Flocculation In Natural And Engineered Environmental Systems - Chapter 1 pot

FLOCCULATION in
NATURAL and ENGINEERED
ENVIRONMENTAL SYSTEMS
Copyright 2005 by CRC Press
CRC PRESS
Boca Raton London New York Washington, D.C.
FLOCCULATION in
NATURAL and ENGINEERED
ENVIRONMENTAL SYSTEMS
Edited by
Ian G. Droppo • Gary G. Leppard
Steven N. Liss • Timothy G. Milligan
Copyright 2005 by CRC Press
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Library of Congress Cataloging-in-Publication Data
Flocculation in natural and engineered
environmental systems/edited by Ian G. Droppo [et al.].
p. cm.
Includes bibliographical references and index.
ISBN 1-56670-615-7 (alk. paper)
1. Flocculation. 2. Water—Purification. I. Droppo, Ian G.
QD547.F584 2004
628.1’622—dc22
2004056933
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Preface
In the history of environmental science, there has probably been no greater struggle
than the attempt to control the impact of the sediment and solids generated by nature
and human influence (including industrial processing) on the terrestrial and aquatic
environment and on socioeconomics in general. Untold billions of dollars are spent
each year on dredging to maintain navigation channels and harbors. Further costs are
added by the need to treat these sediments prior to disposal because of high levels of
contamination resulting from anthropogenic impacts on the environment. Significant
financial burdens arise as a result of the need to remove solids during drinking water
and wastewater treatment processes, a necessity for sustainable development, and the

protection of human and aquatic health. It is now well established that the majority
of particles within natural (freshwater and saltwater) systems are present in a floc-
culated form (i.e., flocs), and that the formation of flocs is essential for the effective
performance of engineering processes such as biological wastewater treatment.
Flocculation is the process of aggregating smaller particles together to form lar-
ger composite particles via various physical, chemical, and biological interactions.
These larger composite particles behave differently in terms of their physical (e.g.,
transport, settling), chemical (e.g., contaminant uptake and transformation), and bio-
logical (e.g., community structure activities and metabolism) behavior relative to
their constituent individual particles due to differences in size, shape, porosity, dens-
ity, and compositional characteristics. Given these significant behavioral differences
between flocs per se and their individual component parts, flocculation influences a
wide array of environmental phenomena related to sediment–water and sediment–
sediment interactions. A few of these include sediment and contaminant transport in
various aquatic ecosystems, remediation of contaminated bed sediments, contamin-
ated bed sediment stability, and habitat destruction resulting from sedimentation (e.g.,
coral reef, salmon spawning grounds, mollusk habitat degradation). These concerns,
coupled with the ubiquitous nature of flocs within natural and engineered systems
and the potential to influence floc properties to control better the environmental and
engineering processes, have generated an increased emphasis on floc research.
The traditional disciplines within saltwater, freshwater, and engineering research
have, however, remained somewhat mutually exclusive in their approach to the study
of flocculation processes. This reality is facilitated by differences in external vari-
ables (e.g., environmental conditions), focus driven research, and discipline bias.
Regardless of differences in discipline or approach, there is great scope and utility for
the sharing of information between scientists who work in these three floc environ-
ments. Often methods used in one environment can, and should, be used in another
to further our understanding of flocculation processes. While new developments in
v
Copyright 2005 by CRC Press

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vi Preface
genomics, nanotechnology, sampling, and modeling permit increasingly revealing
investigations into floc structure, processes, and impact, there is still a fundamental
lack of knowledge related to many aspects of the flocculation process.
In light of the importance of flocculation within natural and engineered systems,
an international workshop was held on September 4 and 5, 2003, at the Canada
Centre for Inland Waters, Burlington, Ontario, Canada. The workshop brought
together academics and government scientists from around the globe to address the
critical issue of sediment flocculation within freshwater, saltwater, and engineered
systems. During the workshop, participants representing these three environments
presented their research findings. Three focus areas were used to structure the
workshop: (a) modeling, (b) physicochemical, and (c) biological aspects of floc-
culation. Following individual presentations, the participants were divided up into
three working groups to address assigned topics in the focus areas. Each focus group
contained researchers from the freshwater, saltwater, and engineered systems to
ensure a cross-communication of ideas between environments and to facilitate an
understanding of the unifying principles of flocculation. Participants ranged from
geographers/geomorphologists who investigate flocculation as it relates to sediment
source, transport, and fate within river systems, sedimentologists interested in floc-
culation’s influence within depositional environments, biologists focusing on the
biopolymeric matrices and microbial consortia of flocs, oceanographers investig-
ating sediment transport and delivery within estuaries and open ocean environments,
and wastewater engineers/biologists interested in floc behavior within engineered
systems.
The peer-reviewed 20 chapters that comprise this text are organized by their envir-
onment of investigation. The final chapter identifies the unifying principles that were
discussed within the workshop focus groups and from the preceding chapters. The
text provides a unique perspective in that it integrates the natural sciences and engin-
eering fields as they relate to the central phenomenon of flocculation. We hope that

the array of information provided in this book will be valuable to all those interested
in flocculation issues within any environment.
Ian G. Droppo
Gary G. Leppard
Steven N. Liss
Timothy G. Milligan
Copyright 2005 by CRC Press
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Acknowledgments
The workshop and this resultant text would not have been made possible without
the generous support of our sponsors. We would like to thank the National Water
Research Institute of Environment Canada, the Department of Fisheries and Oceans,
the Wastewater Technology Centre of Environment Canada, the Brockhouse Institute
for Materials Research of McMaster University, Ryerson University, and the Inter-
national Association for Sediment Water Science for their support. The editors are
particularly grateful to the Natural Sciences and Engineering Research Council of
Canada for their funding support related to research on flocculation.
Each chapter has been peer reviewed by two or three reviewers consistent with
the standards set for international scientific journals. We would like to thank these
reviewers for their efforts in this regard.
Finally, we would like to thank the National Water Research Institute of Envir-
onment Canada for hosting the workshop and John Lawrence, Michel Beland, and
John Preston for their support. The efforts of Elizabeth Wendel, Meenu Pall, Dianne
Crabtree, Allana Manto, Quintin Rochfort, Christina Jaskot, and Brian Trapp of
Environment Canada leading up to, during, and following the workshop are gratefully
acknowledged.
Copyright 2005 by CRC Press
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About the Editors
Ian G. Droppo is a research scientist with the

National Water Research Institute of Environment
Canada and is the current elected vice president of
the International Association for Sediment Water
Science. Dr. Droppo holds adjunct professorships
at McMaster University, School of Geography and
Earth Sciences and at the State University of New
York, College at Buffalo, Department of Geography
and Planning. He holds undergraduate and M.Sc.
degrees in physical geography from McMaster Uni-
versity, Canada and a Ph.D. in physical geography
from the University of Exeter, United Kingdom. He
was a recent recipient of Leverhulme International
Visiting Fellowship held at the University of Exeter in the United Kingdom. Dr.
Droppo’s research interests center around sediment dynamics within natural and
engineered systems with particular emphasis on flocculation processes. He has applied
this knowledge in multiple environments including urban stormwater management,
remediation of contaminated bed sediments, contaminated bed sediment stability,
and in the source, fate, and effect of sediments and associated contaminants within
numerous aquatic environments. His research is supported by awards from Environ-
ment Canada, the Natural Sciences and Engineering Research Council of Canada,
and a range of industrial partners. He has given many invited lectures and seminars at
international conferences, workshops, and universities and has taught many sediment
chemistry monitoring courses in developing countries. Dr. Droppo has carried out
collaborative research in Canada, United States of America, United Kingdom, Japan,
Mexico, Australia and Thailand leading to over 85 peer-reviewed journal publications,
book chapters, and technical reports.
Copyright 2005 by CRC Press
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x About the Editors
Gary G. Leppard is an environmental biochemist and microbiologist who studies

the roles of natural and engineered aquatic aggregates (flocs, biofilms) in the transport
and fate of contaminants. In concert with these activities, he develops electron-optical
means to analyze the colloidal structure of natural dispersing agents and the flocs of
water treatment tanks. He joined the staff of the National Water Research Institute of
Environment Canada at Burlington (ON) in 1975, as a research scientist. While also
holding a professorship at McMaster University and membership in the Brockhouse
Institute for Materials Research (Hamilton, ON), he is a Fellow of the International
Union of Pure and Applied Chemistry and a Consulting Fellow of the World Innov-
ation Foundation. In sequence, he was an invited scientist at the University of Paris
(France), the University of Milan (Italy), Laval Uni-
versity (Quebec City), the National Research Coun-
cil of Canada (Ottawa), the University of Geneva
(Switzerland), the University of Vienna (Austria),
and the Rudjer Boskovic Institute (Croatia).
Dr. Leppard received degrees in several fields
of biology and biological chemistry from the Uni-
versity of Saskatchewan (Saskatoon, SK) and from
Yale University (New Haven, CT, United States).
A Ph.D. in cell biology, with a specialization in
electron-optical methods, was received from Yale in
1968. Research interests then extended into biogeo-
chemistry, wastewater treatment, materials science,
and the activities of natural microbial consortia. His
interdisciplinary research has led to awards from the
North Atlantic Treaty Organization, the Commis-
sion of the European Communities, and the RITE
innovative technology organization in Japan, as well as a role on the editorial board
of the Encyclopedia of Analytical Science. Current scientific interests focus on the
control, by nanoscale phenomena, of macroscale effects in aquatic environments.
These interests are coupled to the development of technology for commercial use,

and include environmental projects for synchrotron laboratories.
Copyright 2005 by CRC Press
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About the Editors xi
Steven N. Liss is a professor of applied microbio-
logy in the Department of Chemistry and Biology
at Ryerson University and is the Associate Dean
(Research, Development and Science Programs)
for the Faculty of Engineering and Applied Sci-
ence. Dr. Liss holds adjunct professorships at the
University of Toronto in the Departments of Chem-
ical Engineering and Applied Chemistry and Civil
Engineering. Dr. Liss holds an undergraduate degree
in microbiology and immunology from the Uni-
versity of Western Ontario (1980) and graduate
degrees in applied microbiology from the Univer-
sity of Saskatchewan (M.Sc, 1983; Ph.D., 1987).
Dr. Liss currently leads research projects on the
microbiology of wastewater treatment, water wells,
and environmental biotechnology. His research is
supported by awards from the Natural Sciences and Engineering Research Council of
Canada, the National Centres of Excellence, Ontario Centres of Excellence, Environ-
ment Canada, Canada Foundation for Innovation (CFI), and a wide range of industry
partners. Specific research activities include microbial floc architecture in engineered
and natural systems, microbial ecology, water quality, filamentous microorganisms
and bulking problems, biofouling and microbial-based tools for studying, and mon-
itoring biological treatment systems including DNA microarrays. His laboratory has
developed expertise related to the physicochemical properties of microbial structures,
their composition and structure, and the application of advanced optical microscopy
in studying microbial structures and physiology. His research in wastewater micro-

biology led to the Ryerson Distinguished Research Award in 1998. Dr. Liss has
supervised 32 graduate students at the masters and Ph.D. levels. He is the author and
co-author of over 100 peer-reviewed journal publications, book chapters, conference
presentations, and technical reports.
Copyright 2005 by CRC Press
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xii About the Editors
Timothy G. Milligan is a researcher with the Mar-
ine Environmental Sciences Division, Fisheries and
Oceans Canada. As head of the Particle Dynamics
Laboratory at the Bedford InstituteofOceanography
he leads the group’s research into the behavior of
fine particulate material in aquatic environments. He
received his B.Sc in geology and M.Sc in ocean-
ography from Dalhousie University and has been
involved with flocs for over 30 years. While his ini-
tial contact was in pulp mill effluent, it was the time
spent with the late Dr. Kate Kranck, a pioneer in
flocculation studies in the marine environment, that
gave him his love of mud. Areas of interest include
the mechanisms governing the loss of sediment from
river plumes, the effect of flocculation on the trans-
port and fate of contaminants, and environmental
impacts of offshore oil and gas and aquaculture. Mr.
Milligan has led research projects in a wide range of geographical areas, from the
Amazon to the Canadian Arctic. While his work concentrates mainly on the marine
environment, the fate of terrestrially derived sediments and associated contaminants
has led him into the study of fluvial transport as well. Mr. Milligan has been involved
in many international ventures, several of which have received funding from the U.S.
Office of Naval Research. His work combines in situ techniques with process-based

parameterization of the size distributions of the component grains in suspended and
bottom sediment to better understand the fate of mud in both marine and freshwater
systems. Over 80 peer-reviewed primary publications, book chapters, and technical
reports have been produced from this work.
Copyright 2005 by CRC Press
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Contributors
D. Grant Allen
Department of Chemical Engineering
and Applied Chemistry
Pulp & Paper Centre
University of Toronto
Toronto, Ontario, Canada
Joseph F. Atkinson
Department of Civil, Structural and
Environmental Engineering
State University at Buffalo
Buffalo, New York, U.S.A.
Adrian B. Burd
Department of Marine Sciences
University of Georgia
Athens, Georgia, U.S.A.
Rajat K. Chakraborti
Department of Civil, Structural and
Environmental Engineering
State University at Buffalo
Buffalo, New York, U.S.A.
Holger Daims
Department of Microbial Ecology
Institute for Ecology and Conservation

Biology
University of Vienna
Vienna, Austria
Patrick J. Dickhudt
University of Maryland Center for
Environmental Science
Horn Point Laboratory
Cambridge, Maryland, U.S.A.
Ian G. Droppo
National Water Research Institute
Environment Canada
Burlington, Ontario, Canada
Ramin Farnood
Department of Chemical Engineering
and Applied Chemistry
University of Toronto
Toronto, Ontario, Canada
Carl T. Friedrichs
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, Virginia, U.S.A.
David D. Fugate
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, Virginia, U.S.A.
Jean-Francois Gaillard
Department of Civil and Environmental
Engineering
Northwestern University
Evanston, Illinois, U.S.A.

Gill G. Geesey
Department of Microbiology
Montana State University
Bozeman, Montana, U.S.A.
Adam P. Hitchcock
Brockhouse Institute for Materials
Research
McMaster University
Hamilton, Ontario, Canada
Copyright 2005 by CRC Press
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xiv Contributors
George A. Jackson
Department of Oceanography
Texas A&M University
College Station, Texas, U.S.A.
Bommanna G. Krishnappan
National Water Research Institute
Environment Canada
Burlington, Ontario, Canada
John R. Lawrence
National Water Research Institute
Environment Canada
Saskatoon, Saskatchewan, Canada
Anne A. Lazarides
MEMS Department
Pratt School of Engineering
Duke University
Durham, North Carolina, U.S.A.
Gary G. Leppard

National Water Research Institute
Environment Canada
Burlington, Ontario, Canada
B.Q. Liao
Department of Chemical Engineering
Lakehead University
Thunder Bay, Ontario, Canada
Steven N. Liss
Department of Chemistry and Biology
Faculty of Engineering and Applied
Science
Ryerson University
Toronto, Ontario, Canada
Bruce E. Logan
Department of Civil and Environmental
Engineering
COE Environmental Institute
The Pennsylvania State University
University Park, Pennsylvania
U.S.A.
Jiri Marsalek
National Water Research Institute
Environment Canada
Burlington, Ontario, Canada
Timothy G. Milligan
Habitat Ecology Section
Bedford Institute of Oceanography
Dartmouth, Nova Scotia, Canada
Fernando Morgan-Sagastume
Department of Chemical Engineering

and Applied Chemistry,
Pulp & Paper Centre
University of Toronto
Toronto, Ontario, Canada
Thomas R. Neu
Department of River Ecology
Magdeburg
UFZ Centre for Environmental Research
Leipzig-Halle, Germany
Ellen L. Petticrew
Department of Geography
University of Northern British Columbia
Prince George, British Columbia
Canada
John M. Phillips
Environment Agency
Blandford Forum
Dorset, U.K.
Alain Reinhardt
Analytical and Biophysical
Environmental Chemistry
University of Geneva
Geneva, Switzerland
Heidi Romine
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, Virginia, U.S.A.
Laura Rubiano-Gomez
University of Maryland Center for
Environmental Science

Horn Point Laboratory
Cambridge, Maryland, U.S.A.
Lawrence P. Sanford
University of Maryland Center for
Environmental Science
Horn Point Laboratory
Cambridge, Maryland, U.S.A.
Copyright 2005 by CRC Press
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Contributors xv
Peter H. Santschi
Laboratory for Oceanographic and
Environmental Research
Department of Oceanography
Texas A&M University
Galveston, Texas, U.S.A.
Steven E. Suttles
University of Maryland Center for
Environmental Science
Horn Point Laboratory
Cambridge, Maryland, U.S.A.
Laurenz Thomsen
School of Engineering and Science
International University Bremen
Bremen, Germany
John E. VanBenschoten
Department of Civil, Structural and
Environmental Engineering
State University of New York at Buffalo
Buffalo, New York, U.S.A.

Fintan Van Ommen Kloeke
Department of Microbiology
Montana State University
Bozeman, Montana, U.S.A.
Desmond E. Walling
Department of Geography
University of Exeter
Exeter, Devon, U.K.
Kevin J. Wilkinson
Analytical and Biophysical
Environmental Chemistry
University of Geneva
Geneva, Switzerland
Johan C. Winterwerp
W.L. Delft Hydraulics
Delft, The Netherlands
Marissa Yates
University of Maryland Center for
Environmental Science
Horn Point Laboratory
Cambridge, Maryland, U.S.A.
Copyright 2005 by CRC Press
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Contents
Chapter 1 Methods for Analyzing Floc Properties 1
Steven N. Liss, Timothy G. Milligan, Ian G. Droppo, and
Gary G. Leppard
I Freshwater Environments 23
Chapter 2 Overview of Flocculation Processes in Freshwater Ecosystems 25
Gary G. Leppard and Ian G. Droppo

Chapter 3 Intra-Storm and Seasonal Variations in the Effective Particle
Size Characteristics and Effective Particle Density of Fluvial
Suspended Sediment in the Exe Basin, Devon, United Kingdom 47
John M. Phillips and Desmond E. Walling
Chapter 4 The Composite Nature of Suspended and Gravel Stored Fine
Sediment in Streams: A Case Study of O’Ne-eil Creek,
British Columbia, Canada 71
Ellen L. Petticrew
Chapter 5 Effects of Floc Size and Shape in Particle Aggregation 95
Joseph F. Atkinson, Rajat K. Chakraborti, and
John E. VanBenschoten
Chapter 6 Mapping Biopolymer Distributions in Microbial Communities 121
John R. Lawrence, Adam P. Hitchcock, Gary G. Leppard, and
Thomas R. Neu
Chapter 7 Contrasting Roles of Natural Organic Matter on Colloidal
Stabilization and Flocculation in Freshwaters 143
Kevin J. Wilkinson and Alain Reinhardt
Chapter 8 An Example of Modeling Flocculation in a Freshwater
Aquatic System 171
Bommanna G. Krishnappan and Jiri Marsalek
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xviii Contents
II Saltwater Environments 189
Chapter 9 Transport of Materials and Chemicals by Nanoscale Colloids
and Micro- to Macro-Scale Flocs in Marine, Freshwater, and
Engineered Systems 191
Peter H. Santschi, Adrian B. Burd,
Jean-Francois Gaillard, and Anne A. Lazarides
Chapter 10 Variability of Suspended Particle Concentrations, Sizes, and

Settling Velocities in the Chesapeake Bay Turbidity Maximum 211
Lawrence P. Sanford, Patrick J. Dickhudt, Laura
Rubiano-Gomez, Marissa Yates, Steven E. Suttles,
Carl T. Friedrichs, David D. Fugate, and Heidi Romine
Chapter 11 Organic Rich Aggregates in the Ocean: Formation, Transport
Behavior, and Biochemical Composition 237
Laurenz Thomsen
Chapter 12 Equilibrium and Nonequilibrium Floc Sizes 249
Johan C. Winterwerp
Chapter 13 Coagulation Theory and Models of Oceanic Plankton
Aggregation 271
George A. Jackson
III Engineered Systems 293
Chapter 14 Extracellular Enzymes Associated with Microbial Flocs
from Activated Sludge of Wastewater Treatment Systems 295
Gill G. Geesey and Fintan Van Ommen Kloeke
Chapter 15 Molecular Analyses of Microbial Community Structure and
Function of Flocs 317
Holger Daims
Chapter 16 Using Atomic Force Microscopy to Study Factors
Affecting Bioadhesion at Molecular to Nanoscale Levels 339
Bruce E. Logan
Chapter 17 Impact of Stresses or Transient Conditions on Deflocculation
in Engineered Microbial Systems 351
Fernando Morgan-Sagastume and D. Grant Allen
Chapter 18 Flocs and Ultraviolet Disinfection 385
Ramin Farnood
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Contents xix

Chapter 19 Surface Thermodynamics and Hydrophobic Properties
of Microbial Flocs 397
B.Q. Liao, Gary G. Leppard, D. Grant Allen, Ian G. Droppo,
and Steven N. Liss
IV Summary 405
Chapter 20 Opportunities, Needs, and Strategic Direction for
Research on Flocculation in Natural and
Engineered Systems 407
Ian G. Droppo, Gary G. Leppard, Steven N. Liss, and
Timothy G. Milligan
Copyright 2005 by CRC Press
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1
Methods for Analyzing
Floc Properties
Steven N. Liss, Timothy G. Milligan,
Ian G. Droppo, and Gary G. Leppard
CONTENTS
1.1 Introduction 1
1.1.1 Floc Size 2
1.1.2 Sample Handling and Stabilization 4
1.2 Floc Settling Velocity 5
1.3 Floc Density and Porosity 7
1.3.1 Floc Structure: Correlative Microscopy 9
1.3.2 Extracellular Polymeric Substances 10
1.4 Surface Charge and Hydrophobicity 11
1.5 Microbial Ecology 13
1.6 Conclusion 14
References 14
1.1 INTRODUCTION

The function–structure relationships of flocs are important to environmentalscientists,
microbiologists, and engineers. Ultimately, their goals include being able to solve
practical problems more effectively, and to provide better information for modeling
ecological processes and contaminant transport in aquatic environments and in the
operation of engineered systems (e.g., wastewater and drinking water). Methods and
analytical tools play a critical role in floc research and in achieving these goals.
These are intended to do one of two things: (i) to provide descriptive and quantitative
information that may lead to a fuller understanding of flocculation and (ii) to have
tools that may be applied to the management of floc processes in engineered and
environmental systems.
At present, few standard methods with good reproducibility are available,
although several physical, chemical, and microbiological measurement and analyt-
ical techniques have been developed. Earlier reviews give a comprehensive review
of the methods and techniques for the measurement of physical characteristics for
activated sludge
1
and an overview of the principles, methods, and applications of
particle size analysis in primarily saltwater systems.
2
Eisma et al.
3
and Dyer et al.
4
1-56670-615-7/05/$0.00+$1.50
© 2005byCRC Press
1
Copyright 2005 by CRC Press
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2 Flocculation in Natural and Engineered Environmental Systems
conducted a comparative study in the Elbe estuary to evaluate several different in situ

methods for determining floc size and settling velocity. More recently, several entries
in the Encyclopaedia of Environmental Microbiology
5
provide overviews of methods,
particularly advanced optical microscopy and molecular tools applied to the study of
microbial structures including flocs.
6
Common to all these reviews is the wide range
of methods employed to determine some of the most basic of parameters that describe
flocs in the environment.
In engineered systems, advances have been achieved primarily in studying floc
properties (ecology, structure, and physicochemical characteristics) of individualflocs
from full-scale systems and from laboratory-scale reactors that were run under well-
controlled conditions. In contrast, studies in the marine and freshwater environments
have concentrated on bulk properties such as gross morphology, size, and settling
velocity in samples collected with an emphasis on in situ measurements. One reason
for the difference between measurements in the natural environment and engineered
systems is the availability of flocs and the ease with which they can be sampled
intact. Those involved with studying natural systems have tended to focuson the gross
properties and behavior of floc. Engineeredsystems are suited to detailed examination
of surface properties and molecular determinants in floc behavior.
In this chapter, we present an overview of the principal methods presently being
used in engineered, freshwater, and marine systems. Some aspects of the methods
presented can be applied to both natural and engineered systems. Ourgoal is to provide
an insight into the work being carried out in the different aquatic environments so that
researchers can consider adapting the techniques presented to their respective fields.
1.1.1 FLOC SIZE
Floc size is a widely measured floc characteristic. Floc size influences properties
such as mass transfer (transport and settling),
7

biomass separation, and sludge
dewatering.
8–10
Flocs are generally observed as two-dimensional (2D) projections,
and there is no simple means of specifying size or shape.
11
However, flocs are
highly irregular in shape, porous, and three-dimensional. Equivalent spherical dia-
meter (ESD), frequently calculated from the two-dimensional area, is often used to
characterize floc size due to its simplicity and its application in Stokes’ law.
11–13
Bache et al.
11
defined the effective diameter as the geometric mean

(d
min
∗
d
max
)
based on the maximum (d
max
) and minimum (d
min
) dimensions across the 2D floc
image. Barbusinski and Koscielniak
14
and Li and Ganczarczyk
15

described floc size
based on the average floc diameter defined as one half of the sum of the longest and
shortest dimensions of the flocs measured.
Flocs in suspension are found over a range of sizes that describe a continuous
distribution. Several standard parameters are available to describe floc size distribu-
tions. Median (d
50
), upper quartile (d
25
), and mode have all been used to describe
the size distribution of flocs in suspension.
13,16,17
Due to the open architecture and
poorly defined association of particles within a floc, researchers use fractal geometry
to describe floc structure.
18−23
Depending on the nature and the sizing technique
employed, there is no evidence to show which definition is the best representation of
floc size. However, researchers should be clear in their definition of floc size when
reporting results.
Copyright 2005 by CRC Press
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Methods for Analyzing Floc Properties 3
In general flocs range in size from a few microns to a few millimeters when
measured by ESD. One exception is large assemblages of diatoms or other biologically
derived material. Sometimes referred to as marine snow to differentiate it from more
inorganic rich flocs, these patches of aggregated organic material can reach ESDs
many orders of magnitude larger than what could be considered normal flocs. When
marine snow becomes buoyant during decomposition, as was observed in the Adriatic
Sea during the mucilage phenomenon, “floc size” can exceed 1 m.

24−27
Many methods and instruments have been developed in the past to measure floc
size distributions in natural and engineered systems. One of the earliest methods was
the Coulter Counter, which determined the size distribution of particles in suspension.
This method was popular in the marine environment as the electrolyte concentration
in seawater permitted samples to be analyzed without alteration. However, stresses
applied during the counting process can disrupt flocs which raises the issue that this
method may be of little value for estimating floc size.
28,29
The determination of floc size has relied primarily on imaging of flocs followed by
image analysis to ascertain the parameters describing the size distribution.
12,30,31
Both
microscopic observations and photographic techniques
13,32–35
have been used. In situ
photography of flocs, although relatively easy to employ, does not allow measurement
of very small flocs due to resolution limits. Often these systems can only image down
from 50 to 100 µm, although a 10 : 1 camera system with a resolution of 10 µm
has been developed.
36,37
Recent advances in digital photography should improve the
resolution of in situ camera systems. The main advantage of these instruments is their
ability to measure floc size with minimal disturbance to the natural stress environment
of the flocs. However, they were developed for the natural environment, and may be
difficult to apply in an engineered system as they are limited by the concentration of
particles in suspension.
Microscopic methods usually incorporate a camera and computerized digitizer
to provide images for analysis. The increased resolution of microscopic systems
allows for accurate, reproducible, and relatively fast estimates of floc morpholo-

gical parameters. Specialized techniques such as confocal microscopy and electron
microscopy (discussed in detail later in the chapter) allow the internal structure of
flocs to be examined. The obvious drawback for microscopic analysis is the require-
ment to remove flocs from their natural environment and the associated instrument
costs.
Common to both photographic and microscopic methods is the requirement to
conduct image processing and analysis on the captured image to determine floc size
and other descriptive parameters. Image processing and analysis comprises several
steps.
38
Different algorithms are applied to the digital image to improve the quality
of the image and to separate a floc from its background. Each area of coherent pixels
with values within a selected range of threshold values is then used to calculate the
differentparameters used to describe floc size. There are differing views on the number
of pixels required to define a particle with values ranging from 3 to 35 pixels.
39,40
Several different image analysis systems are available on the market but all are based
on the same principles for manipulating a matrix of pixel values. Clear explanations
of the methods employed in the analysis are critical for understanding how descriptive
parameters such as ESD are generated.
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4 Flocculation in Natural and Engineered Environmental Systems
Laser based sizing instruments are now being widely used to determine floc size
in situ.
41,42
Two different laser techniques have been used, focused beam reflectance
measurement (FBRM) and laser diffraction. FBRM instruments (modified ParTec)
employ a rotating laser beam to determine the size of particles in the sensing zone.
41

When the laser encounters a particle, the beam is reflected for the period of time it
takes to traverse the particle. Using the angular velocity of the beam and the duration
of the reflected laser pulse, the length of the intersecting particle chord is determ-
ined. A chord correction algorithm is then used to determine the size distribution
of the particles in suspension. FBRM instruments were designed for process control
and are not easily adapted to studies in natural systems. However, they do have the
advantage of working at higher concentrations than instruments that rely on light
transmission.
41
Laser diffractioninstruments were first used by Bale and Morris,
43
who modified a
Malvern particle size analyzer (Malvern Instruments, U.K.) for underwater use. Since
then purpose-designed laser diffraction systems have become available, notably the
LISST (Sequoia Scientific Inc., WA, United States) and the CILAS (CILAS, France).
Laser diffraction instruments are based on the scattering of laser light by particles as
the beam transits a known sample zone. The scattering angle is determined by the
size of the particle with the scattering angle being small for large particles and large
for small particles.
42
A series of concentric ring detectors sense the amount of light
they are receiving. Using the Mie or Fraunhofer theory of scattering for spheres, these
values can then be inverted to yield the particle size distribution.
42
Floc size has also been inferred from the settling behavior of flocculated
suspensions.
4,44
Settling column methods in general measure the equivalent hydraulic
diameter of particles in suspension rather than the actual physical size of the suspen-
ded particles. Floc size is expressed in terms of the diameter of a sphere with the

density of quartz, settling at the same speed as the particle in question.
45
1.1.2 S
AMPLE HANDLING AND STABILIZATION
In situ measurement of floc size is clearly preferable due to the fragile nature of
flocs. Sample handling may break up existing flocs or promote formation of larger
flocs during storage.
46
Gibbs and Konwar
47
showed that common sampling methods
disrupt flocs. Critical to any work with flocs outside their natural environment is
sample handling and preparation. The need for microscopic examination of flocs
and for laboratory experiments with natural flocs has led to the development of new
techniques for removing flocs from their ambient conditions with minimal change in
floc size or structure. Considerable efforts have been given to overcome perturbation
that may be associated with sampling and specimen preparation.
For floc size measurements not performed in situ, samples are collected in
bulk suspension and transported to the laboratory for sizing. Essential to this first
step is minimizing the stress applied to the flocs during sampling. Droppo and
Ongley
12
employed traditional laboratory-used plankton chambers within fluvial sys-
tems. By using the plankton chamber as both the sampling and analytical chamber
for image analysis, potential perturbations are minimized. Depending on the sizing
methods, further floc sampling might be required. Some size measurements using
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Methods for Analyzing Floc Properties 5
image analysis systems or microscopic observation require subsampling of flocs onto

microscope slides. This is normally done using a pipette for which the opening has
to be wide enough (2 to 3 mm) to prevent floc breakage and disaggregation.
48
Floc stabilization prior to further sample handling has been shown to be effective
in preserving floc structural characteristics. Droppo et al.
49,50
described a method
of utilizing low melting point agarose to physically stabilize microbial flocs before
analysis. This technique was found to have no significant effects on floc size distribu-
tions. Ganczarczyk et al.
51
used a similar approach in physically stabilizing microbial
flocs. Optically clear polyacrylamide gels have been used in marine sediment traps
to capture flocs intact for later processing.
52
1.2 FLOC SETTLING VELOCITY
Settling velocity measurements of flocs are important for studying the fate of
sediments within natural systems and for the evaluation of solids removal from treated
effluents and in the estimation of floc wet density. Floc settling velocity has been
found to increase with increasing floc size
53–57
but not necessarily in accordance
with Stokes’ law. Floc settling under gravity has been reported to be affected by a
wide range of factors including the shape and settling orientation of flocs being meas-
ured. The effect of fluid drag force on the settling velocity of a nonspherical particle
is larger than that on a spherical particle.
58,59
The fastest settling rate is for particles
of spherical shape, followed by that of cylindrical, needle-like, and disc-like shape.
58

Floc settling velocity may be affected by the settling orientations of the flocs because
the drag force depends on the floc area facing the settling direction.
53
Fluid flow
through the internal structure of flocs may also be important, as this would reduce
hydrodynamic resistance and increase settling velocity.
15
Zahid and Ganczarczyk
54
stated that the computation of settling velocity by Stokes’ law from the size and dens-
ity measurements has to consider the effect of floc permeability. This, however, is in
contradiction to the usual way of calculating wet density of flocs from the size–settling
velocity measurements. The effect of floc permeability on settling velocity is con-
sidered negligible.
55,60
To complicate this picture from the floc structure viewpoint,
Liss et al.
61
showed that the channels that appeared to be open by conventional optical
microscopy (COM) and confocal laser scanning microscopy (CLSM) were in many
instances filled with extracellular polymeric substance (EPS) fibrils that could be seen
only by transmission electron microscopy (TEM).
Floc settling velocity is most commonly determined by measuring the distance
traveled by a floc over a known time using multiple exposure photographic and video
imaging.
34,35,53,57,62,63
These techniques are effective in measuring floc size and
settling velocity within the resolution limits of the imaging method used. Klimpel
et al.
60

used a cinematographic technique to measure larger flocs (>100 µm), and
the multiple exposure technique to measure smaller flocs (<100 µm). Droppo et al.
57
developed a videographic technique to measure floc settling velocity. This technique
involves using a stereoscopic microscope or 1 ×tellecentric lens and a video camera
to capture images of settling floc in a column filled with a medium similar to the native
environment of the samples. A small quantity (∼1 ml) of floc samples is introduced
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6 Flocculation in Natural and Engineered Environmental Systems
at the top of the column. A sufficient travel distance is allowed for flocs to reach
terminal velocity. Settling images of flocs are then recorded on a VCR as they pass
though the focal plane of the microscope. These images are then analyzed using a
computer imaging software for size and settling velocity.
Similar video imaging techniques have been developed to examine floc size–
settling velocity relationships in situ in marine and freshwater environments.
36,37
All
are based on video imaging of settling flocs within a stilled water column. Missing,
however, from most studies has been an accurate estimation of floc density. A new
instrument called INSSECT (IN situ Size and SEttling Column Tripod) has been
designed to measure all the variables that, at present, are thought to influence the flux
of fine-grained sediment to the bottom.
52
Comprising a rotating sediment trap and
settling column, the rotating tripod is equipped with video and still camera systems,
current meters, and polyacrylamide gels to capture settling flocs.
There is no simple equation relating the settling velocity of flocs to their size.
Stokes’ lawor modified Stokes’ lawbest describes the settling velocity of particles that
approach a perfect sphere. Despite the limitations, estimations of other floc properties

(e.g., density) derived from Stokes’ law have proven useful in floc research. Stokes’
law is defined as follows:
v =
1
18
g ·d
2
(ρ
f
−ρ
w
)
µ
(1.1)
where v is the terminal settling velocity, ρ
f
the wet density of particle, ρ
w
the density
of water (assume settling in water), g the gravitational constant, µ the viscosity of
water (assume settling in water), and d the diameter of particle.
Li and Ganczarczyk
53
used a power function of the form, v = AL
n
, and a linear
function, v = A+BL, to correlate floc settling velocity (v) with its longest dimension
as a characteristic size (L), where A, B, and n are the equation coefficients determined
experimentally. The power function is considered to be a better way to describe the
relationship because the power function predicts that the velocity will be zero when

floc size approaches zero while the linear function does not. However, the measured
settling velocities can yield coefficients lower than that predicted by Stokes’ law
(n = 2).
54,55,64
The power law coefficients (n) calculated from the power function
generally have ranged from 0.55 to 0.88. The number of flocs measured in these
studies were as low as 21 and as high as 343. Lee et al.
56
managed to measure a
total of 1385 flocs for settling velocity and size determinations and reported a power
coefficient of 0.7 to 0.8. A modified linear model incorporating the floc settling shape
factorwasfound to improve the correlation coefficient(R
2
) of the linear relationship.
64
In the marine environment, settling velocities of flocs have been inferred from
clearance rates in enclosed sedimentation tubes.
4,44
Commonly, open-ended tubes
are submerged horizontally to permit free flow of particles in suspension and then
closed, rotated to 90
◦
, and retrieved. Subsamples are removed from the tube at set
intervals during settling and the settling velocity is determined from the change in
concentration with time.
44
Settling velocities calculated from clearance rates were
found to be an order of magnitude less than those determined in situ using camera
techniques during the Elbe Estuary intercalibration program.
4

However, the ease of
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Methods for Analyzing Floc Properties 7
use and the direct application of the results for determining vertical sediment flux
have made settling columns a common instrument for nearshore studies.
In short, floc settling in any environment is not only highly related to size, but also
related to floc shape and density. While Stokes’ law gives a reasonable approximation,
the relationship between floc size and settling velocity is best described by a power
law equation with the value of the exponent close to 1. Recent advances in digital
imaging and image analysis and the ability to collect ancillary data have led to better
understanding of the size–settling velocity relationship for flocculated suspensions.
52
1.3 FLOC DENSITY AND POROSITY
Floc density and porosity are twoimportant floc characteristics in evaluating floc beha-
vior. Along with floc size and shape, floc density plays a strong role in influencing
settling velocity with concomitant transport and industrial efficiency implications.
As the porosity of a floc has consistently been shown to be negatively correlated
to density,
7
it too is an important parameter for floc behavioral assessment. Dens-
ity is usually derived from the settling velocity–size measurements using Stokes’
law or modified Stokes’ law.
34,35,53–57,60,65
The validity of this approach has been
questioned because it usually assumes spherical flocs and the settling velocity and
size relationship do not follow Stokes’ law. Zahid and Ganczarczyk
54
stated that
there were a number of uncertainties involved in the density calculation from Stokes’

law, therefore the approach was regarded only as an approximation. Lee et al.
56
also supported this approach since it provides at least qualitatively valid density
estimation.
The following equation is often used to calculate floc porosity from density:
53
ε =
ρ
s
−ρ
f
ρ
s
−ρ
w
(1.2)
where ρ
s
and ρ
f
are the dried floc density (1.34 to 1.69 g/cm
3
) and wet floc density,
respectively, and ρ
w
is the liquid density.
Andreadakis
66
made use of interference microscopy for floc density determina-
tion and used the above equation to calculate floc porosity. Density determinations

for aggregates are usually based upon observations of terminal velocity, although a
method based upon a series of sucrose solutions of incremented densities has been
presented by Lagvankar and Gemmell.
67
Ozturgut and Lavelle
59
employed a linear-
density stratified column which allows flocs to settle to their isopycnic levels to
measure lowdensity but settleable wastewater effluentflocs. Dammel and Schroeder
68
used a similar density gradient centrifugation technique, which allows the flocs to
settle in a fluid of continuous increasing density until the flocs become stationary,
to measure the density of activated sludge flocs. This technique, however, does not
measure floc size concurrently with its density, thus, a size and density relationship
might not be established easily. In addition, the ionic strength of the suspension
medium and the nature of the medium itself have to be compatible and nontoxic with
the biological flocs.
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8 Flocculation in Natural and Engineered Environmental Systems
A variety of floc density models have been proposed. Magara et al.
34
proposed
the following floc effective density (ρ
e
) model based on Stokes’ law,
ρ
e
= ρ
f

−ρ
w
= 0.003698 ·µ
w
vd
−2
(1.3)
where ρ
f
and ρ
w
are the floc density and liquid density, respectively (g/cm
3
), µ
w
is
the liquid viscosity (g/cm
3
sec
−1
), v is the floc settling velocity (cm/s) and d is the
floc ESD (cm). Tambo and Watanabe
35
suggest a model based on Stokes’ law for
effective floc density and size:
ρ
e
= ρ
f
−ρ

w
=
34µ
w
v
gd
2
f
(1.4)
assuming a drag coefficient of 45/Re and a floc sphericity of 0.8. Andreadakis
66
suggested that the floc density (ρ
f
) is a function of its size (d),
ρ
f
= 1 +0.30 d
−0.82
(1.5)
assuming a dried sludge density of 1.34 g/cm
3
. Glasgow and Hsu
65
developed an
empirical equation for kaolin–polymer aggregate to relate its density (ρ) to diameter
(d) and pH,
ρ = 1.05 ·d
(−0.0038pH+0.00716)
(1.6)
assuming a sphericity of 1.0.

Zahid and Ganczarczyk
54
plotted effective density as a function of average dia-
meter on a logarithmic scale and developed the followingequation for the floc effective
density and the average diameter (D),
ρ
e
=
0.005
D
1.21
(1.7)
where the two constants, 1.21 and 0.005, represent the slope of the straight line and
the effective density of a 1.0 mm diameter particle, respectively. Accordingly, the
size–porosity function was expressed as:
ε = 1.0 −
0.007
D
1.21
(1.8)
Mikkelsen and Perjrup
69
presented a method for determining effective floc density
and calculated settling velocity in coastal marine environments using data collected
by a LISST. Assuming that floc fraction and the amount of material in suspension that
is found in flocs is high then the effective floc density is equal to the total suspended
mass divided by the total volume concentration of the flocs.
Although there are many empirical models available for the estimation of floc
density and porosity, none of them can be considered as a universal model. This is
Copyright 2005 by CRC Press

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Methods for Analyzing Floc Properties 9
simply because all these models were developed from their specific conditions such
as the type of natural or engineered system, the type of microorganisms, the hydro-
dynamic conditions, and the experimental techniques used. Therefore, floc density
and porosity must be experimentally determined in all situations. New instrument-
ation is now available that can determine the size–settling velocity relationship of
flocs in suspension, determine the mass of the flocs, and capture them for micro-
scopic analysis.
52
With these advances, it should be possible to determine densities
in natural environments in situ.
1.3.1 F
LOC STRUCTURE:CORRELATIVE MICROSCOPY
Leppard
70
defined correlative microscopy (CM) as a strategy of using multiple
microscopic techniques which can include conventional optical microscopy (COM),
confocal laser scanning microscopy (CLSM), and transmission electron microscopy
(TEM), and allow one to detect, assess, and minimize artifacts that might arise from
using one technique only. CM has been successfully used by Liss et al.
61
with a min-
imal perturbation approach in studying natural and engineered flocs. Arecent minimal
perturbation approach
49,50
involves the use of sample stabilization in low melting
point agarose and a fourfold multi-preparatory technique. The use of a fourfold
multiple preparatory technique and CM has revealed how to maintain the structural
integrity of the samples through the stabilization, staining, and washing procedures.

The use of only one microscopic technique can bias or limit the information acquired
because of the artifacts that arise in specific sample preparations and the resolution
constraint associated with a particular technique.
The use of COM is the most common microscopic approach in the analysis of
external gross-scale floc structure.
12,14,31,57,71,72
High resolution TEM is often used
to investigate the fine structure of natural and engineered flocs, especially in the
study of EPS distribution within floc structure.
26,27,72−75
This is generally done by
stabilizing samples in a fixing agent such as glutaraldehyde, then embedding in Spurr
resin or alternatively a fixation and embedding in Nanoplast; an ultrathin section is
then obtained from the embedded sample by slicing with an ultramicrotome and a
diamond knife. This ultrathin section (50 to 100 nm) is then placed on a copper grid
for further staining (e.g., uranyl acetate) to give better contrast, although at TEM
resolution, fibrils, bacterial cells, and other components of floc are visible. TEM can
be used in conjunction with energy dispersive spectroscopy (EDS) to detect metal
accumulation and to give element abundance in EPS.
61,76
The thickness constraint
of ultrathin sections (50 to 100 nm or less) in the preparation of TEM images has
restricted the floc sample volume, which has a diameter as large as 1 mm, that can
be examined at high resolution due to the consideration of cost and time. COM and
CLSM images are useful in indicating the number of TEM sections required to be
collected for determining the representative images of flocs.
61
Nanoplast resin is particularly effective as a stabilization medium since it is a
hydrophilic melamine embedding resin that holds the fibrillar EPS in native three-
dimensional disposition. Nanoplast omits the solvent dehydration stage, and it forms

cross-linkages between matrix colloids prior to structural water loss at the end of the
embedding process. Measurements of the dimensions of colloidal matrix material
Copyright 2005 by CRC Press