6.1.2. Photosynthesis

Diatoms and brown algae utilized unique light-harvesting antennas, FCPs, to perform photosynthesis [69]. The pigment compositions and protein organization of FCPs are mostly distinct from those of light-harvesting complexes (Lhcs) in land plants and green algae [70,71]. At least 30 genes related to light harvesting have been identified based on the genome of two diatoms, *Thalassiosira pseudonana* [72] and *Phaeodactylum tricornutum* [73]. The products encoded by these genes were categorized into three groups, Lhcf, Lhcr, and Lhcx proteins. Proteins under the Lhcf family are the main light-harvesting proteins [74]. PSI antennae are composed of proteins from the Lhcr family [3], while Lhcx proteins are involved in non-photochemical quenching mechanisms to protect the photosystem [75]. A high similarity of Lhcx proteins to LI818 or LhcSR proteins of green alga was observed [76].

The FCP trimer, also known as FCPa, is the basic structure of different FCP proteins in the native thylakoid membrane of pennate and diatoms [77]. Diverse populations of FCP trimeric complexes, which differed in polypeptide composition and pigmentation, were identified through the sub-fractionation of FCP complexes of *P. tricornutum* [78]. Four different trimeric subtypes of FCPa in *Cyclotella meneghiniana* were revealed, FCPa1–4. Lhcf4/Lhcf6 proteins were found mainly in the FCPa2 trimer, whereas Lhcf1 was reported to be the major subunit in FCPa1, FCPa3, and FCPa4 [79]. Buchel (2003) [80] discovered that two FCP fractions differed in the polypeptide composition and oligomeric state from *C. meneghiniana*. The first fraction consisted of trimers with mainly 18 kDa polypeptides (FCPa), while the higher oligomers assembled from different trimers (19 kDa subunits) constituted the second fraction (FCPb). Two oligomeric subtypes, FCPb1 and FCPb2, with Lhcf3 being the main subunit in both antenna complexes in *C. meneghiniana* were

revealed [79]. Different FCP complexes were observed using anion chromatography and divided into FCP complexes related to PSI, PSII core complexes, and peripheral FCP complexes. Various Lhcf proteins were detected in FCP complexes associated with PSI and PSII core complexes, whereas peripheral FCP complexes mainly contained Lhcf8 and Lhcf9. Subunits of the PSI core complex composed of Lhcr proteins and Lhcx proteins were the protein subunits that were identified in the PSII core complex [81].

The idea of the FCP trimer as the basic unit of photosynthesis antenna proteins in fucoxanthin-containing algae was contradicted by the findings based on cryo-electron miscopy [82–85] and X-ray crystallography [86]. Wang et al. (2019) [86] unraveled the X-ray crystal structure of an FCP of *P. tricornutum,* which had two monomers held together to form the dimeric structure of FCP within the PSII core. In addition, the cryo-electron microscopy data of the PSII–antenna supercomplex of *Chaetoceros gracilis* revealed a tetrameric organization of FCP proteins associated with the PSII [82]. Furthermore, 24 FCPs surrounding the PSI core of *C. gracilis* were in monomeric form based on cryo-electron microscopy [83].

Important characteristics of pigment organization of isolated FCP and the role of fucoxanthin molecules in excitation energy transfer have been unraveled using steady-state and ultrafast spectroscopic methods [87,88]. Efficient energy transfer was observed from fucoxanthin and chlorophyll *c* (Chl *c*) to Chl *a* based on spectroscopic studies [87,89,90]. At least three forms of fucoxanthin molecules differ in their photophysical and dipolar properties, Fxred, Fxgreen, and Fxblue [91,92] and were confirmed using resonance Raman spectroscopy [93]. The Fxred form transfers energy more efficiently, while the Fxblue form demonstrated less efficiency in transferring excitation energy [92]. The time of energy transfer from fucoxanthin to Chl (around 300 fs) was shorter than the transfer from Chl *c* to Chl *a* (around 500 fs–6 ps), indicating that the fastest energy transfer was between fucoxanthin and Chl *a* [94]. Most of the pump-probe studies examined the dynamical energy transfer process in FCP of fucoxanthin-containing algae. For example, Papagianakis (2005) [87] characterized the energy transfer network in FCP. The energy transfer efficiency from Chl *c* to Chl *a* is 100%, whereas unequal efficiency was observed for fucoxanthins in the FCP. Furthermore, findings based on polarized transient absorption indicated that three fucoxanthin molecules in FCPa transferred their excitation energy directly to Chl *a*. The remaining fucoxanthin molecule was not associated with Chl *a* molecules and might transfer its excitation energy through another fucoxanthin molecule to Chl *a* [89]. In addition, a detailed model was proposed in describing the energy transfer in FCPa upon excitation at two different wavelengths [95]. Another study demonstrated the highly efficient energy transfer from Fx to Chl-a through the S1/ICT state using the pump-probe method [96]. Apart from pump-probe techniques, two-dimensional electronic spectroscopy (2DES) has offered insights into energy change transfer dynamics, exciton diffusion, and molecular system relations [97–99]. The advances in knowledge of the mechanisms and dynamics of energy transfer in the FCPs of diatoms that have been accomplished using two-dimensional electronic spectroscopy (2DES) were reviewed [100].

In addition to FCPs' function as a light-harvesting complex, pigments in the FCPs are also involved in photoprotection. Non-photochemical quenching (NPQ) is the protection mechanism that most algal groups utilize to dissipate excess absorption energy as heat via molecular vibrations. One xanthophyll pigment, diadinoxanthin (Dd), was observed in a peripheral location of FCP [86]. This pigment is converted to diatoxanthin (Dt) under high light intensities [101]. The conversion showed a close relationship with the build-up of the NPQ mechanism [102]. Previously, the amount of diatoxanthin (Dt) was established to influence the NPQ mechanism in vivo, whereas the reduction of fluorescence yield of FCPa complexes in vitro was caused by Dt [103]. In addition, the acidification of thylakoid lumen regulated the ratio between Dd and Dt, which could affect the activities of the epoxidase and de-epoxidase in the NPQ mechanism [104]. Gundermann and Claudia (2012) [105] examined the factors determining the NPQ process in diatoms. The components involved in the NPQ mechanism in C. *meneghiniana* were reported to be heterogeneous and genuinely different from the NPQ type in *P. tricornutum* [106]. Apart from xanthophyll pigments

(Dd and Dt), Lhcx proteins play an essential role in the NPQ mechanism [76,107]. The amount of xanthophyll cycle pigments in various FCP preparations showed a relationship with the existence of Lhcx1 protein [108]. This protein was shown to have short-term photoprotection [107], which induced the conformational changes of the FCPs and reduced fluorescence yield [109]. The molecular structure, the arrangemen<sup>t</sup> of the different Lhc proteins in the complexes, the energy transfer abilities, and the photoprotection of other Lhc systems of Chl *c* containing organisms were reviewed recently [110].

#### 6.1.3. Optimization of Process

The optimization of the process cluster focused mainly on the biosynthesis, biotechnology, extraction, and purification of fucoxanthin. Algal cultivation and fucoxanthin production are the major components of the biosynthesis section. Previous studies demonstrated that the fucoxanthin content of microalgae is higher than that of brown macroalgae [111,112]. The fucoxanthin extracted from fresh brown macroalgae ranged from 0.02 to 0.87 mg/g fresh weight, while the dried form of microalgae showed 1.81–15.33 mg/g dried weight (DW) fucoxanthin [111]. In diatoms, the fucoxanthin content ranged from 0.224% to 2.167% (around 0.224–21.67 mg/g) of dry weight [111,113]. Currently, *P. tricornutum* is the major natural fucoxanthin source in microalgae due to its substantial fucoxanthin [111,114]. Nevertheless, the lower dry well weight (g/L) of this microalgae [114] prompted the searching for alternative fucoxanthin sources via screening. The screening of high-performance microalgae strain is crucial, as it could determine the success of producing the desired amount of fucoxanthin prior to algal cultivation. Previous studies attempted to screen for potential microalgae with a high production of fucoxanthin [115,116]. For instance, Guo et al. (2016) [115] screened 13 diatoms strains to identify a promising strain with desired fucoxanthin production. Among these 13 diatoms strains, the highest fucoxanthin content was *Odontella aurita* (1.50% or 15.00 mg/g DW). A maximum biomass and fucoxanthin concentration of 6.36 g/L and 18.47 mg/g DW were reported for *O. aurita*, respectively [117]. Another study screened *Isochrysis* strains for their potential for concurrent DHA and fucoxanthin production [116]. *Isochyrsis* CCMP1324 demonstrated the comparable biomass concentration (2.72 g/L), DHA content (16.10 mg/g) and fucoxanthin content (14.50 mg/g). The accurate identification of microalgae is essential in the screening process to ensure repeatability, reproducibility, and quality assurance. A comprehensive identification method could assign a precise identity to the microalgae [118]. Successful algal cultivation is affected by the culture parameters. The past studies optimized several culture parameters to obtain the maximum amount of fucoxanthin [114,116]. For instance, McClure et al. (2018) [114] optimized the culture parameters such as light intensity, medium composition, and carbon dioxide addition on the fucoxanthin production of *P. tricornutum*. The authors obtained the maximum concentration of fucoxanthin (59.20 mg/g), which is nearly four times higher than that found by Kim et al. (2012) [111]. These parameters are required to optimize in order to develop a sustainable, feasible, and economically viable cultivation modus operandi for the microalgae [119].

Genetic transformation is one of the critical components of genetic engineering. Several genetic transformation protocols have been established for fucoxanthin-containing microalgae [120–123]. In addition, successful genetic engineering relies on a suitable promoter. Several past researchers searched for the promising promoter in enhancing the gene expression of fucoxanthin-containing algae [122,124]. For example, four different promoters were examined for the genetic transformation of brown algae [122]. The authors concluded that the FCP promoter of *P. tricornutum* was the most suitable promoter for the brown algae, as this promoter induced both integrated and transient expression in the algae. Erdene-Ochir et al. (2016) [124] discovered a potential endogenous promoter of *P. tricornutum*, which is a glutamine synthetase promoter. This promoter induced the gene expression constitutively, and it was at least four times higher than the FCP promoter at the stationary phase.

Furthermore, four additional novel promoters were found in *P. tricornutum* under varied nitrate availability [125]. Moreover, five putative endogenous gene promoters highly expressed in *P. tricornutum* were isolated [126]. The activity of the Vacuolar ATPase (V-ATPase) gene promoter was higher than the other promoters and could drive the gene expression under both light and dark conditions at the stationary phase. The overview of the fucoxanthin synthesis pathway is crucial in genetic engineering. Several review articles have discussed the biosynthetic pathway of fucoxanthin [127–129]. Previous studies adopted the genetic engineering of the carotenoid gene(s) to improve the fucoxanthin content [130,131]. For instance, the introduction of an additional endogenous 1-deoxy-D-xylulose 5-phosphate synthase and phytoene synthase (Psy) gene separately into the genome of *P. tricornutum* resulted in an elevation of carotenoids amounts such as fucoxanthin [130]. In addition, the overexpressing of the phytoene synthase gene (isolated from *P. tricornutum*) in *P*. *tricornutum* also enhanced the fucoxanthin content by around 1.45-fold compared to the wild-type diatom [131]. Recently, Manfellotto et al. (2020) [33] produced *P. tricornutum* transformants with the overexpression of violaxanthin de-epoxidase, Vderelated, and zeaxanthin epoxidase 3. These transformants demonstrated an increased accumulation of fucoxanthin content up to four-fold compared to the wild type.

The production of high-quality fucoxanthin depends on an effective extraction and purification method. Various protocols have been utilized to recover and purify fucoxanthin from algae. These included centrifugal partition chromatography [132], column chromatography [133], microwave irradiation [113], pressurized liquid extraction [134], supercritical carbon dioxide extraction [135], ultrasound-assisted extraction [136], and traditional solvent extraction followed by chromatographic methods [137,138]. Different extraction methods for fucoxanthin have been compared, and the extracted amount was variable [139]. In addition, other parameters such as solvent types, solvent volume, temperature, etc., are crucial in determining the concentration and purity of fucoxanthin. Therefore, optimum conditions of these parameters are necessary for obtaining the highest wield possible [140]. Several review articles have discussed in depth the extraction and purification of fucoxanthin. For example, carotenoids (i.e., fucoxanthin) extracted from algae using different innovative methods were summarized [141]. In addition, Lourenco-Lopes et al. (2020) discussed the available extraction, quantification, and purification methods with the purpose of recovering the highest ratio of fucoxanthin [142].
