Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications PDF
Preview Chlorophyll a Fluorescence in Aquatic Sciences: Methods and Applications
Chlorophyll a Fluorescence in Aquatic Sciences Methods and Applications Developments in Applied Phycology 4 Series Editor: Michael A. Borowitzka School of Biological Sciences & Biotechnology Murdoch University, Murdoch, Western Australia For other titles published in this series, go to www.springer.com/series/7591 David J. Suggett · Ondrej Prášil Michael A. Borowitzka Editors Chlorophyll a Fluorescence in Aquatic Sciences Methods and Applications Editors David J. Suggett Ondrej Prášil Department of Biological Sciences Institute of Microbiology University of Essex Opatovciky mlyn CO4 3SQ Colchester 379 81 Trebon United Kingdom Czech Republic Michael A. Borowitzka Algae R&D Center School of Biological Sciences and Biotechnology Murdoch University Murdoch 6150, WA Australia ISBN 978-90-481-9267-0 e-ISBN 978-90-481-9268-7 DOI 10.1007/978-90-481-9268-7 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2010932001 © Springer Science+Business Media B.V. 2011 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microflming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifcally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Preface It is unquestionable that Chlorophyll a fuorescence is also in how we continue to sustainably exploit our quite literally a global phenomenon. Fluorescence ever-changing environment. merely describes an optical phenomenon where light The history of using fuorescence to investigate bio- absorbed at one wavelength is re-emitted at another mass, photosynthetic physiology and primary produc- (longer) wavelength; it exists passively in nature and tivity has been covered in several comprehensive occurs wherever light exists to be absorbed by publications, most recently by Papageorgiou and Chlorophyll a molecules. These molecules are a Govindjee (2005) (and chapters therein); however, it is common property of all photoautotrophic organisms of course important to note the place of aquatic studies on land and in water; thus Chlorophyll a fuorescence is in this history, at least for the context of the following essentially ubiquitous in nature (Fig. 1). It is incredible chapters. Whilst many major developments in using that such a natural phenomenon has been exploited by variable Chlorophyll a fuorescence have arguably such a wide variety of researchers and across the come from studies on terrestrial (vascular) plants, free- biological and environmental sciences, and perhaps is living microalgae (chlorophytes in particular in par- testament to the importance we place on understanding ticular) and cyanobacteria have proved to be important photoautotrophic activity. We have long known that laboratory organisms in examining principal photobio- Chlorophyll a fuorescence of photosynthetic organi- logical mechanisms. Examining such aquatic organ- sms varies as a result of changes in the amount isms under controlled laboratory conditions is a (biomass), as well as function (quantum yield), of perhaps an obvious step; aside from the relative ease of Chlorophyll a present. At operational temperatures probing photosynthetic machinery of single celled that exist in most natural environments, Chlorophyll a compared to multi-cellular organisms, microalgae and fuorescence is largely derived from the Chlorophyll a cyanobacteria dominate photosynthetic activity of associated with photosystem II (PSII), i.e. the oxygen much of the Earth’s aquatic realm. However, in con- evolving complex; as such, changes in the quantum trast to working on terrestrial plants, extending such yield of fuorescence directly relate to changes in laboratory-based observations to the ‘real world’ has photosynthetic (O evolving) capabilities. Thus, by proven to be the greatest challenge for aquatic scien- 2 actively inducing changes in Chlorophyll a fuorescence tists and one that has been largely led by technology using an actinic light source, we can perturb the and engineering. In overcoming the technical chal- physiological status quo of (PSII) photoautotrophy lenges, exciting and important discoveries, such as the itself. Packaging of technology to enable induction confrmation of iron limitation of ocean productivity and measurement of such Chlorophyll a fuorescence (Behrenfeld et al. 1996) and the discovery of aerobic perturbations has entirely made possible examination anoxygenic bacteria (Kolber et al. 2001), have of processes associated with plant and algal ecology, followed. physiology and productivity, and at scales from the The earliest application of Chlorophyll a fuores- single cell to the entire planet (van Kooten and Snel cence to aquatic system research (in situ) is well recog- 1990). Therefore, it is hard to imagine a future that nized as from Carl Lorenzen (1966) who frst pumped does not continue to exploit the properties of seawater through a shipboard fuorometer. Such a con- Chlorophyll a fuorescence, not only for research but venient, rapid approach was quickly adopted by both v vi Preface Fig. 1 Fluorescence in action: (a–c) Chloroplast fuores- area of the pyrenoid as light intensity increases (Photo: R. cence in the dinofagellate Ceratium sp. (Photo: L. Perkins); (e) Chloroplast fuorescence in the centric diatom Novoveska); (d) False colour high resolution fuorescence Coscinodiscus sp. (Photo: L. Novoveska); (f) Delayed fuo- image of cells of the diatom Nitzschia dubia. Fluorescence rescence in the colonial diatom Rhizosolenia (Photo: M. emanating from the chloroplasts becomes restricted to the Berden-Zrimek) Preface vii oceanographic and limnological communities; this, light source evaluated i ndependently from a relatively not surprisingly quickly led to a wealth of highly novel long yet moderate intensity light pulse; and Pump and studies linking physical and biological processes, in Probe (PP; Mauzerall 1972; Falkowski et al. 1986; but particular, the distribution of phytoplankton with ocean see also Kolber and Falkowski 1993), where variable turbulence (Platt 1972) and the discovery of the deep fuorescence is measured by a weak ‘probe’ actinic chlorophyll maximum of stratifed waters (Cullen and fash before and after a saturating ‘pump’ fash. PP Eppley 1981). The major challenge for aquatic scien- later evolved into Fast Repetition Rate (FRR; Kolber tists to evolve to in situ studies was ‘simply’ to pack- et al. 1998), where a complex fuorescence transient age complex and innovative technology into a system could be induced by initially delivering a series of sub- that could withstand the constraints of working in saturating high intensity fashlets followed by a series water, especially in marine environments where salts of more widely spaced ‘probing’ fashlets that exam- and pressure rapidly build. It wasn’t until the 1970s ined the subsequent fuorescence decay. All subse- that technology caught up with concept and the frst quent variable fuorometers have essentially followed proflable in situ fuorometers were truly developed one (or a combination) of these paths. Importantly, this (see Falkowski and Kolber 1995). Ever since, such new generation of fuorometers not only opened new fuorometers have become smaller and better inte- possibilities for examining photoautotrophic physiol- grated to sensor arrays, and essentially a routine yet ogy but also a potential revolution in how aquatic sci- fundamental tool for aquatic scientists. However, entist would determine primary productivity (Kolber despite their rapid adoption by the aquatic community, and Falkowski 1993, Kromkamp and Forster 2003; these fuorometers were still generally restricted to Suggett et al., Chapter 6, this volume). assaying a single chlorophyll fuorescence yield, which Evolution of both PAM and FRR (PP) was origi- was set according to the excitation intensity of the nally driven from the pioneering laboratory work using instrument in question, and thus could only ever pro- microalgae; as such, the frst in situ variable fuorom- vide some approximate measure of Chlorophyll a bio- eters in the 1980s and 1990s were essentially restricted mass in situ. An important step to aquatic research was to working on natural phytoplankton suspensions in thus in producing fuorometers that induced a variable lakes and oceans. Technical improvements in overall Chlorophyll a excitation (and hence fuorescence emis- signal resolution since then has enabled researchers to sion) protocol (Fig. 2). investigate ever more oligotrophic waters of oceans Numerous laboratory studies by the 1970s and early and nutrient impoverished lakes. However, subtle tech- 1980s had already demonstrated important concepts nological changes in sensitivity and the optical con- linking variable Chlorophyll a fuorescence to photo- fgurations within a few years of PAM and FRR synthetic physiology in aquatic algae (e.g. Mauzerall fuorometer introduction enabled the photophysiology 1972; Ley and Mauzerall 1982; but note an ISI Web of of benthic autotrophs (corals, microphytobenthic mats, Science search yields >125 publications in the 1970s seagrasses and macroalgae) to be examined (see alone!), however, modifcation of these techniques to Chapter 9 by Enríquez and Borowitzka, Chapter 10 by in situ aquatic studies to thus add a physiological com- Warner et al., and Chapter 11 by Shelly et al., this vol- ponent (the variable fuorescence ‘transient’) to mea- ume). More recent additional but relatively small opti- sures of fuorescence yield (biomass) was not cal alterations to the PAM and FRR ‘model’ to examine straightforward. Here, the development of actinic light far red fuorescence (>800 nm) has introduced more sources that could deliver the intensity and/or fre- new research opportunities, e.g. bacteriochlorophyll a quency of excitation required to induce variable fuo- (Kolber et al. 2001) and Photosystem I (PSI) variable rescence remained an even greater technological fuorescence (Dual PAM, e.g. see Sukenik et al. 2009). challenge to the pre-existing in water operational con- Modifcation of the spectral quality of fuorescence straints. Solving this problem essentially had to occur excitation and emission detection has also added the twice since variable fuorescence techniques have potential for variable fuorometers to taxonomically already evolved into two parallel but distinct paths discriminate bulk fuorescence properties (Schreiber (Chapter 3 by Huot and Babin, this volume): Pulse 1998; Beutler et al. 2002; Chapter 7 by MacIntyre Amplitude Modulation (PAM; Schreiber et al. 1986), et al., this volume). All of these advances have unques- where fuorescence is induced by a weak modulated tionably facilitated the explosion of interest in the use viii Preface Fig. 2 Fluorometers in action: (a) Diving PAM used to measure Haematococcus culture grown in solar photobioreactor at the fuorescence signal in corals, Wakatobi Marine National Park, Centre of Biological Technologies, University of South Bohemia Indonesia (Photo: D. Smith); (b) Fluorometer comparisons at in Nové Hrady, Czech Republic (Photo: J. Masojidek); (d) FRRF GAP Workshop, Eilat, Israel, 2008 (Photo: D. Suggett); being deployed in winter, Bedford Basin, Canada (Photo: (c) In-situ measurement of fuorescence quenching using the D. Suggett); (e) Fasttrack II attached to a CTD frame in water fuorometer PAM 101-103 (H.Walz, Germany) in the column sampling in Eilat, Israel (Photo: D. Suggett) Preface ix of fuorometers for aquatic research in recent years; fuorescence data sets, however, we are only recently arguably, compared to 20 years ago, aquatic research arming ourselves with the key knowledge required to investigations are incomplete without some form of interpret and consequently apply these data into fuorescence examination. informed opinion. Examining the growth of citations Variable fuorometers have clearly provided a plat- for (variable) fuorescence-based papers over the past form for aquatic scientists wishing to conveniently decade is perhaps more testament to our confdence in assay photosynthetic physiology non-invasively and interpreting the data as opposed to reduced constraints more accurately scale changes of photosynthesis to the in collecting the data itself. Armed with a decades environment. Current acceleration of environmental worth of what is arguably ‘fuorescence exploration’, variability via climate change perhaps provides very It is really only now that we are beginning to gain max- real justifcation for further investing in tools such as imum beneft of using fuorescence as a tool to address fuorometers that have the capacity to link ecosystem fundamental research questions in the aquatic sciences. processes with environmental regulation. Fluorescence- Why the need for AQUAFLUO? — Rapid growth of based technological development (including delayed using active fuorescence across the aquatic science fuorescence; Chapter 14 by Berden-Zrimec et al., this disciplines has inevitably led to divergence in approach volume) combined with research has publically pro- and terminology (see Chapter 1 by Cosgrove and duced a tool that can potentially inform stakeholders Borowitzka, this volume, for recommended termino- of the photosynthetic ‘viability’ (or ‘health’) of their logy). Even though many of us have been attempting associated aquatic environment; certainly, a tool that is to answer similar questions, this divergence has less labour intensive and costly in the long term than resulted in a lack of consistency required to facilitate conventional (and destructive) assays that require information exchange; consequently, the feld was not water or organisms to be removed and analysed in the evolving as quickly as originally envisaged. Arguably, laboratory. Such applications to those wishing to the aquatic sciences still communicate fuorescence- m onitor and subsequently manage ecosystem function based studies in numerous dialects that are often not was an obvious step in exchanging the knowledge easily inter-comparable or reconcilable. beyond pure research but also necessary for commer- Using fuorescence as a non-invasive means for cial manufacturers to invest further in instrument pro- assaying processes, such as (harmful) bloom detection duction. Key examples to date come from the monitoring and primary productivity, is still heralded as a key of lakes and coastal waters for (harmful) algal blooms breakthrough for aquatic research and not surprisingly (Cullen et al. 1997) and coral reefs for pollution and has attracted much funding and research time invest- coral bleaching (Jones 1999). PAM Fluorometry has ment. However, efforts to capitalise on these larger also been demonstrated in action for two BBC docu- process-scale problems have somewhat overshadowed mentaries – by Prof Ove Hoegh-Guldberg examining our need to understand the fundamental nuances of coral bleaching for the BBC documentary State of the fuorescence measurements using different instrumen- Planet and by Dr Rupert Perkins investigating stromato- tation, protocols and for the array of aquatic primary lites for Oceans) Furthermore, recent developments of producers that exist. On many occasions, the interpre- algae as biofuels will inevitably require application of tation of data sets has been confounded by what is real fuorometers to optimize and also continually monitor in nature versus an artifact of instrument use. yields (Kromkamp et al. 2009; Sukenik et al. 2009) and Conversations amongst the aquatic sciences commu- thus further move fuorometers from a purely ecological nity over recent years have increasingly identifed the to an industrial monitoring tool. need for conformity in the application and operation of Despite the potential growth industry that obviously (active) fuorometers, not only to standardise and rec- exists for chlorophyll fuorescence, it is clear that the oncile existing data sets but also to ensure that fuo- previous growth of fuorometer technological develop- rometry remained ‘accessible’ to the ever-growing ment and the subsequent array of commercially avail- new user community. Such a step is indeed critical if able fuorometers have somewhat superseded our fuorometry is ever to evolve from a purely academic fundamental understanding of the fuorescence signals tool to an everyday, practical and informative manage- generated. It is perhaps quite ironic that technological ment tool. However, despite attempts to call for a com- developments have already enabled us to collect vast mon set of approaches (the best example to date for the x Preface aquatic sciences is Kromkamp and Forster 2003), a fuorometer manufacturers and engineers. By bringing few researchers have adapted their own approach and/ this group together, the needs of all interested or terms to ft. Perhaps the main limitation in adapting s takeholders could for the frst time identify the com- (amongst this rapidly growing and evolving feld) has mon and complimentary needs required to move the been where on earth should we start? feld further forward and identify new opportunities; as The concept of AQUAFLUO (AQUAtic FLUO well, to understand the individual needs of the various rescence) was introduced in 2005 following breakout aquatic disciplines and develop specifc approaches discussions between several of us at a meeting on that may help to bridge gaps in consistency in the fuo- modeling algal growth in Villefranche, France. It was rescence yields that were being measured. A series of then apparent that the immense popularity of fuo- talks and workshops at the AQUAFLUO 2007 meeting rometry for aquatic studies was not a transient phe- led by leaders in key aquatic disciplines successfully nomenon. Both researchers and industry were laid the foundations in exploring these questions. increasingly investing in development of new fuo- Targeted research activities conducted since the meet- rometers; however, the fundamental operational and ing are already beginning to demonstrate that the origi- conceptual issues that were limiting how confdently nal ethos of AQUAFLUO and the outcomes of the trends in variable fuorescence could be scaled to 2007 meeting are being adopted. However, given the biology had become a bottleneck in supporting (and continually evolving nature of using chlorophyll fuo- ultimately rationalising the need for furthering) this rescence in both physiological concept and technologi- industry growth. Therefore, an organizing committee cal approach, AQUAFLUO is seen as the beginning of was established from a cross section of the aquatic a long-term relationship across the aquatic community community and these concerns translated into the and an idea with underlying goals and priority ques- ethos for an international meeting to be held at Nové tions that will inevitably need to be continually Hrady in the Czech Republic in 2007 (Prášil et al. revisited. 2008). Initial priority questions were identifed, The chapters of this book communicate key compo- including: nents of the talks and workshops conducted during the AQUAFLUO 2007. Primarily, they address what mea- 1 . Is it time to step back from trying to focus on using surements can be made and how; the common and variable fuorescence as a substitute for conven- aquatic discipline specifc pitfalls that may be encoun- 14 13 tional productivity ( C, C, O ) primary p roductivity tered in both performing measurements and interpret- 2 techniques? Should we redistribute current research ing the fuorescence yields themselves. In essence the efforts and instead focus on the assessment of algal book provides a guide to making Chlorophyll a fuo- physiology and the general heterogeneity of algal rescence measurements in the various aquatic sciences physiology in nature? and is certainly aimed at experienced and new users 2 . How important (for the end users) are nuances in alike. This book is certainly not intended to be a com- current fuorescence induction methodologies (e.g. prehensive review on the subject of Chlorophyll a fuo- single or multiple turnover fashes; single band vs. rescence; several key aspects are not considered in multi-spectral)? Can we constructively use these depth, notably remote sensing of variable fuorescence differences in experimental techniques to get better and examination of AAPs. insight into algal physiology? Each chapter summarises the progress specifc to 3 . Can we expect successful scaling from direct to that discipline, the journey that Chlorophyll a fuores- remotely sensed (LIDARs and satellites) variable cence has taken in both approach and application; con- fuorescence? sequently, the scientifc information that can be obtained. Importantly, these chapters also mark the It was immediately clear that addressing such prior- output of that meeting: The frst targeted effort to ity questions could only be achieved by bringing amalgamate the concerted efforts of leaders of the var- together researchers from across the aquatic disciplines ious felds/aquatic disciplines to identify what the next (microalgae, macroalgae, submerged vascular plants, major conceptual (physiological, ecological, biogeo- corals and aerobic anoxygenic photoheterotrophs chemical) questions that fuorescence measurements (AAPs); lakes, rivers, coasts and oceans) as well as can contribute? What are the technological challenges
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