How to Extract Carotenoids from Microalgae?

Carotenoids are a class of lipid-soluble isoprenoid compounds widely found in various plants, animals, and microorganisms. They serve as natural antioxidants within organisms and perform important physiological functions. Research has shown that carotenoids possess efficacy in preventing and treating human diseases as well as improving human health, such as preventing cardiovascular diseases, treating cancer, improving vision, and enhancing immune function. In recent years, carotenoids have found widespread application in the pharmaceutical, food, health supplement, cosmetics, and feed industries. The global market demand for carotenoids is growing at an annual rate of 2.9%, with projections reaching 10 million tonnes by 2017 [1]. However, most commercially available carotenoids are derived from chemical synthesis, and their biological effects and safety have been subject to ongoing scrutiny [2–3]. With the continuous enhancement of health awareness, carotenoids derived from natural raw materials are gaining increasing popularity among consumers.

In recent years, microalgae have garnered growing attention as a sustainable and renewable resource for producing biofuels. However, microalgae biofuel technology remains immature, and production costs are prohibitively high, preventing industrial-scale breakthroughs to date. Some researchers have shifted their focus to the production of other high-value-added products from microalgae. Microalgae are considered the best natural source of commercially valuable carotenoids, and extracting carotenoids from microalgae offers significant advantages: first, microalgae grow rapidly, are easy to cultivate, and are suitable for large-scale cultivation; second, microalgae synthesise a wide variety of pigments, such as β-carotene, lutein, and astaxanthin; and the bioactivity and antioxidant properties of these pigments have been confirmed; finally, microalgae growth is not affected by seasonal changes, does not compete for arable land or freshwater resources, and microalgae have strong adaptability, with some species capable of growing and reproducing in wastewater [4–5]. Therefore, researching and developing carotenoids from microalgae can expand the sources of natural carotenoids, enhance the value of algal species, and bring significant economic benefits.

However, currently, the high production cost is the primary constraint on the commercial production of microalgal carotenoids. The production of carotenoids from microalgae involves three stages: microalgal cultivation, algal harvest, and extraction and purification. Among these, algal harvest and carotenoid extraction are the key technologies determining production costs. This paper reviews various extraction technologies for microalgal carotenoids based on published literature from both domestic and international sources, aiming to provide a reference for further research and development of microalgal carotenoids.

1 Common high-yield carotenoid-rich microalgal strains

Research on microalgae as a source of carotenoids began in the 1960s. To date, the microalgae species rich in carotenoids primarily belong to the Chlorophyta division, including Chlorella, Scenedesmus, Chlamydomonas, Dunaliella, Muriellopsis, and Haematococcus. Among these species, Dunaliella salina and Haematococcus pluvialis have been used for commercial production of β-carotene and astaxanthin. Common microalgae species producing various carotenoids and their contents are shown in Table 1.

2   Extraction methods for carotenoids from microalgae

In recent years, how to effectively extract carotenoids from microalgal raw materials has become a key focus of research and exploration by scholars worldwide. The process of obtaining carotenoids from microalgae typically involves the following steps: algal biomass collection → drying → cell disruption → extraction. However, algal biomass collection, drying, and cell disruption require significant energy consumption, leading to high production costs. Some researchers have improved traditional extraction methods by integrating microalgae harvesting, cell wall disruption, and extraction into a single process or omitting the drying step, thereby simplifying the operational steps, reducing energy consumption, and lowering production costs. Currently, the main methods used for extracting carotenoids from microalgae include: organic solvent extraction, pressurised solvent extraction, supercritical/subcritical fluid extraction, in situ extraction, and dual-phase extraction.

2.1 Organic solvent extraction method

The traditional organic solvent extraction method is one of the most commonly used methods for extracting carotenoids from microalgae. However, some high-yield carotenoid-producing algal species, such as Chlorella, Scenedesmus, and Muriellopsis, have extremely hard cell walls, making cell wall disruption difficult and often resulting in incomplete carotenoid extraction. Therefore, prior to carotenoid extraction from microalgae, cell wall disruption is necessary, or auxiliary extraction methods can be employed to simultaneously perform cell wall disruption and extraction operations.

Cerón et al. [15] extracted lutein from Scenedesmus almeriensis and compared the effects of five different cell wall disruption methods (aluminium oxide mortar method, ball milling, aluminium oxide ball milling, ultrasonic disruption, and aluminium oxide ball milling combined with ultrasonic disruption) on lutein extraction efficiency. It was found that cell disruption significantly influenced lutein extraction efficiency, with the optimal cell disruption method being aluminum oxide ball milling for 5 minutes, achieving an extraction rate of 98%, while the extraction rate from unbroken cells was only 40%.

Deenu et al. [16] optimized the process conditions for extracting lutein from Chlorella vulgaris powder using ultrasonic-assisted 90% ethanol extraction. Under optimal conditions of ultrasonic power 35 kHz, ultrasonic intensity 56.58 W/cm², extraction temperature 37.7 °C, extraction time 5 h, and solid-liquid ratio 31 mL/g, the lutein content was (3.16 ± 0.03) mg/g. Zhao Xiaoyan et al. [17] optimised the extraction conditions for astaxanthin from Haematococcus pluvialis using variable-frequency microwave-assisted organic solvents (ethyl acetate : ethanol = 1 : 2; v/v). with the optimal liquid-to-solid ratio, extraction temperature, and extraction time being 200:1, 45°C, and 20 minutes, respectively. The astaxanthin extraction rate was 36.88%. This study demonstrated that variable-frequency microwave-assisted mixed organic solvent extraction can rapidly enhance the astaxanthin extraction rate from Haematococcus pluvialis. Table 2 summarises the principles and advantages/disadvantages of several commonly used methods for microalgal cell wall disruption.

Carotenoids in microalgae primarily exist in two forms: free and fatty acid esters [20]. However, carotenoids extracted using organic solvents often contain impurities such as chlorophyll and oils. The presence of these substances affects the purity of the extracted carotenoids and impacts subsequent processing steps. Saponification of carotenoid samples not only releases bound carotenoids, increasing the content of free carotenoids, but also effectively removes impurities such as chlorophyll and oils, thereby improving the purity of the extracted carotenoid samples [21].

Saponification reagents are typically chosen as a methanol or aqueous solution of KOH, which can be saponified at room temperature or with appropriate heating of the sample to shorten the saponification time; after saponification, extraction is performed using organic solvents with low polarity such as hexane or petroleum ether; finally, the extraction product is washed with water to remove KOH. However, saponification can damage carotenoids and reduce their extraction yield; therefore, saponification conditions must be strictly controlled to minimise losses. Cerón et al. [15] proposed an extraction process suitable for industrial-scale production of lutein from Scenedesmus almeriensis algae and optimised the extraction conditions. This method primarily consists of three steps: cell disruption, alkaline treatment, and solvent extraction. Optimisation results showed that pre-treating algal powder with aluminium oxide ball milling for 5 minutes, followed by treatment with a 4% w/v KOH solution for 100 g/L algal biomass for 5 minutes, and finally extracting with hexane of the same volume as the sample, with six extractions, achieved a lutein recovery rate of 95%.

If traditional extraction methods are used, steps such as harvesting and drying are required before extracting carotenoids from microalgae, thereby increasing production costs. Kang et al. [22] employed a new solvent extraction method to extract free astaxanthin from Haematococcus pluvialis. This method is divided into two stages. In the first stage, they extracted astaxanthin and astaxanthin esters from Haematococcus pluvialis culture medium using dodecane, then separated the dodecane from the culture medium containing cell fragments; in the second stage, the dodecane was continuously mixed with an equal volume of 0.02 mol/L NaOH-methanol solution, During this process, astaxanthin and its esters in the dodecane phase continuously transfer to the methanol phase. Astaxanthin esters are converted into free astaxanthin through saponification in the methanol phase. Finally, the two phases are separated, and the dodecane phase can be reused. The extraction rates of astaxanthin in the two stages were 95% and 85% or higher, respectively. Compared with other extraction methods, this method eliminates the need for microalgae harvesting, is simple to operate, low in energy consumption, and has high development and application value. However, due to the volatility and toxicity of organic solvents, which are detrimental to human health and the environment, green solvents such as plant oils can be used to replace traditional organic solvents for extracting carotenoids from microalgae.

Kang et al. [23] used several common plant oils (soybean oil, corn oil, grape seed oil, and olive oil) to extract astaxanthin from Haematococcus pluvialis. At room temperature, equal volumes of plant oil and Haematococcus pluvialis culture medium were mixed, vigorously stirred to break the algae cells, and allowed to settle to separate the two phases. The plant oil extracted the astaxanthin from the algae cells, with the algae cells remaining in the lower phase and the oil phase achieving a recovery rate of over 88%. This method is environmentally friendly and effectively preserves the stability and natural properties of the oil. After extraction, adsorption methods can be used to separate microalgal carotenoids from plant oil. Baharin[24] employed two types of macroporous adsorption resins to adsorb carotenoids from palm oil. After adsorption, the adsorbent was separated from the palm oil, and the carotenoids on the adsorbent were desorbed using the Soxhlet extraction method.

2.2 Pressurised Solvent Extraction Method

Pressurised Liquid Extraction (PLE), also known as Accelerated Solvent Extraction (ASE), is a novel sample pretreatment technique that has found widespread application in agriculture, food, environment, and medicine [25]. The principle involves increasing the solubility of substances and the diffusion efficiency of solutes under conditions of high temperature (50–200 °C) and high pressure (500–3000 psi), thereby enhancing extraction efficiency [26]. Compared with other extraction methods, PLE offers advantages such as short extraction time, reduced solvent consumption, high extraction efficiency, and high automation.

Castro-Puyana et al. [27] employed PLE to extract carotenoids from the oil-rich green alga Neochloris oleoabundans, simultaneously comparing the extraction efficiency of PLE with that of traditional organic solvent methods. The results showed that under conditions of 100% ethanol extraction at 100°C for 20 minutes, the carotenoid extraction rate was 32.6%, significantly higher than that obtained using acetone containing 0.1% (w/v) butyl hydroxytoluene, which yielded only 28.3%.

Although the PLE method can achieve a high carotenoid extraction rate, Grima et al. [14] noted that this method requires a high extraction temperature, which can cause chlorophyll in microalgae samples to transform into toxic magnesium-depleted chlorophyll, thereby affecting the activity of the extracted carotenoids. Therefore, the PLE method has certain limitations for extracting carotenoids from microalgae. Jaime et al. [28] used the PLE method to extract carotenoids from Haematococcus pluvialis and compared the antioxidant activity of carotenoids extracted at different temperatures (50, 100, 150, 200 °C). The results showed that under conditions of 100% ethanol extraction for 20 minutes, the carotenoid extraction rate increased with higher temperatures, while the antioxidant activity of the extracts decreased accordingly.

However, Cha et al. [29] compared the effects of PLE method with traditional solvent extraction, Soxhlet extraction, and ultrasound-assisted extraction on the content of carotenoids, chlorophyll a, chlorophyll b, and magnesium-depleted chlorophyll a and magnesium-depleted chlorophyll acid a in Chlorella vulgaris. They found that compared with other extraction methods, the PLE method demonstrated better extraction efficiency for carotenoids and chlorophylls. Additionally, they observed that when the extraction temperature was 160°C, the content of magnesium-depleted chlorophyll a in the extract was the lowest, at (0.01 ± 0.00) mg/g, while the traditional solvent extraction method, Soxhlet extraction method, ultrasonic-assisted extraction methods yielded magnesium-depleted chlorophyll a contents of 0.85 ± 0.09, 5.15 ± 0.59, and (2.15 ± 0.71) mg/g, respectively. They speculated that this was due to high temperatures (> 110 °C) causing chlorophyllase inactivation, while other extraction methods were performed under milder conditions at temperatures of 20–80 °C, where chlorophyllase remained active, accelerating the conversion of chlorophyll to magnesium-depleted chlorophyll. Therefore, the PLE method is a highly promising extraction technique for pigments from microalgae.

2.3 Supercritical/Subcritical Fluid Extraction Method

Supercritical fluid extraction (SFE) is an environmentally friendly green extraction technology that uses supercritical fluids as solvents to separate soluble components from the target material. Due to the low viscosity and excellent diffusivity of supercritical fluids, extraction efficiency is faster and more effective. By adjusting the density of the supercritical fluid, active components in microalgae can be selectively extracted. After extraction, by increasing the temperature or reducing the pressure, the supercritical fluid is converted into a common gas and released, leaving no solvent residues in the extracted carotenoids. The extracted algal powder can also be further utilised. Supercritical fluids have advantages such as non-flammability, toxicity, and chemical stability, resulting in safer products.

Kitada et al. [30] used supercritical CO₂ to extract carotenoids and chlorophyll from Chlorella vulgaris, investigating the effects of extraction pressure, temperature, and co-solvents (ethanol and acetone) on pigment content in the extract, and compared the results with those of the traditional Soxhlet extraction method.

The study found that the optimal extraction pressure and temperature were 50 MPa and 80°C. Supercritical CO₂ extraction can selectively extract lutein, but the extraction rate is low. Adding 7.5% ethanol as a co-solvent effectively increases the lutein content in the extract, but also increased the chlorophyll content, resulting in lower purity of the extracted lutein. Compared with the SFE method, the Soxhlet extraction method had the highest pigment extraction rate. In response, some scholars proposed a more effective solution. Bing et al. [31] employed supercritical fluid extraction with an anti-solvent (SFE) method to purify zeaxanthin from the crude extract obtained via the Soxhlet extraction method of the microalgae Nannochloropsis oculata. The results showed that the purity of zeaxanthin reached 93.8%. This method combines the advantages of traditional organic solvent extraction and SFE while effectively avoiding the drawbacks of organic solvents, such as toxicity and low purity of extracts. Therefore, it holds significant potential for development in the field of microalgal carotenoids.

Subcritical fluid extraction (SFE) is a novel extraction technique that uses subcritical fluids as extractants. It transfers lipophilic components from the extractable material to the liquid extractant through molecular diffusion, followed by separation of the extractant and target product via a vacuum evaporation process. Subcritical fluids are fluids at the edge of the supercritical state, with pressure exceeding the critical point pressure and temperature below the critical value, forming a high-pressure liquid. Compared to supercritical fluids, subcritical fluids require lower temperatures, closer to room temperature, eliminating the need for heating equipment, making them more economically viable in terms of equipment investment and energy consumption. Additionally, at the same pressure, subcritical CO₂ has a higher density and stronger solubility than supercritical CO₂. Currently, there are few studies on the extraction of carotenoids from microalgae using subcritical fluid extraction. Only Huang Xingxin et al. [32] have conducted related research. They employed ultrasound-enhanced subcritical CO₂ technology to extract lutein from Chlorella and investigated optimal process conditions, ultimately determining the optimal conditions as follows: extraction temperature 25°C, extraction pressure 11 MPa, fluid flow rate of 30 kg/h, carrier agent (anhydrous ethanol) dosage of 1.5 mL/g, extraction time of 3 hours, and ultrasonic power of 750 W. Under these conditions, the lutein content extracted was 68.85 mg/100 g of Chlorella powder.

2.4 In situ extraction method

In situ extraction refers to continuously mixing algal liquid with a biocompatible organic solvent to extract carotenoids into the organic solvent phase while microalgae cells continue to synthesise carotenoids, thereby achieving simultaneous microalgae cultivation and carotenoid extraction. This eliminates the need for microalgae harvesting, increases carotenoid yield, and reduces production costs.

Hejazi et al. [33] applied the in situ extraction method to the production of β-carotene from Dunaliella salina. After culturing Dunaliella salina cells under normal conditions, they were transferred to a bioreactor as shown in Figure 1. Strong light irradiation induced the production of large amounts of β-carotene, while dodecane was continuously injected into the bottom of the algal solution. Dodecane extracted β-carotene from the algal cells through the aqueous phase, and finally, under the action of a pump, dodecane was recycled from the upper phase back to the bottom to continue the cycle. Experiments demonstrated that under strong light irradiation and in the presence of dodecane, Dunaliella salina could still survive (>47 days), although cell growth slowed, and the extraction rate of β-carotene exceeded 55%. Kleinegris et al. [34] investigated the mechanism of in situ extraction applied to salt algae. They found that contact between salt algae cells and the water-organic phase interface causes cell death, and subsequent cell rupture leads to the release of carotenoids, enabling the extraction process to proceed effectively.

Although the in situ extraction method can eliminate the cumbersome operational steps of traditional extraction methods, Kleinegris et al. [35] found that the volume yield of β-carotene extracted from Dunaliella salina using the in situ extraction method was relatively low, at 8.3 mg/L·d, while the traditional extraction method yielded 13.5 mg/L·d. Additionally, the emulsification of the two-phase solvents and the continuous accumulation of oxygen in the bioreactor inhibit the growth of Dunaliella salina, while strong light exposure causes β-carotene degradation. These drawbacks hinder the further development of the in situ extraction method.

2.5 Aqueous Two-Phase Extraction (ATPE)

Aqueous two-phase extraction (ATPE) originated in the 1960s and is a highly promising solid-liquid separation technology. Similar to the general water-organic extraction principle, it separates components based on their differing distribution behaviours between two phases. ATPE systems hold broad application prospects in the extraction and separation of bioactive substances.

Currently, few studies have investigated the extraction of carotenoids using ATPE. Only Cisneros et al. [36] conducted relevant research, using post-harvest Chlorella protothecoides to study the distribution behaviour of lutein in a PEG-phosphate aqueous two-phase system. They first extracted lutein from the algae slurry using ethanol at 30% wet weight of the algae, followed by extraction of the crude extract in a dual-phase system composed of 22.9% (w/w) PEG 8000 and 10.3% (w/w) phosphate at pH 7.0. The results showed that the majority of carotenoids were distributed in the upper phase, with algal cell fragments in the lower phase, and the carotenoid yield was 81.0% ± 2.8%. This method provides a broader perspective for the research and development of carotenoid extraction methods from microalgae.

3   Outlook

Microalgae are rich in carotenoids, with high content and diverse types, and they possess advantages such as short cultivation cycles, easy-to-control cultivation conditions, and the ability for continuous production, making them an excellent source of carotenoids. However, the preparation of carotenoids from microalgae is a high-cost process, severely limiting the research and development of microalgal carotenoid products. Currently, the extraction of carotenoids from microalgae primarily employs mechanical cell disruption combined with organic solvents. This method is simple to operate and easily scalable for industrial production but requires significant energy consumption and organic solvents. In recent years, with the development of new extraction techniques, such as the ultrasonic extraction method, microwave extraction method, and accelerated solvent extraction method mentioned earlier, it has been possible to effectively improve carotenoid extraction rates, shorten extraction times, and reduce solvent consumption to varying degrees. However, these methods inevitably involve the use of organic solvents, which are not environmentally friendly.

In contrast, supercritical/subcritical fluid extraction aligns with the principles of ‘green chemistry,’ producing carotenoid products with high safety. However, this method has high equipment requirements and achieves lower carotenoid extraction rates compared to solvent-based methods. All of the aforementioned methods require harvesting the algal biomass, which inevitably increases production costs. In contrast, in situ extraction can effectively avoid the harvesting process, enabling simultaneous microalgae cultivation and carotenoid extraction, thereby reducing energy consumption and lowering production costs. However, this method is still in its developmental stage and faces issues such as low extraction rates. In summary, although extensive and in-depth research has been conducted on the extraction of carotenoids from microalgae, with some progress achieved, no method currently exists that simultaneously possesses high extraction efficiency, versatility, rapidity, environmental friendliness, and low cost.

Therefore, to increase carotenoid production and reduce production costs, future development of microalgal carotenoids should focus on the following areas: first, screening fast-growing, economically viable algal strains with high carotenoid content; second, adopting appropriate extraction methods, simplifying extraction steps, optimising extraction processes, and reducing production costs while continuously improving existing methods; research and develop new technologies to achieve industrial-scale production of microalgal carotenoids; third, utilise modern genetic engineering techniques to modify microalgal strains and accelerate the industrialisation of microalgal carotenoid production. With the ongoing development of new microalgal strains and the continuous improvement of extraction processes, large-scale commercial production of microalgal carotenoids is not far off.

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