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 Fundamentals of
BIOFILM RESEARCH
Second Edition

Zbigniew Lewandowski
Haluk Beyenal
 Fundamentals of
BIOFILM RESEARCH
Second Edition
 Fundamentals of
BIOFILM RESEARCH
Second Edition

Zbigniew Lewandowski
Haluk Beyenal
MATLAB® and Simulink® are trademarks of The MathWorks, Inc. and are used with permission. The MathWorks does
not warrant the accuracy of the text or exercises in this book. This book’s use or discussion of MATLAB® and Simulink®
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gogical approach or particular use of the MATLAB® and Simulink® software.

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Contents
Preface........................................................................................................................................... xvii
Authors.......................................................................................................................................... xxi

Chapter 1 Introduction to biofilms and to biofilm research............................................... 1

1.1 Introduction............................................................................................................................ 1
1.2 Terminology...........................................................................................................................3
1.2.1 Biofilms, biofouling, and biofouling deposits..................................................... 3
1.2.2 Biofilm systems, biofilm processes, and biofilm reactors..................................4
1.2.3 Biofilm activity......................................................................................................... 6
1.2.4 Nutrient utilization rate and specific nutrient utilization rate......................... 6
1.2.4.1 Biofilm activity from macroscale measurements: Overall
biofilm activity........................................................................................7
1.2.4.2 Biofilm activity from microscale measurements: Local
biofilm activity........................................................................................8
1.2.5 Biofilm development and the concept of biofilm structure............................... 9
1.3 Characterizing selected parts of biofilm systems........................................................... 14
1.3.1 Characterizing surfaces........................................................................................ 14
1.3.1.1 Surface properties and microbial colonization of surfaces............ 14
1.3.2 Characterizing hydrodynamics and mass transfer in biofilms...................... 18
1.3.3 Characterizing the distribution of nutrients in biofilms................................. 21
1.3.4 Characterizing distribution of effective diffusivity and biofilm density...... 23
1.3.5 Characterizing biofilm structure and its effects............................................... 27
1.3.5.1 Biofilm heterogeneities: Structural, chemical, physiological,
and other................................................................................................ 27
1.3.6 Characterizing the flow of nutrients in biofilms............................................... 28
1.3.7 Characterizing structure and function of EPS.................................................. 30
1.3.7.1 Chemical properties of EPS................................................................. 31
1.3.7.2 Chemical analysis of EPS.....................................................................34
1.3.7.3 Proteomic analyses of EPS................................................................... 36
1.3.7.4 Immunoblot analyses of the EPS proteins........................................ 36
1.3.7.5 Viscoelastic properties of EPS............................................................. 40
1.4 Characterizing microbial growth and biofilm formation............................................. 41
1.4.1 Microbial growth in suspension and in biofilms.............................................. 41
1.4.2 Kinetics of microbial growth...............................................................................42
1.4.2.1 Multiple-nutrient-limited growth.......................................................44
1.4.2.2 Nutrient utilization rate.......................................................................44

v
vi Contents

1.5 Biofilm-based technologies................................................................................................ 46

1.5.1 Biofilm-based technologies in wastewater treatment...................................... 47
1.5.2 Biofilm-based technologies for energy conversion: Microbial fuel cells....... 49
1.6 Strategy of biofilm research............................................................................................... 56
1.6.1 The goal of the study............................................................................................. 57
1.6.2 Hypotheses............................................................................................................. 58
1.6.3 The tasks................................................................................................................. 58
1.6.4 Selecting relevant experimental conditions...................................................... 58
1.6.4.1 Selecting microorganisms and growth media................................. 58
1.6.4.2 Selecting biofilm reactors.................................................................... 59
1.6.4.3 Selecting operational parameters....................................................... 59
1.7 Research program............................................................................................................... 60
1.8 Interpreting results of biofilm studies.............................................................................. 60
Nomenclature................................................................................................................................. 61
Greek symbols...................................................................................................................... 62
Abbreviations....................................................................................................................... 62
References........................................................................................................................................ 62
Suggested readings........................................................................................................................63

Chapter 2 Imaging and characterizing biofilm components............................................ 67

2.1 Microscopy........................................................................................................................... 67
2.1.1 Optical microscopy............................................................................................... 67
2.1.1.1 Resolution of optical microscopes...................................................... 68
2.1.1.2 Examples of optical microscopy applications................................... 70
2.1.2 Fluorescence microscopy...................................................................................... 73
2.1.2.1 Examples of applications..................................................................... 74
2.1.3 Enhanced fluorescence microscopy.................................................................... 81
2.1.3.1 Stimulated emission depletion microscopy...................................... 81
2.1.3.2 Fluorescent resonance energy transfer microscopy........................ 82
2.1.3.3 Two-photon microscopy...................................................................... 82
2.1.3.4 Confocal scanning laser microscopy.................................................83
2.1.4 Electron microscopy.............................................................................................. 88
2.1.4.1 Transmission electron microscopy..................................................... 88
2.1.4.2 Scanning electron microscopy............................................................ 92
2.1.4.3 Environmental scanning electron microscopy................................ 95
2.1.5 Atomic force microscopy...................................................................................... 97
2.1.5.1 Examples of AFM applications........................................................... 97
2.1.6 Microscopy for surface analysis.......................................................................... 99
2.1.6.1 Energy dispersive x-ray spectroscopy............................................. 100
2.1.6.2 X-ray photoelectron spectroscopy.................................................... 100
2.1.6.3 ToF-SIMS.............................................................................................. 103
2.1.7 Nuclear magnetic resonance imaging.............................................................. 105
2.1.7.1 Examples of applications................................................................... 107
References..................................................................................................................................... 111
Suggested readings...................................................................................................................... 111

Chapter 3 Laboratory biofilm reactors and their applications.......................................121

3.1 Introduction........................................................................................................................ 121
3.2 Variables in biofilm reactors............................................................................................. 122
Contents vii

3.2.1 Types of variables................................................................................................ 122

3.2.2 Relating the measured variables to the controlled variables........................ 125
3.2.3 Hydrodynamics in LBRs.................................................................................... 127
3.2.3.1 Flow velocity........................................................................................ 127
3.2.3.2 Momentum.......................................................................................... 127
3.2.3.3 Reynolds number................................................................................ 128
3.2.4 Dimensionless groups quantified in biofilm reactors.................................... 131
3.2.5 Hydrodynamic entry length.............................................................................. 134
3.3 LBRs as scale models......................................................................................................... 136
3.4 Factors affecting biofilm processes in LBRs.................................................................. 137
3.4.1 Stress and strain in the biofilm matrix............................................................. 137
3.4.1.1 Compression and stretching............................................................. 137
3.4.1.2 Shear deformation.............................................................................. 138
3.4.1.3 Elastic and viscoelastic deformations of biofilms.......................... 138
3.4.1.4 Shear stress.......................................................................................... 140
3.4.1.5 Pressure drop...................................................................................... 141
3.4.1.6 Effect of biofilm on the flow in biofilm reactors............................. 145
3.4.2 External mass transport in biofilms................................................................. 146
3.5 Operating modes of LBRs................................................................................................. 149
3.5.1 LBRs without recycle: Once-through mode of operation.............................. 151
3.5.2 LBRs with recycle................................................................................................ 151
3.5.3 LBRs with recycle and a mixing chamber....................................................... 151
3.6 LBRs and their typical applications................................................................................ 151
3.6.1 Flat plate reactors................................................................................................. 151
3.6.1.1 Open-channel flat plate reactors....................................................... 152
3.6.1.2 Flat plate vapor phase reactor........................................................... 161
3.6.1.3 Simplified open-channel flat plate reactors.................................... 162
3.6.2 Rotating reactors.................................................................................................. 167
3.6.2.1 Rotating disc reactor........................................................................... 167
3.6.2.2 Annular reactors................................................................................. 171
3.6.3 Tubular reactors................................................................................................... 173
3.6.3.1 Example of results from a biofilm study using a tubular reactor
to study the effect of biofilm formation on pressure drop............... 173
3.6.3.2 Tubular reactors for quantifying biofilm structure....................... 175
3.6.4 Packed-bed reactors............................................................................................. 175
3.6.5 Hollow fiber biofilm reactors............................................................................. 178
3.7 Using LBRs in conjunction with other tools to explore biofilm processes
at the microscale................................................................................................................ 179
3.7.1 Quantifying mechanisms by which biofilm heterogeneity
affects biofilm activity......................................................................................... 180
3.7.2 Quantifying mechanisms of mass transport in biofilms.............................. 187
3.8 Functionality of LBRs........................................................................................................ 193
Nomenclature............................................................................................................................... 195
Roman symbols.................................................................................................................. 195
Greek symbols.................................................................................................................... 196
Abbreviations..................................................................................................................... 196
References..................................................................................................................................... 196
Suggested readings...................................................................................................................... 197
viii Contents

Chapter 4 Sensors useful in biofilm research...................................................................199

4.1 Fundamental concepts in electrochemistry and principles of electrochemical
sensors................................................................................................................................. 199
4.1.1 Electric charges.................................................................................................... 200
4.1.1.1 Electric potential................................................................................. 200
4.1.1.2 Electrode potential and the Nernst equation................................. 203
4.1.1.3 Reference electrodes........................................................................... 209
4.1.1.4 Electric current.................................................................................... 210
4.1.1.5 Measuring potential and current..................................................... 210
4.1.2 Electrochemical cells........................................................................................... 211
4.1.2.1 Electrodes and the mechanisms of electric charge transfer......... 214
4.1.3 Significance of the electrode potential............................................................. 227
4.1.3.1 Galvanic and electrolytic cells.......................................................... 230
4.1.3.2 Steady state diffusion near the electrode surface........................... 235
4.1.3.3 Membranes of amperometric sensors.............................................. 238
4.1.3.4 Functions of membranes on potentiometric and
amperometric sensors........................................................................ 239
4.1.4 Amperometric sensors........................................................................................ 239
4.1.5 Potentiometric sensor.......................................................................................... 241
4.1.6 Voltammetric sensors.......................................................................................... 241
4.2 Microsensors...................................................................................................................... 242
4.2.1 Potentiometric microelectrodes......................................................................... 247
4.2.1.1 Redox microelectrodes....................................................................... 247
4.2.1.2 Ion-selective microelectrodes............................................................ 249
4.2.1.3 Sulfide microelectrodes..................................................................... 249
4.2.1.4 pH microelectrodes............................................................................ 250
4.2.1.5 Carbon dioxide microelectrodes...................................................... 252
4.2.2 Amperometric microelectrodes......................................................................... 253
4.2.2.1 Dissolved oxygen microelectrodes.................................................. 253
4.2.2.2 Sulfide microelectrodes.....................................................................254
4.2.2.3 Microelectrodes for measuring the concentrations of oxidants....... 255
4.2.2.4 Hydrogen peroxide microelectrodes............................................... 256
4.2.2.5 Hydrogen microelectrode.................................................................. 257
4.2.3 Voltammetric microelectrodes.......................................................................... 258
4.2.3.1 Flavin microelectrode........................................................................ 258
4.2.4 Microbiosensors................................................................................................... 258
4.2.4.1 Glucose microsensors......................................................................... 258
4.2.5 Microelectrodes for quantifying mass transport rates in biofilms.............. 259
4.2.6 Fiber optic microsensors..................................................................................... 262
Nomenclature............................................................................................................................... 265
Subscripts............................................................................................................................ 265
Roman symbols.................................................................................................................. 265
Greek symbols.................................................................................................................... 266
References..................................................................................................................................... 266
Suggested readings...................................................................................................................... 267

Chapter 5 Microsensors: Construction, instrumentation, and calibration.................269

5.1 Introduction........................................................................................................................ 269
5.2 Constructing microelectrodes......................................................................................... 271
Contents ix

5.2.1 Materials............................................................................................................... 271

5.2.1.1 Platinum wire...................................................................................... 271
5.2.1.2 Silver wire............................................................................................ 272
5.2.1.3 Iridium wire........................................................................................ 272
5.2.1.4 Wires for making heating elements................................................. 272
5.2.1.5 Graphite rods....................................................................................... 273
5.2.1.6 Glasses.................................................................................................. 274
5.2.1.7 Liquid ion exchangers........................................................................ 275
5.2.1.8 List of materials we use to construct microelectrodes.................. 276
5.2.2 Tools and instruments for constructing microelectrodes............................. 276
5.2.2.1 Micropipette pullers........................................................................... 276
5.2.2.2 Antivibration tables............................................................................ 278
5.2.2.3 Stereomicroscopes and microscope holders................................... 279
5.2.2.4 Light microscopes............................................................................... 279
5.2.2.5 Grinding wheels................................................................................. 280
5.2.2.6 Micromanipulators............................................................................. 282
5.2.2.7 Microelectrode holders...................................................................... 283
5.2.2.8 Illuminators......................................................................................... 283
5.2.2.9 Power supplies....................................................................................284
5.2.2.10 Storing microelectrodes and parts of microelectrodes.................284
5.2.2.11 Auxiliary tools..................................................................................... 286
5.2.2.12 Vacuum pumps................................................................................... 286
5.2.2.13 Propane torches................................................................................... 286
5.2.2.14 Instruments used to operate the microelectrodes......................... 286
5.2.3 Constructing selected microelectrodes............................................................ 290
5.2.3.1 Constructing hydrogen peroxide microelectrodes........................ 290
5.2.3.2 Constructing dissolved oxygen microelectrodes........................... 298
5.2.3.3 Constructing pH microelectrodes with liquid ion
exchanger membranes....................................................................... 315
5.2.3.4 Constructing redox potential microsensors................................... 319
5.2.3.5 Constructing flavin microelectrodes............................................... 324
5.3 Constructing fiber optic microsensors........................................................................... 330
5.3.1 Tools and procedures for constructing fiber optic microsensors................. 331
5.3.1.1 Construction of the optical microsensor tip................................... 331
5.3.1.2 Constructing fiber optic holders....................................................... 333
5.3.1.3 Cleaving and etching the tip.............................................................334
5.3.1.4 Fiber optic microsensor for measuring backscattered light
intensity in biofilms............................................................................334
5.3.1.5 Fiber optic cables................................................................................. 335
5.3.1.6 Fiber couplers...................................................................................... 336
5.3.1.7 Laser diodes......................................................................................... 337
5.3.1.8 Laser diode drivers............................................................................. 338
5.3.1.9 Photodiodes......................................................................................... 338
5.3.1.10 Detecting signals/amplifiers............................................................. 338
5.3.1.11 Computer interface............................................................................. 339
5.3.1.12 Fiber optic microsensors for measuring fluorescent light
intensity in biofilms............................................................................ 339
5.3.1.13 Constructing the tips..........................................................................340
5.3.1.14 Components of the measurement system.......................................340
x Contents

5.4 Controlling microelectrode movements and data acquisition...................................340

5.4.1 Measuring instruments......................................................................................340
5.4.2 Data acquisition and microsensor movement control................................... 341
5.4.2.1 Data acquisition................................................................................... 341
5.4.3 Microelectrode movement control....................................................................344
5.4.3.1 Linear actuator....................................................................................344
5.4.3.2 Stepper motor controller....................................................................344
5.4.3.3 Limit sensing.......................................................................................346
5.4.3.4 MATLAB program for controlling the stepper motor...................346
5.4.3.5 Simultaneous movement of microsensor and data acquisition........ 347
References..................................................................................................................................... 347
Suggested readings...................................................................................................................... 347

Chapter 6 Quantifying biofilm structure...........................................................................353

6.1 The need to quantify biofilm structure.......................................................................... 353
6.2 Types of images.................................................................................................................. 355
6.2.1 Gray-scale images................................................................................................ 356
6.2.2 Binary images....................................................................................................... 356
6.2.3 Stacks of CSLM images....................................................................................... 356
6.2.4 Converting CSLM image stacks to single images........................................... 358
6.2.5 Converting digital images to data matrices..................................................... 358
6.2.5.1 Images in MATLAB............................................................................ 358
6.2.5.2 Converting RGB images to gray-scale images................................ 359
6.2.5.3 Converting gray-scale images to data matrices.............................. 359
6.3 Computing parameters from biofilm images................................................................ 360
6.3.1 Textural parameters computed from single images....................................... 360
6.3.1.1 Calculating a normalized spatial dependence matrix.................. 360
6.3.1.2 Calculating textural entropy, energy, and homogeneity.............. 362
6.3.1.3 Calculating textural parameters from a biofilm using
MATLAB.............................................................................................. 362
6.3.1.4 Meanings of textural parameters..................................................... 363
6.3.1.5 Examples of textural parameters computed from biofilm
images...................................................................................................364
6.3.2 Areal parameters computed from single images............................................ 365
6.3.2.1 Thresholding biofilm images............................................................ 365
6.3.2.2 The iterative selection method for thresholding biofilm
images................................................................................................... 366
6.3.2.3 Calculating threshold value.............................................................. 367
6.3.2.4 Thresholding using MATLAB for a given matrix.......................... 370
6.3.2.5 Thresholding a biofilm image using MATLAB.............................. 370
6.3.2.6 Thresholding a biofilm image and saving it as a thresholded
image using MATLAB....................................................................... 370
6.3.2.7 Calculating areal parameters............................................................ 371
6.3.2.8 Areal porosity...................................................................................... 371
6.3.2.9 Average run length............................................................................. 372
6.3.2.10 Average and maximum diffusion distances................................... 374
6.3.2.11 Perimeter.............................................................................................. 375
6.3.2.12 Fractal dimension............................................................................... 376
6.3.2.13 Notes about MATLAB calculations.................................................. 379
Contents xi

6.3.3 The meanings of the areal parameters of single images............................... 380

6.3.3.1 Areal parameters computed from biofilm images......................... 381
6.4 Quantifying biomass distribution in 3-D from stacks of biofilm images................. 382
6.4.1 Layers of CSLM images...................................................................................... 382
6.4.1.1 Generating 3-D matrices from stacks of 2-D matrices................... 383
6.4.1.2 Example of generating 3-D matrices from stacks of 2-D
matrices................................................................................................384
6.4.1.3 Interpolation into stacks of biofilm images.....................................384
6.4.1.4 Interpolation of a sample data set using MATLAB....................... 385
6.4.1.5 Interpolation of biofilm images using MATLAB........................... 385
6.5 Computing 3-D parameters from stacks of biofilm images........................................ 386
6.5.1 Textural parameters............................................................................................ 386
6.5.1.1 Calculating a gray-level co-occurrence matrix............................... 386
6.5.1.2 Calculating GLCM from a sample data set using MATLAB........ 388
6.5.1.3 Calculating textural parameters....................................................... 388
6.5.1.4 Calculating textural parameters from a sample data set
using MATLAB................................................................................... 389
6.5.1.5 Calculating textural parameters from a biofilm image using
MATLAB.............................................................................................. 389
6.5.1.6 The meanings of the textural parameters computed from
stacks of biofilm images..................................................................... 390
6.5.2 Volumetric parameters....................................................................................... 391
6.5.2.1 Thresholding layers of biofilm images and generating a
binary 3-D matrix using MATLAB.................................................. 392
6.5.2.2 Average run lengths........................................................................... 392
6.5.2.3 Aspect ratio and its calculation........................................................ 394
6.5.2.4 Average and maximum diffusion distances................................... 395
6.5.2.5 Calculating diffusion distances........................................................ 395
6.5.2.6 Fractal dimension............................................................................... 397
6.5.2.7 Porosity................................................................................................. 402
6.5.2.8 Biovolume............................................................................................ 403
6.5.2.9 Average and maximum biomass thicknesses and biomass
roughness............................................................................................. 405
6.5.2.10 Average and maximum biofilm thicknesses and biofilm
roughness............................................................................................. 409
6.5.2.11 Biomass thickness versus biofilm thickness................................... 411
6.5.2.12 Surface area of the biomass in the biofilm...................................... 412
6.5.2.13 Ratio of biovolume to biomass surface area.................................... 413
6.5.2.14 Meanings of the volumetric parameters......................................... 414
6.5.2.15 Textural and volumetric parameters computed from biofilm
images................................................................................................... 415
6.6 Interfering effects.............................................................................................................. 416
6.6.1 Effects of image orientation and image inverting.......................................... 416
6.6.2 Effects of image filtering..................................................................................... 416
6.6.3 Effects of image filtering on areal parameters................................................ 419
6.6.4 Effects of image filtering on the textural parameters..................................... 419
6.6.5 Effects of image focus......................................................................................... 420
6.6.6 Effects of magnification...................................................................................... 420
6.6.7 Effect of illumination intensity on areal porosity...........................................422
xii Contents

6.7 Testing available software packages quantifying biofilm structure..........................422

6.7.1 Generating images of cubes using MATLAB.................................................. 424
6.7.2 Generating an image of a sphere using MATLAB.......................................... 426
6.7.3 Computing volumetric parameters for known shapes using ISA-2............. 427
6.8 Directions of future research on quantifying biofilm structure................................ 428
Nomenclature............................................................................................................................... 433
References...................................................................................................................................... 435
Suggested readings...................................................................................................................... 436

Chapter 7 Biofilms on metals and other electrically conductive surfaces...................439

7.1 Introduction........................................................................................................................ 439
7.2 Basic electrochemical concepts relevant to understanding the effects of
biofilms on metals.............................................................................................................443
7.2.1 The rate of electrode reactions: The Butler–Volmer equation.......................443
7.2.2 Butler–Volmer equation when the electrode is not polarized....................... 453
7.2.3 Mixed potential....................................................................................................454
7.3 Abiotic corrosion of active metals: Iron.......................................................................... 456
7.3.1 Thermodynamics of iron corrosion.................................................................. 457
7.3.2 Quantifying corrosion potential and corrosion current................................ 459
7.3.2.1 Potentiodynamic polarization techniques for studying
anodic and cathodic reactions.......................................................... 459
7.3.2.2 Visualizing factors affecting the kinetics of corrosion
processes: Evans diagrams................................................................ 460
7.3.2.3 Corrosion rate: Linear polarization resistance............................... 463
7.4 Abiotic corrosion of passive metals................................................................................463
7.4.1 Passive metals and alloys...................................................................................463
7.4.2 Potentiodynamic polarization of passive metals and alloys......................... 466
7.5 Quantifying electron transfer processes in electrochemically active biofilms........ 468
7.5.1 Selecting the material of the electrode on which the biofilms are grown..... 468
7.5.2 Selecting the type of the reactor........................................................................ 470
7.5.3 Selecting the procedure for conditioning the biofilms.................................. 472
7.6 Electrochemical techniques for studying electrochemically active biofilms........... 474
7.6.1 Long-term electrode polarization of electrochemically active biofilms...... 474
7.6.2 Voltammetric techniques.................................................................................... 475
7.6.3 Anodic stripping voltammetry.......................................................................... 476
7.6.4 Cyclic voltammetry............................................................................................. 477
7.6.5 Square-wave voltammetry................................................................................. 479
7.6.6 Use of coupled research techniques to explain the processes in
electrically active biofilms..................................................................................480
7.6.7 Using microelectrodes in electrochemically active biofilms on
electrically polarized electrodes........................................................................ 481
7.6.8 Microscopy...........................................................................................................484
7.6.9 Modeling electrochemically active biofilms.................................................... 485
7.7 Examples of technologically relevant electrochemically active biofilms.................. 486
7.7.1 Microbially influenced corrosion of metals..................................................... 486
7.7.1.1 Mechanisms by which metabolic reactions in biofilms can
affect the processes of corrosion....................................................... 488
7.7.1.2 Further implications........................................................................... 498
7.7.2 Microbial fuel cells..............................................................................................500
Contents xiii

Nomenclature............................................................................................................................... 505
Subscripts............................................................................................................................ 505
Roman Symbols................................................................................................................. 505
Greek symbols.................................................................................................................... 507
References..................................................................................................................................... 507
Suggested readings......................................................................................................................508

Chapter 8 Interpreting results of biofilm studies using the model of stratified

biofilms...................................................................................................................511
8.1 Introduction........................................................................................................................ 511
8.2 Conceptual model of stratified biofilms......................................................................... 514
8.3 Interpreting results of biofilm studies using the conceptual model of stratified
biofilms................................................................................................................................ 516
8.3.1 Measurements at the microscale....................................................................... 516
8.3.2 Interpreting the data using the concept of stratified biofilms...................... 520
8.4 Using the model of stratified biofilms to refine conceptual models of biofilm
structure and activity........................................................................................................ 524
8.4.1 Modeling biofilm processes in stratified biofilms.......................................... 524
8.4.1.1 Nutrient continuity equations in stratified biofilms...................... 524
8.4.1.2 Comparing biofilms using the model of stratified biofilms......... 528
8.4.1.3 Dimensionless equations for stratified biofilms............................. 529
8.4.1.4 Dimensionless equation for biofilms with uniformly
distributed density............................................................................. 531
8.4.1.5 Boundary conditions.......................................................................... 532
8.4.1.6 Effectiveness factors........................................................................... 532
8.4.1.7 Activity of stratified biofilms............................................................ 533
8.5 Computing biokinetics parameters in stratified biofilms............................................ 537
8.5.1 Solution algorithm............................................................................................... 537
8.5.2 Using the concept of stratified biofilms to explain the properties of
uniform biofilms..................................................................................................540
8.6 Mathematical modeling of biofilm processes and experimental verification of
model predictions..............................................................................................................543
Nomenclature...............................................................................................................................545
Roman symbols..................................................................................................................545
Greek symbols....................................................................................................................546
References.....................................................................................................................................546
Suggested readings......................................................................................................................546

Chapter 9 Protocols and procedures....................................................................................551

9.1 Introduction........................................................................................................................ 551
9.2 Preparing the experimental system................................................................................ 551
9.2.1 Selecting the microorganisms........................................................................... 551
9.2.2 Obtaining the microorganisms......................................................................... 552
9.2.3 Preparing and maintaining stock cultures...................................................... 552
9.2.3.1 Preparing a stock culture from a freeze-dried sample from
ATCC.................................................................................................... 553
9.2.3.2 Preparing the growth medium for the selected
microorganisms.................................................................................. 554
9.2.3.3 Inoculating biofilm reactors.............................................................. 554
xiv Contents

9.3 Quantifying microbial growth........................................................................................ 555

9.3.1 Kinetics of microbial growth in suspension.................................................... 555
9.3.1.1 Kinetics of P. aeruginosa (ATCC 700829) growth in suspension........ 555
9.3.1.2 Selecting the agitation rate at which to operate the chemostat......... 560
9.3.1.3 Steady state data for modeling microbial kinetics......................... 561
9.4 Preparing and operating biofilm reactors...................................................................... 566
9.4.1 Operating flat plate biofilm reactors................................................................. 566
9.4.2 Components of the reactor system.................................................................... 566
9.4.3 Common procedures used in operating biofilm reactors.............................. 567
9.4.3.1 Measuring flow rates.......................................................................... 567
9.4.4 Calibrating pumps............................................................................................... 567
9.4.4.1 Calibrating pumps operated at high flow rates.............................. 567
9.4.5 Using flow breakers to measure low flow rates.............................................. 568
9.4.6 Measuring the working volume of a reactor................................................... 570
9.4.7 Sterilizing reactors and other components...................................................... 570
9.4.8 Preparing the surfaces for biofilm growth...................................................... 570
9.4.9 Inoculating the reactors...................................................................................... 571
9.4.10 Sterilizing connectors and tubing..................................................................... 573
9.4.11 Attaching the lid to the reactor.......................................................................... 573
9.4.12 Sterilizing a flat plate flow reactor.................................................................... 574
9.4.13 Removing bleach or alcohol from a flat plate reactor..................................... 574
9.4.14 Replacing water with growth medium in a flat plate reactor....................... 575
9.4.15 Inoculating a flat plate reactor........................................................................... 575
9.4.16 Operating a flat plate reactor............................................................................. 576
9.4.17 Removing suspended microorganisms............................................................ 576
9.4.18 Operating a simplified open-channel flat plate reactor................................. 576
9.4.19 Preparing the inoculum for a simplified open-channel flat plate reactor........ 577
9.4.20 Sterilizing a simplified open-channel flat plate reactor with its
connectors and tubing........................................................................................ 577
9.4.21 Inoculating a simplified open-channel flat plate reactor............................... 577
9.4.22 Operating a simplified open-channel flat plate reactor................................. 578
9.4.23 Operating a modified open-channel flat plate reactor for growing
Geobacter sulfurreducens biofilms and quantifying electron transfer rates...........578
9.4.23.1 Preparing the inoculum..................................................................... 580
9.4.23.2 Assembling the open-channel flat plate reactor............................. 581
9.4.23.3 Sterilizing the reactor, connectors, and tubing.............................. 583
9.3.23.4 Inoculating the reactor....................................................................... 583
9.4.23.5 Operating the reactor......................................................................... 584
9.4.23.6 An example cyclic voltammogram of a G. sulfurreducens
biofilm................................................................................................... 584
9.4.23.7 Using the chronoamperometry script to polarize the
working electrode continuously....................................................... 584
9.4.24 Operating tubular reactors................................................................................. 585
9.4.25 Preparing the inoculum for a tubular reactor................................................. 588
9.4.26 Sterilizing a tubular reactor with its connectors and tubing and
connecting the pressure transducer................................................................. 588
9.4.27 Replacing water with growth medium in a tubular reactor......................... 589
9.4.28 Inoculating a tubular reactor............................................................................. 589
9.4.29 Operating a tubular reactor............................................................................... 589
Contents xv

9.4.30Measuring pressure and pressure drop in a tubular reactor........................ 589

9.4.31Packed-bed reactors............................................................................................. 591
9.4.31.1 Preparing the inoculum for a packed bed reactor......................... 591
9.4.31.2 Preparing a PBR.................................................................................. 591
9.4.31.3 Preparing the packing material for a PBR....................................... 592
9.4.31.4 Calculating the operational parameters of a PBR.......................... 593
9.4.31.5 Growing biofilm in a PBR.................................................................. 594
9.4.32 Hollow fiber biofilm reactors............................................................................. 594
9.4.32.1 Preparing a hollow fiber biofilm reactor......................................... 594
9.4.32.2 EPS extraction...................................................................................... 596
9.5 Common procedures......................................................................................................... 597
9.5.1 Determining viable cell concentration, biomass concentration, and
biofilm activity..................................................................................................... 597
9.5.1.1 Viable cell count.................................................................................. 597
9.5.1.2 Counting bacterial cells in suspension............................................ 597
9.5.1.3 Counting bacterial cells in biofilms................................................. 597
9.5.1.4 Determining biomass concentration................................................600
9.5.1.5 Specific number of viable cells..........................................................600
9.5.2 Testing the efficacy of antimicrobials/biocides against biofilms.................600
9.5.2.1 Treating biofilm samples with antimicrobial agents..................... 601
9.5.2.2 Evaluating the efficacy of antimicrobial agents............................. 601
9.5.3 Using microelectrodes to characterize local mass transport in biofilms.......... 601
9.5.3.1 Inspecting and testing microelectrodes.......................................... 601
9.5.3.2 Replacing the growth medium in a biofilm reactor with an
electrolyte solution.............................................................................. 603
9.5.3.3 Positioning the microelectrode in the reactor................................ 603
9.5.3.4 Selecting the location for the measurement and moving the
microelectrode to this location......................................................... 605
9.5.3.5 Measuring the profile of local limiting current at the
selected location.................................................................................. 605
9.5.3.6 Plotting profiles of local mass transport coefficient and local
effective diffusivity from limiting current profiles....................... 606
9.5.3.7 Presenting the results in a form accepted by the model of
stratified biofilms................................................................................ 608
9.5.4 Live/dead cell imaging in a biofilm.................................................................. 609
9.5.5 Preparing biofilms for scanning electron microscopy................................... 611
9.5.5.1 Fixation protocol................................................................................. 611
9.5.5.2 Dehydration protocol......................................................................... 612
9.6 Disclaimer........................................................................................................................... 612
Nomenclature............................................................................................................................... 612
Roman symbols.................................................................................................................. 612
Greek symbols.................................................................................................................... 613
References..................................................................................................................................... 614
Suggested readings...................................................................................................................... 614
Preface
The seven years that have passed since the publication of the first edition have brought
significant advances in both biofilm research and biofilm engineering, some of which we
have included in the text. As a result, many chapters have been updated and expanded
with the addition of new sections reflecting changes in the status quo in biofilm research
and engineering. We have also emphasized throughout the text that biofilm research and
engineering have matured to the extent that biofilm-based technologies are now being
designed and implemented. Two examples of such technologies are described in the first
chapter: the use of biofilms in reactors for wastewater treatment and the use of biofilms in
microbial fuel cells. The biofilm-based technologies used in wastewater treatment exem-
plify mature technologies: they are in use in large-scale reactors, even though the design
of these reactors is still mostly empirical. Microbial fuel cells, exemplify emerging biofilm-
based technologies that are still at the level of bench- and pilot-scale applications. The
trend is obvious: In the future we will see more and more biofilm-based technologies, and
biofilm research will need to include large-scale reactors and large-scale biofilm-based
processes.
Not all the changes we intended to introduce in the second edition could be accom-
modated by expanding the existing chapters, though, and two chapters have been added:
Chapter 2 (Imaging and Characterizing Biofilm Components) and Chapter 7 (Biofilms
on Metals and Other Electrically Conductive Surfaces). Chapter 2 discusses techniques
that are used to enhance the imaging of components of biofilm systems and includes not
only the imaging of biofilm microorganisms using various types of optical and electron
microscopy but also the imaging of the topography and chemistry of surfaces and of the
hydrodynamics and mass transport in biofilms. Chapter 7 discusses new frontiers in bio-
film research, energy conversion in biofilms, and the biofilm-based conversion of chemical
energy to electrical energy. The effects of depositing biofilms on metals had been studied
by biofilm researchers for a long time, mostly to quantify the mechanisms of microbi-
ally influenced corrosion (MIC). More recently, however, new branches of biofilm research
have emerged, dedicated to the mechanisms of electron transfer between biofilms and
metals, energy conversion in biofilms, and the generation of electricity in microbial fuel
cells (MFC). The biofilms active in MIC and MFC are now seen to be electrochemically
active biofilms (EABs). The processes driven by EABs have a common denominator, the
mechanisms of electron transfer between microorganisms and solids. The studies initially
concerned with electrically conductive surfaces converged with the existing research on
microorganisms growing on minerals. These minerals are not necessarily electrically con-
ductive, but they do serve as electron donors or acceptors in microbial metabolic reac-
tions, giving rise to such terms as “mineral-respiring microorganisms.” Not surprisingly,
these processes are driven by microorganisms attached to the surfaces of minerals, or
biofilms. The mechanisms of electron transfer between minerals and microorganisms are

xvii
xviii Preface

still under study, but the parallels to the mechanisms of electron transfer between micro-
organisms and metals or other electrically conductive surfaces are obvious. The results
of such studies apply to such diverse fields as corrosion protection, new types of power
supplies and advanced techniques of bioremediation. When we were preparing the first
edition of this text, it was the structure and complexity of microbial structures on surfaces
(i.e., the biofilms) that stimulated the imagination and were imaginatively compared to cit-
ies built by microorganisms. Now, the applications of biofilm-based technologies stimulate
the imagination. The edition of Science Daily (http://www.sciencedaily.com) published on
December 15, 2009, extrapolated from a paper presented by a group of scientists from MIT
in the Proceedings of the US National Academy of Sciences and summarized their find-
ings in an article entitled “Rock-Breathing Bacteria Could Generate Electricity and Clean
Up Oil Spills.” We can only hope that by the time we prepare the next edition of this book,
such applications will have become reality.
The main goals for this text remain the same as they were in the first edition.
Specifically, the goals are to serve as

• A compendium of knowledge about biofilms and biofilm processes.

• A set of instructions for designing and conducting biofilm experiments.
• A set of instructions for making and using various tools useful in biofilm research.
• A set of computational procedures useful in interpreting results of biofilm research.
• A set of instructions for using the model of stratified biofilms for data interpretation,
analysis, and biofilm activity prediction.

The book describes biofilm processes and biofilm-based technologies at a level adequate to
help someone with a general knowledge of bioengineering but uninitiated in biofilm research
to learn the tools and techniques used to study biofilm processes. Thus, interested research-
ers can start experimental explorations of biofilm processes armed with a basic knowledge of
these processes and the tools useful for studying them. Once they start their own explorations,
a more thorough literature search will be needed. We provide sources of relevant texts that we
believe are interesting and constitute a good introduction to the subject.
As in the first edition, at the end of the book we attach a CD with software packages
useful for facilitating procedures described in the text. Specifically, the CD contains the
following:

• Computer programs for extracting numerical parameters from images of biofilms.

• Computer programs for computing the growth-limiting nutrient concentration pro-
files in stratified biofilms.
• Computer programs for calculating biokinetic parameters from growth-limiting
nutrient concentration profiles measured using microelectrodes.
• Computer programs for controlling micropositioners and a data acquisition system
for measuring chemical concentrations in biofilms using microelectrodes.

We refer to the use of MATLAB® throughout the book, both for calculations and to
control instruments. The source codes of the MATLAB programs are given on the attached
CD, and the use of the program is described in the corresponding chapters. Before using
the CD, please copy its entire contents to your hard drive. If you use a different directory
structure or folder names different from those used on the CD, you must revise the path
definitions in the corresponding MATLAB programs; otherwise, you will receive error
messages.
Preface xix

Many people contributed to the second edition of the book, most notably the genera-
tions of students and visitors who have worked over the years in the Biofilm Engineering
Research group at Washington State University in Pullman and in the Center for Biofilm
Engineering, Montana State University in Bozeman. Some of their names appear in the
subtitles of figures, some appear in a list of references following a chapter, and some appear
in the first edition of the book. Jerome Babauta provided expert comments on Chapters 4
and 7. His contribution to the subject of electrochemically active biofilms helped us to
address future areas of research on EABs. We both acknowledge support from the Office of
Naval Research, Department of Energy, National Science Foundation, and Department of
Defense, which sponsored projects that allowed us to develop the methodology and results
presented in this book. The expert comments and proofreading of the text by Lois Avci
have improved it considerably. Finally, the generations of our students and visitors who
have worked tirelessly over the years in the Biofilm Structure and Function Laboratory
at the Center for Biofilm Engineering and in the Biofilm Engineering Research group at
Washington State University are gratefully acknowledged.

MATLAB® and Simulink® are registered trademarks of The MathWorks, Inc. For product
information, please contact:

The MathWorks, Inc.

3 Apple Hill Drive
Natick, MA, 01760-2098 USA
Tel: 508-647-7000
Fax: 508-647-7001
E-mail: [email protected]
Web: www.mathworks.com
Authors
Zbigniew Lewandowski has been active in biofilm research for 30 years. He is a professor
in the Department of Civil Engineering at Montana State University in Bozeman and con-
ducts research at the Center for Biofilm Engineering there. Much of his research has been
devoted to studying microscale biofilm processes and their relations to the performance of
macroscale biofilm reactors, relations between microbial and physicochemical processes,
mass transport in biofilm reactors, and energy conversion in biofilms. Most recently, he
has been studying electrochemically active biofilms in such processes as microbially
influenced corrosion and microbial fuel cells. He has published more than 100 papers
and 12 book chapters on biofilms and biofilm processes. He coedited the book Biofouling
and Biocorrosion in Industrial Water Systems. He chaired the Biofilm Specialist Group of the
International Water Association (IWA), organized several international conferences, and
presented short courses on biofilm processes in the US, Ireland, Poland, and China.

Haluk Beyenal is widely known for his biofilm engineering expertise in the areas of
microscale biofilm characterization and electron transfer processes. The research in his
laboratories has focused on a fundamental understanding of biofilm processes, and the
characterization of biofilms and applications of biofilm processes to the environment,
energy, and health. He and his collaborators have developed many research tools for
understanding biofilm processes at the microscale, including microelectrodes for monitor-
ing local chemistry inside biofilms and mathematical models for predicting biofilm activ-
ity, electron transfer rates, and biofilm structure. His research group pioneered the study
of electron transfer processes in biofilms using microelectrodes. He and his collaborators
developed an electrochemical nuclear magnetic resonance microimaging technique to
study electron transfer processes in biofilms. The microscale techniques developed in his
laboratory are crucial to the study of biofilms respiring on electrodes. His research group
and his collaborators also developed technology to power remote sensors using energy
harvested from microbial fuel cells. His research has been funded by the Office of Naval
Research, the National Science Foundation, the Department of Energy, the Department of
Defense, and the National Institutes of Health as well as by industry. He received an NSF-
CAREER award in 2010. He has published more than 80 papers and eight book chapters
on biofilms and biofilm processes. Currently, he is an associate professor at Washington
State University, in the Gene and Linda Voiland School of Chemical Engineering and
Bioengineering.

xxi
chapter one

Introduction to biofilms and

to biofilm research

1.1 Introduction
Many natural and engineered systems are influenced by biofilms, microorganisms firmly
attached to surfaces. From the human perspective, the effects of biofilms vary from desir-
able, through undesirable, to disastrous, depending on the locations where the biofilms
are deposited and on their microbial community structures. Examples of desirable effects
are the activities of biofilms accumulated in wetlands and in the trickling filters used in
wastewater treatment plants. These biofilms remove organics from water. Examples of
undesirable effects are the activities of biofilms accumulated in cooling towers and other
heat exchangers. These biofilms increase heat transfer resistance. Finally, examples of
disastrous effects are the activities of biofilms developing on tampons applied to absorb
bleeding and the activities of biofilms developing on implantable prosthetic devices. The
former biofilms can cause serious illnesses or the death of the infected patients, and sur-
gery may be required to remove the latter. Irrespective of where the biofilms have accumu-
lated and what their microbial compositions are, there is a common desire to control their
activities either to promote their growth or to eradicate them. To facilitate biofilm control
and to optimize biofilm processes, an entirely new branch of bioengineering has emerged:
biofilm engineering. Most subjects covered by this book belong to the domain of biofilm
engineering.
Biofilms affect such disparate processes as bioremediation of toxic compounds, oral
hygiene, souring of oil formations, microbially influenced corrosion (MIC), wastewater
treatment, and the use of implantable prosthetic devices, causing the development of bio-
film research in the related disciplines of science and technology. The two types of biofilm-­
related research activities that have emerged from this complex picture are research
devoted to understanding fundamental biofilm processes and research devoted to devel-
oping biofilm technology. Currently, the most active discipline in biofilm research devoted
to understanding the fundamentals of biofilm processes is microbial ecology, in which bio-
films are seen as highly organized structures of microorganisms having qualities compa-
rable to those of tissues. Microorganisms in biofilms are not randomly distributed; instead,
they aggregate in clusters resembling communities in which various microbial species
have various tasks to accomplish, and they communicate with each other using chemical
signals. This internal organization of bacterial biofilms, documented by confocal images
and molecular probes, has stimulated imaginations and spawned new ideas, sometimes
bordering on science fiction. New Scientist, the British science magazine, published an arti-
cle in August 1996 emphatically comparing biofilms to cities built by microorganisms.
This analogy reflects the popular image of biofilms as self-assembled microbial structures
that are able to optimize their functions. Time has passed, and this paper is now a his-
torical footnote, but the general idea it proposed not only survived but gained even more

1
2 Fundamentals of biofilm research

traction when cell–cell communication in biofilms was discovered. Since the publication of
the paper in 1996, chemical substances have been identified that are used by biofilm micro-
organisms to facilitate the exchange of information, a discovery that implies new possi-
bilities in biofilm control. If these lines of communication could be intercepted, biofilms
could be controlled in an entirely new way, and if such intervention is indeed possible, the
implications go far beyond controlling biofilms. The research is ongoing, and it remains to
be seen to what extent these expectations will be satisfied.
The effects of biofilms on various technological processes constitute the domain of
biofilm engineering, which is mostly devoted to controlling biofilm processes. Among the
various types of research projects oriented toward biofilm control, biomedical research
attracts much attention. This area of biofilm research experiences the most spectacular
development. According to the National Institutes of Health html document, “Guide:
Research on Microbial Biofilms” released on December 20, 2002, biofilms are responsible
for 80% of infections in the body. According to the Guide, these infections include “oral
soft tissues, teeth and dental implants; middle ear; gastrointestinal tract; urogenital tract;
airway/lung tissue; eye; urinary tract prostheses; peritoneal membrane and peritoneal
dialysis catheters, indwelling catheters for hemodialysis and for chronic administration
of chemotherapeutic agents (Hickman catheters); cardiac implants such as pacemakers,
prosthetic heart valves, ventricular assist devices, and synthetic vascular grafts and stents;
prostheses, internal fixation devices, percutaneous sutures; and tracheal and ventilator
tubing.” Graphic images of biofilms on nonhealing wounds and infected implantable
prosthetic devices extracted during revision surgeries are convincing examples of the
importance of biofilms and biofilm processes in biomedical research. However, biomedical
research is not the only, and is perhaps not even the most active, area of research dedicated
to developing biofilm technology. Much has been done to understand and to mitigate the
effects of biofilms in nonbiomedical processes, such as the biodeterioration of stone, MIC,
and the biofouling of filtration membranes. An example of an undesirable biofilm effect
is shown in Figure 1.1, in which a biofilm is accumulating inside silicone tubing used to
deliver nutrients to a biological reactor. As time progresses, the biofilm that has accumu-
lated on the walls may close the conduit entirely.
Biofilms are also used to enhance desirable technological processes, such as the biore-
mediation of contaminated groundwaters and wastewater treatment. Establishing biofilm
research as a discipline provides a common denominator to all these studies and gener-
ates interest in biofilms as a mode of microbial growth that may cause effects that are
difficult to predict using the traditional model of microbial growth in suspension. A host
of biofilm-based technologies have been implemented in biological wastewater treatment.

Figure 1.1 Biofilm formation in silicone tubing.

Chapter one: Introduction to biofilms and to biofilm research 3

The study of biofilms entered the scene relatively late, but our current awareness of bio-
films and knowledge of biofilm processes have changed our perception and our approach
to many problems in everyday life. For example, microbial contaminations of surfaces,
from those in households to those in hospitals, are now seen as biofilm problems. The
rapidly developing interest in controlling biofilm processes, either stimulating or inhibit-
ing them, has generated the field of research activities commonly called biofilm engineer-
ing; but there is no sharp division between the fundamental studies of biofilm processes
and biofilm engineering, as there is no such division between any type of natural science
and engineering, and we will not make such a distinction in this book. Quantifying bio-
film processes at a fundamental level is the prerequisite to understanding biofilm con-
trol, whether for enhancement or inhibition. The physical, conceptual, and virtual tools
described in this text can be used equally well in fundamental research on microbial com-
munity structure and activity and in research on controlling MIC.
The following text will introduce the basic vocabulary needed to discuss biofilm
research. It is unfortunate that many terms used in biofilm research do not have precise
meanings; for example, the term biofilm activity can be defined in more than one way.
Without attempting to rectify this situation, the terms used here will be defined, and every
attempt will be made to use them consistently throughout this text.

1.2 Terminology
1.2.1 Biofilms, biofouling, and biofouling deposits
There is no commonly accepted definition of the term biofilm. Although self-explanatory to
biofilm researchers, it is regarded as controversial by many in other fields of research and
for good reasons. One reason is that film semantically implies a continuous and relatively
thin layer. Despite that implication, many biological structures regarded as biofilms are not
continuous, and many others can hardly be called thin. The lack of a commonly accepted
definition of a biofilm leaves it to the judgment of individual researchers whether, for exam-
ple, microorganisms immobilized in polymers should be considered biofilms or not. In this
text, a biofilm is considered to be an aggregate of microorganisms imbedded in a matrix
composed of microbially produced extracellular polymeric substances (EPS) and attached
to a surface. The aggregate does not have to form a continuous layer, and its thickness is not
limited by any arbitrarily selected number, but it must be attached to a surface. This latter
requirement specifically excludes suspended microbial flocks and microorganisms immo-
bilized in polymer beads from the definition of a biofilm. It accepts immobilized microor-
ganisms, though, as long as the substance in which they are immobilized is attached to a
surface. Briefly, the definition says that biofilms are microorganisms attached to surfaces.
Definitions of biofilms are often challenged, and the definition given here will also be
challenged. Challenging definitions of biofilms by asking apparently provocative ques-
tions, such as “How many microorganisms have to be on the surface to form a biofilm?”
or “Should a deposit of dead microorganisms on a surface be considered a biofilm?” is
more than just a frivolous and sometimes annoying activity. It has a deeper meaning and
significance because it stimulates our understanding of biofilm processes. For example, the
question “How many microorganisms are needed to form a biofilm?” can be answered if
the notion is accepted that microorganisms growing in biofilms are significantly different
from those growing in suspension. Biofilm researchers have demonstrated that microor-
ganisms attached to surfaces consistently express different genes than the same species
growing in suspension. If this notion is accepted, then the answer to the question is, “A
4 Fundamentals of biofilm research

single microbial cell attached to a surface can be called a biofilm, because it expresses the
characteristic features of biofilm microorganisms.” However, this is a rhetorical question,
and our definition of a biofilm as “an aggregate of microorganisms imbedded in a matrix
composed of microbially produced EPS and attached to a surface” does not specify the
number of microorganisms that constitute a biofilm.
Some biofilm researchers argue that biofilms should be regarded as a mode of micro-
bial growth, in the same manner as microbial growth in suspension is, rather than as phys-
ical structures formed by microorganisms attached to surfaces. Following their argument
would produce definitions of biofilms that are different from the one accepted in this book.
Defining biofilms as aggregates of microorganisms attached to surfaces, as is done here, is
practical for those life scientists who study biofilm structure, biofilm activity, and micro-
bial community structure in biofilms, and for those engineers who study rates of biofilm
reactions and mass transfer in microbial aggregates. For others, alternative definitions of
biofilms may be more suitable. Using alternative definitions of biofilms may be quite pro-
ductive in some areas of biofilm research and may help in interpreting biofilm processes
from a point of view other than that assumed in this book. Even though our definition of
biofilm has been designed from the engineering point of view and to satisfy the needs of
biofilm engineering, the definition suggested by the National Institutes of Health in the
html document “Guide: Research on Microbial Biofilms,” released on December 20, 2002,
“A biofilm is an accumulation of microorganisms (bacteria, fungi, and/or protozoa, with
associated bacteriophages and other viruses) embedded in a polysaccharide matrix and
adherent to solid biologic or non-biologic surface,” is quite close to our definition.
It is conceivable to avoid the controversial definition of a biofilm by not using the term
biofilm at all: there are other terms that can be used instead. Another term often used when
referring to attached microbial growth is biofouling (of surfaces). It is derived from the term
fouling of surfaces, which refers to the process of contaminating surfaces with (usually) min-
eral deposits. In principle, the term biofouling refers to the same process as the term fouling
does, but it emphasizes the fact that the deposits on the fouled surfaces are composed
of mineral substances mixed with living microorganisms and macroorganisms and with
EPS excreted by the microorganisms. The term biofouling is less controversial than the term
biofilm, perhaps because fouling does not imply any geometrical form of the deposits, as film
does. Even though the terms biofouling and biofilm are sometimes used interchangeably,
they do not have the same meaning: biofouling is a process, whereas a biofilm is an object.
The term biofouling deposits is considered an equivalent of the term biofilm, particularly in
industrial settings, where it is used to emphasize the presence of scaling deposits or corro-
sion products imbedded in the EPS excreted by the microorganisms attached to surfaces.
When larger organisms accumulate on surfaces, as is common in marine environments,
for example, the term macrofouling is used, without referring to biofilms.

1.2.2 Biofilm systems, biofilm processes, and biofilm reactors

Besides the term biofilm, which refers only to a deposit on a surface, a broader term, biofilm
system, is needed that includes other components affecting the rates of biofilm formation,
biofilm structure, and activity. A biofilm system is defined as a group of compartments and
their components determining biofilm structure and activity.
A biofilm system is composed of four compartments (Figure 1.2):

• The surface to which the microorganisms are attached

• The biofilm (the microorganisms and the EPS matrix)
Chapter one: Introduction to biofilms and to biofilm research 5

(a)

(b)

Gas phase

Solution of nutrients

Microorganisms and EPS

Surface

Figure 1.2 (a) Stainless steel coupons attached to a rack are immersed in a stream. The four com-
partments of this system are (1) all the surfaces on which the biofilms are deposited, including the
stainless steel coupons, (2) the biofilms deposited on these surfaces, (3) the water, and (4) the air.
(Reprinted from Braughton, K. R. et al., Biofouling 17: 241–251, 2001.) (b) Biofilm compartments: gas
phase, solution of nutrients, biofilm and surface.

• The solution of nutrients

• The gas phase (if present)

It is convenient to identify components within biofilm compartments. The number

of components in each compartment may vary, depending on the needs of a particular
description. For example, in one study it may be convenient to identify two components
of the biofilm: (1) the EPS (matrix) and (2) the microorganisms. In another study, it may be
convenient to identify three components of the biofilm: (1) the EPS, (2) the microorganisms,
and (3) the particulate matter trapped in the matrix. Similarly, it may be convenient to
single out two components of the surface: (1) the bulk material and (2) the biomineralized
deposits. Alternatively, if MIC is studied, it may be convenient to describe the surface by
identifying three components: (1) the metal surface, (2) the corrosion products, and (3) the
biomineralized deposits on the surface. The needs of the specific study dictate the number
of components identified in each compartment of the biofilm system, as is exemplified in
Figure 1.2.
The term biofilm processes refers to all physical, chemical, and biological processes in
biofilm systems that affect, or are affected by, the rate of biofilm deposition or the micro-
bial activity in biofilms. Examples of biofilm processes are attachment, detachment, and
growth. In some texts, these three processes are considered the biofilm processes because
other processes occurring in biofilm systems are related to this group of processes. This
tendency to refer to only three biofilm processes originates from the mass balances in
chemical and biological reactors in which the rate of change in the concentration of a
selected component is quantified in the influent, in the effluent, and in the reactor, which
is analogous to quantifying the rates of attachment, detachment, and growth in biofilms
if we assume that the biofilm is the reactor. We do not think it is practical to restrict the
number of biofilm processes to these three, because there are processes occurring in bio-
films that may be difficult to associate with any of them, such as plasmid exchange and the
buildup of resistance to antimicrobial agents.
Biofilm processes are quantified in biofilm reactors. Colloquially, biofilm reactors and
biofilm systems have the same meaning. There is a subtle difference between these two
terms, though: biofilm systems exist with or without our intervention, whereas biofilm
6 Fundamentals of biofilm research

reactors are created by our actions. Whenever a biofilm process is promoted or suppressed
in a biofilm system, or even when a biofilm process in a biofilm system is quantified with-
out its rate being affected, the biofilm system becomes a biofilm reactor. For example, wet-
lands exist in nature whether engineers designed them or not. Wetlands designed and
constructed to remove contaminants are biofilm reactors because the biofilm processes
were used to achieve the goal. However, even natural wetlands, once biofilm performance
in them is monitored, become biofilm reactors. A special subgroup of engineered biofilm
reactors is laboratory biofilm reactors (LBRs), which are constructed to emphasize selected
biofilm processes or to facilitate selected types of measurements in biofilms. Chapter 3 is
entirely dedicated to these biofilm reactors.
It is convenient to identify biofilm reactors with biofilm systems and to define biofilm
reactors as a collection of four compartments and their respective components determin-
ing biofilm processes, e.g., a continuously stirred tank reactor (CSTR), a lake, a wetland,
or a human body, without any implicit or explicit references to natural or engineered pro-
cesses occurring in these systems. One benefit of this definition is that it is not restric-
tive: the reactor whose performance is affected by the presence of biofilms can be affected
by the presence of another agent, biological or not. For example, a CSTR that is used to
grow microorganisms in suspension can be, in addition to its main function of growing
microorganisms in suspension, considered a biofilm reactor—if the researcher decides to
quantify the effect of microbial growth on the walls. Several parameters are quantified in
biofilm reactors, and their meanings must be defined.

1.2.3 Biofilm activity
Biofilm activity is defined here as the rate of nutrient utilization per unit of surface area
covered with biofilm, which is consistent with the definition of biofilm activity in most
biofilm engineering applications. In most studies, biofilm activity is evaluated based on
the rate of utilization of the growth-limiting nutrient. However, in some instances, rates
of change of substances other than the growth-limiting nutrient are selected to compute
biofilm activity. The choice of the substance for evaluating biofilm activity is dictated by
the process under study and sometimes by analytical convenience. If a biofilm reactor is
operated to remove an undesirable substance from the solution, then the rate of removal
of this substance is a natural choice for evaluating biofilm activity. Other choices include
the substances serving as the main electron donor and the main electron acceptor in the
respiration of selected groups of microorganisms residing in the biofilm. Biofilm activity
can be computed from measurements at the macroscale or at the microscale, and we will
call these overall and local biofilm activities, respectively.

1.2.4 Nutrient utilization rate and specific nutrient utilization rate

Nutrient utilization rate (NUR) and specific nutrient utilization rate (SNUR) are two
parameters in common use for quantifying the rates of microbial reactions in suspension.
For a chemostat operated at a steady state with volume V, volumetric flow rate Q, and cell
density X (Equation 1.1), NUR is computed by multiplying the difference in nutrient con-
centration between the influent and the effluent of the reactor by the volumetric flow rate
(Figure 1.3):

NUR = (CInfluent − CEffluent) × Q (1.1)

Chapter one: Introduction to biofilms and to biofilm research 7

CInfluent CEffluent

X, V

Figure 1.3 A chemostat operated at a steady state with volume V, volumetric flow rate Q, and cell
density X.

g m3 g
The units of NUR are × = .
m3 s s
SNUR is NUR divided by the amount of biomass in the reactor (X × V):

(CInfluent − CEffluent ) × Q
SNUR = (1.2)
X ×V

g nutrient
s g nutrient
The units of SNUR are =
g biomass g biomass × s
3
× m3
m
NUR and SNUR have the same meanings in biofilm reactors as they have in reactors
with suspended growth. Computing the amount of biomass accumulated in the biofilm
needed to compute the SNUR is more challenging in a biofilm reactor than it is in a reactor
with suspended microbial growth.

1.2.4.1 Biofilm activity from macroscale measurements: Overall biofilm activity

To evaluate biofilm activity from macroscale measurements (Equation 1.3), the concentra-
tion of the substance selected for evaluating biofilm activity is measured in the influent
and in the effluent of a biofilm reactor, and the difference between these concentrations is
multiplied by the volumetric flow rate and then divided by the surface area covered by the
biofilm (Equation 1.3). The substance selected for the evaluation of biofilm activity must be
actively utilized by the microorganisms, and it should not precipitate, evaporate, be depos-
ited in the biofilm, or otherwise be removed from the reactor in any manner other than by
microbial utilization (Figure 1.4).

(CInfluent − CEffluent ) × Q NUR

Biofilm activity = = (1.3)
A A

g
The unit of biofilm activity is .
s × m2
One common error made when quantifying biofilm activity at the macroscale in LBRs
is not taking into account biofilm growth in the tubing. To avoid this error, it is important
to take the samples of the substance selected for the evaluation of biofilm activity from as
close to the entrance and the exit of the reactor as possible. In particular, evaluating the
concentration of the selected substance in the influent to the reactor based on its concen-
tration in the container with the feed solution should be avoided, if possible. Estimating
biofilm activity from macroscale measurements averages out the differences in biofilm
8 Fundamentals of biofilm research

CInfluent CEffluent

Q
Xf , V, A

Figure 1.4 A continuous flow biofilm reactor. The reactor volume is V, the volumetric flow rate is Q,
the surface area on which the biofilm grows is A, and the biofilm density is Xf .

activity that may exist in various parts of the reactor. Biofilms accumulate on all surfaces
in the reactor, and these surfaces may be made of different materials or differently ori-
ented in space. For example, if the reactor bottom is made of glass but the walls are made
of polycarbonate, the biofilm on the bottom may have different activity than the biofilm
on the walls. Also, the bottom of the reactor is oriented horizontally, whereas the walls
are oriented vertically, which may affect the way the biofilms respond to shear stress. The
differences in material and the differences in flow regime near these surfaces may cause
the differences in activity of the biofilms accumulated on these surfaces. Biofilm activity
estimated from macroscale measurements averages out all these differences and presents
a biofilm of uniformly distributed activity. Macroscale measurements of biofilm activity
can be quite adequate for estimating the overall reactor performance in wastewater treat-
ment, for example; but in experiments designed to quantify the effects of hydrodynam-
ics on biofilm accumulation or the effects of surface chemistry on biofilm accumulation
and activity, biofilm activity should be estimated from microscale measurements, which
deliver information about local biofilm activity, and the locations of the measurements
should be chosen so they are relevant to the type of question addressed by the specific
study.

1.2.4.2 Biofilm activity from microscale measurements: Local biofilm activity

Microbial activity at the microscale is identified with the flux, from the bulk solution to
the biofilm surface, of the substance selected for evaluating biofilm activity. As in the mea-
surements of biofilm activity at the macroscale, the selected substance typically used in
these measurements is the growth-limiting nutrient. Measurements of biofilm activity at
the microscale are determined by the availability of tools to a larger extent than measure-
ments at the macroscale are. Because fluxes at the microscale are quantified locally, rather
than averaged over the entire surface area as is done at the macroscale, the concentration
profiles of the selected substance must be measured with adequate spatial resolution. For
this purpose, microsensors sensitive to the selected substance are used. There is only a
limited number of microsensors that are able to measure concentration, and the availabil-
ity of these tools limits the choices of substances used to evaluate biofilm activity at the
microscale.
The local microbial activity in the biofilm is computed as the flux of the nutrient across
the biofilm surface at the location of the measurement, and the flux is evaluated by mul-
tiplying the slope of the concentration profile near the biofilm/bulk solution interface
(within the diffusion boundary layer) by the diffusivity coefficient of the substance whose
concentration was measured in water:

 dC 
jw = Dw  (1.4)
 dz  w
Chapter one: Introduction to biofilms and to biofilm research 9

where Dw is the diffusivity in water of the substance selected for the evaluation of bio-
film activity, C is the concentration of the substance, z is the distance, and (dC/dz)w is the
slope of the concentration profile measured at the biofilm surface from the water side. The
units of flux are the same as the units of biofilm activity determined from the macroscale
measurements:

m2 g 1 g
× 3 × = 2
s m m m ×s

For each biofilm reactor, biofilm activity may be estimated at two scales of observation:
the macroscale and the microscale. Biofilm activity at the macroscale is reported as a single
number, whereas biofilm activity at the microscale is reported as a set of numbers because
biofilm activity depends on the location of the measurement. The existing mathematical
models of biofilm activity have difficulty reconciling the results of the measurements of
biofilm activity at the macroscale with those at the microscale. It is expected that the two
types of measurements should give the same overall biofilm activity. However, this expec-
tation can be fulfilled only if the biofilm is uniformly distributed in the reactor and the
microbial activity is uniformly distributed in the biofilm. Neither of these two conditions
is satisfied in real biofilms, and the biofilm activity estimated from the microscale mea-
surements, in general, is not equal to that estimated from the macroscale measurements. It
is not clear how to compare these results or how to integrate the results of biofilm activity
measurements at the microscale so that an overall biofilm activity equal to that measured
at the macroscale can be achieved. The concept of stratified biofilms used to interpret the
results of measurements has the potential of bridging the two scales of observation.
Even if it eventually becomes possible to arrive at the same biofilm activity from
results from the two scales of measurements, the measurements at the microscale deliver
information that cannot be extracted from the measurements at the macroscale. For some
biofilm processes, it is important to quantify the extreme values of biofilm activity because
the locations in the biofilm where these extreme values occur exhibit extreme properties.
For example, in studying MIC, which causes highly localized damage to metal surfaces,
it is important to quantify the extreme values of biofilm activity because the extreme, and
highly localized, chemistry in biofilms determines the extent of microbial corrosion. The
average biofilm activity estimated from measurements at the macroscale cannot deliver
this information.

1.2.5 Biofilm development and the concept of biofilm structure

Biofilm formation begins with the adsorption of single cells from the solution. Microbial
cells are transported with the stream of liquid above the surface. Cells from the solution
hit the surface and interact with the conditioning film adhering to the surface. Some cells
attach to the surface, produce EPS, and form a biofilm. Other cells attach and then detach
and leave the surface. The initial colonization of surfaces is supported by three mecha-
nisms of microbial transport: (1) diffusive transport, (2) convective transport, and (3) the
active movement of motile bacteria. The highest rate of microbial transport is by convec-
tion, which may exceed the other two rates by several orders of magnitude. After the
microbial cells are in contact with the surface, they may adhere to it or not. The probability
of lasting adsorption is termed sticking efficiency, which is a parameter that is computed as
the ratio of the number of microorganisms attaching to a surface to the total number of
10 Fundamentals of biofilm research

microorganisms landing on the surface within the same time interval. Sticking efficiency
depends on many factors, including the properties of the surface, the physiological state of
the organisms, and the hydrodynamics near the surface.
Figure 1.5 shows the three characteristic stages of biofilm formation. The first stage,
called attachment, is mostly affected by the surface properties and the rate of microbial
transport to the surface. The second and third stages—colonization and growth—are both
affected by such factors as the mass transport of nutrients to a larger extent than they are
by the properties of the surface or the rate of microbial transport to the surface.
The adherence of bacteria to a surface is followed by the production of slimy adhe-
sive substances, known as EPS. These are predominantly made of polysaccharides and
proteins, and their role is elucidated later in this chapter. Although the association of EPS
with attached bacteria has been well documented in the literature, there is little evidence
to suggest that EPS participates in the initial stages of adhesion, except perhaps in the
cases in which negatively charged bacteria attach to negatively polarized metal surfaces,
in which the involvement of EPS has been hypothesized. However, EPS definitely assists
the formation of mature biofilms by forming a slimy substance called the biofilm matrix.
Figure 1.6a shows an image of bacteria attached to a surface imbedded in a slimy layer
of EPS. The images were acquired using atomic force microscopy, and the biofilm was
partially dried to enhance the visibility of the cells covered with EPS. Drying the samples

Biofilm formation
Attachment Colonization Growth

Surface

Figure 1.5 Stages of biofilm formation.

Height Angle SurfaceNormal Clear Calculator Clear Execute Undo

Flattten
20.0 0.5 v Height 30.0 ˚
10.0

0.3 v 15.0 ˚
7.5

10.0 0.0 v 0.0 ˚

5.0

Digital instruments nanoscope Digital instruments nanoscope

Scan size 20.00 µm 2.5 Scan size 10.67 µm
Scan rate 0.5003 Hz Scan rate 0.5003 Hz
Number of samples 512 Number of samples 512
Image data Amplitude Image data Phase
0 Data scale 500.0 mV Data scale 30.00 ˚
0 10.0 20.0 0
μm 0 2.5 5.0 7.5 10.0
μm

sal2_solution_low_mica_air.002 sal2_solution_low_mica_air.003

(a) (b)

Figure 1.6 (See color insert.) Atomic force microscopy images of bacteria attached to a surface.
The cells are imbedded in EPS that was (a) partially dried to expose the microorganisms and then
(b) further dried. (Both images courtesy of Recep Avci of the Physics Department, Montana State
University, Bozeman.)
Chapter one: Introduction to biofilms and to biofilm research 11

somewhat longer produces an even more convincing image of the microorganisms imbed-
ded in EPS, as seen in Figure 1.6b.
The first conceptual model of microbial biofilms assumed that the microorganisms are
uniformly distributed in the matrix, as shown in Figure 1.7.
This model served the research community well for many years, and it is still use-
ful in the mathematical modeling of some biofilm processes. However, as time passed
and tools were developed for direct probing of the intrabiofilm environment with high
spatial resolution, such as confocal microscopy and microelectrodes, it became obvious
that some of the experimental results acquired using these tools were impossible to inter-
pret using the conceptual model of biofilms in which microorganisms were uniformly
distributed in a continuous matrix of EPS. One of the first signs that something might be
wrong with the model came in 1993, when Drury injected micron-sized fluorescent latex
particles into a biofilm reactor and studied their fate (Drury et al. 1993). The expectation
was that these particles, after settling on the biofilm surface, would be pushed off by the
growing and exfoliating bacteria. One of the popular notions in biofilm engineering at that
time was that faster-growing microorganisms replace slower-growing microorganisms,
analogous to the process occurring in continuous cultures of suspended microorganisms.
The fluorescent beads were used to imitate bacteria having a growth rate equal to zero,
and they were expected to be pushed away by growing biofilm microorganisms. After
the experiment was terminated, the biofilm was sectioned and the beads were recovered.
Surprisingly, many of them were found at the bottom of the biofilm, near the surface sup-
porting biofilm growth. This contradicted one of the assumptions of the model: if the bio-
film was a continuous gelatinous layer, as was stipulated by the model, then how did these
beads get to the bottom of the biofilm? Clearly, the model of homogeneous biofilms could
not explain this result. On the other hand, in a heterogeneous biofilm, this effect is actually
expected: the beads penetrated and were trapped in interstitial voids, which frequently
reach the bottom of the biofilm. Another controversial argument pointing toward the need
to revise the conceptual model of biofilms came from the study of MIC. An argument often
used when discussing MIC was that if biofilms formed continuous layers on metal sur-
faces, then they should decrease the corrosion rate, not increase it, by depleting the oxygen,
the principal cathodic reactant in natural waters, near the surface. In light of that fact, the
experimental observations indicating that biofilms increased the rate of corrosion were
difficult to interpret. In the model of heterogeneous biofilms, oxygen freely penetrates to
the surface through voids, causing the formation of differential aeration cells and acceler-
ating corrosion; the parts of the surface covered by microcolonies are deprived of oxygen
and become anodic, while the parts where oxygen reaches the surface become cathodic. A
serious difficulty in interpreting experimental results arose when concentration profiles of

Bulk liquid

Surface

Figure 1.7 The model of biofilm structure created by early biofilm researchers assumed microor-
ganisms randomly distributed in a continuous layer of EPS.
12 Fundamentals of biofilm research

dissolved nutrients, e.g., oxygen, were measured to verify mathematical models of biofilm
activity. The mathematical models referred to a single profile across the biofilm, and the
profiles measured at different locations in the same biofilm were different to an extent
that could not be ascribed to experimental errors only. As long as single profiles were
analyzed, which was the case in most early publications, everything worked as expected.
Mathematical models of biofilm activity, however, are intended to describe biofilm activ-
ity over a certain surface area, not just at a single location where measurements are taken.
Attempts to find an average nutrient consumption rate over a certain area often generated
surprising variability in that parameter. Because there was no independent technique for
verifying microelectrode measurements, the suspicion was that the microelectrode mea-
surements were not accurate. It is now well known that biofilm heterogeneity strongly
influences the chemical profiles measured by microelectrodes and that measuring differ-
ent nutrient concentration profiles at different locations in a biofilm is to be expected.
The difficulties with interpreting experimental results using the conceptual model
of homogeneous biofilms continued to mount. In another set of experiments, profiles of
flow velocity were measured near the biofilm surface using nuclear magnetic resonance
imaging (NMRI). It was expected that biofilms covering walls of conduits would behave
as if they just decreased the dimensions of the conduits. Flow velocity was expected to
decrease on approaching the biofilm surface and to finally reach zero at the biofilm sur-
face. Careful measurements of flow velocity distribution in biofilms demonstrated that
the flow velocity reached zero at the conduit surface instead of at the biofilm surface as
expected. Again, this effect is expected in heterogeneous biofilms, in which water can
move in the interstitial voids.
All these effects that were impossible to interpret using the model of homogeneous
biofilms—penetration of biofilms by small fluorescent beads, differential aeration cells,
different concentration profiles at different locations, and flow of liquid through the bio-
film—are expected and easily interpreted using the conceptual model of heterogeneous
biofilms. For the purposes of this text we will be using the conceptual model of heteroge-
neous biofilms, i.e., biofilms in which microorganisms are assembled in clusters separated
by interstitial voids (Figure 1.8). A heterogeneous biofilm is composed of (1) a densely com-
pact sublayer, (2) round microcolonies, (3) interstitial voids, and (4) sometimes streamers,

Figure 1.8 Heterogeneous biofilm. Microscopic observations indicate that biofilms are not flat and
that the distribution of microorganisms is not uniform. Instead, multispecies biofilms form highly
complex structures containing voids, channels, cavities, pores, and streamers, with cells arranged
in microcolonies.
Chapter one: Introduction to biofilms and to biofilm research 13

which are long strands of EPS extending from the microcolonies. The sublayer is not con-
tinuous and, in places, exposes the surface. Above the sublayer are dense, often round,
microcolonies that are filled with EPS and densely packed with microorganisms. These
may finish with elongated streamers extending downstream. Microcolonies are sepa-
rated by interstitial voids forming a network of interconnected channels, giving biofilms
their characteristic porous structure. Water can move freely within the network of these
channels.
It is believed that microorganisms in biofilms are organized as communities. It is fur-
ther hypothesized that these communities are assembled following a master plan, so that
the cells of one species associate with those of another species for mutual physiological
benefits. As time passes, the discrete microcolonies of microorganisms deposited on the
surface grow in size, join, and form heterogeneous layers of microorganisms embedded in
the polymer matrix covering the entire surface. Natural biofilms are even more heteroge-
neous than those grown in the laboratory because they contain adsorbed and entrapped
materials including inorganic particles, such as clay or silt. The mixture of all these com-
ponents forms the characteristic biofouling deposits, in which materials of nonbiologi-
cal origin are imbedded in a matrix of EPS excreted by biofilm microorganisms. Images
of mature biofilms are less clear than images of young biofilms because the visibility is
obscured by the EPS excreted by the microorganisms and by the presence of other materi-
als entrapped in the matrix of EPS. Figure 1.8 shows an idealized image of a heterogeneous
biofilm. Such distinct structures are seen only in relatively young biofilms. As biofilms
become older, the semicontinuous sublayer tends to become denser and thicker and to
accumulate various particles from the system, while the upper layer, consisting of the
characteristic round microcolonies and streamers, remains the same. New advances in
understanding biofilm structure show that, as time passes, the bottom layers of biofilms
become dense and compact and their porosity becomes much lower than the porosity of
the upper layers. It is not known whether this decrease in porosity results from biofilm
growth and expansion near the bottom or just from the accumulation of debris and pieces
of biofilm sloughed off upstream.
Once a mature biofilm is established on a surface, it actively propagates and even-
tually covers the entire surface. The mechanisms of propagation in mature biofilms are
more diverse than those of initial attachment; several of these mechanisms are depicted
in Figure 1.9.

Streaming

Detaching
Seeding
dispersal

Rippling

Rolling

Figure 1.9 (See color insert.) Mechanisms of biofilm propagation. (Courtesy of P. Dirckx, MSU
Center for Biofilm Engineering.)
14 Fundamentals of biofilm research

Biofilm researchers see complex modes of biofilm propagation in everyday laboratory

practice and have to take steps against some of them. Biofilms in tubing delivering nutri-
ents to LBRs can spread in the direction opposing the direction of the flow and eventu-
ally invade the container with the nutrients. This fact clearly demonstrates that mature
biofilms propagate in more complex ways—against the flow in this case—than just the
simple attachment of the suspended microbial cells in the incoming stream of nutrients,
which is characteristic of the initial stages of biofilm formation. Specially designed flow
breakers are used to prevent the spread of biofilm in tubing used to deliver nutrients, and
we discuss them in Chapter 9.

1.3 Characterizing selected parts of biofilm systems

In the previous sections we defined a biofilm system as a collection of four compartments:
(1) the surface, (2) the biofilm, (3) the solution of nutrients, and (4) the gas phase. Much of
biofilm research is about characterizing these compartments, their relations to biofilm pro-
cesses, and relations among their components. A set of conceptual models has been devel-
oped to image and to interpret the physicochemical and biological processes occurring in
these compartments. The following section describes these models and these processes.
We characterize selected compartments and selected components of biofilm systems,
those we usually characterize when describing biofilm processes from the engineering
point of view.

1.3.1 Characterizing surfaces
Actually, there are two surfaces of interest in biofilm research: the surface to which the
microorganisms are attached and the surface of the microorganisms—the microbial cell
surface. However, only the surface on which a biofilm accumulates is identified here as a
compartment of the biofilm system. Therefore, throughout this text, when we refer to the
surface we mean the surface to which the microorganisms are attached, unless explicitly
specified otherwise. The surface on which the microorganisms aggregate has acquired
an unfortunate name in biofilm research—the substratum, which is easily confused
with another important name often used in the same context—the substrate, which is
the growth-limiting nutrient. To avoid confusion, it makes sense to use the term nutrient
instead of substrate, whenever possible. We start the analysis of the effects of the surface
on biofilm processes with a description of the effects of the surface on the initial events in
biofilm formation, the microbial colonization of the surface. Despite an intensive research
effort on the relations between the surface properties and the rate of microbial attachment
to the surface, these relations are still not clear.

1.3.1.1 Surface properties and microbial colonization of surfaces

The process of biofilm formation consists of a sequence of steps and begins with the
adsorption of macromolecules (proteins, polysaccharides, nucleic acids, and humic acids)
and smaller molecules (fatty acids, lipids, and pollutants such as polyaromatic hydro-
carbons and polychlorinated biphenyls) onto surfaces. These adsorbed molecules form
conditioning films that may have multiple effects, such as altering the physicochemical
characteristics of the surface, acting as a concentrated nutrient source for microorgan-
isms, suppressing or enhancing the release of toxic metal ions from the surface, detoxi-
fying the bulk solution through the adsorption of inhibitory substances, supplying the
required nutrients and trace elements, and triggering biofilm sloughing. The properties of
Chapter one: Introduction to biofilms and to biofilm research 15

a conditioning film depend on the quality of the water in contact with the surface and may
dramatically affect the surface properties. For example, in 1976, Loeb and Neihof demon-
strated in electrophoretic studies that platinum particles suspended in seawater depleted
of organic matter were electropositive before immersion and became electronegative upon
immersion in natural water. Although there is no doubt that the conditioning film influ-
ences microbial colonization, this effect is difficult to evaluate because the conditioning
film forms on surfaces immediately upon immersion and, consequently, there are no sur-
faces immersed in water that are without such a film to serve as a reference.
The possibility that the adhesion of microorganisms to inert surfaces is affected by
electrostatic interactions has been explored with the hope that such interactions may help
in establishing procedures for preventing biofilm formation on electrically conductive
surfaces. Surfaces immersed in water acquire electrical charges owing to the preferen-
tial release of surface ions, the preferential adsorption of ions from the solution, or the
surface equilibrating redox reactions in water. Also, most bacteria, algae, and detritus in
natural waters carry a net electronegative surface charge. The effects of surface charge on
microbial adhesion can be studied by applying known electrical potentials to metal sur-
faces, polarizing them. The expected consequence of applying a negative charge would be
to repel the negatively charged microorganisms. However, this effect is mitigated by the
presence of the adhesive substances produced by the microorganisms, EPS, which bridge
the microbial cells with the surfaces. As a result of these interactions, negatively charged
bacteria adhere to both negatively and positively charged surfaces.
Much attention has been devoted to studying hydrophobic effects on the attachment
of microbes to each other and to surfaces. It is well known that certain materials can easily
be covered with a film of water, while other materials resist wetting; the first are called
hydrophilic and the latter hydrophobic. A well-known example of hydrophobicity is oil
droplets on a water surface: the surface of oil resists wetting. Hydrophobic particles sus-
pended in water prefer to interact with particles of their own kind, such as other hydro-
phobic particles, rather than with water. Because of this, hydrophobic particles immersed
in water behave as if they were forming “voids” in a bulk solution. This behavior has con-
sequences. Water is a highly structured polar solvent with individual particles connected
by hydrogen bonds. Hydrophobic particles, by forming voids in the bulk solution, deform
that structure, stretching and breaking the hydrogen bonds between water particles. This
effect, the stretching and breaking of hydrogen bonds, promotes the coalescing of hydro-
phobic particles suspended in water through the following mechanism: when two hydro-
phobic particles suspended in water coalesce, the surface area of the combined particle is
smaller than the sum of the surface areas of the coalescing particles. Consequently, the
energy associated with the deformation of the water particles, which is proportional to
the surface area of the particles, decreases as a result of coalescing, making the process of
coalescing thermodynamically spontaneous. When two hydrophobic particles approach
each other and coalesce, the free energy of the system decreases, favoring adhesion.
Conversely, hydrophilic surfaces form structures with water particles that may be stron-
ger than those due to hydrogen bonds between water particles. In summary, the more
hydrophobic surfaces are, the more strongly they adhere to each other. This effect obvi-
ously attracted the attention of those searching for the reason microorganisms attach to
surfaces and to each other and resulted in the hypothesis that hydrophobicity is the reason
for microbial adhesion to surfaces.
Upon closer inspection, there are experimental difficulties in verifying the claim that
microorganisms adhere to surfaces because of hydrophobic interactions. To quantify this
effect, both the microorganisms and the surfaces to which they adhere would need to
16 Fundamentals of biofilm research

be hydrophobic. However, only small parts of microbial cells are hydrophobic: most of
the surface of a microorganism is hydrophilic and for a good reason. If the surface of
a microorganism were entirely hydrophobic, the microorganism would die from starva-
tion because it could not absorb nutrients that were dissolved in water. However, many
biofilm researchers hypothesize that the small hydrophobic areas present on the surfaces
of microbial cells are responsible for the adherence of microorganisms to hydrophobic
surfaces. To test this hypothesis, the hydrophobicity of many surfaces and the surfaces of
many microorganisms have to be evaluated. Evaluating the hydrophobicity of surfaces is
relatively simple, at least in theory, and relevant experimental protocols for evaluating the
hydrophobicity of surfaces by measuring contact angles have been developed.
The hydrophobicity of a surface is evaluated by measuring the contact angle: a drop
of water is deposited on the surface, and the angle between the surface and the drop is
measured (Equation 1.5). If the contact angle is more than 90°, the water forms droplets and
does not wet the surface (image on the left in Figure 1.10) and the surface is called hydro-
phobic. Water does not enter capillary pores in hydrophobic surfaces. If the contact angle
approaches 0°, the water spreads over the surface without forming droplets. The contact
angle can be evaluated precisely using sophisticated equipment provided by specialized
vendors or, perhaps somewhat less precisely, using relatively simple, homemade equip-
ment. The measurements are interpreted using thermodynamics by referring the contact
angle to the interfacial free energies of the liquid, its vapor, and the surface (Figure 1.11).
The results of testing the hypothesis that hydrophobic interactions are responsible for
microbial attachment to surfaces have not been conclusive for several reasons. The current
theory used to evaluate hydrophobicity from measurements of contact angle does not take
into account factors besides hydrophobic interactions affecting the contact angle, such as
surface roughness or the presence or absence of surface films. Interpreting the results of
measurements remains controversial.
The contact angle θ is controlled by three interacting forces, the interfacial tension
of each participating phase (gas [vapor, V], liquid L, and solid S). The relation among

Contact
angle Water drop
θ > 90
θ < 90

Surface Surface

Figure 1.10 Measurement of the contact angle. A line tangent to the curve of the droplet is drawn at
the point where the droplet intersects the solid surface. The angle formed by this tangent line and
the solid surface on the side of the droplet is called the contact angle.

γLV

γSV θ γSL

Surface

Figure 1.11 Forces acting on a drop of a liquid deposited on a surface.

Chapter one: Introduction to biofilms and to biofilm research 17

these factors is quantified by Young’s equation, and the contact angle is sometimes called
Young’s contact angle. Young’s equation presents the horizontal components of the various
surface forces as

γSV = γSL + γLV × cos(θ) (1.5)

From measurements of the contact angle, another parameter, the surface energy, can
be computed. To visualize the meaning of the term, it is convenient to note that when
a drop of water spreads over a surface, the cohesive forces that keep the drop of water
together (surface tension) must be overcome by the adhesive forces between the liquid and
the surface. If water wets the surface, then the energy needed to do the work of expanding
the surface of the drop comes from its interaction with the surface. Consequently, if water
droplets spread over a surface, the surface is referred to as a high-energy surface, and if
water does not spread over the surface, the surface is referred to as a low-energy surface.
Surface energy is measured in Newtons per meter (N/m) and is defined as the force along
a line perpendicular to the surface or as the work done per unit area, which is proportional
to the force γLV cos(θ). Consequently, when θ approaches zero and cos(θ) approaches one,
for a perfectly wettable surface, the surface energy approaches a maximum. In Figure 1.11,
γSL is the surface free energy of the solid surface covered with liquid, γLV is the surface free
energy of the liquid–vapor interface, and γSV is the surface free energy of the solid surface
in contact with vapor.
Despite the effort invested in quantifying the relation among hydrophobicity, surface
energy, and the initial events of biofilm formation, the relation remains unclear. It appears
that the techniques for measuring surface energy are too crude to provide an accurate esti-
mate of hydrophobicity at a scale that is relevant to evaluating microbial attachment. Solid
surfaces have heterogeneous chemistries and heterogeneous distributions of hydropho-
bicity and so do microbial surfaces. Surface chemistries show differences at the submicron
scale, while the linear scale used to evaluate contact angle measurements is much larger
than that. Measuring the contact angle calls for depositing drops of water on the surface.
Drops of water are larger than typical microorganisms attached to surfaces. As a result,
the measurements of contact angle average the properties of a large area of the surface,
rendering the results less useful for evaluating the effects of hydrophobicity on microbial
attachment, because microorganisms attach to a much smaller surface area. Not only are
the individual microorganisms smaller than the droplets of water deposited on the sur-
face, but also the parts of the microorganisms that are actually hydrophobic occupy only
a small part of their surfaces. These differences between the scale of observation and the
scale of the actual event may be one reason the relation between surface energy and bio-
film formation is not as obvious as was initially assumed. As a result, the relation between
microbial colonization and surface energy remains unclear: microorganisms, apparently
unaware of the complexity of the situation, colonize both low- and high-energy surfaces.
Evaluating the hydrophobicity of microbial cells is controversial. There are no estab-
lished rules or experimental protocols for measuring the hydrophobicity of microbial cells.
For obvious reasons, measuring the contact angle on a microbial cell is not possible, so
microbial cell hydrophobicity is evaluated by measuring the kinetics of microbial adhe-
sion to surfaces of immiscible hydrophobic liquids added to water: droplets of a nonpolar
(hydrophobic) solvent are suspended in water, and bacteria adhere to these droplets. The
rate of change in microbial cell count in samples of water under some standardized condi-
tions is a measure of microbial cell hydrophobicity. Measuring the hydrophobicity of micro-
bial cells has been attempted by many researchers, but there is neither a uniform measure
18 Fundamentals of biofilm research

of microbial cell surface hydrophobicity nor any definitive scale of values for comparing
hydrophobicities measured in different systems explored by individual researchers.
Microbially colonized surfaces can be studied from many branches of physical sciences,
including chemistry and solid-state physics. Two characteristics of surfaces that are of par-
ticular interest to biofilm researchers are surface chemistry and surface topography. The
extensive research done to relate surface properties to rates of biofilm accumulation has been
only moderately successful, and the current thinking among biofilm researchers is that sur-
face properties are important only for the initial attachment of microbial cells and, as the
biofilm accumulates, the microbial cells that grow on top of the cells directly attached to
the surface are not affected by the surface properties at all or are affected to a much smaller
extent than the cells directly attached to the surface. This general statement may need to
be modified in some instances. For example, at freely corroding metal surfaces, corrosion
products imbedded in the biofilm matrix may provide additional surface area for microbial
growth and may affect the rates and extents of biofilm growth and accumulation. The notion
that microorganisms deposited within the biofilm matrix and away from the surface do not
sense the properties of the surface is worth remembering when designing so-called anti-
fouling surfaces, which, thus far at least, seem to delay microbial colonization rather than
prevent it. Once microorganisms establish their presence on a surface, biofilm accumulation
accelerates and the surface properties become less relevant, if they remain relevant at all.

1.3.2 Characterizing hydrodynamics and mass transfer in biofilms

In the preceding sections we defined the microbial activity in a biofilm as the rate of nutri-
ent utilization per unit of surface area covered with the biofilm. In a biofilm system, the
nutrient solution occupies the space above and within the biofilm compartment. However,
if the surface is porous, the nutrient solution may also enter the pores of the surface. In
some instances, as in the case of biofilms using electrodes as electron acceptors/donors, as
in electrochemically active biofilms, the surface itself may be the substrate; we will cover
this subject separately in Chapter 7, which is dedicated to biofilms on metals. The nutri-
ent solution serves as the medium for delivering nutrients and other substances such as
antimicrobial agents to the microorganisms imbedded in the biofilm matrix and removes
the products of microbial metabolism from the space occupied by the biofilm. The rates of
nutrient delivery and products removal affect the rates of biofilm processes. Many studies
have quantified the rate of mass transport and the rate of microbial respiration in biofilms,
as these two rates are equal in biofilms. With many simplifying assumptions, the process
of mass transfer in biofilms can be quantified. However, some of the assumptions are not
realistic: they are accepted because of computational convenience, not because they reflect
truth about specific processes. For example, one of the most difficult factors to describe
affecting mass transport in biofilms is hydrodynamics. The effect of hydrodynamics on
biofilms is complicated for many reasons, among them feedback effects: the flow of nutri-
ents affects the structure of the biofilm, and the structure of the biofilm affects the flow of
nutrients. In the following section we will quantify some of the factors in this system, and
we will make simplifying assumptions despite what was said above.
In flowing solutions of nutrients, there are two boundary layers above the biofilm sur-
face: the concentration (mass transport) boundary layer and the hydrodynamic boundary
layer. Above the respective boundary layers, nutrient concentration and flow velocity are
uniformly distributed. Within the respective boundary layers, nutrient concentration and
flow velocity are not constant but instead form gradients: the flow velocity and the nutrient
concentration decrease toward the biofilm surface.
Chapter one: Introduction to biofilms and to biofilm research 19

An axiom commonly accepted by biofilm researchers says that mass transport above
biofilms is due to convection, while that within biofilms is due to diffusion. This axiom has
been imbedded into many mathematical models of biofilm processes. However, several
studies have documented that water actually does move in the space occupied by the bio-
film, demonstrating that this axiom is not accurate. If water moves in the space occupied
by the biofilm, then convective mass transport must be present in that zone. This is some-
what disconcerting, because at the same time many studies have shown profiles of various
substances above and within the biofilms, demonstrating the existence of relatively thick
mass boundary layers above the biofilm. We will return to this apparent controversy later,
when we discuss the modified model of mass transport in biofilm systems. First, we will
discuss the simplified model of hydrodynamics and mass transport in biofilms, shown in
Figure 1.12. Even though we know that the model shown in Figure 1.12 is not accurate, it
is simpler to discuss the meanings and the dimensions of the boundary layers in biofilm
systems using a simplified model, and the conclusions we reach will not change the prin-
ciples of why such layers exist or what they affect in biofilm systems.
In the simplified conceptual model of hydrodynamics and mass transport in biofilms
(Figure 1.12), the flow velocity reaches zero at the surface of the biofilm surface (the nonslip
condition). Because at the same time the flow velocity in the bulk solution is higher than
zero, the flow velocity forms a profile near the biofilm, varying between the average flow
velocity in the bulk solution and zero at the biofilm surface. Experimentally measured
flow velocity profiles near biofilm surfaces look like the one shown in Figure 1.12. The
overall flow velocity in the main stream is considered to be the average flow velocity. The
flow velocity decreases near the surface of the biofilm and then reaches zero at the surface.
The layer of liquid in which the flow velocity decreases as a result of proximity to
the surface is called the hydrodynamic boundary layer. It is marked by the letter ϕ in
Figure 1.12. A direct consequence of the hydrodynamic boundary layer is the formation
of the mass transport boundary layer, also called the diffusion boundary layer. Its exis-
tence is demonstrated by the gradient in nutrient concentration within the hydrodynamic
boundary layer. As the flow velocity decreases near the biofilm surface, the mechanism
of mass transport changes from being dominated by convection at locations away from
the biofilm, where the flow velocity is high, to being dominated by diffusion at locations
near the biofilm surface, where the flow velocity is low. Because the microorganisms in the
biofilm can consume the nutrient at the rate that it is delivered by diffusion, the nutrient

Substrate
concentration Flow velocity
profile profile

Jw = k(Cb – Cs) vb

Dw
k=
δ
Cb

Cs δ

Biofilm

Figure 1.12 Substrate concentration profile and flow velocity distribution near and within an ideal-
ized biofilm.
20 Fundamentals of biofilm research

concentration decreases near the surface, forming a concentration profile within the hydro-
dynamic boundary layer. The layer of liquid above the biofilm surface where the nutrient
concentration decreases as a result of biofilm activity and the diminishing rate of mass
transport is called the mass transport boundary layer or the diffusion boundary layer. It is
marked by the Greek letter δ in Figure 1.12.
There is a relationship between the thicknesses of the mass transport boundary and
the hydrodynamic boundary layer. Both layers are affected by the transport of compo-
nents toward the surface: the hydrodynamic boundary layer is affected by the transport of
momentum, and the mass boundary layer is affected by the transport of nutrients. The rate
of transport of a component is always described as the product of the gradient of the com-
ponent being transported and a proportionality coefficient reflecting the physical nature
of the transport of the component. The thickness of the hydrodynamic boundary layer
is affected by the viscosity of water, and the thickness of the diffusion boundary layer is
affected by the diffusivity of the nutrient; consequently, the proportionality constants in
the relevant equations describing rates of momentum and nutrient transport are viscosity
and diffusivity. The diffusivities of nutrients in water vary within narrow limits, about
2 × 10 –9 m2/s. Meanwhile the kinematic viscosity of water at 20°C is about 1 × 10 –6 m2/s,
which is about 1000× higher than the diffusivity of small particles in water. As a result,
the rate of momentum transfer vastly exceeds the rate of the transfer of nutrient particles
toward the surface, and the hydrodynamic boundary layer is thicker than the mass trans-
port boundary layer. The mass transport boundary layer is effectively buried within the
hydrodynamic boundary layer, as shown in Figure 1.12. Some of the tools we use allow the
experimental measurement of flow velocity gradients and nutrient concentration gradients
and the determination of thicknesses of mass transport and hydrodynamic boundary lay-
ers in biofilm reactors. To understand the entirety of the mass transport and hydrodynam-
ics in biofilm systems, we need to include the intrabiofilm mass transport, and we will do
so in Chapter 2. Just above the biofilm surface, the intensity of mass transport decreases,
because flow velocity decreases near surfaces. As a result, the rate of nutrient consump-
tion by the biofilm is determined by the rate of nutrient delivery by mass transport, and,
in effect, the nutrient concentration decreases near the biofilm surface (Figure 1.12). The
general shape of the nutrient concentration profile is affected simultaneously by the rate
of mass transport and by the rate of microbial respiration. Figure 1.13 shows a profile of
dissolved oxygen above and within a biofilm. In fact, the position of each point on the oxy-
gen concentration profile shown in Figure 1.12 reflects a local equilibrium between these
two processes, oxygen delivery and oxygen removal by microbial respiration. Above the
biofilm surface, where the flow velocity is high, the concentration of microorganisms is
negligible, microbial respiration is negligible, and mass transport is intensive because of
convection. Near the biofilm surface, flow velocity decreases and so does the rate of mass
transport. In that zone, there is no microbial nutrient removal, but the nutrient concentra-
tion is affected by the microbial activity in the biofilm, located just below. To analyze the
shape of the nutrient concentration profile inside the biofilm, microbial respiration and
the nutrient removal that is associated with it must be taken into account. This new factor
changes the curvature of the profile, from concave down, above the biofilm surface, to con-
cave up, below the biofilm surface; the slope of the nutrient concentration profile increases
faster than it would if the nutrient concentration were affected only by the mass transport
limitations. Near the surface, the nutrient concentration is low and may reach zero even in
relatively thick biofilms like the one shown in Figure 1.12. Because the parts of the oxygen
concentration profile that are above and below the biofilm surface are affected by different
factors, mathematically the overall profile shown in Figure 1.12 consists of two profiles,
Chapter one: Introduction to biofilms and to biofilm research 21

Dissolved oxygen concentration (mg/L)

7

1 Biofilm surface

0
0 100 200 300 400 500
Distance from the bottom (µm)

Figure 1.13 Oxygen concentration profile at one location on a biofilm. From the linear part of the
middle section of the profile above the biofilm surface, between 300 and 200 μm, it can be estimated
that oxygen decreases from 6.5 to 2.0 mg/L over a distance of 100 μm, giving the profile a slope of
 4.5 mg 1L 1 µm 
450 mg/Lcm = 0.45 mg/cm4 =  . The slope of the profile at the biofilm
 100 L µm 10 cm 10−4 cm 
3 3

surface is then multiplied by the molecular diffusivity of oxygen in water, 2 × 10 –5 cm2/s, to yield the
oxygen flux to the biofilm, 9 × 10 –6 mg of oxygen/cm2 × s, in accordance with Fick’s first law.

that above the biofilm surface and that below the biofilm surface. The two parts are joined
at the biofilm surface by the requirement of flux continuity across the biofilm surface.
Thus, when characterizing nutrient solutions in biofilm systems, we are mostly inter-
ested in two factors: (1) the distribution of the chemical components in the nutrient solu-
tion and (2) the distribution of the flow velocity in the nutrient solution. If the information
about these two factors is sufficient, we can deduce the biofilm activity and mass transport
in the biofilm system. Consequently, we will focus on evaluating these two factors in such
a manner that the information can be used in mathematical models of biofilm activity
and accumulation. To evaluate the distribution of the chemical components of a growth
medium, we use a variety of microsensors, and to evaluate hydrodynamics, we use a vari-
ety of velocimetry techniques.

1.3.3 Characterizing the distribution of nutrients in biofilms

Two microsensors are particularly useful in determining biofilm activity: the oxygen elec-
trode and the hydrogen sulfide electrode. In natural biofilms, oxygen is the most common
electron acceptor if the solution is aerated, and sulfate is the most common electron accep-
tor in the absence of oxygen. The oxygen and hydrogen sulfide electrodes are used in the
two respective situations. The following text refers to measurements done using oxygen
microelectrodes but almost everything in it applies to hydrogen sulfide microelectrodes as
well, once one adjusts for the fact that oxygen is consumed by microorganisms and hydro-
gen sulfide is produced by sulfate-reducing microorganisms: the hydrogen sulfide profile
is inverted with respect to the oxygen concentration profile.
An oxygen concentration profile measured across a biofilm using an oxygen micro-
electrode is shown in Figure 1.13. Biofilm activity can be computed from these results by
22 Fundamentals of biofilm research

multiplying the slope of the concentration profile near the biofilm surface, from the solu-
tion side, by the diffusivity of oxygen in water. A system of coordinates with its origin at
the biofilm surface is used in these calculations.
A concentration profile such as the one in Figure 1.13 is measured at a selected location
in the biofilm, and the results are representative of that location only. It has been dem-
onstrated that concentration profiles measured at different locations in the same biofilm
are different. This creates a problem: which concentration profile is representative of the
biofilm under study? This question cannot be properly addressed here; we will discuss it
again in Chapter 6, where we use maps of oxygen concentrations at various levels in the
biofilm to arrive at the representative oxygen concentration profile. Meanwhile, to evalu-
ate the distribution of oxygen in a biofilm, the concentration profile can be measured at
several locations. These locations do not have to be randomly selected: it is better if they
are selected to form a pattern, which later helps in interpreting the results. Figure 1.14
shows the locations of a series of measurements; these locations were selected to be along
a straight line transecting a microcolony. As a result, the profiles measured at the locations
indicated in Figure 1.14 can be arranged and interpreted as a two-dimensional (2-D) distri-
bution of oxygen across the selected transect, as shown in Figure 1.15.

#1 #2 #3 #4 #5 #6 #7 #8

200 micrometers

Figure 1.14 The strategy for taking microelectrode measurements if the data are to be presented
in 2-D images. This approach is taken here to show the direction of the oxygen flux to the biofilm.

Cell clusters
300
240 O2 (mM)
Depth (µm)

0.142
180 0.114
0.086
120
0.010 0.058
60 0.010 0.029
0.020
0 0.020
0.010
0 120 240 360 480 600 720 840
Horizontal distance (µm)

Figure 1.15 Oxygen concentration around a microcolony. The oxygen flux, which always follows
the steepest gradient, is not always perpendicular to the surface. (Reprinted from deBeer, D. et al.,
Biotechnology and Bioengineering 43: 1131–1138, 1994.)
Chapter one: Introduction to biofilms and to biofilm research 23

Z
Y

Figure 1.16 The strategy for taking microelectrode measurements if the data are to be presented in
3-D images. This approach is convenient when the data are to be compared with confocal micros-
copy images of the biofilm. First, the images, taken at the same distances from the bottom as the
maps of the measured parameter, are quantified by computing appropriate parameters character-
izing biofilm structure; then, the values of these parameters are plotted against the average concen-
tration of each measured substance for each layer in the biofilm.

Continuous lines indicate isobars, and arrows indicate the directions of oxygen fluxes,
which are always perpendicular to the active surface. Notice that the microcolony is anoxic
in the middle, while oxygen is still detectable at the bottom, which demonstrates that the
oxygen near the bottom was transported there via channels and voids, not just by dif-
fusion through the microcolony. This conclusion demonstrates that the assumptions of
one-dimensional biofilm models, that the direction of mass transport in biofilms is per-
pendicular to the surface and that it is entirely due to molecular diffusion, may not hold
in heterogeneous biofilms. Two-dimensional images show a much more realistic picture
of oxygen concentration in biofilms than individual profiles of oxygen concentration like
the one shown in Figure 1.13. However, the true image of the oxygen distribution is 3-D.
To provide the data for imaging the 3-D distribution of a component of a biofilm, measur-
ing along a transect, as shown in Figure 1.15, is not adequate. A new approach needs to
be implemented, and this approach is shown in Figure 1.16. To image the 3-D distribution
of the oxygen concentration in a biofilm, we use measurements based on the concept of
stratified biofilms. In this approach, a biofilm is subdivided into a finite number of dis-
crete, uniform, and continuous layers. This procedure is explained in detail along with
experimental measurements in Chapter 9.

1.3.4 Characterizing distribution of effective diffusivity and biofilm density

The most striking differences between the models of homogeneous and heterogeneous bio-
films are related to the mechanisms of mass transport in the two systems: h ­ eterogeneous
biofilms are porous, water moves within the space occupied by them, and their mass trans-
port rates vary from one place to another. Particularly large differences in the mass trans-
port rate are measured between the microcolonies and the voids because mass transport
is controlled in the microcolonies by diffusion and in the voids by convection. These dif-
ferences make using a single mass transport coefficient for the entire biofilm questionable.
Whether using a single mass transport coefficient for the entire biofilm is justified or not
depends on how much it varies from place to place. To evaluate the variation in mass trans-
port rate among locations in biofilms, we introduce the concept of the local mass transport
coefficient, the mass transport coefficient at a single point within the biofilm.
24 Fundamentals of biofilm research

Evaluating the diffusivities of various substances in the matrix of EPS delivers informa-
tion about mass transport within the EPS, but not necessarily about the mass transport within
the biofilm. Because the biofilm is composed of microcolonies separated by interstitial voids,
mass transport in the voids needs to be accounted for as well. In Chapter 2, we describe a
technique we used to measure diffusivity in biofilms. It was based on injecting a tiny amount,
measured in picoliters, of a fluorescent dye into a microcolony and measuring the rate of its
dispersion using confocal microscopy. From such a set of data, diffusivity can be calculated.
This technique was suitable for measuring diffusivity within individual microcolonies. For
measurements of diffusivity in a biofilm composed of microcolonies and interstitial voids, the
microinjection technique is not suitable because the plume of the injected substance disperses
faster in the interstitial voids than it does in the microcolonies; in fact, in interstitial voids the
tracer disperses faster than it is possible to monitor using microscopy. As a remedy, the con-
cept of the local mass transport coefficient was introduced to quantify the distribution of the
mass transfer coefficient and of the diffusivity in biofilms. The tools for evaluating the local
mass transport coefficient are relatively simple and available in every microsensor laboratory:
they are amperometric microelectrodes without membranes.
The idea of using amperometric microsensors without membranes goes against the
usual principles of constructing such microsensors, as explained in Chapter 4. Amperometric
microsensors are covered with membranes to mitigate the effects of mass transport known
as the “stirring effect,” because amperometric sensors without membranes are sensitive to
mass transport in the vicinity of the sensor tips. This is an undesirable feature because the
response of the sensor depends on two variables, the concentration of the solute and the
rate of transport of the solute to the tip of the electrode. Applying a membrane with a dif-
fusivity coefficient a few orders of magnitude lower than the diffusivity coefficient of the
solute in water practically confines the entire mass transport resistance to the membrane
and makes the sensor insensitive to the mass transport resistance in the vicinity of the sen-
sor tip. Using amperometric sensors without membranes defeats this purpose and makes
such sensors useless for determining the concentration of the solute. The principle of con-
structing sensors to determine the mass transfer rate includes keeping the concentration of
the solute constant; then the sensor responds to the mass transport rate only. Such sensors
are well known: rotating electrodes are built on this principle. Electrochemical sensors are
also used to quantify mass transport rates to surfaces. To measure the distribution of the
local mass transport coefficient in biofilms, we use mobile microsensors that can measure
profiles of the mass transport coefficient across the biofilm or the mass transport coeffi-
cient at any selected location in the biofilm.
The choice of electrochemical reactant for the measurement of local mass transport
coefficients in biofilm systems is narrowly restricted by the following criteria: (1) chemical
stability; (2) high solubility; (3) low cost; (4) redox potential sufficiently different from that
of hydrogen evolution (or oxygen evolution) to give long, well-defined limiting current
plateaus within the water stability limits; and (5) low toxicity and negligible destructive-
ness to biofilm structure. One electrolyte system (Fe(CN)63–/Fe(CN)4– 6 ) meets most of the
listed criteria and is selected for these measurements. Prior to measurement, the nutrient
solution in the reactor is replaced with the electrolyte. After the electrolyte equilibrates
with the biomass, the local mass transport coefficient is measured at selected locations on
a grid. The local mass transport coefficients measured at the intersections of the grid lines
are reported as maps of the distribution of the local mass transport coefficient at various
distances from the bottom, as shown in Figure 1.17.
Studying the distribution of the local mass transport coefficient, particularly in com-
parison with the local biofilm activity, has proved to be very productive and explained
Chapter one: Introduction to biofilms and to biofilm research 25

2.00e–4

Horizontally averaged mass

c

transfer coefficient (m/s)

1.50e–4
b a

Top of the cluster

e d
1.00e–4
f

5.00e–5

0.00
0 200 400 600 800 1000 1200
Distance from the bottom (µm)

Figure 1.17 Distribution of local mass transport coefficient above and within a biofilm. Surprisingly,
the local mass transport coefficient does not follow the shape of the oxygen concentration curve.
We will explain this effect in Chapter 2. (Reproduced from Yang, S. N., and Lewandowski, Z.,
Biotechnology and Bioengineering 48: 737–744, 1995.)

some anomalies we had been measuring in biofilms (see Chapter 9 for details). However,
biofilm modeling frequently uses effective diffusivity, and it is important to know how
this parameter is distributed in biofilms. Biofilm reactions are heterogeneous, and in such
reactions the rate of mass transport is balanced with the rate of reaction, such as the nutri-
ent removal rate. Measuring local rate of reaction and local effective diffusivity at the same
locations helps in understanding the relations between activity and mass transport at the
microscale. We related local limiting current density to local effective diffusivity using
layers of agar of different and known densities, and known effective diffusivities of fer-
ricyanide. The effective diffusivity of ferricyanide in the agar layers was measured in a
diffusion cell. A calibration graph was constructed to relate the limiting current density
measured by the microelectrode and the effective diffusivity measured in the diffusion
cell. This calibration graph was further used to calculate local effective diffusivity in bio-
films from local limiting current density measurements, and prepare maps of local diffu-
sivity distribution at various distances from the surface, as shown in Figure 1.18. Different
biofilms were used to identify the effects of the growth conditions and the type of

0.9

0.6
D1

0.3
1000
Y
(µ 500 500
m
)
0 0 )
µm
X(

Figure 1.18 An example of local relative effective diffusivity (Dl) distribution at a selected distance
from the bottom. (Reprinted from Beyenal, H. and Lewandowski, Z., Water Research 34: 528–538,
2000.)
26 Fundamentals of biofilm research

­ icroorganisms on the local effective diffusivity. Finally, we used an empirical equation

m
reported in the literature to relate the local effective diffusivity to the local cell density.
The use of microelectrodes operated at the limiting current condition can be extended
to quantifying other factors affecting rates of nutrient removal in biofilm reactors. If prop-
erly designed and calibrated, such electrodes can be used to measure profiles of diffusivity
across biofilms. The variation of diffusivity in biofilms is a direct effect of the variation in
local biomass density. Using simple equations relating effective diffusivity and biomass
density (see Chapter 9 for details), profiles of effective diffusivity can be translated into
profiles of biomass density across biofilms like those shown in Figure 1.19. These reveal a
large difference in biofilm density between locations near the bottom and locations near
the biofilm surface. The biomass density near the bottom approached 100 g/L.

(a)
100
Pseudomonas aeruginosa
(v = 3.2 cm/s)
80 Mixed culture (v = 3.2 cm/s)
Mixed culture (v = 1.6 cm/s)
Biofilm density (g/L)

60

40

20

0
0 100 200 300 400 500
Distance from the bottom, z (µm)

(b)
0.50
Surface averaged relative effective diffusivity

Pseudomonas aeruginosa
(v = 3.2 cm/s)
0.45
Mixed culture (v = 3.2 cm/s)
Mixed culture (v = 1.6 cm/s)
0.40

0.35

0.30

0.25

0.20
0 100 200 300 400 500
Distance from the bottom, z (µm)

Figure 1.19 Variation of (a) biofilm density and (b) surface-averaged local effective diffusivity in the
biofilms. (Reprinted from Beyenal, H. et al., Water Science and Technology 38: 171–178, 1998.)
Chapter one: Introduction to biofilms and to biofilm research 27

1.3.5 Characterizing biofilm structure and its effects

Biofilm structure is defined as the distribution of biomass in the space occupied by a bio-
film. Different biofilms have different structures (see Chapter 2), and the structure of the
same biofilm varies over time. It has been hypothesized that the biofilm structure reflects
certain functions of the biofilm and that a biofilm can optimize its structure to control the
rate of nutrient delivery to the deeper layers of the biofilm. These and similar hypotheses
based on visual analysis of various biofilms generated the need for quantifying biofilm
structure. Large sections of this book are dedicated to quantifying biofilm structure.

1.3.5.1 Biofilm heterogeneities: Structural, chemical, physiological, and other

Biofilm heterogeneity is the extent of nonuniform distribution of any selected component
in any of the compartments of the biofilm system, such as the distribution of the biomass,
selected nutrients, selected products of microbial metabolism, or selected groups of micro-
organisms. Because there are many choices for the components selected to evaluate biofilm
heterogeneity, the term biofilm heterogeneity is usually combined with an adjective refer-
ring to the selected component, such as structural heterogeneity, chemical heterogeneity, or
physiological heterogeneity. The term biofilm heterogeneity was initially used exclusively
for referring to the nonuniform distribution of the biomass in a biofilm. As time has passed,
more types of heterogeneity have been described, and the term biofilm heterogeneity is not
self-explanatory anymore. Colloquially, however, the original meaning of biofilm hetero-
geneity is often assumed: if no indication is given as to which component is referred to, the
term heterogeneity refers to structural heterogeneity, defined as the nonuniform distribu-
tion of the biomass in the space occupied by the biofilm. Figure 1.20 gives an example of
chemical heterogeneity in biofilms, a nonuniform distribution of oxygen.
Because the component used to evaluate heterogeneity is oxygen, this type of hetero-
geneity qualifies as chemical heterogeneity. There are important consequences of such het-
erogeneity. That there are different concentrations of oxygen at equal distances from the
bottom indicates the existence of large horizontal gradients in the concentration of oxygen,
which is the necessary precondition to horizontal mass transport in biofilms. The existence
of horizontal mass transport in biofilms, in its turn, exposes the weakness of the assump-
tion of many mathematical models of biofilm activity that the mass transport in biofilms
is only from the bulk solution toward the bottom. To remedy this, it is important to relate
biofilm heterogeneity numerically to the rates of mass transport. Quantifying biofilm
heterogeneity is equivalent to quantifying the extent of nonuniform distribution. Several
tools from a statistical toolbox are available for evaluating the extent of nonuniform dis-
tribution; the most popular is the standard deviation. For example, oxygen concentrations
at locations equidistant from the surface are measured, and the differences among them
are ascribed to chemical heterogeneity. The procedure for estimating the extent of hetero-
geneity of a selected component of a biofilm is identical with the procedure for evaluating
the standard deviation of a set of experimental data, with one important difference: the
deviations from the average are not due to errors in measurement but reflect a feature of
the biofilm— heterogeneity. Using standard deviation as the measure of heterogeneity has
a disadvantage: the standard deviation has the same units as the original measurement.
Therefore, we prefer using the coefficient of variation instead, which is the standard devia-
tion expressed as a percentage of the average (see Chapter 6). Still, every time biofilm het-
erogeneity is reported, even if it is expressed as a percentage of the average, the component
chosen for the measurements should be reported as well. If the component is not reported,
we assume that the measured heterogeneity refers to the distribution of the biomass.
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