Pharmaceutical applications and consequent environmental impacts of Spirulina (Arthrospira): An overview

W. Shao^a*, R. Ebaid^a*, M. El-Sheekhc, A. Abomohra^b,^c,^* and H. Eladel^d

^aSchool of the Environment and Safety Engineering, Jiangsu University, 212013 Jiangsu, China

^bSchool of Energy and Power Engineering, Jiangsu University, 212013 Jiangsu, China.

^cBotany Department, Faculty of Science, Tanta University, 31527 Tanta, Egypt

^dBotany Department, Faculty of Science, Benha University, 13518 Benha, Egypt

^*The first and second authors contributed equally to the present work.

*Corresponding author: abomohra@ujs.edu.cn; abomohra@science.tanta.edu.eg

1. INTRODUCTIONTOP

Microorganisms have unique characteristics and therefore, they are considered good pillars for the large-scale production of many useful metabolites. Microbial cells have a high area/volume ratio, which results in high efficiency of nutrient uptake for the biosynthesis of different metabolites through different metabolic pathways. Microalgae are a wide group of photosynthetic microorganisms that can be isolated from various habitats and can grow efficiently in commercial large-scale raceway ponds or photobioreactors (Figure 1), where cells can grow on inexpensive nutrient sources to produce valuable compounds. Cyanophytes are a group of microalgae commonly grown in rice paddies and have a positive impact on growth, yield and nitrogen levels of the crop plants (Whitton, 2000). This group of microorganisms also known as cyanobacteria, as they resemble bacteria in that they are prokaryotes and lack the internal structures such as nucleus and other organelles found in eukaryotic microalgae. From the practical and economical point of view, microalgae offer numerous advantages, such as high photosynthetic efficiency which results in high growth rates, high CO₂ mitigation rate, possibility for cultivation on arid lands using marine-, fresh- or wastewater (Abomohra et al., 2018).

Although many previous studies focused on different cyanophytes as nitrogen-fixing organisms, Spirulina is gaining attention due to its high nutritional value and extensive pharmaceutical applications (Table 1). The correct identification of a certain species is a fundamental requisite for research study and/or developmental applications. The genus Spirulina has been incorrectly used to describe two different genera, Spirulina and Arthrospira. However, Stizenberger (1854) and Gomont (1892–1893) classified the forms with visible septa within the genus Arthrospira Stizenberger 1852; while coiled filaments with invisible septa were classified as belonging to the genus Spirulina Turpin 1829 (Tomaselli, 1997). The view of Arthrospira and Spirulina as two separate genera has been officially accepted by Bergey’s Manual of Systematic Bacteriology (Castenholz, 1989). The separation between these two genera has been repeatedly affirmed on the basis of many other characteristics such as cell wall structure, helicity, trichome size, motility, gas vesicles, thylakoid pattern, GC analysis and phylogenetically using 16S rRNA. Depending on the later, 16S rRNA of two Arthrospira strains (PCC 7345 and PCC 8005) and one Spirulina strain (PCC 6313) confirmed that the two Arthrospira strains form a tight cluster not closely related to the Spirulina strain, which supports the separation of these two genera (Tomaselli, 1997). Therefore, a molecular approach is not only useful for the precise identification of a certain species, but it also helps to understand the evolutionary relationships occurring among various microorganisms.

Spirulina is a photosynthetic autotrophic organism containing the blue pigment phycocyanin as the main photosynthetic pigment in addition to the green pigment chlorophyll a, which results in the blue-green color of the cells. Spirulina is one of multicellular unbranched non-heterocystous filamentous microalgae which are recognizable by the unique open left-handed helix along the entire length of the filament (Figure 2). The filaments are 50-500 μm in length and 2-4 μm wide, composed of only vegetative cells containing gas vacuoles. The long size filaments and strong cells allow successful filtration comparing to unicellular microalgae which results in the cost-effective harvest of Spirulina compared to other microalgal species. Currently, microalgae cultivation takes place in two major cultivation systems, namely closed photobioreactors (PBR) and open ponds. Both approaches have their own advantages and disadvantages which were discussed in details by Soni et al., (2017). However, Spirulina is mainly reported to be grown commercially in open ponds. Recently, new hybrid cost-effective techniques such as poly bags can also be used to increase algal biomass productivity (Soni et al., 2017).

Many microorganisms are used as a massive sustainable resource for bio-pharmaceuticals which can be produced by direct extraction or after biomass bioprocessing. During secondary metabolism, microorganisms produce special secondary metabolites which possess special chemical structures and are not critical for the growth of the producing organism. In general, such metabolites are produced under unfavorable conditions in defense against predation, herbivory and competition for space. So far, some secondary metabolites from different microorganisms were isolated and identified in pure form. For example, chlorellin is a fatty acid produced by the green microalga Chlorella vulgaris and was first identified as an active anti-bacterial substance in 1940s (Centeno and Ballentine, 1999). On the other hand, the limited availability of fossil fuels and their environmental issues, especially participation in global warming, enhanced the extensive worldwide search for new renewable energy sources with combined CO₂ mitigation. Spirulina was discussed as one of microalgae capable of producing different kinds of bioenergy with high efficiency of CO₂ fixation (Sumprasit et al., 2017; Shao et al., 2018; Rempel et al., 2018), which provides the advantage of an eco-friendly utilization of Spirulina. The main aim of the present work was to provide a critical overview of the current status of the pharmaceutical applications of Spirulina, including antioxidant, anti-inflammatory, anti-cancer, anti-microbial, anti- diabetes & obesity and anti-toxicity activities. In addition, future perspectives in genetic engineering technologies to enhance valuable products from Spirulina were discussed. The present work shed light on the environmental impacts of large-scale cultivation of Spirulina as one of the economically important microalgae.

2. NUTRITIONAL VALUE OF SPIRULINATOP

Compared to other foods, Spirulina shows extraordinary nutritional value as it provides high levels of many essential nutrients and minerals (Table 2). However, the biochemical composition of Spirulina is highly dependent on growth conditions and the methods used for harvest and drying. In general, Capelli and Cysewski (2010) reported that the amount of calcium in Spirulina is 1.8 times higher than that in whole milk, total protein is 6.7 times that of tofu, iron is 51 times greater than that of spinach and β-carotene is 31 times more abundant than in carrots. Therefore, Spirulina was given the label of ‘super food’ by The World Health Organization (WHO). In addition, it was sent to space by National Aeronautics and Space Administration (NASA) due to its high nutritional value (Khan et al., 2005). Spirulina is produced commercially in open ponds by many producers (Table 3). Earthrise Farms, located in USA, covering 437060 m², is the world’s largest producer of Spirulina. The company produces dried Spirulina in the form of tablets and powder in over 20 countries around the world with an average annual production of 550 tons. Other large Spirulina production plants are Cyanotech and Hainan DIC Microalgae located in USA and China, covering 364217 and 100000 m² with annual production of 400 and 350 tons, respectively (Table 3). However, other companies sell Spirulina special products such as nutraceuticals, e.g. Myanmar Spirulina Factory (Yangon, Myanmar) sells tablets, chips, pasta and liquid extract.

3. PHARMACEUTICAL APPLICATIONS OF SPIRULINATOP

Spirulina is the most highly consumed microalga (Bleakley and Hayes, 2017), which has been widely used as human healthy food centuries ago; and it is currently produced commercially as healthy food and valued additives in a hugely profitable business. In addition to its high nutritional value, Spirulina shows many biological activities such as antioxidant, anti-inflammatory, anti-cancer, anti-microbial, anti- diabetes & obesity and anti-toxicity.

3.1. Antioxidant activitiesTOP

Spirulina extracts show relatively high antioxidant activities that have attracted the attention of many researchers. Manoj et al., (1992) confirmed the activity of alcoholic extract of Spirulina in lipid peroxidation inhibition more significantly (65%) than some chemical antioxidants like α-tocopherol (35%) and β-carotene (48%). In addition, they reported that a hot-water extract of Spirulina has a greater antioxidant effect (76%) than chlorogenic acid (56%) and gallic acid (54%). Miranda et al., (1998) studied the in vivo and in vitro antioxidant activity of S. maxima methanolic extract. In vivo antioxidant activity as lipid peroxidation was studied in the plasma, brain and liver homogenates of experimental rats using a daily dose of 5 mg of methanolic extract. The extract contained total tocopherol, β-carotene and total phenolic compounds of 18 mg·L^–1, 27.5 mg·L^–1 and 96.3 mg·L^–1, respectively. Upon treatment, the antioxidant activity increased in the plasma of the Spirulina-treated group by 31.5% over the control group. The antioxidant effect was mainly associated with the individual or synergetic effect of tocopherol, β-carotene and phenolic compounds. In another study, the antioxidant activity was attributed to the phenolic compounds found in the methanolic alga extract including salicylic, chlorogenic, trans-­cinnamic, caffeic acids, quimic and synaptic; individually or in a synergistic action (Baicus and Baicus, 2007). However, the caffeic acids and chlorogenic acid present in the Spirulina extract recorded more antioxidant efficiency than other acids (Henrikson, 1994); which also recorded a potential preventive action against cancer (Subhashini et al., 2004). Recently, Abomohra et al., (2016) concluded that the exposure of A. platensis to gamma radiation induces the cellular reactive oxygen species (ROS) by purinergic signaling, stimulating cells to produce more antioxidants under the induced oxidative stress.

3.2. Anti-inflammatory activitiesTOP

The light-harvesting pigment “phycocyanin” is one of phycobiliproteins along with phycoerythrin and allophycocyanin, and found exclusively in cyanobacteria, cryptophytes and red algae (Hu et al., 2018). The presence of phycocyanin gives the characteristic blue color of cyanophytes, and hence the name of blue green algae. Eriksen (2016) concluded that Spirulina is the sole efficient source of natural phycocyanin production under photoautotrophic conditions. Despite the challenges facing Spirulina cultivation and phycocyanin extraction, there are no other particular sources of natural phycocyanin (Hu et al., 2018). In addition to the autotrophic growth of Spirulina, it is also capable of heterotrophic growth in the presence of glucose, with a phycocyanin content of 57 mg·g⁻¹ dry weight, which represents half of that obtained in mixotrophic cultivation (Marquez et al., 1993). Phycocyanin can also be produced from other microalgae, e.g. from the unicellular red alga Galdieria sulphuraria under heterotrophic conditions. However, the phycocyanin content in Spirulina grown phototrophically is much higher than that of G. sulphuraria grown heterotrophically (Table 4), which gives the advantage of a lower cost production of phycocyanin using Spirulina.

In a previous study, Ebaid et al., (2017) estimated the phycocyanin content of 46.57 mg·g⁻¹ dry weight in A. platensis grown in a Zarrouk medium for 18 days at 25 ± 3 °C and light intensity of 2500 Lux with a photoperiod of 18:6 h light: dark cycle. It is worth noting that the relative content of phycocyanin showed significant changes under different conditions of prevalent light, which was confirmed as a scavenging mechanism to prevent or reduce photoinhibitory damage (Mohanty et al., 1997). Romay et al., (1998) studied four in vivo experimental inflammation models (n = 7) and confirmed the activity of pure phycocyanin as an anti-inflammatory agent in all the studied models. Using 50, 100 and 200 mg·kg⁻¹ of phycocyanin significantly reduced ear oedema induced by tetradecanoylphorbol acetate and arachidonic acid in mice by 47.6, 60.3 and 66.6%, respectively. In addition, the application of 50-200 mg·kg⁻¹ of phycocyanin in experimental rats inhibited leukotriene B4 (LTB4), prostaglandin E2 (PGE2) levels and oedema induced by arachidonic acid (Romay et al., 2000). Vázquez-Velasco et al., (2014) concluded that Spirulina protects against negative proinflammatory effects induced by glucomannan in Zucker Fa/Fa rats due to the high phycocyanin content.

3.3. Anti-cancer activitiesTOP

Many cancer forms result from cellular DNA damage, leading to uncontrolled cellular growth. Cellular endonucleases frequently repair the damaged DNA in order to keep the cell alive and healthy. The deactivation of these enzymes by oxidation, radiation or toxins results in errors in DNA, and eventually leads to the development of cancer. Many studies confirmed the anti-cancer activities of Spirulina extracts, either alone or in combination with other compounds. In vitro studies suggested the unique composition of Spirulina polysaccharides which enhance enzymatic activity and DNA repair. Decades ago, Schwartz and Shklar (1987) studied the effect of microalgae extracts on the induced squamous cell carcinoma of hamster buccal pouches in 20 animals. The results showed that Spirulina extract (Phycotene) resulted in total tumor regression by 30% of treated animals; while β-carotene and canthaxanthin resulted in total tumor regression by 20% and 15% of treated animals, respectively. This confirmed the higher anti-cancer activity of Spirulina extract than β-carotene or canthaxanthin. One year later, Schwartz et al., (1988) reported that the anti-cancer activity of the Spirulina extract was attributed to stimulation of the immune response to selectively destroy the small foci of developing malignant cells with no negative impacts on the normal cells. Liu et al., (2000) found that 160 mg·L⁻¹ of c-phycocyanin from S. platensis significantly inhibited the in vitro growth of human leukemia K562 cells as colony numbers reduced from 4.8 in the control to 2.3 in the phycocyanin-treated cells after 12 days of incubation. In addition, Subhashini et al., (2004) reported that treatment with 50 μM of highly purified c-phycocyanin from S. platensis up to 48 h results in the inhibition of K562 cell proliferation by 49%. In another in vivo study, Simsek et al., (2008) suggested that S. platensis supplementation is useful in the treatment of anemia and leukemia induced by lead and cadmium toxicity. Moreover, Choi et al., (2013) investigated the anti-cancer activity of the marine cyanophyte S. maxima grown in deep seawater and reported effective suppression in the over-expression of the Bcl2 gene in A549 cells leading to the inhibition of various human cancer cells.

3.4. Anti-microbial activitiesTOP

A large number of algal extracellular products and/or extracts have been reported as antimicrobial (anti-viral, anti-bacterial, anti-fungal, anti-algal, anti-protozoal) agents, although the detailed structure and identity of the active constituents of many of these are not yet known (Borowitzka 1995). Among microalgae, Spirulina is gaining more and more consideration as a natural antimicrobial agent. Kokou et al., (2012) confirmed the anti-bacterial activity of Spirulina against 6 Vibrio strains; V. anguillarum, V. splendidus, V. parahaemolyticus, V. scophthalmi, V. lentus and V. alginolyticus. The anti-microbial activity of acrylic acid, which is found in Spirulina in relatively high quantities, was confirmed at the end of 1970s. In addition to acrylic acid, probionic, benzoic, and mandelic organic acids were also reported as antimicrobial active compounds. Özdemir et al., (2004) also tested different extracts of S. platensis against bacteria and yeasts. They found that methanol extract was the most potent anti-microbial fraction. Santoyo et al., (2006) confirmed the anti-microbial activity of S. platensis using ethanol, hexane and petroleum ether as extracting solvents against Escherichia coli, Staphylococcus aureus, Aspergillus niger and Candida albicans. Moreover, purified c-phycocyanin extracted from S. ­platensis showed substantial inhibition in the growth of many multidrug-resistant bacteria. For example, purified c-phycocyanin showed minimum inhibitory concentrations (MIC) in vitro of 125, 100, 75 and 50 mg·L⁻¹ against S. aureus, E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa (Sarada et al., 2011).

In addition, Arun et al., (2012) confirmed the antifungal activity of S. platensis methanolic extract. According to Hayashi et al., (1993), S. platensis water-soluble extract also significantly inhibited the in vitro HSV−1 replication in HeLa cells with ED₅₀ (the lowest phycocyanin concentration for reducing plaque number by 50%) at 0.173 mg·mL⁻¹. The addition of 1 mg·mL⁻¹ of S. platensis extract 3 h before viral infection resulted in 50% inhibition of virus protein synthesis without affecting the host. Subsequently, Hayashi et al., (1996) isolated calcium spirulan (Ca-SP) from S. platensis as a novel sulfate polysaccharide. The isolated Ca-SP inhibited the in vitro replication of several enveloped viruses such as the human cytomegalo virus, HSV−1, influenza A virus, measles virus, HIV−1 virus and mumps virus.

3.5. Anti-diabetes and anti-obesity activitiesTOP

The disease diabetes mellitus results from abnormalities in carbohydrate metabolism and is characterized by deficiencies in insulin secretion, resulting in hyperglycemia. The later was widely reported as the main reason for diabetic complications. Recently, the activity of Spirulina as anti-obesity and anti-diabetes was confirmed. The administration of 2 g·kg^-1·day^-1 of Spirulina for 21 days to alloxanized-induced diabetic rats (n = 10) resulted in a significant reduction in plasma glucose levels by 13.7% below the diabetic group (El-Desouki et al., 2015). They attributed the antihyperglycemic action of Spirulina to the potentiation of the pancreatic secretion of insulin from islet β-cells, or to enhancement of blood glucose transport to the peripheral tissue. In addition, Spirulina supplementation promotes vasodilation and restricts vasoconstriction which decreases the levels of plasma lipids and reduces blood pressure (Schwartz et al., 1988). Han et al., (2006) reported that glycolipid H-b2 extracted from Spirulina was found to reduce postprandial triglyceride (TGs) levels and inhibit pancreatic lipase activity in a dose-dependent way; while phycocyanin inhibited the pancreatic lipase activity, which resulted in higher excretion of TGs in experimental animals’ feces and reduced serum TGs.

3.6. Anti-toxicity activitiesTOP

Shastri et al., (1999) investigated the protective effect of S. fusiformis against lead toxicity in albino mice and recorded a pronounced increase in survival rate by administration of Spirulina. Recently, Ebaid et al., (2017) studied the protective effect of A. ­platensis against liver toxicity produced by treatment with copper nanoparticles. They confirmed the beneficial role of A. platensis as antioxidant markers which enhanced the hepatic functional parameters. Numerous mechanisms of cellular toxicity induction under stress conditions were suggested. Lipid peroxidation is one of the main mechanisms to induce cell damage due to the reaction of free radicals with lipids (Rajesh and Kala 2015). It results in malondialdehyde (MDA), which leads to oxidative stress by further interaction with many other molecules. Nigma et al., (1999) attributed ROS production under copper stress to antioxidant depletion or the direct effect of copper on the peroxidation reaction. However, Kim et al., (2011) reported that ROS alter various signal-transduction pathways in addition to the oxidation of vital cellular macromolecules such as DNA and lipids. ROS also increases the cytosolic Ca⁺² levels by activation of non-selective cation channels in cell membranes (Mukherjee et al., 2002), promoting cell damage. The protective effect of Spirulina against oxidative stress was attributed to the relatively high contents of phycocyanin and carotenoids which result in antioxidant activity (Ebaid et al., 2017). In addition, S. platensis was found to positively influence the antioxidant enzyme level and oxidative stress markers under deltamethrin toxicity (Abdel-Daim et al., 2016), urethane and cisplatin (Premkumar et al., 2004) and mitomycin C and cyclophosphamide (Premkumar et al., 2001). In addition to its antioxidant properties, Spirulina showed a strong heavy metal chelating activity which reduced its toxicity as well (Rangsayator et al., 2002).

4. ENVIRONMENTAL IMPACTSTOP

The scarcity of clean environments for the safe livelihood of human beings has gained global concerns. Continuous growth of the human population has resulted in an increase in petroleum consumption and growing energy demands all over the world. Due to human population growth, the world energy consumption is predicted to grow by 8.1% between 2015 and 2040, from 97.8 quadrillion British thermal units (Btu) to 105.7 quadrillion Btu (Fig. 3A). The extensive use of fossil fuels contributes to high atmospheric CO₂ levels, which are predicted to increase by 2.2% between 2015 and 2040 (Figure 3B). Increases in atmospheric CO₂ raise the global air temperature, which is known as “global warming”. If this development continues, the planet will be threatened with the catastrophic changes in the earth’s climate. Global warming does not only mean the earth’s getting warmer, but it is associated with massive negative impacts on economic, social, and physical health which are of great distress. The melting of polar ice, violent surges of hurricanes and collapse of wildlife are all examples of how climate change can affect the planet. Microalgae are widely discussed to diminish the greenhouse effect by CO₂ sequestration and abundant oxygen production. A stoichiometric amount of 1.8 kg CO₂·kg⁻¹ of microalgal biomass can be consumed which makes Spirulina cultivation using flue gas more economically and environmentally useful (Doucha et al., 2005). Based on the biomass produced by the area of Earthrise Farms (550 tons in 0.437 km²), about 990 tons of CO₂ can be mitigated annually by Earthrise Farms. Thus, 28.9% of USA land area would be theoretically used to mitigate the global CO₂ produced in 2015, which could be increased to 29.5% by the expected increase in CO₂ emission in 2040 (Figure 3B). In addition, environmental pollution might result from the corruption of human resources by various micropollutants including pharmaceutical contaminants (Xiong et al., 2018). The distribution of these synthetic chemicals through the air, water and soil develop multidrug-resistant microorganisms. Therefore, the use of naturally synthesized products with the limitation of using chemical compounds will enhance the optimal utilization of pharmaceuticals.

Water and land use are two important environmental factors that need to be considered. Interestingly, Spirulina cultivation can be done in arid lands where no other crops can survive (Soni et al., 2016). In addition, it needs relatively lower amount of water, as water can be recycled after harvest. After the extraction of essential compounds from the biomass, the residual biomass can be used in many other applications in what is called refining process (Figure 4). For example, sugar-based and cellulosic biorefining results in the accumulation of protein by-products, which can be used as animal feed (Soni et al., 2016). In addition, after the extraction of essential compounds, the residual biomass can be used as energy feedstock for biodiesel production from lipids and/or bioethanol production from carbohydrate residues. Thus, integrated food and energy systems are designed to combine, intensify, and increase the productivity of food and energy simultaneously through sustainable land management (Soni et al., 2016). Using Spirulina as bioenergy feedstock showed promising results in many studies. Sumprasit et al., (2017) studied energy recovery in the form of biodiesel and biogas from ubiquitous S. platensis and recorded a biodiesel yield of 7.1% of dry weight. The results also confirmed that lipid-free residue can be used as feedstock for biogas production with an average methane yield of about 290 mL·g⁻¹ VS. In addition, Shao et al., (2018) reported the efficiency of Cd²⁺-sorbed Arthrospira platensis for bioethanol production due to the accumulation of carbohydrates. Moreover, Chagas et al., (2016) studied the catalytic pyrolysis of Arthrospira platensis and Arthrospira maxima using pyrolysis/GC–MS. They found that the high acidity of HZSM-5 catalysts could maximize the conversion of biomass to aromatic hydrocarbons and the aromatic yields increased as the catalyst/biomass ratio increased from 1:1 to 10:1.By food-energy integration, Spirulina products are even more economically competitive with high diversification. Consequently, large-scale cultivation of Spirulina will not only have healthy and economic benefits, but will also help to save our planet.

5. GENETIC ENGINEERING OF SPIRULINATOP

Genetic engineering facilitates the duplication and manipulation of specific fragments of DNA for industrial, medical and research purposes. Recently, genetic engineering led to a revolution in molecular biology with many benefits in different fields, especially in medicine. The cloning of recombinant DNA facilitates the use of bacteria to produce important proteins such as human insulin which is of great importance and now marketed throughout the world. It was revealed that biosynthetic pathways for the production of different cellular macromolecules are complex processes which include many genes (Bhunia et al., 2018). Many industrial compounds can be produced by several molecular techniques such as rational and non-rational mutagenesis, including site-specific mutagenesis, random mutagenesis, and site-saturation mutagenesis. In the last few decades, extensive research has been carried out to understand the biosynthesis genetic regulations of different valuable products in cyanophytes. For example, enhanced alcohol production via genetic engineering has been implemented in two independent cyanobacterial strains through the over-expression of pyruvate decarboxylase and alcohol dehydrogenase II genes (Deng and Coleman, 1999). In addition, previous studies on the regulation of desaturase gene expression in Spirulina platensis revealed that the mRNA level and mRNA stability of the D6D gene increased drastically by decreasing the growth temperature from 35 to 22 °C, which led to an increase in the cellular γ-linolenic acid level by approximately 30% (Deshnium et al., 2000; Honsthong et al., 2003). Further, site-specific mutation studies by Subudhi et al., (2008) demonstrated that the sequence TATAAT located at −33 bp was essential for the D6D promoter function and suggested a complex regulation of D6D gene expression in Spirulina.

Currently, most cloned genes are introduced into bacteria or fungi on heterotrophic growth media for industrial enzyme production. However, developing a protocol for using Spirulina instead involves an economic and procedural reason for considering it as an alternate producer of industrial enzymes. Spirulina can grow autotrophically by converting light energy into biomass rich in minerals, protein, vitamin B₁₂, essential fatty acids, amino acids and β-carotene. In addition, Spirulina does not require physical or chemical pre-treatment in order to become digestible due to the absence of a cellulosic cell wall (Toyomizu et al., 2001a). Moreover, residual biomass after the extraction of valuable molecules can be used as feed for fish, farm animals and poultry (Toyomizu et al., 2001b; Abomohra et al., 2014). Therefore, the development of a method for stable gene cloning into Spirulina as a biological factory for recombinant enzyme production will have many advantages. Unfortunately, the genetic engineering methods for Spirulina have been almost non-existent due to the absence of plasmids in Spirulina (Vachhani and Vonshak, 1997; Toyomizu et al., 2001a). Some Spirulina platensis genes involved in fundamental processes such as protein synthesis, carbon dioxide fixation and nitrogen metabolism were successfully cloned (e.g. Bini et al., 1992; Nelissen et al., 1994; Murata et al., 1996). However, the development of gene transfer systems is a big challenge. Using the plasmid pKK232–8 carrying a promoterless chloramphenicol acetyltransferase (CAT) gene, Cheevadhanarak et al., (1993) transformed S. ­platensis to stably express the CAT gene which maintained under selection pressure for several generations. However, the stability of the transformants was a major problem, as the transformation system developed for Spirulina must be efficient and allow not only a good degree of expression of the introduced gene but also to maintain itself in a fast-growing culture. Toyomizu et al., (2001a) studied the optimization of electroporation of plasmid pHSG399 in S. platensis. The plasmid contained a chloramphenicol resistance gene which produces an E. coli protein, Cm acetyltransferase (CAT). They reported that Spirulina is a suitable organism for the expression of genetically engineered proteins, but the gene transfer system has not yet been established. Later, Kawata et al., (2004) employed a new approach using natural transposase, mini-Tn5 transposon, and a cation liposome to transform Spirulina platensis as a stable recombinant DNA methodology. According to Toyomizu et al., (2001a), more research is needed to investigate other requirements for a ‘stable’ S. platensis gene transfer system, which include; (1) cloning and assessment of Spirulina promoters using a plasmid carrying a promoterless CAT reporter gene, (2) developing a simple and rapid method for sensitive fluorometric determination of Spirulina transformants, and (3) subjecting Spirulina transformants to protoplast fusion in glycine, for cloning. Thus, genetic engineering techniques currently used in plant and microbial biotechnology are required to enable Spirulina, as an autotrophic factory, to produce desired molecules in order to incorporate algal biotechnology products into industrial technologies.

6. CONCLUSIONSTOP

Spirulina is considered as a valuable pharmaceutical agent which offers many unique features with many environmental and health benefits. Despite their extensive applications for nutrition, the industrial production of Spirulina is still more or less limited to relatively small areas. In addition, the molecular engineering of Spirulina seems as a neglected research field in spite of its importance to resolving many challenges, and any piece of work accumulated at this stage will be undoubtedly of great value. There is much more research which needs to be done, including innovative cultivation systems and development of successful genetic and molecular biology tools for Spirulina. The development of a reliable genetic engineering methodology will open the way, not only for better basic research, but also will enable a better program for the development of new and better strains to be used by the industry. This could integrate Spirulina into the industrial sector for the production of essential industrial products while producing valuable nutrient-rich biomass. In addition, nutritional awareness and increasing the acceptance level of Spirulina are required in order to develop such Spirulina-fortified products. At the same time, further scientific, clinical and toxicological research is required to evaluate the safety of large-scale production of Spirulina with harvest at minimum contamination rates, since contamination with harmful elements or toxic substances might potentially cause several serious human health effects.

ACKNOWLEDGMENTSTOP

This work was financially supported by the Key Program of Science and Technology of the Jiangsu Province, China (grant number BE2016353) and Egyptian Ministry of Higher Education and Scientific Research (MHESR).

REFERENCESTOP