**Abstract**

Pigments, as a vital part of phytoplankton, act as the light harvesters and protectors in the process of photosynthesis. Historically, most of the previous studies have been focused on chlorophyll *a*, the primary light harvesting pigment. With the advances in technologies, especially High-Performance Liquid Chromatography (HPLC) and satellite ocean color remote sensing, recent studies promote the importance of the phytoplankton accessory pigments. In this chapter, we will overview the technology advances in phytoplankton pigment identification, the history of ocean color remote sensing and its application in retrieving phytoplankton pigments, and the existing challenges and opportunities for future studies in this field.

**Keywords:** phytoplankton, pigments, remote sensing, ocean color, satellite

## **1. Introduction**

Phytoplankton live near the water surface to capture sufficient light for photosynthesis and act as the primary producer of the plankton community. They form the bottom levels of the marine and aquatic food webs, and their existence not only makes life in the water possible but also makes the ocean an important food source for mankind. Phytoplankton play a crucial role in the biogeochemical cycles of many important chemical elements, not only carbon but also of other elements, such as silica and nitrogen [1–4]. The release and uptake of CO2 and CH4, and the excretion of dimethylsulphide by phytoplankton influence the atmosphere and climate [5]. As a result of the changes in their living condition, their composition and concentration vary over space and time, which in turn can influence the whole ecosystem, such as through the changes in the size structure, formation of harmful algal blooms and development of hypoxic regions. Blooms and hypoxia can disrupt food-webs and threaten human health.

Phytoplankton pigments capture sunlight. The resulting photosynthesis and its products, especially the oxygen and organic compounds, all rely on the light energy captured by the different phytoplankton pigments [6–8]. Chlorophyll *a* is the major pigment for light harvesting. Accessory pigments (e.g. chlorophylls *b* and *c*, carotenoids, and phycobiliproteins) also play a significant role in photosynthesis and photoprotection, by extending the light collection window and protecting the cell from damage of high irradiance levels or high ultraviolet light exposure. With the commercial availability of fluorometers, routine measurements of chlorophyll *a* became possible. That single technology to measure chlorophyll *a* fluorescence made the measurement a universal parameter for estimating phytoplankton

biomass and productivity. As a result of improvements in culturing, microscopy, HPLC and molecular methods, rapidly separating and quantifying pigments from different phytoplankton has become possible [9–11]. These new measurements make it possible to use phytoplankton pigments as indicators to elucidate the composition and fate of phytoplankton in the world's oceans [12].

Light absorbed by phytoplankton pigments provides the initial energy for carbon cycles, and is also one of the major factors influencing the appearance of water color [13–16]. To study this important water column phenomenon, ocean color remote sensing was first proposed in late 1970s. Satellite-based ocean color remote sensing provides unique observational capability to scientists for phytoplankton studies by providing synoptic views of the ocean with high spatial and temporal resolution. Since the Coastal Zone Color Scanner (CZCS) mission, chlorophyll *a* retrieval has been the principle focus of ocean color remote sensing research (e.g., [17]). Whereas this focus continues to the present [18–20], an evolving interest in retrieving other pigments, has emerged in recent years.

What follows, based on the most recent research findings from the ocean color community, is a brief review of how phytoplankton pigments are estimated from water samples, how pigment maps are derived from satellite measurements and what are the existing challenges and opportunities for the estimates and application of remote sensed pigments. This chapter is not meant to present a comprehensive list of all possible topics related to satellite-based pigment observations, but rather its focus is on the history of pigment retrievals with several examples showing major findings. For interested readers, a full breadth and depth knowledge in this field can be obtained by reading the refereed literature and technical reports compiled on the National Aeronautics and Space Administration ocean color website (https://oceancolor.gsfc.nasa.gov) and by International Ocean Color Coordinating Group (http://www.ioccg.org).

## **2. Phytoplankton and pigment properties**

#### **2.1 Optical properties**

#### *2.1.1 Absorption properties*

Optical properties of phytoplankton, especially the absorption coefficients of the pigments inside them (**Figure 1**), play a key role in determining not only the use of this radiant energy for photosynthesis, but also the penetration of the radiant energy within water. These pigment absorption coefficients are important for identifying and quantifying phytoplankton groups [12] and size class distributions (IOCCG report 15 and references therein), understanding of photosynthetic rate [11, 21], and in particular for ocean color interpretation.

Light absorption properties of phytoplankton cells from laboratory cultures as experimental materials have received a great deal of attention in fundamental photosynthesis research [22, 23]. However, the phytoplankton pigment absorption properties from natural water is the information needed in ocean color remote sensing. The collection of phytoplankton pigment information has been obtained from measurement of the spectral absorption of phytoplankton, usually through filtration onto a filter pad because of the low *in situ* concentrations of phytoplankton in the water [24].

Using data on pigment concentrations and their absorption properties, Kirkpatrick *et al*. [25] used the specific pigment absorption peaks for identification of phytoplankton types. This method has been integrated into spectral shape-based

**Figure 1.**

*Weight-specific (or pigment-specific) in vitro absorption spectra of various pigments derived from measuring the absorption spectra of individual pigments in solvent and shifting the maxima of the spectra according to Bidigare et al. [14]. Data obtained courtesy of Annick Bricaud (See Bricaud et al. [15]). Credit to Moisan et al. [30].*

remote sensing algorithms [26, 27]. However, the absorption of phytoplankton is more complicated than a simple sum of the absorption properties of individual pigments. Differences in pigment composition and the pigment package effect influence not only the magnitude but also the shape of the spectra of phytoplankton absorption [14, 15, 28–30]. All these introduce variabilities in the specific absorption coefficients and increase the uncertainties in the application of such information.

Hoepffner and Sathyendranath [29] proposed Gaussian decomposition of phytoplankton absorption spectra. For the first time, this method decomposed the absorption spectra into Gaussian curve components and linked them to the light absorption coefficients of multiple pigments inside phytoplankton cells. Several studies followed this proxy to estimate multiple phytoplankton pigments for different water bodies [31–33] but were limited to using only *in situ* measured absorption coefficients. Wang et al. [34, 35] proposed a semi-analytical algorithm to obtain these Gaussian curves and pigment absorption coefficients from ocean color remote sensing data.

#### *2.1.2 Fluorescence*

A portion of the light absorbed by phytoplankton pigments can be emitted at a longer wavelength in a physical process called fluorescence [36]. The energy dissipated in fluorescence is secondary to the amount absorbed and used for photosynthesis, but it is still significant enough to be observed in ocean color remote sensing data. Chlorophyll *a* fluorescence has been the most significantly used fluorescence feature (**Figure 2**), and the detection and products from satellite ocean color sensors have been widely used [37, 38]. Several other phytoplankton pigments (pheopigments and phycobilins) can also fluoresce.

**Figure 2.** *Chlorophyll* a *fluorescence emission. Data from Du et al. [42] and Dixon et al. [43].*

Several factors influence phytoplankton fluorescence: nutrient conditions, stage of growth, physiological state of phytoplankton, pigment content and ratios, taxonomic position of algae, and photoadaptation [39–41]. *In situ* chlorophyll fluorescence has been the most frequent method for describing the chlorophyll and phytoplankton variation and distribution in the ocean [41], but all the uncertainties from the pigment properties make the interpretation of the chlorophyll fluorescence data a challenge.

#### **2.2 Pigment measurements**

Historically, chlorophyll *a* has been routinely derived from filtered fluorometric measurements following standard methods using commercially availability of fluorometers. However, even standard methods yield varying results depending on the composition of pigments within the phytoplankton, and errors can be on the order of 50% [44–46]. The presence of significant amount of chlorophyll *b* and/or chlorophyll *c*, causes fluorometric techniques to under- or over-estimate Chlorophyll *a* with respect to fluorometric measurements [44–47]. The pigment package effect is also a major source of concern.

The introduction of pigment analyses by high-pressure liquid chromatography (HPLC) [48, 49] facilitated easy and accurate separation, identification, and quantification of phytoplankton pigments. Pigment detection based on HPLC methods enables quantification of over 50 phytoplankton pigments [11, 50]. Some of the pigments can be used as diagnostic pigments for phytoplankton groups (e.g., fucoxanthin for diatoms, peridinin for dinoflagellates, alloxanthin for cryptophytes, chlorophyll *b* for chlorophytes, 19′-hex-fucoxanthin for haptophytes, and 19′-but-fucoxanthin for pelagophytes) [51, 52]. Moreover, diadinoxanthin and diatoxanthin are generally found in dinoflagellates (Phylum Miozoa, Class Dinophyceae) and diatoms (Phylum Bacillariophyta, Class Bacillariophyceae), whereas lutein, prasinoxanthin, neoxanthin, and violaxanthin are found in class Chlorophyceae (Phylum Chlorophyta) and class Prasinophyceae (Phylum Chlorophyta). Chlorophyll *a*, *c*, and β-carotene are used as general indicators of

total algal biomass. Phytoplankton are also often categorized into three different groups: micro-phytoplankton (20–200 μm), nano-phytoplankton (2–20 μm), and pico-phytoplankton (0.2–2 μm) [53]. The contribution of each group can also be calculated using its pigment signatures [54].
