Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are five-carbon (C${}_5$) ubiquitous precursor metabolites, required for the biosynthesis of carotenoids. In living systems, two different pathways: (1) MVA (mevalonate) pathway and (2) MEP (methylerythritol phosphate) pathway, participates in biosynthesis of these precursor metabolites [20]. The MVA pathway was discovered in the 1950s and was considered as the sole path for the biosynthesis of IPP in all living organisms [55]. But results of MVA pathway enzyme inhibition and isotopic labeling studies on prokaryotes, algae and higher plants, indicated the absence of some key enzymes of the MVA pathway. However, IPP was well incorporated in different compounds of these organisms under study. These studies pointed out the presence of another route of IPP biosynthesis. This new route of IPP biosynthesis is called a non-mevalonate pathway or 1-deoxy-D-xylulose 5-phosphate (DXP) pathway or methylerythritol phosphate (MEP) pathway [56]. Animals and fungi use the MVA pathway for biosynthesis of IPP while the MEP pathway is identified in plants and algae. In higher plants, both MVA and MEP pathways operates simultaneously. While, in the case of algae, the MVA pathway has been lost in several groups like: Chlorophyceae, Prasinophyceae, Trebouxiophyceae and Cyanidioschyzon merolae (red alga). Therefore, the MEP pathway is the key pathway, which supplies the majority of IPP/DMAPP for carotenoid biosynthesis in these algal groups. In other groups of algae, like Glaucophyta and Heterokontophyta, both MVA and MEP pathways participate in the synthesis of IPP/DMAPP isoprene molecules [57]. In the case of red algae, Lohr et al. [55] reported that both MVA and MEP pathways participate in the biosynthesis of IPP molecules. On the other hand, Deng et al. [58] suggested that bioinformatics analyses are not able to characterize the genes for MVA pathway enzymes in red algae. Based on the existing ESTs, genome data, and phylogeny clustering analysis, Du et al. [59] also reported that green algae and red algae received their plastid by primary endosymbiotic events with cyanobacteria, while brown algae obtained their plastid via secondary endosymbiotic event with red algae.

Geranylgeranyl diphosphate (GGPP) is an immediate metabolic predecessor of the carotenoid biosynthesis pathway. Formation of GGPS (geranylgeranyl phosphate synthase) is a three-step process. In the initial step, 10-carbon compound sesquiterpene is synthesized by the addition of one IPP and one DMAPP molecule. In subsequent steps, FPP is synthesized by the addition of one IPP molecule to sesquiterpene and then GGPP is formed by the addition of one more IPP molecule to FPP (farnesyl pyrophosphate) [67, 68]. This process involves three different types of enzymes: GPPS—geranyl diphosphate synthase, FPPS—farnesyl diphosphate synthase, and GGPPS—geranylgeranyl diphosphate synthase, sequentially [65]. Ruiz-Sola et al. [69] reported 12 paralogues genes for GGPPS in Arabidopsis. They also suggested that, out of different GGPPS isozymes, GGPPS11 isozyme behave like a hub isozyme and it interacts with other proteins required for the biosynthesis of carotenoids. These enzymes are rarely studied in algae in comparison to higher plants. Yang et al. [70] cloned and characterized the GGPP synthase gene (PuGGPS) in a red alga Pyropia umbilicalis (Bangiales). They reported that a polypeptide sequence of 345 amino acids with transit peptide sequence (N-terminal) is encoded by this gene. Lao et al. [67] reported that, GGPPS of an alga Haematococcus pluvialis (HpGGPPS) have tri-functional catalytic activities, which catalyzes all three steps of GGPP biosynthesis. Deng et al. [58] cloned and characterize the bfGGPPS from the red alga Bangia fuscopurpurea. They also report that GGPP is the only product of this enzyme and it also interacts with psy gene, which is the rate-limiting enzyme of the carotenoid biosynthesis pathway. Deng et al. [58] also performed the phylogeny analysis and suggest that the red algal and diatoms share a common ancestor for GGPPS but green algae and higher plants show an early divergence of GGPPS during evolution. It has been reported that, the supply of GGPP and its precursors decides the rate and subsequent flux of carotenoid biosynthesis in algae [68].

Biosynthesis of phytoene is the first entry step reaction towards the carotenoid biosynthesis. It is the first colorless carotenoid (40-carbon) compound, which is synthesized by condensation of two GGPP molecules. This reaction is catalyzed by enzyme phytoene synthase (PSY) [65, 71]. PSY is one of the important rate-limiting and key flux controlling enzyme, which decides the pool size of carotenoids. Some studies indicate that several isoforms of PSY exist in different plant species and these are regulated by alternative splicing and protein modifications under the influence of different abiotic and biotic signals [20]. Recently, various genomic and phylogenetic studies are conducted using genomic sequences of some algae belonging to different groups like red algae, green algae, brown algae and diatoms to identify the PSY genes [52]. Tran et al. [72] reported the two orthologous copies of the PSY gene in green algae Micromonas and Ostreococcus. These studies indicate the gene duplication events of the PSY gene during ancient evolution, which produces the two classes of PSY gene in algae. But presently, these two classes, PSY I and PSY II are only retained in members of Prasinophyceae (Chlorophyta). Green algae (other than Prasinophyceae) and higher plants have lost PSY II and reported to have only the PSY I class. In contrast to this, the members of algae belong to Rhodophyta, Heterokontophyta and Haptophyta have only the class PSY II gene [52]. Due to the major flux controlling enzyme of the carotenoid biosynthesis pathway, PSY has been recognized as a major target for metabolic engineering [73]. Recently a novel protein CPSFL1 has been identified, which is bound with phytoene and modulates the accumulation of carotenoids in the chloroplast [74].

Biosynthesis of lycopene is a multistep process in which phytoene is converted to lycopene via sequential desaturation and isomerization reactions. This whole process includes four conserved enzymes: PDS, Z-ISO, ZDS and CrtISO [75]. Phytoene, which is synthesized in the previous step, is desaturated to $\zeta$-carotene. This occurs in two steps and both steps are catalyzes by an enzyme PDS. In the first step, phytoene is converted to 9,15-di-cis-phytofluene, and then in the next step, this phytofluene is converted to 9,15,9-tri-cis-$\zeta$-carotene. This 9,15,9-tri-cis-$\zeta$-carotene is yellow in color. The 9,15,9-tri-cis-$\zeta$-carotene is then converted to lycopene via multiple steps. In the first step, 9,15,9-tri-cis-$\zeta$-carotene is converted to 9,9-di-cis-$\zeta$-carotene and then to prolycopene. These two reactions are catalyzes by the enzyme Z-ISO and ZSO, respectively. This prolycopene, which is orange in color, is converted to red colored compound lycopene. This conversion is catalyzed by the enzyme CrtISO [20, 76]. Wang et al. [52] have identified and characterized the gene of these enzymes in different groups of algae-like Chlorophyta, Phaeophyta, and Rhodophyta. Various studies reported that, out of these four genes; the gene for PDS, ZDS and CRTISO are present in all three groups of algae, while gene for Z-ISO is absent in red algae (Rhodophyta). Wang et al. [52] also suggested that the absence of this gene in red algae does not affect the carotenoid biosynthesis.

Lycopene is the first link of carotenogenesis, which allows the biosynthesis of both $\alpha$-carotene and $\beta$-carotene in algae. It is a most important branching point, where the ratio of $\alpha$-carotenoids (lutein) to $\beta$-carotenoids ($\beta$-carotene) is to be decided. In $\alpha$-carotene, one $ϵ$-ring and one $\beta$-ring are present at the extremity of lycopene. While, in $\beta$-carotene, two $\beta$-rings are present at the extremity of lycopene. Carotene with two $ϵ$-ionone rings rarely occurs in nature. This branch point reaction is catalyzed by two different enzymes; lycopene $ϵ$-cyclase (LCYE) and lycopene $\beta$-cyclase (LCYB) [77]. Formation of $\alpha$-carotene from lycopene occurs in two sequential steps: in the first step, enzyme LCYE catalyzes the cyclization at the one open end and form $\delta$-carotene; in the next step, LCYB catalyzes the $\beta$-ionone ring formation at another end and ultimately $\alpha$-carotene is synthesized. In further sequential steps this $\alpha$-carotene is converted to lutein. Similarly, $\beta$-carotene is also synthesized in two sequential steps, but here, both steps are catalyzes by the same enzyme LCYB. In the first step lycopene converted to $\gamma$-carotene and in the next step this $\gamma$-carotene is converted to $\beta$-carotene [58].

Based on chemical, GSMN and proteomic studies, it has been elucidated that algae are different in their lycopene cyclase enzyme compositions. Some algae contain both LCYE and LCYB, while other algae have only one class of LCY. Cui et al. reported that in green alga (except for C. reinhardtii (LCYE), and Chlorella sp. NC64A (LCYE), H. pluvialis (LCYB), D. salina (LCYB),) two distinct LCY (beta- and epsilon-type) enzymes are present, on the other hand, heterokontophyta have only lycopene beta-cyclase (LCYB) [77]. Recently, Inoue et al. [78] also reported the LCYB activity in brown alga Undaria pinnatifda. Macrophytic red algae have both LCYB and LCYE enzymes while microphytic algae have only the enzyme LCYB [52]. Some other studies also suggested that, synthesis of $\delta$-carotene, $\alpha$-carotene and lutein are absent in some groups of algae-like Bacillariophyceae, Chrysophyceae, Phaeophyceae, Xanthophyceae, and some red algae, while the $\beta$-carotene occurs in majority groups of algae and other photosynthetic organisms [18, 58]. Liang et al. [79] reported that the amino acid sequences of LCYB and LCYE are significantly similar. This report indicates that the gene duplication events in a common ancestor may be the possible reason behind the presence of two classes of LCY enzymes in algae. Deng et al. [58] suggested that LCYE, in green and red algae, evolved separately. Both enzyme LCYB and LCYE plays a major role in the metabolic flux of carotenes. Sathasivam and Ki [80] reported that treatment of redox-active heavy metals enhances the expression level of enzymes PSY, PDS, and LCYB and ultimately enhance the accumulation of lutein and $\beta$-carotene in brown alga Tetraselmis suecia.

In algae, different types of xanthophylls like: lutein, cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, fucoxanthin, diadinoxanthin, diatoxanthin, canthaxanthin, astaxanthin etc., are synthesized. Types and nature of xanthophyll molecules differ in various algal groups. Both $\alpha$- and $\beta$-carotene serves as precursor molecules for the biosynthesis of these xanthophyll compounds.

Lutein is a derivative of $\alpha$-carotene. It is synthesized in two successive steps: firstly $\alpha$-carotene is converted to zeinoxanthin or $\alpha$-cryptoxanthin and then in second step zeinoxanthin is converted to lutein. Both of these steps are catalyzed by P450-type enzymes $ϵ$-hydroxylase and $\beta$-hydroxylase. Yang et al. [81] reported that red algal enzyme CYP97B29 has both $ϵ$- and $\beta$-ring hydroxylase activity. Liang et al. [82] functionally identify the genes of carotene hydroxylases from alga Dunaliella bardawil. Various studies reported that, biosynthesis of lutein mainly occurs in green algae and some of the red algae. While other group of algae-like Bacillariophyceae, Chrysophyceae, Phaeophyceae and Xanthophyceae cannot synthesize lutein and other derivatives of $\alpha$-carotene.

Zeaxanthin is a double hydroxylation derivative of $\beta$-carotene. $\beta$-carotene is converted to $\beta$-cryptoxanthin and then $\beta$-cryptoxanthin is converted to zeaxanthin. Both of these hydroxylation reactions are catalyzed by the enzyme $\beta$-carotene hydroxylase (BCH). Biosynthesis of zeaxanthin occurs in all groups of algae as well as higher plants. Further, zeaxanthin is converted to violaxanthin in a two-step process. In the first step, zeaxanthin is converted to antheraxanthin and in the next step, antheraxanthin is converted to violaxanthin. Both of these steps are catalyzed by the enzyme ZEP. Enzyme ZEP catalyzes the epoxidation reaction of $\beta$-rings of zeaxanthin molecule [83]. Another enzyme VDE has also been reported which led to the formation of $\beta$-rings and ultimately reverses these reactions. This whole process of violaxanthin biosynthesis is also known as xanthophyll cycle-I. This cycle occurs in all members of green and brown algae while in the case of red algae it occurs partially [84].

In the case of brown algae some commercially important carotenoids like fucoxanthin and diadinoxanthin are also synthesized. Scientists have proposed two different pathways for the biosynthesis of these carotenoid compounds. According to the first pathway, violaxanthin is converted to neoxanthin and then neoxanthin is used as a precursor molecule for the biosynthesis of both fucoxanthin. An enzyme neoxanthin synthase (NXS) catalyzes the conversion of violaxanthin to neoxanthin in higher plants. In the case of algae, recently, Dautermann et al. [85] reported a new enzyme violaxanthin de-epoxide-like (VDL), which is responsible for the conversion of violaxanthin to neoxanthin. They also reported that VDL is also involved in the synthesis of peridinin and vaucheriaxanthin. According to the second pathway, violaxanthin is used as a precursor for the biosynthesis of diadinoxanthin and then, the diadinoxanthin is converted into the fucoxanthin. The enzymes involved in this process are also still unknown. In some alga, diadinoxanthin can also be converted to diatoxanthin. This reaction is catalyzed by an enzyme de-epoxidase (DDE). This conversion of diadinoxanthin to diatoxanthin can be reversed by an enzyme diatoxanthin epoxide (DEP) under low light intensity. This pathway is called xanthophyll cycle-II. This cycle mainly occurs in members of Chrysophyceae, Bacillariophyceae, Phaeophyceae, and Xanthophyceae [54].

Astaxanthin, is another important carotenoid compound, which has strong antioxidant activity as compared to vitamin C and E, is synthesized by several bacteria, fungi, algae and higher plants. In algae several species have been reported which are involved in de novo synthesis of astaxanthin. The scientist has identified the two biochemical pathways for the biosynthesis of astaxanthin. According to the first pathway, $\beta$-carotene is converted to canthaxanthin and then, canthaxanthin is converted to astaxanthin. These two reactions are catalyzes by enzyme BKT and BCH, respectively. According to another pathway, zeaxanthin is converted to adonixanthin and then adonixanthin is converted to astaxanthin. In this second pathway, both of the reactions are catalyzed by the same enzyme BKT. Mao et al. [75] reported that, under sulfur stress conditions, genes LCYE and ZEP are down-regulated while gene LCYB, BCH and BKT are up-regulated. This regulation of gene expression enhances the accumulation of astaxanthin in green alga Chromochloris zofingiensis. Among the algae, Haematococcus pluvialis considered as the richest source of natural astaxanthin. Astaxanthin in algae is present in esterified form in contrast to non-esterified form in yeast and synthetic form.

Carotenoids are extracted from microalgae utilizing conventional solvent extraction methods using organic solvents. Conventional extraction using organic or aqueous solvents depends on the polarity, solubility, and chemical stability of carotenoids to be extracted. Therefore, the selection of a suitable solvent system is necessary which can selectively and efficiently extract carotenoids with high purity. Non-polar solvents (n-hexane, dichloromethane, dimethyl ether, diethyl ether) and polar solvents (acetone, methanol, ethanol, biphasic mixtures of several organic solvents) can be used based on the polarity of the target carotenoid. The use of green solvents (environmentally safe and non-toxic solvents) such as ethanol, limonene, and biphasic mixtures of water and organic solvents has been investigated for the recovery of carotenoids from microalgae. However, the commercial reality of carotenoid extraction from microalgal species is still challenging due to the high cost of production, and usage of enormous amounts of solvents. The uses of non-conventional extraction methods are therefore gaining interest in recent years. These non-conventional extraction methods have several advantages including rapid extraction, low solvent consumption, better recovery, and higher selectivity. These different extraction approaches for various carotenoids along with their relative yield are mentioned in Table 2 (Ref. [97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112]).

Microalgal cells are difficult to disrupt due to algaenan and sporopollenin within their cell wall [113]. Further, conventional techniques used for cell disruption and extraction methods have low efficiencies. MAE is an efficient method that takes advantage of microwave irradiation to accelerate the extraction of a diversity of compounds from natural matrices. MAE generates high-frequency waves (ranging from 300 MHz to 300 GHz) with wavelengths of 1 mm to 1 m. Microwave radiation when applied at a frequency near 2.45 GHz causes vibration of polar molecules resulting in inter and intra-molecular friction. The friction, together with the movement and collision of a large number of charged ions, results in the rapid heating (within few seconds) of the matrix. Intracellular heating leads to the breakdown of cell walls and membranes and therefore there is a faster transfer of the compounds from the cells into the extracting solvent. There are two major types of microwaves; closed and open vessels. In open vessels microwave application is performed at atmospheric pressure while in closed vessels, samples are irradiated by microwave under controlled pressure and temperature. The extraction temperature depends on the polarity of the solvent. Solvents with higher dielectric constant ($\epsilon {}^{\prime }$) absorb greater energy and thus achieve faster extraction and therefore polar solvents are better extractants than non-polar solvents. Pasqueta et al. [110] investigated the performance of microwave irradiation compared to conventional processes to extract pigments from two marine microalgae Dunaliella tertiolecta(Chlorophyta) and Cylindrotheca closterium (Bacillariophyta). All processes performed on D. tertiolecta led to rapid pigment extraction. Though the presence of frustule in the diatom C. closterium acted as a mechanical barrier to pigment extraction. MAE was found to be the best extraction process for C. closterium pigments with advantages like rapidity, reproducibility, homogeneous heating and high extraction yields. Fabrowska et al. [114] examined the efficiency of microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE) and conventional soxhlet extraction in three freshwater green algae species: Cladophora glomerata, Cladophora rivularis, and Ulva flexuosa. MAE and UAE were proved to be cost-effective techniques with higher yield compared to traditional solvent extraction techniques. Microwave-assisted extraction (MAE) has been applied for the extraction of astaxanthin from Haematococcus pluvialis which has the highest astaxanthin content [115]. For optimal extraction of astaxanthin, parameters like microwave power (W), extraction time (s), solvent volume (mL), and the number of extractions, were optimized using response surface methodology. The study suggested that optimized conditions of MAE viz. microwave power 141 W, extraction time 83 sec, solvent volume 9.8 mL, the number of extraction four times led to the extraction of about 594 $\pm$ 3.02 $\mu$g astaxanthin per 100 mg of dried powder.

Ultrasonic assisted extraction is based on ultrasonic cavitation. Ultrasonic extraction has been used to extract bioactive compounds like vitamins, polyphenols, pigments and other phytochemicals. UAE is cost-effective and significantly reduces the extraction time, whilst resulting in increased extraction yields. The ultrasound can be divided into two distinct categories: low intensity-high frequency (100 kHz–1 MHz) and high intensity-low frequency (20–100 kHz). Ultrasonic extraction is achieved with high intensity and low-frequency ultrasound waves. Ultrasound waves when traveling through liquid creates alternating high-pressure and low-pressure cycles resulting in the production of cavitation bubbles in the solvent. Cavitation bubbles form in the liquid during the expansion phase. The ability to cause cavitation depends on the frequency of the ultrasound wave, the solvent properties, and the extraction conditions. During the compression cycle cavitation bubble implodes on the surface of the matrix (cell, tissue or any particle) and a high-speed micro-jet is created leading to the generation of effects like surface peeling, particle breakdown, sonoporation and cell disruption. Sonoporation (perforation in cell walls and membranes) exerts a mechanical effect, allowing greater penetration of solvent into the sample matrix. This leads to increased extraction efficiency in less time. Using ultrasound-assisted extraction, 4.66 mg $\beta$-carotene per g of dry weight has been obtained from microalgae Spirulina platensis [116]. Various parameters (amplitude, duty cycle, sonication time, and depth of horn immersed into the solution) were optimized for intensified extraction. The optimized condition for the maximum extraction of $\beta$-carotene from this alga was 80% amplitude, 33% duty cycle, 0.5 cm depth of horn immersed in the solution, and 10 min ultrasonication time. UAE has also been applied for the extraction of lutein, $\beta$-carotene, and $\alpha$-carotene from Chlorella vulgaris [117]. The maximum extraction achieved were 4.844 $\pm$ 0.780, 0.258 $\pm$ 0.020, and 0.275 $\pm$ 0.040 mg/g of dry weight biomass, respectively.

Electrotechnologies, such as pulsed electric field (PEP), moderate electric field (MEF), high-voltage electric discharges (HVED) are emerging, non-thermal, and green extraction techniques for targeting intracellular compounds from bio-suspensions. In the pulsed electric field (PEP), the sample matrix is exposed to repetitive electric frequencies (Hz–kHz) with an intense (0.1–80 kV/cm) electric field for very short periods (from several nanoseconds to several milliseconds). In the moderate electric field (MEF), the sample matrix is exposed to low electric fields (between 1 and 1000 V/cm) with electric frequencies in the range of Hz up to tens of kHz. While high-voltage electric discharges (HVED) typically have 40–60 kV/cm for 2–5 $\mu$s electrical property. All of these electrotechnologies have their mechanism of delivering electrical current through the processed biomaterial, they all promote electroporation or electro-permeabilization allowing the extraction of analytes. The selective extraction of intracellular compounds can be achieved by controlling the pore formation, which is dependent on various factors such as the intensity of the applied electric field, pulse duration, treatment time, and the cell characteristics (i.e., size, shape, orientation in the electric field) [118]. Besides these factors, the temperature is another critical parameter affecting the efficacy of the electrotechnology assisted extraction [104]. Pulses of milliseconds (5 kV/cm-40 ms) or microseconds (20 kV/cm-75 $\mu$s) have improved the efficiency of carotenoids extraction from Chlorella vulgaris by 80%. In the microalgae Heterochlorella luteoviridis the application of MEF combined with ethanol as solvent (180 V, 75 mL/100 mL of ethanol solution) resulted in up to 73% of carotenoid extraction [119]. Moderate electric field (MEF) (0–180 V) has been evaluated as a pre-treatment for carotenoid extraction at different temperatures followed by extraction step using ethanol/water as solvent (75% of ethanol, v/v). The highest extraction yield, 86% of the total carotenoid content was achieved at both 40 and 50 ${}^{\circ }$C with the MEF pre-treatment [120]. HVED treatment has been applied and favored the selective recovery of intracellular compounds from algae Parachlorella kessleri and Nannochloropsis oculate [121, 122].

The main purpose of using PLE is that it allows rapid extraction and reduces solvent consumption; therefore, it is sometimes referred to as accelerated solvent extraction. PLE involves the extraction using solvents at elevated temperature and pressure but always below their critical points. This normally falls in the ranges of 50–200 ${}^{\circ }$C and 35–200 bars. During PLE, the interaction between the solvent and the biological sample is increased compared to common solvent extraction methods. Therefore, less solvent is required for extraction. PLE has been applied for the extraction of carotenoids from freeze-dried microalgae and macroalgal biomass. In Phaeodactylum tricornutum pressurized liquid extractions resulted in exceptional amounts of fucoxanthin up to 26.1 mg/g dw [123]. Pressurized liquid extraction has been successfully applied in the case of Neochloris oleoabundans for the recovery of bioactive carotenoids lutein, carotenoid monoesters and violaxanthin [124]. Pressurized liquid extraction (PLE) has been optimized for the extraction of carotenoids and chlorophylls from the green microalga Chlorella vulgaris and showed higher extraction efficiencies than maceration (MAC), soxhlet extraction (SOX), and ultrasound-assisted extraction (UAE) [49]. PLE has been optimized for Nannochloropsis oceanica with ethanol as extraction solvent. A total carotenoids content of 115.1 $\pm$ 0.6 mg/g extract was obtained at the optimum extraction conditions of 57 ${}^{\circ }$C and 3 extraction cycles [125].

SFE involves extraction using supercritical fluids i.e., fluids at a temperature and pressure above its critical limit. Supercritical fluids provide better solvating and transport properties than liquids due to their low viscosity and high diffusivity. For the selective extraction of a broad range of compounds, the solvating power of supercritical fluid can be adjusted by manipulating the temperature and pressure of the fluid. Carbon dioxide is the preferable solvent which can easily achieve supercritical conditions and has benefits like high purity, low toxicity and low flammability compared to other fluids. Supercritical carbon dioxide is non-polar and its polarity can be modified by using co-solvents. Besides CO${}_2$, ethane and ethylene are other SFE solvents that have been used for the extraction of carotenoids in some studies. In Scenedesmus obliquus the highest carotenoid yield was achieved at 250 bar and 60 ${}^{\circ }$C using SFE [126]. Supercritical CO${}_2$ extraction has been applied in Spirulina to obtain carotenoids, chlorophylls, and phycocyanins. The SFE method resulted in 3.5 $\pm$ 0.2 mg/g total carotenoid contents in Spirulina [127].

Subcritical fluid extraction is similar to SFE, where subcritical (liquefied) fluids are used as extraction solvent. Subcritical fluid extraction works at relatively low temperature and pressure than supercritical fluid extraction. In various studies, subcritical CO${}_2$, 1,1,1,2-tetrafluoroethane and dimethyl ether (DME) have shown the potential to extract carotenoids from algae. Subcritical fluid extraction has been performed to extract fucoxanthin from Phaeodactylum tricornutum. The highest fucoxanthin content (0.69 mg/g) was achieved with a solvent-to-solid ratio of 200 : 1, 20 MPa, 35 ${}^{\circ }$C at 120 rpm 60 min by subcritical extraction [128]. The carotenoids and chlorophyll-a from Laminaria japonica have been isolated using ethanol modified subcritical 1,1,1,2-tetrafluoroethane [129].

Microalgal cells are difficult to disrupt, therefore, a physical or enzymatic pre-treatment before extraction can be opted to promote the recovery of carotenoids. High-pressure homogenization (HPH) is one such method, where, cell disruption is achieved by applying high intensity fluid stress (50–400 MPa). In comparison with other physical milling processes, it offers significant advantages such as ease of operation, commercial applicability, reproducibility and high throughput. High-pressure homogenization (HPH) found to be very effective in microalgae with a recalcitrant cell wall such as Nannochloropsis [130]. Cell disruption by high-pressure homogenization has been shown to increase carotenoid and $\omega$3-LC-PUFA bio accessibility [131]. Enzyme-assisted extraction (EAE) methods use hydrolytic enzymes like cellulase and pectinase for improved extraction. Cellulase hydrolyzes the 1,4-$\beta$-d-glycosidic links of the cellulose, whereas, pectinase breaks down the pectic substances and pectin found in cell wall components. In a study, enzyme type (cellulase and pectinase), pH values, hydrolysis temperature, and time on the release of astaxanthin from Haematococcus pluvialis were evaluated. The results showed that enzymatic pre-treatment improve the separation yield of astaxanthin. Pectinase release rate of astaxanthin from H. pluvialis was found to be significantly higher than cellulase [132].

Astaxanthin is generally known as ‘Super Vit-E’ due to its compelling antioxidant properties [134]. Kishimoto et al. [135] bestowed it with ‘King of antioxidants’ because of its better antioxidant properties as it emulates Vit C, resveratrol, CoQ10, green tea catechins, and Vit E. Lipid peroxidation, LDL oxidation, and peroxide-induced cytotoxicity, etc. are inhibited by astaxanthin [136]. Their roles as anti-inflammatory, hepatoprotector and protective influence on retinal, brain, and spinal cord neurons and in the prevention of cardiovascular disease have been reported [137]. Aquaculture feed; poultry and food industries use this tremendously as red colorant [138]. Astaxanthin favorably lowers the level of plasma reactive protein C, down regulate inflammatory cytokines, modulate mitogen-induced lymphoproliferation, and boost the immune response [139]. It protects the skin from the detrimental effects of UV-A photo ageing and is therefore widely used in cosmetics [140]. The inability of astaxanthin to convert into Vit A in the human body safeguards from hypervitaminosis A toxicity even after its overconsumption [141].

The role of $\beta$-carotene as anti-inflammatory, antioxidant, immunoprotector, dermoprotector, hepatoprotecter, retinoprotective are well known [142]. The food colorant and cosmetic industries use this pigment on a large scale [143]. It has been reported that the beneficial role of this pigment in breast, colon, lung, liver, and skin cancers. $\beta$-carotene can trigger better functioning of gap junction intercellular communications and can diminish the harmful effect of H${}_2$O${}_2$ on the same and maintain the processes like cell differentiation, growth, and apoptosis [144]. Its regular consumption in the daily diet protects from night blindness and liver fibrosis.

Fucoxanthin has well-marked use as antioxidant, anti-angiogenic, anti-inflammatory, photo- and neuroprotective, which makes it commercially valuable. Peng et al. [145] reported its role in hindering DNA damage and H${}_2$O${}_2$induced apoptosis. In HL-60, PC-3, HT-29, DLD-1, and Caco-2 human cell lines it acts as an antiproliferative [146]. It reduces obesity by up-regulating uncoupling protein-1 (UCP 1) to speed up metabolism and energy expenditure [147]. Heo et al. [148] reported its role in reducing nitric oxide, prostaglandin, TNF-$\alpha$, histamine levels in in-vivo models.

The role of zeaxanthin in ophthalmological fields is significant and crucial. It is naturally present in the central macula of the eye, and protects the visionary organ from blue, near UV radiations, age-related macular degeneration [149]. Its promising property as natural color mark it use in pigmentation of poultry and fishes, in food, and as an antioxidant in cosmetics [44]. Regular consumption of zeaxanthin might prove useful in avoiding lung and pancreatic cancers in diabetic patients and found to be effective in cardiovascular problems [150]. This xanthophyll has the ability to trigger apoptosis in melanoma cells and therefore useful in adjuvant therapy.

Lycopene has antioxidant, photo-protective properties. It proves to be anti-cancerous against cell lines from the human colon, breast, prostate, liver, and lymphocytes [151]. It prevents from early arthrosclerosis and high blood pressure problems by boosting endothelial functioning and lowering oxidative stress. Lycopene, also act as hypolipidemic in a way similar to statin and maintain blood cholesterol levels [152]. The sufficient daily doses of lycopene strengthen the skeleton system, enhance gap junction intercellular communications, and maintain glucose homeostasis, which in turn prevent type-2 diabetes. Viuda-Martos et al. [151] reported lycopene-induced inhibition of tumor metastasis because of modulated cell cycle progressions. The poultry farms use this as feed to improve the health of poultry birds and it also relieves heat stress in commercial poultry. Lycopene is commercially available in form of capsules, tablets, and in gel forms. It has profound role in cosmetics and coloring agent in food sectors.

Marigold is the chief source of lutein production at the industrial level. Like zeaxanthin, it protects eye from pernicious UV, blue light and therefore, referred to as eye vitamin. It lowers the level of plasma factor D and in turn plays a defensive role against age-related macular degeneration and cataract. It is effectual as a chemotherapeutic agent [153]. It is an active anti-tumor substance against prostate, breast, and colon cancer. Lutein consumption can also lessen the chance of early atherosclerosis and lung cancer as well. It alleviates the effects of neurodegenerative disorders arise due to inflammation. It has prodigious use in poultry as yolk color enhancer, feather coloration through feed and as an additive in infant food, drug and cosmetics [154].

Canthaxanthin is a $\beta$-carotene derived product, is remarkably used in food and as an additive in animal feed to poultry and fish (salmon, rainbow trout). It is an FDA approved compound to be used as a fish color intensifier [44]. Its doses along with $\beta$-carotene prevent idiopathic photodermatosis. Capsanthin is another pigment that shows antioxidant activity and is used as a natural colorant in food and cosmetic. Nowadays, it has been widely used in chicken feed to intensify color of egg yolk. The food sector uses one more yellow-orange pigment i.e., Annatto, to provide color to meat products, beverages, and smoked seafood. Echineone is a by-product of astaxanthin, possess pro-vitamin A and antioxidant property. Another pigment, peridinin presents anti-carcinogenic activities. The neoxanthin might lessen the lung cancer risk through regular consumption of 9-(Z) neoxanthin [143].