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Review

Microalgae-Based PUFAs for Food and Feed: Current Applications, Future Possibilities, and Constraints

by
Anna Santin
1,†,
Sergio Balzano
2,3,†,
Monia Teresa Russo
1,
Fortunato Palma Esposito
2,
Maria Immacolata Ferrante
1,
Martina Blasio
2,
Elena Cavalletti
2 and
Angela Sardo
1,4,*
1
Stazione Zoologica Anton Dohrn, Department of Integrative Marine Ecology, Villa Comunale, 80121 Naples, Italy
2
Stazione Zoologica Anton Dohrn, Department of Ecosustainable Marine Biotechnology, Via Acton 55, 80133 Naples, Italy
3
Department of Marine Microbiology and Biogeochemistry (MMB), Netherland Institute for Sea Research (NIOZ), Landsdiep 4, 1793 AB Texel, The Netherlands
4
Istituto di Scienze Applicate e Sistemi Intelligenti “Eduardo Caianiello” (ISASI), Via Campi Flegrei, 34, 80078 Pozzuoli, Italy
*
Author to whom correspondence should be addressed.
†
These authors equally contributed to this work.
J. Mar. Sci. Eng. 2022, 10(7), 844; https://doi.org/10.3390/jmse10070844
Submission received: 1 June 2022 / Revised: 17 June 2022 / Accepted: 18 June 2022 / Published: 21 June 2022
(This article belongs to the Special Issue Research and Advance in Marine Biotechnology)
Browse Figure
Review Reports Versions Notes

Abstract

:
Microalgae are currently considered an attractive source of highly valuable compounds for human and animal consumption, including polyunsaturated fatty acids (PUFAs). Several microalgae-derived compounds, such as ω-3 fatty acids, pigments, and whole dried biomasses are available on the market and are mainly produced by culturing microalgae in open ponds, which can be achieved with low setup and maintenance costs with respect to enclosed systems. However, open tanks are more susceptible to bacterial and other environmental contamination, do not guarantee a high reproducibility of algal biochemical profiles and productivities, and constrain massive cultivation to a limited number of species. Genetic engineering techniques have substantially improved over the last decade, and several model microalgae have been successfully modified to promote the accumulation of specific value-added compounds. However, transgenic strains should be cultured in closed photobioreactors (PBRs) to minimize risks of contamination of aquatic environments with allochthonous species; in addition, faster growth rates and higher yields of compounds of interest can be achieved in PBRs compared to open ponds. In this review, we present information collected about the major microalgae-derived commodities (with a special focus on PUFAs) produced at industrial scale, as well genetically-engineered microalgae to increase PUFA production. We also critically analyzed the main bottlenecks that make large-scale production of algal commodities difficult, as well as possible solutions to overcome the main problems and render the processes economically and environmentally safe.

1. Introduction

Agriculture and animal breeding account for about 11% of total greenhouse gas (GHG) emissions, totaling 9.3 GtCO2eq in 2018, 5.3 of which consisted of methane and nitrous oxide derived mostly from livestock (https://www.c2es.org/content/international-emissions; access date: 4 April 2022). The massive GHG emissions from livestock indicate that human consumption of dairy, meat, eggs, and other animal-based products should drastically decline. The environmental impact of animal breeding is also exacerbated by livestock diets, which can occasionally include animal-based products. In particular, fishmeal and fish oils have been supplemented in feed for both terrestrial and aquatic animals for many years. The widespread usage of fish-derived meal in aquaculture and livestock feed is due to easy availability as fishing bycatches, as well as processing residues of the fishing industry [1]. The nutritional value of animal feed can be improved by adding fish-based products that typically contain high proportions of polyunsaturated fatty acids (PUFAs). PUFA-rich food supplied to terrestrial and aquatic animals contributes to the development of PUFA-rich meat for human consumption. Because of their antioxidant properties, PUFAs exert beneficial effects on health, decreasing the risks of a number of diseases [2,3,4].
Because of their beneficial effects, PUFAs are also present in a number of nutraceutical products for direct human consumption. Analogously to animal feed, the main source of PUFAs in nutraceuticals are fish oils. Overall, it has been estimated that most PUFA market volume originates from fish oils [5] mostly derived from wild-caught fish, posing serious issues to the sustainability of fishery activities. Fish assimilate PUFAs mostly through their diet by feeding on microalgae or microalgal grazers, such as zooplankton or other PUFA-rich organisms. Alternative PUFA sources include terrestrial plants, microalgae, and some heterotrophic protists. Plants are typically rich in short-chain PUFAs, such as C18:2ω6 (linoleic acid), C18:3ω3 (α-linolenic acid), and C18:3ω6 (γ-linolenic acid), and they are particularly abundant in linseeds and nuts. In contrast, microalgae are particularly rich in two long-chain PUFAs: C20:5ω3 (eicosapentaenoic acid, EPA) and C22:6ω3 (docosahexaenoic acid, DHA). Microalgae and other protists, such as Thraustochytrids or heterotrophic dinoflagellates, are the major PUFA producers [6]. Most marine species contain both EPA and DHA in varying proportions, with the former typically more abundant in diatoms, Haptophyta, and green algae and the latter more present in dinoflagellates and Thraustochytrids. In addition, Eustigmatophyceae, including Nannochloropsis spp., are rich in EPA and do not contain DHA. Within this contest, the food and feed industries are increasingly interested in using and promoting products obtained from plants and microalgae. The latter are receiving considerable attention because of their culturing easiness, their fast growth rates, and the diversity of metabolites the different taxa can produce. Moreover, some microalgae have already received the “generally recognized as safe” (GRAS) status by the Food and Drug Administration (FDA). These include cyanobacteria, such as Arthrospira (Spirulina) sp., and Aphanizomenon flos-aquae; Chlorophyta, such as Chlamydomonas reinhardtii, Auxenochlorella protothecoides, Chlorella vulgaris, Chlorella protothecoides, Dunaliella bardawil, and Haematococcus pluvialis; Rhodophyta, such as Porphyridium cruentum; Heterokontophyta, such as Schizochytrium sp.; Dinophyta, such as Crypthecodinium cohnii; Euglenophyta, such as Euglena gracilis; and Bigyra, such as Ulkenia sp. [7,8].
Most industrial plants that produce microalgae-derived lipid commodities are based on the employment of open ponds rather than enclosed systems (e.g., photobioreactors). This is mainly due to the lower system and management costs [9,10,11]. Unfortunately, open systems do not guarantee high levels of purity of algal commodities, as cultures could be affected by bacterial contamination and/or meteoric contributions. Moreover, because of the weekly and seasonal variability in temperatures and light exposure, the maintenance of microalgal cultures in outdoor systems requires phenotypes to achieve fast growth under continuously changing physical conditions. This drastically limits the number of species capable of growing in outdoor ponds. In contrast, the employment of photobioreactors (PBRs)—and thus of enclosed culture chambers—allows for better control of culture conditions, reduces risks of contamination and evaporation of the culture media, and likely allows for massive growth of a wider range of algal species. In indoor systems, the reproducibility of biomass yields and biochemical composition is enhanced by the lack of temperature and irradiance fluctuations. However, the true bottleneck of PBRs are the capital expenditure (CAPEX) and the operating expense (OPEX) associated with their construction and maintenance, respectively [12].
Despite the considerable difficulties related to the economic feasibility of these technologies, several patents exalting the potential of genetically modified microalgae to enhance PUFA yields have been approved in the last decade, paving the way for future applications for the production of microalgae-derived lipids at an industrial scale.
In this review, we provide information about the current market for microalgae-based commodities, genetic transformations aimed at increasing lipid productivities from model microalgal species, the main bottlenecks, and the possible solutions to promote the production and the marketing of microalgal specialties.

2. Current Market for Microalgae-Derived Products, with a Special Focus on PUFAs

Microalgae-based products are still niche commodities, the production of which could be encouraged by environmental sustainability policies. Microalgae are suitable alternatives to fossil fuels for the production of biofuels and as agricultural crops for human and animal food applications [13,14,15,16,17]. However, in contrast to research or review articles regarding the microalgal potential for biotechnological purposes, which are easily accessible through scientific search engines, it is difficult to get a precise idea of the true microalgal market and some of its sectors.
In this review, we made a substantial effort to collect information available on microalgal commercialization, with special attention on marketable products derived from microalgal lipids and particularly unsaturated FAs (Table 1 and Figure 1).
Many Countries invest in the microalgal bio-based industry. Such countries are mainly located in North America (United States, Canada, and Mexico), accounting for the majority share of the global ω-3 PUFA market, both in terms of value and volume [18]. However, but investments in the microalgae industry have also been made in Europe (Germany, UK, France, Italy, and Spain), the Asia–Pacific area (China, Japan, India, Australia, and Taiwan), South America, the Middle East, and Africa. The main genera employed for large-scale production are Arthrospira, Dunaliella, and Chlorella, as expected, given the plethora of research articles focused on these species [19,20,21,22,23,24].
An accurate analysis available online established that global microalgae market is expected to grow, reaching USD 4049.6 million by 2026, from USD 3391.5 million in 2020, at a compound annual growth rate (CAGR) of 3.0% between 2020 to 2026 (https://southeast.newschannelnebraska.com/story/46278524/microalgae-market-size-in-2022-30-cagr-with-top-countries-data-which-product-segment-is-expected-to-garner-highest-traction-within-the-microalgae; access date: 15 June 2022). The microalgal market segments are food and beverage, animal feed, pharmaceuticals and nutraceuticals, and personal care.
The global ω-3 market was estimated to be worth USD 2.49 billion in 2019 and is expected to expand at a compound annual growth rate (CAGR) of 7% between 2020 and 2027 [26]. FAO statistics and, in particular, FAO report n° 978/2010 [27] reported a market size of EPA and DHA of USD 300 million and 1.5 billion and prices of USD 0.2–0.5/gm and USD 18–22 /gm, respectively [18]. The driving forces for the expected growth of the FA market and, in particular, ω-3, are mainly related to the demographic increase, the effectiveness of PUFAs against cardiovascular diseases (EFSA 2010), and the growing market for functional ingredients [26].
However, information on the main microalgae-derived products with commercial value is still scarce, although our research suggests that they are mainly employed as food supplements and animal feed. In our opinion, this could be due both to a real necessity of healthy and viable alternatives to animal-derived nutritional products and to the long times required for preclinical and clinical trials, which do not encourage the employment of these commodities as pharmaceuticals. Preclinical and clinical trials to demonstrate the safety and the beneficial effects of microalgal products can last approximately ten years [28].
In Table 1, we summarized the main information regarding industrial societies with a website or cited in literature papers that produce microalgae-based products for food and/or feed. Companies that commercialize microalgal products are mostly based in the US, EU, China, and Japan (Figure 1). Although this survey does not cover the whole market of microalgal nutraceuticals due to the difficulty of finding exhaustive information, it seems clear that Arthrospira is, by far, the most employed microalgal genus for the industrial sector of food supplements for both human and animal nutrition. Given the large number of culturable microalgal species worldwide, few species are commercially produced. This is probably due to the strict food safety regulations and difficulty of adapting algae for large-scale production. Several companies also produce other microalgal commodities for different market sectors. For example, Cellana LLC produces biofuels from microalgae, whereas Photonz Corporation exploits microalgae for pharmaceutical purposes, producing high-purity EPAs to treat cardiovascular diseases.
Microalgal products are mainly commercialized as dried algae, mostly obtained from Chlorella and Arthrospira spp. Dried algae are rich in vitamins and are usually sold as dietary supplements and specialty products, such as ω-3 FAs, astaxanthin, β-carotene, and phycocyanin, that can be added to food and feed to improve their nutritional value [29].
Regarding industrial plants for microalgal culture exploitation, the information gathered in Table 1 clearly shows that open ponds are the most widespread cultivation systems, being less expensive than enclosed systems. Photobioreactors require a high power supply to maintain constant growth conditions but guarantee low risks of contamination.

3. Microalgae-Based Genetic Engineering Technology for PUFA Production: Most Relevant Patents

Different strategies have been studied in order to improve PUFA production from microalgae in recent decades. These include the screening and selection of more productive species and strains, as well as the modulation of growth conditions, such as nutrients, light, or CO2 [30]; and the most recent and most promising strategy to increase PUFA production is represented by genetic engineering. Thanks to the rapid development of high-throughput sequencing technologies and genetic engineering techniques, several microbes, including microalgae, have been successfully transformed. Manipulation of metabolic microbial pathways can often result in transformed genotypes with valuable biotechnological properties, such as increased PUFA content, resulting in a dry biomass more suitable for commercialization compared to the same dry biomass harvested from the wild-type genotype. The number of patents released annually has increased sharply in the last 10 years, and this demonstrates the need to understand the molecular mechanisms that regulate PUFA production in microalgae and to exploit this knowledge to manipulate microorganisms in order to find more sustainable solutions.
Despite the lack of solid regulation in many countries and the large gap between production costs and ultimate benefits, genetic engineering research groups have identified interesting strains and genes that can be modified to produce more PUFAs.
As regards genetic engineering, the model microalgae are the freshwater green algae Chlorella and Chlamydomonas, the eustigmatophycean Nannochloropsis, and the diatom Phaeodactylum. These species have been studied for many years, their genomes are fully available, and transformation techniques have been developed in order to easily obtain transgenic strains. However, in recent years, the genomes of many other microalgal species have been sequenced and studied in order to broaden knowledge and identify new opportunities for biotechnological applications. An example is the diatom Cyclotella, which has attracted increasing interest for biofuel development because of its high TAG content [31]. Moreover, new projects are ongoing to sequence a large number of microalgal genomes (https://jgi.doe.gov/csp-2021-100-diatom-genomes/; access date: 8 February 2022). In addition, information on the most frequently expressed genes and the resulting predicted proteins is available in transcriptome datasets, such as EukProt [32] and MMETSP [33,34].
Various transformation techniques are currently available and have been successfully applied to different microalgal species, from electroporation to microparticle bombardment [30,35]. Furthermore, various editing approaches are possible, for example, overexpressing genes encoding for enzymes that represent critical steps of PUFA production pathways or expressing a gene in another organism (called heterologous expression).
The overexpression of endogenous genes is particularly useful when encoded enzymes are directly involved in fatty acid biosynthesis, with the aim of increasing the final metabolite production (Table 2). For example, beta-ketoacyl-acyl carrier-protein synthase (KAS) and thioesterases (FAT or TE) were overexpressed in the microalgae Chlorella or Prototheca spp. to increase the production of C8 and C16 fatty acids [36]. Alternatively, genes coding for glycerol-3-phosphate acyltransferase (GPAT) or diacylglycerol acyl-transferase (DGAT), both involved in triacylglycerols (TAGs), can be overexpressed to alter lipid equilibrium within cells, increasing the production of TAGs and/or fatty acids in the diatom Phaeodactylum tricornutum [37,38], the green microalga Chlamydomonas reinhardtii [39] or the oleaginous alga Nannochloropsis oceanica [40]. Other interesting examples of gene overexpression in microalgae are represented by transcription factors, such as bHLH2 in Nannochloropsis salina [41] and DOF in C. reinhardtii [42]; these two studies demonstrated how acting on a single transcription factor can lead simultaneous modification of multiple steps of biosynthetic pathways, altering global cellular metabolism and increasing lipid production.
On the other hand, heterologous expression is particularly productive when an organism can be easily cultured in the laboratory, achieving fast growth under standard culturing conditions, although resulting in low content of specific metabolites, such as PUFAs. PUFA content is selected and transformed with a transgene, which increases its PUFA production. Often, these model organisms are bacteria [43] or yeasts [44], for which extraction and purification techniques are better defined and less costly than for microalgae, as well as plants, which can be used as PUFA-enriched, plant-based biomass without requiring many purification steps [45]. Genes encoding FAT, GPAT, or DGAT can also be expressed through heterologous expression [43,46] (Table 2). Other interesting studies have involved genes encoding elongases and desaturases of the PUFA biosynthetic pathway; manipulation of these genes can lead to the alteration of PUFA proportions, increasing the levels of PUFAs with more interesting nutraceutical applications, such as EPA and DHA [44]. Moreover, Hu et al. [47,48,49] expressed C. ellipsoidea nuclear factor Y (NF-Y) into plant A. thaliana; these factors, which were firs described here for C. ellipsoidea, lead to increased biomass and fatty acid production in plants (Table 2).
In other cases, silencing is a useful strategy when targets are genes involved in PUFA catabolism or alternative pathways [30]. For example, Huang et al. [42] silenced the acetyl-CoA synthetase (ACS) of C. reinhardtii, increasing fatty acid excretion (Table 2). Although some interesting applications of silencing techniques have been reported, this approach is often preferred to characterize gene function in basic research and less used for biotechnological applications. With the expansion of this field in recent years, the next step is the application and regulation of such products in the market.
Although the most exploited genera for large-scale production ensure medium to high biomass and lipid productivities, their genetically engineered clones could significantly increase the yields of microalgae-derived commodities with respect to their wild types, as shown in Table 3.

4. Main Bottlenecks for PUFA-Derived Microalgae for Large-Scale Production and Possible Solutions

Although commercial-scale cultivation of microalgae is quickly improving, production costs of microalgal biomass are still far from competitive. The main reasons behind the high production costs are related to the low yields of PUFAs within microalgal biomass [80], which may depend on the cell growth and on the extraction methods. Novel growth strategies aimed at achieving economic viability of mass culturing foresee the employment of waste carbon sources in heterotrophically growing cultures [81] in order to recycle and minimize reject goods, as well as the improvement of harvesting and dewatering technologies, which account for up to 20–30% of the whole biomass cost [71,82,83,84]. However, the high costs related to artificial illumination and energy requirements of air and/or gas sparging inside photobioreactors actually make their use economically unsustainable; thus, open ponds rather than enclosed chambers are more feasible as cultivation systems. On the other hand, open ponds are more prone to contamination compared to PBRs, potentially compromising growth and final product purity. The latter aspect should not be neglected in the food and feed sector, as it represents one of the main issues with respect to microalgal products commercialized for human and, to a lesser extent, animal consumption.
The patents described in Table 2 with the aim of optimizing the yields of highly valuable lipids underline the urgent need to grow microalgae in enclosed systems to increase their productivity; in addition, for the cultivation of allochthonous species, enclosed systems also minimize risks of biological contamination of aquatic environments compared to open ponds. Striking a balance between cost-effectiveness and cutting-edge technologies is crucial from the perspective of large-scale production of microalgae-derived commodities.
Another strategy to reduce costs of microalgal mass culturing is based on minimizing the amount of waste, thus exploiting a larger fraction of the microalgal biomass generated by the whole production process. In addition to PUFAs, other fractions, such as TAGs, sterols, carotenoids, and alcohols, can be exploited from the lipid pool. Moreover, both the protein and carbohydrate fractions of microalgal biomass can be exploited to obtain other microalgae-derived commodities. Microalgae are a potential source of several protein ingredients, such as whole-cell proteins, protein concentrates, isolates, hydrolysates, and bioactive peptides [85], as well as a source of recombinant proteins mainly derived from chloroplasts [86]. Carbohydrates, on the other hand, are mainly used as source of bioenergy [87] and, recently, as an eco-sustainable source of prebiotics [88]. In diatoms, frustules could be recovered and used for biomedical applications—e.g., as drug carriers and matrices for tissue regeneration [89,90,91], as well as for pollutant removal [92]. Finally, alternative methods for PUFAs extraction foreseeing the use of a low or null solvent amount could be appreciable from the perspective of an environmentally and economically sustainable route. Conventional extraction methods with organic solvents are still the main techniques used to extract lipids for microalgae [93,94] due to the high percentages of final products obtained, but they require more subsequent purification steps to remove as much solvent as possible from the final product. Therefore, other methods, such as enzymatic disruption, ultrasonication, microwave methods [95,96], or supercritical fluid extraction [97,98,99], should be taken into account and optimized to reach a good compromise between lipid yield costs and environmentally safe procedures.
One of the main challenges is represented by the adaptation of legislations concerning the production and the market introduction of microalgal products, in particular with regard to genetically engineered strains for food and feed applications. European countries seem to be generally more reticent than other countries to consider genetically modified organisms (GMO) as nutraceuticals. The US Department of Agriculture (USDA) has decided that all the regulations should relate to the final product or the organism itself rather than the process. This means that the USA tends to rule in favour of gene-edited organisms with regard to approval for growth or entry into the food chain [100]. This is in contrast to the EU and the UK, where current policy and GMO guidelines continue tightly control plants, animals, fungi, and microorganisms modified by gene editing, with severe limitations on their applications. For example, the new European regulatory framework based on the precautionary principle is composed of Directive 2001/18/EC on the deliberate release into the environment of GMOs, taking into account the risks to the environmental and human health. Furthermore, two regulations, 1829 and 1830/2003/EC [101,102], govern the authorization and traceability of food and feed consisting of GMOs or their derivatives. They are subject to extensive, case-by-case, science-based food evaluation by the European Food Safety Authority (EFSA). However, more than half the 28 countries in the European Union (EU) have decided to ban GMO production for food and feed applications, particularly because their risk/benefit balance is perceived as very unfavorable and because the general public often lacks confidence in their promoters and the regulatory process. Despite the majority of the recent innovations around gene editing in microalgae are coming from groups based in the EU [30], the majority of the commercial applications of these technologies occur outside of the EU, with companies not investing in research and development around this area in the EU, causing an economic stalemate across all sectors.

5. Concluding Remarks

The main issues that are currently limiting large-scale production of PUFA-derived microalgae are: (1) the economic unsustainability of some industrial processes, especially those based on the employment of enclosed systems, which are the most suitable systems for the production of genetically modified strains; (2) the inadequacy of open systems for species vulnerable to contamination, temperature, and/or irradiance fluctuations; and (3) a certain reticence to exploit gene-edited microalgae as food or feed supplements. In our opinion, a biorefinery approach aimed at exploiting the whole biomass enhancing the co-production of other commodities and/or minimizing waste is the basic requirement to promote the industrial production of microalgal PUFAs and other high-value compounds. Large-scale production of microalgae to obtain specialty products could be further enhanced by higher confidence in the potential of genetically engineered strains and their official recognition as nutraceuticals, as well as technological advances to completely exclude risks of contamination of aquatic environments with GMOs.

Author Contributions

A.S. (Anna Santin), S.B. and A.S. (Angela Sardo): conceptualization and original draft preparation; M.T.R., F.P.E., M.I.F., M.B. and E.C.: review and editing; A.S. (Angela Sardo): supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

The authors did not require any signed informed consent.

Data Availability Statement

Not applicable.

Acknowledgments

Anna Santin, Martina Blasio, and Elena Cavalletti were supported by a PhD fellowship funded by the Stazione Zoologica Anton Dohrn (Open University–Stazione Zoologica Anton Dohrn PhD Program).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. FAO. The State of World Fisheries and Aquaculture 2020; FAO: Rome, Italy, 2020. [Google Scholar]
  2. Briggs, M.A.; Petersen, K.S.; Kris-Etherton, P.M. Saturated Fatty Acids and Cardiovascular Disease: Replacements for Saturated Fat to Reduce Cardiovascular Risk. Healthcare 2017, 5, 29. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Dessì, M.; Noce, A.; Bertucci, P.; Manca di Villahermosa, S.; Zenobi, R.; Castagnola, V.; Addessi, E.; Di Daniele, N. Atherosclerosis, dyslipidemia, and inflammation: The significant role of polyunsaturated Fatty acids. ISRN Inflamm. 2013, 2013, 191823. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Gu, Z.; Shan, K.; Chen, H.; Chen, Y.Q. n-3 Polyunsaturated Fatty Acids and Their Role in Cancer Chemoprevention. Curr. Pharmacol. Rep. 2015, 1, 283–294. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Shepherd, C.J.; Jackson, A.J. Global fishmeal and fish-oil supply: Inputs, outputs and markets. J. Fish Biol. 2013, 83, 1046–1066. [Google Scholar] [CrossRef]
  6. Blasio, M.; Balzano, S. Fatty Acids Derivatives From Eukaryotic Microalgae, Pathways and Potential Applications. Front. Microbiol. 2021, 12, 718933. [Google Scholar] [CrossRef]
  7. Garcia, J.L.; de Vicente, M.; Galan, B. Microalgae, old sustainable food and fashion nutraceuticals. Microb. Biotechnol. 2017, 10, 1017–1024. [Google Scholar] [CrossRef] [Green Version]
  8. Torres-Tiji, Y.; Fields, F.J.; Mayfield, S.P. Microalgae as a future food source. Biotechnol. Adv. 2020, 41, 107536. [Google Scholar] [CrossRef]
  9. Richardson, J.W.; Johnson, M.D.; Outlaw, J.L. Economic comparison of open pond raceways to photo bio-reactors for profitable production of algae for transportation fuels in the Southwest. Algal Res. 2012, 1, 93–100. [Google Scholar] [CrossRef]
  10. Shen, Y.; Yuan, W.; Pei, Z.J.; Wu, Q.; Mao, E. Microalgae Mass Production Methods. Trans. ASABE 2009, 52, 1275–1287. [Google Scholar] [CrossRef]
  11. Li, T.; Chen, Z.; Wu, J.; Wu, H.; Yang, B.; Dai, L.; Wu, H.; Xiang, W. The potential productivity of the microalga, Nannochloropsis oceanica SCS-1981, in a solar powered outdoor open pond as an aquaculture feed. Algal Res. 2020, 46, 101793. [Google Scholar] [CrossRef]
  12. Salama, E.-S.; Kurade, M.B.; Abou-Shanab, R.A.I.; El-Dalatony, M.M.; Yang, I.-S.; Min, B.; Jeon, B.-H. Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation. Renew. Sustain. Energy Rev. 2017, 79, 1189–1211. [Google Scholar] [CrossRef]
  13. Benvenuti, G.; Ruiz, J.; Lamers, P.P.; Bosma, R.; Wijffels, R.H.; Barbosa, M.J. Towards microalgal triglycerides in the commodity markets. Biotechnol. Biofuels 2017, 10, 188. [Google Scholar] [CrossRef] [PubMed]
  14. Dineshbabu, G.; Goswami, G.; Kumar, R.; Sinha, A.; Das, D. Microalgae-nutritious, sustainable aqua- and animal feed source. J. Funct. Foods 2019, 62, 103545. [Google Scholar] [CrossRef]
  15. Draaisma, R.B.; Wijffels, R.H.; Slegers, P.M.; Brentner, L.B.; Roy, A.; Barbosa, M.J. Food commodities from microalgae. Curr. Opin. Biotechnol. 2013, 24, 169–177. [Google Scholar] [CrossRef] [PubMed]
  16. Bortoleto, G.G.; de Miranda, H.L.; de Campos, R.H. Biodiesel from Microalgae: Third Generation Biofuel. In Plant-Based Genetic Tools for Biodiesel Production; Bentham Science Publishers: Sharja, United Arab Emirates, 2017; pp. 169–198. [Google Scholar]
  17. Gong, Y.M.; Jiang, M.L. Biodiesel production with microalgae as feedstock: From strains to biodiesel. Biotechnol. Lett. 2011, 33, 1269–1284. [Google Scholar] [CrossRef] [PubMed]
  18. Rahman, K.M. Food and High Value Products from Microalgae: Market Opportunities and Challenges. In Microalgae Biotechnology for Food, Health and High Value Products; Alam, M.A., Xu, J.-L., Wang, Z., Eds.; Springer: Singapore, 2020; pp. 3–27. [Google Scholar]
  19. Angulo, L.D.M.; Merida, L.G.R. Current status of microalgae producers companies for food and food supplements in Latin America. Rev. Venez. Cienc. Tecnol. Aliment. 2017, 8, 130–147. [Google Scholar]
  20. Farag, M.R.; Alagawany, M.; Abd El-Hack, M.E.; Dhama, K. Nutritional and Healthical Aspects of Spirulina (Arthrospira) for Poultry, Animals and Human. Int. J. Pharmacol. 2016, 12, 36–51. [Google Scholar] [CrossRef] [Green Version]
  21. Wachda; Hadiyanto, H.; Harjanto, G.D.; Huzain, M.L.; Aji, R.W. Iop. Production of antioxidant C-phycocyanin using extraction process of Spirulina platensis in large scale industry. In Proceedings of the 2nd International Conference on Food Science and Engineering (ICFSE), Surakarta, Indonesia, 25–26 September 2018. [Google Scholar]
  22. Sui, Y.X.; Vlaeminck, S.E. Effects of salinity, pH and growth phase on the protein productivity by Dunaliella salina. J. Chem. Technol. Biotechnol. 2019, 94, 1032–1040. [Google Scholar] [CrossRef]
  23. Borowitzka, M.A. Commercial production of microalgae: Ponds, tanks, tubes and fermenters. J. Biotechnol. 1999, 70, 313–321. [Google Scholar] [CrossRef]
  24. Rasheed, R.; Saadaoui, I.; Bounnit, T.; Cherif, M.; Al Ghazal, G.; Al Jabri, H. Sustainable Food Production and Nutraceutical Applications from Qatar Desert Chlorella sp. (Chlorophyceae). Animals 2020, 10, 1413. [Google Scholar] [CrossRef]
  25. Ratledge, C. Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 2004, 86, 807–815. [Google Scholar] [CrossRef] [PubMed]
  26. Oliver, L.; Dietrich, T.; Maranon, I.; Villaran, M.C.; Barrio, R.J. Producing Omega-3 Polyunsaturated Fatty Acids: A Review of Sustainable Sources and Future Trends for the EPA and DHA Market. Resources 2020, 9, 148. [Google Scholar] [CrossRef]
  27. World Health Organization. Report of the Joint FAO/WHO Expert Consultation on the Risks and Benefits of Fish Consumption. Rome, 25–29 January 2010; World Health Organization: Geneva, Switzerland, 2011.
  28. Jaspars, M.; De Pascale, D.; Andersen, J.H.; Reyes, F.; Crawford, A.D.; Ianora, A. The marine biodiscovery pipeline and ocean medicines of tomorrow. J. Mar. Biol. Assoc. UK 2016, 96, 151–158. [Google Scholar] [CrossRef] [Green Version]
  29. Ashour, M.; Elshobary, M.E.; El-Shenody, R.; Kamil, A.W.; Abomohra, A. Evaluation of a native oleaginous marine microalga Nannochloropsis oceanica for dual use in biodiesel production and aquaculture feed. Biomass Bioenergy 2019, 120, 439–447. [Google Scholar] [CrossRef]
  30. Santin, A.; Russo, M.T.; Ferrante, M.I.; Balzano, S.; Orefice, I.; Sardo, A. Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture. Molecules 2021, 26, 7697. [Google Scholar] [CrossRef]
  31. Traller, J.C.; Cokus, S.J.; Lopez, D.A.; Gaidarenko, O.; Smith, S.R.; McCrow, J.P.; Gallaher, S.D.; Podell, S.; Thompson, M.; Cook, O.; et al. Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnol. Biofuels 2016, 9, 258. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Richter, D.J.; Berney, C.; Strassert, J.F.H.; Poh, Y.-P.; Herman, E.K.; Muñoz-Gómez, S.A.; Wideman, J.G.; Burki, F.; de Vargas, C. EukProt: A database of genome-scale predicted proteins across the diversity of eukaryotes. BioRxiv 2020, 1–13. [Google Scholar] [CrossRef]
  33. Keeling, P.J. The Number, Speed, and Impact of Plastid Endosymbioses in Eukaryotic Evolution. Annu. Rev. Plant Biol. 2013, 64, 583–607. [Google Scholar] [CrossRef] [Green Version]
  34. Johnson, L.K.; Alexander, H.; Brown, C.T. Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes. GigaScience 2018, 8, giy158. [Google Scholar] [CrossRef] [Green Version]
  35. Sreenikethanam, A.; Raj, S.; Rajesh Banu, J.; Gugulothu, P.; Bajhaiya, A.K. Genetic Engineering of Microalgae for Secondary Metabolite Production: Recent Developments, Challenges, and Future Prospects. Front. Bioeng. Biotechnol. 2022, 10, 836056. [Google Scholar] [CrossRef]
  36. Davis, D.; Rudenko George, N.; Somanchi, A.; Casolari, J.; Franklin, S.; Ewing, A. Novel Ketoacyl Acp Synthase Genes and Uses Thereof. U.S. Patent 2018/0230442 A1, 16 August 2018. [Google Scholar]
  37. Jiang, P.; Yang, G.; Jin, Z.; Huaxin, C. Method for Increasing Lipid Content of Diatom Through Metabolism Gene Engineering. CN Patent 105219649 A, 6 January 2016. [Google Scholar]
  38. Guiheneuf, F.; Leu, S.; Zarka, A.; Khozin-Goldberg, I.; Boussiba, S. Acyl-Coa: Diacylglycerol Acyltransferase 1-Like Gene (Ptdgat1) And Uses Thereof. U.S. Patent 2014/0196177 A1, 10 July 2014. [Google Scholar]
  39. Deng, X.; Fei, X.; Gu, B.; Luo, Q. Chlamydomonas reinhardtii Lipid Metabolism Gene CrDGAT2-5, Encoding Protein Thereof, and Application Thereof. CN Patent 102321642 A, 18 January 2012. [Google Scholar]
  40. Xin, Y.; Xu, J. Gene with Triglyceride (TAG) Synthesis Function and Construction Method and Application Thereof. CN Patent 110305883 A, 8 October 2019. [Google Scholar]
  41. Yang Ji, W.; Kang Nam, K.; Jeong Byeong, R.; Park Min, S.; Lee Bong, S.; Choi Gang, G.; Jeon Seung, J.; Kwon So, H. Method for Increasing Lipid Productivity and Growth of Microalgae by Using an Overexpression of bHLH Transcription Factors. KR Patent 20160142024 A, 12 December 2016. [Google Scholar]
  42. Huang, Y.; Song, Y.; Hu, Z.; Jia, B.I.N.; Wu, M.I.N.; Lin, B. Chlamydomonas reinhardtii Acyl Coenzyme A Synthetase Anti-sense RNA Expression Vector and Structuring Method and Application Thereof. CN Patent 105647957 A, 8 June 2016. [Google Scholar]
  43. Ozaki, T. Lipid Production Method Using Acyl-Acp Thioesterase. JP Patent 2016007154 A, 18 January 2016. [Google Scholar]
  44. Jiang, M.; Xia, W.; Gong, Y.; Liang, Z.; Zhang, Y.; Bing, G.; Zhang, Y. Isochrysis sphaerica Delta 4-Fatty Acid Desaturase Gene and Cloning Method Thereof. CN Patent 102559710 B, 30 October 2013. [Google Scholar]
  45. Benedetti, M.; Vecchi, V.; Barera, S.; Dall’Osto, L. Biomass from microalgae: The potential of domestication towards sustainable biofactories. Microb. Cell Factories 2018, 17, 173. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Hu, Z.; Guo, X.; Chen, Y.; Yin, W. CeDGAT1 (Chlorella ellipsoidea Diacylglycerol Acyltransferase 1) Gene and Application Thereof. CN Patent 103397007 A, 20 November 2013. [Google Scholar]
  47. Hu, Z.; Fan, C.; Sun, P.; Chen, Y. Chlorella ellipsoidea NF-YA Gene and Application Thereof. CN Patent 107936098 A, 20 April 2018. [Google Scholar]
  48. Hu, Z.; Fan, C.; Chen, Y. Chlorella ellipsoidea NF-YB Gene and Application Thereof. CN Patent 108003226 A, 8 May 2018. [Google Scholar]
  49. Hu, Z.; Fan, C.; Chen, Y. Chlorella ellipsoidea NF-YC Gene and Application Thereof. CN Patent 108101973 A, 1 June 2018. [Google Scholar]
  50. Jeong Won, J.; Ahn Joon, W.; Hwangbo, K.; Park Youn, I.; Liu Jang, R. Method For Increasing Total Fatty Acid Content And Useful Fatty Acid Ratio In Transgenic Microalgae Overexpressing Fab2 Gene. KR Patent 20140005001 A, 14 January 2014. [Google Scholar]
  51. Yang, J.-W.; Shin, S.-E.; Jeong, B.-R.; Kang Nam, K.; Choi Gang, G.; Jun Seung, J.; Kwak, M.-S. Recombinant Vector For Increasing Biomass And Lipid Productivity Of Microalgae And Use Thereof. WO Patent 2015/105233 A1, 16 July 2015. [Google Scholar]
  52. Chang Yong, K.; Koh Hyun, G.; Jeong Byeong, R.; Kang Nam, K.; Jeon Seung, J.; Shin Won, S. Recombinant Vector for Targeting Chloroplast of Microalgae and Uses Thereof. KR Patent 101855739 B1, 9 May 2018. [Google Scholar]
  53. Huang, Y.; Xie, X.; Jia, B.; Hu, Z. Recombinant Expression Vector of Chlamydomonas reinhardtii Dof (DNA Binding with One Finger) Gene as Well as Construction Method and Application of Recombinant Expression Vector. CN Patent 105755034 A, 13 July 2016. [Google Scholar]
  54. Suetsugu, M.; Ogawa, A. Gene For Improving Fatty Acid Productivity. JP Patent 2017127278 A, 27 July 2017. [Google Scholar]
  55. Hu, Z.; Zhang, D.; Li, S.; Fan, C.; Chen, Y. Cem GPDH (Glycerol-3-Phosphate Dehydrogenase) Gene and Application Thereof. CN Patent 104357415 A, 18 February 2015. [Google Scholar]
  56. Trimbur, D.; Im, C.-S.; Dillon, H.; Day, A.; Franklin, S.; Coragliotti, A. Recombinant Microalgal Cell And Method Of Producing Lipids Using Said Cell. EP Patent 3546588 A2, 2 October 2019. [Google Scholar]
  57. Xu, Y. New Delta12 Desaturase Gene Derived from Pavlova viridis. CN Patent 104388442 A, 4 March 2015. [Google Scholar]
  58. Xu, Y. Delta 8 Desaturase New Gene Derived from Pavlova viridis. CN Patent 104293769 A, 21 January 2015. [Google Scholar]
  59. Fan, J.; Li, Y.; Xu, H.U.I.; Run, C.; Fang, L.E.I.; Li, S. Method for Increasing Content of Fatty Acid and Oil and Fat in Microalgae by Using Type III NAD Kinase. CN Patent 105316358 A, 10 February 2016. [Google Scholar]
  60. Oh, H.M.; Lee, H.G.; Woo, L.J.; Kim, H.S.; Ha, S.J. DGTS Neutral Lipid-Rich Microalgae Transformants through Membrane Lipid DGTS Modulation and Uses Thereof. KR Patent 101893522 B1, 31 August 2018. [Google Scholar]
  61. Huang, Y.; Yin, J.; Jia, B. High-Oil-Yield Engineering Algae Constructed by Polygene Co-Silencing Technology as Well as Preparation Method and Application of High-Oil-Yield Engineering Algae. CN Patent 110564623 A, 13 December 2019. [Google Scholar]
  62. Zaki, M.A.; Ashour, M.; Heneash, A.M.M.; Mabrouk, M.M.; Alprol, A.E.; Khairy, H.M.; Nour, A.M.; Mansour, A.T.; Hassanien, H.A.; Gaber, A.; et al. Potential Applications of Native Cyanobacterium Isolate (Arthrospira platensis NIOF17/003) for Biodiesel Production and Utilization of Its Byproduct in Marine Rotifer (Brachionus plicatilis) Production. Sustainability 2021, 13, 1769. [Google Scholar] [CrossRef]
  63. Morales-Sanchez, D.; Schulze, P.S.C.; Kiron, V.; Wijffels, R.H. Production of carbohydrates, lipids and polyunsaturated fatty acids (PUFA) by the polar marine microalga Chlamydomonas malina RCC2488. Algal Res.-Biomass Biofuels Bioprod. 2020, 50, 102016. [Google Scholar] [CrossRef]
  64. Hang, L.T.; Mori, K.; Tanaka, Y.; Morikawa, M.; Toyama, T. Enhanced lipid productivity of Chlamydomonas reinhardtii with combination of NaCl and CaCl2 stresses. Bioprocess Biosyst. Eng. 2020, 43, 971–980. [Google Scholar] [CrossRef] [PubMed]
  65. Work, V.H.; Radakovits, R.; Jinkerson, R.E.; Meuser, J.E.; Elliott, L.G.; Vinyard, D.J.; Laurens, L.M.L.; Dismukes, G.C.; Posewitz, M.C. Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains. Eukaryot. Cell 2010, 9, 1251–1261. [Google Scholar] [CrossRef] [Green Version]
  66. Nascimento, I.A.; Marques, S.S.I.; Cabanelas, I.T.D.; Pereira, S.A.; Druzian, J.I.; de Souza, C.O.; Vich, D.V.; de Carvalho, G.C.; Nascimento, M.A. Screening Microalgae Strains for Biodiesel Production: Lipid Productivity and Estimation of Fuel Quality Based on Fatty Acids Profiles as Selective Criteria. Bioenergy Res. 2013, 6, 1–13. [Google Scholar] [CrossRef]
  67. Chen, C.Y.; Kao, A.L.; Tsai, Z.C.; Chow, T.J.; Chang, H.Y.; Zhao, X.Q.; Chen, P.T.; Su, H.Y.; Chang, J.S. Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus. Biotechnol. J. 2016, 11, 336–344. [Google Scholar] [CrossRef]
  68. Mehta, P.; Rani, R.; Gupta, R.; Mathur, A.S.; Puri, S.K. Biomass and lipid production of a novel freshwater thermo-tolerant mutant strain of Chlorella pyrenoidosa NCIM 2738 in seawater salinity recycled medium. Algal Res.-Biomass Biofuels Bioprod. 2018, 36, 88–95. [Google Scholar] [CrossRef]
  69. Zhang, X.; Rung, J.F.; Chen, H.; He, C.L.; Wang, Q. Current status and outlook in the application of microalgae in biodiesel production and environmental protection. Front. Energy Res. 2014, 2, 32. [Google Scholar] [CrossRef] [Green Version]
  70. Xue, J.; Wang, L.; Zhang, L.; Balamurugan, S.; Li, D.W.; Zeng, H.; Yang, W.D.; Liu, J.S.; Li, H.Y. The pivotal role of malic enzyme in enhancing oil accumulation in green microalga Chlorella pyrenoidosa. Microb. Cell Factories 2016, 15, 120. [Google Scholar] [CrossRef] [Green Version]
  71. Mata, T.M.; Martins, A.A.; Caetano, N.S. Microalgae for biodiesel production and other applications: A review. Renew. Sustain. Energy Rev. 2010, 14, 217–232. [Google Scholar] [CrossRef] [Green Version]
  72. Yuan, Y.; Xuyang, L.; Quanyu, Z. Enhancing growth and lipid productivity in Dunaliella salina under high light intensity and nitrogen limited conditions. Bioresour. Technol. Rep. 2019, 7, 100211. [Google Scholar] [CrossRef]
  73. Talebi, A.F.; Tohidfar, M.; Bagheri, A.; Lyon, S.R.; Salehi-Ashtiani, K.; Tabatabaei, M. Manipulation of carbon flux into fatty acid biosynthesis pathway in Dunaliella salina using AccD and ME genes to enhance lipid content and to improve produced biodiesel quality. Biofuel Res. J. 2014, 1, 91–97. [Google Scholar] [CrossRef]
  74. Guerra, I.; Pereira, H.; Costa, M.; Silva, J.T.; Santos, T.; Varela, J.; Mateus, M.; Silva, J. Operation Regimes: A Comparison Based on Nannochloropsis oceanica Biomass and Lipid Productivity. Energies 2021, 14, 1542. [Google Scholar] [CrossRef]
  75. Han, X.; Song, X.; Li, F.; Lu, Y. Improving lipid productivity by engineering a control-knob gene in the oleaginous microalga Nannochloropsis oceanica. Metab. Eng. Commun. 2020, 11, e00142. [Google Scholar] [CrossRef]
  76. Patel, A.; Matsakas, L.; Hrůzová, K.; Rova, U.; Christakopoulos, P. Biosynthesis of Nutraceutical Fatty Acids by the Oleaginous Marine Microalgae Phaeodactylum tricornutum Utilizing Hydrolysates from Organosolv-Pretreated Birch and Spruce Biomass. Mar. Drugs 2019, 17, 119. [Google Scholar] [CrossRef] [Green Version]
  77. Daboussi, F.; Leduc, S.; Marechal, A.; Dubois, G.; Guyot, V.; Perez-Michaut, C.; Amato, A.; Falciatore, A.; Juillerat, A.; Beurdeley, M.; et al. Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat. Commun. 2014, 5, 3831. [Google Scholar] [CrossRef]
  78. Byreddy, A.R.; Gupta, A.; Barrow, C.J.; Puri, M. Comparison of Cell Disruption Methods for Improving Lipid Extraction from Thraustochytrid Strains. Mar. Drugs 2015, 13, 5111–5127. [Google Scholar] [CrossRef] [Green Version]
  79. Li, Z.; Meng, T.; Ling, X.; Li, J.; Zheng, C.; Shi, Y.; Chen, Z.; Li, Z.; Li, Q.; Lu, Y.; et al. Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production. J. Agric. Food Chem. 2018, 66, 5382–5391. [Google Scholar] [CrossRef]
  80. da Silva, T.L.; Moniz, P.; Silva, C.; Reis, A. The Dark Side of Microalgae Biotechnology: A Heterotrophic Biorefinery Platform Directed to omega-3 Rich Lipid Production. Microorganisms 2019, 7, 670. [Google Scholar] [CrossRef] [Green Version]
  81. Kreft, J.; Moe, E.; Garcia, N.; Ross, A.; Seames, W. Comparative scoping study report for the extraction of microalgae oils from two subspecies of Chlorella vulgaris. Clean Energy 2020, 4, 233–246. [Google Scholar] [CrossRef]
  82. Singh, G.; Patidar, S.K. Microalgae harvesting techniques: A review. J. Environ. Manag. 2018, 217, 499–508. [Google Scholar] [CrossRef] [PubMed]
  83. Grima, E.M.; Belarbi, E.H.; Fernandez, F.G.A.; Medina, A.R.; Chisti, Y. Recovery of microalgal biomass and metabolites: Process options and economics. Biotechnol. Adv. 2003, 20, 491–515. [Google Scholar] [CrossRef]
  84. Verma, N.M.; Mehrotra, S.; Shukla, A.; Mishra, B.N. Prospective of biodiesel production utilizing microalgae as the cell factories: A comprehensive discussion. Afr. J. Biotechnol. 2010, 9, 1402–1411. [Google Scholar]
  85. Soto-Sierra, L.; Stoykova, P.; Nikolov, Z.L. Extraction and fractionation of microalgae-based protein products. Algal Res.-Biomass Biofuels Bioprod. 2018, 36, 175–192. [Google Scholar] [CrossRef]
  86. Ahmad, N.; Mehmood, M.A.; Malik, S. Recombinant Protein Production in Microalgae: Emerging Trends. Protein Pept. Lett. 2020, 27, 105–110. [Google Scholar] [CrossRef]
  87. de Carvalho Silvello, M.A.; Severo Gonçalves, I.; Patrícia Held Azambuja, S.; Silva Costa, S.; Garcia Pereira Silva, P.; Oliveira Santos, L.; Goldbeck, R. Microalgae-based carbohydrates: A green innovative source of bioenergy. Bioresour Technol. 2022, 344, 126304. [Google Scholar] [CrossRef]
  88. Gouda, M.; Tadda, M.A.; Zhao, Y.; Farmanullah, F.; Chu, B.; Li, X.; He, Y. Microalgae Bioactive Carbohydrates as a Novel Sustainable and Eco-Friendly Source of Prebiotics: Emerging Health Functionality and Recent Technologies for Extraction and Detection. Front. Nutr. 2022, 9, 806692. [Google Scholar] [CrossRef]
  89. Sardo, A.; Orefice, I.; Balzano, S.; Barra, L.; Romano, G. Mini-Review: Potential of Diatom-Derived Silica for Biomedical Applications. Appl. Sci. 2021, 11, 4533. [Google Scholar] [CrossRef]
  90. Ruggiero, I.; Terracciano, M.; Martucci, N.M.; De Stefano, L.; Migliaccio, N.; Tatè, R.; Rendina, I.; Arcari, P.; Lamberti, A.; Rea, I. Diatomite silica nanoparticles for drug delivery. Nanoscale Res. Lett. 2014, 9, 329. [Google Scholar] [CrossRef] [Green Version]
  91. Dalgıç, A.; Atila, D.; Karataş, A.Y.; Tezcaner, A.; Keskin, D. Diatom shell incorporated PHBV/PCL-pullulan co-electrospun scaffold for bone tissue engineering. Mater. Sci. Eng. C Mater. Biol. Appl. 2019, 100, 735–746. [Google Scholar] [CrossRef] [PubMed]
  92. Abdelhamid, M.A.A.; Pack, S.P. Biomimetic and bioinspired silicifications: Recent advances for biomaterial design and applications. Acta Biomater. 2021, 120, 38–56. [Google Scholar] [CrossRef] [PubMed]
  93. Lee, S.Y.; Khoiroh, I.; Vo, D.-V.N.; Senthil Kumar, P.; Show, P.L. Techniques of lipid extraction from microalgae for biofuel production: A review. Environ. Chem. Lett. 2021, 19, 231–251. [Google Scholar] [CrossRef]
  94. Lee, J.-Y.; Yoo, C.; Jun, S.-Y.; Ahn, C.-Y.; Oh, H.-M. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 2010, 101, S75–S77. [Google Scholar] [CrossRef]
  95. Rokicka, M.; Zieliński, M.; Dudek, M.; Dębowski, M. Effects of Ultrasonic and Microwave Pretreatment on Lipid Extraction of Microalgae and Methane Production from the Residual Extracted Biomass. BioEnergy Res. 2021, 14, 752–760. [Google Scholar] [CrossRef]
  96. Rahman, M.M.; Hosano, N.; Hosano, H. Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies. Molecules 2022, 27, 2786. [Google Scholar] [CrossRef]
  97. Michalak, I.; Dmytryk, A.; Wieczorek, P.P.; Rój, E.; Łęska, B.; Górka, B.; Messyasz, B.; Lipok, J.; Mikulewicz, M.; Wilk, R.; et al. Supercritical Algal Extracts: A Source of Biologically Active Compounds from Nature. J. Chem. 2015, 2015, 597140. [Google Scholar] [CrossRef]
  98. Molino, A.; Mehariya, S.; Di Sanzo, G.; Larocca, V.; Martino, M.; Leone, G.P.; Marino, T.; Chianese, S.; Balducchi, R.; Musmarra, D. Recent developments in supercritical fluid extraction of bioactive compounds from microalgae: Role of key parameters, technological achievements and challenges. J. CO2 Util. 2020, 36, 196–209. [Google Scholar] [CrossRef]
  99. Daintree, L.S.; Kordikowski, A.; York, P. Separation processes for organic molecules using SCF technologies. Adv. Drug Deliv. Rev. 2008, 60, 351–372. [Google Scholar] [CrossRef]
  100. Spicer, A.; Molnar, A. Gene Editing of Microalgae: Scientific Progress and Regulatory Challenges in Europe. Biology 2018, 7, 21. [Google Scholar] [CrossRef] [Green Version]
  101. EU 1829/2003/EC; E.U. Regulation (EC) No. 1829/2003 of the European Parliament and of the Council on Genetically Modified Food and Feed. Official Journal L 268; European Union: Brussels, Belgium, 18 October 2003; pp. 1–23.
  102. EU 1830/2003/EC; E.U. Regulation (EC) No. 1830/2003 of the European Parliament and of the Council Concerning the Traceability and Labelling of Genetically Modified Organisms and the Traceability of Food and FEED products Produced from Genetically Modified Organisms and Amending Directive 2001/18/EC. Official Journal L 268; European Union: Brussels, Belgium, 18 October 2003; pp. 24–28.
Jmse 10 00844 g001 550
Figure 1. Map representing the main companies that produce PUFA-rich microalgae and other microalgae-derived commodities.
Figure 1. Map representing the main companies that produce PUFA-rich microalgae and other microalgae-derived commodities.
Jmse 10 00844 g001
Table
Table 1. List of commercially available microalgae-derived commodities. The table includes names and locations of companies, species cultivated and their final products, types of cultivation systems, and links to company websites (n.f.: not found). Websites access date: 15 October 2021.
Table 1. List of commercially available microalgae-derived commodities. The table includes names and locations of companies, species cultivated and their final products, types of cultivation systems, and links to company websites (n.f.: not found). Websites access date: 15 October 2021.
CompanyCountrySpecies/CategoryProduct(s) SectorType of Plant/Growth ConditionWebsite or Reference(s)
CyanotechHawaiiArthrospira sp. (Spirulina)Hawaiian BioAstin
Hawaiian spirulina 		Dietary supplements for human consumption Open pondshttps://www.cyanotech.com/our-purpose/
Cellana, LLCHawaiiMarine microalgae,
Staurosira sp.		ReNew™ (ω-3 rich oils)Human and animal food
Whole algae enriched with EPA and DHA (as animal feed)		Open pondshttp://cellana.com/
AlltechUnited StatesSchizochytrium sp.All-G-RichTMDehydrated whole algae for poultry nutrition rich in DHA, biofuelsn.f.https://www.alltech.com/
TerraVia Holdings, Inc. (formerly Solazyme)United StatesChlorella sp.Golden Chlorella, AlgaVia (algal powder, food ingredient line recognized as GRAS), AlgaWise (food oils),
AlgaPrime DHA (aquaculture)		Dietary supplements (high-value oils and whole-algae ingredients) for human and animal (aquaculture) consumptionHeterotrophic growth in stainless-steel containershttps://www.solazyme.com/
AlgenolUnited StatesArthrospira sp. (spirulina)
(other strains available)		Whole algae or protein isolates, phycocyaninFood and food colorantsOpen pondshttps://www.algenol.com/
Omega TechUnited StatesSchizochytrium sp.DHA Gold (oil)Food supplementsn.f. [25]
Martek Biosciences CorporationUnited StatesCrypthecodinium cohniiω-3, ω-6, ARAFood, beverages, dietary supplements, and early-life nutritionn.f.https://www.dsm.com/corporate/home.html
https://www.linkedin.com/company/martek-biosciences/
GCI NutrientsUnited StatesChlorella, Arthrospira sp. (Spirulina)DHA3Sure™ DHA Algae 35% oil complex,
organic Chlorella, organic broken-cell Chlorella, 		Nutrientsn.f. https://gcinutrients.com/
AZBIOUnited StatesMarine microalgaeAlgaeBio Omega-3 Origins™EPA and DHA for food and feedAutotrophic growthhttps://www.azbio.org/tag/algae-biosciences
Arizona Algae Products, LLCUnited StatesMarine microalgaeProtein+omega3 powder, EPA extractFood, dietary supplements, and wellness productsPhotobioreactorshttps://www.azalgae.com/
Taau Australia Pty Ltd.AustraliaArthrospira sp. (spirulina)Tabs and powderHuman consumptionOpen pondshttps://www.taau.com.au/company.html
Photonz CorporationNew ZealandMarine microalgaeEPAAquaculture (fish oil replacement purposes), pharmaceuticalsFermentation processeshttps://pureadvantage.org/photonz-corporation/
Blue Biotech International GmBHGermanyNannochloropsis, HaematococcusAlgal concentrate, frozen paste, freeze-dried cells, phycocyanin, astaxanthin, microalgae powderHatchery, feedPhotobioreactorshttps://www.bluebiotech.de/com/index.html
NutrinovaGermanyUlkenia sp.DHActiveTMAquacultureFermentation processes (80 m3)[25]
Corbion N.V.NetherlandsSchizochytrium sp.AlgaPrimeTMAquaculture, pet, and livestock industriesFermentation processeshttp://www.corbion.com
VeramarisNetherlandsMarine microalgaeVeramaris® Pets (algal oil rich in EPA and DHA; GRAS product)Dog food n.f.https://pets.veramaris.com/
DuplacoNetherlandsChlorellaPowder, food ingredients, and supplementsFoodPhotobioreactors and fermentershttps://duplaco.com/
NeoalgaeSpainSpirulina, ChlorellaPowders and capsulesFoodPhotobioreactorshttps://neoalgae.es/?lang=en
AlgaEnergySpainArthrospira sp. (spirulina) Dietary supplements, feed for aquaculture, biostimulants for agricultureFood and feedOutdoor open ponds and outdoor photobioreactorshttps://www.algaenergy.it/
AlgAlimentoSpainTetraselmis sp., Dunaliella salina, Arthrospira sp.Powder for antioxidant food supplements and cosmeticsFoodOutdoor open pondshttp://www.algalimento.com/
Fitoplancton marino Spain Tetraselmis chuiiCosmetics, food supplements, feed as Easyreefs® and Easyalgae®Food, feed, and cosmeticsOpen ponds and photobioreactorshttp://www.fitoplanctonmarino.com/index.html
MicrophytFranceArthrospira sp. (spirulina), Haematococcus pluvialisPowders and pastes, dietary supplementsFood, feed, and cosmeticsPhotobioreactors (indoor and outdoor systems)http://www.microphyt.eu/
InalveFranceBiofilm-forming microalgaePowders and pastes for food and feed supplements (e.g., FEALTM)Food and feedPhotobioreactorshttps://www.inalve.com/
FermentAlgFranceSchizochytrium sp.Powder and pastes for food supplements (e.g., DHA-ORIGIN TM)FoodFermentation processhttps://www.fermentalg.com/
AlgenuityUnited KingdomChlorella vulgarisFood ingredientsFoodPhotobioreactorshttps://www.algenuity.com/
AllMicroalgaePortugalChlorella vulgaris, Tetraselmis chui, Nannochloropsis oceanica, Scenedesmus obliquusPowders and pastes Food and feedPhotobioreactors (outdoor systems)https://www.allmicroalgae.com/en/
A4F-AlgafuelPortugalDunaliella salina (pilot), Lobosphaera incisa, Prorocentrum casubicum, Raphidonema sp., Food colorants, ω-6 food additives for children, and dietary supplementsFood and feedOpen ponds and photobioreactorshttps://a4f.pt/pt
NorsanNorway, GermanySchizochytrium sp.ω-3 oilFood supplementsn.f. https://www.norsan-omega.com/
Algaria SpireatItalyArthrospira sp. (spirulina)Snack and dietary supplementsFood supplementsn.f. https://spireat.it/
Biospira SrlItalyArthrospira sp. (spirulina)Capsules, powders, flakesFood supplementsMonitored tanks that isolate the algae from the external environmenthttps://www.biospira.it/
BiosyntexItalyArthrospira sp. (spirulina), Haematococcus pluvialisFood ingredients, beverages, nutraceuticals, cosmetics, feed for aquaculture, and biostimulants for agricultureMainly food, feed, and biostimulantsIndoor and outdoor photobioreactorswww.biosyntex.com/
Micoperi Blue GrowthItalyPhaeodactylum tricornutum, Arthrospira platensis, Euglena gracilisCapsules, powdersFood supplementsPhotobioreactors and indoor pondshttp://www.micoperibg.eu/?page_id=101
TOLO Green SrlItalyArthrospira sp. (spirulina)Food and feed supplements, biostimulantsFood, feed, and agricultureOpen ponds and monitored tanks isolated from the external environmenthttps://www.tologreen.it/
AlghitalyItalyArthrospira sp. (spirulina)Powders and pastesFood and feed supplementsOutdoor photobioreactorshttps://www.alghitaly.it/
BrevelIsraeln.f.Natural salmon feedFeed Indoor photobioreactorshttps://brevel.co.il/
AlgatechIsraelNannochloropsis sp., Porphyridium cruentumFood supplementsFoodOutdoor photobioreactorshttps://www.algatech.com/
Chlorella Industry Co., Ltd.JapanChlorellaTablets, extracts for food ingredientsFood and feedOpen pondshttps://www.chlorella.co.jp/
Yaeyama SyokusanJapanChlorellaPowder, tablets, food, and feed supplementsFood and feedOpen pondshttps://www.yaeyamachlorella.com/
Japan AlgaeJapanArthrospira sp. (spirulina)Powder and tablets for food ingredients and supplementsFood Open pondshttp://www.sp100.com/
Taiwan Chlorella Manifacturing Co., Ltd.TaiwanChlorella sorokinianaTablets and food supplementsFoodOpen pondshttp://www.taiwanchlorella.com/
Far East Microalgae Industries Co., Ltd.TaiwanChlorella, Arthrospira sp. (spirulina)Powder, dietary supplements, skin care systems, aquaculture feedsFood, feed, and cosmeticsOpen pondshttp://www.femico.com.tw/
Bluetec Naturals Co., Ltd.ChinaChlorella, Arthrospira sp. (spirulina)DHA-rich powder and oilFood supplementOpen ponds monitored and isolated from the external environmenthttps://www.bestphycocyanin.com/
Tianjin Norland Biotech Co., Ltd.ChinaChlorella, Spirulina, Haematococcus pluvialisPowder, oils, and tabletsFood Open pondshttp://www.norlandbiotech.com/
Hangzhou OuQi Food co., Ltd.ChinaSpirulina, Chlorella, Dunaliella salinaOrganic Spirulina, organic Chlorella, broken Chlorella, organic DunaliellaFood ingredients and supplementsIndoor and outdoor systemshttp://www.onlygreen.cn/webEn/LM_about%20us
Shaanxi Rebecca Bio-Tech Co., LTDChinaDunaliella salina, Haematococcus pluvialisPowder, extractsNatural foodFermentation processhttp://it.rebeccabio.com/
Seagrass Tech Private Limited IndiaDunaliella salina, Chlorella salina, Arthrospira subsalsaSeaCarotene®, SeaLipro®, SeaProtein®Food n.f.https://seagrasstech.com/company-overview/
E.I.D.-Parry LimitedIndiaArthrospira sp. (spirulina)Organic Chlorella, spirulina, Phycocyanin, natural astaxanthinNutraceuticalsn.f.https://www.eidparry.com/
Table
Table 2. List of patents of microalgae that have been genetically modified to enhance their PUFA production. ACS: ADP-forming acetyl-CoA synthetase; bHLH2: basic helix–loop–helix transcription factor 2; BTA: betaine lipid synthase; CAO: Chlorophyllide a oxygenase; DES: desaturase (the number near the Greek letter Δ indicates that the double bond is created at a fixed position from the carboxyl end of a fatty acid chain); DGAT: diacylglycerol acyltransferase; DOF: DNA binding with one finger-type transcription factor; FAB: stearoyl-acyl carrier protein desaturase; GAPDH/GPDH: glyceraldehyde-3-phosphate dehydrogenase; GPAT: glycerol-3-phosphate acyltransferase; KAS: beta-ketoacyl-acyl carrier-protein synthase; NADK: NADH kinase; NF: nuclear factor; TE/FAT: thioesterase/acyl-ACP thioesterase.
Table 2. List of patents of microalgae that have been genetically modified to enhance their PUFA production. ACS: ADP-forming acetyl-CoA synthetase; bHLH2: basic helix–loop–helix transcription factor 2; BTA: betaine lipid synthase; CAO: Chlorophyllide a oxygenase; DES: desaturase (the number near the Greek letter Δ indicates that the double bond is created at a fixed position from the carboxyl end of a fatty acid chain); DGAT: diacylglycerol acyltransferase; DOF: DNA binding with one finger-type transcription factor; FAB: stearoyl-acyl carrier protein desaturase; GAPDH/GPDH: glyceraldehyde-3-phosphate dehydrogenase; GPAT: glycerol-3-phosphate acyltransferase; KAS: beta-ketoacyl-acyl carrier-protein synthase; NADK: NADH kinase; NF: nuclear factor; TE/FAT: thioesterase/acyl-ACP thioesterase.
TransformationGeneSpeciesResultsPatentSubmission Date Reference
OverexpressionKASI-KASIV-FATA-FATBChlorella or Prototheca spp.+C8-C16 FAsUS 20180230442 A1July 2014[36]
OverexpressionFAB2C. reinhardtii+FAsKR20140005001 AJuly 2012[50]
OverexpressionGPATP. tricornutum+TAGsCN105219649 AOctober 2015[37]
OverexpressionDGAT1P. tricornutum+TAGsUS2014196177 A1November 2010[38]
OverexpressionDGATN. oceanica+TAGs, +PUFAsCN110305883 AMarch 2018[40]
OverexpressionDGAT2-5C. reinhardtii+biomass, +neutral lipidsCN102321642 ASeptember 2011[39]
OverexpressionGAPDHmicroalgae+biomass, +lipidsWO2015105233 A1January 2014[51]
OverexpressionCAOC. reinhardtii+biomass, +lipidsKR101855739 B1June 2017[52]
OverexpressionbHLH2N. salina+lipidsKR20160142024 AJune 2015[41]
OverexpressionDOFC. reinhardtii+lipidsCN105755034 AMarch 2016[53]
Heterologous expression8 nucleotide sequencesfrom C. bastropiensis to bacteria and mi-croalgae+FAsJP2017127278 AJanuary 2016[54]
Heterologous expressionAcyl-ACP TEfrom E. siliculosus to bacteria and microalgae +lipidsJP2016007154 AJune 2014[43]
Heterologous expressionDGAT1from C. ellipsoidea to yeasts, plants or microalgae +FAsCN103397007 AJuly 2013[46]
Heterologous expressionGPDHfrom C. ellipsoidea to yeasts, plants or microalgae +FAsCN104357415 BOctober 2014[55]
Heterologous expressionSucrose invertasebacteria, yeasts and microalgae+lipidsEP3546588 A3June 2007[56]
Heterologous expressionΔ12-DESfrom P. viridis+LA, +EPACN104388442 ANovember 2014[57]
Heterologous expressionΔ8-DESfrom P. viridis+DHA, +EPACN104293769 AOctober 2014[58]
Heterologous expressionΔ4-DESfrom E. sphaerica to yeast P. pastorisfunction confirmedCN102559710 BJune 2011[44]
Heterologous expressionNADK3from plant A. thaliana to C. pyrenoidesa+FAsCN105316358 AOctober 2015[59]
Heterologous expressionNF-YA from C. ellipsoidea to plant A. thaliana+44.9% weight, +22.4% FAsCN107936098 ADecember 2017[47]
Heterologous expressionNF-YBfrom C. ellipsoidea to plant A. thaliana+51% weight, +11.2% FAsCN108003226 ADecember 2017[48]
Heterologous expressionNF-YC from C. ellipsoidea to plant A. thaliana+44.9% weight, +15.4% FAsCN108101973 ADecember 2017[49]
SilencingACS1-ACS2C. reinhardtii+fatty acid excretionCN105647957 AJanuary 2016[42]
SilencingBTA1Chlamydomonas sp.−glycolipids, 2.5-3X TAGsKR101893522 B1April 2017[60]
Polygene co-silencingCarbon metabolism genesC. reinhardtii+biomass, +lipidsCN110564623 ASeptember 2019[61]
Table
Table 3. Comparison between biomass and lipid productivities of the main species with a commercial value and their genetically modified clones. WT: wild type; GMO: genetically modified organisms.
Table 3. Comparison between biomass and lipid productivities of the main species with a commercial value and their genetically modified clones. WT: wild type; GMO: genetically modified organisms.
SpeciesWT BiomassWT Lipid ProductivityReferenceNumber of GMO StrainsGMO Lipid YieldsReference
Arthrospira platensis0.14 g/L/day14.37 mg/L/day[62]--
Chlamydomonas malina and Chlamydomonas reinhardtii0.53 g/L/day (C. malina); 0.014 g/L/day (C. reinhardtii)Total lipid 161.3 (C. malina); 10.9 mg/L/day (C. reinhardtii)
PUFAs 85.4 mg/L/day (C. malina)		[63,64]26 (of which 4 are patented)Total lipids ~3.2X more than C. reinhardtii WT (~118.3 μg/mL culture)[65]
Scenedesmus obliquus0.16 g/L/day26.77 mg/L/day[66]4Biomass ~17% higher than S. obliquus WT (~234.3 mg/L/day) and lipid productivity ~2.2X more than WT (~42.4 mg/L/day)[67]
Chlorella vulgaris
Chlorella pyrenoidosa		0.73 mg/L/day (C. vulgaris) and 0.34 g/L/day (C. pyrenoidosa)204.91 mg/L/day (C. vulgaris) and 66 mg/L/day (C. pyrenoidosa)[68,69]6 (of which 2 are patented)Neutral lipid content ~3.2X and total PUFAs > 34% higher than C. pyrenoidosa WT[70]
Tetraselmis sp.0.30 g/L/day43.4 mg/L/day[71]--
Dunaliella
salina		1.03  g/L/day0.24 mg/L/day[72]3Oil content ~13% higher than D. salina WT (~25%)[73]
Nannochloropsis oceanica0.427 g/L/day39.6 mg/L/day[74]11 (of which 2 are patented)Lipid production 110.6% higher than N. oceanica WT (~1.15 g/L) and TAG 148.6% higher than WT (~0.80 g/L)[75]
Phaeodactylum tricornutum 0.254 g/L/day 99.23 mg/L/day[76]35 (of which 2 are patented)TAG ~45X more than P. tricornutum WT[77]
Schizochytrium sp. S310.81 g/L/day100.74 mg/L/day[78]3Total lipid yield ~39.6% higher than Schizochytrium sp. WT (~110.5 g/L)[79]
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Santin, A.; Balzano, S.; Russo, M.T.; Palma Esposito, F.; Ferrante, M.I.; Blasio, M.; Cavalletti, E.; Sardo, A. Microalgae-Based PUFAs for Food and Feed: Current Applications, Future Possibilities, and Constraints. J. Mar. Sci. Eng. 2022, 10, 844. https://doi.org/10.3390/jmse10070844

AMA Style

Santin A, Balzano S, Russo MT, Palma Esposito F, Ferrante MI, Blasio M, Cavalletti E, Sardo A. Microalgae-Based PUFAs for Food and Feed: Current Applications, Future Possibilities, and Constraints. Journal of Marine Science and Engineering. 2022; 10(7):844. https://doi.org/10.3390/jmse10070844

Chicago/Turabian Style

Santin, Anna, Sergio Balzano, Monia Teresa Russo, Fortunato Palma Esposito, Maria Immacolata Ferrante, Martina Blasio, Elena Cavalletti, and Angela Sardo. 2022. "Microalgae-Based PUFAs for Food and Feed: Current Applications, Future Possibilities, and Constraints" Journal of Marine Science and Engineering 10, no. 7: 844. https://doi.org/10.3390/jmse10070844

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