Biofouling Methods

Biofouling Methods provides a cook book for both established workers and those new to the field. The methods included in this important new book range from tried and tested techniques to those at the cutting edge, encompassing the full diversity of this multidisciplinary field.

The book covers methods for microbial and macrofouling, coatings and biocides, and ranges from methods for fundamental studies to methods relevant for industrial applications. There is an emphasis on answering questions and each chapter provides technical methods and problem-solving hints and tips.

Bringing together a wealth of international contributions and edited by three internationally known and respected experts in the subjectBiofouling Methods is the essential methodology reference in the field for all those working in the antifouling industry including those involved in formulation of antifouling products such as paints and other coatings. Aquatic biologists, ecologists, environmental scientists and lawyers, marine engineers, aquaculture personnel, chemists, and medical researchers will all find much of interest within this book. All universities and research establishments where these subjects are studied and taught should have copies of this important work on their shelves.

Dr. Sergey Dobretsov has worked for more than 20 years on biofouling, is widely published, and is the co-inventor on four international antifouling patents. He trained as a biologist in St Petersburg State University, Russia, and has worked in leading biofouling research centers in Russia, Hong Kong, Germany, and the USA. He is currently an Assistant Professor at Sultan Qaboos University, Oman. He is on the editorial boards of the journalsMarine Ecology Progress Series andBiofouling.

Dr. David N. Williams is the RD&I Director for AkzoNobel Marine& Protective Coatings. Based in the North East of England he originally trained as a chemist at Durham University and at Lausanne University, Switzerland. His specific expertise is in the area of nonbiocidal antifouling technologies and he is the co-inventor on a number of patents on silicone foul-release coatings and applications.

Dr. Jeremy C. Thomason is a marine biologist, a former academic at a British University and Royal Society Industrial Research Fellow, and now runs a scientific and technical consultancy, Ecoteknica, from the Yucatán, México. He has worked in the field of biofouling for more than 20 years, is co-inventor on several patents, and is a co-editor of the bookBiofouling also published by Wiley Blackwell in 2010.

Introduction xvi

Guide to Methods xviii

Part I Methods for Microfouling 1

Part Editor: Sergey Dobretsov

1 Microscopy of biofilms 3

Section 1 Traditional light and epifluorescent microscopy 4Sergey Dobretsov and Raeid M.M. Abed

1.1 Introduction 4

1.2 Determination of bacterial abundance 8

1.3 Catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) 9

1.4 Suggestions, with examples, for data analysis and presentation 12

Acknowledgements 13

References 13

Section 2 Confocal laser scanning microscopy 15Koty Sharp

1.5 Introduction 15

1.6 Materials, equipment, and method 18

1.7 Image acquisition 21

1.8 Presentation 21

1.9 Troubleshooting hints and tips 21

1.10 Notes 23

References 23

Section 3 Electron microscopy 26Omar Skalli, Lou G. Boykins, and Lewis Coons

1.11 Introduction 26

1.12 Transmission electron microscopy (TEM) 27

1.13 Scanning electron microscopy (SEM) 35

References 40

2 Traditional and bulk methods for biofilms 44

Section 1 Traditional microbiological methods 45Hans-Uwe Dahms

2.1 Introduction 45

2.2 Enrichment culture, isolation of microbes 45

2.3 Counting methods 48

2.4 Troubleshooting hints and tips 49

References 50

Section 2 Bulk methods 52Sergey Dobretsov

2.5 Introduction 52

2.6 Measurement of biofilm thickness 53

2.7 Biofilm dry weight determination 54

2.8 Biofilm ATP content 55

2.9 Troubleshooting hints and tips 56

Acknowledgements 57

References 57

3 Biocide testing against microbes 58

Section 1 Testing biocides in solution: flow cytometry for planktonic stages 59Tristan Biggs, Tom Vance, and Glen Tarran

3.1 Introduction 59

3.2 Method introductions 60

3.3 Pros and cons 66

3.4 Materials and equipment 67

3.5 Methods 68

3.6 Troubleshooting hints and tips 70

3.7 Suggestions 71

References 72

Section 2 Biocide testing using single and multispecies biofilms 76Torben Lund Skovhus

3.8 Introduction 76

3.9 Questions to answer when applying biocides 76

3.10 Laboratory methods for testing biocide effect 78

3.11 Field methods for testing biocide effect 81

3.12 Troubleshooting hints and tips 83

Acknowledgements 84

References 84

4 Molecular methods for biofilms 87

Section 1 Isolation of nucleic acids 88Isabel Ferrera and Vanessa Balagué

4.1 Introduction 88

4.2 Materials 89

4.3 Isolation of DNA from a biofilm 90

4.4 Troubleshooting hints and tips 91

References 91

Section 2 PCR and DNA sequencing 93Christian R. Voolstra, Manuel Aranda, and Till Bayer

4.5 PCR and DNA sequencing: General introduction 93

4.6 PCR 93

4.7 Microbial marker genes 16S 94

4.8 DNA sequencing 95

4.9 454 16S amplicon pyrotag sequencing 95

4.10 Protocol 1: DNA extraction using the Qiagen DNeasy Plant Mini Kit 96

4.11 Protocol 2: Full-length 16S PCR using the Qiagen Multiplex Kit 98

4.12 Protocol 3: Analysis of full-length 16S genes 100

4.13 Protocol 4: 16S amplicon PCR for 454 sequencing using the Qiagen Multiplex Kit 102

4.14 Protocol 5: Trimming and filtering of 454 16S pyrotag sequencing 106

4.15 Protocol 6: Taxon-based analyses 108

4.16 Protocol 7: Phylogeny-based analyses 109

References 111

Section 3 Community comparison by genetic fingerprinting techniques 114Raeid M.M. Abed and Sergey Dobretsov

4.17 Introduction 114

4.18 History and principles of the methods 115

4.19 Advantages and limitations of fingerprinting techniques 116

4.20 Materials and equipment 116

4.21 Suggestions for data analysis and presentation 121

4.22 Troubleshooting hints and tips 121

Acknowledgements 122

References 122

Section 4 Metagenomics 125Sarah M. Owens, Jared Wilkening, Jennifer L. Fessle, and Jack A. Gilbert

4.23 Introduction and brief summary of methods 125

4.24 Overview of metagenomics methods 125

4.25 Method introduction 126

4.26 Overview of DNA handling for BAC library construction 127

4.27 BAC and Fosmid library construction 127

4.28 Library handling, archiving, and databasing 128

4.29 Facilitating library screening 128

4.30 Time frame considerations 129

4.31 Materials and equipment 129

4.32 Detailed methods: DNA handling and BAC library construction 130

4.33 Troubleshooting tips 131

4.34 Suggestions for data analysis 132

4.35 Suggestions for presentation of data 134

Acknowledgements 135

References 135

5 Methods for biofilm constituents and turnover 138

Section 1 Destructive and nondestructive methods 139Arnaud Bridier, Florence Dubois-Brissonnet, and Romain Briandet

5.1 Introduction 139

5.2 Pros and cons of destructive and nondestructive M-LSM methods for biofilm analysis 140

5.3 Materials and equipment required for M-LSM 140

5.4 Example of questions than can be answered with the method 140

5.5 Suggestions for data analysis and presentation 148

References 149

Section 2 Biofilm formation and quorum sensing bioassays 153Clayton E. Cox, William J. Zaragoza, Cory J. Krediet, and Max Teplitski

5.6 Introduction 153

5.7 Materials and equipment 157

5.8 Methods 157

Acknowledgements 165

References 165

6 Sampling and experiments with biofilms in the environment 168

Section 1 Field trials with biofilms 169Jeremy C. Thomason

6.1 Introduction 169

6.2 Materials and equipment 170

6.3 Method 170

6.4 Troubleshooting hints and tips 171

6.5 Suggestions for data analysis and presentation 172

References 173

Section 2 Sampling from large structures such as ballast tanks 175Robert L. Forsberg, Anne E. Meyer, and Robert E. Baier

6.6 Introduction 175

6.7 Materials and equipment 178

6.8 Troubleshooting hints and tips 180

6.9 Analytical methods 180

6.10 Suggestions for data analysis and presentation 182

References 182

Section 3 Sampling from living organisms 184Christina A. Kellogg

6.11 Introduction 184

6.12 Historical background 185

6.13 Advantages and limitations of collection techniques 185

6.14 Protocols 186

6.15 Suggestions for data analysis 187

6.16 Troubleshooting hints and tips 187

Acknowledgment 188

References 188

Section 4 Optical methods in the field 190Richard J. Murphy

6.17 Introduction 190

6.18 Examples of the use of optical methods 191

6.19 Spectral characteristics of biofilms 192

6.20 The use of chlorophyll-a as an index of biomass of biofilm 193

6.21 Multi-versus hyperspectral measurements (CIR imagery versus field spectrometry) 194

6.22 Calibration of data to reflectance 195

6.23 Suggestions for data analysis and presentation 195

6.24 Methods 197

6.25 Troubleshooting hints and tips 201

References 202

7 Laboratory experiments and cultures 204

Section 1 Static, constant depth and/or flow cells 205Robert L. Forsberg, Anne E. Meyer, and Robert E. Baier

7.1 Introduction 205

7.2 Portable Biofouling Unit 207

7.3 Pros and cons of the method 207

7.4 Materials and equipment 208

7.5 Suggestions for data analysis 209

7.6 Benchmark bacteria and biofilm characterization 210

7.7 Troubleshooting hints and tips 212

References 212

Section 2 Mixed population fermentor 214Jennifer Longyear

7.8 Introduction 214

7.9 Pros and cons 215

7.10 Fermentor 215

7.11 Mixed species microfouling culture 215

7.12 Utilizing the fermentor test section 218

7.13 Troubleshooting, hints and tips 218

References 219

Part II Methods for Macrofouling, Coatings and Biocides 221

Part Editors: Jeremy C. Thomason, David N. Williams.

8 Measuring larval availability, supply and behavior 223

Section 1 Larval availability and supply 224Sarah Dudas and Joe Tyburczy

8.1 Introduction to measuring larval availability and supply 224

8.2 Measuring settlement and recruitment 235

References 238

Section 2 Larval behavior 241Jeremy C. Thomason

8.3 Introduction 241

8.4 Method for tracking larvae 242

8.5 Troubleshooting hints and tips 245

8.6 Suggestions for data analysis and presentation 246

References 249

9 Assessing macrofouling 251

Section 1: Assessing fouling assemblages 252João Canning-Clode and Heather Sugden

9.1 Introduction 252

9.2 A note on taxonomy 253

9.3 Field methods 253

9.4 Digital methods 258

9.5 Functional groups 261

9.6 Predicting total richness: from the known to the unknown 264

References 267

Section 2 Assessment of in-service vessels for biosecurity risk 271Francisco Sylvester and Oliver Floerl

9.7 Introduction 271

9.8 Surveys of vessel hulls 272

9.9 Sample and data analysis 277

Acknowledgements 279

References 279

Section 3 Experiments on a global scale 281Mark Lenz

9.10 Experiments in ecology: the need for scaling up 281

9.11 GAME a program for modular experimental research in marine ecology 281

9.12 Marine macrofouling communities as model systems 282

9.13 Chronology of a GAME project 283

Acknowledgements 289

References 289

10 Efficacy testing of nonbiocidal and fouling-release coatings 291Maureen E. Callow, James A. Callow, Sheelagh Conlan, Anthony S. Clare, and Shane Stafslien

10.1 Introduction 291

10.2 Test organisms 293

10.3 Test samples 294

10.4 Antifouling settlement assays 295

10.5 Fouling-release assays 299

10.6 Adhesion assays for high-throughput screening 304

10.7 Apparatus 310

Acknowledgements 313

References 314

11 Contact angle measurements 317

Section 1 Surface characterization by contact angle measurements 318Doris M. Fopp-Spori

11.1 Introduction 318

11.2 Liquids in contact with solids 318

11.3 Reproducible contact angle measurements 320

11.4 Surface energy calculations 323

References 324

Section 2 Underwater contact angle measurement by the captive bubble method 326Pierre Martin-Tanchereau

11.5 Introduction 326

11.6 Materials and requirements 327

11.7 Method 329

11.8 Surface energy 330

Acknowledgements 330

References 331

12 Efficacy testing of biocides and biocidal coatings332Christine Bressy, Jean-François Briand, Chantal Compère, and Karine Réhel

12.1 Introduction 332

12.2 Laboratory assays for biocides 333

12.3 Field test methodology for biocidal coatings 337

References 343

13 Commercialization 346

Section 1 Processing a new marine biocide from innovation through regulatory approvals towards commercialization 347Lena Lindblat

13.1 Introduction 347

13.2 Basics about the regulatory landscape from the academic perspective 349

13.3 Risk, risk assessment and risk management 349

13.4 Future directions 353

13.5 Conclusions 355

References 356

Section 2 From laboratory to ship: pragmatic development of fouling control coatings in industry 358Richie Ramsden and Jennifer Longyear

13.6 Introduction 358

13.7 Laboratory coating development 358

13.8 Laboratory bioassay screening 359

13.9 Fitness for purpose (FFP) testing 360

13.10 Field antifouling performance testing 361

13.11 Test patch and vessel trials 363

13.12 Performance monitoring 364

13.13 Summary 365

References 365

Index 366