Process intensification for the protein production from microalgae: a comprehensive review

Abstract

Microalgae (predominantly unicellular photosynthetic eukaryotes) has been recognized as a “protein bio-factory” because they may produce up to 70% of protein (dry basis). Advantageous aspects of proteins from microalgae compared to conventional animal- and plant-based proteins were proven. Numerous review articles have addressed the effects of composition of cultivation medium and operational parameters to obtain high protein yield from microalgae. Nevertheless, several critical limitations remain for the industrial expansions of the production of proteins from microalgae. Presently, process intensification is considered a strategic approach for the scalable production of proteins from microalgae, achieved by implementing mutagenic and recombinant strains, increasing productivity through high-cell-density cultivation strategies, using cold-adapted microalgae and minimizing equipment footprint via integrating multiple unit operations into a more efficient and compact system. Different strategies of random mutagenesis (chemical: ethyl methane sulfonate-mediated, physical: heavy-ion irradiation-mediated and ultraviolet-mediated) have been employed to get the desired phenotype of microalgae for higher protein yield. Emerging genetic engineering approaches, including the design of expression vectors, identification of genetic regulatory elements, and genetic transformation methods, along with bioinformatics algorithms, have been employed to develop recombinant strains of microalgae with higher protein yield. Combined cultivation systems, such as sequential heterotrophic-autotrophic system, switching of nitrogen-rich cultivation medium from nitrogen-deficient medium and mixotrophic system have come to the forefront. In this review article, process intensification strategies for the production of proteins from microalgae are comprehensively discussed, with special emphasis on the development of mutant and recombinant strains, strategies of cultivation and decisive operational parameters of bioreactor operation.

Keywords

Protein from microalgae / Mutant phenotype / Recombinant strain / Operational strategy of cultivation / Operating parameters / Process intensification

References

[1] Abiusi F, Sampietro G, Marturano G, et al.. Growth, photosynthetic efficiency, and biochemical composition of Tetraselmis suecica F&M-M33 grown with LEDs of different colors. Biotechnol Bioeng, 2014, 111: 956-964

[2] Abiusi F, Moñino Fernández P, Canziani S, et al.. Mixotrophic cultivation of Galdieria sulphuraria for C-phycocyanin and protein production. Algal Res, 2022, 61: 102603

[3] Ahmad I, Hellebust JA. Transport and assimilation of nitrogen by stichococcus bacillaris grown in the presence of methionine sulfoximine. Plant Physiol, 1985, 79: 1125-1126

[4] Ahmad I, Hellebust JA (1986) Assimilation in the soil alga stichococcus bacillaris Naeg pathways of ammonium. New Phytol 103:57–68. https://doi.org/https://doihttps://doi.org/10.1111/j.1469-8137.1986.tb00596.x

[5] Ahmad N, Mehmood MA, Malik S. Recombinant protein production in microalgae: emerging trends. Protein Pept Lett, 2020, 27: 105-110

[6] Ahmad Kamal AH, Mohd Hamidi NF, Zakaria MF, et al.. Genetically engineered microalgae for enhanced bioactive compounds. Discov Appl Sci, 2024, 6: 482

[7] Álvarez-González A, Uggetti E, Serrano L, et al.. The potential of wastewater grown microalgae for agricultural purposes: contaminants of emerging concern, heavy metals and pathogens assessment. Environ Pollut, 2023, 324: 121399

[8] Alzahmi AS, Daakour S, Nelson D, et al.. Enhancing algal production strategies: strain selection, AI-informed cultivation, and mutagenesis. Front Sustain Food Syst Volume, 2024

[9] Aparicio PJ, Quiñones MA. Blue light, a positive switch signal for nitrate and nitrite uptake by the green alga monoraphidium braunii 1. Plant Physiol, 1991, 95: 374-378

[10] Baek K, Kim DH, Jeong J, et al.. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep, 2016, 6: 30620

[11] Banerjee A, Ward V. Production of recombinant and therapeutic proteins in microalgae. Curr Opin Biotechnol, 2022, 78: 102784

[12] Barahimipour R, Neupert J, Bock R. Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker. Plant Mol Biol, 2016, 90: 403-418

[13] Barati B, Zeng K, Baeyens J, et al.. Recent progress in genetically modified microalgae for enhanced carbon dioxide sequestration. Biomass Bioenergy, 2021, 145: 105927

[14] Barboza-Rodríguez R, Rodríguez-Jasso RM, Rosero-Chasoy G, et al.. Photobioreactor configurations in cultivating microalgae biomass for biorefinery. Bioresour Technol, 2024, 394: 130208

[15] Barros A, Guerra LT, Simões M, et al.. Mass balance analysis of carbon and nitrogen in industrial scale mixotrophic microalgae cultures. Algal Res, 2017, 21: 35-41

[16] Barros A, Pereira H, Campos J, et al.. Heterotrophy as a tool to overcome the long and costly autotrophic scale-up process for large scale production of microalgae. Sci Rep, 2019, 9: 13935

[17] Bassani JC, Martins VFR, Barbosa J, et al.. Exploring the development of a clean-label vegan burger enriched with fermented microalgae. Foods, 2025, 14: 2884

[18] Becker EW. Micro-algae as a source of protein. Biotechnol Adv, 2007, 25: 207-210

[19] Bekheet IA, Syrett PJ. Urea-degrading enzymes in algae. Br Phycol J, 1977, 12: 137-143

[20] BIRDSEY EC, LYNCH VH. Utilization of nitrogen compounds by unicellular algae. Science, 1962, 137: 763-764

[21] Bleakley S, Hayes M (2017) Algal proteins: extraction, application, and challenges concerning production. Foods (Basel Switzerland). https://doi.org/10.3390/foods6050033

[22] Bleisch R, Freitag L, Ihadjadene Y, et al.. Strain development in microalgal biotechnology—random mutagenesis techniques. Life, 2022, 12: 961

[23] Borodin VB. Effect of red and blue light on acclimation of Chlamydomonas reinhardtii to CO2-limiting conditions. Russ J Plant Physiol, 2008, 55: 441-448

[24] Borowitzka MA. High-value products from microalgae—their development and commercialisation. J Appl Phycol, 2013, 25: 743-756

[25] Bromke MA. Amino acid biosynthesis pathways in diatoms. Metabolites, 2013, 3: 294-311

[26] Cao L, Wang J, Zhou T, et al.. Evaluation of ammonia recovery from swine wastewater via a innovative spraying technology. Bioresour Technol, 2019, 272: 235-240

[27] Černá M (2011) Chap. 24: Seaweed proteins and amino acids as nutraceuticals. In: Kim S-KBT-A in F and NR (ed) Marine medicinal foods. Academic Press, pp 297–312

[28] Chacón-Lee TL, González-Mariño GE. Microalgae for healthy foods—possibilities and challenges. Compr Rev Food Sci Food Saf, 2010, 9: 655-675

[29] Charrier B, Rolland E, Gupta V, Reddy CRK. Production of genetically and developmentally modified seaweeds: exploiting the potential of artificial selection techniques. Front Plant Sci, 2015, 6: 127

[30] Chen C, Tang T, Shi Q, et al.. The potential and challenge of microalgae as promising future food sources. Trends Food Sci Technol, 2022, 126: 99-112

[31] Chen H, Qi H, Xiong P (2022b) Phycobiliproteins-A Family of Algae-Derived Biliproteins: Productions, Characterization and Pharmaceutical Potentials. Mar Drugs 20:450. https://doi.org/10.3390/md20070450

[32] Chen S, Zhu H, Radican E, et al.. Engineering approaches for sustainable protein production in microalgae: a comprehensive review. Compr Rev Food Sci Food Saf, 2025, 24: e70308

[33] Chitolina L, Tatiele S, Marcele N et al (2026) Designing the future of food: challenges to innovation in derived blue protein systems. Sustain Food Proteins. https://doi.org/10.1002/sfp2.70057

[34] Correa-Porcel V, Piedra-Muñoz L, Galdeano-Gómez E (2021) Water–energy–food nexus in the agri-food sector: research trends and innovating practices. Int J Environ Res Public Health. https://doi.org/10.3390/ijerph182412966

[35] Cruz JD, Vasconcelos V (2024) Legal Aspects of Microalgae in the European Food Sector. Foods 13:124

[36] Cullimore JV, Sims AP. Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii. Phytochemistry, 1981, 20: 933-940

[37] da Fontoura Prates D, Duarte JH, Vendruscolo RG, et al.. Role of light emitting diode (LED) wavelengths on increase of protein productivity and free amino acid profile of Spirulina sp. cultures. Bioresour Technol, 2020, 306: 123184

[38] Dasan YK, Lam MK, Yusup S, et al.. Cultivation of chlorella vulgaris in sequential flow photobioreactor system: influence of recycled culture medium on growth, lipid and protein content. IOP Conf Ser Earth Environ Sci, 2021, 721: 12013

[39] Davis A, Crum LT, Corbeil LB, Hildebrand M. Expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana. Appl Microbiol Biotechnol, 2017, 101: 5313-5324

[40] de Mendonça HV, Ometto JPHB, Otenio MH, et al.. Microalgae-mediated bioremediation and valorization of cattle wastewater previously digested in a hybrid anaerobic reactor using a photobioreactor: Comparison between batch and continuous operation. Sci Total Environ, 2018, 633: 1-11

[41] de Mendonça HV, Otenio MH, Marchão L, et al.. Biofuel recovery from microalgae biomass grown in dairy wastewater treated with activated sludge: the next step in sustainable production. Sci Total Environ, 2022, 824: 153838

[42] de Vries M, de Boer IJM. Comparing environmental impacts for livestock products: a review of life cycle assessments. Livest Sci, 2010, 128: 1-11

[43] Delgado CL (2003) Rising Consumption of Meat and Milk in Developing Countries Has Created a New Food Revolution. J Nutr 133:3907S–3910S. https://doi.org/10.1093/jn/133.11.3907S

[44] Deprá MC, Dias RR, Zepka LQ, Jacob-Lopes E. Tackling old challenges in microalgal biotechnology: the role of photobioreactors to advance the technology readiness level. Processes, 2025, 13: 51

[45] Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta, 2009, 229: 873-883

[46] El-Ayouty Y, El-Manawy I, Nasih S, et al.. Engineering Chlamydomonas reinhardtii for expression of functionally active human interferon-α. Mol Biotechnol, 2019, 61: 134-144

[47] Esteves AF, Pardilhó S, Gonçalves AL, et al.. Unravelling the impact of light spectra on microalgal growth and biochemical composition using principal component analysis and artificial neural network models. Algal Res, 2025, 85: 103820

[48] Fabris M, Abbriano RM, Pernice M, et al.. Emerging technologies in algal biotechnology: toward the establishment of a sustainable, algae-based bioeconomy. Front Plant Sci, 2020, 11: 1-22

[49] Fajardo C, De Donato M, Carrasco R, et al.. Advances and challenges in genetic engineering of microalgae. Rev Aquac, 2020, 12: 365-381

[50] Fan J, Huang J, Li Y, et al.. Sequential heterotrophy–dilution–photoinduction cultivation for efficient microalgal biomass and lipid production. Bioresour Technol, 2012, 112: 206-211

[51] Fasaei F, Bitter JH, Slegers PM, van Boxtel AJB. Techno-economic evaluation of microalgae harvesting and dewatering systems. Algal Res, 2018, 31: 347-362

[52] Ferenczi A, Pyott DE, Xipnitou A, Molnar A. Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA. Proc Natl Acad Sci U S A, 2017, 114: 13567-13572

[53] Fernandez E, Galvan A. Inorganic nitrogen assimilation in Chlamydomonas. J Exp Bot, 2007, 58: 2279-2287

[54] Ferreira de Oliveira AP, Bragotto APA (2022) Microalgae-based products: Food and public health. Futur Foods. https://doi.org/10.1016/j.fufo.2022.100157

[55] Florencio FJ, Vega JM. Utilization of nitrate, nitrite and ammonium by Chlamydomonas reinhardii: photoproduction of ammonium. Planta, 1983, 158: 288-293

[56] Flores E, Herrero A. Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem Soc Trans, 2005, 33: 164-167

[57] Freitas BCB, Cassuriaga APA, Morais MG, Costa JAV. Pentoses and light intensity increase the growth and carbohydrate production and alter the protein profile of Chlorella minutissima. Bioresour Technol, 2017, 238: 248-253

[58] Gantar M, Svirčev Z. Microalgae and cyanobacteria: food for thought1. J Phycol, 2008, 44: 260-268

[59] Gao L, Qin Y, Zhou X, et al.. Microalgae as future food: Rich nutrients, safety, production costs and environmental effects. Sci Total Environ, 2024, 927: 172167

[60] García-Domínguez M, Reyes JC, Florencio FJ. Purification and characterization of a new type of glutamine synthetase from cyanobacteria. Eur J Biochem, 1997, 244: 258-264

[61] George B, Pancha I, Desai C, et al.. Effects of different media composition, light intensity and photoperiod on morphology and physiology of freshwater microalgae Ankistrodesmus falcatus – A potential strain for bio-fuel production. Bioresour Technol, 2014, 171: 367-374

[62] Goel P, Singh AK. Abiotic stresses downregulate key genes involved in nitrogen uptake and assimilation in Brassica juncea L. PLoS ONE, 2015, 10: e0143645

[63] Gonçalves CF, Menegol T, Rech R. Biochemical composition of green microalgae Pseudoneochloris marina grown under different temperature and light conditions. Biocatal Agric Biotechnol, 2019, 18: 101032

[64] Grant BR, Turner IM. Light-stimulated nitrate and nitrite assimilation in several species of algae. Comp Biochem Physiol, 1969, 29: 995-1004

[65] Grechanik VI, Tsygankov AA. The relationship between photosystem II regulation and light-dependent hydrogen production by microalgae. Biophys Rev, 2022, 14: 893-904

[66] Grossmann L, Hinrichs J, Weiss J. Cultivation and downstream processing of microalgae and cyanobacteria to generate protein-based technofunctional food ingredients. Crit Rev Food Sci Nutr, 2020, 60: 2961-2989

[67] Grützner T, Ziegenbalg D, Güttel R. Process intensification: an unbroken trend in chemical engineering. Chemie Ing Tech, 2018, 90: 1823-1831

[68] Gutierrez J, Kwan TA, Zimmerman JB, Peccia J. Ammonia inhibition in oleaginous microalgae. Algal Res, 2016, 19: 123-127

[69] Harnedy PA, FitzGerald RJ. Bioactive proteins, peptides, and amino acids from macroalgae1. J Phycol, 2011, 47: 218-232

[70] He Y, Peng H, Liu J, et al.. Chlorella sp. transgenic with Scy-hepc enhancing the survival of Sparus macrocephalus and hybrid grouper challenged with Aeromonas hydrophila. Fish Shellfish Immunol, 2018, 73: 22-29

[71] Hellebust JA, Ahmad I (2013) Regulation of nitrogen assimilation in green microalgae. Biol Oceanogr6:241–255. https://doi.org/10.1080/01965581.1988.10749529

[72] Hellebust JA, Soto C, Hutchinson TC. Effect of naphthalene and aqueous crude-oil extracts on the green flagellate Chlamydomonas angulosa. VII. Nitrate and methylamine uptake and retention. Can J Bot, 1985, 63: 834-840

[73] Hempel F, Maier UG. Microalgae as solar-powered protein factories. Adv Exp Med Biol, 2016, 896: 241-262

[74] Hernández-Ramírez J, Wong-Arce A, González-Ortega O, Rosales-Mendoza S. Expression in algae of a chimeric protein carrying several epitopes from tumor associated antigens. Int J Biol Macromol, 2020, 147: 46-52

[75] Hirschl S, Ralser C, Asam C, et al.. Expression and characterization of functional recombinant Bet v 1.0101 in the chloroplast of chlamydomonas reinhardtii. Int Arch Allergy Immunol, 2017, 173: 44-50

[76] Hu J, Wang D, Chen H, Wang Q (2023) Advances in genetic engineering in improving photosynthesis and microalgal productivity. Int J Mol Sci. https://doi.org/10.3390/ijms24031898

[77] Ibrahim Mze SA, Azmi AS, Mohd Puad NI, et al.. Microalgae cultivation: from CO2 fixation to single-cell protein production. IOP Conf Ser Earth Environ Sci, 2023, 1281: 12049

[78] Isleten Hosoglu M. Aroma characterization of five microalgae species using solid-phase microextraction and gas chromatography-mass spectrometry/olfactometry. Food Chem, 2018, 240: 1210-1218

[79] Ismail BP, Senaratne-Lenagala L, Stube A, Brackenridge A. Protein demand: review of plant and animal proteins used in alternative protein product development and production. Anim Front, 2020, 10: 53-63

[80] Janssen M, Wijffels RH, Barbosa MJ. Microalgae based production of single-cell protein. Curr Opin Biotechnol, 2022, 75: 102705

[81] Jareonsin S, Pumas C (2021) Advantages of heterotrophic microalgae as a host for phytochemicals production. Front Bioeng Biotechnol 9:628597. https://doi.org/10.3389/fbioe.2021.628597

[82] Jarquín-Cordero M, Chávez MN, Centeno-Cerdas C, et al.. Towards a biotechnological platform for the production of human pro-angiogenic growth factors in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol, 2020, 104: 725-739

[83] Jin H, Chuai W, Li K, et al.. Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production. Biotechnol Bioeng, 2021, 118: 4138-4151

[84] Juneja A, Ceballos RM, Murthy GS. Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies, 2013, 6: 4607-4638

[85] Kamalanathan M, Chaisutyakorn P, Gleadow R, Beardall J. A comparison of photoautotrophic, heterotrophic, and mixotrophic growth for biomass production by the green alga Scenedesmus sp. (Chlorophyceae). Phycologia, 2018, 57: 309-317

[86] Karabulut G, Purkiewicz A, Goksen G. Recent developments and challenges in algal protein and peptide extraction strategies, functional and technological properties, bioaccessibility, and commercial applications. Compr Rev food Sci food Saf, 2024, 23: e13372

[87] Kaur M, Bhatia S, Bagchi D, et al.. Enhancing microalgal proteins for nutraceutical and functional food applications. Futur Foods, 2025, 11: 100564

[88] Kerby NW, Rowell P, Stewart WDP. Cyanobacterial ammonium transport, ammonium assimilation, and nitrogenase regulation. New Zeal J Mar Freshw Res, 1987, 21: 447-455

[89] Khalid S, Chaudhary K, Aziz H, et al.. Trends in extracting protein from microalgae Spirulina platensis, using innovative extraction techniques: mechanisms, potentials, and limitations. Crit Rev Food Sci Nutr, 2025, 65: 4293-4309

[90] Khan MI, Shin JH, Kim JD (2018) The promising future of microalgae: Current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products. Microb Cell Fact 17:1–21 https://doi.org/10.1186/s12934-018-0879-x

[91] Kiataramgul A, Maneenin S, Purton S, et al.. An oral delivery system for controlling white spot syndrome virus infection in shrimp using transgenic microalgae. Aquaculture, 2020, 521: 735022

[92] Kopilovic B, Valente AI, Ferreira AM, et al.. Towards the sustainable extraction and purification of non-animal proteins from biomass using alternative solvents. RSC Sustain, 2023, 1: 1314-1331

[93] Koyande AK, Chew KW, Rambabu K, et al.. Microalgae: a potential alternative to health supplementation for humans. Food Sci Hum Wellness, 2019, 8: 16-24

[94] Krishna Koyande A, Tanzil V, Murraly Dharan H, et al.. Integration of osmotic shock assisted liquid biphasic system for protein extraction from microalgae Chlorella vulgaris. Biochem Eng J, 2020, 157: 107532

[95] Kruger JS, Wiatrowski M, Davis RE, et al.. Enabling production of algal biofuels by techno-economic optimization of co-product suites. Front Chem Eng Volume, 2022

[96] Kselíková V, Singh A, Bialevich V, et al.. Improving microalgae for biotechnology: from genetics to synthetic biology—moving forward but not there yet. Biotechnol Adv, 2022, 58: 107885

[97] Kudelala MK, Cochlan WP. Nitrogen and carbon uptake kinetics and the influence of irradiance for a red tide bloom off southern California. Aquat Microb Ecol, 2000, 21: 31-47

[98] Kumar A, Bera S. Revisiting nitrogen utilization in algae: a review on the process of regulation and assimilation. Bioresour Technol Rep, 2020, 12: 100584

[99] Kumar D, Singh B. Algal biorefinery: An integrated approach for sustainable biodiesel production. Biomass Bioenergy, 2019, 131: 105398

[100] Kumar G, Nguyen DD, Huy M, et al.. Effects of light intensity on biomass, carbohydrate and fatty acid compositions of three different mixed consortia from natural ecological water bodies. J Environ Manage, 2019, 230: 293-300

[101] Kumar G, Shekh A, Jakhu S et al (2020) Bioengineering of microalgae: recent advances, perspectives, and regulatory challenges for industrial application. Front Bioeng Biotechnol. https://doi.org/10.3389/fbioe.2020.00914

[102] Lee RA, Lavoie J-M. From first- to third-generation biofuels: challenges of producing a commodity from a biomass of increasing complexity. Anim Front, 2013, 3: 6-11

[103] Leftley JW, Syrett PJ. ATP: Urea amidolyase activity in anicellular algae. J Gen Microbiol, 1973, 77: 109-115

[104] Lehmuskero A, Skogen Chauton M, Boström T. Light and photosynthetic microalgae: a review of cellular- and molecular-scale optical processes. Prog Oceanogr, 2018, 168: 43-56

[105] León-Bañares R, González-Ballester D, Galván A, Fernández E. Transgenic microalgae as green cell-factories. Trends Biotechnol, 2004, 22: 45-52

[106] Levine JM, Adler PB, Yelenik SG. A meta-analysis of biotic resistance to exotic plant invasions. Ecol Lett, 2004, 7: 975-989

[107] Li X, Li W, Zhai J, et al.. Effect of ammonium nitrogen on microalgal growth, biochemical composition and photosynthetic performance in mixotrophic cultivation. Bioresour Technol, 2019, 273: 368-376

[108] Li X, Manuel J, Slavens S, et al.. Interactive effects of light quality and culturing temperature on algal cell size, biomass doubling time, protein content, and carbohydrate content. Appl Microbiol Biotechnol, 2021, 105: 587-597

[109] Liang L, Wang Z, Ding Y, et al.. Protein reserves elucidate the growth of microalgae under nitrogen deficiency. Algal Res, 2023, 75: 103269

[110] Lima S, Webb CL, Deery E et al (2018) Human Intrinsic factor expression for bioavailable vitamin B(12) enrichment in microalgae. Biology (Basel). https://doi.org/10.3390/biology7010019

[111] Lingg N, Zhang P, Song Z, Bardor M. The sweet tooth of biopharmaceuticals: importance of recombinant protein glycosylation analysis. Biotechnol J, 2012, 7: 1462-1472

[112] Liu N, Li F, Ge F, et al.. Mechanisms of ammonium assimilation by Chlorella vulgaris F1068: Isotope fractionation and proteomic approaches. Bioresour Technol, 2015, 190: 307-314

[113] Lonnie M, Hooker E, Brunstrom JM et al (2018) Protein for life: review of optimal protein intake, sustainable dietary sources and the effect on appetite in ageing adults. Nutrients. https://doi.org/10.3390/nu10030360

[114] Lucakova S, Branyikova I, Hayes M (2022) Microalgal proteins and bioactives for food, feed, and other applications. Appl Sci 12:4402

[115] Ma R, Wang B, Chua ET et al (2020) Comprehensive utilization of marine microalgae for enhanced co-production of multiple compounds. Mar Drugs 18:467

[116] Maeda Y, Tsuru Y, Matsumoto N, et al.. Prostaglandin production by the microalga with heterologous expression of cyclooxygenase. Biotechnol Bioeng, 2021, 118: 2734-2743

[117] Malla A, Rosales-Mendoza S, Phoolcharoen W, Vimolmangkang S. Efficient transient expression of recombinant proteins using DNA viral vectors in freshwater microalgal species. Front Plant Sci, 2021, 12: 650820

[118] Mandal S, Shurin JB, Efroymson RA, Mathews TJ. Functional divergence in nitrogen uptake rates explains diversity–productivity relationship in microalgal communities. Ecosphere, 2018, 9: e02228

[119] Markou G, Vandamme D, Muylaert K. Microalgal and cyanobacterial cultivation: the supply of nutrients. Water Res, 2014, 65: 186-202

[120] Markou G, Wang L, Ye J, Unc A. Using agro-industrial wastes for the cultivation of microalgae and duckweeds: contamination risks and biomass safety concerns. Biotechnol Adv, 2018, 36: 1238-1254

[121] Márquez-Escobar VA, Bañuelos-Hernández B, Rosales-Mendoza S. Expression of a Zika virus antigen in microalgae: towards mucosal vaccine development. J Biotechnol, 2018, 282: 86-91

[122] Matos ÂP (2019) Chap. 3 - Microalgae as a potential source of proteins. In: Galanakis Processing and applications CMBT-PSS (ed). Academic Press, pp 63–96

[123] Matos ÂP, Cavanholi MG, Moecke EHS, Sant’Anna ES. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour Technol, 2017, 224: 490-497

[124] Matos ÂP, Novelli E, Tribuzi G. Use of algae as food ingredient: sensory acceptance and commercial products. Front Food Sci Technol, 2022, 2: 1-8

[125] Milrad Y, Mosebach L, Buchert F (2024) Regulation of microalgal photosynthetic electron transfer. Plants 13:2103

[126] Molin WT, Cunningham TP, Bascomb NF, et al.. Light requirement for induction and continuous accumulation of an ammonium-inducible NADP-specific glutamate dehydrogenase in chlorella 1. Plant Physiol, 1981, 67: 1250-1254

[127] Mosibo OK, Ferrentino G, Udenigwe CC (2024) Microalgae proteins as sustainable ingredients in novel foods: recent developments and challenges. Foods 13: 733

[128] Mouritsen OG, Duelund L, Petersen MA, et al.. Umami taste, free amino acid composition, and volatile compounds of brown seaweeds. J Appl Phycol, 2019, 31: 1213-1232

[129] Mussgnug JH, Thomas-Hall S, Rupprecht J, et al.. Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion. Plant Biotechnol J, 2007, 5: 802-814

[130] Nahdliah NL, Joudi L, Yousefi S, et al.. Microalgae production and utilization as an alternative protein source in poultry nutrition: a comprehensive review. J Appl Poult Res, 2026, 35: 100696

[131] Nedaei L, Shokrkar H. Influence of process factors and photobioreactor design on microalgae cultivation. Biofuels Bioprod Biorefining, 2025, 19: 2433-2456

[132] NEILSON AH, LARSSON T. The utilization of organic nitrogen for growth of algae: physiological aspects. Physiol Plant, 1980, 48: 542-553

[133] Nitsos C, Filali R, Taidi B, Lemaire J. Current and novel approaches to downstream processing of microalgae: a review. Biotechnol Adv, 2020, 45: 107650

[134] Norsker N-H, Barbosa MJ, Vermuë MH, Wijffels RH. Microalgal production: a close look at the economics. Biotechnol Adv, 2011, 29: 24-27

[135] Norziah MH, Ching CY. Nutritional composition of edible seaweed Gracilaria changgi. Food Chem, 2000, 68: 69-76

[136] Nova P, Martins AP, Teixeira C, et al.. Foods with microalgae and seaweeds fostering consumers health: a review on scientific and market innovations. J Appl Phycol, 2020, 32: 1789-1802

[137] Novoveská L, Nielsen SL, Eroldoğan OT, et al.. Overview and challenges of large-scale cultivation of photosynthetic microalgae and cyanobacteria. Mar Drugs, 2023, 21: 445

[138] Nunes E, Odenthal K, Nunes N, et al.. Protein extracts from microalgae and cyanobacteria biomass. Techno-functional properties and bioactivity: a review. Algal Res, 2024, 82: 103638

[139] Ogbonna JC, Masui H, Tanaka H. Sequential heterotrophic/autotrophic cultivation: an efficient method of producing Chlorella biomass for health food and animal feed. J Appl Phycol, 1997, 9: 359-366

[140] Olsen MFL, Pedersen JS, Thomsen ST, et al.. Outdoor cultivation of a novel isolate of the microalgae Scenedesmus sp. and the evaluation of its potential as a novel protein crop. Physiol Plant, 2021, 173: 483-494

[141] Onn SM, Koh GJ, Yap WH, et al.. Recent advances in genetic engineering of microalgae: bioengineering strategies, regulatory challenges and future perspectives. J Appl Phycol, 2025, 37: 247-264

[142] Onwezen MC, Bouwman EP, Reinders MJ, Dagevos H. A systematic review on consumer acceptance of alternative proteins: Pulses, algae, insects, plant-based meat alternatives, and cultured meat. Appetite, 2021, 159: 105058

[143] Ortega-Berlanga B, Bañuelos-Hernández B, Rosales-Mendoza S. Efficient expression of an Alzheimer’s disease vaccine candidate in the microalga Schizochytrium sp. using the algevir system. Mol Biotechnol, 2018, 60: 362-368

[144] Pan-utai W, Iamtham S (2023) Chap. 24: Techno-functional properties of microalgae in food products. In: Jacob-Lopes E, Queiroz MI, Maroneze MM, Zepka LQBT-H of F and F from M (eds). Academic Press, pp 293–304

[145] Peers GS, Milligan AJ, Harrison PJ (2000) Assay optimization and regulation of urease activity in two marine diatoms. J Phycol 36:523–528. https://doi.org/10.1046/j.1529-8817.2000.99037.x

[146] Prasad W, Wani AD, Khamrui K, et al.. Green solvents, potential alternatives for petroleum based products in food processing industries. Clean Chem Eng, 2022, 3: 100052

[147] Prates DdaF, Radmann EM, Duarte JH, et al.. Spirulina cultivated under different light emitting diodes: enhanced cell growth and phycocyanin production. Bioresour Technol, 2018, 256: 38-43

[148] Prunkard DE, Bascomb NF, Molin WT, Schmidt RR. Effect of different carbon sources on the ammonium induction of different forms of NADP-specific glutamate dehydrogenase in chlorella sorokiniana cells cultured in the light and dark. Plant Physiol, 1986, 81: 413-422

[149] Qian X, Kim MK, Kumaraswamy GK, et al.. Flux balance analysis of photoautotrophic metabolism: uncovering new biological details of subsystems involved in cyanobacterial photosynthesis. Biochim Biophys Acta - Bioenerg, 2017, 1858: 276-287

[150] Qin S, Lin H, Jiang P. Advances in genetic engineering of marine algae. Biotechnol Adv, 2012, 30: 1602-1613

[151] Raja K, Kadirvel V, Subramaniyan T. Seaweeds, an aquatic plant-based protein for sustainable nutrition: a review. Futur Foods, 2022, 5: 100142

[152] Rajauria G, Yuan YV (2021) Algae: a functional food with a rich history and future superfood. In: recent advances in micro and macroalgal processing. pp 1–13

[153] Ramazanov A, Ramazanov Z. Isolation and characterization of a starchless mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high protein and polyunsaturated fatty acid content. Phycol Res, 2006, 54: 255-259

[154] Rasala BA, Mayfield SP. Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res, 2015, 123: 227-239

[155] Redekop P, Sanz-Luque E, Yuan Y, et al.. Transcriptional regulation of photoprotection in dark-to-light transition-More than just a matter of excess light energy. Sci Adv, 2022, 8: eabn1832

[156] Rivera-Sánchez E, Villaró-Cos S, Salinas-García M, Lafarga T. Increasing the sustainability of photoautotrophic microalgae production by minimising freshwater requirements. N Biotechnol, 2025, 86: 14-24

[157] Safi C, Frances C, Ursu AV, et al.. Understanding the effect of cell disruption methods on the diffusion of Chlorella vulgaris proteins and pigments in the aqueous phase. Algal Res, 2015, 8: 61-68

[158] Sarkar S, Purkait MK, Gayen K, Bhowmick TK (2026) Chap. 47: Process intensification applied to microalgae based processes and products. In: Jacob-Lopes E, Maroneze MM, Queiroz MI, Zepka LQBT-H of M-BP and P (Second E (eds). Academic Press, pp 843–872

[159] Schüler L, de Morais E, Trovão M, et al.. Isolation and characterization of novel chlorella vulgaris mutants with low chlorophyll and improved protein contents for food applications. Front Bioeng Biotechnol, 2020

[160] Schulze PSC, Hulatt CJ, Morales-Sánchez D, et al.. Fatty acids and proteins from marine cold adapted microalgae for biotechnology. Algal Res, 2019, 42: 101604

[161] Scott SA, Davey MP, Dennis JS, et al.. Biodiesel from algae: challenges and prospects. Curr Opin Biotechnol, 2010, 21: 277-286

[162] Seidler CA, Zeindl R, Fernández-Quintero ML, et al.. Allergenicity and conformational diversity of allergens. Allergies, 2024, 4: 1-16

[163] Severo IA, de Lira GS, Ambati RR, et al.. Disruptive potential of microalgae proteins: shaping the future of the food industry. Futur Foods, 2024, 9: 100318

[164] Seyfabadi J, Ramezanpour Z, Amini Khoeyi Z. Protein, fatty acid, and pigment content of Chlorella vulgaris under different light regimes. J Appl Phycol, 2011, 23: 721-726

[165] Shamriz S, Ofoghi H. Expression of recombinant PfCelTOS antigen in the chloroplast of chlamydomonas reinhardtii and its potential use in detection of malaria. Mol Biotechnol, 2019, 61: 102-110

[166] Shi C, Liu X, Han K et al (2021) A database and comprehensive analysis of the algae genomes. https://doi.org/10.1101/2021.10.30.466624. bioRxiv 2021.10.30.466624

[167] Show PL, Chew KW, Khoo KS, Tawai A (2022) Effective solvents for proteins recovery from microalgae. 02009:1–4

[168] Singh RN, Sharma S. Development of suitable photobioreactor for algae production: a review. Renew Sustain Energy Rev, 2012, 16: 2347-2353

[169] Singh SP, Singh P. Effect of CO2 concentration on algal growth: a review. Renew Sustain Energy Rev, 2014, 38: 172-179

[170] Singh SP, Singh P. Effect of temperature and light on the growth of algae species: a review. Renew Sustain Energy Rev, 2015, 50: 431-444

[171] Singh Chauhan D, Sahoo L, Mohanty K. Acclimation-driven microalgal cultivation improved temperature and light stress tolerance, CO2 sequestration and metabolite regulation for bioenergy production. Bioresour Technol, 2023, 385: 129386

[172] Slegers PM, Olivieri G, Breitmayer E et al (2020) Design of value chains for microalgal biorefinery at industrial scale: process integration and techno-economic analysis. Front Bioeng Biotechnol. https://doi.org/10.3389/fbioe.2020.550758

[173] Soeder CJ, Pabst W. Gesichtspunkte für die Verwendung von Mikroalgen in der Ernährung von Mensch und Tier. Ber Dtsch Bot Ges, 1970, 83: 607-625

[174] Solomon CM, Collier JL, Berg GM, Glibert PM. Role of urea in microbial metabolism in aquatic systems: a biochemical and molecular review. Aquat Microb Ecol, 2010, 59: 67-88

[175] Song X, Wang J, Wang Y, et al.. Artificial creation of Chlorella pyrenoidosa mutants for economic sustainable food production. Bioresour Technol, 2018, 268: 340-345

[176] Soto-Sierra L, Stoykova P, Nikolov ZL. Extraction and fractionation of microalgae-based protein products. Algal Res, 2018, 36: 175-192

[177] Spicer A, Molnar A. Gene editing of microalgae: scientific progress and regulatory challenges in Europe. Biology (Basel), 2018, 7: 21

[178] Sproles AE, Fields FJ, Smalley TN, et al.. Recent advancements in the genetic engineering of microalgae. Algal Res, 2021, 53: 102158

[179] Stavridou E, Karapetsi L, Nteve GM, et al.. Landscape of microalgae omics and metabolic engineering research for strain improvement: an overview. Aquaculture, 2024, 587: 740803

[180] Stilgoe J, Owen R, Macnaghten P. Developing a framework for responsible innovation. Res Policy, 2013, 42: 1568-1580

[181] Stoffels L, Taunt HN, Charalambous B, Purton S. Synthesis of bacteriophage lytic proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J, 2017, 15: 1130-1140

[182] Sui Y, Harvey PJ (2021) Effect of light intensity and wavelength on biomass growth and protein and amino acid composition of dunaliella salina. Foods. https://doi.org/10.3390/foods10051018

[183] Sui Y, Muys M, Vermeir P, et al.. Light regime and growth phase affect the microalgal production of protein quantity and quality with Dunaliella salina. Bioresour Technol, 2019, 275: 145-152

[184] Suttle CA, Fuhrman JA, Capone DG. Rapid ammonium cycling and concentration-dependent partitioning of ammonium and phosphate: implications for carbon transfer in planktonic communities. Limnol Oceanogr, 1990, 35: 424-433

[185] Syrett P, Leftley J. Nitrate and urea assimilation by algae. Perspect Expr Biol, 2016, 2: 221-234

[186] Szyjka SJ, Mandal S, Schoepp NG, et al.. Evaluation of phenotype stability and ecological risk of a genetically engineered alga in open pond production. Algal Res, 2017, 24: 378-386

[187] Tenzin G, Schipper K, Rathore H, et al.. Analytical framework to navigate microalgae-based product development—aligning commercialization and regulatory pathways. Mar Drugs, 2026, 24: 66

[188] Teoh M-L, Chu W-L, Marchant H, Phang S-M. Influence of culture temperature on the growth, biochemical composition and fatty acid profiles of six Antarctic microalgae. J Appl Phycol, 2004, 16: 421-430

[189] Terrado R, Monier A, Edgar R, Lovejoy C. Diversity of nitrogen assimilation pathways among microbial photosynthetic eukaryotes. J Phycol, 2015, 51: 490-506

[190] Tevatia R, Payne S, Allen J, et al.. A synthetic cdo/csad taurine pathway in the green unicellular alga Chlamydomonas reinhardtii. Algal Res, 2019, 40: 101491

[191] Thacker ANN, Syrett PJ (1972) Disappearance of nitrate reductase activity from chlamydomonas reinhardi. New Phytol 71:435–441. https://doi.org/https://doi.https://doi.org/10.1111/j.1469-8137.1972.tb01943.x

[192] Thompson PA, Guo M, Harrison PJ (1992) Effects of Variation in temperature. I. on the biochemical composition of eight species of marine phytoplankton1. J Phycol 28:481–488. https://doi.org/10.1111/j.0022-3646.1992.00481.x

[193] Tibocha-Bonilla JD, Zuñiga C, Godoy-Silva RD, Zengler K. Advances in metabolic modeling of oleaginous microalgae. Biotechnol Biofuels, 2018, 11: 241

[194] Tischner R. Evidence for the participation of NADP-Glutamate dehydrogenase in the ammonium assimulation of Chlorella sorokiniana. Plant Sci Lett, 1984, 34: 73-80

[195] Tischner R, Hüttermann A. Regulation of glutamine synthetase by light and during nitrogen deficiency in synchronous chlorella sorokiniana. Plant Physiol, 1980, 66: 805-808

[196] Torres-Tiji Y, Fields FJ, Mayfield SP. Microalgae as a future food source. Biotechnol Adv, 2020, 41: 107536

[197] Trovão M, Schüler LM, Machado A et al (2022) Random mutagenesis as a promising tool for microalgal strain improvement towards industrial production. Mar Drugs. https://doi.org/10.3390/md20070440

[198] Uma VS, Usmani Z, Sharma M, et al.. Valorisation of algal biomass to value-added metabolites: emerging trends and opportunities. Phytochem Rev, 2022

[199] Van KMM, van Der PB et al (2013) Cultivation, Processing and nutritional aspects for pigs and poultry of european protein sources as alternatives for imported soybean products. Lelystad Wageningen UR Livest Res

[200] Van De Walle S, Broucke K, Baune M-C, et al.. Microalgae protein digestibility: How to crack open the black box?. Crit Rev Food Sci Nutr, 2024, 64: 7149-7171

[201] van der Spiegel M, Noordam MY, van der Fels-Klerx HJ. Safety of novel protein sources (insects, microalgae, seaweed, duckweed, and rapeseed) and legislative aspects for their application in food and feed production. Compr Rev food Sci food Saf, 2013, 12: 662-678

[202] Veaudor T, Cassier-chauvat C, Chauvat F (2019) Genomics of urea transport and catabolism in cyanobacteria: biotechnological implications. 10:1–14. https://doi.org/10.3389/fmicb.2019.02052

[203] Vendruscolo RG, Fagundes MB, Maroneze MM, et al.. Scenedesmus obliquus metabolomics: effect of photoperiods and cell growth phases. Bioprocess Biosyst Eng, 2019, 42: 727-739

[204] Venkataraman LV, Becker WE, Shamala TR. Studies on the cultivation and utilization of the alga Scenedesmus acutus as a single cell protein. Life Sci, 1977, 20: 223-233

[205] Wang J, Yang H, Wang F. Mixotrophic cultivation of microalgae for biodiesel production: status and prospects. Appl Biochem Biotechnol, 2014, 172: 3307-3329

[206] Wang S-K, Stiles AR, Guo C, Liu C-Z. Microalgae cultivation in photobioreactors: an overview of light characteristics. Eng Life Sci, 2014, 14: 550-559

[207] Wang K, Cui Y, Wang Y, et al.. Chloroplast genetic engineering of a unicellular green alga haematococcus pluvialis with expression of an antimicrobial peptide. Mar Biotechnol (NY), 2020, 22: 572-580

[208] Wang Y, Tibbetts SM, McGinn PJ (2021) Microalgae as sources of high-quality protein for human food and protein supplements. Foods (Basel Switzerland). https://doi.org/10.3390/foods10123002

[209] Wannathong T, Waterhouse JC, Young REB, et al.. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol, 2016, 100: 5467-5477

[210] Weger HG, Birch DG, Elrifi IR, Turpin DH. Ammonium assimilation requires mitochondrial respiration in the light 1: a study with the green alga selenastrum minutum. Plant Physiol, 1988, 86: 688-692

[211] Wesseler J, Kleter G, Meulenbroek M, Purnhagen KP. EU regulation of genetically modified microorganisms in light of new policy developments: possible implications for EU bioeconomy investments. Appl Econ Perspect Policy, 2023, 45: 839-859

[212] Wheeler PA (1980) Use of methylammonium as an ammonium analogue in nitrogen transport and assimilation studies with cyclotella cryptica (Bacillariophyceae). J Phycol 16:328–334. https://doi.org/10.1111/j.1529-8817.1980.tb03041.x

[213] Woodley JM. Bioprocess intensification for the effective production of chemical products. Comput Chem Eng, 2017, 105: 297-307

[214] Wu G. Dietary protein intake and human health. Food Funct, 2016, 7: 1251-1265

[215] Wu M, Zhang H, Sun W, et al.. Metabolic plasticity of the starchless mutant of Chlorella sorokiniana and mechanisms underlying its enhanced lipid production revealed by comparative metabolomics analysis. Algal Res, 2019, 42: 101587

[216] Wu C, Zheng C, Wang J, Jiang P (2021) Recombinant expression of thrombolytic agent reteplase in marine microalga tetraselmis subcordiformis (Chlorodendrales, Chlorophyta). Mar Drugs 19. https://doi.org/10.3390/md19060315

[217] Wu-Scharf D, Jeong B, Zhang C, Cerutti H. Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Sci (80-), 2000, 290: 1159-1162

[218] Wulandari I, Putri RAE, Mudrikah S, et al.. Comparison of two different visible light electromagnetic spectrum to the metabolites production of Euglena anabaena. Korean J Microbiol, 2025, 61: 126-140

[219] Xiao X, Zhou Y, Liang Z, et al.. A novel two-stage heterotrophic cultivation for starch-to-protein switch to efficiently enhance protein content of Chlorella sp. MBFJNU-17. Bioresour Technol, 2022, 344: 126187

[220] Xie T, Xia Y, Zeng Y, et al.. Nitrate concentration-shift cultivation to enhance protein content of heterotrophic microalga Chlorella vulgaris: Over-compensation strategy. Bioresour Technol, 2017, 233: 247-255

[221] Xu F, Pan J. Potassium channel KCN11 is required for maintaining cellular osmolarity during nitrogen starvation to control proper cell physiology and TAG accumulation in Chlamydomonas reinhardtii. Biotechnol Biofuels, 2020, 13: 129

[222] Xu L, Weathers PJ, Xiong XR, Liu CZ. Microalgal bioreactors: challenges and opportunities. Eng Life Sci, 2009, 9: 178-189

[223] Xu K, Zou X, Wen H, et al.. Effects of multi-temperature regimes on cultivation of microalgae in municipal wastewater to simultaneously remove nutrients and produce biomass. Appl Microbiol Biotechnol, 2019, 103: 8255-8265

[224] Xu Q, Hou G, Chen J, et al.. Heterotrophically ultrahigh-cell-density cultivation of a high protein-yielding unicellular alga chlorella with a novel nitrogen-supply strategy. Front Bioeng Biotechnol, 2021, 9: 1-9

[225] Xue B, Dong C-M, Hu H-H, et al.. Chlamydomonas reinhardtii-expressed multimer of ToAMP4 inhibits the growth of bacteria of both Gram-positive and Gram-negative. Process Biochem, 2020, 91: 311-318

[226] Yan N, Fan C, Chen Y, Hu Z (2016) The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci. https://doi.org/10.3390/ijms17060962

[227] Yang C, Hua Q, Shimizu K. Energetics and carbon metabolism during growth of microalgal cells under photoautotrophic, mixotrophic and cyclic light-autotrophic/dark-heterotrophic conditions. Biochem Eng J, 2000, 6: 87-102

[228] Young EB, Reed L, Berges JA. Growth parameters and responses of green algae across a gradient of phototrophic, mixotrophic and heterotrophic conditions. PeerJ, 2022, 10: 1-27

[229] Zabochnicka M, Krzywonos M, Romanowska-Duda Z, et al.. Algal biomass utilization toward circular economy. Life, 2022, 12: 1480

[230] Zhang Y, Li X, Li Y, et al.. Regulation of different light conditions for efficient biomass production and protein accumulation of Spirulina platensis. J Oceanol Limnol, 2024, 42: 174-186

[231] Zhang J, Zhu Y, Hawkins TR, et al.. Saline microalgae cultivation for the coproduction of biofuel and protein in the United States: an integrated assessment of costs, carbon, water, and land impacts. Sustain Energy Fuels, 2025, 9: 1859-1870

[232] Zhao X-C, Tan X-B, Yang L-B, et al.. Cultivation of Chlorella pyrenoidosa in anaerobic wastewater: the coupled effects of ammonium, temperature and pH conditions on lipids compositions. Bioresour Technol, 2019, 284: 90-97

[233] Zheng J, Geng J, Li J, et al.. Harnessing microalgae for protein production: advances, functional properties, and industrial potential. Futur Foods, 2025, 12: 100776

[234] Zhubi-Bakija F, Bajraktari G, Bytyçi I, et al.. The impact of type of dietary protein, animal versus vegetable, in modifying cardiometabolic risk factors: a position paper from the International Lipid Expert Panel (ILEP). Clin Nutr, 2021, 40: 255-276

Funding

Hungarian University of Agriculture and Life Sciences

Rights & permissions

The Author(s)