Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications (eBook)

Preface 5
References 11
Contributors 15
Contents 13
Chapter 1: Chlorophyll Fluorescence Terminology: An Introduction 19
1 Introduction 19
2 Light and Absorption 19
3 Fluorescence 22
3.1 Fast Phase (O-J-I-P) 23
3.1.1 Additional Features 24
3.2 Slow Phase (S-M-T) 25
3.3 The Saturation Pulse Method 26
3.4 Quantum Yield for PSII (FPSII) 28
3.5 Quenching 29
4 Conclusion 31
References 31
Chapter 2: In Situ Measurement of Variable Fluorescence Transients 36
1 Introduction 36
2 Phytoplankton Variable Fluorescence In Situ 37
2.1 Dynamical Protocols for Stimulating Variable Fluorescence 37
2.2 The Practical Relevance of the Single-turnover Time Scale In Situ 38
2.3 Issues Related to the Marine Light Field 41
2.4 Apparent Effects Resulting from Assemblage Composition 43
2.5 Effects Due to Optical Properties of Natural Waters 44
3 Conclusions and Future Directions 45
References 45
Chapter 3: Overview of Fluorescence Protocols: Theory, Basic Concepts, and Practice 48
1 Introduction 48
2 Theoretical Background 49
2.1 The Fluorescing Properties of Chlorophyll a 49
2.2 Source of Fluorescence in Seawater and Mathematical Description of Fluorescence Emission 50
2.3 The Functional Organization of the Photosynthetic Apparatus 53
2.3.1 Photosystem II 53
2.3.2 The Photosynthetic Chain 55
2.4 Adaptation, Acclimation, Regulation of Phytoplankton 56
2.5 Fates of Absorbed Photons Within PSII 56
2.6 A Simple Model of In Vivo Processes In PSII At Room Temperature 58
2.6.1 Quantum Yield of Fluorescence 58
2.7 Charge Separation at PSII 59
2.8 Photochemical Quenching of Fluorescence 59
2.9 Non-photochemical Quenching of Fluorescence 60
2.9.1 Energy-dependent Non-photochemical Quenching 60
2.9.2 Quenching Due to State Transitions 61
2.9.3 Quenching Linked to Inhibition 61
2.9.4 Reaction Center Quenching 61
2.10 Transient Changes in Fluorescence 61
3 Protocols for Measurement of In Vivo Phytoplankton Fluorescence, and the Use of Chl a Fluorescence to Study Phytoplankton 62
3.1 The Determination of Biomass In Vivo 62
3.1.1 Basic Principle 63
3.1.2 Instruments and Protocols 63
3.1.3 Validity of the Underlying Assumptions 64
3.1.4 Examples 65
3.2 Spectrofluorometry 67
3.2.1 Basic Principle 67
3.2.2 Instruments and Protocols 68
3.2.3 Validity of the Underlying Assumptions 68
3.2.4 Examples 69
3.3 Sun-induced Chlorophyll Fluorescence 69
3.3.1 Validity of the Underlying Assumptions 71
3.3.2 Examples 72
3.4 Flow Cytometry 73
3.5 Laser Excitation and LIDAR Fluorometry 73
3.6 Variable Fluorescence 74
3.6.1 Basic Principle 74
3.6.2 Instruments and Protocols 76
Use of DCMU 76
Pulse Amplitude Modulation 76
Pump-and-Probe 76
Fast Repetition Rate 77
3.6.3 Validity of the Underlying Assumptions 77
3.6.4 Examples 79
4 The Use of Chlorophyll Fluorescence to Estimate Primary Production 81
4.1 Variable Fluorescence 81
4.1.1 If is Available (FRRF and Pump and Probe Protocol) 81
4.1.2 When is not Available (PAM Protocol) 83
4.2 Sun-induced Chlorophyll Fluorescence 83
5 Conclusion 84
6 List of Symbols 84
References 86
Chapter 4: Fluorescence as a Tool to Understand Changes in Photosynthetic Electron Flow Regulation 92
1 Introduction 92
2 Electron Usage in Photosynthesis 92
2.1 Alternative Electron Cycling (AEC) 93
2.2 Electron Usage to Produce New Biomass 94
3 Effect of Light Stress on Fluorescence Signatures and their Interpretation 95
4 Use of Chemicals for the Differentiation of Photosynthetic Processes 99
4.1 Inhibitors of Linear Electron Transport 99
4.2 Inhibitors of Cyclic Electron Transport 101
4.3 Inhibitors of Alternative Electron Cycling (AEC) 101
4.4 Inhibitors of CO2 Fixation 102
4.5 Electron Transport Uncouplers 102
4.6 Electron Acceptors 103
References 103
Chapter 5: Microscopic Measurements of the Chlorophyll a Fluorescence Kinetics 107
1 Introduction 107
2 Fluorescence Techniques in High Resolution 109
3 Applications of Fluorescence Kinetic Microscopy 113
References 116
Chapter 6: Estimating Aquatic Productivity from Active Fluorescence Measurements 118
1 Fluorescence as a Probe for Photosynthesis 118
2 Overview of the Theory of Calculating ETRPSII 119
2.1 Measuring fPSII¢ and Calculating ETR 121
2.2 Examining Changes to the Quantum Yield Under Actinic Light 122
3 Light Absorption by Photosystem II 123
3.1 Bio-Physical Measures of PSII Absorption and Calculationof Chlorophyll-Specific ETR 123
3.2 Bio-Optical Based Determinationsof PSII Absorption 126
4 Reconciling Active Fluorescence-based Estimates of Productivity with Gas Exchange 128
4.1 Practical Constraints in Comparing Fluorescence- and Gas Exchange-Based Productivity Measurements 131
4.2 Are ETRs Indicative of Gross O2 Evolution? 135
4.3 Estimating Net O2 Production and C-Fixation from ETRs 136
4.4 Reconciliation of ETRPSII : O2 : CO2 Estimates 137
5 Future Application of ETRs to Primary Productivity Studies 137
References 138
Chapter 7: Taxonomic Discrimination of Phytoplankton by Spectral Fluorescence 143
1 Introduction 143
2 The Principles of Taxonomy by Spectral Fluorescence 144
2.1 Energy Transfer Between Pigments 145
2.2 Taxonomic Differences in Fluorescence Spectra 146
2.3 Taxonomic Discrimination by Spectral Fluorescence 148
3 Variation in Chlorophyll-specific Fluorescence, FChl 152
3.1 Inter-Specific Variability 152
3.2 Intra-specific Variability 154
3.3 Short-Term Quenching 160
4 Optical Indices and Application of the SFS Approach in the Field 163
4.1 Bias in SFS by Background Absorption and Scattering 165
4.2 Quenching In Situ and Taxonomic Assessment 167
5 A Field Test of the SFS Approach 170
6 Conclusion 178
References 178
Chapter 8: Flow Cytometry in Phytoplankton Research 184
1 Introduction 184
2 Background and Historical Perspective 184
3 Select Research Applications 186
3.1 Picophytoplankton Community Structure and Dynamics 186
3.2 Time Resolved Pulses for Physiological and Ecological Studies 187
3.3 Cell Sorting for Physiology and Diversity 188
3.4 Interpretation of Optical Variability in the Ocean 190
4 Emerging Approaches and Applications 191
References 193
Chapter 9: The Use of the Fluorescence Signal in Studies of Seagrasses and Macroalgae 199
1 Introduction 199
2 Major Achievements Using the Chlorophyll a Fluorescence Signal in Seagrass and Macroalgae Studies 201
2.1 Quenching Analysis 204
2.2 Analysis of Quenching Components: Use of Chemicals 205
3 Protocols Used, Limitations and Specific Modifications for Aquatic Macrophytes 207
3.1 Determination of the Variation in Fv/Fm and DF/Fm¢ 207
3.2 Limitation of the Use of Rapid Light Curves (RLC) 208
3.3 The Importance of Photosynthesis Induction 209
3.4 Determination of Absorptance, PSII Effective Absorption Cross-Section and the Number of Reaction Centers 210
3.5 Use of Relative ETR Values (rETR) 212
3.6 The Use of Electron Transport Rates Values (ETR) as Descriptors of Gross Photosynthesis (GPS) 213
4 Final Comments 216
References 216
Chapter 10: Chlorophyll Fluorescence in Reef Building Corals 221
1 Introduction 221
2 Natural Patterns of Fluorescence 222
2.1 Multiple and Single Turnover Instrumentation 223
2.2 Non-Photochemical Quenching 224
3 Detecting Stress 226
4 Protocols and Pitfalls 228
4.1 Dark Acclimation, Sample Area and Related Matters 228
4.2 Electron Transport Rate 229
5 Conclusion 231
References 231
Chapter 11: Assessing Nutrient Status of Microalgae Using Chlorophyll a Fluorescence 235
1 Introduction 235
2 Defining Nutrient Limitation 236
3 The Effects of Nutrient Limitation on Phytoplankton 236
3.1 Nitrogen 236
3.2 Phosphorus 236
3.3 Iron 237
4 Measuring Nutrient Limitation 238
4.1 Nutrient Enrichment Bioassays 238
4.2 Chlorophyll a Fluorescence as a Measure of Nutrient Stress 239
4.3 Natural Population Enrichments and Chlorophyll a Fluorescence 239
5 NIFTS 240
5.1 What is a NIFT? 240
5.2 How to Measure NIFTs 240
5.3 The Characteristics of the NIFT Response are Dependent on the Limiting Nutrient 241
5.4 NIFT Responses of Different Taxa 242
5.5 Mechanisms Behind NIFTs 242
6 Conclusion 244
References 244
Chapter 12: The Application of Variable Chlorophyll Fluorescence to Microphytobenthic Biofilms 248
1 Introduction to Benthic Biofilms 248
2 The Effects of Subsurface Signal 249
2.1 Microphytobenthic Biofilms on Soft Sediments 249
2.2 Stromatolites – the effect of “layered” biofilms 252
2.3 Deconvolution of Depth Integrated Signals 253
3 Down Regulation Through Non-photochemical Quenching 254
3.1 NPQ and the Xanthophyll Cycle in Diatoms 254
3.2 NPQ in the Dark 257
4 The Quantification of the Microalgal Biomass Using Fluorescence 258
5 Calculation of Electron Transport Rate: ETR v rETR 259
5.1 Multiple and Single Turnover Methods 259
5.2 The MT-method 259
5.3 The ST-method 260
5.4 Assumptions and Uncertainties 261
5.5 Calculation of ETR in Microphytobenthos Studies 262
6 Light Response Curves 263
6.1 A Brief Overview of Methodology 263
6.2 Steady State Light Curves 264
6.3 Rapid Light Curves 265
6.4 Non-sequential Light Curves 268
6.5 Light Curves Summary 268
7 Comparison of Fluorescence with Other Methodologies 269
8 General Summary 281
References 281
Chapter 13: Chlorophyll Fluorescence Applications in Microalgal Mass Cultures 287
1 Preface 287
2 Historical Overview of Using Chl Fluorescence in Microalgal Mass Cultures 287
3 Microalgae Grown for Commercial Purposes and Cultivation Systems 288
4 Principles of Microalgae Mass Culturing 290
4.1 Culture Maintenance 291
5 Interpretation of Chl Fluorescence Parameters in MicroalgaeMass Cultures 291
6 Chlorophyll Fluorescence Monitoring in Microalgal Mass Cultures 295
7 Light Adaptation – Non-photochemical Fluorescence Quenching 298
8 Major Achievements in Microalgal Mass Culture Monitoring 299
9 Concluding Remarks 299
References 300
Chapter 14: Delayed Fluorescence 303
1 Introduction 303
2 Historical Overview 303
3 Basic Characteristics of Delayed Fluorescence 304
3.1 Delayed Fluorescence Decay Kinetics and Intensity 304
3.2 Physiology 306
3.2.1 Temperature and Illumination Intensity Dependance 306
3.2.2 Influence of Toxins 309
3.2.3 Nutrients 309
3.3 Delayed Fluorescence Excitation Spectroscopy 312
3.4 Photosynthetic Activity Index (PhAI) 313
3.5 Delayed Fluorescence Visualisation 313
4 Delayed Fluorescence Applications 313
4.1 Description of the Instrument: What is Needed 313
4.2 Toxicity Tests 314
4.3 Primary Production 315
4.4 Delayed Fluorescence Excitation Spectroscopy 315
References 317
Chapter 15: The Study of Phytoplankton Photosynthesis by Photoacoustics 320
1 Introduction 320
2 The Photoacoustic Method 320
3 Efficiency of Photosynthesis and Photosynthesis Versus Energy Relationship 321
4 The Effect of Nutrient Limitation on Photosynthesis 322
5 The Effect of Lead Poisoning on Photosynthesis 322
6 Conclusions 323
References 323
Index 325