Chlorophylls are necessary for photosynthesis, and are present in all photoautotrophic organisms (Saini et al 2020). They are commonly used in the food industry (as green dyeing agents, additive E140(i)), and in pharmaceutical and cosmetic preparations for their antioxidant, antimutagenic and antimicrobial properties, among other health benefits (Levasseur et al 2020). Although green microalgae can be a source of more chlorophyll content than terrestrial plants (e.g. 6.7% of the dry weight (DW) of Chlorella vulgaris (Chlorophyta) (Nakanishi and Deuchi 2014)), plants (mainly grass or alfalfa) remain the major natural sources of chlorophyll, as their production is the most cost-effective (Deepika et al 2022). Thus, the production of microalgae chlorophylls is of limited commercial interest, and should only be considered as part of a multiple compound’s valorization during biorefinery processes (Nakanishi and Deuchi 2014; Levasseur et al 2020; Chen et al 2022).

Chlorophylls consists of four pyrroles (4 carbons and 1 nitrogen ring, C4H5N) connected by one-carbon bridges to form a conjugated tetrapyrrole ring, with a fused modified cyclopentanone. The center of this tetrapyrrole ring holds a magnesium ion (Cao et al 2023). The chemical structure of the five existing forms of chlorophyll (Chl a, b, c, d, and f) differs according to their C17-C18 carbon bond, and to five side chain substituents (R1, R2, R3, R4, and R5) in the tetrapyrrole ring, respectively at the C2, C3, C7, C8 and C17 carbon positions (Fig. 1).

The structure and the maximum absorption bands of chlorophylls

Chl a, the predominant form of chlorophyll, is present in the photosynthetic reaction center of photosynthetic organisms, and in the antenna complex. Chl b is also present, as auxiliary pigment, in the antenna complexes of all green microalgae (Chlorophyta), but absent in red microalgae (Rhodophyta), which instead contain Chl d (da Silva and Lombardi 2020). Other microalgae (Heterokontophyta), particularly including brown microalgae (Phaeophyceae) and diatoms (Bacillariophyceae) use Chl c as an auxiliary photosynthetic harvesting pigment. It is worth noting that Chl d has been reported to be present in a Cyanobacteria (Acaryochloris marina) accounting for more than 95% of its total Chl content. Chl f was discovered in 2010 in the Cyanobacteria Halomicronema hongdechloris (Cao et al 2023).

All chlorophylls absorb the light in two main regions of the visible spectrum, exhibiting bands of absorption in the blue (≈400–470 nm) and the red regions (≈630–700 nm), respectively called the Soret and Q bands. The lack of absorption in the green region (between blue and red) explains their greenish color (Li and Chen 2015). The structural variances between the different forms of chlorophylls shift their respective absorption spectra (Deepika et al 2022) (Fig. 1). The maximum chlorophyll absorption in the Q band (dissolved in methanol) is around 665 (Chl a), 652 (Chl b), 630 (Chl c), 697 (Chl d), and 707 nm (Chl f) (da Silva and Lombardi 2020). Chl f is the one form of chlorophyll that absorbs at the higher (Q band) and the lower (Soret band) wavelengths. This wider absorption spectrum of Chl f can increase its photon utilization and thusly increase the biomass (Cao et al 2023).

Carotenoids are produced by all photosynthetic organisms, including all microalgae, and by some non-photosynthetic organisms (several types of bacteria and fungi) (Ashokkumar et al 2023; Simkin et al 2022). They are the most diverse class of pigments, with more than 600 natural compounds identified, including 50 varieties in algae (Levasseur et al 2020; Saini et al 2020). Carotenoids are grouped into two families, the carotenes which contain carbon and hydrogen, and the xanthophylls, which also contain oxygen. Only a few carotenoids are actually commercialized, including two carotenes (β-carotene and lycopene) and four xanthophylls (astaxanthin, canthaxanthin, lutein and zeaxanthin), all of which are present in microalgae (Ashokkumar et al 2023; Gong and Bassi 2016; Razzak 2024). Together, β-carotene and astaxanthin represent nearly 50% of the current carotenoid market (Gong and Bassi 2016).

Carotenoids, the most commercialized class of biological pigments, have many applications. First of all, β-carotene, a vitamin A precursor, is essential in our diet, to support vision, skin health, and immunity (Ashokkumar et al 2023). Furthermore, some pharmaceutical formulations include carotenoids to provide protection from the harmful effects of oxidative stress (Razzak 2024). Other related health benefits include the prevention of compromised immune response, of premature aging, and of certain cancers (Saini et al 2020). More specifically, lycopene, lutein and canthaxanthin are recognized for their respective regulation of the formation of atheromatous, cardiovascular and blood-related disorders (Saini et al 2020). In the nutraceutical eye health market, lutein and zeaxanthin are gaining importance, as they can prevent cataract and macular degeneration (Gong and Bassi 2016). Carotenoids are also widely used as colorant additives for human food, with the E160 international numbering being applicable to carotenes (carotene: E160a; lycopene: E160d), and the E161 numbering being applicable to xanthophylls (lutein: E161b; zeaxanthin: E161h; canthaxanthin: E161g; astaxanthin: E161j) (Coulombier et al 2021). Additionally, lutein, zeaxanthin, canthaxanthin and astaxanthin have been added to animal feed formulations (for poultry and farm-raised salmonids), to enhance the color of the egg-yolks and muscle tissue (Gupta et al 2021).

As an alternative to synthetic pigments, natural carotenoids are also used in cosmetic products. Particularly, some lipsticks contain lycopene and astaxanthin to provide bright red and vibrant red–orange colors (Razzak 2024). The antioxidant properties of lycopene have been exploited by the cosmetics industry, particularly for so-called anti-ageing formulations (Levasseur et al 2020). Similarly, cosmetic foundations containing β-carotene or lutein, with alleged skin rejuvenating properties, provide yellow-to-orange and light yellow-golden tones, respectively. Moreover, astaxanthin (the most potent antioxidant present in nature (Saini et al 2020)), has photoprotective properties that have been exploited via its use as sunscreen and UV protection ingredients (Razzak 2024).

Microalgal carotenoids can be classified as primary or secondary via reference to whether they have a photosynthetic or a non-photosynthetic role. Notwithstanding, lycopene plays a peculiar role, as it is the precursor of all primary and secondary carotenoids (Mulders et al 2015). Primary carotenoids (e.g. α and β-carotene, lutein and zeaxanthin), which are greater in content under normal cell growth conditions (Shi et al 2020), are necessary for adequate photosynthesis and cell survival, as structural and functional components of the photosynthetic apparatus (Eonseon et al 2003; Pagels et al 2020a). Actually, carotenoid/chlorophyll binding protein complexes are present in thylakoid membranes of Cyanobacteria and eukaryotic algae. Particularly, β-carotene is found in the core complexes of photosystem I and II (Chen et al 2020; Deepika et al 2022), while lutein and zeaxanthin are bound to the antenna proteins of the light harvesting complexes (Zheng et al 2022; Simkin et al 2022). Besides regulating and stabilizing the photosystems structures of microalgae, primary carotenoids have key photosynthetic roles (Eonseon et al 2003; Deepika et al 2022). First of all, carotenoids are indispensable for harvesting light and transferring its energy during photosynthesis (Chen et al 2020). With a light absorption range between 400 and 550 nm, including the green window not absorbed by chlorophylls (Deepika et al 2022) (Table 1), carotenoids act as accessory photosynthetic pigments. They broaden the spectrum of collected light, absorbing photons and finally transferring the excitation energy to the primary Chl a (Simkin et al 2022; Pagels et al 2020a). As such it is estimated that carotenoids are responsible for ~20–30% of the light harvested during this process (Deepika et al 2022). Additionally, primary carotenoids can also protect the photosynthesis apparatus from photooxidative damage (Chen et al 2020; Cano et al 2021). When chlorophylls receive more light than they can transfer to the PETC, they can elevate their energy from a basal singlet spin state to a triplet excited state. In turn, such triplet Chl can transfer its energy to ground state molecular oxygen, forming reactive oxygen species (ROS), among which is highly reactive singlet oxygen (Mulders et al 2014). ROS cause cell damage in the vicinity of their production area, generating protein oxidations, and chloroplasts and thylakoids membranes disruptions (Coulombier et al 2021). Thus, in excessive light, damage to the photosystems occurs, reducing photosynthetic capacity, a process known as photoinhibition (Simkin et al 2022). Some carotenoids can directly neutralize singlet oxygen when formed (Coulombier et al 2021). For instance, the β-carotene present in the core complex of photosystems quenches triplet Chl and singlet oxygen (Simkin et al 2022; Chen et al 2020). Carotenoids are also involved in a process called “non-photosynthetical quenching” (NPQ), which is conducive to lowering the energy level of singlet excited Chl and to the formation of triple state carotenoids, which can safely release energy via thermal dissipation, preventing the formation of triplet Chl, ROS and singlet oxygen (Simkin et al 2022; Pagels et al 2020a). Three primary xanthophylls present in the antenna complexes of the PSII, namely violaxanthin, antherazanthin, and zeaxanthin, abbreviated as VAZ, are particularly active in NPQ. A reversible interconversion between violaxanthin and zeaxanthin (involving antheraxanthin) occurs, known as the ‘VAZ cycle’, through a series of enzymatic reactions (Simkin et al 2022). Under oversaturating light, violaxanthin is converted to antheraxanthin and subsequently to zeaxanthin, via the effects of the deepoxidase enzyme, with the reverse epoxidation reactions occurring under low light (Mulders et al 2014). The induction of these cycles allows for the avoidance or reduction of cellular damage, as zeaxanthin is used for the dissipation of excess energy from excited chlorophylls during NPQ (Zittelli et al 2023). Thus, the zeaxanthin content of microalgae is usually regulated by light irradiance, and unstressed photosynthetic organisms contain lower zeaxanthin (Eonseon et al 2003). This cycle, which dissipates an excess of light energy as heat occurs in all species of green and brown microalgae (Gupta et al 2021).

In contrast to primary carotenoids, secondary carotenoids (e.g. astaxanthin and canthaxanthin) are not bound to the photosystems, as they are only produced when cells are exposed to a stress stimulus, under adverse growth or stress conditions, also known as carotenogenesis conditions (Gupta et al 2021; Mulders et al 2015). They accumulate outside the thylakoids, in cytoplasmic lipid globules, to form a protective layer (Pagels et al 2020a). In some green algae (Chlorella and Scenedesmus), secondary carotenoids can also accumulate in the outer cell wall layer (Cano et al 2021). When located in oil droplets outside the photosynthetic apparatus, β-carotene accumulates under stress conditions and acts as a secondary pigment (Mulders et al 2014; Shi et al 2020).

Structurally, most carotenoids have a common 40 carbon structure derived from 8 consecutive isoprene (C5H8) units, forming a set of conjugated double bonds (Gupta et al 2021) (Fig. 2). Carotenoids are fat-soluble pigments of a yellow, orange or red color (Levasseur et al 2020). Lycopene, which has an acyclic structure, is an intermediate in the biosynthesis of all major carotenes and xanthophyls (Ashokkumar et al 2023). Without altering the molecular formula (C40H56), microalgae cyclase enzymes mediate the formation of a 9 carbons ring at each end of the lycopene molecule, generating the two main isomers of carotene (α and β). The position of a double bond in one of the rings of these isomers differs, with a final structure incorporating the presence of 10 conjugated double bonds in α-carotene, and 11 in β-carotene (Ashokkumar et al 2023) (Fig. 2). In turn, such carotenes are precursors for the synthesis of microalgal xanthophylls. Lutein and zeaxanthin are formed adding a –OH function via the hydroxylation of α- and β-carotene, respectively (Patel et al 2022; Ashokkumar et al 2023; Kou et al 2020). β-carotene ketolases induce the further formation of the ketocarotenoids, with the introduction of a keto group (C=O) in α-carotene and zeaxanthin, to form canthaxanthin and astaxanthin, respectively (Saini et al 2020) (Fig. 2). Astaxanthin can also be formed by addition of (–OH) via the action of the hydroxylase enzyme on canthaxanthin (Patel et al 2022).

The structure of the carotenoids of main commercial importance

Table 1 shows a non-exhaustive list of the most commonly studied microalgal strains for carotenoid production (Ambati et al 2018; Liang et al 2019; Saini et al 2020; Bourdon et al 2021; Patel et al 2022; Razzak 2024). Among them only two green microalgae, Dunaliella salina and Haematococcus lacustris (formerly Haematococcus pluvialis), are widely used for commercialized microalgal carotenoids, namely β-carotene and astaxanthin, respectively (Rajput et al 2022). As observed in Table 1, the green microalgae possess the largest variety of carotenoid pigments (Mulders et al 2015). Particularly the chlorophyceae family (Chlorella, Dunaliella, Haematococcus, Chromochloris) is one particularly important source of carotenoids (Razzak