Introduction

This chapter presents a diverse collection of perspectives covering recent discoveries and ‘crystal ball gazing’ on future directions. Detection and characterisation from a molecular level is covered through to monitoring phytoplankton dynamics and climate change at a regional and global Earth observation level. At a molecular level, perspectives are provided on our basic understanding of the role of pigments in photosynthesis and photoprotection incorporating the development of new analytical and ‘omics’ techniques. Applied perspectives are included on HAB detection, aquaculture and algal biotechnology. Phytoplankton pigment research continues to develop opening up many fascinating and exciting possibilities. These perspectives highlight how research on pigments acts as a linchpin across a diverse range of disciplines including microbial ecology, oceanography, limnology, remote sensing and applied phycology.

Description and role of MAAs

It is now generally believed that natural ultraviolet-A radiation (UV-A, 315–400 nm) and ultraviolet-B radiation (UV-B, 280–315 nm) are strong environmental factors affecting both productivity and community structure in marine and terrestrial ecosystems (de Mora et al., 2000). Reduction of the stratospheric ozone layer, which has caused an increase in the UV-B flux to the Earth's surface in recent years (Farman et al., 1985), could result in increased levels of UV-induced damage for most living organisms (Vincent and Neale, 2000), producing a great impact on the photosynthetic carbon fixation by plants and, consequently, on the global climate change (UNEP, 2006). At the beginning of the evolution of life on Earth, UV-B flux rates clearly exceeded the present values (Cockell and Horneck, 2001) resulting in the evolution of several protection strategies to counteract the negative effects of UV radiation (Roy, 2000). One of the adaptations whereby phytoplankton can reduce UV-induced damage is the synthesis of compounds that can absorb the damaging wavelengths and dissipate the absorbed energy without generating phototoxic reactive intermediates. A variety of such compounds have been found in aquatic and terrestrial plants (Rozema et al., 2002).

As early as 1938 there were observations of UV-absorbing compounds in marine algae (Kalle, 1938; referenced in Sivalingam et al., 1974). This was followed in 1969 by reports of UV-absorbing substances (named S-320) in water extracts from several species of corals and a cyanobacterium (most likely Trichodesmium) from the Great Barrier Reef (Shibata, 1969). Mycosporine-like amino acids (MAAs) from marine organisms were first isolated and characterized by Hirata and co-workers (Hirata et al., 1979). They isolated and characterized mycosporine-glycine from the tropical zoanthid Palythoa tuberculosa (Ito and Hirata, 1977), a compound previously isolated from mycelia of sporulating fungi (Favre-Bonvin et al., 1976), and then described several related imine derivatives of mycosporines (Hirata et al., 1979). Since then, more than 20 closely related MAA compounds have been isolated and characterized from several plants and marine animals (Figure 10.1 and Table 10.1). Wide ranging studies indicate that these compounds occur in virtually all taxa of marine and freshwater cyanobacteria and algae, in invertebrate–microbial symbioses and in metazoans (Karentz et al., 1991; Gröniger et al., 2000; Karentz, 2001).

LC-MS analysis of chlorophylls and carotenoids: introduction

Because of improvements in HPLC methods for the analysis of phytoplankton pigments, pigments that are unidentified but spectrally related to known compounds are frequently reported in microalgal cultures as well as in natural distributions (Jeffrey and Wright, 1994; Vaulot et al., 1994; Garrido and Zapata, 1998; Airs et al., 2001a; Carreto et al., 2001; Zapata et al., 2004). Distinctions between common chlorophylls and carotenoids can be ascertained during HPLC from on-line UV/visible (UV/Vis) spectra, or co-elution with authentic standards. Several chlorophyll types however occur as suites of compounds. A diverse array of chlorophylls c have been observed in marine microalgal cultures as free acids or esterified by a range of non-polar groups (Garrido and Zapata, 1998; Garrido et al., 2000; Zapata et al., 2001, 2006). Photosynthetic bacteria belonging to the genera Chlorobiaceae produce suites of bacteriochlorophylls differing both in the degree of alkylation at positions C-8 and/or C-12 (Smith and Bobe, 1987; Airs and Keely, 2002), and/or the alcohol esterified to the propionic acid group at C-17 (Caple et al., 1978; Otte et al., 1993; Airs et al., 2001b). Furthermore, chlorophyll a undergoes a range of transformation and alteration reactions when the phytoplankton cell is compromised and as detrital material is transported through the water column; these reactions are potentially indicative of specific environmental conditions or processes (Head and Horne, 1993; Head et al., 1994; Veldhuis et al., 2001; Walker and Keely, 2004). Several carotenoid types also exist as suites of compounds, for example fucoxanthin esters (Airs and Llewellyn, 2006). Characterisation of novel compounds involves rigorous chemical and analytical techniques following preparative isolation of individual components (Egeland et al., 2000). Such an approach can be impractical when unknowns are present in low relative abundance. Liquid chromatography-mass spectrometry (LC-MS) permits acquisition of structural data during a single chromatographic run. Molecular mass information, used in conjunction with PDA UV/vis spectra is often sufficient for the assignment of components. HPLC coupled to tandem MS (LC-tandem MS) adds a further dimension and can be used to identify structural differences that do not affect the UV/Vis absorption properties, or to distinguish ions with the same mass-to-charge ratio (isobaric ions). Furthermore, LC-tandem MS is particularly powerful if MS/MS spectra of an unknown compound are compared with a structurally related, identified compound.

Introduction

A quality assurance plan (QAP) describes a process to ensure an analytical method fulfils the agreed upon accuracy objectives at all points during the analysis of samples. A QAP includes such things as standardized procedures, method validation, quality control (QC) measurements, and quality assessment (QA); the latter quantitatively describes how results of QC measurements are used to determine whether a method is performing within expectations. By analogy, a QAP describes what can be considered, in a more general sense, a ‘holistic’ approach to sample analysis, whereby the ‘whole is considered to be a result of the interdependence of all parts’. Here, the ‘whole’ represents the overall combined uncertainty of a final data product, and ‘the interdependence of all parts’ represents the uncertainties contributed by the many individual procedures required to produce that final data product.

An in depth discussion of uncertainty analysis in the chemical laboratory, given in EURACHEM (2000), is beyond the scope of this chapter, but the importance of a so-called holistic approach to sample analysis, whether this includes a formalized QAP or not, is necessary to provide knowledge of the uncertainties associated with measured values and, thus, facilitate confidence in the data products. Such knowledge is important, because pigment data are often compiled in increasingly large databases, and end users of the data are remote (in space and time) from the data providers. Knowing the accuracy of the data facilitates their wider utility, for both current and unanticipated future applications. This chapter describes components of a QAP in the context of knowledge gained during intercomparisons sponsored by the National Aeronautics and Space Administration (NASA). A primary objective of these activities was to determine if the myriad providers of HPLC analyses to NASA researchers were satisfying the accuracy requirements for field observations.

UV-Vis spectra

HPLC chromatogram of Chroomonas salina (system 1)

Preface

In 1997, the Scientific Committee on Oceanic Research (SCOR) (with support from the United Nations Educational, Scientific and Cultural Organization (UNESCO) and the editors' institutions) sponsored a volume on phytoplankton pigments entitled Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods. This volume was edited by Drs S. W. Jeffrey, R. F. C. Mantoura and S. W. Wright and resulted from the activities of SCOR Working Group 78. The 1997 volume went out of print a few years after publication (about 2000 copies were sold), which prompted UNESCO Publishing to print another 500 copies in 2005.

In April 2006, SCOR sponsored a workshop of pigment specialists from around the world to examine updates in this field. This workshop was hosted by Dr R. Fauzi C. Mantoura and the International Atomic Energy Agency's Marine Environmental Laboratory in Monaco. The updates that were identified include new advances in the taxonomy of marine phytoplankton (several new algal groups have been described since 1997), improved analytical techniques (notably HPLC-linked mass spectrometry, not generally used for pigment analysis before 1997), and new applications for pigments. The outcome of this meeting was a consensus that an update of the original 1997 volume was urgently needed, and a new editorial team was nominated. The present volume is the result of this update. Two of the three former editors of the 1997 volume contributed to the present volume (S. W. Jeffrey and S. W. Wright). Their collaboration ensures a smoother transition between the two volumes and prevents repetition, focusing instead on developments since the 1997 volume.

Introduction

The microalgae that make up the extensive phytoplankton pastures of the world's oceans originated in ancient evolutionary times. They obtained their primitive ‘plastids’ from an unknown ancestral cyanobacterium with photosynthetic oxygen-evolving capabilities (Bhattacharya, 1997; Delwiche, 1999; McFadden, 2001; Palmer, 2003; Keeling, 2004a, b). Serial symbioses within heterotrophic hosts gave rise to the present wide diversity of photosynthetic microalgae, which evolved a range of photosynthetic pigments capable of collectively harvesting most of the wavelengths of light available to them in underwater marine habitats (Jeffrey and Wright, 2006).

At the present time, the marine phytoplankton contribute at least a quarter of the biomass of the world's vegetation, and constitute the base of the food web that supports either directly or indirectly all the animal populations of the open sea. Some microalgae also contribute significantly to climatic processes, providing nuclei for atmospheric water condensation (Aiken et al., 1992). All microalgae, by their photosynthetic activities, contribute to atmospheric carbon dioxide ‘draw-down’ (Jeffrey and Mantoura, 1997), thus helping to ameliorate green-house gases, by removing nearly a third of the anthropogenic carbon released to the atmosphere (Sabine and Feely, 2007).

Filtration

In Chapter 10 of Jeffrey et al. (1997), Whatman GF/F (or equivalent) filters (0.7 μm nominal pore size) were recommended for sample filtration. With the exception of targeted studies, GF/F filters remain the most commonly used media for routine filtration and accompanying in vitro analyses by HPLC, fluorometry and spectrophotometry. As indicated in the above-mentioned Chapter 10 (p. 284–287), many studies have compared the effectiveness of different filter types and highlighted their advantages/disadvantages. Of particular note are three relatively recent papers highlighting the limitations of GF/F filters. Knefelkamp et al. (2007) compared six different filter types and concluded that Whatman nylon membranes (0.2 μm pore size, 47 mm diameter) provided the most consistent results with respect to chlorophyll a analyses. Nucleopore filters (0.2 μm) have been reported to retain as much as four times the amount of chlorophyll a as GF/F filters in open ocean samples (Dickson and Wheeler, 1993). Furthermore, Lee et al. (1995) reported that GF/F filters retained only 13–51% of small bacterioplankton (< 0.8 μm diameter) in natural samples. In contrast, recent comparisons of filter types have reported no differences in pigment concentrations obtained using GF/F and membrane filters in a variety of aquatic habitats (Chavez et al., 1995; Morán et al., 1999). The choice of filter type, whether glass-fibre or membrane, should be determined by the individual investigator for their particular application. However, once a filter type is selected, it should be used uniformly for sample filtrations to insure consistency between samples.

In estuarine and coastal waters, particulate matter (seston) may result in rapid saturation and ‘clogging’ of filters. Continued vacuum filtration of ‘clogged’ filters may promote mechanical stress and induce cell lysis, potentially resulting in underestimation of the actual pigment concentrations in the sample (Goldman and Dennett, 1985; Taguchi and Laws, 1988; Richardson and Pinckney, 2004). The total time for sample filtration should not exceed 5–10 min to minimize filter saturation (Wasmund et al., 2006). Filters should be removed as soon as the passage of water through the filter is undetectable and the vacuum should never exceed 50 kPa. Although not commonly used, positive pressure filtration (7–14 kPa) reportedly allows the filtration of larger volumes of water with reduced filtration times (Gibb et al., 2001; Bidigare et al., 2002). Regardless of the filtration method used, multiple filters can be pooled to achieve the biomass necessary for HPLC analyses. After filtration, filters should be folded in half, blotted on absorbent paper to remove excess water, and immediately flash frozen and stored in liquid nitrogen or at −80 °C (Wright and Jeffrey, 2006).

Introduction

Carotenoids are among the natural products with the highest diversity. To date more than 700 different naturally occurring carotenoids have been described (Britton et al., 2004), and they are virtually ubiquitous in living organisms. Carotenoids belong to the compound class of isoprenoids, and the majority of carotenoids are tetraterpenoids with a C40 skeleton as the basic molecular structure. Formally, they can be divided into the carotenes, which are pure hydrocarbons, and the xanthophylls, which are derived from carotenes by introduction of oxygen functions. The ability of de novo biosynthesis of carotenoids is not limited to land plants and algae, but is frequently encountered among prokaryotes (both eubacteria and archaebacteria) and fungi (Britton, 1998). To meet their metabolic demands, animals rely on food-borne uptake of carotenoids which then, however, can be further metabolized. An important example is β-carotene (provitamin A; trivial names for carotenes are used in this chapter – see Data sheets for alternative names such as β,β-carotene in this case) as a precursor of the visual pigment retinal in mammals and animals in general (Goodwin, 1984; von Lintig et al., 2005) or – together with its hydroxylated derivatives – as precursors of ketocarotenoids used as colourants by many crustaceans, various carp species and birds like flamingos or finches (Goodwin, 1984; McGraw et al., 2006). This chapter summarizes our current view on the biosynthesis of carotenoids, termed carotenogenesis, in land plants, cyanobacteria and algae with a focus on recent advances in the genetics of carotenoid biosynthesis. The scope of this review will be limited to oxygenic phototrophs; for details on the carotenogenesis of anoxygenic phototrophic bacteria, readers are referred to an excellent recent review by Maresca, Graham and Bryant (Maresca et al., 2008).

When the chapter on carotenoids in the volume by Jeffrey et al. (1997) was written, only the enzymes of the cytosolic mevalonic acid pathway of isoprene formation and those catalysing the early steps of carotenoid biosynthesis up to the carotenes were known. During the last decade, most of the genes involved in carotenogenesis in cyanobacteria and in land plants have been identified, and a new pathway of isoprene formation that is operative in many bacteria and in plastids has been discovered. Most of our current knowledge about the biosynthesis of carotenoids in oxygenic phototrophs stems from work on seed plants and cyanobacteria, and to some extent on green algae. Almost no experimental data are available from other algal groups, but increasingly more whole-genome data are generated enabling homology-based identification of potential carotenogenic genes in these algae. As will be discussed, the emerging picture is rather complex with a significant number of reactions being catalysed by multiple enzymes which are often phylogenetically unrelated. Other enzymes share the same ancestor, but yet catalyse different reactions. In the following, the known enzymatic steps of carotenogenesis in oxygenic phototrophs will be summarized, with special reference to carotenoid biosynthesis in algae and cyanobacteria.

The most direct way to measure phytoplankton growth/production rate is to quantify changes in biomass over a period of time, something difficult to achieve methodologically in nature. A second and serious problem is the choice of the biomass unit for measurements. Many different macromolecules have been suggested (e.g. proteins, nucleic acids, ATP, lipids and pigments). However, carbon (C) is commonly the standard unit for biomass and the desired currency for productivity models (MacIntyre et al., 2000). Because carbon is common to all organisms, estimation of phytoplankton rates based on carbon measurement is not straightforward. Among macromolecules, pigments are specific to phytoplankton. Their specificity and distinct optical properties make pigments a useful tool for studying phytoplankton processes.

Phytoplankton dynamics reflect the balance between production and losses, notably through population growth and grazing. It is, however, difficult to determine these rates simultaneously, and significant differences can be observed in the short term (hours, days), on the scale of phytoplankton generation times. Active growth is accompanied by a parallel grazing pressure exercised mostly by microzooplankton. This chapter reviews the pigment-based methods used to measure both production and grazing rates, focusing on incubation-dependent methods: the 14C-pigment labeling technique and the serial dilution bioassay. Finally we will briefly comment on other approaches such as satellite imagery and fast repetition rate fluorometry (FRRF), which are free of incubation-derived artifacts.