Physicochemical Characterisation of Microalgal Biomass: Paving the Way for Industrial Exploitation

TL;DR

This study compares six microalgae strains to identify their biochemical properties, finding some as promising sources of protein, lipids, and unique compounds for food and feed.

Contribution

The study provides a unified biochemical characterization of six underexplored microalgal strains, identifying novel candidates for industrial use.

Findings

P. tricornutum and T. chuii have high protein content (31% and 41%) with essential amino acids matching commercial standards.

N. oceanica has elevated levels of beneficial fatty acids like palmitic and eicosapentaenoic acids.

Volatile organic compound analyses revealed unique aroma profiles in less-exploited strains.

Abstract

Arthrospira platensis and Chlorella vulgaris are popular commercialised microalgae due to their benefits and relatively easy large-scale cultivation. However, recent advances in biotechnology have revealed a new range of promising strains with industrial potential but limited current markets. To bridge the gap in the existing literature, this study provides a comprehensive and simultaneous biochemical characterisation within a unified analytical framework of six additional strains: Phaeodactylum tricornutum, Tetraselmis chuii, Nannochloropsis oceanica, Scenedesmus almeriensis, Tisochrysis lutea, and Skeletonema costatum. The analyses included macromolecular composition, amino acid and fatty acid profiles, and volatile organic compound composition. Key results identified P. tricornutum and T. chuii as high-quality protein alternatives, reaching protein concentrations of 31% and 41% (dw), respectively, with essential amino acid profiles (arginine and tryptophan) that match commercial standards. Additionally, specific carbohydrate and lipid strengths were identified: P. tricornutum showed a high carbohydrate content (37%), while N. oceanica exhibited elevated levels of palmitic, palmitoleic, eicosapentaenoic, and arachidonic acids, marking them as versatile candidates for nutritional applications. Finally, volatile organic compound analyses revealed distinct aroma profiles, highlighting the potential of less-exploited microalgal strains for the food and feed sectors.

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1. Introduction

Thousands of microalgal strains are preserved in culture collections worldwide, yet only a few have been extensively studied. Even fewer are being produced on a commercial scale, primarily for food applications. These include Arthrospira platensis (AP; commercially known as Spirulina) and Chlorella vulgaris (CV), both of which are mainly marketed as human food [1,2,3]. Commercial examples of products containing AP and CV include Spirulina BLU water (FUL Foods, Amsterdam, The Netherlands) and Bio. Algen cracker (FELIX Austria GmbH, Mattersburg, Austria). Other examples are Dunaliella salina, a source of β-carotene, and Haematococcus pluvialis, which produces and accumulates astaxanthin—both widely used as ingredients in food supplements and functional foods [4,5]. Commercial products include Natural Source Oceanic Beta Carotene capsules (Solgar, Leonia, NJ, USA) and Astaxanthin capsules (BioProphyl, Nitz, Germany). Today, most of the microalgal biomass is produced using open systems, which are cheaper to build and operate. In general, being open to the environment, these reactors support the production of only extremophile or fast-growing strains [6]. For example, AP is produced in alkaline media (pH 9.5–11.0) [7] and D. salina can grow well in media with a conductivity higher than 150 mS·cm^−1^ [8], limiting the growth of unwanted microorganisms. H. pluvialis is an exception. It is not an extremophile, but its capacity to produce and accumulate astaxanthin renders its production using controlled closed photobioreactors operated using artificial illumination economically viable [9]. Indeed, one of the main producers of H. pluvialis in Europe (Algalif, Reykjanesbær, Iceland) produces biomass in optimised controlled environments using renewable geothermal energy.

Recent advances in biomass production, including the optimisation of tubular photobioreactors, production indoors using artificial illumination or heterotrophic production using conventional fermenters, open novel opportunities to produce strains that are currently being understudied. These include Phaeodactylum tricornutum (PT) and Tisochrysis lutea (TL). Both strains are produced using tubular photobioreactors [10], but their market is still limited. The former is known to produce and store valuable products such as fucoxanthin or eicosapentaenoic acid (EPA) [11], while the latter has been suggested as a potential source of fucoxanthin and docosahexaenoic acid (DHA) [12]. However, in-depth characterisations of the composition of these strains have yet to be conducted. The same potential for industrial expansion applies to other emerging strains, such as Skeletonema costatum (SC), Nannochloropsis oceanica (NO), and Scenedesmus almeriensis (SA). While these are currently limited to niche markets like aquafeed, they show significant promise for broader applications. A key example of this transition is Tetraselmis chuii (TC), which was authorised in the European Union (EU) as a novel food in 2014 and as a food supplement in 2017, paving the way for its inclusion in the human diet.

Microalgae can be used in the food and feed industries as a source of proteins rich in essential amino acids [13], polyunsaturated fatty acids including EPA and DHA [14], bioactive pigments such as chlorophylls, phycobiliproteins, and carotenoids [15], and other valuable biomolecules including volatile organic compounds (VOCs) [16]. This study bridges a critical knowledge gap by performing a comprehensive biochemical profile of six promising, yet under-exploited, microalgae with significant potential for large-scale production. By comparing these against industrial strains (AC and CV) through a detailed analysis of nutritional and volatile organic compositions, the research identifies key functional attributes that will drive their future industrial adoption.

2. Materials and Methods

2.1. Microalgae Used

For a complete biochemical characterisation, the following eight microalgal strains were produced at CIESOL (Solar Energy Research Centre) microalgae demonstration plant, University of Almería (Spain): PT, TC, CV; NO, SA, TL, AP, and SC. The biomass production was carried out using 3.1 m^3^ tubular photobioreactors located inside a greenhouse (36°50′03.3″ N 2°24′09.8″ W). Three identical photobioreactors were used simultaneously, each photobioreactor being an experimental unit. Each photobioreactor was equipped with a 0.7 m^3^ bubble column 2.3 m tall with a diameter of 0.64 m. The bubble columns were continuously aerated with a constant airflow of around 150 L·min^−1^. The tubing systems was 28 m long with a tube diameter of 9 cm, arranged in seven parallel conduits on each side. The tubes were fabricated from methacrylate and had a total horizontal length of 400 m. The culture medium consisted of 1.25 g·L^−1^ NaNO_3_ (SQM, Santiago, Chile), 0.16 g·L^−1^ K_2_HPO_4_ (Yara, Oslo, Norway), 0.2 g·L^−1^ MgSO_4_ (I.M.S.A, Albacete, Spain), 0.1 g·L^−1^ CaCl_2_ (Tetra, Spring, TX, USA), and 5 mg·L^−1^ of a commercial mixture of micronutrients (Karentol®, Konegard, Barcelona, Spain) [17]. SA, CV and AP were produced using freshwater while the other strains were produced using seawater collected directly from the Mediterranean Sea. All the chemicals used were agricultural grade (fertilisers). The biomass production was done in batch mode until the stationary phase was reached. The biomass was harvested using a continuous centrifuge (Ortoalresa, Madrid, Spain), washed using tap water, freeze-dried and stored at −20 °C until further use. Figure 1 shows the colour of the eight freeze-dried samples.

2.2. Macromolecular Composition

Crude protein was measured employing the Kjeldahl method [18], using a protein conversion factor of 5.95. Lipid content was determined gravimetrically following the Folch method using chloroform/methanol (2:1 v/v) as solvent [19]. Moisture and ash content were determined following the standard EN 17605:2022 [20]. Carbohydrate content was calculated as the difference between 100% and the sum of proteins, lipids, ash, and moisture.

2.3. Amino Acid Profile

Total amino acid analysis of the microalgal biomasses was performed using a previously described methodology [21]. Briefly, 100 mg of dried biomass was hydrolysed using 6 N HCl at 110 °C for 24 h. The hydrolysate was then filtered using 0.2 μm Captiva nylon syringe filters (Agilent Technologies, Santa Clara, CA, USA), dried under a nitrogen stream, and resuspended in 2 mL of ultrapure water obtained from a Milli-Q system (Merck Millipore, Darmstadt, Germany). Then, 20 μL of this mixture was analysed using a HPLC system (Perkin Elmer Series 200; Perkin Elmer, Waltham, MA, USA) equipped with a fluorescence detector (Perkin Elmer Altus A-10; Perkin Elmer, Waltham, MA, USA). The separation used a linear gradient over 75 min (Phase A: methanol:acetonitrile, 12:1; Phase B: 23 mM NaOAc, pH 5.95) at flow rate 1 mL·min^−1^. The amino acid quantification was done in duplicate per natural replicate. Methanol was purchased from Honeywell (Morristown, NJ, USA) and acetonitrile and NaOAc were purchased from Sigma-Aldrich (Madrid, Spain).

2.4. Fatty Acid Profile

Fatty acid methyl esters (FAMEs) were prepared for the characterisation of the fatty acid profiles of the different microalgal samples as described in a previous work [22]. Briefly, approximately 1 g of the sample, 100 μL of internal standard (IS) solution (C23:0 methyl ester in heptane (Sigma-Aldrich, Madrid, Spain), 10 mg/mL), and 10 mL of potassium hydroxide in methanol (2.5%, w/v) were saponified (130 °C, 4 min) in a microwave system (MARS 6 Express 40, CEM Corporation, Matthews, NC, USA). Methanol was purchased from Honeywell (Morristown, NJ, USA) and heptane and potassium hydroxide were purchased from Sigma-Aldrich (Madrid, Spain). The samples were cooled to room temperature, and 15 mL of an acetyl chloride solution in methanol (5%, v/v) (Sigma-Aldrich, Madrid, Spain) was added to each sample to perform their methyl esterification in the microwave system (120 °C, 2 min). The samples were cooled to room temperature, followed by the addition of 10 mL pentane and 20 mL of saturated salt solution to allow the separation of the pentane layers containing FAMEs for analysis. Pentane was purchased from Sigma-Aldrich (Madrid, Spain). FAMEs were separated and quantified using gas chromatography with a flame ionisation detector (GC-FID) using a Clarus 580 gas chromatograph and a capillary column CP-Sil 88 (Agilent, Santa Clara, CA, USA; length: 100 m × 0.25 mm ID, thickness of film: 0.2 µm). The injection volume was 0.5 µL and hydrogen was used as the carrier gas at a flow rate of 1.25 mL·min^−1^. The initial oven temperature was 80 °C, which was increased to 220 °C (6.2 °C·min^−1^) and held at this temperature for 3.2 min before ramping to 240 °C (6.3 °C·min^−1^) and holding this final temperature for 6.5 min. Supelco^TM^ FAME mix (Sigma Aldrich, Arklow Co., Wicklow, Ireland) was used as the certified material for the identification of fatty acids, and the integration of the peaks was performed using TotalChrom 6.3.2 (PerkinElmer, Waltham, MA, USA). The final quantification of each fatty acid was performed based on the IS.

2.5. Volatile Organic Compound Profile

The VOC profiles were determined using a 7890 B gas chromatograph coupled to a 5977 A mass spectrometer (GC-MS), both from Agilent (CA, USA), equipped with an HP-FFAP capillary column (50 m × 200 µm × 0.33 µm; Agilent, CA, USA). The column flow was 1.44 mL·min^−1^. The oven was operated at 40 °C for 6 min and heated at a rate of 10 °C·min^−1^ to 250 °C and held for 3 min. The transfer line and ion source temperature were set to 250 °C, and the mass spectrometry was operated in full-scan mode in the 40–400 m/z range with hydrogen as carrier gas. The gas phase of the samples was extracted following a previous study [23]. The vacuum pump (V-300; Büchi, Flawil, Switzerland) was operated at 5 mbar. VOC desorption into the cooled injection system (CIS) equipped with a Tenax filled liner (both from Gerstel, Sursee, Switzerland) was done by heating the ITEX trap (BGB Analytik, Rheinfelden, Germany) to 300 °C with a desorption flow of 150 mL·min^−1^ for 4 min. The solvent vent flow for CIS operation was 20 mL·min^−1^. The CIS temperature programme started at 10 °C, was held for 4 min during desorption, and was then increased to 300 °C with a rate of 12 °C·s^−1^. After each sample, the ITEX trap was cleaned at 300 °C for 10 min under hydrogen flow. VOC identification with the mass spectrometry data was performed based on spectral comparison with the NIST database (Version NIST17, National Institute of Standards and Technology, Gaithersburg, MD, USA). Match factors above 80% were accepted for identification. Experimental retention indices (RIs) were determined by using an alkane mix (Merck, Buchs, Switzerland) and comparing the experimental RIs to the literature RIs listed in the NIST database as recommended by the Metabolomics Standard Initiative [24].

The detection of volatile sulphur compounds was performed with a 5380 pulsed-flame photometric detector (PFPD; OI Analytical, College Station, TX, USA) operated in sulphur mode at 250 °C. The GC column flow was split at a 2:1 (MS:PFPD) ratio. The PFPD detector is more sensitive to sulphur compounds than mass spectrometry. However, it does not produce spectra, and sulphur compound standards (Merck, Buchs, Switzerland) were measured for compound identification.

2.6. Statistical Analysis

All the samples were chemically analysed in triplicate (n = 3). The data were calculated and reported as the mean ± standard deviation. All statistical analyses were performed using R Studio software (version 2024.09.01.394). Differences in physicochemical attributes between microalgae were studied by one-way analysis of variance (ANOVA). When significant differences were obtained (p < 0.05), strain means were differentiated using a multiple range test (Tukey’s HSD post hoc test).

3. Results and Discussion

3.1. Macromolecular Composition

The macromolecular composition of the eight strains of microalgae studied is shown in Table 1. Overall, proteins were the most abundant macromolecule, followed by carbohydrates, and lipids, which is in line with what is known for most microalgal strains [25]. The only exception was PT, whose most abundant macromolecules were carbohydrates. Together with NO, PT has been suggested a promising source of lipids, including omega-3 oil for use in the food and feed industries [26,27]. ANOVA results showed significant differences in the protein content of the different strains (p < 0.001). For instance, AP (Arthrospira platensis) and CV (64.8%) had the highest protein content (p < 0.001). This was not unexpected as both strains are mainly produced for human consumption because of their high protein content and concentration of essential amino acids [28]. The protein concentrations of AP and CV are in line with the data reported in previous studies [25]. The other strains had a lower protein content, ranging from 31.2% (PT) to 41.1% (TC). Despite having lower protein content than AP and CV, levels in these microalgae (PT, TC, NO, SA, TL, and SC) are similar to or even higher than that of most common plant-based protein-rich foods according to the data available in the USDA FoodData Central (https://fdc.nal.usda.gov). For example, their protein levels are higher than those of lentils (~24%), chickpeas (~21%), almonds (~21%), peanuts (~23%), oats (~13%) or quinoa (~14%). This supports the role of microalgae as a remarkable protein source.

Carbohydrates were the second most abundant macromolecule in most strains (p < 0.001). The highest carbohydrate levels were found in PT, SA and SC, all of which had a similar carbohydrate content of approximately 30%. The lowest concentration (~15%) was found in AP, NO, and CV (p < 0.05). Microalgae-derived carbohydrates are actively being widely studied as a promising feedstock for producing third-generation biofuels, partially because microalgal carbohydrate metabolism can be modulated to promote their accumulation [29]. Moreover, significant differences were also observed in the lipid content (p < 0.001). The strains NO, TL, and TC showed the highest lipid content (~20–30%), whereas the biomass of CV and AP had a lipid content of 13.3% and 12.9%, respectively. No significant differences were found in the lipid concentrations of AP, CV, SC, SA, and PT. Microalgal lipids are a hot trend in different markets [30]. They were first identified as a feedstock to produce biodiesel; however, the yield from large-scale production facilities was far from the theoretical values and their production process remains under investigation [31]. Most of the production processes being developed today aim at using microalgal lipids in the food, feed, chemical and pharmaceutical/cosmetical industries [32]. Finally, significant differences were also observed in the ash content of the biomasses (p < 0.001), with values ranging from 6.8% (CV) to 17.7% (SC). The ash content of SC was significantly different from that of the other strains. These values were in line with previous work, such as a hierarchical Bayesian analysis of data compiled from the literature estimating the median content of ash in microalgae growing in nutrient-sufficient media as 17.3% [25].

3.2. Amino Acid Composition

Overall, as mentioned above, proteins were the most abundant macromolecules in all of the studied strains. A complete description of the essential and non-essential amino acid content of the studied strains is presented in Table 2. Regarding essential amino acids, arginine and tryptophan predominated, accounting for 14.3–26.8% of the total protein content (7.6–17.3% on a dry-weight basis). AP and CV contained ~10% arginine and ~6% tryptophan, respectively. These two strains are mainly used as food, especially as protein sources [33,34]. Their arginine and tryptophan levels were the highest (p < 0.05); however, no significant differences were found for the comparison of tryptophan levels of TC with both AP and CV. CV also presented a high isoleucine content (6.68%). This amino acid, with values ranging from 2.48% (TL) to 4.92% (AP), was the third most abundant in all strains except PT and TC, in which valine ranked third. The content of the remaining essential amino acids ranged from 0.65% lysine (SA) to 3.42% leucine (AP). Essential amino acids are crucial for survival because the human body cannot synthesise them; hence, they must be obtained through the diet. They serve as building blocks of proteins and play key roles in numerous physiological processes. Given that essential amino acids are mainly present in animal proteins, microalgae rich in essential amino acids are being commercialised as protein supplements for vegan consumers. Microalgae have also been previously used as a source of essential amino acids in wheat-based products, with small proportions of microalgae (1–4%) supplying a higher content of essential amino acids [13].

Among the non-essential amino acids, aspartic and glutamic acids were the most abundant in the microalgal strains, accounting for 18–23% of the total protein content (6–15% on a dry-weight basis). The biomass of AP and CV had the highest content of these amino acids, mainly because of their higher protein content. The biomass of AP contained 5.09% aspartic acid and 9.98% glutamic acid (21.7% of the total protein content), while CV had 4.24% and 8.13% (12.4% of the total protein content), respectively. A similar study reported a comparable content of these amino acids in AP and CV, at 24.5% and 18.8% of the total protein content, respectively [35]. The content of all non-essential amino acids in AP, with the exceptions of glycine (which showed no significant differences compared to CV, PT, and TL) and alanine (no significant differences compared to CV), were significantly different (p < 0.05) from those of other strains. Alanine was the third most abundant non-essential amino acid in all strains, except for NO and SC, where tyrosine and serine were more abundant.

3.3. Fatty Acid Composition

In this study, 12 major fatty acids (>0.5% of the total composition in any strain on a dry-weight basis) and 26 minor fatty acids (<0.5%) were identified. The results of the fatty acid determination are provided in Table 3. Overall, among the major fatty acids, myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1n7c), linolelaidic acid (C18:2n6t), α-linolenic acid (C18:3n3c), and eicosapentaenoic acid (C20:5n3c) were present at concentrations exceeding 1% in at least one strain. Meanwhile, oleic acid (C18:1n9c), vaccenic acid (C18:1n7c), linoleic acid (C18:2n6c), γ-linolenic acid (C18:3n6c), arachidonic acid (C20:4n6c), and docosahexaenoic acid (C22:6n3c) were detected at concentrations between 0.5 and 1.0% in at least one strain. Several minor fatty acids, such as caproic acid (C6:0), stearic acid (C18:0), lauric acid (C12:0), and lignoceric acid (C24:0), were detected, each contributing to less than 0.5% of the total composition on a dry-weight basis. These minor fatty acids were not included in the comparative analysis. Concerning the saturated fatty acids (SFAs), TL exhibited the highest concentration of myristic acid (1094.7 mg·100 g^−1^), followed by SC (983.4 mg·100 g^−1^) and NO (902.7 mg·100 g^−1^). The myristic acid concentration in TL was significantly different (p < 0.001) from that of all other strains. Its concentrations in SC and NO were also statistically different (p < 0.001). Palmitic acid was the most abundant in NO (3980.7 mg·100 g^−1^), with significant differences found compared to all the studied strains (p < 0.001). The second highest concentration of palmitic acid was found in CV (1926.5 mg·100 g^−1^). Moreover, significant differences were observed between the content of monounsaturated fatty acids (MUFAs) in the different strains studied (p < 0.001). For example, the highest concentration of palmitoleic acid was observed in the biomass of NO (3683.8 mg·100 g^−1^). The second highest concentration of palmitoleic acid was found in PT (1295.6 mg·100 g^−1^), followed by SC (927.7 mg·100 g^−1^). Oleic acid showed the highest concentration in CV (931.2 mg·100 g^−1^), which was significantly different from the other strains (p < 0.001). The second and third strains with the highest content of oleic acid were PT (659.9 mg·100 g^−1^) and NO (540.6 mg·100 g^−1^), with significant differences between them (p < 0.001). No significant differences were observed in the oleic acid content of TL and SA (~500 mg·100 g^−1^). CV also had the highest content of vaccenic acid (828.5 mg·100 g^−1^), with significant differences found compared to the other strains (p < 0.001).

Polyunsaturated fatty acids (PUFAs) are a type of dietary fat that are considered essential for humans [30]. They are characterised by having two or more carbon–carbon double bonds giving them unique properties. Several PUFAs were identified. Linolelaidic acid is an isomer of linoleic acid and is included in the group of omega-6 fatty acids. The highest concentration of this compound was observed in CV (2707.5 mg·100 g^−1^; p < 0.001). The remaining strains had very low levels of this fatty acid, ranging from 0 to 5.8 mg·100 g^−1^. For α-linolenic acid, SA exhibited the highest concentration (2374.7 mg·100 g^−1^; p < 0.001). The second, third, and fourth highest concentrations of α-linolenic acid were found in CV (973.8 mg·100 g^−1^), TC (636.0 mg·100 g^−1^), and TL (556.9 mg·100 g^−1^). α-Linolenic acid belongs to the omega-3 family and is a precursor of other essential fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. These two fatty acids also belong to the omega-3 family and are important for their health-promoting properties, including brain development and function. In this study, NO showed the highest concentration of eicosapentaenoic (3424.7 mg·100 g^−1^; p < 0.001), followed by PT (1776.9 mg·100 g^−1^) and SC (341.1 mg·100 g^−1^). Marine microalgae including Nannochloropsis strains are widely investigated as a source of eicosapentaenoic acid [36]. SA exhibited the highest concentration of linoleic acid (697.0 mg·100 g^−1^) and AP showed the highest γ-linolenic acid concentration (567.7 mg·100 g^−1^). The highest concentration of arachidonic acid was observed in NO (849.48 mg·100 g^−1^; p < 0.001). In the other strains the concentration of arachidonic acid ranged from 0 mg·100 g^−1^ (TL) to 26.6 mg·100 g^−1^ (SC). Finally, TL exhibited the highest docosahexaenoic acid concentration, with a marked difference compared to the remaining strains (0–53.7 mg·100 g^−1^).

Overall, NO showed the highest concentrations of palmitic, palmitoleic, eicosapentaenoic, and arachidonic acids, while TL had the highest concentrations of myristic and docosahexaenoic acids. In addition, CV was distinguished for its linolelaidic, oleic, and vaccenic acid content, whereas SA exhibited the highest α-linolenic and linoleic acid concentrations. AP had the highest concentration of γ-linolenic acid. The fatty acid composition of the studied strains, particularly in NO, reinforces their potential as a high-value source of PUFAs for nutraceutical applications [37].

3.4. Volatile Organic Compounds

Microalgae are widely recognised for their high nutritional value, offering a rich source of proteins, carbohydrates, and lipids [38]. However, aroma plays a pivotal role in consumer acceptance. Certain molecules impart unpleasant aromas to the dried biomass of algae and microalgae [39]. These include, among others, geosmin (earthy, musty), dimethyl sulphide (fishy, marine), and oxidised lipids (rancid) [40]. As aroma is shaped by a wide array of different VOCs, the analysis of VOCs is essential for both aroma characterisation and the evaluation of nutritional value. Additionally, the VOC profile provides valuable insights into the metabolic processes of microalgae. VOCs in microalgae can arise from several metabolic pathways, predominantly amino acid and fatty acid metabolism, and can serve as biomarkers for growth phase and culture fitness [41].

In this study, a diverse range of VOCs was identified in the different microalgae samples. The wide diversity of VOCs identified establishes a unique ‘sensory fingerprint’ for each strain, making it possible to strategically select microalgae based on the desired aromatic profile of the final food product [42]. These compounds were classified into various substance groups, including alkanes, alkenes, aldehydes, ketones, alcohols, acids, esters, amides, sulphur compounds, and unknown compounds. A comprehensive list of the identified compounds is provided in Table 4 and specific aroma and analysed properties are listed in Supplementary Table S1. The distribution of these substance classes among the microalgae is visualised in Figure 2. Sulphur compounds were examined separately using a pulsed-flame photometric detector, as their scale was not directly comparable to the mass spectrometry results. Alkanes, alkenes and alkynes are known to have a low aroma impact. Alkanes exhibited a wide distribution ranging from 1 to 48% among the microalgae, with the lowest proportion in SC and the highest in AP. By contrast, alkenes were present at levels below 12%. Aldehydes contributing fresh, green, herbal, spicy and fruity notes [42] were consistently present at levels of <4% across all samples, indicating a limited influence on the overall aroma profile. Ketones, typically associated with sweet, woody, fruity, floral, and herbal notes perceived as pleasant [41], ranged from 1 to 20%, with the highest abundance in TC and the lowest in NO.

Alcohols, which can impart alcoholic, ethereal, medicinal, fermented, and occasionally fruity or green notes, were detected in varying amounts, with the highest level of 35% in SC and the lowest (<1%) in AP. Although alcohol is often perceived as pleasant when moderate in concentration, its strong character can occasionally be overwhelming [38,43]. Acids exhibited the most extensive variation among the microalgae, ranging from <1% to 90% (SA vs. TL). Their characteristic sharp, sour and cheesy notes can be agreeable at low levels but become unappealing at higher concentrations. The presence of acids in dried microalgal powders can also influence the pH of aqueous solutions. Esters, known for their pleasant sweet, fruity and sometimes ethereal and creamy aromas reminiscent of grape, cherry, milk or vanilla, were generally present at low abundances (<5%) in TL, NO, PT, and SC. By contrast, significantly higher levels (44–55%) were found in SA, CV, and AP, contributing to their aromatic profile. The amide group was represented solely by acetamide with a generally low abundance (<1%); the molecule has no specifically known aroma. Sulphur compounds, despite their low abundance in NO, SA, CV, and AP (<1%), were notably more prevalent in other samples (17–30%). As the human olfactory system is highly sensitive to sulphur compounds (odour thresholds in water of 6.65–80 ppb; see Supplementary Table S1), even small amounts can have a significant impact on the perceived aroma. Sulphur compounds can evoke aromas similar to those in cabbage, onion/garlic, fish, and rotten eggs [38]. The influence of the VOCs we categorised as unknown compounds is not discussed, as their structures and substance classes are unclear.

The high abundance of esters and the low abundance of sulphur compounds (especially dimethyl sulphide, which is associated with a fishy taste) in CV and AP could explain why they have a long history of use as food [44]. Moreover, SA is identified as providing a pleasant aroma with a low amount of sulphur compounds comparable to the aroma of CV. TC has been found to possess a balanced, complex aroma with many pleasant compound groups; however, it also has a high level of sulphur compounds and acids, which could affect the pleasant aroma.

4. Conclusions

Interest in microalgae as a sustainable food source is rapidly increasing; however, to date, industrial-scale exploitation has been predominantly limited to two species: A. platensis and C. vulgaris. This study investigated six alternative microalgal strains (Phaeodactylum tricornutum, Tetraselmis chuii, Nannochloropsis oceanica, Scenedesmus almeriensis, Tisochrysis lutea, and Skeletonema costatum) demonstrating their significant potential for industrial-scale applications in the food sector by offering a nutritional density comparable to these established commercial standards.

Proteins were the most abundant macromolecule for most of these organisms, with concentration values ranging from 31.2 to 65.9% and high levels of essential amino acids, notably arginine and tryptophan. In addition to their protein profile, several microalgae showed carbohydrate contents of approximately 30%. Furthermore, the strains NO, TL, and TC exhibited the highest lipid content, highlighting their potential for diversified applications in the food sector.

Different volatile organic compounds were observed among the strains studied, playing an important role in determining the aromatic potential for industrial applications. Strains belonging to the genera Chlorella, Arthrospira, and Scenedesmus presented high ester and low sulphur compound levels, contributing to a pleasant, fruity aromatic profile favourable for sensory acceptance. In contrast, the complex aroma with high acid and sulphur contents of other strains could negatively impact palatability, requiring specific technological processing, such as deodorization, to improve their organoleptic properties. Finally, while strains like T. chuii are already authorised in the EU, the industrial exploitation of the other promising strains must still address regulatory and safety considerations.

The detailed physicochemical profiling provided here establishes a robust analytical baseline of alternative microalgal strains, positioning these less-exploited microalgae as high-value alternatives and paving the way for their formal recognition as novel foods in the global market.