Factors Influencing Phycocyanin Synthesis in Microalgae and Culture Strategies: Toward Efficient Production of Alternative Proteins

Abstract

Global population growth makes an increase in food production inevitable, and protein plays a vital role as an essential nutrient. However, as the proportion of land used for agriculture and animal protein production decreases, the search for sustainable, low-cost alternatives to proteins has become a research priority. Microalgae can synthesize a wide range of proteins, among which phycocyanin is of interest due to its unique biological activity. It has a complete amino acid profile, contains essential amino acids, and is a high-quality source of protein. Most of the existing studies have focused on single influencing factors, improved methods, or specific culture conditions for the synthesis of phycocyanin in microalgae and have not yet analyzed the culture conditions, influencing factors, and improved strategies for the synthesis of phycocyanin in microalgae in a systematic and integrated manner, and the studies lacked comprehensiveness and consistency. In this paper, the key factors, mechanisms of action, and improvement strategies affecting the accumulation of phycocyanin in microalgae are reviewed. The growth of microalgae under autotrophic, heterotrophic, and mixed culture conditions and their effects on phycocyanin synthesis were systematically described. The aim is to accelerate the application of phycocyanin in the food industry and alternative proteins by improving the production efficiency of microalgae, promoting their comprehensive utilization, and injecting a new impetus into the development of a sustainable protein industry.

1. Introduction

According to data released by the United Nations, the global population has grown by 33 percent since 2000 (6 billion in 1999 to 8 billion in 2021) and will likely reach 9.5 billion by 2050 [1]. This will pose a huge food challenge for the world. As an essential nutrient for the human body, increasing the proportion and total amount of protein in food is necessary to cope with population growth. However, due to the reduction in agricultural land and environmental issues such as water pollution caused by livestock farming, finding a nutritious and sustainable natural food source is an effective way to meet human dietary needs [2]. As a result, alternative proteins have emerged as a promising solution and have quickly become the focus of much discussion, bringing new light to the protein supply dilemma. In recent years, algae have been promoted as a novel natural source of proteins and bioactive compounds due to their extensive and remarkable ecological adaptability [3]. Microalgae are highly adaptable to the environment in which they grow and therefore have more relaxed environmental requirements. This has led to the development of more complex physiological structures, which are suitable for the exploitation of a variety of biological resources [4]. More than 40,000 microalgae species have been counted to date [5]. These microalgae are rich in a variety of nutrients such as pigments, oils, vitamins, and proteins, and are therefore widely used in the field of food and nutritional supplements [6]. They are especially important for protein, a key nutrient indispensable for human health, especially in facilitating the transportation of substances, participating in immune responses, and repairing damaged cells [7].

The protein content of microalgae ranges from 51 to 71%, which is not only rich in essential and non-essential amino acids but also exhibits excellent protein utilization [8]. Moreover, microalgae do not require the use of arable land, when grown in ocean salt water, and do not compete with the environment and economic development in a mutually constraining manner, which is recognized as a green solution for the protein revolution. Among many microalgae, Spirulina is the most widely studied [9], which is not only rich in vitamins, chlorophyll, and unsaturated fatty acids but also in proteins, especially light-harvesting pigment proteins such as phycobiliproteins, which are unique to algae [10]. The range of nutrients contained in Spirulina is demonstrated in Figure 1 [11].

In addition, the general quality of plant proteins is considered to be lower than the amount of animal proteins. One of the key factors in evaluating this metric is whether the protein source contains sufficient amounts of essential amino acids. According to the current study, it was found that protein samples from the studied microalgae species exceeded both the plant and animal protein sources, and most of the microalgae proteins contained all the essential amino acids [12]. Therefore, synthesizing proteins using microalgae is one of the best options for alternative protein development.

Table 1. Amino acid profile of proteins found in microalgae.

Microalgae	Essential Amino Acid Content g/100 g Protein						Non-Essential Amino Acid Content g/100 g Protein											References
	Ile	Leu	Lys	Met	Phe	Thr	Trp	Val	Ala	Arg	Asp	Cys	Glu	Gly	His	Pro	Ser	Tyr
Chlorella vulgaris	3.38	7.30	12.32	2.47	3.57	4.37	-	5.70	8.97	10.30	14.11	1.22	13.35	7.03	2.21	7.95	4.49	2.81	[13]
Spirulina sp.	0.46	0.76	0.26	0.19	0.44	0.36	-	0.55	0.50	0.43	0.72	0.07	0.77	0.41	0.18	0.34	0.36	0.28	[14]
Genus Spirulina	4.48	9.81	7.11	1.93	7.85	4.57	1.16	7.81	11.48	6.02	10.12	1.94	14.36	5.25	2.19	5.17	3.31	7.85	[15]
Tetraselmis chui	2.65	5.08	4.11	1.79	3.56	3.71	-	3.87	5.52	8.29	11.40	1.06	10.61	4.55	1.22	4.46	2.59	2.10	[13]
Nannochloropsis oceanica	4.18	7.11	5.93	2.18	5.04	4.96	-	5.56	6.56	5.78	10.96	0.58	10.62	5.89	1.71	4.91	3.56	2.93	[13]
Nostoc sp.	3.68	9.41	6.47	2.23	7.15	5.31	1.02	7.15	9.88	6.15	9.18	1.54	12.38	6.54	2.01	5.28	3.16	6.84	[15]
Dunaliella salina	4.09	9.58	5.99	2.79	6.98	5.16	0.18	7.23	10.99	8.16	9.56	1.63	12.41	8.71	1.73	5.23	4.81	4.86	[15]
Pleurochrysis carterae	4.22	9.93	7.24	2.41	7.69	5.67	1.14	7.55	11.51	6.88	9.19	2.03	15.17	7.02	1.89	5.12	3.48	7.69	[15]
Haematococcus pluvialis	1.93	4.47	2.54	0.53	2.20	2.35	-	2.23	5.00	2.80	5.38	ND	7.12	3.45	ND	-	3.37	1.36	[16]
Acutodesmus acuminatus	4.34	6.69	4.46	1.31	4.51	4.29	-	3.20	8.00	3.83	7.54	ND	9.20	5.71	ND	-	4.80	2.74	[16]
Botryococcus braunii	2.55	5.35	3.01	0.67	3.69	2.94	-	2.38	5.64	3.58	3.12	ND	7.91	3.65	0.43	-	3.69	1.74	[16]
Skeletonema costatum	6.74	3.37	2.91	1.05	4.07	6.05	-	3.84	3.02	7.91	7.44	ND	10.58	3.49	1.28	-	4.19	14.65	[16]
Nannochloropsis oculata	1.00	2.00	1.50	0.43	1.20	1.20	0.41	1.50	1.60	1.50	2.10	0.25	2.60	1.40	0.44	2.40	1.10	0.88	[17]
Tetraselmis sp.	4.06	9.45	6.52	2.78	5.62	5.17	1.61	5.73	9.39	5.01	-	1.39	-	6.40	2.01	6.22	4.39	3.63	[18]
Chloromonas cf. reticulata	1.33	-	3.16	4.08	1.98	-	-	1.97	-	2.13	1.47	-	2.38	-	0.61	5.17	-	-	[19]
Pseudopediastrum boryanum	1.34	-	3.62	13.72	2.00	-	-	2.02	-	0.71	1.14	-	1.95	-	0.56	4.99	-	-	[19]
Chloroidium saccharophilum	1.14	-	3.65	2.99	1.49	-	-	1.85	–	0.60	0.34	-	3.05	-	0.18	3.17	-	-	[19]
Laurencia filiformis	2.73	4.37	5.46	1.64	2.73	3.28	1.09	2.73	3.83	3.28	8.20	0.55	7.65	-	1.09	2.73	3.28	3.28	[20]
Gracilaria crassa	3.83	5.16	3.83	0.82	-	3.64	-	2.12	4.49	4.04	11.76	0.40	8.59	3.43	1.20	2.65	4.23	1.99	[21]
Phaeodactylum tricornutum	3.23	2.42	-	2.49	2.87	3.28	2.25	1.63	3.67	2.89	2.22	2.88	2.52	2.53	ND	1.00	3.66	1.42	[22]

“ND” means undetected, and “-” means not included in the determination. Adapted from [12]. Ile—isoleucine; Leu—leucine; Lys—lysine; Met—methionine; Phe—phenylalanine; Thr—threonine; Trp—tryptophan; Val—valine; Ala—alanine; Arg—arginine; Asp—aspartic acid; Cys—cysteine; Glu—glutamic acid; Gly—glycine; His—histidine; Pro—proline; Ser—serine; Tyr—tyrosine.

demonstrates the content of various types of proteins in different microalgae. From the table, it can be seen that there are obvious differences in the content of essential and non-essential amino acids in different species of microalgae. These differences indicate that various microalgae are specific to certain protein amino acid compositions, which may be related to their unique physiological metabolism and ecological adaptation strategies. These differences also reflect the diversity of amino acid metabolism and accumulation patterns in microalgae during their long-term evolutionary process, which provides a direction for the subsequent exploration of unique amino acid resources. Among them, tryptophan was not detected in most microalgae, which may reflect the specificity of its synthesis pathway or its non-critical position in microalgal growth metabolism. These findings can provide a key reference for the development of the microalgae industry. In food processing, microalgae raw materials can be precisely screened according to the amino acid requirements of the human body; in feed production, suitable microalgae can be selected according to the nutritional requirements of farm animals to develop efficient feed additives.

Phycocyanin, one of the more abundant phytochromes in blue-green and red algae [23], is a light-trapping pigment–protein complex that is blue and possesses significant antioxidant, anticancer, and anti-inflammatory properties. In the field of medical applications, phycocyanin exhibits unique advantages over traditional anticancer drugs. Conventional anticancer drugs often have serious side effects on the body and have a short effective half-life. However, research data show that when used in combination with anticancer drugs or radiotherapy, phycocyanin not only significantly reduces the dosage of anticancer drugs, thereby reducing their side effects, but also enhances the therapeutic effect and improves the patient’s tolerance and response to treatment [24]. In addition, it can be used as a natural blue functional food coloring agent or for cosmetic dyes. As a hydrophilic protein, phycocyanin contains fluorescent pigments, which are divided into three main categories: phycocyanin (dark blue), phycoerythrin (red), and alloxanin (light blue) [25]. Phycocyanin is usually produced by Spirulina [26], which can be categorized into several subtypes, with the C-PC type being the most studied [27], as shown in Figure 2.

The protein components of phycocyanin include α-subunits and β-subunits, (αβ)-monomers as basic components that polymerize to form (αβ)3-trimers or (αβ)6-hexamers with a relative molecular mass of about 20 kDa, and a tetrapyrrole chromophore that is covalently bonded to a cysteine residue of the apolipoprotein structure [29]. Two subunits covalently bind three phycocyanin chromophores, two of which are located in the β-subunit and the other in the α-subunit, which gives the alginate a characteristic absorption peak in the 610–620 nm wavelength range [30]. Figure 3 illustrates the mechanism of microalgae synthesizing phycocyanin. The process of microalgae synthesizing phycocyanin begins with photosynthesis, in which the microalgae capture light energy through the photosystem and convert it into chemical energy to produce ATP and NADPH, which provide energy and reducing power for the subsequent metabolism. Based on photosynthesis, microalgae use the Calvin cycle to fix carbon dioxide into organic carbon compounds and at the same time improve photosynthetic efficiency with the help of the carbon concentration mechanism (CCM). This generates organic carbon in the metabolic pathway, providing carbon skeletons for the synthesis of phycocyanin. At the same time, microalgae take up nitrogen (e.g., nitrate or ammonium) from the environment, which is converted into amino acids by assimilation, of which specific amino acids (e.g., tryptophan and tyrosine) are the key precursors for phycocyanin synthesis. Under the regulation of gene expression, the α-subunit and β-subunit of phycocyanin are synthesized in the cytoplasm and form functional phycocyanin monomers by covalent bonding to the tetrapyrrole chromophore. These are further assembled into trimers or hexamers to form stable phycocyanin complexes.

As a potential bioactive substance in Spirulina, phycocyanin occupies an important position in the field of microalgae active ingredient research due to its unique molecular structure and diverse biological functions. However, the synthesis efficiency of phycocyanin in microalgae is easily constrained by multiple factors such as environmental factors, nutritional conditions, and culture modes. The systematic review and integration of existing single-factor research results and in-depth analysis of the key technical pathways to enhance the production of phycocyanin can not only help to optimize the microalgal culture process but also significantly improve the industrial production efficiency of phycocyanin. In this paper, we systematically review the research progress of the factors affecting phycocyanin synthesis in recent years. This paper also summarizes the key regulatory strategies and looks forward to future research directions. The goal of this study is to provide a theoretical basis and practical guidance for the development of efficient production technology for phycocyanin. This work is expected to provide practical references for phycocyanin production practitioners and effectively reduce the research and development time and economic costs.

2. Culture Conditions for Algal Blue Protein Synthesis

2.1. Light

Phycocyanin is an important phycobiliprotein widely found in cyanobacteria and some red algae, which acts as a light-trapping pigment complex in photosynthesis and is capable of absorbing and transferring light energy. It efficiently transfers light energy to photosynthetic reaction centers by absorbing light wavelengths that are underutilized by chlorophyll (e.g., blue and green light), thus improving the photosynthetic efficiency of algae under different light conditions [31]. However, the effects of light intensity on algal growth and phycocyanin synthesis are more complex. Although high light intensity can enhance the growth rate of Spirulina, too high light intensity will cause the growth rate to reach the upper limit and then no longer increase. In contrast, a low-light-intensity environment, although not conducive to rapid growth, promotes the synthesis of pigment- and protein-rich biomass, which in turn enhances the concentration of cultured cells. However, high cell concentration triggers the self-shading phenomenon, which hinders light penetration and ultimately leads to the growth rate of Spirulina being significantly inhibited. Therefore, both spectral composition and intensity levels of light are key environmental factors that regulate algal biomass accumulation [32]. Upon investigation, it was found that there are different views of light intensity on the accumulation of phycocyanin. It has been found that the content of phycocyanin increased in Arthrospira platensis ATCC 29,408 between 750 and 3000 μmol m⁻² s⁻¹, that is, at higher light intensities [33]. In the same vein, it is also believed that higher light intensity favors the accumulation of phycocyanin and phycoerythrin [34]. Similarly, researchers have experimentally discovered that the content of phycocyanin in Spirulina platensis UTEX 1962 increases with increasing light intensity [35]. However, some scholars hold different views. No significant relationship between the proteins and lipids within Spirulina platensis and light intensity was observed in the experiment [36]. Another scholar’s study also showed that light intensity had little effect on the content of proteins within Spirulina under light-saturated conditions [37]. In addition, the content of phycocyanin is closely related to light-quality conditions. According to the results of related literature research, red light and blue light are the two main light qualities that promote the synthesis of phycocyanin. In the early studies, it was found that the highest phycocyanin production rate of Spirulina was found under blue light irradiation [34]. This conclusion was confirmed by a follow-up study, in which researchers found that the phycocyanin content of Arthrospira platensis SAG 21.99 was highest under blue LED light. However, in comparison, the lowest phycocyanin content was found under pink and red light conditions [38]. In addition, other researchers have evaluated the effects of blue, orange, and white light on the growth and phycocyanin production of Arthrospira platensis. The results showed that orange light was able to enhance the growth rate and phycocyanin productivity of Arthrospira platensis, while blue light contributed to the increase in phycocyanin content [39]. In a study regarding the effect of LED spectra on biomass and phycocyanin production in Spirulina platensis, red light was found to be one of the best spectra to maximize the rate of phycocyanin production [40]. Similarly, a researcher cultured Arthrospira in low-light-intensity, high-light-intensity, and room-temperature watercourse tanks and found that the phycocyanin content was the highest under red light conditions in all culture conditions [41]. A study of red and blue luminescent solar concentrators (LSCs) in outdoor waterway ponds found that phycocyanin productivity increased by 44% when red LSCs were used, whereas the biomass and phycocyanin content of the algae did not show a significant increase in blue light conditions [42].

For mixed spectra, experimentalists used mixed red–blue, red–green, and red–green–blue spectra to study the culture of Arthrospira platensis FACHB-882 and found that the mixed spectra not only increased its growth rate and biomass but also increased the photosynthetic pigments and the main nutrients to a certain extent. Specifically, the phycocyanin content was increased by 14.38% under the mixed spectrum of 5% blue, 15% green, and 80% red compared to that of Arthrospira platensis UTEXEXB-882 under white culture [43]. The peak production of phycocyanin was achieved when the cells of Spirulina platensis UTEXLB 2340 were incubated under mixed spectral conditions consisting of 75% blue and 25% red light [44]. The increase in phycocyanin content was significant in Arthrospira platensis, cultured under a mixed spectrum of 30% blue light and 70% red light [45].

In summary, the relationship between light intensity and protein, as well as phycocyanin synthesis, is complex and has not yet shown a clear and uniform regularity. In terms of the effect of light quality on the synthesis of phycocyanin, both blue light and red light showed a promotion effect, but there is no conclusive evidence to show which light quality has a more significant promotion effect. In practical studies, the use of mixed spectra is one of the effective ways to optimize the performance of a single spectrum. It is worth noting that the effects of light quality and light intensity on algal physiological processes are closely related to the algal species selected. Typically, there is a specific saturation threshold for light intensity on Spirulina biomass accumulation, and when the light intensity is above or below this saturation point, it may trigger the phenomenon of photoinhibition, thus affecting the normal growth of algae. Comparatively speaking, blue light, red light, and a reasonable proportion of mixed spectra have positive effects in promoting the accumulation of phycocyanin and other pigments. Therefore, when carrying out relevant experiments, researchers need to fully consider the biological characteristics of algae based on specific experimental conditions, carry out targeted experimental design, and strictly follow the scientific experimental norms to ensure experimental rigor, to ensure that the experimental results have a good reliability and reproducibility and to provide solid data support for the subsequent in-depth research and practical applications.

2.2. pH

Light conditions have a complex effect on the synthesis of phycocyanin, and in addition to light, pH is also an important factor affecting phycocyanin. It is well known that the stability of phycocyanin is poor, and pH is one of the key factors for its denaturation. With the gradual advancement of the stability study of phycocyanin, many studies have been devoted to enhancing its stability by combining phycocyanin with other substances or adding stabilizers, and sugar is one of them. Sugar solution can significantly enhance its stability. Because sugar is easy to hydrate with water molecules to increase the viscosity of the solution, this viscous effect can reduce the speed of the movement of phycocyanin molecules and reduce collisions between molecules and aggregation, reducing the degradation rate of phycocyanin. However, the stabilizing effect of sugars on phycocyanin has a direct relationship with pH. In addition, solution pH is a central environmental factor regulating the dissociation and aggregation behavior of phycocyanin oligomers (monomers, trimers, etc.) [46]. It has been found that extreme changes in the pH at which the phycocyanin solution is located may lead to disturbances in the electrostatic properties and hydrogen bonding involved in protein conjugation, which may result in changes in the structure of the chromophore. Moreover, in an extremely low pH environment, it may lead to the dissociation of the trimer of phycocyanin into monomers, the decomposition of monomers into individual subunits, and partial subunit unfolding [47]. Therefore, it is extremely critical to precisely control the pH range during experiments. The color of phycocyanin is sensitive to pH changes due to the pH-triggered protein unfolding and disassembly mechanism that affects the energetics and spatial arrangement of the chromophore [48]. It has been shown that when the pH value dropped to about 3.0, the phycocyanin solution showed a tendency to aggregate and precipitate, and the color of the solution changed to lime green [49]. For pH > 8.0, the solution had a purple color [50]. This implies that the molecular conformation and electronic state of phycocyanin will be different under different pH conditions, which in turn will show different optical properties and physical states. To enhance the stability of phycocyanin in a low pH environment, the ultrasonic method was used to prepare phycocyanin–okra polysaccharide complexes under different pH conditions. The results showed that the solution at pH = 3.4 was the darkest; this suggests that the tetrapyrrole chromophore to which the phycocyanin subunit is attached suffers the least damage and the solution is more stable, forming a stable colloidal complex [51]. In addition to the use of sugars to enhance the stability of phycocyanin in low pH environments, significant progress has been made in recent years to reach this goal with the help of whey proteins. Typically, when the pH is lowered to 3.0, phycocyanin molecules tend to aggregate and form insoluble complexes, so the researchers explored the effect of the three on the stability of phycocyanin at this pH using pea protein, egg albumen protein, and whey protein. Only the whey protein–phycocyanin mixtures were kept in suspension, before and after heat treatment, with very little precipitate at the bottom of the tube. By combining all the experimental data and analysis, it is clear that whey protein provides the best protection for phycocyanin [49]. Previous studies have shown that phycocyanin solutions become unstable near their isoelectric point due to the weakening of electrostatic repulsion between phycocyanin molecules [52]. In another study, it was noted that Arthrospira platensis was able to achieve a maximum growth rate at a pH of 9.5–9.8. In contrast, at a pH of 7.0, the growth rate was significantly inhibited to only 20% of the optimal conditions [53]. This may be because, at higher pH, carbon dioxide dissolves to produce bicarbonate ions (HCO₃⁻), which provide a more efficient source of inorganic carbon for the cells [54]. However, the concentration of phycocyanin produced by Spirulina decreased under higher pH conditions [55]. Therefore, the pH adjustment of the culture system should be rationalized according to the demand of microalgae growth conditions at each stage of the experiment to achieve the optimal condition.

Because the stability of phycocyanin is significantly affected by pH, it is easy to denature and aggregate at low pH, and although sugars can enhance its stability, it is closely related to pH. Since whey protein is effective in enhancing the stability of phycocyanin at low pH, we can analyze the molecular mechanism of the interaction between whey protein and phycocyanin and precisely analyze the binding sites and types of forces between the two to optimize the design of formulations. At the same time, multiple comparative experiments can be carried out to explore the differences in the stabilizing effect of whey proteins of different origins and purities on phycocyanin to identify the most suitable products. We can also try to compound whey protein with proven stabilizers such as okra polysaccharides to explore the synergistic effect, develop more innovative products based on stabilized phycocyanin systems, and promote the development of phycocyanin-related industries.

2.3. Temperature

Phycocyanin is highly sensitive to temperature, and when used as a functional food material, it is often treated with microencapsulation, which reduces the loss of active compounds within the material during processing. For high-temperature-sensitive substances, a commonly used microencapsulation method is freeze-drying, which realizes the drying process through an extremely low-temperature environment and can effectively avoid thermal degradation of the substance. It was found that when Spirulina platensis powder was heated at 40 °C and 100 °C for 30 min, the degradation rate of phycocyanin was calculated to be 9.37% and 57.23%, respectively. In contrast, the degradation rates of phycocyanin were 4.28% and 17.40% when Spirulina microcapsules were heated at the same temperature and time. This suggests that microencapsulation of Spirulina can reduce the degradation of phycocyanin during heating [56]. Phycocyanin structures are denatured above 50 °C, resulting in altered chromophore stability and color changes [57,58]. These denaturation reactions may be related to modifications of the secondary, tertiary, and quaternary structures promoted by high temperatures [59]. When the environment is a high-temperature situation, hydrophobic amino acid residues promote protein aggregation, which tends to cause color change in phycocyanin due to the denaturation or aggregation of the APAs. It is hypothesized that the thermal instability and consequent loss of color of phycocyanin are due to the aggregation or denaturation of its peptide subunits. This can be assessed based on the changes in the photophysical properties of the peptide subunits of phycocyanin [60]. To date, the largest commercial source of phycocyanin is Spirulina, but it is noteworthy that phycocyanin obtained from Spirulina exhibits poor thermal stability, which limits its application in the food industry [61]. To solve this problem, researchers have started to explore other microalgae that can produce phycocyanin with good thermal stability. Among them, thermophilic microorganisms have been widely considered. Phycocyanin synthesized by thermophilic microorganisms exhibits better stability than that synthesized via mesophilic biosynthesis. Thermosynechococcus is one of the most widely studied strains in this regard [62]. Although techniques such as microencapsulation and the application of thermophilic microorganisms provide effective solutions to the problem of the thermal stability of phycocyanin, there is still room for further optimization.

2.4. Salinity

Salt is one of the key factors influencing the growth and biochemical composition of microalgae and can also cause stress damage to their growth and metabolism, altering metabolic pathways and leading to an increase or decrease in their bioactive compounds [22]. The interaction between salinity and nutrient effectiveness significantly exacerbates the complexity of osmoregulation, enzyme activity regulation, and structural integrity of microalgal cellular components in a mixed-trophic paradigm. Salinity stress induces the synthesis of compatible solutes and stress-responsive proteins in microalgae, which in turn leads to altered metabolic fluxes of secondary metabolites, including phycocyanin [63]. Therefore, salinity levels are extremely important for the synthesis of phycocyanin. In the case of Spirulina maxima, it was found that under normal culture conditions (0.02 M NaCl), its biomass reached a maximum value of 139.5 mg/L in one day, whereas under salt stress conditions (0.1 M NaCl), the biomass increased significantly, reaching a maximum value of 221.33 mg/L. This suggests that salt stress can promote the algae’s growth. In further studies, the purification of phycocyanin was carried out by ammonium sulfate precipitation. The results showed that the A₆₂₀/A₂₈₀ ratio in the protein fraction of Spirulina maxima purified by ammonium sulfate precipitation under 0.10 M NaCl stress was 2.25, which was significantly higher than that of phycocyanin in crude extracts. This indicated that the phycocyanin content of Spirulina was significantly increased under salt stress, suggesting that salt stress not only promotes the growth of algae but also improves the accumulation of phycocyanin [64].

However, microalgal biomass and synthesis of target metabolites can be affected by algal species type, medium composition, and environmental factors. The use of abiotic stresses such as nutrient deficiencies, high light intensity, and temperature and salinity extremes to regulate microalgal metabolites is an accepted strategy to achieve low-cost production of microalgae-derived products [65]. In response to abiotic stresses, microalgae make metabolic adjustments to increase the production of specific metabolites to resist the stresses, but this process usually results in a reduction in biomass accumulation [65,66].

One of the strategies to address this problem is the use of phytohormones, which can simultaneously increase the productivity and resilience of valuable microalgal biomass-derived metabolites [67]. Glycine betaine (GB) can significantly enhance cellular tolerance to adversities such as high salinity, nutrient deficiencies, temperature extremes, and drought by regulating osmotic homeostasis, stabilizing photosynthetic mechanisms, and enhancing antioxidant systems. In addition, it is one of the substances of choice for enhancing cellular resistance due to its low cost of synthesis and high economics [68,69]. In these experiments, the effects of salt stress and glycine betaine on its biomass and phycocyanin production were investigated, using Arthrospira platensis. First, by comparing the culture conditions without salt and different salt concentrations, it was found that the phycocyanin production showed a tendency to first increase and then decrease with the increase in salt concentration. When the salt concentration reached 100 mmol/L NaCl, the phycocyanin yield reached its highest. In further experiments, different concentrations of glycine betaine were applied in combination with 100 mmol/L NaCl to assess the synergistic effects on biomass and phycocyanin production. The results showed that biomass production reached a maximum value of 2.58 g/L at a glycine betaine concentration of 150 μM, which was 1.37 times higher than that of the control group; phycocyanin content and productivity also peaked under this condition, which increased by 37.38% and 88.27%, respectively, compared with the control group. This suggests that glycine betaine can effectively alleviate the inhibitory effect of salt stress on Arthrospira platensis and significantly increase its biomass and phycocyanin accumulation, which is a highly efficient microalgae metabolism regulation strategy [70].

Phytohormone regulators have shown great potential in mitigating the adverse effects of salt stress, significantly increasing biomass and phycocyanin production under salt stress conditions. But there are fewer studies on these conditions. The potential synergistic effects of combining GB or other hormones with other stress-relieving compounds or environmental regulators can be explored in the future, which may lead to more optimized strategies for microalgal culture. In addition, generalization of these laboratory findings to industrial applications will require studies on the economic feasibility and utility of these hormones in large-scale microalgae production systems. Ultimately, a comprehensive understanding and strategic application of these findings will provide a solid foundation for realizing sustainable and cost-effective production of high-value microalgae products. In turn, this will contribute to the broader goals of bioenergy development, biotechnology innovation, and environmental sustainability. Similarly, in our opinion, screening microalgae from seawater for high salt tolerance, studying their gene sequences in depth, pinpointing the target genes, and introducing them into other microalgae is also a viable solution to salt stress.

3. Cultivation Strategies

3.1. Self-Supporting Mode

The autotrophic mode is characterized by environmental friendliness, efficient resource utilization, and wide adaptability of algal species. Autotrophic algae only need some inorganic compounds, such as carbon dioxide, light energy, and inorganic salts. Photosynthetic autotrophy is a more economical and environmentally friendly way to convert CO₂ and energy into organic components through photosynthesis. And compared with other modes, under photoautotrophic conditions, microalgae tend to exhibit the strongest carbon uptake capacity for the same cell density, which can effectively reduce the carbon dioxide concentration in the air [71]. However, the photoautotrophic mode has significant limitations: The level of microalgal biomass accumulation is relatively low, with starch content typically ranging from 18% to 25% and lipid content fluctuating from 5% to 50%. The maximum biomass of this mode under outdoor pond culture conditions was about 86.7 tons/(yr/ha), which was significantly lower than the other culture modes [72]. As a commonly used means of large-scale microalgal biomass cultivation, the photoautotrophic mode faces many challenges in maintaining high-density microalgal cultures. On the one hand, the insufficient supply of carbon dioxide limits the growth rate and reproduction scale of microalgae; on the other hand, the light conditions show significant uncertainty due to weather conditions, where too much light may inhibit microalgae growth on sunny days, and insufficient light on cloudy or rainy days fails to satisfy their photosynthesis needs. These factors greatly increase the difficulty of realizing stable and efficient high-density microalgae culture. There are two main types of autotrophic algal cultures: one is carried out in an open pond system, and the other is carried out in a closed photobioreactor; both of these methods have advantages and disadvantages.

Table 2. Comparison of factors in open and closed systems.

Factor	Open Systems	Photobioreactors or Closed Systems
Space required	High	Low
Evaporation	High	No evaporation
Water losses	Extremely high	Almost none
CO2 sequestration rate	Low	High
CO2 losses	High	Almost none
Temperature	Highly variable	Required cooling
Weather dependence	Production is impossible during rain	Insignificant because they allow production in any weather conditions
Process control	Difficult	Easy
Shear	Low	High
Cleaning	None	Required
Contamination risk	High	None
Algal species variability	Restricted microalgae species may be cultivated	Nearly all microalgae species may be cultivated
Biomass quality	Not susceptible	Susceptible
Population density	Low	High
Harvesting efficiency	Low	High
Cost of harvesting	High	Lower
Light utilization capability	Poor	Good
Most costly parameters	Mixing	Oxygen and temperature control
Energy requirement (W)	High	Low
Capital investments	Low	High
Biomass concentration	Low during production, approx. 0.1–0.2 g/L	High, approx. 2–8 g/L

Adapted from [71].

provides statistics on open and closed culture systems in terms of various factors. Open ponds are simple to construct and relatively inexpensive, but they are more susceptible to environmental factors and can be easily contaminated by bacteria or other pathogens. Closed photobioreactors reduce this risk to some extent because they are largely closed monoculture systems and also achieve higher algal cell densities due to higher surface area and volume ratios, but this method is generally considered to be more costly [73].

From Table 2, it can be inferred that the choice of a closed culture system may be more favorable for improving the yield and quality of phycocyanin in the synthesis of phycocyanin by microalgae, although its initial investment cost may be higher. Closed systems can provide more stable environmental conditions, such as temperature control, reduced water loss and evaporation, improved CO₂ utilization efficiency, as well as being less affected by weather changes than open systems. In addition, closed systems help reduce the risk of contamination and improve the accuracy of process control, resulting in higher-quality biomass and higher phycocyanin yields. While open systems may be more advantageous in terms of space requirements and cost, in the case of phycocyanin production, the stability and controllability provided by closed systems are critical to ensuring product quality and productivity, helping to reduce long-term operating costs and improving the economic viability of the overall production process.

The technology of microalgae culture in open channel ponds appeared as early as the 1950s and has been applied since then; today, this technology is mature and well-established. Open waterway ponds are usually categorized into two types: artificial ponds and natural ponds [74]. Artificial ponds can be categorized in terms of their form into shallow ponds, runway ponds, and circular ponds [75]. The main designs can be categorized into watercourse systems, inclined or cascade systems, and circular ponds with a central pivot rotation system. The most commonly used of these is the waterway system [74]. Typically, open pond applications are prioritized as the most cost-effective aquaculture system due to their simple operating procedures, low maintenance difficulties, low energy consumption, and high scalability, which makes it easy to achieve large-scale applications. Although the area of water used for microalgae culture is considerable, the cell concentration of microalgae harvested from natural water bodies is relatively low. This calls for the development of an efficient microalgae harvesting method to address this challenge. Unlike open ponds, closed culture systems apply artificial light, which can be varied in intensity, spectral composition, and duration [76] to better suit the growth needs of microalgae. In addition, closed systems can be used for sterilizing the culture medium, introducing organic nutrients for different nutrient pathways, and resisting changing environmental conditions [77]. PBRs typically consist of a four-phase system: microalgae (solid phase), culture medium (liquid phase), O₂ or CO₂ (gaseous phase), and superimposed light radiation fields [78]. The lighting equipment, the culture tank, and the control unit constitute the three main components necessary for the manipulation of the PBR system [77]. Depending on the illumination surface, PBRs can be categorized as flat, tubular, or columnar. Based on the liquid flow pattern, PBRs can be categorized as stirred reactors, bubble tower reactors, or air-lift reactors. An ideal PBR needs to have a highly transparent surface, minimize non-illuminated parts, and have a high mass transfer rate for high biomass growth [78]. In summary, open and closed culture systems have their advantages and limitations. Therefore, there is great potential to explore the balance between the two, and to innovate methods and ideas to achieve the dual goals of economic and sustainable development.

3.2. Heterotrophic Mode

Unlike the autotrophic mode, microalgae in the heterotrophic mode have a unique way of growth and metabolism. In this mode, microalgae do not rely on photosynthesis but acquire energy and carbon by absorbing external organic carbon sources (e.g., glucose, sucrose, glycerol, etc.).

Figure 4 demonstrates the mechanism of synthesizing phycocyanin by microalgae in the heterotrophic mode.

Heterotrophy has the advantages of high biomass productivity, low risk of pollution, and easy control. However, not all algae can grow under heterotrophic conditions. There is a wide variety of microalgae species, and there are significant differences in the function of the organelles of different species, which will directly or indirectly affect the ability of microalgae to adapt to heterotrophic conditions. Some of these are because some microalgae still have imperfect mechanisms for extracellular organic carbon uptake and utilization [79]. Specifically, for some microalgae, organic matter is difficult to enter through the cell membrane or they cannot concentrate organic matter [80]. In addition, in some microalgae, the enzyme systems required for the metabolism of intracellular organic matter are poorly developed; therefore, organic matter is not efficiently utilized, which makes it difficult for some microalgal species to be heterotrophic [81], and respiration does not provide enough energy to sustain them, which are some of the reasons why microalgae cannot be heterotrophic [82].

However, in contrast to the photoautotrophic mode, external inorganic carbon sources and light conditions are not dependent elements for microalgal growth in the heterotrophic mode. As a result, they typically achieve higher cell densities and biomass, about four to eight times higher than in the photoautotrophic mode. In addition, their starch content occupies 32–53% of their dry weight, and lipid accumulation can reach 58% [83]. However, microalgae heterotrophic culture techniques have encountered serious challenges at the level of protein output: microalgae cultured using heterotrophic culture generally have a protein content of less than 40%, which is significantly lower than that of microalgae obtained from photoautotrophic culture methods [84]. Therefore, researchers have also explored ways to increase the protein content synthesized by microalgae under heterotrophic conditions.

For example, a novel culture strategy of “sequential heterotrophy–dilution–photoinduction” was developed. Chlorella was first cultured heterotrophically to reach a high cell density state and then photo-induced under a light environment, and under this strategy, the intracellular proteins of Chlorella rapidly increased to 50.87% [80]. Similarly, a mixed-nutrient culture of Chlorella in an illuminated glass bioreactor followed by scaling up the process to a 1000 L bioreactor resulted in a protein content of 60% on a dry weight basis [85]. A heterotrophic microalgae “compensatory growth” strategy has been proposed to increase protein content. The specific process is as follows: firstly, the accumulation of microalgae biomass is promoted under the condition of unlimited supply of nitrogen; next, the microalgae are made to go through a phase of nitrogen deficiency and then re-supplied a large amount of nitrogen to induce and strengthen the nitrogen “compensatory uptake” and accelerate the process of protein synthesis. The “compensatory uptake” of nitrogen is induced and strengthened to accelerate the process of protein synthesis. The experimental data showed that the protein content of common red algae cultivated by this method reached 44.3% [86].

For phycocyanin, under heterotrophic conditions, the synthesis of phycocyanin relies primarily on available ammonium [31], whereas excess glucose inhibits the synthesis of phycocyanin. Therefore, researchers have begun to explore the potential of culturing microalgae under conditions of limited carbon but adequate nitrogen sources to optimize algal biomass and the accumulation of high-value protein products. In the case of G. sulphuraria 074G, for example, researchers investigated the production of isolated phycocyanin under glucose-limited/nitrogen-sufficient, heterotrophic, high cell density, batch replenishment, and continuous flow-addition culturing conditions; it was found that the highest value of phycocyanin production was observed in the stabilization period when the environmental conditions provided were carbon-limited and nitrogen-sufficient [87].

Although the methods mentioned above have achieved an increase in protein content, they are difficult to realize in practice for two main reasons:

(1) It is relatively easy to carry out autotrophic or mixed-nutrient cultures of heterotrophically cultured microalgae in a laboratory environment, but it is difficult to carry out large-scale cultures because, due to the high density of algae under heterotrophic or mixed-nutrient cultures, the supplemental light intensity can decrease rapidly. How to distribute sufficient light energy to each algal cell became a major challenge.

(2) In the heterotrophy–dilution–photoinduction process, light autotrophic cultivation facilities are an indispensable presence. As the incubation time increases, not only does the complexity of the whole incubation technique increase significantly, but the risk of microbial contamination also increases dramatically, posing a great challenge for the stable operation of large-scale cultures [84]. Overall, heterotrophic microalgae can achieve high cell densities but face the problem of low protein content. Many of the reported methods to increase protein content are more or less problematic. Therefore, we believe that using advanced biotechnological means to induce directed mutations in microalgal genes, combined with fine optimization of culture conditions under the heterotrophic culture mode, is expected to achieve a significant increase in the protein content of algae. Thus, this will provide a feasible and efficient methodological pathway for the efficient and economic production of algal proteins.

3.3. Mixed Nutrition

The mixed-trophic mode includes both photoautotrophic and heterotrophic conditions. Microalgae can absorb both organic and inorganic carbon for energy metabolism using solar energy [72]. It is worth noting that this mode is not a mechanical superposition of the photoautotrophic and heterotrophic modes but a synergistic mode in which the functions of the two are complementary [88]. In some cases, microalgal productivity, biomass, and percentage of active substances were higher in the mixed-trophic mode than in the combination of the photoautotrophic and heterotrophic modes [89]. This makes the mixed-trophic mode an important microalgae culture mode in the fields of aquaculture, industrial production of microalgae, medicine, and food [90]. In addition, it has been noted that the uptake of both inorganic and organic carbon by microalgae was reduced in the mixed-trophic mode [91]; e.g., the glucose consumption of Chromochloris zofingiensis was about 0.166 g/h in the heterotrophic mode and 0.137 g/h in the mixed-trophic mode, whereas in the photoautotrophic mode, the carbon dioxide consumption was about 0.82 g/h, while it was 0.67 g/h in the mixed-trophic mode [88]. Similarly, Chrysophyllum showed a similar pattern. This phenomenon may be considered to be related to the synergistic effects of carbon and energy metabolism in microalgae in mixed-trophic modes, although the exact mechanisms have not been fully elucidated [92]. Significantly higher biomass and lipid production were produced during mixed nutrition compared to photoautotrophy and heterotrophy [93]. For example, some researchers conducted a mixed-nutrient culture of Chlorella vulgaris using 4 g/L glucose as an organic carbon source and air as an inorganic carbon source. The results showed that under the same culture conditions, the biomass obtained from this mode was 2.5 times higher than that of autotrophic culture and 87% higher than that of heterotrophic culture; its lipid content increased by 32% compared to heterotrophic culture and 77% compared to autotrophic culture [94].

Mixed-nutrient cultivation provides an effective strategy to achieve high growth rates and productivity, which is desirable in many applications such as microalgae cultivation and the production of high-value products. Utilizing organic resources from food industry waste to sustain microalgae growth is one of the most promising directions for the development of mixed-nutrient culture. In recent years, some researchers have explored the effects of brewery wastewater and salinity on the mixed-nutrient cultivation of A. platensis. They utilized a proportional mix of brewery wastewater as a salt-stressed organic carbon source and seawater to optimize phycocyanin production and biomass composition. They found that A. platensis biomass reached a maximum value of 3.70 g/L, with a significant increase in the concentration of phycocyanin under mixed-nutrient conditions, with brewery wastewater and seawater at 2% [95].

On the whole, in the field of phycocyanin production, the three culture modes of autotrophic, heterotrophic, and mixed-nutrient present different characteristics and advantages, which significantly affect the key factors of algal cyanin yield, quality, and production cost.

The autotrophic culture mode relies mainly on light energy and carbon dioxide to synthesize phycocyanin through photosynthesis. This mode has a significant cost advantage in phycocyanin production because it does not require the addition of additional organic carbon sources, which reduces production costs. For example, in large-scale open pond cultures, the autotrophic mode can fully utilize solar energy for efficient algal growth and high phycocyanin accumulation. However, the growth rate of the autotrophic mode is relatively slow, and the production and quality of phycocyanin may be limited by factors such as light intensity and carbon dioxide supply. It was shown that the production of phycocyanin in the autotrophic mode could be significantly improved by optimizing the light conditions and carbon dioxide supply, but its upper limit was still lower than that of the heterotrophic mode.

The heterotrophic culture mode excelled in biomass production and product quality of phycocyanin. Algae grow and metabolize by consuming organic carbon sources and can rapidly accumulate large amounts of phycocyanin with stable product quality. This mode is suitable for industrial applications that require high yield and purity of phycocyanin, such as pharmaceuticals and food additives. However, heterotrophic culture requires a large organic carbon source and strict aseptic operation conditions, which not only increases the production cost but also may lead to environmental pollution problems. In addition, the high demand for nutrients during heterotrophic culture may be limited by the availability of resources, thus affecting the feasibility of its large-scale application.

The mixed-nutrient cultivation mode synthesizes the advantages of both autotrophic and heterotrophic modes and shows greater comprehensive performance potential. Algae can utilize light energy for photosynthesis and consume organic carbon sources for heterotrophic growth, thus achieving efficient resource utilization and phycocyanin production. This mode is particularly suitable for resource recovery and efficient production scenarios, such as in wastewater treatment, where algae can utilize both organic matter and nutrients in wastewater for growth, achieving pollutant removal and phycocyanin accumulation. However, the complexity of the mixed-trophic mode also poses challenges, and more research investment is needed to optimize culture conditions, reduce costs, and improve system stability and reproducibility.

Table 3. Phycocyanin production by microalgae under heterotrophic and mixed-nutrient conditions.

Microalga	Carbon Source	Cultivation Mode	Phycocyanin Content	Reference
Galdieria sulphuraria 074G	Glucose 50 g/L	Heterotrophic	3.6 mg/g	[96]
	Fructose 50 g/L	Heterotrophic	3.4 mg/g	[96]
	Sucrose 50 g/L	Heterotrophic	4.3 mg/g	[96]
	Molasses 7.5 g/L plus Glucose 45 g/L	Heterotrophic	11.2 mg/g	[96]
	Sugar beet molasses sucrose 50 g/L, total sugar up to 750 g/L	Heterotrophic	350 mg/L	[96]
G. sulphuraria 074G	Glucose 500 g/L	Heterotrophic	1.4–2.9 g/L	[87]
	Glucose, fructose, glycerol 5 g/L	Heterotrophic	2–4 mg/g	[97]
		Heterotrophic, carbon-limited, nitrogen-replete	8–12 mg/g	[97]
	Glucose 5 g/L	Heterotrophic	18 mg/g	[98]
	Restaurant waste with glucose 5 g/L	Heterotrophic	20 mg/g	[98]
	Bakery waste with glucose 5 g/L	Heterotrophic	21.8 mg/g	[98]
G. sulphuraria strain 074G	Glucose 5 g/L	Heterotrophic	25–30 mg/g	[99]
Spirulina platensis	Glucose 2 g/L−1	Mixotrophic	0.279 g/L−1	[100]
Spirulina platensis	Glucose 2 g/L−1, Se 250 mg L−1	Mixotrophic	0.295 g/L−1	[100]
Spirulina platensis	Glucose 0.67 g/L	Mixotrophic	26.93 mg/L/d	[101]

Adapted from [102].

shows the synthesis and production of phycocyanin by some microalgae under heterotrophic and mixed-trophic conditions.

Future research and applications should be based on specific needs and conditions; the culture modes should be rationally selected and optimized to achieve sustainable development of the microalgae industry.

Table 4. Comparison of the characteristics of microalgae in different culture modes.

Cultivation Methods	Carbon Source	Energy	Bioaccumulation	Growth Rate	Price	Specificities
Photoautotrophic mode	Inorganic	Light energy	Low	Slower	Low	Low cost, slow growth, and limited bioaccumulation
Heterotrophic mode	Organic	Organism	High	Quick	High	Rapid growth rate, high biomass and yield accumulation, and susceptibility to contamination
Mixotrophic mode	Inorganic and organic	Light energy and an organism	High	Quick	Moderate	High cost, combines the advantages of autotrophs and heterotrophs, and prone to contamination

illustrates the characteristics of microalgae under different culture modes.

4. Prospects

Against the backdrop of continuous global population growth, intensifying pressure on resources and the environment, and soaring consumer concern for healthy and sustainable food, the field of alternative proteins has become a hot focus of current scientific research exploration and industrial deployment. The synthesis of phycocyanin by microalgae has significant advantages as a promising alternative protein source. From the perspective of resource utilization, microalgae can utilize biomass wastewater and atmospheric carbon dioxide for growth. This not only realizes the recycling of resources and reduces the production cost but also plays a positive role in mitigating the greenhouse effect and reducing environmental pollution. This is highly compatible with the concept of sustainable development currently advocated globally and is also an effective strategy to cope with resource shortages and environmental challenges. However, there are both opportunities and challenges for the future development of microalgae synthesizing phycocyanin. Through in-depth exploration and practice in the optimization of culture environment, research on influencing factors, and innovation of culture mode, it is expected that efficient and low-cost production of phycocyanin will be achieved, their wide application in various industries will be promoted, and an important contribution will be made to solving the global protein supply problem and promoting sustainable development. In addition, metabolomics and proteomics, as well as transcriptomics, can be used to comprehensively analyze the metabolites and protein expression changes of microalgae under different cultivation conditions and to excavate the key regulatory targets to provide a basis for precise regulation. In future research on protein synthesis in microalgae, especially in the field of alternative proteins, we can focus on the precise nitrogen regulation strategy. The precise synthesis of specific proteins can be realized by accurately controlling the amount of added nitrogen sources and meticulously screening different kinds of nitrogen sources. Specific genetic modification engineering can also be carried out to enable microalgae to form stable and excellent genes. At the same time, the optimization of process conditions will be carried out in depth to promote the expansion of relevant technology from the laboratory environment to the industrial factory production environment. We will further focus on the research of protein synthesis in large-scale factory cultivation of microalgae, accumulate practical cases, and provide solid support and reference for the wide application of this technology in the alternative protein industry.

It should be noted, however, that the future development of microalgae-synthesized phycocyanin presents both opportunities and challenges. Despite the sizable potential for the application of phycocyanin in the food industry, its actual promotion is still faced with real barriers such as regulatory approvals, consumer safety concerns, and market acceptance. Currently, the regulatory standards for phycocyanin as food additives or in other uses have not yet formed a unified system in various countries; the compliance assessment process of the extraction process is complicated, and the approval cycle and cost may constrain its commercialization; in addition, there are cognitive biases in consumer perceptions of the safety of microalgae-derived ingredients. Breakthroughs in these dimensions are also crucial for assessing its practical application value in the sustainable food sector.