A groundbreaking study by researchers at the Laboratory of Sustainable Food Processing, ETH Zurich (Proietti Tocca et al., 2025), has revealed the remarkable photoformatotrophic capacity of the extremophilic microalga Galdieria sulphuraria SAG 108.79, marking the first documented case of a wild-type microalgal species capable of efficiently utilising formic acid as a carbon source.
This research demonstrates complete conversion of organic acids into protein-rich biomass, achieving biomass yields of 89-100% whilst maintaining exceptional nutritional quality. The discovery of native formate dehydrogenase enzymes in this acidophilic organism opens new avenues for sustainable biotechnology and the emerging formate bioeconomy.
Introduction
The urgent need for sustainable protein production technologies has intensified as global carbon emissions reached 35.8 Gt CO₂ in 2023, with agrifood systems contributing approximately one-third of greenhouse gas emissions. Microalgae cultivation represents a promising solution, offering protein productivity an order of magnitude higher than conventional crops whilst potentially containing up to 80% protein by weight. However, traditional photoautotrophic cultivation faces significant challenges related to CO₂ mass transfer and infrastructure requirements for concentrated CO₂ supply.
The concept of the formate bioeconomy – encompassing biotechnological processes that employ formate (the salt of formic acid) as a key molecule for the production of biomass, proteins, biofuels, and other industrially relevant compounds – has emerged as an innovative approach to overcoming these limitations. Formic acid (FA) and acetic acid (AA) can be synthesised directly from CO₂ through electrochemical reduction or biological processes, offering liquid, transportable alternatives to gaseous CO₂. Despite considerable interest in formatotrophic metabolism, previous attempts to engineer microalgae for formic acid utilisation have required extensive genetic modifications and resulted in modest yields.
Galdieria sulphuraria, a polyextremophilic red microalga, thrives in highly acidic environments (pH < 3) and elevated temperatures (up to 56°C), making it uniquely suited for industrial applications. This study investigates the unprecedented mixotrophic cultivation capabilities of G. sulphuraria SAG 108.79 using organic acids as carbon sources, with implications for single-cell protein production and sustainable biotechnology.
Methodology
Experimental design and strain selection
The research employed Galdieria sulphuraria SAG 108.79, purchased from the algae culture collection of Gottingen University (SAG), which was selected for its high protein bioaccessibility (69%) and fully sequenced genome. Toxicity assessment was conducted using 24-well microplates at pH 2 and 4, testing eight concentrations (0.0-0.7 g L⁻¹) of both formic and acetic acids. The experimental design recognised that organic acid toxicity significantly increases below their respective pKa values (3.75 for FA, 4.75 for AA) due to passive membrane diffusion and cytoplasmic acidification.
Photobioreactor configuration
Mixotrophic experiments were conducted in 2.0 L flat-panel photobioreactors with 1.8 L working volume. The systems maintained constant temperature (42°C), pH 2.0 ± 0.1, and photosynthetically active photon flux density (PPFD) of 250 μmol photons m⁻² s⁻¹.
Continuous feeding strategies were implemented to avoid organic acid accumulation, with feeding rates calculated based on photoautotrophic CO₂ fixation rates using arbitrary factors (F = 0.5, 1, 1.25 for FA; F = 0.75, 1.25 for AA).
Analytical methods
Biomass productivity (rₓ) was determined through linear regression of dry weight measurements over time. Carbon-based productivity (rₓᶜ) calculations incorporated biomass carbon content measured via CHNS analysis.
Biomass yields on substrate (Yᶜₓ/ₛ) were calculated according to the ratio of carbon-based productivity to daily substrate supply rate. Amino acid profiling utilised HPLC analysis following acid hydrolysis and o-phthaldialdehyde derivatisation.
Bioinformatic analysis
Genomic investigation focused on identifying formate-metabolising enzymes through comprehensive searches of KEGG and BRENDA databases.
Enzyme Commission (EC) numbers, representing enzymes involved in known formate metabolic pathways, were compared with UniProt entries for G. sulphuraria to identify matches between characterized enzymes and those present in the microalga.
Horizontal gene transfer analysis employed tBLASTn searches against GenBank sequences to determine the evolutionary origin of formate dehydrogenase genes.
Results and discussion
Toxicity profiles and feeding strategy development
Organic acid toxicity testing revealed significant pH-dependent effects on G. sulphuraria growth. At pH 2, formic acid proved lethal at concentrations as low as 0.1 g L⁻¹, whilst acetic acid was tolerated up to 0.3 g L⁻¹.
These findings align with previous research on acidophilic bacteria, where half-maximal inhibitory concentrations of 0.03 g L⁻¹ for formic acid have been reported.
The extreme toxicity necessitated the development of continuous feeding protocols to maintain substrate concentrations below inhibitory thresholds.
Mixotrophic growth performance
Photoautotrophic benchmarking established that G. sulphuraria productivity increased substantially with CO₂ enrichment, from 0.11 ± 0.02 g L⁻¹ day⁻¹ (atmospheric CO₂) to 0.96 ± 0.02 g L⁻¹ day⁻¹ (2% CO₂). However, biomass yields on CO₂ remained low (3.0%) due to mass transfer limitations, validating the need for alternative carbon delivery systems.
Formic acid mixotrophy demonstrated remarkable efficiency, achieving complete substrate conversion (Yᶜₓ/ₛ = 106.4 ± 2.3%) when feeding rates matched photoautotrophic carbon fixation capacity (F = 0.5). Increasing substrate supply to exceed fixation rates (F = 1.25) resulted in productivity enhancement to 1.06 ± 0.03 g L⁻¹ day⁻¹, though biomass yields decreased to 89.1 ± 4.1%. The heterotrophic yield on formic acid (31.8 ± 11.2%) indicated efficient carbon utilisation but limited energy generation, confirming formic acid’s role as a carbon source rather than primary energy substrate.
Acetic acid cultivation yielded superior energetic performance, with heterotrophic yields approaching theoretical maxima (70.9 ± 34.1%) and significant productivity increases (19% above photoautotrophic reference at F = 1.25). These results demonstrate acetic acid’s dual functionality as both carbon and energy source, consistent with established microalgal metabolism.
Genomic basis for formatotrophic capacity
Bioinformatic analysis revealed the presence of two homologous formate dehydrogenase (FDH) enzymes (M2WYT6_GALSU and M2XS58_GALSU) within the G. sulphuraria genome. This represents the first identification of native FDH in microalgae, explaining the organism’s unique formatotrophic capabilities. Phylogenetic analysis suggests horizontal gene transfer from thermophilic fungi, consistent with previous findings that approximately 5% of G. sulphuraria protein-coding genes originated through such mechanisms.
The proposed metabolic pathway involves formic acid passive diffusion across cell membranes, cytoplasmic deprotonation to formate, and subsequent FDH-catalysed oxidation to CO₂ with concomitant NADH generation. The produced CO₂ enters chloroplasts for Calvin cycle fixation, whilst NADH supports anabolic processes or oxidative phosphorylation. This mixotrophic mechanism elegantly couples organic acid oxidation with photosynthetic carbon fixation.
Biomass quality and nutritional assessment
Protein content remained consistently high (52% w/w) across all cultivation conditions, unaffected by carbon source selection. Elemental composition analysis revealed stable carbon (48.2 ± 0.4%) and nitrogen (11.3 ± 0.3%) contents, though sulfur content exhibited greater variability (0.64-1.01%), likely reflecting G. sulphuraria‘s adaptation to sulfur-rich environments.
Amino acid profiling demonstrated that all cultivation conditions met FAO dietary requirements for adults (2013), with essential amino acid compositions remaining stable regardless of organic acid supplementation. This nutritional consistency, combined with previously reported protein bioaccessibility of 69% for this strain, confirms G. sulphuraria‘s potential as a premium single-cell protein source.
Industrial implications and future perspectives
The discovery of native photoformatotrophic capacity in G. sulphuraria addresses critical limitations in current microalgae production systems. Formic acid and acetic acid production through CO₂ electrocatalysis offers scalable pathways for converting atmospheric or industrial CO₂ into concentrated, transportable carbon sources. This approach circumvents expensive CO₂ capture and transport infrastructure whilst enabling distributed microalgae production independent of high-concentration CO₂ sources.
Economic modelling suggests that current direct air capture costs (500-1000 $ ton⁻¹ CO₂) could be offset by the superior biomass yields and simplified infrastructure requirements of organic acid-based cultivation systems. As electrochemical CO₂ reduction technologies mature and costs decline, mixotrophic cultivation using electrochemically derived organic acids may become economically competitive with traditional approaches.
The environmental sustainability of this system depends critically on renewable energy integration for electrochemical processes. Life cycle assessment studies will be essential to quantify net carbon and energy balances, particularly considering the energy requirements for organic acid synthesis versus CO₂ concentration and transport.
Regulatory pathway for Galdieria sulphuraria as a novel food in the European Union
The commercialisation of Galdieria sulphuraria protein in Europe is subject to stringent requirements under the EU Novel Food Regulation (EU) 2015/2283, which covers foods not consumed to a significant degree before 15 May 1997, including microalgae-derived products.
G. sulphuraria cannot yet be used in food production in the EU, as the application for authorisation of its dried biomass as a novel food, submitted back in 2019 (European Commission, 2019), is still pending evaluation by EFSA.
QPS status
The species was not granted Qualified Presumption of Safety (QPS) status due to insufficient evidence of safe use in the food and feed chain. This necessitates a complete novel food application with comprehensive safety data and scientific substantiation, rather than the streamlined QPS route.
Production method considerations
Formatotrophic cultivation, which uses formic acid and acetic acid as carbon sources, would necessitate a new novel food application, thereby introducing additional regulatory complexity. Although both substances are authorised food additives under Regulation (EC) No 1333/2008 (E236 and E260), their role in microalgae growth requires targeted evaluation, including potential process-related contaminants, residual acids, and metabolic by-products.
Safety and nutritional data
The novel food process requires extensive toxicological, nutritional, and allergenicity testing. Evidence of stable nutritional composition across cultivation conditions, such as the consistent essential amino acid profile reported by Proietti Tocca et al. (2025), provides important contribution for regulatory submissions.
Market implications
Successful authorisation would position G. sulphuraria as a premium protein source, combining high protein content (52% w/w) with a complete amino acid profile, potentially commanding price premiums over conventional microalgae. Its non-GMO status may further facilitate approval by avoiding additional biosafety evaluations required under Directive 2001/18/EC.
FARE/Wiise expertise
The FARE (Food and Agriculture Requirements) unit of our Wiise benefit company provides specialised expertise in novel food and health claims applications, with in-depth knowledge of EFSA regulatory pathways. Its participation in the European ProFuture project on microalgae proteins highlights the ability to connect innovative research with regulatory validation.
Technological challenges and research directions
Despite promising initial results, several technical challenges must be addressed for commercial implementation. Organic acid toxicity at low pH necessitates sophisticated process control systems to maintain optimal feeding rates. Scale-up studies will be crucial to validate continuous feeding strategies in larger production systems whilst maintaining the high biomass yields demonstrated at laboratory scale.
Metabolic engineering approaches could potentially enhance formatotrophic efficiency through increased FDH expression or improved organic acid transport mechanisms. However, the discovery of native formatotrophic capacity reduces the complexity typically associated with genetically modified microorganisms, potentially facilitating regulatory approval for food and feed applications.
Process optimisation research should focus on developing integrated systems that couple electrochemical CO₂ reduction with microalgae cultivation. Real-time monitoring and control of organic acid concentrations, coupled with advanced bioprocess automation, will be essential for maintaining optimal growth conditions whilst maximising substrate conversion efficiency.
Economic and environmental considerations
Techno-economic analysis of formate-based microalgae production must consider the complete value chain from CO₂ capture through organic acid synthesis to biomass harvesting and processing. Current electrochemical conversion efficiencies for CO₂ to formic acid range from 48.7-96.5%, whilst acetic acid production remains less mature technologically. Process integration strategies that couple renewable electricity generation with electrochemical reduction could significantly improve economic viability.
Carbon footprint assessment should account for the energy requirements of organic acid synthesis versus traditional CO₂ concentration methods. The distributed production model enabled by organic acid feedstocks could reduce transportation emissions whilst providing rural economic opportunities in regions with abundant renewable energy resources.
Market positioning of protein produced through this route will depend on demonstrating sustainability credentials and nutritional equivalence to conventional protein sources. The non-GMO status of wild-type G. sulphuraria may provide market advantages in regions with restrictive biotechnology regulations.
Conclusions
This pioneering research by ETH Zurich researchers demonstrates the revolutionary potential of Galdieria sulphuraria for sustainable protein production through photoformatotrophic cultivation. The achievement of complete organic acid conversion into high-quality biomass, combined with the identification of native formate dehydrogenase enzymes, establishes this extremophilic microalga as a unique platform for next-generation biotechnology.
The mixotrophic cultivation approach using electrochemically derived organic acids offers a compelling alternative to conventional CO₂-based systems, potentially enabling distributed microalgae production with reduced infrastructure requirements. Biomass yields of 89-100% and consistent protein quality meeting FAO nutritional standards validate the commercial potential of this technology.
Future research priorities should focus on process scale-up, economic optimisation, and life cycle assessment to quantify environmental benefits. The integration of renewable energy-driven electrochemical CO₂ reduction with formatotrophic microalgae cultivation represents a promising pathway towards circular carbon economy implementation.
The discovery that G. sulphuraria can efficiently utilise formic acid without genetic modification addresses key regulatory and consumer acceptance challenges associated with engineered microorganisms. This natural metabolic capacity, combined with the organism’s extremophilic characteristics and exceptional nutritional profile, positions G. sulphuraria as a leading candidate for sustainable protein production in the emerging bioeconomy.
Dario Dongo
Cover art copyright © 2025 Dario Dongo (AI-assisted creation)
References
- European Commission. (2019). Public summary of the dossier: Dried biomass of Galdieria sulphuraria (Application No. 2019/1342). Novel Food application under Regulation (EU) 2015/2283. Retrieved from https://food.ec.europa.eu/document/download/b165acbd-199a-4046-b1be-f97831891d3e_en
- Joint FAO/WHO/UNU Expert Consultation on Protein and Amino Acid Requirements in Human Nutrition. (2007). Protein and amino acid requirements in human nutrition: Report of a Joint WHO/FAO/UNU Expert Consultation. WHO Technical Report Series No. 935. World Health Organization. ISBN: 978-92-4-120935-9
- Proietti Tocca, G., Abiusi, F., Macken, D., Fraterrigo Garofalo, S., Tommasi, T., Fino, D., & Mathys, A. (2025). Complete conversion of organic acids into protein-rich biomass: Discovering the photoformatotrophic capacity of Galdieria sulphuraria. ACS Sustainable Chemistry & Engineering. https://doi.org/10.1021/acssuschemeng.5c02338
Dario Dongo, lawyer and journalist, PhD in international food law, founder of WIISE (FARE - GIFT - Food Times) and Égalité.
