.A.J. Letti Bioprocess Engineering and Biotechnology Department, Federal

O. Loera-Corral Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, DF,

A.I. Magalhães, Jr. Bioprocess Engineering and Biotechnology Department,

A.B.P. Medeiros Bioprocess Engineering and Biotechnology Department, Federal

J.D.C. Medina Bioprocess Engineering and Biotechnology Department, Federal

F. Miranda-Hernández Universidad Autónoma Metropolitana-Iztapalapa, Mexico

N.R. Nair CSIR-National Institute for Interdisciplinary Science and Technology

S. Nair Dow Chemicals GmBH, Dubai, UAE

K.M. Nampoothiri CSIR-National Institute for Interdisciplinary Science and

A. Nandan CSIR-National Institute for Interdisciplinary Science and Technology

S. Negi Motilal Nehru National Institute of Technology, Allahabad, India

M.G.B. Pagnoncelli Bioprocess Engineering and Biotechnology Department,

A. Pandey Center of Innovative and Applied Bioprocessing, (a national institute

V. Rajulapati Indian Institute of Technology Guwahati, Guwahati, Assam, India

S. Ramachandran Insight Professional Institute, Dubai, UAE

A. Rani Indian Institute of Technology Guwahati, Guwahati, Assam, India