Native proteins from Galdieria sulphuraria to replace fetal bovine serum in mammalian cell culture (original) (raw)

Abstract

The use of fetal bovine serum (FBS) in cell culture applications causes high costs and unacceptable animal suffering when FBS is extracted from fetal calves. Despite efforts, the exact composition of FBS still remains partially unresolved. Native proteins in FBS, such as growth factors, and their binding to cell receptors seem to be crucial for cell proliferation and differentiation. Recently, algal extracts with high protein content were considered to reduce the FBS demand. Algae extracts yielded promising results as growth serum in mammalian cell culture. Nevertheless, the dependence on residual FBS and the undefined composition of algae extracts are challenges. In this study, we aimed to yield highly concentrated extracts of native proteins from mixotrophically grown Galdieria sulphuraria to replace FBS in mammalian cell culture. Crude extracts and native proteins were concentrated by ammonium sulfate precipitation, and all extracts underwent heat inactivation (HI) for selective protein inactivation. The remaining proteins’ native conformation was verified by enzyme activity assays. All extracts were used to replace FBS during the cultivation of Chinese hamster ovary (CHO) cells, and proliferation was tested. We found that G. sulphuraria crude and protein extracts depended on HI to promote CHO cell growth to a similar extent as FBS. CHO cells grown with 5% or 10% heat-treated algal extracts had a relative proliferation of 260 to 230% compared to FBS controls with 210% and 300%, respectively. We anticipate our findings will help replace FBS in mammalian cell culture, increasing sustainability and consumer acceptance.

Key points

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Introduction

Cell and tissue culture is becoming increasingly important for basic research, as well as biotechnological and medical applications. Mammalian cell and tissue cultures are crucial for the evaluation of toxic substances and can reduce and replace the necessity of animal testing (Zhu et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR52 "Zhu X, Trehan R, Xie C (2024) Primary liver cancer organoids and their application to research and therapy. J Natl Cancer Cent 4:195–202. https://doi.org/10.1016/j.jncc.2024.06.002

            ")). Chinese hamster ovary (CHO) cells represent the most common expression platform for the large-scale production of therapeutic proteins, such as monoclonal antibodies (Dhara et al. [2018](/article/10.1007/s00253-025-13507-0#ref-CR12 "Dhara VG, Naik HM, Majewska NI, Betenbaugh MJ (2018) Recombinant antibody production in CHO and NS0 cells: differences and similarities. BioDrugs 32:571–584. 
              https://doi.org/10.1007/s40259-018-0319-9
              
            ")). Recently, cell culture has also established itself as an effective solution for culture-based fish and meat products entering the market (Eibl et al. [2021](/article/10.1007/s00253-025-13507-0#ref-CR16 "Eibl R, Senn Y, Gubser G, Jossen V, van den Bos C, Eibl D (2021) Cellular agriculture: opportunities and challenges. Annu Rev Food Sci Technol 12:51–73. 
              https://doi.org/10.1146/annurev-food-063020-123940
              
            "); Quek et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR37 "Quek JP, Gaffoor AA, Tan YX, Tan TRM, Chua YF, Leong DSZ, Ali AS, Ng SK (2024) Exploring cost reduction strategies for serum free media development. npj Sci Food 8:1–10. 
              https://doi.org/10.1038/s41538-024-00352-0
              
            ")).

A limitation for in vitro cultivation of animal cells is their need for growth factors. The most common supplement is fetal bovine serum (FBS), which is rich in native proteins. For example, growth factors and hormones bind to cell receptors according to the key-lock principle and help the cell to remain within the cell cycle or prevent apoptosis (Wang [2021](/article/10.1007/s00253-025-13507-0#ref-CR50 "Wang Z (2021) Regulation of cell cycle progression by growth factor-induced cell signaling. Cells 10:3327. https://doi.org/10.3390/cells10123327

            ")). FBS is usually added as a sterile filtered extract so that proteins and other components do not denature and remain in their natural state (Francis [2010](/article/10.1007/s00253-025-13507-0#ref-CR17 "Francis GL (2010) Albumin and mammalian cell culture: implications for biotechnology applications. Cytotechnology 62:1–16. 
              https://doi.org/10.1007/s10616-010-9263-3
              
            ")).

There are substantial ethical and scientific concerns about the use of FBS in cell culture. The product is generated under precarious conditions, and the quality of the serum fluctuates between batches (van der Valk et al. [2004](/article/10.1007/s00253-025-13507-0#ref-CR48 "van der Valk J, Mellor D, Brands R, Fischer R, Gruber F, Gstraunthaler G, Hellebrekers L, Hyllner J, Jonker FH, Prieto P, Thalen M, Baumans V (2004) The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicol Vitr 18:1–12. https://doi.org/10.1016/j.tiv.2003.08.009

            ")). Furthermore, there are safety concerns due to the risk of contamination with viruses or _Mycoplasma_ species, respectively (Ozturk and Hu [2005](/article/10.1007/s00253-025-13507-0#ref-CR35 "Ozturk S, Hu WS (2005) Cell culture technology for pharmaceutical and cell-based therapies (1st edition). CRC Press, Boca Raton, FL, USA. 
              https://doi.org/10.1201/9780849351068
              
            ")). Finally, the price of FBS has doubled within the last 5 years, ranging from 1000 to 2000$ L−1 depending on the source and quality (ThermoFisher Waltham, MA, USA, and Gibco, Grand Island, NY, USA). Thus, a chemically defined serum-free medium would allow for a stable and reproducibly high productivity for the large-scale production of biopharmaceuticals or other cell-based therapeutics. In the search for alternatives, different chemically synthesized and bio-derived products were identified as possible replacements for FBS, but these are not standard serum-free media, and each cell type requires its own composition of the medium (van der Valk et al. [2018](/article/10.1007/s00253-025-13507-0#ref-CR47 "van der Valk J, Bieback K, Buta C, Cochrane B, Dirks WG, Fu J, Hickman JJ, Hohensee C, Kolar R, Liebsch M, Pistollato F, Schulz M, Thieme D, Weber T, Wiest J, Winkler S, Gstraunthaler G (2018) Fetal bovine serum (FBS): past – present – future. ALTEX 99–118. 
              https://doi.org/10.14573/altex.1705101
              
            "); Subbiahanadar Chelladurai et al. [2021](/article/10.1007/s00253-025-13507-0#ref-CR43 "SubbiahanadarChelladurai K, SelvanChristyraj JD, Rajagopalan K, Yesudhason BV, Venkatachalam S, Mohan M, ChellathuraiVasantha N, SelvanChristyraj JRS (2021) Alternative to FBS in animal cell culture - an overview and future perspective. Heliyon 7:e07686. 
              https://doi.org/10.1016/j.heliyon.2021.e07686
              
            "); Quek et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR37 "Quek JP, Gaffoor AA, Tan YX, Tan TRM, Chua YF, Leong DSZ, Ali AS, Ng SK (2024) Exploring cost reduction strategies for serum free media development. npj Sci Food 8:1–10. 
              https://doi.org/10.1038/s41538-024-00352-0
              
            ")).

The composition of commercially available serum-free media is often not disclosed, prompting scientists to seek accessible alternatives (Gstraunthaler and Lindl [2017](/article/10.1007/s00253-025-13507-0#ref-CR23 "Gstraunthaler G, Lindl T (2017) Auf der Suche nach brauchbaren Serumalternativen. Biospektrum 23:724–725. https://doi.org/10.1007/s12268-017-0843-z

            ")). Human thrombocyte lysates, heat-inactivated coelomic fluid from earthworms, and sericin from raw silk have contributed to replacing FBS (Terada et al. [2002](/article/10.1007/s00253-025-13507-0#ref-CR44 "Terada S, Nishimura T, Sasaki M, Yamada H, Miki M (2002) Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma. Cytotechnology 40:3–12. 
              https://doi.org/10.1023/A:1023993400608
              
            "); Cao and Zhang [2015](/article/10.1007/s00253-025-13507-0#ref-CR8 "Cao TT, Zhang YQ (2015) Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute. Appl Microbiol Biotechnol 99:7219–7228. 
              https://doi.org/10.1007/s00253-015-6576-3
              
            ")). So far, plant-based extracts have not matched FBS’s effectiveness in promoting cell growth (Pazos et al. [2004](/article/10.1007/s00253-025-13507-0#ref-CR36 "Pazos P, Boveri M, Gennari A, Casado J, Fernandez F, Prieto P (2004) Culturing cells without serum: lessons learnt using molecules of plant origin. Altex 21:67–72")). However, research into the use of microalgae extracts in cell culture media appears to be worthwhile. Microalgae can grow in seawater and on wastewater nutrients, with higher productivity per unit area (Zakaria and Kamal [2016](/article/10.1007/s00253-025-13507-0#ref-CR51 "Zakaria SM, Kamal SMM (2016) Subcritical water extraction of bioactive compounds from plants and algae: applications in pharmaceutical and food ingredients. Food Eng Rev 8:23–34. 
              https://doi.org/10.1007/s12393-015-9119-x
              
            ")). They are used for biofuels, cosmetics, and food supplements (Occhipinti et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR33 "Occhipinti PS, Russo N, Foti P, Zingale IM, Pino A, Romeo FV, Randazzo CL, Caggia C (2024) Current challenges of microalgae applications: exploiting the potential of non-conventional microalgae species. J Sci Food Agric 104:3823–3833. 
              https://doi.org/10.1002/jsfa.13136
              
            ")). In the latter, their ability to produce large quantities of digestible protein may render them a major source of protein to feed a growing population. The use of algal extracts in cell culture has been conceived for more than a decade (Song et al. [2012](/article/10.1007/s00253-025-13507-0#ref-CR42 "Song SH, Kim IH, Nam TJ (2012) Effect of a hot water extract of Chlorella vulgaris on proliferation of IEC-6 cells. Int J Mol Med 29:741–746. 
              https://doi.org/10.3892/ijmm.2012.899
              
            "); Okamoto et al. [2020](/article/10.1007/s00253-025-13507-0#ref-CR34 "Okamoto Y, Haraguchi Y, Sawamura N, Asahi T, Shimizu T (2020) Mammalian cell cultivation using nutrients extracted from microalgae. Biotechnol Prog 36:1–9. 
              https://doi.org/10.1002/btpr.2941
              
            "); Amorim et al. [2021](/article/10.1007/s00253-025-13507-0#ref-CR3 "Amorim ML, Soares J, Coimbra JS dos R, Leite M de O, Albino LFT, Martins MA, Bhat ZF, Bhat H, Pathak V, Canelli G, Abiusi F, Vidal Garcia A, Canziani S, Mathys A, Subbiahanadar Chelladurai K, Selvan Christyraj JRSJD, Rajagopalan K, Yesudhason BV, Venkatachalam S, Mohan M, Chellathurai Vasantha N, Selvan Christyraj JRSJD, Duque P, Gómez E, Díaz E, Facal N, Hidalgo C, Díez C, Ng JY, Chua ML, Zhang C, Hong S, Kumar Y, Gokhale R, Ee PLR, Geada P, Moreira C, Silva M, Nunes R, Madureira L, Rocha CMR, Pereira RN, Vicente AA, Teixeira JA, Hirooka S, Itabashi T, Ichinose TM, Onuma R, Fujiwara T, Yamashita S, Jong LW, Tomita R, Iwane AH, Miyagishima SY, Song SH, Kim IH, Nam TJ, Mosser M, Chevalot I, Olmos E, Blanchard F, Kapel R, Oriol E, Marc I, Marc A, Haraguchi Y, Shimizu T, Okamoto Y, Shimizu T, Yamanaka K, Haraguchi Y, Takahashi H, Kawashima I, Shimizu T, Soto-Sierra L, Stoykova P, Nikolov ZL, Vingiani GM, De Luca P, Ianora A, Dobson ADW, Lauritano C, Waghmare AG, Salve MK, LeBlanc JG, Arya SS (2021) A circular cell culture system using microalgae and mammalian myoblasts for the production of sustainable cultured meat. Arch Microbiol 36:1–11. 
              https://doi.org/10.1007/s00203-022-03234-9
              
            "); Defendi-Cho and Gould [2023](/article/10.1007/s00253-025-13507-0#ref-CR11 "Defendi-Cho G, Gould TM (2023) In vitro culture of bovine fibroblasts using select serum-free media supplemented with Chlorella vulgaris extract. BMC Biotechnol 23:1–11. 
              https://doi.org/10.1186/s12896-023-00774-w
              
            "); Dong et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR14 "Dong N, Xue C, Yang Y, Chang Y, Wang Y, Guo H, Liu Y, Wang Y (2023) Auxenochlorella pyrenoidosa extract supplementation replacing fetal bovine serum for Carassius auratus muscle cell culture under low-serum conditions. Food Res Int 164:112438. 
              https://doi.org/10.1016/j.foodres.2022.112438
              
            "); [2024](/article/10.1007/s00253-025-13507-0#ref-CR13 "Dong N, Jiang B, Chang Y, Wang Y, Xue C (2024) Integrated omics approach: revealing the mechanism of Auxenochlorella pyrenoidosa protein extract replacing fetal bovine serum for fish muscle cell culture. J Agric Food Chem 72:6064–6076. 
              https://doi.org/10.1021/acs.jafc.4c00624
              
            "); Sibinčić et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR40 "Sibinčić N, Krstić Ristivojević M, Gligorijević N, Veličković L, Ćulafić K, Jovanović Z, Ivanov A, Tubić L, Vialleix C, Michel T, Srdić Rajić T, Nikolić M, Stojadinović M, Minić S (2024) Screening algal and cyanobacterial extracts to identify potential substitutes for fetal bovine serum in cellular meat cultivation. Foods 13 
              https://doi.org/10.3390/foods13233741
              
            ")). Hot water extracts of _Chlorella vulgaris_ (CVE) increased IGF-1 receptor expression and phosphorylation of focal adhesion kinase and Src in rat intestinal epithelial cells (Song et al. [2012](/article/10.1007/s00253-025-13507-0#ref-CR42 "Song SH, Kim IH, Nam TJ (2012) Effect of a hot water extract of Chlorella vulgaris on proliferation of IEC-6 cells. Int J Mol Med 29:741–746. 
              https://doi.org/10.3892/ijmm.2012.899
              
            ")). CVE also increased MAPK and PI3 K/Akt pathway when tested with 10% FBS. Later, FBS reduction to 2% in cell culture medium was achieved using _C. vulgaris_ hot water extracts, referred to as _Chlorella_ growth factor (CGF) (Ng et al. [2020](/article/10.1007/s00253-025-13507-0#ref-CR32 "Ng JY, Chua ML, Zhang C, Hong S, Kumar Y, Gokhale R, Ee PLR (2020) Chlorella vulgaris extract as a serum replacement that enhances mammalian cell growth and protein expression. Front Bioeng Biotechnol 8:1–11. 
              https://doi.org/10.3389/fbioe.2020.564667
              
            ")). The major CGF components were proteins and carbohydrates, with the protein fraction being highest at 67%. A hot water extract from _Auxenochlorella pyrenoidosa_ (APE) revealed to be effective in fish cell cultures and promoted cell proliferation in low-serum culture medium; a complete FBS replacement was not possible (Dong et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR14 "Dong N, Xue C, Yang Y, Chang Y, Wang Y, Guo H, Liu Y, Wang Y (2023) Auxenochlorella pyrenoidosa extract supplementation replacing fetal bovine serum for Carassius auratus muscle cell culture under low-serum conditions. Food Res Int 164:112438. 
              https://doi.org/10.1016/j.foodres.2022.112438
              
            "); [2024](/article/10.1007/s00253-025-13507-0#ref-CR13 "Dong N, Jiang B, Chang Y, Wang Y, Xue C (2024) Integrated omics approach: revealing the mechanism of Auxenochlorella pyrenoidosa protein extract replacing fetal bovine serum for fish muscle cell culture. J Agric Food Chem 72:6064–6076. 
              https://doi.org/10.1021/acs.jafc.4c00624
              
            ")). In a similar approach, _Chlorococcum littorale_ compounds soluble in water (CW) showed to be an effective FBS replacement in three different mammalian cell lines (Ghosh et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR18 "Ghosh J, Akiyama Y, Haraguchi Y, Yamanaka K, Asahi T, Nakao Y, Shimizu T (2024) Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support. Biotechnol Prog 40:1–11. 
              https://doi.org/10.1002/btpr.3402
              
            ")). Additionally, extracts obtained from the cyanobacterium _Anabaena_ PCC 7120 were used as a media supplement to cultivate mouse and quail muscle cells (Ghosh et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR19 "Ghosh J, Haraguchi Y, Asahi T, Nakao Y, Shimizu T (2023) Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 for sustainable cultured meat production. Biochem Biophys Res Commun 682:316–324. 
              https://doi.org/10.1016/j.bbrc.2023.10.018
              
            ")). In a screening study, water extracts of _Arthrospira platensis_ and _Dunaliella tertiolecta_ successfully replaced up to 90% of FBS in cell culture medium (Sibincˇic et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR40 "Sibinčić N, Krstić Ristivojević M, Gligorijević N, Veličković L, Ćulafić K, Jovanović Z, Ivanov A, Tubić L, Vialleix C, Michel T, Srdić Rajić T, Nikolić M, Stojadinović M, Minić S (2024) Screening algal and cyanobacterial extracts to identify potential substitutes for fetal bovine serum in cellular meat cultivation. Foods 13 
              https://doi.org/10.3390/foods13233741
              
            ")). Published results indicate that the water-soluble fraction of microalgae promotes growth in mammalian and fish cell cultures. However, the composition of these algae-derived replacements is largely unidentified. To develop a defined serum substitute, it is crucial to identify the active algae fractions and analyze individual molecules and their targets in mammalian cells.

In this study, we set out to produce standardized, highly concentrated native protein extracts from microalgae to reliably replace FBS in cultured mammalian cells. We selected Galdieria sulphuraria, a protein-rich, thermo-acidophilic red alga, for high biomass production. This well-characterized species can produce highly concentrated protein extracts and grow on various organic carbon sources in a (photo)bioreactor (Schmidt et al. [2005](/article/10.1007/s00253-025-13507-0#ref-CR39 "Schmidt RA, Wiebe MG, Eriksen NT (2005) Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria. Biotechnol Bioeng 90:77–84. https://doi.org/10.1002/bit.20417

            "); Sloth et al. [2017](/article/10.1007/s00253-025-13507-0#ref-CR41 "Sloth JK, Jensen HC, Pleissner D, Eriksen NT (2017) Growth and phycocyanin synthesis in the heterotrophic microalga Galdieria sulphuraria on substrates made of food waste from restaurants and bakeries. Bioresour Technol 238:296–305. 
              https://doi.org/10.1016/j.biortech.2017.04.043
              
            "); Abiusi et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR1 "Abiusi F, MoñinoFernández P, Canziani S, Janssen M, Wijffels RH, Barbosa M (2022) Mixotrophic cultivation of Galdieria sulphuraria for C-phycocyanin and protein production. Algal Res 61:102603. 
              https://doi.org/10.1016/j.algal.2021.102603
              
            "); Lang et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR28 "Lang I, Bashir S, Lorenz M, Rader S, Weber G (2022) Exploiting the potential of Cyanidiales as a valuable resource for biotechnological applications. Appl Phycol 3:199–210. 
              https://doi.org/10.1080/26388081.2020.1765702
              
            ")). Optimal growth conditions for _G. sulphuraria_ are a pH of 1–2.5 and temperatures between 35 and 45 °C (Gross and Schnarrenberger [1995](/article/10.1007/s00253-025-13507-0#ref-CR22 "Gross W, Schnarrenberger C (1995) Heterotrophic growth of two strains of the acido-thermophilic red alga Galdieria sulphuraria. Plant Cell Physiol 36:633–638. 
              https://doi.org/10.1093/oxfordjournals.pcp.a078803
              
            ")). This reduces contamination risk and cooling costs in closed photobioreactors (PBRs). Additionally, _G. sulphuraria_ is scalable for industrial production and is being evaluated as a novel food ingredient (Fermentalg, Libourne, France; Galdieria Blue Extract, 26.03.2025, 10:27 a.m.). Here, we set a mixotrophic cultivation of _G. sulphuraria_ (strain 074G) to gain a high biomass yield. We recovered native protein fractions and validated the stability of native proteins by enzyme activity assessment of typical cytosolic and chloroplast enzymes. Then, raw algae extracts, as well as native and heat-inactivated protein fractions, were tested as possible substitutions for FBS in mammalian cell culture.

Material and methods

Strain and culture conditions

The strain G. sulphuraria 074G was kindly provided by Christine Oesterhelt (also available as CCCryo 120–00, Fraunhofer Institute for Cell Therapy and Immunology, Postdam-Golm, Germany). For all experiments, the culture medium was M Allen (Allen [1959](/article/10.1007/s00253-025-13507-0#ref-CR2 "Allen MB (1959) Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch Mikrobiol 32:270–277. https://doi.org/10.1007/BF00409348

            ")) with pH 2.5 and supplemented with 0.28 M glucose and 37.84 mM additional ammonium sulfate for mixotrophic growth. Pre-cultures were grown in 50 mL medium in a 100-mL sterile Erlenmeyer flask. The cultures grew mixotrophically at an average photon flux density (PFD) of 77 µmol photons m−2 s−1 provided by fluorescent bulbs with a 12:12 h light/dark cycle on a shaker (200 rpm) in a 40 °C incubator. After 1 week, the pre-cultures were transferred into a 1-L bottle with 900 mL medium. Cultures grew at 358 µmol photons m−2 s−1 with a 12:12 h light/dark cycle and at 200 rpm (9-cm stir bar) at room temperature (RT). Once the cultures reached an optical density (OD) of around 6 at 750 nm, inoculation of a 6-L flat-panel airlift PBR (LS6 Subitec GmbH, Köngen, Germany) followed. The experiments were conducted under 24 h or 12 h illumination with a 320-W light-emitting diode (LED) at an average PFD of 150 µmol photon m−2 s−1 from one side. Mixing in the PBR was achieved by a continuous airflow of 150 NL h−1. The culture temperature was 38 °C. For inoculation, medium was pumped through a membrane filter into the reactor. Then, the pre-culture was pumped into the reactor to reach a starting culture density of approximately 0.5 g L−1. The experiments were conducted in batch mode and lasted for 5 days.

Determination of cell growth

Growth of Galdieria cultures was monitored by measures of OD at 750 nm and biomass dry weight (DW) in g L−1. For DW, a respective volume of culture suspension was filtered through pre-dried glass-fiber round filters, Whatman™, 45 mm diameter (Cytiva, Global Life Sciences Solutions USA LLC, Marlborough, MA, USA) and then washed twice with distilled water. The filter was placed in an aluminum tin and dried at 50 °C for 3 days before the weight was taken. The values of OD and DW were used for a calibration between the two measures. The conversion factor was 1 OD mL−1 = 0.468 g dry biomass L−1 for G. sulphuraria 074G under the given mixotrophic conditions.

Optimization of cell disruption

The biomass was harvested by centrifugation at 4302 g for 15 min at 4 °C. The supernatant was discarded, and the cell pellet was washed twice with distilled water. The cell disruption was carried out by using a bead mill (Mini-BeadBeater 96, Biospec products, INC., Bartlesville, USA) for 30 s at 2100 rpm, with 2 min of cooling at − 20 °C in between. To achieve cell lysis and optimal recovery of the soluble protein, the properties of the wet biomass (frozen, thawed) and the size and quantity of the glass beads (GB) were investigated. For the first approach, 3 g of thawed biomass was resuspended in 30 mL of 50 mM phosphate buffer saline (PBS) + 500 mM NaCl pH 7.0 (PBS + NaCl). GB of 0.25–0.5 mm or 0.75–1 mm were added to the sample at a ratio of 1:10 (w/w). To the frozen wet biomass, an equal weight of GB was added, and only after cell disruption, the biomass was resuspended in 10 mL PBS + NaCl buffer. In the second approach, thawed biomass was used to study GB ratios of 1:10 (w/w) and 1:5 (w/w) as well as three consecutive runs of cell disruption with a GB ratio of 1:3.3 (w/w) in 10 mL buffer. After two cycles of bead milling, the Falcons for the consecutive preparation were centrifuged at 4302 g for 10 min at 4 °C, the supernatant was collected, and the cell pellet was resuspended again in 10 mL PBS + NaCl buffer. This was repeated two more times, for a total of three cycles.

Protein extraction and quantification

The harvest of biomass was conducted as described for the cell disruption optimization experiments. After washing the pellet, 3 g of fresh biomass was resuspended in 30 mL of PBS + NaCl. With the addition of 10 g of GB (0.25–0.5 mm), cells were disrupted in six consecutive runs of 30-s bead milling (Mini-BeadBeater 96, Biospec products, INC., Bartlesville, USA) at 2100 rpm, with 2-min cooling in between. After every second run, three times in total, the biomass was centrifuged at 4302 × g for 10 min at 4 °C. Supernatant was collected and stored on ice, and the pellet was resuspended in 10 mL PBS + NaCl. After centrifugation of the collected supernatant at 11,000 × g for 2 h at 4 °C, aliquots of the supernatant (crude extract) were further ultra-centrifuged at 61,973 × g for 20 min at 4 °C (Sigma 3-30 KS, Sigma Laborzentrifuge GmbH, Osterode am Harz, Germany). Native proteins were precipitated by the addition of ammonium sulfate (100% saturated solution) up to a saturation of 60% at 4 °C and incubation at 4 °C overnight. The proteins were recovered by centrifugation (11,000 × g, 2 h, and 4 °C), resuspension in 50 mM PBS buffer pH 7, and subsequent desalination using a PD-10 desalting column/Sephadex G-25 resin desalting column (Cytiva, Global Life Sciences Solutions USA LLC, Marlborough, MA, USA). If the extracts were heat-inactivated, they were incubated at 70 °C for 20 min, and the supernatant was collected after centrifugation (13,000 × g, 20 min, and 4 °C). The protein content of the crude extract and protein eluants were assayed following Bradford and Lowry (Lowry et al. 1951; Bradford [1976](/article/10.1007/s00253-025-13507-0#ref-CR6 "Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3

            ")). Total protein after culture harvest was followed by the protocol of de Marsac and Houmard ([1988](/article/10.1007/s00253-025-13507-0#ref-CR10 "de Marsac NT, Houmard J (1988) Complementary chromatic adaptation: physiological conditions and action spectra. Methods Enzymol 167:318–328. 
              https://doi.org/10.1016/0076-6879(88)67037-6
              
            ")) with the subsequent Lowry assay protocol for protein quantification.

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed to obtain a rough insight into the protein content of the algae extracts (Laemmli [1970](/article/10.1007/s00253-025-13507-0#ref-CR27 "Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0

            ")). All samples were mixed with 4:1 (v/v) 4 × SDS sample buffer (Roti-Load 1, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and incubated at 95 °C for 5 min. Insoluble protein was first resuspended in an equal volume of deionized water corresponding to the total volume of the respective crude extract. Either 20 µL of undiluted samples were loaded or a protein amount of 10, 30, or 50 µg, and protein marker (PageRuler Prestained Protein Ladder, Thermo Scientific, Darmstadt, Germany) to a 10% acrylamide gel. Protein separation was performed in a running buffer containing TRIS base (0.25 M), glycine (1.92 M), and SDS (1% (w/v)) (Rotiphorese 10 × SDS-PAGE, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for approx. 50 min at 25 mA per gel. The gel was subjected to the fixing solution (40% v/v methanol, 10% acetic acid) and stained (0.2% (w/v) with Serva blue G-250 in 90% methanol mixed 1:1 to acetic acid) and subsequently destained (methanol/acetic acid/H2O (2:1:7)).

Enzyme activity assays

To determine the specific activities of enzymes, 10 or 20 µL of the extracts were combined with pyruvate kinase (PK) and phosphoenolpyruvate carboxylase (PEPC) reaction mixtures in wells of microtiter plates at room temperature. The reaction mixture for the PEPC activity contained a total volume of 300 µL 10 mM MgCl, 30 mM NaHCO3, 5 mM phosphoenolpyruvic acid (PEP), 0.5 U mL−1 magnesium hydroxide (MDH), 0.2 mM NADH, and 100 mM HEPES/NaOH buffer, pH 7.4. The increase of absorbance at 340 nm was monitored over 15 min. The reaction mixture for the PK activity contained a total volume of 300 µL 10 mM MgCl2, 50 mM KCl, 1 mM ADP, 5 mM PEP, 2 U mL−1 lactate dehydrogenase (LDH), 0.2 mM NADH, 50 mM Tris/HCl buffer, and pH 8.0. The activities obtained were normalized to the protein content per well to calculate the specific activities.

Mammalian cell culture and 3-[4,5-dimethylthiazol-2-yl]−2,5 diphenyl tetrazolium bromide (MTT) assay

CHO cells (CHO-K1| 603480, Cell lines Service GmbH, Eppelheim, Germany) were cultivated in 75-cm2 flask until sub-confluency of approximately 80% in proliferation medium (Th. Geyer GmbH and Co. KG, Höxter, Germany, BioWest Ham’s F-12) containing 10% FBS (Sigma-Aldrich, Taufkirchen, Germany, Lot. BCCC4867, F0804) and 1% penicillin–streptomycin (Sigma-Aldrich, Taufkirchen, Germany). They were incubated at 37 °C with 5% CO2 and renewed every second day. Subcultivation was achieved by removing the medium and using 2.5% (w/v) trypsin (Sigma-Aldrich, Taufkirchen, Germany) in PBS (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37 °C. Trypsinization was stopped by the addition of 10 mL of proliferation medium and centrifugation at 800 g for 7 min. After resuspension in fresh proliferation medium, 10,000 viable cells were seeded per well of a 96-well plate for the proliferation assay. Ten percent FBS and 5% FBS were used as positive controls. For assessing changes in proliferation, 5 µL of Rotitest Vital (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) was applied to each well using a multichannel pipette. Cells were then incubated for 90 min, and the absorption was measured at 450 nm using Gen5 Microplate Reader and Imager Software by BioTek and Epoch Microplate Reader (Biotek Instruments, Winooski, VT, USA). For the remaining plates, the medium was changed every 24 h during the experiment.

Results

Protein recovery from G. sulphuraria biomass

To establish biomass for protein extraction, G. sulphuraria 074G was cultured mixotrophically in batch mode in a PBR. Batches that were grown until the stationary phase yielded 74.3 g dry biomass (data not shown). This biomass showed a large insoluble protein fraction after cell disruption (Fig. 1A). Biomass harvested during the late exponential or linear phase had a larger fraction of soluble protein (Fig. 1A). Thus, subsequent cultivations were harvested at approximately 10 g L−1. Continuous illumination for batches 2 and 3 resulted in higher final biomass (9.5 g L−1 and 9 g L−1) compared to batch 1’s 12-h photoperiod (4.5 g L−1).

Fig. 1

Fig. 1

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Optimization of cell disruption and protein extraction from wet biomass of G. sulphuraria. A SDS page showing soluble (s) and insoluble (is) protein fractions in the CE (crude extract) of biomass harvested in exponential (E) and stationary (S) phases. B Growth of three batches of G. sulphuraria in a 6 L flat-panel PBR under 12 h (batch 1) and 24 h (batches 2 and 3) illumination. Cells were grown in the presence of 0.28 M glucose and 37.84 mM ammonium sulfate. Growth is expressed as DW (g L−1) over the course of 5 days. C Cell disruption optimization via bead mill, varying GB size between 0.25 and 0.5 mm and 0.75 and 1 mm on either frozen biomass or thawed biomass. Protein yields relative to DW indicated cell disruption. The results shown represent one test run; a repetition showed comparable results. The second experiment represents the cell disruption using 0.25–0.5 mm GB in different amounts, 15 and 30 g, and three consecutive extraction cycles of bead milling (BM). D Table summarizing the microscopically observed percentage of lysed cells for the different cell disruption conditions

G. sulphuraria cells differ in size at different life cycle stages (Hirooka et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR24 "Hirooka S, Itabashi T, Ichinose TM, Onuma R, Fujiwara T, Yamashita S, Jong LW, Tomita R, Iwane AH, Miyagishima SY (2022) Life cycle and functional genomics of the unicellular red alga Galdieria for elucidating algal and plant evolution and industrial use. Proc Natl Acad Sci U S A 119:1–12. https://doi.org/10.1073/pnas.2210665119

            ")), affecting cell disruption. Using frozen and thawed biomass, we tested various GB sizes for their ability to produce high amounts of soluble protein (Fig. [1](/article/10.1007/s00253-025-13507-0#Fig1)C). Beads of 0.25–0.5 mm produced higher protein yields, 17% with frozen and 30% with thawed, compared to 0.75-to 1-mm beads. Cell lysis was estimated microscopically, with the best results for 0.25- to 0.5-mm beads (90% for frozen, 85% for thawed, Fig. [1](/article/10.1007/s00253-025-13507-0#Fig1)D). Further tests with thawed biomass using different GB amounts and a cascade extraction (CAE) of three consecutive cycles showed the best results with a 1:10 (w/w) ratio and 50% protein yield (Fig. [1](/article/10.1007/s00253-025-13507-0#Fig1)C, Table [1](/article/10.1007/s00253-025-13507-0#Tab1)). Thus, for all subsequent experiments, we used this method to extract native proteins from _G. sulphuraria_. The protein yield of the three _G. sulphuraria_ culture batches ranged from 0.37 to 0.39 g g−1 DW (Table [1](/article/10.1007/s00253-025-13507-0#Tab1)). The quantity of soluble protein in the crude extract (CE) was 2.2 mg mL−1 (batch 1), 5.9 mg mL−1 (batch 2), and 4.2 mg mL−1 (batch 3) with partly huge deviations between extractions (Table [1](/article/10.1007/s00253-025-13507-0#Tab1)).

Table 1 Amount of soluble protein determined for the respective Galdieria extracts after each step of processing. CE, crude extract; HIE, heat-inactivated crude extract; ASE, protein concentrated by ammonium sulfate precipitation; HIASE, heat-inactivated ASE. Total protein: values are given as mean of 16 technical replicates. Soluble protein: values represent the mean of 3–8 technical replicates, depending on the amount of biomass available

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Since FBS is commonly heat-treated (55–60 °C for 10–30 min) to inactivate complement proteins of the innate immune system (ThermoFisher [2025](/article/10.1007/s00253-025-13507-0#ref-CR45 "ThermoFisher S (2025) Heat-inactivated FBS (HI FBS). https://www.thermofisher.com/de/de/home/life-science/cell-culture/mammalian-cell-culture/fbs/heat-inactivated-fetal-bovine-serum.html

            . Accessed 26 Mar 2025")), we did the same for all algae samples. A heat inactivation (HI) resulted in less soluble protein being present in the samples (Table [1](/article/10.1007/s00253-025-13507-0#Tab1)). For a concentrated native protein fraction, an ammonium sulfate precipitation was conducted to separate proteins from other cellular components. The precipitated protein fraction (ASE) was resuspended, desalted, and the soluble protein concentration determined. ASE had slightly higher concentrations compared to CE, with 4.9 mg mL−1 (batch 1), 6.7 mg mL−1 (batch 2), and 5.2 mg mL−1 (batch 3). Heat-treated samples (HIASE) had lower protein contents of 2.4 mg mL−1 (batch 1), 5.8 mg mL−1 (batch 2), and 3 mg mL−1 (batch 3).

Enzyme activity as a measure of native protein

FBS is a complex biological product, and most of the known molecules active in cell culture are native proteins and peptides. Consequently, we decided to extract native protein from Galdieria and confirm the nativity of extracts by determining the specific activity of two marker enzymes, PK and PEPC. At least two PK isozymes exist for plants and microalgae: a cytosolic and a chloroplast one, both catalyzing the dephosphorylation of PEP to pyruvate (Unterlander et al. [2017](/article/10.1007/s00253-025-13507-0#ref-CR46 "Unterlander N, Champagne P, Plaxton WC (2017) Lyophilization pretreatment facilitates extraction of soluble proteins and active enzymes from the oil-accumulating microalga Chlorella vulgaris. Algal Res 25:439–444. https://doi.org/10.1016/j.algal.2017.06.010

            ")). PEPC resides in the cytosol and is involved in carbon fixation and regulation by catalyzing the carboxylation of pyruvate with bicarbonate (HCO3−) to oxaloacetate and inorganic phosphorus (e.g., Wang et al. 2017). PK activity was relatively similar across samples within each batch, despite heat treatment, indicating the native nature of the protein samples (Fig. [2](/article/10.1007/s00253-025-13507-0#Fig2)). ASE protein extracts generally had lower enzymatic activity. In batch 1, PK activity ranged from 0.036 units µg−1 protein in CE to 0.033 units µg−1 protein in HIASE. For batch 2, PK activity ranged from 0.047 units µg−1 protein in CE to 0.042 units µg−1 protein in HIASE. Batch 3 had the lowest PK activity, from 0.02 units µg−1 protein in CE to 0.03 units µg−1 protein in HIASE. The different crude and protein extracts did not show a loss of PEPC activity within each batch. In all three batches, PEPC activity in CE was above 0.045 units µg−1 protein, in HIE above 0.5 units µg−1 protein, and in ASE and HIASE around 0.3 and 0.4 units µg−1 protein, respectively (Fig. [1](/article/10.1007/s00253-025-13507-0#Fig1)D).

Fig. 2

Fig. 2

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Specific enzyme activity of pyruvate kinase (PK) and phosphoenolpyruvate carboxylase (PEPC). Activity was measured for three distinct batches (batch 1, batch 2, batch 3) of crude extracts (CE), heat-inactivated extracts (HIE), ammonium sulfate extract (ASE), and heat-inactivated ammonium sulfate extracts (HIASE). Bars represent the enzyme activity (units µg.−1 protein), with PK groups shown in grey and PEPC groups in light grey. Data present a mean ± standard deviation with n = 18

Assessing the effects of algal proteins on CHO cell growth

The viability of CHO cells was tested using _Galdieria_-derived protein fractions as a FBS replacement over 24, 48, and 72 h. CHO cells were directly inoculated into the serum-free medium without acclimation, and their proliferation rate was monitored via MTT assay and confirmed by cell counts and microscopy (supplemental data). Cells were supplemented with 5% and 10% (v/v) protein samples (CE, HIE, ASE, HIASE) from batches 1–3, and the results were compared to control cultures containing 5% and 10% FBS. CHO cells grew successfully in serum-free medium supplemented with Galdieria extracts from culture batch 1 (Fig. 3). Heat inactivation of extracts (HIE-1, HIASE-1) supported viable proliferation comparable to control cultures with 5% and 10% FBS. In contrast, native protein extracts CE-1 and ASE-1 did not induce cell growth. While 5% HIE-1 promoted CHO growth more than 10% HIE-1, the opposite was seen with 5% and 10% HIASE-1. The addition of 5% FBS to 5% CE-1 and 5% ASE-1 resulted in a twofold increase in the CHO proliferation rate. Adding 5% FBS to 5% HIE slightly reduced growth, while adding 5% FBS to 5% HIASE increased cell growth. The amount of protein in the respective samples was 1.5- to twofold higher in CE-1 and ASE-1 compared to HIE-1 and HIASE-1 (Table 2), and the addition of 5% FBS to 5% samples increased the amount of protein in the CHO treatments by about tenfold.

Fig. 3

Fig. 3

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Proliferative effect of native protein extracts from batch 1 of G. sulphuraria 074G on CHO cell proliferation. Cells were treated with 10% FBS, 5% FBS, 0% FBS, CE, HIE, ASE, HIASE, and 5% extract + 5% FBS from batch 1. Relative proliferation was calculated as a percentage by setting absorbance values at 24 h, 48 h, and 72 h in relation to the initial value. Results are shown as relative cell proliferation in % ± standard deviation with n = 4

Table 2 Amount of soluble protein (g L−1) in the respective Galdieria extracts (CE-1, HIE-1, ASE-1, and HIASE-1) and soluble protein (µg) in the proliferation medium for CHO cells to determine the viability by MTT assay. For the addition of 10% and 5% of extracts, 10 µL and 5 µL were mixed into a total reaction volume of 100 µL

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The data were generated from one extraction of biomass from batch 1 and should be replicated in the following. We generated native protein extracts from three Galdieria batches, producing four independent extracts from each batch. CHO cells successfully grew in serum-free medium with Galdieria extracts, but on average at rates five- to twofold lower than control cultures (Fig. 4A, B). Extracts from the three batches varied significantly, with batch 1 being the most consistent. Heat treatment of protein extracts was confirmed to be necessary for proliferation, e.g., 10% HIE and HIASE of batch 1 significantly increased proliferation, while 10% CE and ASE had no effect (Fig. 4A). The effect of HI was more evident at 5%, where HIASE better promoted growth than HIE (Fig. 4B). The considerable variation in the proliferation rate between samples might be explained by differences in the composition of the protein extracts and the amount of protein. Galdieria extracts had 5–10 times lower protein content than FBS, with 248 µg (10% approach) and 130 µg (5% approach, Table 3). The higher proliferation rate with 10% HIE and HIASE (batch 1) compared to 5% suggests protein concentration may account for the reduced rate versus the FBS control.

Fig. 4

Fig. 4

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Proliferative effect of native protein extracts from G. sulphuraria 074G on cell proliferation of CHO cells. The cells were treated with 10% FBS, 5% FBS, and 0% FBS, as well as 10% (A) and 5% (B) of the algae extracts in the form of crude extracts (CE), heat-inactivated extracts (HIE) ammonium sulfate extracts (ASE), and heat-inactivated ammonium sulfate extracts (HIASE) from three different batches (1, 2, 3). The relative proliferation was calculated as a percentage by setting the measured absorbance values at the respective time points (24 h, 48 h, and 72 h) in relation to the initial value determined before the start of treatment. Results are shown as relative cell proliferation in % ± standard deviation with n = 18

Table 3 Amount of soluble protein (µg) in the proliferation medium for CHO cells to determine the viability by MTT assay. For the addition of 10% and 5% of extracts, 10 µL and 5 µL were mixed into a total reaction volume of 100 µL

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Discussion

In this study, we present a production process for obtaining native protein from wet biomass of G. sulphuraria 074G, which was intended as an FBS substitute. The protein extracts were tested on serum-free CHO cell cultures and showed proliferative activity after HI. To our best knowledge, this is the first time that several independent extractions of three different cultivation batches were used to evaluate the potential of native protein from G. sulphuraria as an FBS replacement.

We first produced algae biomass mixotrophically in a PBR and were able to harvest up to 9.5 g L−1 of dry G. sulphuraria 074G biomass within 5 days. This was higher than reported for other mixotrophic batch cultivations with the same strain and is possibly explained by the PBR system and general growth conditions (Graverholt and Eriksen [2007](/article/10.1007/s00253-025-13507-0#ref-CR20 "Graverholt OS, Eriksen NT (2007) Heterotrophic high-cell-density fed-batch and continuous-flow cultures of Galdieria sulphuraria and production of phycocyanin. Appl Microbiol Biotechnol 77:69–75. https://doi.org/10.1007/s00253-007-1150-2

            "); Abiusi et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR1 "Abiusi F, MoñinoFernández P, Canziani S, Janssen M, Wijffels RH, Barbosa M (2022) Mixotrophic cultivation of Galdieria sulphuraria for C-phycocyanin and protein production. Algal Res 61:102603. 
              https://doi.org/10.1016/j.algal.2021.102603
              
            ")). We found that harvesting in the late exponential to linear growth phase yielded higher soluble protein compared to high cell densities. The protein content and extractability are affected by older cultures producing more polymers that trap soluble proteins, causing precipitation after cell disruption and leaving membrane-associated proteins insoluble. Besides culture age, the chemical and physical properties of the cell wall impact the success of cell disruption and protein solubility (Safi et al. [2013](/article/10.1007/s00253-025-13507-0#ref-CR38 "Safi C, Charton M, Pignolet O, Silvestre F, Vaca-Garcia C, Pontalier P-Y (2013) Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors. J Appl Phycol 25:523–529. 
              https://doi.org/10.1007/s10811-012-9886-1
              
            ")). The cell wall of _Galdieria_ species is quite rigid, and the closely related _Cyanidium caldarium_ has up to 55% proteins in the cell wall (Bailey and Staehelin [1968](/article/10.1007/s00253-025-13507-0#ref-CR4 "Bailey RW, Staehelin LA (1968) The chemical composition of isolated cell walls of Cyanidium caldarium. J Gen Microbiol 54:269–276. 
              https://doi.org/10.1099/00221287-54-2-269
              
            ")). _Galdieria_ cells are commonly disrupted using a bead mill, high-pressure homogenizer (HPH), or French press (Carfagna et al. [2018](/article/10.1007/s00253-025-13507-0#ref-CR9 "Carfagna S, Landi V, Coraggio F, Salbitani G, Vona V, Pinto G, Pollio A, Ciniglia C (2018) Different characteristics of C-phycocyanin (C-PC) in two strains of the extremophilic Galdieria phlegrea. Algal Res 31:406–412. 
              https://doi.org/10.1016/j.algal.2018.02.030
              
            "); Imbimbo et al. [2019](/article/10.1007/s00253-025-13507-0#ref-CR26 "Imbimbo P, Romanucci V, Pollio A, Fontanarosa C, Amoresano A, Zarrelli A, Olivieri G, Monti DM (2019) A cascade extraction of active phycocyanin and fatty acids from Galdieria phlegrea. Appl Microbiol Biotechnol 103:9455–9464. 
              https://doi.org/10.1007/s00253-019-10154-0
              
            "); Abiusi et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR1 "Abiusi F, MoñinoFernández P, Canziani S, Janssen M, Wijffels RH, Barbosa M (2022) Mixotrophic cultivation of Galdieria sulphuraria for C-phycocyanin and protein production. Algal Res 61:102603. 
              https://doi.org/10.1016/j.algal.2021.102603
              
            ")). We found bead milling and HPH to be efficient; however, HPH resulted in more suspended matter in the cell extract (data not shown). Thus, we decided on bead milling. Despite 90% of cells being lysed, protein solubility remained an obstacle. Various extraction matrices were tested, but a fraction of protein remained insoluble. A potential solution could be cascade extraction, which might also allow the extraction of additional cellular fractions (Imbimbo et al. [2019](/article/10.1007/s00253-025-13507-0#ref-CR26 "Imbimbo P, Romanucci V, Pollio A, Fontanarosa C, Amoresano A, Zarrelli A, Olivieri G, Monti DM (2019) A cascade extraction of active phycocyanin and fatty acids from Galdieria phlegrea. Appl Microbiol Biotechnol 103:9455–9464. 
              https://doi.org/10.1007/s00253-019-10154-0
              
            ")).

The protein data on mixotrophic Galdieria cultures show great variability and range from 22 to 72% proteins of DW (Graziani et al. [2013](/article/10.1007/s00253-025-13507-0#ref-CR21 "Graziani G, Schiavo S, Nicolai MA, Buono S, Fogliano V, Pinto G, Pollio A (2013) Microalgae as human food: chemical and nutritional characteristics of the thermo-acidophilic microalga Galdieria sulphuraria. Food Funct 4:144–152. https://doi.org/10.1039/C2FO30198A

            "); Wan et al. [2016](/article/10.1007/s00253-025-13507-0#ref-CR49 "Wan M, Wang Z, Zhang Z, Wang J, Li S, Yu A, Li Y (2016) A novel paradigm for the high-efficient production of phycocyanin from Galdieria sulphuraria. Bioresour Technol 218:272–278. 
              https://doi.org/10.1016/j.biortech.2016.06.045PM-27372006M4-Citavi
              
            "); Massa et al. [2019](/article/10.1007/s00253-025-13507-0#ref-CR31 "Massa M, Buono S, Langellotti AL, Martello A, Russo GL, Troise DA, Sacchi R, Vitaglione P, Fogliano V (2019) Biochemical composition and in vitro digestibility of Galdieria sulphuraria grown on spent cherry-brine liquid. N Biotechnol 53:9–15. 
              https://doi.org/10.1016/j.nbt.2019.06.003
              
            "); Abiusi et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR1 "Abiusi F, MoñinoFernández P, Canziani S, Janssen M, Wijffels RH, Barbosa M (2022) Mixotrophic cultivation of Galdieria sulphuraria for C-phycocyanin and protein production. Algal Res 61:102603. 
              https://doi.org/10.1016/j.algal.2021.102603
              
            "); Canelli et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR7 "Canelli G, Abiusi F, Vidal Garcia A, Canziani S, Mathys A (2023) Amino acid profile and protein bioaccessibility of two Galdieria sulphuraria strains cultivated autotrophically and mixotrophically in pilot-scale photobioreactors. Innov Food Sci Emerg Technol 84:103287. 
              https://doi.org/10.1016/j.ifset.2023.103287
              
            ")). In addition to the different _Galdieria_ strains that were evaluated, the quantification methods employed are the main reason for large deviations (Barbarino and Lourenço [2005](/article/10.1007/s00253-025-13507-0#ref-CR5 "Barbarino E, Lourenço SO (2005) An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae. J Appl Phycol 17:447–460. 
              https://doi.org/10.1007/s10811-005-1641-4
              
            "); Abiusi et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR1 "Abiusi F, MoñinoFernández P, Canziani S, Janssen M, Wijffels RH, Barbosa M (2022) Mixotrophic cultivation of Galdieria sulphuraria for C-phycocyanin and protein production. Algal Res 61:102603. 
              https://doi.org/10.1016/j.algal.2021.102603
              
            ")). Bradford and Lowry assays are commonly used, with Lowry providing consistent results and Bradford usually giving lower values (Barbarino and Lourenço [2005](/article/10.1007/s00253-025-13507-0#ref-CR5 "Barbarino E, Lourenço SO (2005) An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae. J Appl Phycol 17:447–460. 
              https://doi.org/10.1007/s10811-005-1641-4
              
            ")), in our case with a factor of 2.39\. We used Bradford to quantify the proteins in the extracts, while the total protein was measured using the Lowry assay.

In initial experiments, the proliferation rate of CHO cells in the presence of HI protein extracts matched that of FBS controls. To exclude artifacts from a single experiment, we repeated the whole process with three additional extractions from three independent culture batches. An admixture of 5% of the enriched protein samples (HIASE) appeared to promote CHO growth better than crude extracts (HIE), indicating either a reduction of growth-inhibiting factors or an enrichment of growth-promoting factors. The effect could only be replicated with the 10% batch 1 approach, supporting the hypothesis of a non-uniform protein and peptide composition in the extracts. The protein concentration of Galdieria extracts tested was around five- to tenfold lower than that of FBS. However, with 8–37 µg protein in HIE and 12–58 µg protein in HIASE supplemented to serum-free medium, the CHO proliferation rate was significantly higher than 0% FBS cultures, but around twofold lower than the respective 5 and 10% FBS cultures. Either the lower protein levels were the cause of the reduced proliferation rate, or Galdieria extracts do not completely replace FBS, and additional growth-promoting molecules should be added to the serum-free medium. If we look at the results of the first test of batch 1, growth of the CHO cells, like FBS controls, seems to be possible even without further additives. Therefore, the identification of the protein and peptide composition of the different extracts seems to be a reasonable solution. In addition, an acclimation of CHO cells to Galdieria extracts prior to the actual tests might also improve results. Algae extracts with 1 µg mL−1 and 10 µg mL−1 protein content showed similar or slightly higher cell viability of myoblast cells from Japanese quail (QM7) when supplemented with 1% FBS (Sibinčić et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR40 "Sibinčić N, Krstić Ristivojević M, Gligorijević N, Veličković L, Ćulafić K, Jovanović Z, Ivanov A, Tubić L, Vialleix C, Michel T, Srdić Rajić T, Nikolić M, Stojadinović M, Minić S (2024) Screening algal and cyanobacterial extracts to identify potential substitutes for fetal bovine serum in cellular meat cultivation. Foods 13  https://doi.org/10.3390/foods13233741

            ")). Higher protein concentrations of algae extracts led to growth-inhibiting effects for most samples. CW of _C. littorale_ (15 mg protein mL−1) was efficient in replacing FBS in the serum-free culture medium of CHO and other tested cell lines, with similar or slightly lower viabilities than controls (Ghosh et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR19 "Ghosh J, Haraguchi Y, Asahi T, Nakao Y, Shimizu T (2023) Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 for sustainable cultured meat production. Biochem Biophys Res Commun 682:316–324. 
              https://doi.org/10.1016/j.bbrc.2023.10.018
              
            ")). However, this was shown for one extract with three replicates in total.

Supplementing algae extracts with growth factors or low FBS amounts promoted cell growth significantly (Ng et al. [2020](/article/10.1007/s00253-025-13507-0#ref-CR32 "Ng JY, Chua ML, Zhang C, Hong S, Kumar Y, Gokhale R, Ee PLR (2020) Chlorella vulgaris extract as a serum replacement that enhances mammalian cell growth and protein expression. Front Bioeng Biotechnol 8:1–11. https://doi.org/10.3389/fbioe.2020.564667

            "); Defendi-Cho and Gould [2023](/article/10.1007/s00253-025-13507-0#ref-CR11 "Defendi-Cho G, Gould TM (2023) In vitro culture of bovine fibroblasts using select serum-free media supplemented with Chlorella vulgaris extract. BMC Biotechnol 23:1–11. 
              https://doi.org/10.1186/s12896-023-00774-w
              
            "); Dong et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR14 "Dong N, Xue C, Yang Y, Chang Y, Wang Y, Guo H, Liu Y, Wang Y (2023) Auxenochlorella pyrenoidosa extract supplementation replacing fetal bovine serum for Carassius auratus muscle cell culture under low-serum conditions. Food Res Int 164:112438. 
              https://doi.org/10.1016/j.foodres.2022.112438
              
            "); [2024](/article/10.1007/s00253-025-13507-0#ref-CR13 "Dong N, Jiang B, Chang Y, Wang Y, Xue C (2024) Integrated omics approach: revealing the mechanism of Auxenochlorella pyrenoidosa protein extract replacing fetal bovine serum for fish muscle cell culture. J Agric Food Chem 72:6064–6076. 
              https://doi.org/10.1021/acs.jafc.4c00624
              
            "); Sibinčić et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR40 "Sibinčić N, Krstić Ristivojević M, Gligorijević N, Veličković L, Ćulafić K, Jovanović Z, Ivanov A, Tubić L, Vialleix C, Michel T, Srdić Rajić T, Nikolić M, Stojadinović M, Minić S (2024) Screening algal and cyanobacterial extracts to identify potential substitutes for fetal bovine serum in cellular meat cultivation. Foods 13 
              https://doi.org/10.3390/foods13233741
              
            ")). CVE and CGF showed up to 80% growth-promoting activity in serum-free media with added growth factors such as basic fibroblast growth factor (FGF), transforming growth factors (TGF)-β1, and insulin (Defendi-Cho and Gould [2023](/article/10.1007/s00253-025-13507-0#ref-CR11 "Defendi-Cho G, Gould TM (2023) In vitro culture of bovine fibroblasts using select serum-free media supplemented with Chlorella vulgaris extract. BMC Biotechnol 23:1–11. 
              https://doi.org/10.1186/s12896-023-00774-w
              
            ")). Similar viability and proliferation were observed in fish and mammalian cell cultures using water extracts in serum-reduced medium (Dong et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR14 "Dong N, Xue C, Yang Y, Chang Y, Wang Y, Guo H, Liu Y, Wang Y (2023) Auxenochlorella pyrenoidosa extract supplementation replacing fetal bovine serum for Carassius auratus muscle cell culture under low-serum conditions. Food Res Int 164:112438. 
              https://doi.org/10.1016/j.foodres.2022.112438
              
            "); [2024](/article/10.1007/s00253-025-13507-0#ref-CR13 "Dong N, Jiang B, Chang Y, Wang Y, Xue C (2024) Integrated omics approach: revealing the mechanism of Auxenochlorella pyrenoidosa protein extract replacing fetal bovine serum for fish muscle cell culture. J Agric Food Chem 72:6064–6076. 
              https://doi.org/10.1021/acs.jafc.4c00624
              
            "); Sibinčić et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR40 "Sibinčić N, Krstić Ristivojević M, Gligorijević N, Veličković L, Ćulafić K, Jovanović Z, Ivanov A, Tubić L, Vialleix C, Michel T, Srdić Rajić T, Nikolić M, Stojadinović M, Minić S (2024) Screening algal and cyanobacterial extracts to identify potential substitutes for fetal bovine serum in cellular meat cultivation. Foods 13 
              https://doi.org/10.3390/foods13233741
              
            ")). Furthermore, phycocyanin C (C-PC), which was present in our extracts, was found to positively regulate the cell cycle of human fibroblast WI-38 cells (Madhyastha et al. [2006](/article/10.1007/s00253-025-13507-0#ref-CR30 "Madhyastha HK, Radha KS, Sugiki M, Omura S, Maruyama M (2006) C-Phycocyanin transcriptionally regulates uPA mRNA through cAMP mediated PKA pathway in human fibroblast WI-38 cells. Biochim Biophys Acta - Gen Subj 1760:1624–1630. 
              https://doi.org/10.1016/j.bbagen.2006.08.012
              
            "); Dranseikienė et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR15 "Dranseikienė D, Balčiūnaitė-Murzienė G, Karosienė J, Morudov D, Juodžiukynienė N, Hudz N, Gerbutavičienė RJ, Savickienė N (2022) Cyano-phycocyanin: mechanisms of action on human skin and future perspectives in medicine. Plants 11:1249. 
              https://doi.org/10.3390/plants11091249
              
            ")) and promoted growth in the mouse myoblast cell line C2 C12 and QM7 cells in serum-free medium (Ghosh et al. [2023](/article/10.1007/s00253-025-13507-0#ref-CR19 "Ghosh J, Haraguchi Y, Asahi T, Nakao Y, Shimizu T (2023) Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 for sustainable cultured meat production. Biochem Biophys Res Commun 682:316–324. 
              https://doi.org/10.1016/j.bbrc.2023.10.018
              
            ")). All findings indicate the protein fraction of microalgae is key in replacing FBS, making the identification of effective molecules in protein extracts crucial.

The cultured meat and fish industry seeks serum-free media to reduce costs and address ethical and regulatory concerns associated with FBS. Currently, serum-free media account for about 50% of the variable operating costs in cultivated meat production, with growth factors and recombinant proteins being the primary cost drivers (Quek et al. [2024](/article/10.1007/s00253-025-13507-0#ref-CR37 "Quek JP, Gaffoor AA, Tan YX, Tan TRM, Chua YF, Leong DSZ, Ali AS, Ng SK (2024) Exploring cost reduction strategies for serum free media development. npj Sci Food 8:1–10. https://doi.org/10.1038/s41538-024-00352-0

            ")). Microalgae present a promising cost-effective alternative; however, it is crucial to establish an affordable production process for microalgal serum, considering the demand for FBS. Mixotrophic or heterotrophic production with _Galdieria_ can be advantageous, as its preference for a very low pH reduces the risk of contamination under heterotrophic conditions compared to, e.g., _Chlorella_, and it contains PC, which is discussed as a key substance (Hirooka and Miyagishima [2016](/article/10.1007/s00253-025-13507-0#ref-CR25 "Hirooka S, Miyagishima S (2016) Cultivation of acidophilic algae Galdieria sulphuraria and Pseudochlorella sp. YKT1 in media derived from acidic hot springs. Front Microbiol 07:2022. 
              https://doi.org/10.3389/fmicb.2016.02022
              
            "); Dranseikienė et al. [2022](/article/10.1007/s00253-025-13507-0#ref-CR15 "Dranseikienė D, Balčiūnaitė-Murzienė G, Karosienė J, Morudov D, Juodžiukynienė N, Hudz N, Gerbutavičienė RJ, Savickienė N (2022) Cyano-phycocyanin: mechanisms of action on human skin and future perspectives in medicine. Plants 11:1249. 
              https://doi.org/10.3390/plants11091249
              
            ")). Nevertheless, continuous passage of CHO and other mammalian cell lines on the _Galdieria_ protein extracts HIE and HIASE is necessary to prove long-term feasibility. Thus, improving the composition and quality of native protein extracts requires an improved extraction method from high-density _Galdieria_ cultures.

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No datasets were generated or analysed during the current study.

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Acknowledgements

Our appreciations go to the entire SerAZel team. We also want to thank Linda Meissner and Peer Jakob Müntinga for their support in the laboratory when conducting algae and CHO cultivations and analytical measurements.

Funding

Open Access funding enabled and organized by Projekt DEAL. This work was funded by the German Federal Ministry of Education and Research (BMBF) FKZ 031B1363 A.

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  1. Institute EcoMaterials, Bremerhaven University of Applied Sciences, An Der Karlstadt 8, 27568, Bremerhaven, Germany
    Hanna Eisenberg, Svenja Hütker, Felicitas Berger & Imke Lang

Authors

  1. Hanna Eisenberg
  2. Svenja Hütker
  3. Felicitas Berger
  4. Imke Lang

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HE, SH, FB and IL wrote the original manuscript, revised the manuscript, and conducted the literature review. IL and FB conceived and conceptualized the experiments. HE and SH conducted experiments and prepared Figures and Tables. All authors reviewed and approved the final version of the manuscript.

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Correspondence toImke Lang.

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Eisenberg, H., Hütker, S., Berger, F. et al. Native proteins from Galdieria sulphuraria to replace fetal bovine serum in mammalian cell culture.Appl Microbiol Biotechnol 109, 119 (2025). https://doi.org/10.1007/s00253-025-13507-0

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