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 Current Developments
in Biotechnology and
Bioengineering
Biological Treatment of
Industrial Effluents

Edited by
Duu-Jong Lee, Veeriah Jegatheesan, Hao Huu Ngo,
Patrick C. Hallenbeck, Ashok Pandey

AMSTERDAM l BOSTON l HEIDELBERG l LONDON l NEW YORK l OXFORD

PARIS l SAN DIEGO l SAN FRANCISCO l SINGAPORE l SYDNEY l TOKYO
Elsevier
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List of Contributors
A. Asghar University of Malaya, Kuala Lumpur, Malaysia

N. Balagurusamy Universidad Autónoma de Coahuila, Torreón, Coahuila, Mexico

E.R. Bandala Universidad de Las Américas Puebla, Cholula, Puebla, Mexico

A. Buthiyappan University of Malaya, Kuala Lumpur, Malaysia

P. Champagne Queen’s University, Kingston, ON, Canada

C.-C. Chen Chung Chou University of Science and Technology, Changhwa, Taiwan

W.M.A.W. Daud University of Malaya, Kuala Lumpur, Malaysia

A. Daverey Doon University, Dehradun, India

C. Fu Centre for Water Research, National University of Singapore, Singapore

V. Gadhamshetty South Dakota School of Mines and Technology, Rapid City, SD,
United States

M. García Lozano Universidad Autónoma de Coahuila, Torreón, Coahuila, Mexico

B.D. Gebrewold UNESCO-IHE Institute for Water Education, Delft, The Netherlands

B. Giri University of Hawai’i at Manoa, Honolulu, HI, United States

J.-D. Gu The University of Hong Kong, Hong Kong, China

W. Guo University of Technology Sydney, Sydney, NSW, Australia

F.I. Hai University of Wollongong, Wollongong, NSW, Australia

B.S. Herath Western Sydney University, Penrith, NSW, Australia

M. Howell Queen’s University, Kingston, ON, Canada

xiii
xiv List of Contributors

S. Hu Advanced Water Management Centre, The University of Queensland, Brisbane,

Australia

G. Kastl Western Sydney University, Penrith, NSW, Australia

S.K. Khanal University of Hawai’i at Manoa, Honolulu, HI, United States

M. Kumar Indian Institute of Technology Madras, Chennai, India

C.-H. Lay Feng Chia University, Taichung, Taiwan

D.-J. Lee National Taiwan University, Taipei, Taiwan; National Taiwan University of
Science and Technology, Taipei, Taiwan

C.-Y. Lin Feng Chia University, Taichung, Taiwan

J.-G. Lin National Chiao Tung University, Hsinchu, Taiwan

J.C. Liu National Taiwan University of Science and Technology, Taipei, Taiwan

L. Liu Queen’s University, Kingston, ON, Canada

S.L. Low Centre for Water Research, National University of Singapore, Singapore

Y. Lu Advanced Water Management Centre, The University of Queensland, Brisbane,

Australia

L.E. Montañez Hernandez Universidad Autónoma de Coahuila, Torreón,

Coahuila, Mexico

P. Mullai Annamalai University, Tamil Nadu, India

H.Y. Ng Centre for Water Research, National University of Singapore, Singapore

K.K. Ng Centre for Water Research, National University of Singapore, Singapore

L.D. Nghiem University of Wollongong, Wollongong, NSW, Australia

H.H. Ngo University of Technology Sydney, Sydney, NSW, Australia

S. Nitayavardhana Chiang Mai University, Chiang Mai, Thailand

 List of Contributors xv

R. Oropeza Navarro Universidad Nacional Autnoma de México, Cuernavaca,

Morelos, Mexico

K. Pakshirajan Indian Institute of Technology Guwahati, Guwahati, Assam, India

Y. Peña Garcia Universidad Autónoma de Coahuila, Torreón, Coahuila, Mexico

W.E. Price University of Wollongong, Wollongong, NSW, Australia

G. Qiu National University of Singapore, Singapore

A.A.A. Raman University of Malaya, Kuala Lumpur, Malaysia

M.E. Raynal Gutierrez Universidad de Las Américas Puebla, Cholula, Puebla,

Mexico

E.R. Rene UNESCO-IHE Institute for Water Education, Delft, The Netherlands

J.L. Sanchez-Salas Universidad de Las Américas Puebla, Cholula, Puebla, Mexico

A. Sathasivan Western Sydney University, Penrith, NSW, Australia

S.T.M.L.D. Senevirathna Charles Sturt University, Bathurst, NSW, Australia

X. Shi Centre for Water Research, National University of Singapore, Singapore

K.-Y. Show Zhejiang Juneng Co. Ltd., Tongxiang, China

Y. Song Chinese Research Academy of Environmental Sciences, Beijing, PR China

D.S. Srinivasa Raghavan National University of Singapore, Singapore

S. Su Commonwealth Scientific and Industrial Research Organisation (CSIRO),

Pullenvale, Queensland, Australia

M.M. Tejas Namboodiri Indian Institute of Technology Guwahati, Guwahati,

Assam, India

R. Thiruvenkatachari Commonwealth Scientific and Industrial Research

Organisation (CSIRO), Pullenvale, Queensland, Australia

Y.-P. Ting National University of Singapore, Singapore

xvi List of Contributors

T.P. Tram Vo University of Technology Sydney, Sydney, NSW, Australia

J. Virkutyte Environmental Consultant, Cincinnati, OH, United States

S. Vishali SRM University, Tamil Nadu, India

R. Wickham University of Wollongong, Wollongong, NSW, Australia

M.K. Yogeswari Vivekanandha College of Arts and Sciences for Women,

Tamil Nadu, India
About the Editors

Duu-Jong Lee
Professor Duu-Jong Lee is currently a Life-Time Specially
Appointed Professor of National Taiwan University (NTU)
and chair professor and vice president of the National
Taiwan University of Science and Technology. He obtained
his bachelor (1984) and Ph.D. (1989) degrees both at the
Chemical Engineering Department, NTU. Professor Lee has
a strong research interest in the areas of industrial waste-
water treatment, biomass to energy, and environmental
management, especially in microscale transport and reac-
tion processes in biomass matrix. Since 1989, he has
completed several projects successfully and developed
various processes with full-scale applications. His research
has credited him with more than 1000 publications and
communications. The citations in SCOPUS on Professor Lee’s work have exceeded
14,000 with an h index of 57. Professor Lee is the recipient of many national and in-
ternational awards and fellowships. He was also the president of the Taiwan Institute of
Chemical Engineers, with 3500 members in Taiwan and overseas.

Veeriah Jegatheesan
Professor Veeriah Jegatheesan (Jega) received his Ph.D. in
Water Engineering and has 20 years of experience in water
research. He has worked as a consultant at Sydney Water
Corporation and has collaborated with several other water
authorities in Australia. Currently he is attached to the
School of Engineering at the Royal Melbourne Institute of
Technology, Australia. His research focuses on sustainable
catchment management through the application of novel
treatment processes, resource recovery, and mathematical
modeling. He has coedited four books and was a managing guest editor for 15 special
issues in peer-reviewed journals and has published 90 journal articles. He is the Chief
Editor of a book series entitled Applied Environmental Science and Engineering for a
Sustainable Future published by Springer. He is an associate editor for the Journal of
Water Sustainability and an editorial board member of a number of journals. His core

xvii
xviii About the Editors

expertise is in membrane system design, aquaculture, desalination, forward osmosis,

resource recovery, and water distribution maintenance management. Professor
Jegatheesan is the cofounder and the chair of an international conference on the
“Challenges in Environmental Science and Engineering,” which has been held annually
since 2008 around the world.

Hao Huu Ngo

Professor Hao Huu Ngo is an academic with more than
30 years of professional experience in Australia and in Asian
countries. He is currently a professor of Environmental
Engineering and serving as Deputy Director and Research
Theme CoordinatordSustainable Water: Treatment and
Reuse Technologies and Management at the Centre for
Technology in Water and Wastewater, School of Civil and
Environmental Engineering, University of Technology,
Sydney.
Professor Ngo is well known for his activities in the
areas of water and wastewater and waste treatment and
reuse technologies, which include advanced biological waste treatment (aerobic and
anaerobic membrane bioreactors, specific attached- and/or suspended-growth bio-
reactors, biosorption), membrane technologies (membrane hybrid systems, desalina-
tion), and physicalechemical separation technologies as pretreatment or posttreatment
(adsorption, flocculation, and filtration). His expertise and practical experience also
cover the areas of solid waste management, alternative water resources, water man-
agement and impact assessment, desalination, and specific green technologies:
waterewasteebioenergy. He has published more than 300 publications including two
books and several book chapters and patents while receiving quite a number of highly
recognized honors and awards. He has served as an editor of Bioresource Technology and
an editorial board member of several esteemed ISI/SCI international journals.

Patrick C. Hallenbeck
Professor Patrick C. Hallenbeck, Microbiologist, obtained a
Ph.D. in Biophysics from the University of California at
Berkeley (United States) and held postdoctoral positions at
the French Nuclear Energy Center, Grenoble (France), and
the University of California at Davis (United States) before
taking up a position as professor in the Department of
Microbiology and Immunology, University of Montreal,
and a senior scientist at the US Air Force Academy Life
Sciences Research Center. Professor Hallenbeck’s research
interests are in microbial physiology, applied microbiology
and biotechnology, biological energy production, anaer-
obic metabolism, and nitrogen fixation. Professor
 About the Editors xix

Hallenbeck is President of the Biohydrogen Subdivision of the International Hydrogen

Association. He has served on the organizing committees of numerous international
conferences, was chair of ISPP2009, and was chair of BioH2 2013. He is the author of over
60 original research publications, 22 reviews and book chapters, and numerous abstracts
in conference proceedings. He is the editor of two books. Professor Hallenbeck won the
Outstanding Scientist Award for 2012 from the International Forum on Industrial
Bioprocesses (www.ifibiop.org). He is currently researching the role of metabolite
sensing and signaling in the regulation of microbial metabolism and using metabolic
engineering to increase biofuel production. He is an editorial board member for
Bioresource Technology, Journal of Bacteriology, Frontiers in Microbiology, and Marine
Biotechnology and a section editor of Biotechnology for Biofuels.

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, pro-
biotics 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 technologies 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
xx About the Editors

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
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.
Preface

This book is a part of the comprehensive series Current Developments in Biotechnology and
Bioengineering (Editor-in-Chief: Ashok Pandey), comprising nine volumes. To this series, the
present volume brings an extensive and thorough treatment of various waste and wastewater
treatment processes by internationally recognized experts. Up-to-date coverage of recent
advances is given for each specific subject area, and the remaining challenges over the wide
range of processes are highlighted. As will be evident, enormous quantities of municipal,
industrial, and agricultural wastewaters are generated globally each year.
With increasing global urbanization, municipal wastewater treatment has taken on
worldwide importance. This is no small problem, with domestic wastewater production
estimated to be 330 km3/year. This is enough to provide, if all were treated, sufficient water
and nutrients for millions of acres of food crop production and enough energy, through
anaerobic digestion, for millions of households. Of course, we are far from achieving total
recovery of the valuable resources present. This waste stream is particularly well character-
ized and of rather similar composition everywhere. However, even here there are treatment
challenges related to climatic variations or nutrient removal requirements. These subjects are
treated in part by several of the chapters in the present volume, including one on wastewater
treatment in cold climates and another on the recently developed anammox process for
effective tertiary treatment through the removal of fixed nitrogen.
The quantity of annual agricultural wastewater produced globally is enormous because
of the large water usage requirement: on average 1000 L/kg for plant crops and at least three
times this amount for meat production. Agricultural wastewater, largely runoff, is difficult to
treat because it is a non-point-source emission, but has substantial pollution potential due to
its content of fertilizers, leading to eutrophication of natural water bodies, and to its content
of herbicides and pesticides. Agricultural runoff is causing enormous problems worldwide,
with more than 70% of rivers and 50% of lakes in the United States being affected. Some types
of agricultural wastewaters can be effectively treated using anaerobic digestion, a topic that is
discussed in several chapters in the present volume: microbiology and biochemistry of
anaerobic treatment; anaerobic bioreactors/digesters, design and development; and by-
products of anaerobic treatmentsdmethane and manure.
Industrial wastewater effluents have a significant potential for pollution of local water
resources with important consequent impacts on human health and ecosystems, problems
that have been encountered in actual fact over the past century with the direct discharge of
enormous amounts of wastes into lakes, rivers, and oceans. In addition, many industrial
processes in current use have very high water demands. Coupled with increasing water
scarcity in many parts of the world, this means that effective wastewater treatment will
become imperative to return useable water to local environments for reuse.
Some treatment technologies have already been brought into practice and are currently
deployed in at least some developed countries, but their widespread adoption by developing
countries remains to be implemented. With increased industrialization of developing coun-
tries, effective treatment of industrial wastes will become a challenge of ever greater
importance in the future. In addition, the efficiency of many present treatment technologies,

xxi
xxii Preface

both in terms of energy usage and in terms of treatment efficacy, has room for significant
improvement. This, together with the large scope of treatment required, presents ample
challenges for dedicated research and development efforts, as detailed by a number of au-
thors in this volume.
Unlike municipal and agricultural wastewaters, which are fairly uniform in composition
throughout the world, industrial wastewaters are highly variable in both the quantity of
polluting materials present and their composition. Each type of industry produces a waste
stream with a distinct chemical composition that is a direct reflection of the particular
chemical/biochemical processes involved. Hence, treatment processes need to be specifically
tailored for each type of wastewater that is to be discharged into the environment. This
volume contains chapters specifically discussing treatment of low-strength and high-strength
wastewaters. As well, a large number of examples of treatment processes for many important
industrial sectors are presented, including effluents from the food and beverage industries,
the textile industry, aquaculture, the pharmaceutical and personal care products industries,
the petroleum industry, the pulp and paper industry, mining, and the electronic and elec-
trochemical industries. Additional chapters discuss some specific treatment aspects relevant
to industrial wastewaters, including dechlorination processes, treatment of recalcitrant
wastes, and removal of toxic components of wastewaters.
Finally, two chapters discuss topics of general interest in wastewater treatment, the
advantages and disadvantages of anaerobic treatments versus aerobic treatments and the
application of molecular biological tools to monitor process efficiency. Taken together, we
believe this volume presents an authoritative and comprehensive review of selected topics in
wastewater treatment that should be of use to practitioners, researchers, and teachers and
students.
We would like to acknowledge the reviewers for their valuable comments to improve the
final quality of the chapters included in this volume. In addition, we would also like to thank
Dr. Kostas Marinakis, Book Acquisition Editor; Ms. Anneka Hess; and the entire production
team at Elsevier for their help and support in bringing out this volume. Without their
commitment, efficiency, and dedicated work, this volume could not have ever been
accomplished.
Editors
Duu-Jong Lee
Patrick C. Hallenbeck
Hao Huu Ngo
Veeriah Jegatheesan
Ashok Pandey
 1
Aerobic Treatment of Effluents
From Textile Industry

P. Mullai1, *, M.K. Yogeswari2, S. Vishali3,

M.M. Tejas Namboodiri4, B.D. Gebrewold5, E.R. Rene5,
K. Pakshirajan4
1
ANNAMALAI UNIVERS ITY, TAMIL NADU, INDIA; 2 VIVEKANANDHA COLLEGE OF ARTS AND
SCIENCES FOR W OMEN, TAMIL NADU, INDIA; 3 SRM UNIVERSITY, TAMIL NADU, INDIA;
4
INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI, GUWAHATI, ASSAM, INDIA; 5 U N E S CO - I H E
INSTITUTE FOR WATER EDUCATION, DELFT, THE NETHERL ANDS

1.1 Introduction
Rapid industrialization of the developing world has contributed to unsustainable
pollution levels [1,2]. In the past few decades, an increase in the demand for textile
products has led to a steep rise in water pollution [3,4]. Textile effluent is tagged as the
most polluting as it consumes a large quantity of water and chemicals for the processing
of fabrics throughout the world and in turn these industries generate pollution by dis-
carding the wastes [5e7]. Similarly, increasing financial constraints pave the way for
discharge of untreated effluents into the environment [8]. Globally, 280,000 tons of dye is
discharged into textile industry wastewater every year [9,10].
Although the use of textile dyes is important, it causes serious environmental prob-
lems. Textile wastewater contains a mixture of inorganic and organic compounds, which
are complex in nature [11]. According to a recent report from China, each year about
70 billion tons of wastewater from the textile and dyeing industry is generated and needs
adequate treatment before ultimate discharge into the environment. Surprisingly, about
10e15% of the dyes used in the dyeing process do not fix with the textile fibers and,
therefore, they are carried by the wastewater in their original forms and concentrations
[12]. The major pollutants present in textile wastewater are recalcitrant organics, residues
of reactive dyes, aerosols, leveling agents, acids, alkalis, amines, heavy metals, chlor-
ophenol, chlorine, halogen carriers, formaldehyde, biocides, and softeners [13e16].
Table 1.1 presents the major pollutants and chemical types present in textile wastewater
and their main processes of origin. Accordingly, the various unit processes, such as sizing,
desizing, bleaching, mercerizing, dyeing, and printing, generate high levels of biochemical
*
Corresponding Author.

Current Developments in Biotechnology and Bioengineering: Biological Treatment of Industrial Effluents

http://dx.doi.org/10.1016/B978-0-444-63665-2.00001-1 3
Copyright © 2017 Elsevier B.V. All rights reserved.
4 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

Table 1.1 Major Pollutants Present in Textile Wastewater

Pollutant Major Chemical Types Main Processes of Origin
Organic load Starches, enzymes, fats, grease, waxes, surfactants, acetic acid Desizing, bleaching, dyeing
Color Dyes, scoured wool impurities Dyeing
Nutrients Ammonium salts, buffers, sequestrants Dyeing
pH and salt NaOH, mineral/organic acids, NaCl, silicate, sulfate, carbonate Scouring, desizing, bleaching,
effects mercerizing, dyeing
Sulfur Sulfate, sulfide, hydrosulfide salts, sulfuric acid Dyeing
Toxicants Heavy metals, reducing agents, oxidizing agents, biocides, Desizing, bleaching, dyeing, finishing
quaternary ammonium salts
Refractory Surfactant dyes, resins, chlorinated organic compounds, carrier Bleaching, desizing, dyeing, finishing
organics organic solvents

Adapted from H. Patel, R.T. Vashi, Characterization and Treatment of Textile Wastewater, Butterworth Heinemann, Elsevier,
USA, 2015 [18].

oxygen demand (BOD), chemical oxygen demand (COD), suspended solids, dissolved
solids, alkalinity, pH, and strong odorous conditions in the wastewater stream [17].
The low biodegradability of textile wastewater is generally attributable to the exis-
tence of recalcitrant organics including dyes, sizing agents, and dyeing aids. These
pollutants, directly or indirectly, are known to cause several chronic diseases to living
beings. Also, their dark color disrupts the penetration of sunlight, resulting in the pre-
vention of photosynthesis of the ecosystem, leading to eutrophication of water bodies
[19]. In addition, odorous compounds such as hydrogen sulfide may be produced under
anaerobic conditions. The biological activity of the receiving water body will be affected
by the presence of dissolved sulfide species. Again, in the textile industry, volatile aro-
matic hydrocarbons are also emitted owing to the use of fossil fuels and other industrial
chemicals [20]. Many dyes and their breakdown products have evidenced harmful effects
on humans and in mammalian assays. In addition, they can be mutagenic or carcino-
genic in nature and they tend to bioaccumulate in the food chain [21e24]. The release of
textile wastewater into the environment not only affects the water resources, but also
alters the soil productivity, marine life, and ecosystem.
The textile industries are constantly identifying, tracking, and addressing their
pollution-related vulnerabilities to satisfy the increasing demands from regulatory
boards and policy makers [25]. Globally about 7  105 tons of dye is produced per year.
Of this, 10e15% ends up in the effluent during the dyeing process as mentioned earlier
[26,27]. Dyes like triphenyl methane and azo dyes are extensively used in textile in-
dustries for dyeing of nylon, wool, silk, and cotton [28]. The dyes employed in textile
industries are composed mainly of aromatic organic compounds [29]. The aromatic
benzene structures are usually complex in nature and highly resistant to light, biological
activity, ozone, and other environmental conditions. Because of these limitations, the
application of conventional wastewater treatment processes still remains a major chal-
lenge for the textile industry.
 Chapter 1  Aerobic Treatment of Effluents From Textile Industry 5

1.2 Dye Composition and Classification

Dye is a mixture of two main components, namely, a chromophore and an auxochrome.
The colored compound that contains a certain unsaturated group is called the chro-
mophore. The usual chromophores seen in ancient dyes are nitro, azo, keto, thioketo,
ortho-, and para-quinoid ring chromophores. A compound with a chromophore is
described as a chromogen. The basic meaning of the word auxochrome is color
enhancer. When a chromogen contains groups other than those mentioned earlier, it is
called an auxochrome. The auxochrome may be either acidic or basic, like eOH or
eNH2; however, other auxochromes include eSO3H, eNR2, and eNHR. In general, dyes
can be classified into natural dyes and synthetic dyes based on their origin (Fig. 1.1). The
use of natural dyes was practiced during the bronze age. Natural dyes are a group of dyes
that are extracted from biological material like plant and animal residues. Even though
they are extracted from biological material, such dyes are considered mordant dyes
because they need addition of one or more of copper, aluminum, chromium, iron, and
other metallic salts. Such mordant components are required for ensuring the fastness of
the dyes when exposed to light and during the washing process. On the other hand,
synthetic dyes were commercialized only in the middle 1860s. Synthetic dye production
rapidly replaced the use of conventional natural dyes as they impart a variety of colors to
fabrics [30]. Based on the chemical composition and mode of application in the dyeing
process, synthetic dyes can be classified as follows.

1.2.1 Basic Dyes

These are also called cationic dyes and usually they are water soluble. Basic dyes along
with a mordant are primarily employed to dye acrylic fibers like polyesters, and nylon,
among others. These basic dyes are not appropriate for other fibers than acrylic. Hence,
they are normally used for subsequent treatment of fabric that was previously dyed with
acid dyes.

1.2.2 Direct Dyes

Direct dyes or substantive dyes are used to color cellulose-based fibers without the help
of a mordant. These dyes are used to color wool, silk, and rayon, among others. These
dyes are dull and have meager fastness to washing.

1.2.3 Mordant Dyes

Mordant dyes are acidic in nature. They are also called as chrome dyes. Sodium or
potassium salts are used to treat fabrics during or after the completion of the process of
dyeing for strong binding of the chrome. They are mainly used for wool. They are less
effective for dyeing cotton, linen, silk, rayon, and nylon.
6 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

FIGURE 1.1 Representative chemical structures of various dyes used in the textile industry. (A) Natural, (B) basic,
(C) direct, (D) mordant, (E) vat, (F) reactive, (G) disperse, and (H) sulfur.

1.2.4 Vat Dyes

Vat dyes are naturally water insoluble. To make them soluble and to fix with fabrics, they
are reduced with alkali salts. Further oxidation tends to restore their insoluble nature.
Without the addition of mordant, these dyes are used to color cotton, linen, and rayon,
whereas with the addition of mordant, they are used to color wool, nylon, polyesters, and
acrylics.
 Chapter 1  Aerobic Treatment of Effluents From Textile Industry 7

1.2.5 Reactive Dyes

Reactive dyes were initially used for cellulose-based fibers. Occasionally, heat treatment
is required for these reactive dyes for generating various shades. Once the dyeing process
is completed, the fabrics are washed using soap to take away every unfixed dye.
Applications include dyeing wool materials, silk fibers, and nylon blends.

1.2.6 Disperse Dyes

Disperse dyes are insoluble in water and marketed in the form of powder or paste.
Traditionally, disperse dyes were prepared to dye cellulose materials. These days, they
are employed to dye acrylic and nylon fibers.

1.2.7 Sulfur Dyes

The insoluble nature of sulfur dyes is made soluble by treating them with soda and
sodium salts. Usually, treatment at high temperature and the addition of salts are
required to impart color to the fabrics. Once the dyeing process is completed, the
unfixed salts and dyes are removed. These dyes are mainly employed to impart color to
cotton and linen materials.

1.3 Main Processes Involved in the Textile Industry

The four main stages of textile processing involve yarn production, fabric production,
wet processing, and garment manufacturing [31]. Yarn fabrication is the process in
which the conversion of raw fiber into yarn or thread occurs. The second step, fabric
production, involves weaving, knitting, and tufting. The next step is wet processing,
which includes pretreatment, dyeing, printing, and finishing. In this process, the sizing
elements are detached from the gray fabric by treating them with acids or enzymes. The
scouring process is done to remove impurities such as oils, waxy materials, and fatty
acids, among others, seen in the fabric, under alkaline conditions, high pressure, and
high temperature. Bleaching is performed to whiten the fabrics and yarns using
bleaching agents like hypochlorite and hydrogen peroxide. During the mercerizing
process, the fabrics or yarns are treated with a sodium hydroxide (cold) solution, which
increases the tensile potency, gleam, and dye uptake by making the fabric swell. The
dyeing process is carried out to give a finished texture to the fabric by diffusion.

1.4 Aerobic Processes to Treat Textile Industry Effluents

Aerobic treatment systems are high-rate oxidizers of soluble organic and nitrogenous
compounds. Commercially available aerobic treatment reactors promote the removal of
color, suspended solids, and pathogens and the reduction of COD, BOD, and other
8 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING

toxins from textile wastewater. According to Khelifi et al. [32], aerobic processes for the
treatment of textile wastewater are efficient and cost-effective. Most of the aerobic re-
actors operate as constant volume reactors with complete mixing and intermittent flow,
as the influent is not continuous in small-scale textile industries. Complete mixing of
contents is ensured in the aeration chamber to promote and maximize the contact
between dissolved oxygen, microbes, and wastewater. The effluent from the aeration
chamber moves into a clarifier. The rate of discharge of the effluent is a direct response
to the influent flow rate. Typical bioprocesses that will be discussed in this chapter for
the treatment of wastewater from textile industries include the extended aeration pro-
cess and suspended and attached growth processes.

1.4.1 Extended Aeration

In practice, most aerobic reactors operate as extended aeration units. The term extended
is associated with the duration of aeration. It is characterized by long-term aeration, long
retention times, low food-to-microorganism (F/M) ratio, and low biomass accumulation.
To ensure the endogenous growth phase of microorganisms, excess dissolved oxygen
and minimal soluble organic matter are provided, and microbes will readily deplete the
bioavailable organic carbon, including biomass. The focus is to establish the right bal-
ance between the amount of new cells produced and the amount of cells endogenously
degraded per day [33].

1.4.2 Suspended-Growth Bioreactors

The suspended-growth aerobic units are termed as scaled-down activated sludge plants
(Fig. 1.2). The nature of activated sludge is a heterogeneous microbial culture comprising
bacteria, protozoa, rotifers, and fungi. Assimilation of organic matter is achieved by
the presence of the bacteria [34]. The segregation of dispersed bacteria from the bio-
aerated unit treated effluent is gained by the existence of protozoa and rotifers, which
serve as the predators. The biomass is thoroughly blended with the biodegradable
organic fraction and individual organisms agglomerate together (flocculate) to form a

FIGURE 1.2 Schematic of an aerobic suspended-growth process.

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"Indeed, I shall be happy to do anything I can for him," said that
spinster with indubitable sincerity. "I have had to do with sick people
before now."

Having thus easily rid herself of responsibility, Paulina was soon off
on her bicycle for Chelmsford. She found time to call at the doctor's,
for he arrived at "Gay Bowers" a little later. He did not think seriously
of his patient, but said he needed care. Aunt and Miss Cottrell were
busy for some time carrying out the doctor's instructions. Aunt Patty
told me afterwards that Miss Cottrell was most useful in a sickroom.
All her little vanities and affectations vanished in the presence of a
need which she could relieve, and she showed herself a sensible,
capable, helpful woman.

When Paulina got back in the evening she found her father no longer
in pain, and sound asleep.

"Say, didn't I tell you he would soon be better? He always thinks he

is going to die when he gets these attacks."

"I must say that when I saw him this morning, I felt very uneasy,"
replied my aunt.

"Ah, you do not know him as well as I do," was her rejoinder. "I never
let these attacks alarm me. See now, I called at the post-office, and
found this letter for you."

The letter proved to be from my Aunt Clara, and interested me

considerably. She wrote to ask if aunt could find room in her house
for my cousin Agneta. Manchester did not suit her. She was out of
health, suffering from general depression, and needed a thorough
change. "I thought it would be nice for her to stay in your house while
Annie is there," she wrote; "they are about the same age, and will
enjoy being together."

I received this proposal with mingled emotions. I hardly knew my

cousin, and was by no means sure that I should enjoy having her at
"Gay Bowers." Her upbringing had been so different from mine, that I
fancied we should have little in common. Aunt Clara had never
before shown any desire that her children should become
acquainted with her sister's family. I wondered that she should now
deem it "nice" that I and Agneta should meet.

"There is one thing to be said about it, Nan," remarked my aunt. "I
have no room to give her; if she comes she must share yours."

As soon as I heard that, I was certain that I did not wish Agneta to
come. I hated the idea of having to share my pleasant room with
another girl, and the fact that the girl in question was my cousin did
not reconcile me to it. It seemed essential to my happiness that I
should have some place, however small, for my very own, to which I
could retreat when I wanted to possess my soul in peace.

"Oh, auntie," I said, "could you not tell Miss Cottrell that you will not
longer have room for her?"

"Impossible, Nan; I could not treat her so unhandsomely, especially

since she has been so kind and helpful with poor Mr. Dicks. Never
mind, dear; you shall not share your room with your cousin if you
would rather not."

"Oh, I do not mind if there is no other way," I felt constrained to say;

but I did mind very much, and when Aunt Patty said that she would
write to Mrs. Redmayne, and explain that this was the only
arrangement she could make, I devoutly hoped that Aunt Clara
would object to Agneta's sharing a room with me.

CHAPTER IX
 MISS COTTRELL'S ALIAS

MY hopes were doomed to disappointment. Aunt Clara wrote that

Agneta would be only too pleased to share my room, as she had a
nervous dread of sleeping alone in a strange place. So I had to
resign myself to the inevitable, and I tried to do it with as good a
grace as possible. Aunt Clara said that she would like Agneta to join
us in the following week; thus my room had soon to be prepared for
another occupant. There was ample space in it for two bedsteads,
and aunt had everything arranged very comfortably, but for me, its
charm had gone when it was no longer my own sanctum.

"It may not be for long, Nan," my aunt said, reading my thoughts, as
together we inspected the new arrangements. "I cannot tell how long
these guests will remain with me. I naturally hope they will stay all
the summer, but I shall be exceptionally fortunate if they do. When
there is another room vacant Agneta shall have it."

"You forget that she does not like to sleep alone," I said. "Oh, it will
be all right, auntie. I dare say we shall get on nicely together, and it
will be better for you, for then you can let the vacant room to some
one else."

"If any one else wants it," said Aunt Patty smiling. "I am really very
thankful to have all my rooms occupied. It makes things much easier,
and it might have been so different."

That day I received a box from home. It struck me as a curious

coincidence, when I perceived that it contained the evening gown
that had belonged to my cousin, but was now to be worn by me. I
had asked Olive to send it as soon as possible, for I felt altogether
too dowdy of an evening in contrast with Paulina's splendour, to say
nothing of Miss Cottrell's tasteless efforts at display. Veiled with black
grenadine, and finished with dainty frills and furbelows by Olive's
clever fingers, the pink gown was so transformed, that I doubted if
even its former wearer would recognise it. As I examined it, I
remembered the spots we had seen on the bodice, which Olive had
declared to be tears. Surely she was mistaken!

Then I saw that Olive had pinned a little note to the sleeve.

"Dearest Nan," she wrote, "I do hope you will like this
frock. I really feel that I have succeeded beyond my
hopes. Last night I tried it on, and even mother said it
was very pretty, while Peggy grew green with envy,
and declares she shall ask Aunt Patty to have her
another year. Is it not funny to think that you will wear it
before the eyes of its former owner? I hardly think,
though, that she will know it again. We were so
surprised to hear of Aunt Clara's plan, and only think,
Agneta is to break her journey in London, and will stay
a night here, so we shall all see her! Aunt Clara gives a
poor account of her though, says she is nervous,
depressed, excitable, and difficult to manage at home,
and hints that it is all owing to a 'foolish fancy' for a
man who is a 'sad detrimental.' Poor little cousin to
have lost her heart so soon, and to one of whom her
parents disapprove! Peggy says that it makes her as
interesting as the heroine of a penny novelette. We all
look forward to seeing her, and mother is going to write
and ask her to stay a few days with us. Do write soon
and say how you like the frock and tell us the latest
about Pollie Dicks."

"Your loving sister,"

"OLIVE."

"So those were tears!" I said to myself, as I folded up Olive's letter.

"Poor Agneta! I wonder what the 'sad detrimental' is like. She is to be
sent down here to be well out of his way. Probably it is not so much
that Manchester does not suit her, as that it does not suit Aunt Clara
to keep her in Manchester just now. I wonder how she likes being
banished to this rural solitude." And I, as well as my sisters, began to
look forward with some curiosity to making our cousin's
acquaintance.

We expected Agneta about the middle of the week, but mother

succeeded in keeping her for a day or two, and sent us word that
she would come on Saturday. An early train was named, and Aunt
Patty asked me to drive into Chelmsford in the "sociable" and meet
my cousin at the station. It was a lovely day, and I ran cheerfully to
get ready. I was so glad that Agneta should arrive on a day when
"Gay Bowers" was looking its best. Already there were roses
opening their pink petals against the wall of the house, and the
flower-beds were bright with scarlet geraniums and verbenas.
Through the staircase window I caught sight of Miss Cottrell's garden
hat away in the corner to which she devoted her energies, and was
glad to think that she was engaged in gardening. Paulina, I knew,
was busy with letters, which she meant to "mail" later, so I hoped to
get off by myself on this occasion.

But I congratulated myself too soon. When I came downstairs the

carriage was not yet at the door, but Miss Cottrell came hurrying in
from the garden.

"Are you going to Chelmsford to meet your cousin?" she asked.

"Yes," I answered, putting on my gloves with an air of haste.

"Can I go with you?" she asked. "I want to change my book at the
railway stall."

"You had better let me do that for you," I said. "I must be off in three
minutes."

"Oh, but I can be ready in that time," she said, "and there are other
things I want to do in town."

"Very well, if you will not make me late for the train," I said coldly.
I felt sure that nothing short of telling her I did not want her would
deliver me from her company.

She was back again almost as soon as the conveyance drove up.
She had changed her hat and added a gauzy ruffle, which was
rather incongruous, to the morning blouse which so ill-became her
heavy thick-set figure. She looked an odd individual, and I could not
help wondering what Agneta would think of her.

The fresh air and the sweet scent of the hedges, on which the may
blossom still lingered, soon soothed my ruffled feelings, and I tried to
respond amiably to Miss Cottrell's remarks.

"Now where do you wish to be put down?" I asked as we drove into

the town. "If you are coming back with us, you must not be very long,
or we shall all be late for luncheon."

"Oh, I will go straight to the station," she replied. "I like to be there
when the train comes in. One sees life at a railway station, as dear
Lady Mowbray used to say."

So it seemed that her business was a mere pretext for assisting at

the meeting between me and the cousin of whom, as she had
already discovered, I knew so little! I felt both cross and
contemptuous; but my vexation vanished when the train came in, for
a delightful surprise awaited me. One of the first persons to step on
to the platform was my father!

"Oh, father, it is never you!" I cried, feeling ready to hug him. "How
nice of you to come!"

"Nice for myself you mean," he said. "I felt it high time I came and
saw how my Nan was getting on—to say nothing of your Aunt Patty
and her 'paying guests'—so I thought I would bring your cousin down
and have a peep at you all."

So saying he turned and gave his hand to a fair, slender girl, who
stepped lightly from the carriage. I saw at a glance that she was very
pretty, but her face was colourless, and, though she shook hands
with me pleasantly enough, her manner showed a strange lack of
animation. She wore a grey travelling gown, and a red hat which
made me think of Olive's riddle.

"I am so glad you have such a nice day," I said. "It is really warm at
last. I hope you like the country."

"Oh, yes, I like it well enough," she said indifferently. "It seems rather
pretty about here."

"How well you look, Nan!" said my father. "You are not like the same
girl whom I saw off from Liverpool Street four months ago. I hope
'Gay Bowers' will do as much for your cousin; she needs some roses
badly. Why, Miss Smith, who would have thought of seeing you
here? How are you?"

To my amazement I saw that it was Miss Cottrell whom he thus

addressed. She shrank back, her face crimson.

"You are mistaken," she stammered, "my name is not Smith."

"Then you have changed it since I last saw you; you are married, I
suppose," he said pleasantly; "for we certainly called you Miss Smith
at the 'Havelock Arms.'"

"The ''Avelock Arms!'" she stammered. Her h's always dropped when
she was agitated.

"Why, father, this is Miss Cottrell," I said, pitying her embarrassment

as she grew redder and redder.

"Then she has changed her name," said my father, looking at her in
astonishment, "for it was as Miss Smith I knew her in Devonshire. I
used to stay sometimes at her uncle's inn, a very pleasant place of
sojourn on the border of Dartmoor, where I went for the sake of
fishing. But it must be nearly twenty years since I was last there. I
heard only the other day that John Smith and his wife were both
dead and the inn had changed hands. That is true, I suppose?"

He looked keenly at her as he spoke, and her eyes fell beneath his
gaze. She was crimson. Her face was the picture of misery and
shame. But it was clear that my father had not the least doubt of her
identity with Miss Smith, and she dared not deny it.

"Yes, my uncle and aunt have passed away," she said awkwardly. "I
do not remember that I ever saw you at their house. There are so
many Smiths in the world that I thought I should like another name
and took that of Cottrell, which was my mother's. I hope there is no
harm in that?"

"Not at all; this is a free country, and it is not an unheard-of thing for
people to change their names," said my father, anxious now to
relieve the embarrassment which he had innocently caused. "I must
see about your luggage, Agneta. Is there a conveyance outside,
Nan?"

"John and the wagonette are there," I said. "Come, Agneta, we may
as well take our seats."

Father was about to shake hands with Miss Cottrell when I said
hurriedly:

"Miss Cottrell is coming with us, father; she is staying at 'Gay

Bowers.'"

"Oh, that is right," he said quickly, but I saw a gleam of amusement

leap into his eyes, and the corners of his mouth twitched.

Poor Miss Cottrell looked utterly bewildered and crestfallen as she

followed us to the wagonette. She hardly said a word as we drove
homewards. Father and I had a great deal to say to one another. I
wanted to hear all the home news, but I tried to draw Agneta into the
talk. As I observed her, it struck me that she was more like my
mother than her own. I could trace no resemblance in her features to
Aunt Clara, but something in her face reminded me of mother.

For some time Miss Cottrell's tongue was absolutely still, a thing I
could hardly have believed possible, until father said:

"By the way, Miss Smith—Cottrell, I mean—I remember that it was

only in the summer that you were at the 'Havelock Arms.' You lived
with a lady—all, I have forgotten her name—who had an afflicted
daughter whose nurse you were."

"Excuse me, sir," said Miss Cottrell angrily, "I was her companion."

"That would be the same thing under the circumstances, would it

not?" he asked gently.

Miss Cottrell vouchsafed no reply, but her eyes flashed fire. I pitied
the uncomfortable position into which false pride had led her, and
hastily drew father's attention to the beauty of the common across
which we were driving.

"So you have Professor Faulkner at 'Gay Bowers,'" father said

presently. "I am looking forward to making his acquaintance."

I started and felt my colour rise.

"Why, what do you know of him?" I asked eagerly.

"No more than all the world may know," he said. "That he is a very
brilliant young scholar and has written a scientific criticism of
Shakespeare's plays which promises to become a standard work."

"Oh, father, you fairly frighten me!" I said; yet somehow I was very
glad. "I know he writes and studies a great deal. He spends all the
mornings in his room at work, yet he is so simple and human in his
ways that auntie and I had almost forgotten that he is a learned
professor."
"Don't you know yet, Nan, that greatness and simplicity are generally
combined?" my father asked, with a smile. "It is your shallow-pated
man who gives himself airs."

Aunt Patty was delighted to welcome father, for she had no more
expected to see him than I had. We seemed a large party at
luncheon, and there was plenty of talk, although Miss Cottrell was
unusually silent. I was terribly afraid that father would call her "Miss
Smith," but happily, he never addressed a remark to her, being much
absorbed in talk with Colonel Hyde and Professor Faulkner. He
seemed to get on exceedingly well with the latter, and I longed to
hear what they were saying, but with Agneta beside me demanding
my attention and Paulina chattering to me across the table, I could
never catch more than a word or two. Paulina made various attempts
to draw out Agneta, but with only partial success.

"Say," she said to me aside, after luncheon, "what is the matter with
that cousin of yours? Is she shy, or sick, or what?"

"I don't think she is shy," I said, "but she has not been well lately and
is rather depressed."

"I believe you," said Paulina; "but, do you know, it strikes me that she
is not so meek as she looks and has a will of her own."

"Very likely," I said, reflecting that the curves of Agneta's mouth and
chin were similar to mother's, and mother had never shown any lack
of spirit and determination.

I was rather sorry to see Mr. Faulkner go off on his bicycle soon after
luncheon, for I wanted father to know more of him. The rest of us
spent the afternoon in the garden. Paulina and her father, Colonel
Hyde and Agneta had a game of croquet, while Aunt Patty, father
and I sat and chatted in the summer-house at the end of the lawn.

"Oh, father, do tell auntie about Miss Cottrell!" I said, after first
looking cautiously round to be sure that the spinster was not within
earshot, but for once she had taken herself out of the way.
"Alias Miss Smith," he said.

"Was it really an inn in which you used to see her?" I asked.

"Yes, a good old country inn, much frequented by fishermen in the

season. I went there several summers in succession, till the cares of
a family shackled my movements. John Smith and his wife were
homely, honest folk who made us very comfortable in rustic fashion.
They did not call the 'Havelock Arms' an hotel, nor speak of their
boarders as 'paying guests,'" said my father with a mischievous
glance at Aunt Patty.

"Their niece would have done so, if she had ever alluded to the
business," I remarked. "I imagine John Smith was the uncle whose
money she inherited."

"She told me he was in the tea trade," said my aunt.

Father laughed.

"Why, so he was," he said. "A good many tourists and picnic-parties

used to come to the inn for tea. I believe he sold as much tea as
beer."

"And was she really a nurse?" I asked.

"Well, yes, in a way, but not like a modern trained nurse," he replied.
"Lady—let me see—"

"Mowbray," I suggested.

"Mowbray! that's the name," he said. "Well, Lady Mowbray had a

daughter who was sadly afflicted—I believe she was almost an idiot
—and Miss Smith used to take care of her—was her 'companion' as
you heard her say. I suppose she thinks that word is more genteel
than nurse. Lady Mowbray lived somewhere near Bath."

"And had also a house in Bryanston Street," I said.

"Ah, I see you know all about it," said my father.

"With a difference," I rejoined. "Lady Mowbray was Miss Cottrell's

dearest friend and could not bear to be separated from her."

"Really! Well, I believe she was very grateful for Miss Smith's
devotion to her child. Miss Smith was generally with them except that
she came to the 'Havelock Arms' for a month or so in the summer,
and then used to help her aunt look after her customers. So she has
been posing here as a fine lady! How droll!"

And father quietly laughed with an air of the utmost amusement.

"She has tried to do so," said my aunt dryly.

"I am afraid she is sorely mortified to think that you have revealed so
much to us," I said.

"We will not talk of it," said my aunt quickly. "Her vanity is foolish and
paltry, but we will spare her feelings. I must ask Agneta not to
mention it. Oh, dear, how white that girl looks!"

So my cousin became the topic of conversation, and father told Aunt

Patty that Mrs. Redmayne begged that she would not allow Agneta
to go up to London, on any pretext whatever, unless she or I could
accompany her. I could see that aunt did not like the injunction.

"My guests are free to do as they like," she said. "This is not a
boarding school."

CHAPTER X
 COUSIN AGNETA'S LOVE STORY

WE sat down to dinner rather earlier than usual that evening

because father had to catch a train which left Chelmsford a little
before nine. Mr. Faulkner's place at the table was vacant. I kept
expecting that he would drop in, but he did not appear. It vexed me
that father should go away without having another word with him, for
although we were comparatively near London, I knew it might be a
very long time ere father came again. He was a busy man and rarely
gave himself a holiday.

I got ready to drive with father to the station, and no one offered to
accompany us. Miss Cottrell was not visible when he took his
departure. It struck me that she must have slipped away to avoid
saying good-bye to him, fearing that he might address her as Miss
Smith in the hearing of the others.

"It's a pretty place," said father, looking back at "Gay Bowers," as we

drove away in the fair, sweet dusk of the evening, "and I am glad that
your aunt can stay there, if she is happy at least. How does it
answer, Nan? Do the 'paying guests' bother her much?"

"I think not, father," I answered. "Miss Cottrell was rather a worry; but
we are beginning not to mind her peculiarities."

Father laughed.

"Poor thing!" he said. "What a pity she should try to pass herself off
as other than she is! It is an attempt fore-doomed to failure. Do you
know Emerson's words? 'Don't say things. What you are stands over
you the while, and thunders so that I cannot hear what you say to the
contrary.' She does herself an injury, for she is really an excellent
woman in many respects."

"Aunt says she is a capital nurse," I replied. "She was very helpful
when Mr. Dicks was ill."
"What a man he is!" said father; "but a genuine one, I think. I wish I
could have had a longer talk with Professor Faulkner. He is a fine
man. Do you read much now, Nan?"

"You forget that books are forbidden me," I said, "though I must
confess I do not pay much heed to Dr. Algar's prohibition when
something good comes in my way. The difficulty is to get time for
reading."

"If Dr. Algar could see you, I don't think he would be afraid of your
reading," said my father. "Do you get any headaches now?"

"I have almost forgotten what a headache is like," I replied joyfully.

"Even after I had worked out a mathematical problem which Jack
Upsher could not master, my head did not ache."

"What, you presumed to beat Jack?" said father, smiling. "How did
he stand that assumption of feminine superiority?"

"He was very grateful to me for helping him," I said. "Jack is not in
the least ashamed of his feeble scholarship."

"Do you think he will get through his exam?" he asked.

"I hope so," I said. "He is working harder than he has ever worked
before."

"I wish he might," said father. "I should like to see him in the Artillery,
for I believe there is in him the making of a good soldier."

"He is tempted to spend too much of his time at 'Gay Bowers,'" I said
laughingly; "but aunt is very severe with him. He flirts with Paulina, or
rather, I believe, it would be more correct to say that she flirts with
him."

Father laughed.

"That is harmless enough," he said, "since she must be several

years older than he. Well, Nan, I am glad the experiment has
answered so well as far as you are concerned. Your mother will be
delighted to hear how much better you are. You must run up to town
and see us all one of these days. You deserve a little change, for
your aunt says you are the greatest comfort to her, and she does not
know what she would do without you."

"Oh, father!" I exclaimed. "Why, I do nothing!"

Yet I knew that the nothings I did—cycling into Chelmsford to give

orders, answering letters, seeing to the entertainment of the guests,
and the like—filled up my days and were not exactly what I should
have chosen to do had I been free to choose.

I saw father off in the train and started homeward, sitting alone in the
wagonette. We were getting clear of the houses when John suddenly
pulled up, and I saw Mr. Faulkner standing on the path.

"Will you take me home with you, please?" he asked smilingly.

"Why, of course," I said. "But what have you done with your bicycle?"

"It has come to grief," he said. "I had a spill—oh, don't be alarmed, it
was nothing serious! I was coming down a hill near Maldon; there
was a sharp bend, and rounding it incautiously, I came into collision
with a wagon which was right across the road."

"Oh, how dangerous!" I exclaimed. "Are you sure you were not
hurt?"

"Oh, I wrenched my shoulder a bit and got a few bruises," he said;

"but I jumped off, you see, and the machine got the brunt of it. Of
course, I could not ride it afterwards, so I had to get back to
Chelmsford in a roundabout way by rail, and I have now left my
machine to be repaired."

"How fortunate that I can give you a lift!" I said. "I have just been
seeing father off."
"I am sorry Mr. Darracott has gone," he said. "I like your father, Miss
Nan."

"And he likes you," was on the tip of my tongue, but I did not say it.
His remark, however, so set me at ease that I began to talk to him
about my home and my people as I had never done before.

"Oh, you can't think," I said with a sudden burst of confidence, "how I
long to see them all again. Father says I must go up one day soon."

"I wonder you have not been before," said Alan Faulkner; "it is so
easy to run up to town from here. And your sisters—why do they not
come and spend the day sometimes?"

"OH, I WRENCHED MY SHOULDER A BIT," HE SAID.

"It is because we are all so busy," was my reply, "and moreover have
little superfluous cash. We can afford neither the time nor the money
for such pleasant little trips."

Then I felt the blood mount to my forehead, and was thankful that in
the twilight, he could not see how I blushed for my outspokenness.
Why do people find it harder to avow poverty than to confess to
grave faults? Few, except those who are really comfortably off, can
talk with ease of being poor. I was not to blame, nor were they, that
my parents' income was so limited, yet I felt ashamed of the fact that
the small sum required for the railway fare to and from London was
of importance to us.

"I understand," said Alan Faulkner quietly. "Indeed, I have had to

practise that kind of self-denial a good deal myself, and know well
how irksome is the effort to keep one's expenditure within narrow
limits, yet it is good for one to learn how easily one may do without
many of the things that seem desirable."

With that he began to tell me about his early life. His father had died
when he was a little boy. When he was twelve years old, his mother
married again. Up to that time they had been everything to each
other, and he could by no means welcome this change in their life.
But his stepfather was good to him, and he became very fond of the
little sisters who were born later. Before they were grown-up, their
father died, and Alan found himself the sole guardian of his mother
and his sisters. Very simply, he told the story, saying little of the part
he had played. Not till long afterwards did I know that the self-denial
of which he had spoken had been voluntarily practised in order that
he might secure for his sisters a first-class education.

"So," I said, "the sister who has lately left school and gone to Paris to
perfect her French is your half-sister merely."

"But a very real and dear sister all the same," he said.
"And your other sister, where is she?"

"She is a governess in a school in Yorkshire," he replied. "I hope that

you and my sisters may know each other some day, Miss Nan."

"Oh, I should like so much to know them!" I said earnestly, while I

wondered how it could come about. There was another thing I
wanted to know, but I did not like to question him. Perhaps he
divined my thoughts, for after a few moments' silence he said in a
low tone:

"There are only the three of us now. The saddest thing about my
return to England was that there was no mother to welcome me."

"Oh, I am very sorry!" was all I could find to say.

The words came from my very heart, for I did not need to be told all
that this meant for him.

Hardly another word passed between us then, for we had reached

the gate of "Gay Bowers." I ran into the house, feeling that the past
day had been a golden one for me. Each hour had been full of quiet
pleasure, and not least should I prize the memory of the confidential
talk with Alan Faulkner, which seemed to have made us true friends.

Aunt told me that Agneta had complained of being tired, and had
gone to bed. I soon followed her example, though I was far from
feeling sleepy.

When I entered our room, Agneta was already in bed. She lay with
her head almost hidden by the bed-clothes, and when I wished her
"Good-night," she responded in a muffled tone. She did not raise her
face for me to kiss, and I could divine the reason. Her face was wet
with tears.

I felt very sorry for my cousin as I lay down and gave myself up, not
to sleep, but to the delight of recalling every word that had passed
between me and Alan Faulkner. I thought I knew how full of pain her
heart was, and I longed to assure her of my sympathy, but did not
like to open the subject.

On the following Wednesday some friends of the Colonel's, who

were staying at Chelmsford, were expected to dine with us, so I
arrayed myself in my new evening frock. I saw Agneta looking at me
as I put it on, and when the last hook was fastened, she said
admiringly:

"What a sweet frock, Nan!"

"I am glad you like it," I said as I turned slowly round before the
mirror. "It is Olive's contrivance. Don't you think she is very clever?"

"Indeed I do. She has quite a genius for dressmaking. The girls
showed me some of her masterpieces when I was at your home."

"And do you mean to say that you do not recognise this gown?" I
asked.

"No, how should I?" She came nearer, and looked closely at it. Then
her face changed. "Why, it is—never! Yes, it is my pink ball-dress!
Oh, Nan, I wish you had not told me! Why did you remind me of that
night?"

She threw up her hands with a tragic, despairful gesture, and I saw
she was struggling with strong emotion.

"Oh, Agneta, what about it? What is it that makes you so unhappy?
Tell me about that night."

"Indeed, I am unhappy—never anything but unhappy now," said

Agneta with tears, and the whole story came out.

It seemed that she had last worn this frock at a ball, where she met
Ralph Marshman, and said farewell to him. He was a junior clerk in a
bank, and Mr. Redmayne had been indignant at his presumption in
thinking to wed his daughter. He had forbidden him to address
Agneta again, and, in order to make obedience easy, had used his
influence to get the young man removed from the Manchester bank
to a branch bank at Newcastle.

In spite of every precaution, however, the two had managed to

secure a few minutes' quiet talk at this ball on the night prior to
Marshman's departure for Newcastle. They had vowed to be faithful
to one another, and to meet, in spite of Mr. Redmayne's prohibition,
whenever opportunity offered. They had even arranged to carry on a
secret correspondence; but, through the treachery, as Agneta
described it, of a servant whom she had bribed to secrete her letters,
one of them had fallen into her mother's hands. A painful scene
ensued, and her mother, after extorting from her a promise that she
would not write to Marshman again, had finally arranged to send her
to "Gay Bowers." And now, at a distance from her lover, and fearful,
in spite of her protestations that she would never give him up, lest
her parents should succeed in finally separating her from him,
Agneta was in a miserable frame of mind.

I pitied her greatly as she opened her heart to me, and yet I listened
with a sense of revulsion. There seemed to me something ignoble
and degrading in the way this courtship had been conducted. It hurt
me to think that my cousin could stoop to practise such
dissimulation, and I found it hard to believe that the man could be
worthy of a woman's love who wooed her in this clandestine fashion.
The beautiful crown of love was tarnished and defiled by being thus
dragged in the dust.

I was shocked, too, by the way Agneta spoke of her parents. She
seemed to regard them as her natural enemies. It was clear to me
that the atmosphere of her home must be very different from that of
ours. We girls had no secrets from our mother. Our parents were not
afraid to trust us, nor we to trust them.

"Mother cares for nothing but money," Agneta said, and I was afraid
there might be some truth in this statement. "Because Ralph is poor,
she cannot say a good word for him. If he were rich, she would not
mind what his past had been."