1 Introduction

Vitamin B12, also referred to as cobalamin, is a chemical compound possessing vital vitamin properties. B12 cannot be synthesized by animals, fungi, or plants. Instead, the exclusive producers of vitamin B12 are microorganisms, primarily anaerobic species, and archaebacteria. The most prevalent natural and biologically active forms of B12 include adenosylcobalamin, also known as coenzyme B12, and methylcobalamin, often used as a dietary supplement [1].

Vitamin B12 plays pivotal roles in various biological functions. Adenosylcobalamin functions as a co-factor for methylmalonyl-coenzyme A (CoA) mutase, which operates within the mitochondria to convert methylmalonyl-CoA into succinyl-CoA; a reaction necessary for the breakdown of cholesterol and fatty acids [2]. Methylcobalamin serves as a co-factor for the enzyme methionine synthase, facilitating the conversion of homocysteine into methionine within the cytosol. These reactions are integral to the synthesis of neurotransmitters, phospholipids, DNA, and RNA [3].

As opposed to other vitamins which may be absorbed directly, and predominantly, from plant-based sources (e.g., vitamin A in the form of provitamin A carotenoids, vitamin C, vitamin E, vitamin K), the only sources of vitamin B12 in human diets are animal source foods (ASF), alongside fortified foods with synthetic B12 that is produced industrially, for instance by fermenting Pseudomonas denitrificans and Propionibacterium freudenreichii [4]. This is due to the structure of the human digestive system and intestinal microbiota [5].

Specifically, ruminant-derived foods (beef, lamb, mutton meat, and bovine milk) serve as a primary source of B12 (adenosylcobalamine and methylcobalamin), owing to bacteria within the gastrointestinal tract of ruminants that produce cobalamin which is then stored in muscle tissues and the liver. Therefore, consumption of ruminant-derived meat and dairy products plays a crucial role in meeting the official B12 intake recommendation of 2.4 µg/day (recommended vitamin B12 intake for pregnant women and breastfeeding stands at 2.6 and 2.8 µg/day, respectively; see Table 1) [6].

Table 1 Vitamin B12 Recommended Dietary Allowances
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Dietary vitamin B12 deficiency is a common and deleterious medical condition. B12 deficiency is marked by megaloblastic anemia and neuropathy. Megaloblastic anemia may present with features such as anemia, decreased white blood cell count, a painful tongue, and infertility. Neuropathy symptoms are characterized by spinal cord degeneration, loss of proprioceptive sensation, spastic weakness in the lower limbs, and, in some cases, the presence of memory loss [7]. Infants breastfed by mothers deficient in vitamin B12 face a heightened risk of severe developmental abnormalities, growth impediments, tissues and organ dysfunction, and anemia.

B12 deficiency has long been acknowledged as a concern in both developed and developing regions. In developing regions, vitamin B12 deficiency is prevalent due to malnutrition and limited consumption of ASF, leading to insufficient dietary intake. Globally, individuals following strict vegan and vegetarian diets may be at risk of cobalamin deficiency because of ASF avoidance [8].

Previous surveys have indicated that the prevalence of vitamin B12 deficiency is approximately 6% in people below the age of 60 in the UK and the US, approximately 40% in children and adults in Latin America (clinical or subclinical deficiency), approximately 70% among Kenyan schoolchildren, and 80% among Indian preschool children [9].

However, increasing the availability of ASF, to mitigate vitamin B12 deficiency, involves a nutritional-environmental trade-off, since the production of beef, lamb, and mutton meat, as well as bovine milk, are prime emitters of greenhouse gases (GHGs).

The GHG emissions related to the production of one kilogram of beef have been reported in a landmark study [10] as 99.48 kg CO2-eq with the figures for lamb and mutton, cheese, and milk being 39.72 kg CO2-eq, 23.88 kg CO2-eq, and 3.15 kg CO2-eq, respectively.

Moreover, ruminant agriculture involves considerable environmental impacts, including deforestation and water scarcity. Deforestation is often a result of clearing land for grazing or feed production for livestock [11]. Previous research indicated that the expansion of pastureland to raise ruminants is responsible for over 40% of tropical deforestation [12]. This leads to the loss of biodiversity and habitat for an abundance of species [13].

In addition, the water footprint of livestock production is relatively high, with large amounts of water required for feed production and animal consumption [14]. One seminal study estimated a global average of 15,400 m3 required to produce one ton of meat from beef cattle [15].

In recent years, the environmental ramifications associated with ASF have motivated the development of novel meat and milk alternatives considered safer, nutritious, and more sustainable. These alternatives are referred to in literature as novel and future foods (NFFs) [16,17,18,19,20]). NFFs include products from cell cultures such as cultured meat and milk (also known as meat and milk analogues), microbial foods processed from yeasts and bacteria, and algae-based foods including from Chlorella microalgae (Chlorella vulgaris) and Spirulina.

Within NFFs, Spirulina blue-green algae (Arthrospira platensis) has been deemed safe for human consumption and has garnered substantial attention as an appropriate ASF alternative, in both science and industry with mass production of the algae [21].

Spirulina is a microscopic cyanobacterium regarded as a superb provider of macro- and micro-nutrients [22, 23], including essential amino acids, alpha-linolenic acid (ω-3 fatty acid) and linoleic acid (ω-6 fatty acid), various minerals, including calcium, potassium, magnesium and iron, and vitamins, such as beta-carotene (β-Carotene). It has been shown to have antioxidant and anti-inflammatory properties, which can reduce the risk of cardiovascular diseases, improving gut health, and supporting immune function [24,25,26,27].

However, previous analyses have shown that the predominant form of vitamin B12 found in commercially available sources of Spirulina is mostly an inactive form (cobamide or pseudo-vitamin B12), which is unavailable to humans [28]. This renders traditional Spirulina a limited alternative to ASF.

In Cobamide, the pseudo-vitamin B12, the lower ligand of the molecule (5,6-dimethylbenzimidazole (DMB)) is replaced by adenine as a base. This affects its ability to be absorbed and utilized by the body [29, 30]. Furthermore, pseudo-B12 cobamide in naturally occurring Spirulina is structurally akin to cobalamin, and therefore competes with cobalamin sources for absorption [31].

As a response, in this exploratory in vitro study, we ask whether light conditions may enhance active vitamin B12 production in Spirulina. Accordingly, we describe the cultivation of Spirulina in a novel algal biotechnology facility, using light-emitting diodes (LED), and report to activate natural metabolic pathways to produce a nutritious biomass containing unopposed, biologically active vitamin B12.

This experimental approach was inspired by previous research [32,33,34] that showed that photonic management of Spirulina—without genetic modifications—can affect its metabolomics profile to achieve and enhance desirable bioactive compounds (e.g., C-phycocyanin) with therapeutic properties, such as antioxidant and anti-inflammatory activities.

Research spanning decades has shown that Spirulina may be cultured in a variety of conditions, including photobioreactors where photon management (i.e., controlling the light collected by the algae) may enhance photosynthesis [35, 36], while substantially minimizing the land and water footprint of production [37,38,39,40]). If cultivated in photobioreactors that rely on renewable energy sources, algae production—and to the extent of this study, unopposed vitamin B12 —can prove to be carbon neutral [41].

Here, we report that unopposed, active concentration of vitamin B12, in Photosynthetically Controlled Spirulina (hereinafter: PCS) is viable and was found to be at concentrations similar to active B12 in beef meat. Both pseudo- and active- forms of B12 for PCS were analyzed, and the unopposed, net bioactive vitamin B12 concentration was calculated (amount of bioactive compounds minus non-bioactive compounds). This finding was consistent over 9 months of continuous cultivation.

2 Materials and methods

2.1 Spirulina culture, cultivation, and sampling

Information concerning Spirulina (Arthrospira platensis, UTEX 3086) cultivation process was obtained in situ from the Hellisheidi Spirulina facility in Hellisheiði (Hellisheidi) geothermal power park, in the region of Hengill, Iceland (N64°2′12″ W21°23′53″).

Spirulina was cultivated in a modified Zarrouk's medium, designed for culturing algae and cyanobacteria, consisting of the several components in varying concentrations (per Liter of solution), including sodium bicarbonate (NaHCO3) at 16.8 g, sodium nitrate (NaNO3) at 2.5 g, sodium chloride (NaCl) at 1 g, potassium sulfate (K2SO4) and potassium phosphate dibasic (K2HPO4) at 1 g and 0.5 g respectively, trace amounts of magnesium sulfate heptahydrate (MgSO4·7H2O), iron(II) sulfate heptahydrate (FeSO4·7H2O), and calcium chloride dihydrate (CaCl2·2H2O), EDTA, at 0.08 g. The medium is completed with 1000 millilitres of distilled water to dissolve and distribute these components evenly [42,43,44]. Cultivation of Spirulina was conducted in three enclosed, segmented, contamination-free units, each comprising a bundle of 80, flat panel airlift photobioreactors (FPA-Photobioreactors, Subitec GmbH), 180 L each (totalling 14,400 L) [45].

Culture concentration was maintained at 12.0 ± 0.2 g/l by continuous harvest. The Spirulina biomass concentration was monitored at optical density at a wavelength of 670 nm, using a digital spectrophotometer (QUIMIS Q798DRM, Diadema–SP–Brazil), controlling the harvest rate. A biomass standard growth curve was generated to correlate the optical density with the dry weight biomass [46]. Fresh medium was introduced, at the harvest rate, to compensate for the harvested volume and the evaporative loss.

Cultures were maintained under agitation pneumatically induced by CO2-enriched air, with flow aeration of 0.5 vvm (air volume/medium volume/minute). CO2 inclusion rate was adjusted to maintain pH 10.8 ± 0.2. The temperature of the culture was maintained at 31 ± 2 °C. Mechanical pumps are not used, to prevent shearing of the Spirulina.

Cultures were grown using broad-spectrum OZRAM (Osram Opto Semiconductors) mid-power LED lights, with total irradiance of 1,750 μmol/(m2s) [41], distributed per Table 2. LED were fixed on a support 5 cm beside the photobioreactor (both sides). Irradiance and photosynthetically active radiation (PAR) were measured on the surface of the panel using a LI-250A light meter (Nebraska, USA) and a Li-Cor quantum sensor (LI-190R).

Table 2  Experimental LEDs colours and photoperiods
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Lighting conditions remained consistent during the entire experimental period. Samples were analysed at two different times: after 3 months to establish a baseline composition under the specific lighting conditions, and after 9 months to assess any changes or consistency in composition. Detailed lighting parameters, including light intensity, light colours, their relative intensities, and the photoperiod for each light colour, are provided in Table 2. UV light was included in the regime, as previous studies have shown it can enhance metabolic pathways in Spirulina [34]. Future studies could explore additional lighting conditions to enhance our understanding of how light affects the metabolic pathways of vitamin B12 production in Spirulina.

Figures 1 and 2 provide internal and external views of the PCS cultivation system described and utilized in this experimental research. Figure 1 (internal view) is taken from between the flat-panel airlift photobioreactors, Image 2 (external view) is taken from the facility floor.

Fig. 1
figure 1

Internal view of the photobioreactors described in this study

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Fig. 2
figure 2

External view of the photobioreactors described in this study

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Figures 1 and 2. The PCS cultivation system described in this research, flat-panel airlift photobioreactors.

PCS biomass samples were freeze dried, to preserve bioactive composition, and analyzed for pseudo- and active- B12 composition. Unopposed B12 was calculated as active B12 minus pseudo B12. Results are presented in µg per 100g of edible biomass in line with previous research [34].

2.2 Extraction of cyanocobalamin and pseudo-cyanocobalamin

Analysis of biomass composition was conducted at the Institute of Food Science, Vienna, Austria. For sample preparation, purification, elution, and analysis, we apply the methods described in van den Oever and Mayer [28].

After transport, freeze-dried samples were stored at 4°C until chemical analysis. Table 3 describes the main nutritional content of PCS after harvest, based on third-party laboratory analysis (conducted by Eurofins Scientific SE), highlighting water, protein, essential amino acids (EAAs), and iron, per 100 g.

Table 3 Biomass composition of the PCS after harvest
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Since vitamin B12 is sensitive to light, further operations were conducted using amber glassware, covered tubes using aluminum foil or under subdued light. Sample preparation was performed according to the manufacturer’s instructions of the purification kit (EASI EXTRACT®, R-Biopharm; Glasgow, UK) and Van den Oever and Mayer [28] with slight modifications regarding these Spirulina samples. Approximately 1 g of freeze-dried samples were accurately weighted with an analytical balance into a laboratory glass bottle, and thoroughly mixed with 50 mL of sodium acetate buffer (50 Mm; pH 4.0) [28].

Afterwards, enzymatic digestion was performed to free protein-bound vitamin B12. Hence, pepsin (1 g) and taka-diastase (0.25 g) were weighted to the samples, mixed, and shaken on a Heidolph Unimax 1000 (Walpersdorfer, Germany) for 10 min. Subsequently, all possible vitamers of vitamin B12 were converted into the stable cyano-form (cyanocobalamin) by adding 1 mL of a fresh potassium cyanide solution (1% w/v).

Samples were then incubated for 1 h at 37 °C under continuous shaking on the Heidolph Unimax 1000. Enzymatic reaction was stopped by incubating samples for 30 min at 120 °C in a Heraeus Oven (Hanau, Germany) with shaking after 10 min. Samples were further cooled to room temperature using a water bath, and transferred quantitatively into 100 mL volumetric flasks, which were then brought to volume using the sodium acetate buffer. Afterwards, samples were filtered through a Whatman S&S 595 ½ filter paper (Maidstone, UK). Clear sample solutions were stored (under light protection) for 3 days at room temperature prior to purification procedure [28].

Purification was performed using EASI EXTRACT® immunoaffinity columns (R-Biopharm, Glasgow, UK), which were acclimated to ambient conditions before use. Samples were centrifuged at 8000 × g for 10 min or filtered again immediately before purification. Immunoaffinity column was attached to the corresponding adapter, and column storage buffer was removed with a flow rate of 2 droplets per second. Ten millilitres of clear sample solution were passed through immunoaffinity column with a flow rate of 1 droplet per second [28].

To ensure adequate capture of the vitamin by the antibody, a slow and steady flow rate is essential. For washing the immunoaffinity column, 10 mL of UHQ water was passed through (2 droplets per second), followed by complete drying with enhanced air pressure. Subsequently, cyanocobalamin as well as its pseudo-form were eluted from the column with 5 mL methanol (LC–MS grade) having a flow rate of 1 droplet per second, and eluted vitamin B12 was collected in an appropriate tube with screw cap [28].

During elution procedure, back-flushing was performed 6 times as well as resting of 1 min after each backflush in order to increase contact time of solvent with the antibody to ensure complete elution of the vitamin present in the sample. The captured eluate was then dried overnight at 40 ℃ under reduced pressure using a SpeedVac Concentrator (SVC-200H, Savant Instruments Inc., Farmingdale, NY, USA).

Prior to chromatographic analysis, dried residue was reconstituted in 500 μL of solvent A (UHQ water modified with 0.1% acetic acid) containing 6% of methanol and filtered through a 0.20 μm syringe filter (Sartorius, Goettingen, Germany). If necessary, filtered injection solution was further diluted with reconstitution solution in order to fit in the calibrated range [28].

A total of six determinations were performed for each sample by performing two separate extractions of the freeze-dried sample. The immunoaffinity columns were then determined in triplicate for each of the two clear extracts.

2.3 UHPLC analysis of (pseudo-) vitamin B12 and data preprocessing

All reagents and chemicals were of analytical or LC–MS grade. Water (Ultra-pure, ≥ 18.2 mΩ—UHQ) was provided by a SG Ultra Clear UC System (SG-Water, Barsbüttel, Germany), acetic acid glacial (Optima® LC–MS grade) was acquired from Fischer Chemicals (Geel, Belgium), and LC–MS grade methanol was obtained from Chem-Lab NV (Zedelgem, Belgium). Hydroxocobalamin ≥ 98 was purchased from USP (Rockville, MD, USA), and cyanocobalamin ≥ 98%, 5′-deoxyadenosylcobalamin ≥ 97%, and methylcobalamin ≥ 97%, were purchased from Sigma-Aldrich (St. Louis, MO, USA) [28].

For calibration, a stock solution of cyanocobalamin of 100 μM was completely dissolved in UHQ-water in a light-protected flask, and used for dilution of the standard calibration curve with the following levels: 2, 3, 4, 6, 8, 10, 12.5, 25, 50, 75, 100, 150 and 200 nM. Since there is no certified reference material available yet for pseudo-cyanocobalamin, quantification was performed using the calibration curve for cyanocobalamin as suggested by Edelmann et al. [47].

Method validation was performed following the EURACHEM guide [48] as described by Van den Oever and Mayer [28]. Chromatographic analysis of cyanocobalamin and pseudo-cyanocobalamin was performed on a Waters Acquity™ Ultra Performance LC (UPLC™) H-class system (Waters, Milford, MA, USA) equipped with an Acquity™ photodiode array (PDA) eλ detector for quantification as well as an Acquity™ QDa single quadrupole mass detector for correct identification of different forms.

Cyanocobalamin and pseudo-cyanocobalamin were separated using an Acquity UPLC™ column (BEH C18, 50 × 2.1 mm i.d., 1.7 μm). Gradient elution was performed using UHQ-water modified with 0.1% acetic acid as solvent A, and LC–MS grade methanol with 0.1% acetic acid as solvent B as follows: initial–3.0 min/2–20% B; 3.0–4.0 min/20–20% B; 4.0–5.0 min/20–100% B; 5.0–7.0 min/100–100% B; 7.0–7.5 min/100–2% B. Reequilibration at initial conditions was performed for another 3 min. Column temperature was set to 45 °C, flow rate was 0.65 mL/min, and injection volume was set to 50 μL [28].

PDA detector was operated at 361 nm for (pseudo-)cyanocobalamin at a resolution of 1.2 nm and a sampling rate of 40 points/s in order to monitor column eluates. QDa parameters for correct identification of cyanocobalamin and pseudo-cyanocobalamin were as follows: sampling rate of 20 points/s, electro spray ionization (ESI) in positive ion mode, cone voltage: 15 V, capillary voltage: 0.8 kV, probe temperature 600 ◦C in selected ion recording (SIR) with m/z 678.2914 for cyanocobalamin, and m/z 672.7749 for pseudo-cyanocobalamin both as [M + 2 H]2+ [49]. Chromatographic data were collected and processed using Waters Empower™ software version 3.

2.4 Statistical analysis

Data were obtained from two production lots, after month 3 and month 9 of continuous PCS cultivation in the Hellisheidi Spirulina facility (Exp1 and Exp2, respectively). Three samples per lot were analysed. Analysis was performed in six replicates per sample, yielding eighteen data points per lot. Statistical significance was determined by one-way analysis of variance (ANOVA) and post hoc Tukey, using the Statistical Analysis System program, PRISMA 8. The difference between the significance levels was set to p < 0.05.

For the statistical analysis of PCS composition, we employed a multilevel model in the JMP Pro 17 program, an advanced statistical software. Variations between repetitions (a random effect) and within each repetition (a random effect) were accounted for, as well as the main factors of interest (fixed effects). Our analysis shows a high level of statistical significance, with P = 0.9942.

3 Results

The analysis of vitamin B12 (measured as cyanocobalamin) of PCS showed a novel and consistent composition (Table 4). Although both pseudo- and active forms of vitamin B12 were observed, the active form largely dominates the composition (> 98%).

Table 4 Pseudo, active, unopposed B12 composition of Photosynthetically Controlled Spirulina (PCS) biomass, and standard deviations (SD), after 3 and 9 months of continuous cultivation in photobioreactors in the Hellisheidi Spirulina facility
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While the composition of traditional, naturally occurring Spirulina is dominated by pseudo-vitamin B12, with a net-negative content of active B12, the results obtained here illustrate the effects of photonic management on enhancing natural metabolic pathways for achieving active, and net-active B12 in Spirulina.

Each sample analysis was performed in six repetitions. Consequently, for each lot (Exp1 and Exp2), we acquired a total of 18 measurements, calculated by multiplying the number of samples (3) by the number of repetitions per set (6). A visual statistical evaluation for the consistency of the unopposed B12 is shown in Figs. 3 and 4, using arbitrary units. A comparison of the two lots yields an insignificant difference (paired t-test, p = 0.9773, n = 42).

Fig. 3
figure 3

Results of unopposed vitamin B12 (arbitrary units) from two production lots: Exp1 and Exp2, taken after 3 and 9 month of continuous PCS cultivation, respectively; three samples per lot (samples A, B and C per Exp1; samples D, E and F per Exp2). The green diamond indicates statistical characteristics, the middle line indicates the average, and the top and bottom lines indicate the 95% confidence level

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Fig. 4
figure 4

Visualization of the experimental unopposed vitamin B12 values (arbitrary units) of the two lots: Exp1 and Exp2, taken after 3 and 9 months of continuous PCS cultivation, respectively. The green diamond indicates statistical characteristics, the middle line indicates the average, and the top and bottom lines indicate the 95% confidence level

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Table 5 expands on Table 4 and compares the ratio of cyanocobalamin to pseudo-cyanocobalamin found in PCS (at the ninth month of cultivation, see Table 4) with 13 previously analysed Spirulina-derived nutritional supplements [28]. A ratio smaller than one (< 1) indicates a net-pseudo vitamin B12 content.

Table 5 Comparison of vitamin B12 to pseudo-vitamin B12 ratios of PCS and 13 other Spirulina-derived nutritional supplements, analysed in van den Oever and Mayer (2022) [28]
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4 Discussion

4.1 Nutritional benefits of replacing ASF with PCS

To consider nutritional and health benefits of replacing ASF with PCS, beef meat is used as a benchmark due to the nutritional parity of the two in terms of protein content, essential amino acids, and bioavailable Iron.

The current research highlights an additional key nutritional aspect: unopposed active vitamin B12 content, which was found to be statistically consistent over 9 months of continues cultivation. At a concentration of 1.64 µg/100g, PCS is comparable and slightly superior to beef meat with a vitamin B12 concentration of 0.7–1.5 μg/100g [50].

While PCS shows comparable concentrations of protein, iron and vitamin B12 to meat from beef cattle (27.2 g/100 g protein in PCS versus 25.8 in beef, 0.03 g/100 g iron in PCS versus 0.02 in beef, see Tzachor et al. [41]), its integration into daily diets—instead of beef products—has the potential to mitigate, if not prevent, additional deleterious health implications.

Excessive consumption of beef has been associated with several health risks, including cardiovascular diseases and type 2 diabetes. This is primarily due to the high levels of saturated fatty acids (myristic, palmitic, and stearic) and low-density lipoprotein (LDL) cholesterol found in beef, which can contribute to the development of such chronic conditions [51]. The consumption of high amounts of red and processed meats has been further linked to an increased risk of colon cancer [52]. Additionally, the industrial production of beef has been linked to the use of antibiotics, such as tetracyclines [53], and growth promoting hormones that may have negative impacts on human health [54, 55].

4.2 Environmental benefits and GHG intensities of vitamin B12

Beef meat is also used as a benchmark to account for the environmental implications of replacing ASF with PCS as a source for vitamin B12 in diets. Replacing each kg of beef meat with a kg of PCS (at a comparable nutritional content)—when Spirulina is either consumed as whole food or as an ingredient (to fortify other foods)—would save 99.48 CO2-eq kg and 15.4m3 of water.

As a thought experiment, considering the GHG intensity of beef meat (99.48 kg CO2-eq per kg), and its concentration of vitamin B12 (1.5 μg/100g; taking the upper band), if all emissions are allocated to beef-derived vitamin B12 its GHG intensity would be approximately 6.632 million tonne CO2​-eq per kg of vitamin B12 (or 6.63 kg CO2​-eq per μg B12). In contrast, in the Hellisheidi facility, vitamin B12 is carbon neutral. This is a thought experiment, as beef serves more than a single dietary requirement in human diets and is not processed solely for satisfying vitamin B12 needs, nor is the vitamin extracted from beef for food fortification purposes.

Indeed, obtaining vitamin B12 from an autotroph (Spirulina), as opposed to a heterotroph (ruminants), is a resource-efficient strategy, especially if the former is cultured using a low environmental footprint technique, with no runoff and no environmental contamination.

4.3 Scale-up potential of vitamin B12 production in Iceland

Iceland, where the Hellisheidi facility is located, and where 99% of electricity is generated from renewable and non-intermittent resources (hydroelectric and geothermal), has a potential to scale up production of PCS-based vitamin B12 in order to satisfy the recommended dietary allowance of vitamin B12 (2.4 μg/day) for regional and global populations.

To assess this potential, this paper draws on a previous feasibility study that integrated data related to Spirulina cultivation in the Hellisheidi facility together with data related to installed and potentially installed energy generation in Iceland (including renewable energy sources), and accordingly calculated six production scale-up scenarios [56].

Table 6 shows six PCS production scale-up scenarios. In each scenario the energy allocated to biomass production is presented (in MWh) alongside annual biomass that could be produced, and the recommended dietary allowance figures for vitamin B12 to determine the populations whose demands can be met once scale up has been realized.

Table 6 Six scale up scenarios, including electricity allocations, PCS biomass production, and people provided with the recommended dietary allowance (RDA) of vitamin B12 (per year). For further data see Tzachor et al. [56]
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Exploring realistic scale-up scenarios, without the development of new renewable energy sources in Iceland, and by re-allocating 100% of the electricity currently consumed by heavy industry (mainly aluminium smelting), totalling 15,146,411 MWh per year, to production units such as the one operating in Hellisheidi, Iceland could produce 277,950 tonnes of Spirulina biomass per year, which translates into approximately 4555 g per year of active vitamin B12, able to satisfy the RDA of over 13.8 million children aged 1–3 (scenario 4 in Table 5).

In a more ambitious production scenarios (scenario six, where 8,711 g/year vitamin B12 are produced) could see Iceland providing the RDA of over 26.5 million children aged 1–3. This is approximately 44% of the 60 million children in the same age group across all of India (with 119.9 million children aged 0–4 in 2021) [57].

Reallocating 100% of Iceland's heavy industry electricity to PCS production would involve significant government intervention and policy adjustments, as well as substantial industrial repercussions. Governmental policy adaptations would include legislative changes for energy redirection, provision of economic incentives, and infrastructure investments. Such a shift stands to disrupt existing industries, namely aluminum smelting, leading to potential industry closures and job displacement. Government support would be essential for transitioning affected workers to emerging biotech roles, where possible. Concomitantly, the transition would alter national energy consumption patterns. The industrial impact would include a major economic shift from traditional manufacturing to biotechnology, potentially positioning Iceland as a global leader in sustainable bioproduct innovation. These issues, along with the need for government intervention to facilitate the proposed transition, justify further ex-ante techno-economic studies.

In terms of financing this scale-up, and mitigating vitamin B12 deficiency, one option is to consider the GHG emissions reduction associated with replacing ASF, specifically beef meat (a primary B12 source in daily diets) with PCS. In this option, the value of carbon emissions reduction arising from the replacement of beef with PCS could be used (as credits) to finance B12 production from cultivation through downstream processing outside factory gates to distribution in low- and middle-income countries.

Based on conservative figures for comparing PCS with beef meat, the production of 1 kg of PCS in the Hellisheidi facility can prove carbon neutral (− 0.008 CO2-eq per kg), while the production of 1 kg beef meat is estimated to emit 99.48 CO2-eq kg. By replacing 1 kg beef meat with 1 kg Spirulina, individuals may save nearly 100 kg CO2-eq of GHG emissions.

4.4 Reservations and future research

This study presents initial results of an in vitro experiment and sample analyses. Future studies should explore the mechanisms that increase the active form of Vitamin B12 with omics approaches, such as RNAseq and metabolomics. Moreover, in vivo research, that employs direct dietary interventions over time, is necessary to understand the impacts of PCS on human health. Considering Spirulina is FDA approved for human nutrition, such dietary intervention trials should meet no obstacles (for instance, if PCS is integrated as an ingredient in other foods so that these foods provide 20% or more of the recommended daily value of vitamin B12, the FDA should approve these foods as natural sources of active vitamin B12).

Furthermore, the usage of PCS as a potent vitamin and mineral source could support the transition from animal-based to well-balanced plant-based diets. This could be either in the form of nutritional supplements or as fortification in food products. However, due to possible unintended effects on organoleptic properties of the foods in which PCS is incorporated, further product development of such products is needed before reaching a mass-market.

Used to fortify foods, including traditional foods, PCS-based active vitamin B12 could be distributed extensively without risking the health of the general population. Drawing on these in vitro results the need for in vivo studies is emphasized to evaluate the effectiveness of PCS to mitigate vitamin B12 deficiency.

The feasibility of consuming 100 g of PCS daily, compared to the same quantity of beef, involves several technical considerations, particularly in terms of palatability, nutrition, and practicality. From a palatability perspective, consuming 100 g of PCS daily may be challenging due to its taste and texture, which may not be easily adaptable to a wide variety of dishes as beef. Moreover, a high intake could also pose risks of excessive micronutrient accumulation, such as iodine, which Spirulina can contain in large amounts. While PCS could be utilized to fortify other foods, achieving the RDA for certain nutrients solely through fortification may require careful formulation to avoid under-nutrition, thus complicating its use on a large scale. Therefore, while PCS offers substantial nutritional benefits, relying on it as a primary source to meet specific nutritional requirements like vitamin B12 through high daily intake or fortification may present challenges.

4.4.1 Directions for future research

Further research is needed to fully understand the implications of substituting animal-based vitamin B12 sources with PCS. This includes investigating potential nutritional deficiencies and the bioavailability of vitamin B12 from PCS in human diets. For an in vivo study we propose an 18–24 month randomized controlled dietary intervention trial to evaluate the residual beneficial effect of a Mediterranean diet enriched with PCS containing active vitamin B12 and reduced meat content. The trial could involve 300 adult volunteers, aged 30–60, willing to follow assigned dietary guidelines, and provide informed consent, randomized into three groups: a PCS-enhanced Mediterranean diet group, a standard Mediterranean diet group, and a control group following general healthy dietary guidelines. Conducted in a controlled workplace the trial will ensure close dietary monitoring and compliance. Participants will undergo regular health check-ups, and should not receive financial compensation. Such research will aim to assess the scalability of PCS as a sustainable B12 source and its integration into diets, potentially establishing it as a key component in improving public health outcomes.

In the same vein, there is need for future work to assess the bioavailability of vitamin B12 from PCS. This study could be designed as a controlled, crossover dietary intervention trial, involving healthy adult volunteers who will undergo a washout diet low in vitamin B12 prior to the intervention. Participants would then be randomly assigned to receive either PCS-based supplementation containing a quantified amount of vitamin B12 or a standard B12 supplement as a control. Each intervention phase would be followed by a washout period, and then participants would cross over to the alternate treatment. Blood samples would be collected at baseline, post-intervention, and at the end of each washout period to measure serum B12 levels and relevant biomarkers of B12 activity, such as methylmalonic acid and homocysteine, using standardized biochemical assays. The primary outcome would be the change in serum B12 levels, while secondary outcomes would assess changes in metabolic markers that reflect B12 utilization. This study design allows for a direct comparison of the bioavailability of B12 from PCS against a known standard, thereby providing a clear measure of its efficacy as a dietary source of the vitamin.

While comparing the nutritional and environmental benefits of PCS with beef, we recognize that direct vitamin B12 supplementation and fortification—through tablets or liquids—may be equally effective. These supplements often contain bioactive forms of the vitamin, such as methylcobalamin and cyanocobalamin, which ensure effective absorption and physiological utility. Although not the focus of this study, this approach could help achieve adequate vitamin B12 intake, particularly in diets that limit or exclude animal products. Understanding this is crucial for making informed dietary decisions, especially for those at risk of B12 deficiency.

While the current study focused on PCS cultivation in one specific site, and country, identical production systems may be implemented in other regions and settings where renewable energy sources (to maintain carbon neutrality) and concentrated CO2 (for improved photosynthesis) are available to support cultivation. Owing to the engineering of such systems, arable-land and favourable climate are not expected to limit production [58]. This would support the decentralization of production and bring the algae-based micronutrient closer to consumers, enhancing the food and nutritional security of food networks in low- and middle-income countries, where vitamin B12 deficiency is most acute.