Development in health-promoting essential polyunsaturated fatty acids production by microalgae: a review

Abstract

Polyunsaturated fatty acids (PUFAs) found in microalgae, primarily omega-3 (ω-3) and omega-6 (ω-6) are essential nutrients with positive effects on diseases such as hyperlipidemia, atherosclerosis, and coronary risk. Researchers still seek improvement in PUFA yield at a large scale for better commercial prospects. This review summarizes advancements in microalgae PUFA research for their cost-effective production and potential applications. Moreover, it discusses the most promising cultivation modes using organic and inorganic sources. It also discusses biomass hydrolysates to increase PUFA production as an alternative and sustainable organic source. For cost-effective PUFA production, heterotrophic, mixotrophic, and photoheterotrophic cultivation modes are assessed with traditional photoautotrophic production modes. Also, mixotrophic cultivation has fascinating sustainable attributes over other trophic modes. Furthermore, it provides insight into growth phase (stage I) improvement strategies to accumulate biomass and the complementing effects of other stress-inducing strategies during the production phase (stage II) on PUFA enhancement under these cultivation modes. The role of an excessive or limiting range of salinity, nutrients, carbon source, and light intensity were the most effective parameter in stage II for accumulating higher PUFAs such as ω-3 and ω-6. This article outlines the commercial potential of microalgae for omega PUFA production. They reduce the risk of diabetes, cardiovascular diseases (CVDs), cancer, and hypertension and play an important role in their emerging role in healthy lifestyle management.

Introduction

Several fatty acids with an unbranched chain and an even number of carbon atoms, ranging from 4 to 28, can be found in nature. The fatty acids can be monounsaturated (MSFA), polyunsaturated (PUFA), or saturated (SFA), depending on the type of hydrocarbon chain. Although PUFAs are a necessary component of the human diet, people cannot synthesize them independently. Therefore, PUFAs must be obtained externally through foods. Humans can produce a wide variety of fatty acids, but some polyunsaturated fatty acids (PUFAs), such as ω-3 and ω-6 fatty acids, cannot be produced and hence are classified as essential PUFAs. Linoleic acid (C18:2 n6, LA) and alpha-linolenic acid (C18:3 n3, ALA) are the parent ingredients of ω-6 and ω-3 fatty acids, respectively (Patel et al. 2019a, b). Humans can produce essential ω-3 fatty acids like eicosapentaenoic acid (C20:5 n3; EPA), docosapentaenoic acid (C22:5 n3, DPA), and docosahexaenoic acid (C22:6 n3, DHA) from ALA, but they cannot make ω-6 fatty acids like arachidonic acid (C20:4 n6; AA) from LA. Nevertheless, ALA is also converted into EPA, DPA, and DHA (Patel et al. 2022b). Healthy human diets typically contain a 1:1 ratio of ω-3 to ω-6 (Katiyar and Arora 2020). Researchers are paying attention to microalgae’s functional lipids/PUFAs to treat diabetes, chronic respiratory diseases, CVDs, and malnutrition. This is to decrease the onset of these diseases and their risk factors. PUFAs are believed to lower cholesterol levels and prevent hyperlipidemia, atherosclerosis, and coronary artery disease. DHA and EPA, two types of ω-3 PUFAs, are exceptionally nutrient-dense compounds linked to improved eye and brain development in newborns and heart disease prevention in adults (Katiyar and Arora 2020).

Microalgae are mostly unicellular, photosynthetic eukaryotic (including prokaryotic cyanobacteria) microorganisms. They are highly diverse; some may possess heterotrophic, mixotrophic, and photoheterotrophic growth potential (Patel et al. 2019a, b). Thraustochytrids (formerly heterotrophic microalgae) are reported as leading heterotrophic producers of essential PUFAs (Chauhan et al. 2023a, b, c). Microalgae are the second-best PUFA producers. However, low biomass yield is the major bottleneck for algal PUFA production. They can survive in several different environments and generate biomass containing many components that may be utilized in the food, pharmaceutical, and cosmetic sectors. In addition, they may be used in value-added products or biofuel production (Patel et al. 2020b). Microalgae produces lipids, it contains a several fatty acids, including polyunsaturated fatty acids (PUFAs), monosaturated fatty acids, and saturated fatty acids. Among them, PUFAs claim great importance in several health applications. It also produces high-value carotenoids, like β-carotene, astaxanthin, and lutein, which are of considerable interest to the feed and food industry (Vadrale et al. 2023; Patel et al. 2022c, Maltsev et al. 2021a, b). About 40% of the total fatty acids produced by the Porphyridium genus are PUFAs, primarily eicosapentaenoic acid (EPA, 20:5, 3) and arachidonic acid (ARA, 20:4, 6) and they are basically known for several health benefits: lowering blood cholesterol, reducing inflammation, or acting as precursors to antiviral and antihyperglycemic activity (Ardiles et al. 2020). Several researchers have investigated the ω-3 PUFA production potential of selective microalgae strains. Several genera of marine microalgae exhibited high PUFA concentrations. They are commonly photoautotrophs. However, some species can grow mixotrophically and heterotrophically, including Brachiomonas, Chlorella, Chlorococcum, Cyclotella, Euglena, Haematococcus, Nannochloropsis, Navicula, Nitzschia, Ochromonas, Phaeodactylum, Rhodomonas, Scenedesmus, and Amphora, Ankistrodesmus, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Dunaliella, Euglena, Nannochloropsis, Nitzschia, Ochromonas, Tetraselmis, respectively (Russo et al. 2021). Mixotrophic cultivations have tested several strategies to increase biomass and PUFA yields. However, the mixotrophic potential largely depends on the potential of microalgal strains. Also, strain modification to enhance the metabolic capacities of microalgal strains are required for improvement in commercial production. Moreover, advancement in biomass harvesting to bypass costly and energy-intensive methods will lead to realization of above algal product biorefining (Kumar et al. 2023). However, with bioengineering approaches, optimization must be carried out for critical abiotic parameters, e.g., light intensity, light cycle, temperature, salinity, nutrient availability, and operation mode, e.g., batch, fed-batch, continuous along with involved stages (Liyanaarachchi et al. 2021).

Large-scale PUFA production faces several challenges. The main challenges include low biomass and lipid production, PUFA ratio, long bioprocessing time, and economic downstream. The optimal cultivation conditions for producing ω-PUFAs are still being researched. These obstacles must be overcome to make microalgae compatible with ω-PUFAs production and health applications (Perdana et al. 2021). The current review focuses on compiling recent updates in algal PUFA production, existing bottlenecks, and probable solutions for developing sustainable production routes of omega PUFA and their bioactivities assessment towards establishing potential health applications. Sustainable PUFA production emphasizes mixotrophic cultivation advancements. These advancements show sustainable features such as carbon capturing, environment footprint upgrade, greenhouse gas emission reduction, cost-effective production, and environmentally friendly process.

Culture conditions of microalgae to promote PUFA production

Microalgae use light, inorganic carbon, and organic/organic carbon to grow in the photoautotrophic, heterotrophic, and mixotrophic modes, respectively. Microalgae grow in the photoautotrophic mode, which is a conventional low-yielding route. It uses light as energy and inorganic carbon (CO₂) as a carbon source for PUFA synthesis (Barta et al. 2021). On the other hand, organic carbon sources for heterotrophic and mixotrophic cultivation are high-yielding routes due to fast biomass generation and bioconversion rates. Mixotrophic cultivation employs a dual pathway for higher yield with sustainable attributes (Patel et al. 2020a, 2022a). The contaminants found in crude glycerol (methanol, ethanol, salts, metals, and soaps) might prevent microbial development, delaying crude glycerol biological conversion. Traditionally, pure glycerol was employed as a carbon source in microbial medium formulations (Silva et al. 2021). The carbon sources utilized by microalgae in different growing conditions are shown in Table 1.

Table 1.

The characteristics of different trophic modes, merits, and demerits

Trophic mode	Carbon source	Energy source	Light presence & metabolism	Top advantages	Main disadvantages
Photoauto-trophic	Inorganic carbon (CO2)	Light	Obligatory; No switch between sources	Convenient & low-cost sources CO2 sequestration Low contamination risk	dependency Low biomass & lipid productivity Affected by environmental factors
Heterotrophic	Organic carbon	Organic carbon	Not required; Switch between sources	Light-independency lower harvesting cost high biomass & lipid productivity	High cost of carbon source, High contami-nation risk, disable to induce photodepen-dent metabolites
Photo-hetero-trophic	Organic carbon	light	Obligatory; switch between sources	Light-independency lower harvesting cost high biomass & lipid productivity	High cost of carbon source, High contamination risk, enable to induce photo-dependent metabolites
Mixotrophic	Inorganic & organic carbon	Light & organic carbon	No obligatory; Simultaneous utilization	Top growth rate & high biomass yield, high lipid productivity, capable against photo-oxidative damage, lower substrate photo-inhibition, improved trans-esterification due to lower chlorophyll synthesis	Light & organic carbon High contamination risk High cost of carbon source

Photoautotrophic cultivation and PUFA production

The most popular method for producing microalgae on a large scale for different applications is phototrophic culture. Most algae species that produce EPA use phototrophic or autotrophic modes of growth, where microalgae get their energy from light and CO₂. Because of its low cost of scale-up, least adverse environmental effect, and reduced risk of contamination, autotrophic culture has been widely researched for PUFA synthesis (Saxena et al. 2020).

The most popular conventional technique for producing microalgae is photoautotrophic culture, which mostly used closed outdoor photobioreactors or open ponds. The earliest, most widely used, and the easiest platform is open-pond cultivation. A closed autotrophic photobioreactor system continued to be the center of microalgae biotechnology research from the 1980s through the 1990s due to a stable environment. It was also the site for commercial microalgae biomass production and valuable biomolecules. A closed photobioreactor offers various benefits over an open-pond system, including a lesser acreage need, control over operating and growth conditions, a greater surface area, a higher rate of nutrient consumption, and a closed environment also supports aseptic culture conditions (Ren et al. 2022).

Photoautotrophy has some limitations, along with its many advantages. Light intensity and photoperiod play a crucial role in microalgae growth, as they affect photosynthesis rate and, thus, biomass production. However, excessive light can cause photoinhibition and damage the photosynthetic apparatus, decreasing microalgae growth. Moreover, the photoperiod also affects microalgae growth; short light and long dark periods reduce biomass yield. Moreover, photoautotrophic microalgae cannot assimilate organic carbon. However, microalgae depend on inorganic nutrients, such as carbon, nitrogen, and phosphorus, to grow and survive (Patel et al. 2021a). Phototrophic microalgae utilize CO₂ during photosynthesis to produce organic compounds such as lipids, proteins, and carbohydrates. However, nutrient assimilation can be affected by light invasion and opaque or unhygienic environmental conditions, limiting microalgae growth and biomass productivity. The nutrient-to-biomass conversion rate is slower than the other two organic carbon-supported trophic modes (Patel et al. 2021b). PUFA yield is directly proportional to biomass and lipid yield; once biomass and lipid yield is hampered or declined, PUFA yield is significantly affected (Chauhan et al. 2023a, b).

Microalgal PUFA production under heterotrophic condition

Heterotrophic microalgae may grow in the dark and utilize organic carbon (such as glucose, acetate, crop flour, and wastewater) to achieve significant cell densities, rapid growth rates, and greater biomass production. A few species, however, can grow heterotrophically, and there is a substantial danger of contamination during culture by other heterotrophic microorganisms (e.g., bacteria, yeasts, and fungi). To prevent contamination, during heterotrophic cultivation, microalgae must be grown under strict sterile condition (Morales-Sánchez et al. 2015). Some microalgal genera, for example, Phaeodactylum and Scenedesmus, are obligate autotrophs, but others, like Nannochloropsis, Dunaliella, and Tetraselmis species, can grow in organic substrates as facultative or obligate heterotrophs similar to dinoflagellates Crypthecodinium and Gyrodinium (Russo et al. 2021). Some algae species, such as Galdieria sulphuraria, had comparatively higher PUFA content under heterotrophic condition than that of phototrophic condition. It also possesses higher concentration of saturated and monounsaturated FAs. Metabolic flux is higher for lipid bioconversion when excessive energy and carbon is available as readily utilizable organic source such as glucose. Similar findings were made with the marine diatom Cyclotella cryptica. It was discovered that heterotrophic conditions (i.e., glucose-enriched culture medium) were successful in raising EPA yields in comparison to the autotrophic cultivation (Cupo et al. 2021). Microalgae heterotrophic cultivation can vastly enhance total lipids and cell biomass. Heterotrophic fermentation produces 3–4 times higher lipid yield than autotrophic mode. Galdieria sp. USBA-GBX-832 exhibited the higher biomass and PUFA yield under heterotrophic conditions than in autotrophic conditions (Lopez et al. 2019).

Heterotrophic microalgae growth is faster than photosynthesis-supported microalgal growth. Adequate amounts of nutrients are needed to maintain fast growth in heterotrophic environments. Among macronutrients, carbon, nitrogen, and phosphorus are more needed. Typically, heterotrophic cultivation uses glucose, yeast extract (or ammonia), and phosphates as carbon, nitrogen, and phosphorus sources. Because heterotrophic cultivation does not use alternative organic carbon sources, its cost is the biggest concern. The biggest disadvantage of producing DHA in heterotrophic circumstances from an economic standpoint is that glucose may account for 80% of the entire cost of heterotrophic production. Therefore, the production of cost-effective and sustainable carbohydrate-rich biomass to produce bioethanol and value-added compounds such as vitamins, pigments, proteins, lipids, and antioxidants is made possible by the mixotrophic microalgae. As Spirulina platensis is cultivated with cheese whey in a mixotrophic environment, it may yield significant biomass (Patel et al. 2020b). The effects of various glycerol concentrations on Spirulina sp. LEB 18 biomass production, as well as the biomass makeup in terms of fatty acids and proteins, were examined. When 0.05 mol/L of glycerol was added to the culture, compared with the control, the concentration of PUFAs increased by 20%, while saturated fatty acids decreased by 60% (Morais et al. 2019).

Several PUFA producing microalgae were examined in previous study. Thraustochytriacea and Crypthecodiniacea were major families identified as ω-3 PUFA producers in heterotrophic environments (Oliver et al. 2020). The highest ω-3 PUFA, mainly DHA, is determined in Crypthecodinium from the Crypthecodiniacea family. In addition, Schyzochitrium and Ulkenia are found promising from the Thraustochytriacea family. Thraustrochytrium aureum 28,210 could produce 48.3–58.2% DHA of total fatty acids. Moreover, Thraustrochytrium aureum 38,304 strain exhibited 41–75% DHA that of total fatty acids. While Ulkenia showed approx. 5–13% DHA yield (Oliver et al. 2020). In a recent study Chlorella sp., Planophila sp., and Nannochloropsis sp. produced 50, 36, and 50% ALA fractions in total fatty acids, respectively (Jain et al. 2022).

Microalgal PUFA production under mixotrophic condition

Mixotrophic microalgae have both photoautotrophic and heterotrophic characteristics. They may utilize light as an energy source, and CO₂, and sugar/acid as carbon sources. Therefore, as compared to phototrophic culture, they exhibit combined benefits of photoautotrophic and heterotrophic growth modes to produce additional biomass and lipid under the same cultivation period that of phototrophic mode offer alone. Only some microalgae species have the potential to grow mixotrophically and utilize organic and inorganic carbon sources simultaneously. This mode is most promising for larger-scale production due to carbon capturing ability and higher product yield than other trophic modes (Patel et al. 2020a). Microalgal growth in sugar-containing media needs strict sterilization (similar to heterotrophs) to avoid fast bacterial growth (Ananthi et al. 2021). Mixotrophic cultivation favours high biomass and lipid yields. For example, Scenedesmus obliquus produces more lipids (11.6–58.6%) during mixotrophic cultivation than under photoautotrophic cultivation (7.14%). Compared to photoautotrophic culture, mixotrophic cultivation of Chlorella vulgaris yielded 4.43 times higher biomass than phototrophic cultivation (Katiyar et al. 2018). Compared to photoautotrophic culture, the PUFAs e.g., arachidonic (ARA; C20:4 -6), eicosapentaenoic (EPA; 20:5 -3), and docosahexaenoic (DHA; 22:6 -3), are essential to human health. Therefore, concern for its cost-effective production has grown significantly in the pharmaceutical and nutraceutical sectors (Jiao et al. 2018). The conventional sources of these PUFAs are marine fish oils; however, their PUFA content are varies (Shahidi and Wanasundara 1998). The most affecting factors are heavy metals or pesticides contaminations. Moreover, owing to overfishing, these sources are not considered as environmentally friendly and sustainable for long term goal. Microalgal oil is a different choice. Microalgae mostly produces these fatty acids, and a number of growing techniques have been examined for their production, with mixotrophic cultivation being one of the alternatives (Castillo et al. 2021). Mixotrophic growth employs sunlight and organic carbon sources to generate extra biomass through dual pathways: photosynthesis and respiration, respectively. Mixotrophic cultivation increases the EPA production of some microalgae strains (Xu et al. 2004). The Nannochloropsis sp. biomass concentration was 1.4-fold higher with 0.03 mol L⁻¹ glucose in a mixotrophic mode than in a phototrophic mode. Nonetheless, there were only minor variations in the EPA yield (Sivakumar et al. 2022). Polyunsaturated fatty acids mainly EPA, can improve health by reducing neuroinflammatory and cardiovascular risks. A previous study examined the EPA yield in N. laevis under glucose (5 g/L) supported by heterotrophic and mixotrophic cultivations. The EPA concentration was 50% higher in mixotrophic cultivations of N. laevis than in heterotrophic cultivations. The N. gaditana microalgal EPA concentration greatly increased in continuous mixotrophic growth with glucose (5 g/L) or glycerol (1 g/L) than in autotrophic conditions (Menegol et al. 2019). In Fig. 1, the sustainability features of mixotrophic cultivation of microalgae PUFAs are illustrated.

Fig. 1.

Comparative production strategies of microalgal PUFAs with promising mixotrophic cultivation approach and its sustainability features

ARA is a very prominent polyunsaturated fatty acid for the human brain. This makes it a significant source of polyunsaturated fatty acids in the nutraceutical and pharmaceutical industries. The batch production of ARA in Porphyridium purpureum CoE1 under mixotrophic conditions was assessed to measure its potential and determined it as a good ARA producer. The potential carbon sources examined were acetate, glycerol, and glucose. The ARA content rose by 43% and 52% with acetate (0.25%) or glycerol (0.5%) under mixotrophic mode as compared to autotrophic conditions (Castillo et al. 2021).

Advantages of heterotrophy and mixotrophy over photoautotrophy

Heterotrophic growth conditions involve using organic carbon sources, such as sugars, organic acids, and glycerol, as energy and carbon sources. Under heterotrophic conditions, microalgae can bypass carbon fixation, resulting in faster growth rates and higher biomass productivity. Mixotrophic growth conditions combine both phototrophic and heterotrophic growth conditions, allowing microalgae to utilize both light and organic carbon sources simultaneously. This enables microalgae to grow faster and offers higher yields than phototrophic growth conditions (Patel et al. 2021a). Under mixotrophic growth conditions, microalgae generate 3–fivefold higher biomass than phototrophic culture, resulting in increased CO₂ fixation rates (Sim et al. 2019; Patel et al. 2022a). Besides photosynthesis, mixotrophic microalgae can use organic carbon to generate extra biomass via oxidative phosphorylation (Patel et al. 2020a, b). Additionally, mixotrophic growth conditions can enhance the microalgal photosynthetic efficiency by providing additional energy and reducing photoinhibition (Sim et al. 2019; Patel et al. 2021b). Heterotrophic and mixotrophic growth conditions are often carried out in closed cultivation systems such as fermenters or bioreactors, which minimize the risk of contamination by unwanted microorganisms (Sim et al. 2019). The use of closed systems also allows for better control of environmental parameters, such as temperature, pH, and nutrient levels, which can further increase growth and productivity (Patel et al. 2022a).

Effect of organic carbon sources

Several authors have identified various carbon sources for the heterotrophic cultivation of Chlorella pyrenoidosa, Auxenochlorella protothecoides, Schizochytrium mangrovei, or S. limacinum, including glycerol, acetate, prehydrolyzed whey permeate, forest biomass, or food waste hydrolysates. Reports frequently focus on biodiesel development. Studies on the utilization of alternative carbon sources in ω-3 PUFA production techniques have recently been reported. In several studies, Schizochytrium sp. growing with alternate carbon sources is one of the most promising options for ω-3 PUFA synthesis (Oliver et al. 2020). Mixotrophically cultivated Dunaliella salina, showed promising potential to be used in commercial PUFA production. The organic sources helped to determine their effect on PUFA yield at heterotrophic and mixotrophic conditions compared to autotrophic microalgae. Inorganic carbon supported autotrophic growth at 25 mM NaHCO₃. Mixotrophic growth was assessed using glucose (20–100 mM) and/or acetate (50–200 mM). In a comparative study, acetate increased the number of cells, their 100 mM or higher concentrations resulting in maximum cell growth (50–106 cells/mL). Similarly, there was a rise in cell population for glucose concentrations over 20 mM in mixotrophy with the highest cell concentration (90–106 cells/mL) with 60 mM glucose. Both heterotrophic and mixotrophic cell counts and PUFA content were higher than autotrophic (Castillo et al. 2021). PUFA enhancement in organic carbon-induced cultures was more prominent than in autotrophic cultures, for example, Phaeodactylum tricornutum yielded EPA 45.52 ± 2.72% of total fatty acid in crude glycerol-supported mixotrophic growth (Penhaul Smith et al. 2023). Various microalgal strains are mixotrophically cultivated using different organic carbon sources to produce ω 3 and ω 6 PUFAs discussed in Table 2.

Table 2.

Microalgal polyunsaturated fatty acids production from various organic carbon sources

Microalgae	Carbon source	PUFAs	Yield	References
T. pseudonana	Crude glycerol	EPA	8.41 ± 0.15%	Baldisserotto et al. (2021)
Chlorella pyrenoidosa	Glucose	Linolic acid	16.3 ± 0.99%	Ratnapuram et al. (2018)
Porphyridium purpureum	Sodium acetate	ARA	0.38% (v/v)	Jiao et al. (2018)
P. tricornutum	Crude glycerol	EPA	45.52 ± 2.72%	Penhaul Smith et al. (2023)
C. vulgaris	Glucose and industrial oleaginous residue	γ-linolenic acid	4.9%	Rosa et al. (2020)
Phaeodactylum tricornutum	Glucose	EPA	23.6%	Hamilton et al. (2016)
P. tricornutum	Glycerol	EPA	31.36 ± 5.4 mg/L	Kim et al., (2023)
Tetradesmus obliquus	Beet molasses	Linoleic acid	20.19%	Piasecka et al. (2020)
Chlorococcum sp.	Dairy effluent	Linoleic acid	10.80%	Ummalyma and Sukumaran (2014)
P. tricornutum	Spruce (SH) hydrolysates	DHA	4.89%	Patel et al. (2019a, b)
I. galbana	1% D-glucose	Linoleic acid	87.9 mg/g	Nicodemou et al. (2022)
Scenedesmus obliquus	Glucose	Linoleic acid	26.9 ± 0.0%	Shen et al. (2020)
Spirulina platensis	Sodium acetate	Linoleic acid	1.38 ± 0.21%	Lu et al. (2021a, b)
C. pyrenoidosa	Hydrolysate	C16:2, C16:3, C18:2, C18:3 & C18:4	35.3%	Zhang et al. (2020)
Crypthecodinium cohnii	Lignocellulosic biomass	DHA	2.2 g/L	Karnaouri et al. (2020)
Phaeodactylum tricornutum	Spruce hydrolysate mixed with f/2 medium	EPA	19.69 mg/L/day	Patel et al. (2019a, b)
Fistulifera sp	–	EPA	17.1%	Liang et al. (2013)
Chlorococcum humicola	–	LA	10.45% of TFA	Hena et al. (2015)
Nannochloropsis oculata	Glucose	EPA	30%	Menegol et al. (2019)
Chlorella vulgaris	Glucose	Linoleic	5.8 ± 0.1 g /100 g	Canelli et al. (2022)
T. obliquus	Acetate + glycerol	Linoleic	11.2 ± 0.4%	Patnaik and Mallick (2020)
Chlorella pyrenoidosa	Glucose + NaHCO3	Arachidonic acid	1.2 ± 0.01	Ratnapuram et al. (2018)
Chlorella sp	Glucose	Linolenic Acid	11.7–19.8%	Gao et al. (2019)
N. oceanica	Glucose	EPA	28.09%	Mitra and Mishra (2018)
C. vulgaris	Dextrose + Bicarbonate	Linolenic Acid	10.44%	Whang chai et al. (2021)
C. kessleri	Glucose	Alpha linolenic	22.2%	Deng et al. (2019)

Enhancement strategies for PUFA yield

Microalgae PUFA production and content have been improved through various methodological approaches. Using suitable statistical equations to represent the various metabolic pathways of production is one of these approaches. In addition, using the dynamics of interactions between various species or changing the growth conditions.

Co-cultivation strategy

Mixed culture systems have been examined in another research for algal PUFA synthesis. This study aimed to examine the synergistic coexistence between species and multi-PUFA fractions. This method involves cultivating multiple microalgae species in a single reactor. This technique has received much attention and has been shown to accumulate more PUFA than in monoculture systems. Research has shown that increasing CO₂ concentrations in mixed cultures improves biomass lipid fraction. Moreover, PUFA-producing microalgae consortiums might be simultaneously used for CO₂ bioremediation from the atmosphere (Soto-Sánchez et al. 2023). The cultivation strategy for enhanced PUFA may consider crucial factors: light distribution, nitrogen absorption, growth, and respiration rates, based on temperature and production scale. The dilution rate and inorganic nitrogen content substantially affect microalgal development in continuous culture systems (Yuan et al. 2020).

Effect of different abiotic stress factors

Microalgal lipids are often divided into two groups: storage lipids, which are non-polar lipids like saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), and structural lipids, which are polar lipids mostly composed of polyunsaturated fatty acids (PUFAs). The development of lipids and PUFAs is also stimulated in microalgae cultures under stress conditions such as high salinity, low P and N, and low temperature. By altering environmental growth conditions at specific levels or by having a certain influence on a particular species, stress conditions are created, enhancing PUFA yield. Studies have been conducted on chemicals with various modes of action to achieve favorable outcomes in PUFA production, including exposure to certain nutritional levels (missing or excess nutrients). For instance, microalgae exposed to high salinity produced more PUFAs, while cultures exposed to residues of mutagens like ethyl methane sulphonate produced surprising amounts of lipids (Soto-Sánchez et al. 2023).

Triacylglycerols (TAGs), which make up most storage lipids, are transesterified to create biodiesel, which fuels metabolic functions. Being the matrix for several metabolic activities, PUFAs sustain membrane functioning. It has been shown that TAGs participate in polar lipid synthesis throughout the day and accumulate in the cytosolic lipid bodies at night (Thompson 1996). Several microalgal species accumulate PUFA in TAG, including Nannochloropsis sp., Pavlova lutheri, P. tricornutum, and others. Microalgal lipid content improves when growth conditions are changed. For survival against growth-limiting stress situations (such as light intensity, temperature, salinity, nutrients, pH, and UV radiation), microalgae began to accumulate lipids and/or starch (Paliwal et al. 2017). n Fig. 2, combination of abiotic factor and other improvement for lipid enhancement are illustrated.

Fig. 2.

Combination of abiotic and other factor for microalgal lipid enhancement

Effect of salinity

It is an essential component of culture media that may alter the osmotic pressure of algal cells and affect their cellular growth and biomass yield. Salinity can also affect the fatty acid composition of marine algal lipids. Chlamydomonas reinhardtii was cultured in 0.1–0.3 M sodium chloride (NaCl) supplemented production media and showed a reduction in certain PUFA content under given salinity. Algal cells died in 0.2 and 0.3 M NaCl-supplemented media due to high salinity. At 0.1 M of low dose NaCl, microalgae grew well. However, a decrease in PUFA percentages in total fatty acids was observed (Hounslow et al. 2016).

Salt stress stimulated a higher yield of lipids, whereas PUFA ratios in total fatty acids were lowered. It has been found that increased total lipid yield in tested growth conditions usually compensates for decreased PUFA percentages, thereby increasing the overall yield of algal PUFAs (Lu et al., 2021a, b).

Effect of pH

One of the key factors during microalgal culture is pH, which determines CO₂ and nutrient solubility, and nutrient availability for cell growth. To improve inorganic carbon intake during microalgae growth, pH increases. This might limit cell growth depending on the type of microalgae species. Both biomass yield and lipid accumulation depend on pH (Qiu et al. 2017). Besides CO₂, bicarbonate (HCO₃⁻) based salts are used as an inorganic carbon source for microalgae to regulate pH and fulfill CO₂ needs.

A previous study assessed Desmodesmus sp. (MAS1) and Heterochlorella sp. (MAS3) tolerance at various Cd concentrations (Abinandan et al. 2019). The findings showed that at low pH 3.5, microalgae responded better at Cd concentrations of 1–5 mg L⁻¹ than at 10–20 mg L⁻¹. The scientists also discovered around a 10% increase in lipid accumulation compared to the control (without Cd), resulting in a relatively lipid-rich biomass. However, one study noted an increase in Pavlova lutheri cell density at pH 7 to 9 (Shah et al. 2014). In addition, over the same pH range, there was also a large buildup of lipids (32–35%), corresponding to one third of unsaturated fatty acids.

Effect of temperature

To increase microalgal PUFA yield, temperature is a crucial parameter. Leptocylindrus danicus produced more PUFAs when grown at 14 °C than at 26 °C (PUFAs: 39.80%; EPA: 13.80%; and DHA: 3.96% of TFA). The culture of Nannochloropsis salina, Isochrysis galbana, Rhodomonas salina, and Dixioniella grisea also revealed the beneficial effects of low temperature on PUFA production. Freshwater microalgae treated at low temperatures to increase PUFA accumulation are more interesting than marine microalgae. The EPA levels of Scenedesmus sp. grown at 10 °C were significantly higher than those grown at 20 °C and 30 °C at the growth stage (from 3 to 9 days) in recent research. Another study employed Crypthecodinium cohnii, cultivated at various temperatures (15–40 °C) to produce DHA. Below are a few examples demonstrating the significance of low-temperature settings for PUFA synthesis in microalgae (Lu et al. 2021a, b).

Another study examined whether Trachydiscus minutus’s PUFA composition and EPA generation were influenced by temperature and other factors. This study focused on cultivating plants outdoors in moderate summer weather since EPA output peaked at 28 °C (0.03 g L/ day), even though EPA content in fatty acids was the highest at 20 °C. When Porphyridium cruentum cells were kept at 20 °C, more EPA production and a lower ARA to EPA (ARA/ EPA) ratio were observed. With cultivation at 20 °C instead of the optimal temperature (25 °C), an increase in EPA content from 20 to 45% (w/w) of total fatty acids was determined. As reduced temperature stress may enhance microalgae EPA concentration, biomass and the production of EPA may require distinct ideal temperatures (Sivakumar et al. 2022).

Effect of nitrogen

Microalgae biochemistry is influenced significantly by nutrients. As a result, altering the nutritional profile during microalgae development might alter the final content and composition of lipids. When specific nutrients are withheld from microalgae, they exhibit a greater lipid content. Microalgae often produce carbohydrates instead of lipids when conditions are favourable. Most microalgae, however, increase the amount of lipids in their bodies when deprived of nitrogen, and other nutrients e.g., iron (to a lesser extent), cobalt etc. Nitrogen deprivation increases total lipid biosynthesis but has no direct effect on ω-3 PUFA accumulation. The ω-3 fatty acid amounts in microalgae vary depending on the amount of nitrogen utilized for biomass production. Recent research investigated the impact of nitrogen deprivation on lipid compositions and discovered that different nitrogen stress sources affected the fatty acid makeup of microalgae (Sajjadi et al. 2018). The nitrogen stress conditions reduced polyunsaturated fatty acids while raising saturated fatty acids (Perdana et al. 2021).

Effect of light

Taxonomic specialization and environmental factors affect fatty acid content, reconstruction, and light intensity fluctuations. Fatty acids directly affect photosynthetically active membrane characteristics. Thus, they play a role in microalgal biochemical responses to changes in illumination (Maltsev et al. 2021a, b). The duration of illumination and light wavelength may also influence PUFA accumulation in algal cells. Chlorella vulgaris, Chlorella pyrenoidosa, Scenedesmus quadricauda, and Scenedesmus obliquus were grown at various light wavelengths. Blue light significantly increased LA formation in all species over red light. Moreover, Chlorella and Scenedesmus cultivated under blue light exhibited ω-6/3 ratios lower than those produced under red light, indicating that light’s wavelength is crucial to alter the PUFA profile in microalgae (Lu et al. 2021a, b). Chlorella sp. was grown at 500 mol photons m/s, which led to a 26% increase in fatty acid synthesis compared to 200 mol photons m/s. In Lobosphaera incisa (Parietochloris incisa), at 400 mol photons m/s, a similar finding of a rise in TFA concentration and arachidonic acid (ARA) was observed. The reason for this enhancement was the significant rise in biomass under such illumination. Moreover, nitrogen deprivation and 2000 mol photons m/s of light exposure induce Lobosphaera incisa to produce higher ARA (Maltsev et al. 2021a, b).

Application of PUFAs

PUFAs exhibit a wide range of molecular and cellular biological activities that impact cell and tissue function and, thus, health outcomes and disease risks. The ω-3 PUFAs have essential antioxidant and anti-inflammatory effects known to reduce disease risk and severity (i.e., better health). Moreover, these characteristics provide therapeutic options in situations characterized by high levels of inflammation and oxidative stress. ω-6 PUFA LA lowers LDL-cholesterol, while ω-3 PUFAs EPA and DHA lower triglycerides, enhance blood flow, support cardiac and vascular function, and control thrombosis and inflammation. ω-6 and ω-3 PUFAs lower CVD risk through multiple interrelated mechanisms (Djuricic and Calder 2021). In Fig. 3 and Table 3 summarizes some key health applications of Omega PUFA.

Fig. 3.

Effect of microalgal PUFAs on human health

Table 3.

Application of different PUFAs in health maintenance and disease prevention

PUFA	Source	Health performance	References
Arachidonic acid (AA)	Porphyridium purpureum, P. cruentum, Parietochloris incisa	improves normal growth, visual and functional development in infants	Su et al. (2016)
Eicosapentaenoic acid (EPA)	Nannochloropsis sp., Phaeodactylum tricornutum, Porphyridium cruentum	Cardiovascular benefits, mental development and support, anti-inflammatory, protection against atherosclerosis	Asgharpour et al. (2015)
EPA + DHA	–	Cardiovascular benefits, improves nervous system development and function of the brain	Bernasconi et al. (2021)
GLA	Arthrospira sp.	GLA can kill tumor cells without damaging any healthy cells	Sathasivam et al. (2019)
DHA	Schizochytrium sp.	PUFA-rich microalgae enhanced innate immunological responses in shrimp, such as antioxidant enzyme activity	Allen et al. (2019)

Application of linoleic acid to blood cholesterol and cardiovascular diseases (CVDs)

Linoleic acid (LA) reduces LDL cholesterol, leading to a decrease in the risk and mortality of CVDs. It is fast becoming known that ω-3 PUFAs have several positive benefits, including reducing blood pressure, triglyceride levels, and platelet aggregation. These benefits minimize the CVDs risk. Two meta-analyses published in 2021 provided comprehensive data on the cardiovascular system and advantages of EPA and DHA, the effective dosing rate, and the effect of EPA alone versus EPA and DHA together. According to the meta-analysis, taking EPA and DHA supplements is an excellent lifestyle for preventing CVDs, and the effectiveness seems to rise with increasing dosage (Bernasconi et al. 2019). It was discovered that adverse cardiovascular effects, and revascularization was linked to ω-3 PUFA dosing, and showed significant reduction in cardiovascular mortality, non-fatal myocardial infarction, coronary heart disease. EPA monotherapy reduced the random-effects rate ratios of all the aforementioned cardiovascular events more than EPA and DHA combined treatment (Liu et al. 2022).

Application of ω-3 PUFAs for obesity and diabetes

EPA and DHA could also prevent obesity by blocking specific lipid-synthesis-related enzymes, which affect serum lipids and lipoproteins. Prescriptions for EPA and DHA for Type 2 diabetes mellitus considerably lower the levels of triglycerides, believed to be the cause of fat deposits, reducing the risk of hypertriglyceridemia. The EPA-only prescribed, however, looks to be more effective than the DHA-only prescription since it lowers overall cholesterol levels, has fewer gastrointestinal side effects, and did not result in an increase in low-density lipoprotein cholesterol (Tajuddin et al. 2016).

Application of ω-3 PUFAs to brain development and tumors

Polyunsaturated fatty acids are also helpful for brain development. DHA is one of the major building blocks of nerve cells that help in transmit signals that maintain brain flexibility. Arachidonic acid (ARA—20:4n-6) facilitates neuronal transmission and long-term potentiation is important for brain function. Both of them protect it from oxidative damage. DHA is essential for vision since it is found in photoreceptor cells. DHA supplementation improves brain functions, such as learning and memory, in rodent and human models (Garg et al. 2017).

Additionally, ω-3 PUFAs help to develop synapses, neuron growth, and nerve fibres differentiation and extension. DHA has also been investigated as a beneficial component to slow brain aging and fight Alzheimer’s disease (AD). Patients with schizophrenia may have lower amounts of ω-3 PUFAs and ARA in their body tissues than individuals with other brain illnesses. Evidence indicates that therapy with EPA and/or DHA supplements could reduce violent behavior in schizophrenic patients (Remize et al. 2021).

Limitations and bottlenecks of microalgal PUFAs production

A significant drawback of using algae as an alternative to traditional fish oil feedstock involves production costs, including high capital expenditures, energy consumption, and consumables, including water, fertilizer, and CO₂. water, nitrogen, phosphorus, organic carbon, and other nutrients may feed microalgae using wastewater as a growth medium. Study shows wastewater culture lessen lipid accumulation, still microalgae’s fatty acid composition was not affected and was useful for both biofuel and bio-lubricant (Calijuri et al. 2022).

There are several challenges to be overcome for the industrial production of high-value microalgal products such as EPA and DHA. It was necessary to optimize numerous processes to scale up, including strain selection, culture development, product induction, and extraction technologies. Suitable carbon sources for PUFA synthesis have a significant impact on algal bioprocess development (Khan et al. 2018). Though microalgal cultivation on an industrial scale is growing rapidly, microalgal biomass production costs are still very high. Poor PUFA yields in microalgal biomass are largely influenced by cell growth and extraction techniques. These are the primary causes of the excessive production costs. From a sustainable economic and environmental perspective, PUFA extraction with a low solvent quantity may be significant. Due to the high percentage of final products obtained, conventional extraction methods using organic solvents are still the primary methods used to extract lipids from microalgae; however, these methods require more purification steps to remove as much solvent from the final product as possible. To achieve an appropriate equilibrium between lipid yield costs and environmentally safe practices, alternative techniques such as enzymatic disruption, ultrasonication, microwave technologies, or supercritical fluid extraction should be developed (Santin et al. 2022).

Conclusions

As a sustainable platform for PUFA synthesis, mixotrophic algae cultivation has emerged as a promising alternative to autotrophic and heterotrophic algae cultivation. Mixotrophic microalgae research advances have made significant advances in increasing essential PUFA yields and productivities as well as scale-up potential. The development of sustainable PUFA bioprocesses also requires economically viable and environmentally friendly production methods. The microalgae process parameters must be optimized for maximum biomass and lipid fraction and cultivation cost per unit of PUFA output. This review provides a thorough analysis of key factors, including temperature, nutrients, light, pH, and salinity, among others, for PUFA enhancement. Most studies focus on the impacts of individual elements. However, this work collated and combined several aspects to provide a more effective method for increasing essential PUFA synthesis.

Author contributions

SD: Writing-original draft, literature review; CC: Supervision, draft preparation; AKP: Supervision, Writing—review and editing.; SKB: Literature review, draft preparation; Supervision.; RRS: Supervision, Writing—review, and editing, CD: Supervision, Writing—review and editing, validation.

Funding

AKP is grateful to NSTC, Taiwan, for funding support (Ref. No. NSTC 111-2222-E-992-006) and the National Kaohsiung University of Science and Technology 112 Annual Marine Characteristics Sustainable Development Research Program (Program code 112A13).

Data availability

Not applicable.

Code availability

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Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

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