Microalgae as sources of green bioactives for health-enhancing food supplements and nutraceuticals: A review of literature

Antioxidants

Antioxidants inhibit oxidation and prevent damage caused to cells by free radicals. Flavonoids and carotenoids are the two primary antioxidant pigments. Flavonoids are among the most common polyphenols, further divided into flavones, flavanols, flavanones, and isoflavones [Figure 1].^[16] Common examples of flavonoids include catechins and proanthocyanidins. Carotenoids are fat-soluble pigments copious in plants and algae, including lycopene, α-carotene, β-carotene, lutein, and zeaxanthin.^[21] Both carotenoids and flavonoids are essential in lowering the risk of diseases due to their antioxidant and anti-inflammatory properties. For instance, lutein and zeaxanthin are the carotenoids present in the retina and prevent retinal degeneration by acting as photoprotectants.^[22] Furthermore, some vitamins and minerals act as antioxidants, namely, retinol, ascorbic acid, tocopherols, magnesium, manganese, selenium, chromium, and zinc.^[13,23,24]

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Figure 1:

Major classes of flavonoids and some examples of their compounds (with structures).

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Antioxidants are crucial for optimal cellular health and to prevent diseases associated with free radical damage, such as cancer, Alzheimer’s disease, Parkinson’s disease, and aging.^[25-28] Among the various treatments against Alzheimer’s disease, is a promising method where the aggregation of the 42-mer amyloid β protein (Aβ42)is prevented, which a desired condition. The latter was explored with the flavonoid kaempferol, which successfully suppressed the Aβ42 aggregation by preventing amyloid fibril elongation.^[29] The potential of this compound was further evaluated on Drosophila flies expressing human amyloid beta-42 and resulted in a delay in memory loss, reduced oxidative stress, and acetylcholinesterase activity with 40 µM of kaempferol.^[30]

Other medical conditions such as hyperlipidemia, a group of metabolic disorders, are denoted by hypercholesterol in the blood circulation, which may lead to cardiovascular disease (CVD). A naturally occurring anthocyanin called cyanidin-3-rutinoside has been recently identified as a lipid-lowering agent by inhibiting the formation of cholesterol micelles and inhibiting pancreatic cholesterol esterase by 5–18%.^[31] A different metabolic disorder, Type 1 diabetes, is a prevalent health issue without a cure but can be controlled. The flavonoid naringin was identified as a beneficial compound considering its ability to mitigate complications of diabetic ketoacidosis by improving the fasting plasma insulin, hepatic glycogen content, and other related factors.^[32]

Neurological diseases have been have been constantly and increasingly under study, sharing similar concerns regarding the lack of cure for metabolic disorders. A widespread disease in this category is status epilepticus (which can be described as a failure to terminate or initiate mechanisms leading to abnormally prolonged seizures).^[33] A study that used astaxanthin on rats with status epilepticus indicated an alleviation of neuroinflammation in the brain and alleviated damage caused to their hippocampus.^[33] Even neuropsychiatry medical conditions such as depression can be managed with lutein due to its antidepressant-like effect following corticosterone treatment in mice to induce depression.^[34] Other conditions such as spinal cord injuries can also be managed (reduced oxidative damage, mitochondrial dysfunction, and cell apoptosis) with the use of different carotenoids such as lycopene due to its antioxidant capacity, as demonstrated in a study on rats administered with the compound (10 and 20 mg/kg).^[35]

Carbohydrates and dietary fiber

Carbohydrates are essential primary metabolites that may occur as free sugars such as glucose, short-chain carbohydrates, or polysaccharides such as starch. They are the main source of energy to fuel the human body. Glucose acts as a short-term but immediate source of energy, while fiber, an indigestible carbohydrate, helps with digestion and maintains normal blood cholesterol levels. Important sources of carbohydrates are milk, sugarcane, tubers, rice, whole grains, fruits, and beans, among many others. Plants synthesize glucose during photosynthesis which is then converted to starch, cellulose, and hemicellulose.^[36] Starch is the most commonly consumed carbohydrate by humans, while animals such as ruminants can also digest cellulose. Furthermore, carbohydrates are utilized in producing oral nutritional supplements as taste enhancers or inhibitors, energy sources in prolonged fasting, and color, flavor, and texture enhancers.^[37] In aquaculture practices, carbohydrates in bread flour, cornflour, and cassava starch are used as energy sources and as binders in the feed production process.^[38] Indian major carp is an extensively cultured fish in countries such as Bangladesh, and its feed formulation can contain up to 30% carbohydrates, as a cost lowering measure.^[39]

Dietary fibers are non-digestible carbohydrates that the human body cannot break down. They exist as either insoluble fibers (cellulose and hemicellulose) or soluble fibers (pectin and gum) and are mostly present in fruits, vegetables, and whole grains.^[5] Dietary fiber is crucial for maintaining a healthy digestive system. In a study conducted by Hou et al.,^[40] dietary fibers help reduce inflammatory bowel diseases, namely, Crohn’s disease and ulcerative colitis. Furthermore, soluble fibers have also been found to help regulate blood glucose and fat levels.^[5]

Lipids and fatty acids

Fatty acids are the building blocks of lipids, a group of fats or fat-like substances characterized by their insolubility in water. Due to the diversity of lipids and their structural variations, a standard classification of lipids has been developed containing eight categories: Fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids, and phenol lipids.^[41] This classification includes lipids from various sources such as animals, plants, bacteria, microalgae, and fungi. The intake of lipids can be achieved by consuming plants and plant-based products (walnut, fruits, vegetable oils, and grains), aquatic organisms (fish, crustaceans, and molluscs), or even animal-derived products such as milk, meat, and eggs.^[42]

Fatty acids can be classified as either saturated fatty acids, monounsaturated fatty acids, or polyunsaturated fatty acids (PUFAs). The latter are widely used as nutraceuticals due to their positive impacts on overall health.^[7] There are fatty acids synthesized in very low amounts by the body, called essential fatty acids, which must be supplied through the diet. Those are essentially the main n-3 PUFAs α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). These n-3 PUFAs have positive effects against cardiac disorders, Type 2 diabetes, and autoimmune diseases,^[43-45] and they are also crucial for the growth and the healthy development and functioning of the brain and nervous system.^[46] Fatty acids are highly involved in metabolic pathways, such as linoleic acid (LA) and ALA, which are the precursors to other PUFAs. LA is the most common and highly consumed PUFA in the diet and helps in growth and development. One of the primary uses of LA is its physiological ability to maintain the transdermal water barrier of the epidermis, and deficiency of this PUFA would lead to skin lesions.^[47]

Moreover, n-6 PUFAs have also been found to be involved in health. For instance, arachidonic acid is vital in the healthy development of the central nervous system and retina of newborns and promotes healthy muscle growth in humans.^[48] Gamma-linolenic acid, another n-6 fatty acid that can be obtained in human milk, plant seed oils, blackcurrant, and borage,^[49] is successful in treating rheumatoid arthritis as it may increase the E-levels of prostaglandins, which, in turn, increase cAMP levels, thus suppressing tumor necrosis factor-α (TNF-α) synthesis.^[50] Thus, the dietary intake of PUFAs, especially the n-3 and n-6 ones, is crucial for proper growth and optimal health.

Proteins and bioactive peptides

Proteins are macromolecules that consist of amino acid chains. Proteins and amino acids play dominant roles in the maintenance of body homeostasis. They are critical components for the muscles, enzymes involved in metabolic pathways, immune system (as immunoglobulins), and hormones that control various activities such as growth and many other functions.^[51] Therefore, sufficient amounts of proteins in the diet are needed to meet the daily physical requirements.^[52]

Bioactive peptides are organic compounds produced when proteins are fermented or digested by proteolytic enzymes.^[5] Most bioactive peptides are derived from milk proteins, and they are believed to improve human health. For instance, lactoferrin possesses antimicrobial, anti-inflammatory, anticarcinogenic, antioxidant, and immunostimulatory activities.^[53] Furthermore, some bioactive peptides have an opioid-like activity, and they are involved in different processes in the nervous system and the regulation of cytokines in immunomodulatory activity, decreasing blood pressure, and preventing hypertension.^[54]

Probiotics and prebiotics

In general, the gut has an abundance of beneficial bacteria. However, intestinal dysbiosis may occur due to antibiotic therapies, poor diets, and stress.^[55] Probiotics and prebiotics can help restore the gut microbiota healthy balance and, in turn, improve intestinal health. Probiotics are living microorganisms that benefit the gut microbiota when administered adequately. Probiotic organisms most commonly used include Bifidobacterium, Lactobacillus, and Saccharomyces.^[24] It has been shown that probiotics can contribute to the prevention of diarrheal infections and reduce the length of the diarrheal spell in outpatient treatments.^[56] On the other hand, prebiotics is non-digestible compounds on which intestinal microbes feed themselves for growth while beneficially impacting the host.^[57] The most common prebiotics include oligosaccharides present in fruits, vegetables, and whole grains. Hence, probiotics and prebiotics work together in symbiosis to maintain healthy gut health.

Vitamins and minerals

Vitamins and minerals are considered micronutrients, required in smaller quantities by the body compared to macronutrients. Both are required for the body’s normal functioning, except that vitamins are organic substances while minerals are inorganic ones. According to the World Health Organization (WHO), it is estimated that more than 2 billion people worldwide suffer from micronutrient deficiency. The latter can greatly increase the risk of several diseases and health complications: Anemia, reduced cognitive functions, increased risk of infections, and perinatal complications.^[58] These micronutrients are mainly obtained from external food sources since most of them cannot be synthesized by the body and play pivotal roles in maintaining health [Table 2] and preventing many diseases.^[59]

Strengthening the immune system with nutraceuticals

Viral diseases are presently one of the leading causes of death worldwide, and their ability to spread is escalating, which could result in pandemics, especially in the case of COVID-19. A healthy diet consisting of nutraceuticals can significantly boost the immune system, optimize cell functions, and fight against several viral infections.^[20] Nutraceuticals that have been shown to have antiviral properties include resveratrol, quercetin, curcumin, and epigallocatechin gallate.^{[14,15,60,61]}

In a research study, these nutraceuticals had demonstrated antiviral activities against coronaviruses by inhibiting the viral replication process through regulation of the protease, Mpro of the virus.^[62] Moreover, Vitamins A, D, B, C, and E and minerals such as calcium, magnesium, and zinc have significantly strengthened and supported the immune system, allowing the body to combat viral invasions.^[20,62-66]

Nutraceuticals against CVDs

CVDs are disorders related to the heart and blood vessels, including coronary heart diseases, cerebrovascular diseases, peripheral arterial diseases, rheumatic heart diseases, congenital heart diseases, and deep vein thrombosis.^[67] According to the latest study published by the WHO, an estimated number of 17.9 million people died from CVDs worldwide in 2019. This alarming finding raises concerns and calls authorities to find effective ways in preventing and treating this disease. There is growing evidence showing that some nutraceuticals can lower the risks associated with CVDs. For instance, regular consumption of n-3PUFAs, especially LAs, EPA, and DHA, has been proven to lower the risks of CVDs and diabetes and can also enhance brain development.^[7,68]

A study conducted in Japan with 18,645 patients demonstrated that EPA, supplied as 1.8 g in the diet, reduced coronary diseases by 18%.^[69] Results were similarly reported in another study, where patients with percutaneous coronary intervention were given early EPA treatment, resulting in a significantly lower death rate at 0.8% than 4.2% in the control group.^[70] Another study reported that patients who consumed Annurca apple polyphenol extract increased fecal cholesterol excretion by 35%.^[71] Therefore, diets rich in nutraceuticals can potentially be used to prevent and treat CVDs.

Nutraceuticals for cancer prevention

Cancer is the second leading cause of mortality worldwide, and around 35% of these deaths are associated with diet.^[1] Nutraceuticals in the categories of antioxidants, bioactive peptides, minerals, and vitamins have demonstrated the ability to prevent certain cancers. For instance, the carotenoids lycopene and β-carotene have antioxidant activity and can prevent cancer by decreasing oxidative stress and damage to DNA.^[23,72] Moreover, it has been found that bioactive peptides can reduce the risk of developing certain tumors and enhance the effectiveness of anticarcinogenic therapies.^[73] For example, the bioactive peptide lactoferrin possesses antitumor activities without damaging healthy body cells.^[74,75] Therefore, a healthy lifestyle and a diet rich in these nutraceuticals mentioned above, along with selenium, Vitamin C, and Vitamin E, can help prevent cancer.^[6] However, it should be noted that these studies have demonstrated that nutraceuticals mainly participate in preventing cancer rather than treating the disease, and hence, more research is still needed in this area.

Importance of proteins and microalgae

Microalgae diversity facilitates multiple combinations of primary and secondary metabolites.

Among these combinations, are proteins and amino acids, which play dominant roles in maintaining body homeostasis. Traditional protein sources include meat, eggs, milk, and soybean. From investigations, protein levels from microalgae are comparable to levels from traditional sources.^[79] Furthermore, since proteins are composed of amino acids, the composition comparison between some microalgae species and some basic conventional food items share similar patterns.^[95] Acquiring protein from microalgae is deemed more beneficial than traditional high-protein crops in productivity and nutritional value. Microalgae can produce a protein yield of 4–15 tons/Ha/year compared to soybean (0.6–1.2 tons/Ha/year) and wheat (1.1 tons/Ha/year).^[106] Since microalgae are being prospected for biofuel production, the residual biomass would contain the proteins useful for feed formulations, following lipids and carbohydrate extraction.^[88,107]

Commercially important microalgae for protein

Despite the production advantage, microalgae culture on a large scale as sources of food/feed is limited to a few countries due to the costs involved. However, research has identified suitable microalgae candidates that are suitable sources of proteins and amino acids and are currently being cultured at the industrial level. One popular microalgae is Spirulina platensis, a non-toxic cyanobacterium, often regarded as a complete food containing all essential amino acids and can reach up to 62% of protein levels.^[108,109] More recent studies on S. platensis produced a protein concentrate that contained 75.97% proteins on a dry weight basis which could help in food/feed formulations.^[110]

Chlorella spp. is also a very good source of proteins that can reach a content of 51–58% as dry weight. The amino acid composition of this Chlorophyta microalgae makes it an appealing source of proteins as it contains nearly all of the essential ones.^[95] Scenedesmus spp. microalgae, typically investigated for biofuels, tend to yield protein contents between 30 and 55%, and the variations are due to type of strain, culture medium, among other factors.^[111] Another commercially produced microalgae is Dunaliella spp., yielding up to 57% protein content.^[95] However, this microalgae tends to be overlooked as a source of protein due to its importance in β-carotene production.^[112]

High-value protein metabolites

Nonetheless, the microalgae proteins are released in various forms following biomass processing, such as glycoproteins and phycobiliproteins.^[113] Early research on Chlorella vulgaris led to the discovery of a water-soluble glycoprotein with antitumor activity.^[114,115] An investigation on C. vulgaris glycoproteins indicated a rare occurrence of modified oligomannosidic glycans that have not been found in either plant or vertebrate N-glycans, prompting the need for further investigations as these microalgae are sold as a source of protein.^[116]

Other glycoproteins such as lectins are involved in various biological processes, including cell-cell communication, antiviral activities, and antibacterial activities, among many others.^[117] In the phycobiliproteins group, phycocyanin and phycoerythrin are the most common compounds produced by microalgae. Phycocyanin is mainly derived from S. platensis and is a natural dye for food products and cosmetics. Phycoerythrin is used as a fluorescent agent for diagnostic purposes and is mainly derived from red algae such as Porphyridium cruentum.^[118]

Mode of nutrition

Due to the diversity of microalgae species and the need to acquire commercially valuable ones, research has been able to help identify their mode of nutrition. These have been identified as follows: Photoautotrophy, heterotrophy, and mixotrophy. The mode of nutrition is an important aspect of microalgae culture to consider because it dictates the whole process of the culture from the choice of the microalgae, type of culture vessel, culture conditions (light, temperature, agitation, and among others), source of nutrients, and ultimately, the overall production costs. Therefore, understanding these modes of nutrition is crucial from an economic point of view to judge how much investment is necessary.

Photoautotrophy-based culture is considered the most utilized method and is extensively used in open ponds and photobioreactor (PBRs) systems.^[147] It uses light as an energy source, CO₂ from the surface air, and aerators in artificial ponds.^[148] The cells grow and multiply through photosynthesis, where solar energy is stored as adenosine 5’-triphosphate and nicotinamide adenine dinucleotide phosphate hydrogen. The Calvin cycle processes these to generate glucose as an energy source. The extensive use of this culture method is judged most economical since it utilizes sunlight as an energy source.

However, despite being abundantly available, sunlight is also considered a limiting factor as photosynthesis will only occur in the presence of light. To counter this issue, using artificial lightning shows potential, but it may prove very costly to implement and run. In addition, recent investigations on this method resulted in creating droplet-based PBRs that allow monitoring the algae growth under various concentrations of CO₂ and light intensities. This results in the rapid evaluation of the photoautotrophic growth of microalgae species and finding the most appropriate conditions.^[149]

The second mode of nutrition is through heterotrophy, where an organic compound (consisting of carbon) is present in the media. It is then absorbed by the microalgae and subsequently metabolized. Through this method, biomass production does not require light, and the yield is much higher. Glucose is generally considered a primary carbon source, but other alternatives such as peptone and acetate have also been used.^[150] The main disadvantages of this type of culture are the costs required to supply the carbon source and potential contamination with bacteria/fungi, resulting in waste of resources.

The growth of diatom Cyclotella cryptica was investigated under heterotrophic conditions while using glucose as the sole carbon source. The resulting biomass produced cellular carbohydrate, protein, and lipid contents of 360 mg/g, 260 mg/g, and 165 mg/g, respectively, showing promising levels of carbohydrates.^[132] Another study with Chlorella spp. HS2 showed a 3-fold increase (from 5.58 g/L to 18.13 g/L) in biomass production when the media were supplemented with glucose at 72 g/L.^[151] Such findings showcase the potential of this method of culture. However, recent studies have shown wastewater as a cost-effective organic source to culture microalgae.^[152] A variation of heterotrophy is a photoheterotrophic culture where the condition is that light is needed to activate photosystem I while using sugar as the exclusive carbon source, but this leads to an expensive production.

Mixotrophic systems are designed to allow the occurrence of photosynthesis in the presence of different carbon sources, such as organic compounds and CO₂.^[150] This method employs both methods described above in two stages. The preliminary stage is performed under heterotrophy in the presence of high organic carbon content. The second stage would then be performed by photoautotrophy when the supplied organic carbon source is no longer available.^[153] However, mixotrophy does not always follow this pattern. When cultured under such conditions, C. vulgaris recorded yields of 140% and 170% of biomass and lipid productions, respectively, compared to autotrophic growth.^[154] The authors pointed out that culture under such conditions was not a simple combination of heterotrophy and autotrophy. One of these metabolic pathways (either heterotrophy or autotrophy) was dominant, leaving the other one active at a much lower rate.

Each of these culture conditions has its advantages and disadvantages regarding the light and carbon sources, biomass yield, and costs. However, depending on the culture’s goal, whether for feed production, biofuel production, value-added secondary metabolite production, or other applications, the culture parameters will depend on the microalgae strain/species, level of investments, and the desired end product.

Light

Light energy is necessary for fundamental biological processes such as synthesizing the cell’s protoplasm. Microalgae thus require light for optimal growth and productivity. Light intensity and photoperiod are both important lighting parameters in microalgae culture. Light directly relates to the photosynthesis rate.^[155] It has been established that microalgal growth increases with light intensity to a saturation point, beyond which a significant decline in growth is noted. This decline is attributed to photodamage and photoinhibition by intense and prolonged light.^[156]

Besides biomass production, light also affects the biochemical composition of microalgae. A study investigating the effect of light intensity on pigment accumulation in Phaeodactylum tricornutum has revealed that low light intensity favored the light-harvesting pigment, fucoxanthin.^[157] In fact, at low light intensities, microalgae respond by enhancing light-harvesting pigments such as phycobilins, primary carotenoids, and chlorophylls, probably to maximize the light utilization by the microalgae. This has been evidenced in a study where the accumulation of beta-carotene and lutein at low light intensity (140 µmolm^-2s^-1) was demonstrated.^[158] The same study showed an increase in lipid production under low light intensity.

Besides light intensity, photoperiod also impacts biomass productivity and biochemical composition. Continuous illumination, light flashing, and alternate light and dark cycles are used during microalgae cultivation.^[159] Rhodomonas spp. showed enhanced production of EPA and DHA under 16:8 h (light: dark) light regime compared to continuous lighting and achieved greater biomass.^[160] Decreasing the light duration from 16 h to 8 h promoted the production of phycoerythrin, phycocyanin, and allophycocyanin while increasing the light period enhanced the accumulation of carbohydrates and carotenoids in Nostoc calcicola.^[161] Prolonged lighting is not always beneficial for biomass and metabolites production as excess light is dissipated as heat and causes damage to cells.^[162] Excessive light unnecessarily contributes to the production cost, an important aspect when considering the profitability of the process.

Light source is equally important. Recent investigations on cost-effective light sources for the profitable production of microalgae included the use of red and blue light-emitting diodes (LEDs). A study investigated the effect of various combinations of red and blue LEDs in the culture of Chlorella spp., and the researchers recorded the highest biomass when light intensity was 500 lux.^[163] They also recorded a significant increase from 30% to 60% (w/w) in lipid content when the light source was altered from white to red/blue LEDs. Another study with C. vulgaris using blue LEDs indicated a lipid content of 34.06% as the highest lipid content instead of cool white and red LEDs.^[164] Such experiments signify the importance of using wavelength-based light sources as an economical step in microalgae culture.

Nitrogen and phosphorus

Nitrogen and phosphorus are critical macronutrients required for microalgae growth and play an essential role in the biosynthesis of important biomolecules. An inadequate supply of these nutrients can be detrimental to the cells’ morphology, physiology, and biochemical composition. Microalgae can assimilate nitrogen in the form of nitrates, nitrites, ammonium, and urea. In general, microalgae’s preferred form of nitrogen is ammonium as its assimilation is more energetically favorable, although some microalgae such as Dunaliella spp. and Botryococcus spp. prefer nitrates.^[165] However, it has been established that microalgae do not tolerate high concentrations of ammonium (above 25 µM).^[166] For this reason, nitrates and urea are most commonly used in culture media.

Although nitrogen deficiency limits the growth of microalgae, it is known to shift the system toward the accumulation of carbohydrates and lipids. Researchers have reported that nitrogen depletion considerably decreased the growth of Isochrysis galbana and protein content, significantly increasing carbohydrate content and saturated fatty acids.^[167] Similarly, Rhodomonas spp. showed reduced cell growth but enhanced PUFAs accumulation under nitrogen starvation.^[168] Nitrogen deficiency shifts the metabolism to the lipid metabolic pathway, leading to the accumulation of TAGs as energy sources. In terms of pigment production, chlorophyll content decreased while carotenoid content increased significantly in Pycocystis salinarum under nitrogen limitation.^[169] Other researchers revealed similar findings where a decrease in phycobilins in Rhodomonas spp. and an increase in carotenoid content in D. salina were noted.^[168,170] The impact of nitrogen concentration on key light-harvesting pigments also relates to the rate of photosynthesis, which is adversely affected under nitrogen limitations. Undeniably, nitrogen plays a critical role in biomass production and productivity of microalgae. Depending on the target compound, the nutrient levels should be adjusted accordingly.

Temperature

The temperature of the system strongly influences the growth and accumulation of biomolecules. Temperature affects crucial enzyme-mediated metabolic reactions.^[171] Depending on the strain, microalgae have an optimum temperature ranging from 15 to 50°C, beyond which growth declines due to denaturation of enzymes and instability of pigments.^[172]

Biochemical pathways for biosynthesis and accumulation of unsaturated and saturated fatty acids and lipids are susceptible to thermal variations.^[173] Microalgae respond by increasing total fatty acids and omega-3-fatty acid content at low-to-moderate temperatures (10–20°C).^[174] A study investigating the effect of temperature on eight microalgae strains demonstrated that EPA and DHA are produced at greater amounts at 14°C than 26°C.^[175] PUFA content increased under low temperature (18°C) in Nannochloropsis oceanica.^[176] In another study, a 40.7–52.9% increase in PUFAs was produced by Heterochlorella luteoviridis, when the temperature was increased from 22 to 27°C.^[177] Enhanced lipid synthesis, especially of PUFAs, is an adaptive mechanism of microalgae at low temperatures aimed at maintaining the fluidity, functionality, and flexibility of the cell membrane.^[178]

CO₂ supply

Carbon dioxide is a key requirement for photosynthesis in microalgae. In PBRs, the CO₂ concentration supplied through spargers can be easily controlled, while in open raceway ponds, microalgae can utilize atmospheric CO₂. The tolerance to CO₂ levels is species specific.^[179] CO₂ concentration significantly affects microalgae growth as it influences the pH of the medium and the availability of bicarbonates.^[180]

A study investigating the effect of CO₂ on polyculture of microalgae reported that growth of the cells increased with increasing CO₂, but beyond 400 mgL^-1, the growth rate reduced to half.^[181] Similar observations were made by researchers who reported that the growth rates of Desmodesmus spp. and Scenedesmus spp. increased until a saturation point of 60 µM and 30 µM, respectively.^[182] CO₂ influences enzymes involved in carbon metabolisms such as Rubisco and carbonic anhydrase.^[183] Hence, increasing the concentration of CO₂ results in enhanced rates of photosynthesis, which has a positive effect on the growth of the cells. Consequently, elevated concentration may act as a stress factor and impede the growth rate. Increased dissolved CO₂ levels in the media decrease the system’s pH, making it unfavorable for microalgae growth. It should be noted that optimal pH for microalgae lies between 7 and 9, above or below which inhibition of key enzymes involved in carbon fixation may occur.^[184]

In terms of biochemical composition, CO₂ affects lipid, protein, and pigment contents. A different study suggested that high CO₂ favors the accumulation of chlorophylls, carotenoids, and lipids. Interestingly, one strain under study showed the accumulation of beneficial omega-6 fatty acid, LA.^[180] In Scenedesmus bajacalifornicus, the maximum protein content was produced at 15% CO₂ and decreased beyond this CO₂ level.^[185] The same study showed a significant increase in lipid and pigment content at 25% CO₂. Similar results were obtained in a different study.^[181] In this study, a maximum protein content (30.41%) was obtained at 400 mgL^-1 of CO_2, which decreased to 8.92% when CO₂ concentration was increased to 600 mgL^-1. Lipid content increased from 26.39% to 56% when CO₂ concentration increased from 200 mgL^-1 to 800 mgL^-1. Increased levels of lipids at increased CO₂ were associated with stress-induced lipid synthesis due to a possible disruption in the system’s pH.^[186]

Open systems

Open cultivation systems include natural and artificial ponds such as the open raceway and circular ponds.^[187] Open raceway ponds, also known as high-rate algal ponds (HRAPs), consist of a looped and closed channel driven by a paddle and baffles which guide the continuous liquid flow and promote agitation of the medium, preventing sedimentation.^[188] These ponds are shallow (15–30 cm deep) to maximize light penetration and prevent self-shading of the microalgae.^[189] Atmospheric CO₂ is the source of aeration, although aerators may be added as an additional CO₂ source in some cases.^[190,191] Open ponds are ideal for pilot and commercial scale productions because of their low operation and maintenance cost and easy set-up.^[192]

The set-up of raceway ponds does not require arable land and this is an important aspect as it does not compromise on land aimed at agricultural production. In fact, this culture system is widely utilized for the industrial production of microalgae cells for producing pigments, single-cell proteins, and beta-carotenes and accounts for 90% of microalgae biomass production worldwide.^[193,194] HRAPs have been reportedly established in the USA, India, Australia, Israel, and China to produce nutraceuticals commercially.^[195] An Australian firm achieved about 13 ta^-1 productions of beta-carotene from microalgae grown in open unmixed ponds.^[196]

However, the open ponds are limited by evaporation losses, the variation in weather conditions in terms of temperature and duration of sunlight, and contamination by predators and other heterotrophic microorganisms that compete with microalgae for nutrients and produce toxins that may contaminate the final product.^[197] Furthermore, atmospheric CO₂ (0.04–0.06%) is suboptimal for microalgae growth, which impedes biomass production and productivity.^[198]

The circular pond consists of a circular tank (30–70 cm deep) with a central rotating agitator to mix and prevent sedimentation.^[199] This type of open system has the same demerits as the raceway pond.

Closed PBRs

Closed PBRs have been designed to counter the shortcomings of open ponds and represent an attractive option due to high biomass production and low contamination. Tubular PBR, flat panel PBR, and vertical column PBR are commonly used in nutraceutical production. The design, merits, and demerits of each PBR are described in [Table 4].^{[193,199,201]} The commercial application of PBRs is manifold. For instance, Israel and Germany have employed the tubular PBR to cultivate Haematococcus spp. and Chlorella spp.^[200] The flat panel PBR is used in the Czech Republic to produce astaxanthin.^[199]

Overall, these PBRs offer better control and optimize culture parameters such as light intensity, temperature, agitation, low-energy consumption, and high photosynthetic efficiency.^[189] For this reason, PBRs demonstrate significantly higher biomass production. The productivity potential of Nannochloropsis spp. in flat panel PBR was found to range between 3800 and 13,000 tkm^{-2 year-1}, which was higher when compared to the open algal pond.^[202] Chlorella salina could achieve 9.73 mgL^{-1 day}-1 of lutein when cultured in an 8 L lab-scale air-lift PBR at optimized conditions.^[203]

Another essential aspect of PBRs is a lower likelihood of contamination, favoring microalgae axenic cultures.^[204] Prevention of contamination is critical in producing high-end nutraceuticals and biopharmaceuticals. The compactness of the design of PBRs is another advantage, as it does not take up as much space as open raceway ponds. Overheating, bio-fouling, and cleaning difficulties are some of the limitations of PBRs.^[205] Another demerit of PBRs is difficulty in up-scaling due to the high costs associated with superior material and support systems.^[206]