Docosahexaenoic acid (DHA, C22:6n-3) is an omega-3 polyunsaturated fatty acid (PUFA) mainly found in human’s brain and eyes exhibiting several beneficial health effects mainly in prevention and potential treatment of neurodegenerative diseases such as Alzheimer’s and Parkinson’s in addition to eyes’ diseases (Hachem and Nacir, 2022; Hachem et al., 2016; Belkouch et al., 2016; Hachem et al., 2020). DHA can be found in several food sources including fish and salmon. Moreover, DHA can be produced from microalgae.

Indeed, algae are a group of photosynthetic organisms found in various aquatic ecosystems, such as rivers, oceans, and ponds. They can tolerate the variation of several environmental parameters, including temperature, salinity, pH values, and light intensity. They can also endure harsh conditions in deserts, reservoirs and grow alone or in symbiosis with other organisms (Khan et al., 2018). Moreover, algae encompass various groups of photosynthetic organisms including microscopic unicellular microalgae and complex macroalgae, also known as seaweed. More recently, algae have received substantial attention due to several features, including remarkable nutritional profiles and potentials in food and pharmaceutical applications (Wu et al., 2023; Kumar et al., 2021; Kent et al., 2015).

Similar to other photosynthetic organisms, algae can synthesize various bioactive compounds known as secondary metabolites (Cardozo et al., 2007; Pereira et al., 2021). Metabolites are produced at the end of the growth phase or due to abiotic/biotic stress induced by the surrounding environmental conditions. These bioactive natural metabolites produced from algae including phycobiliprotein pigments, phenolic compounds, carotenoids and polysaccharides exhibit a wide range of biological properties, such as anticancer, antimicrobial, and antioxidant activities (Shalaby, 2011; Roleira et al., 2015; Avila-Roman et al., 2021; Hou and Cui, 2018). Additionally, microalgae can produce several lipids-based compounds, including fatty acids (FAs) and PUFAs such as DHA (Remize et al., 2021). FAs are considered as one of the major groups of microalgae biomass and generally constitute 5%–60% of cell dry weight (Morales et al., 2021).

Among those microalgae, Thraustochytrids, categorized as Stramenopiles, are heterotrophic marine protists capable of producing vast quantities of PUFA including DHA (Nyunoya et al., 2022). Additionally, Crypthecodinium cohnii is another heterotrophic marine dinoflagellate considered as a rich producer of DHA. DHA’s purification process from C. Cohnii is appealing since it significantly produces DHA, rather than any other PUFAs, utilized in pharmaceutical and nutraceutical applications (Mendes et al., 2009). Moreover, Isochrysis galbana is an example of microalgae producing DHA. Indeed, I. galbana can grow in harsh environmental conditions and possess a high growth rate and productivity. It is considered also as a rich source for other valuable products significant for human health such as vitamins, polysaccharides, sterols and carotenoids (Señoráns et al., 2020). Likewise, Nannochloropsis oculata is one more rich source of DHA and EPA (Nuño et al., 2013). Furthermore, Aurantiochytrium limacinum (A. Limacinum), a thraustochytrid strain, is considered an ideal source of DHA constituting 1.43%–29.6% of total FAs (Sirirak et al., 2020) where the composition of FAs is generally determined as fatty acids methyl esters (FAMEs) using Gas Chromatography coupled with Flame Ionisation Detection (GC-FID) (Ichihara and Fukubayashi, 2010).

While GC-FID approach was extensively applied for FAs profiling in microalgae (Visudtiphole et al., 2021; Dillon et al., 2019; Jarle Horn and Klüver, 2025), the combination of the GC power with the sensitivity of Isotope Ratio Mass Spectrometry (IRMS) was recently introduced to microalgal systems, through analysis of stable isotope ratios (carbon-13, hydrogen-2, etc.) of individual molecules. This approach is known as Compound-Specific Isotope Analysis (CSIA) (Pilecky et al., 2021; Xin et al., 2024). In CSIA, the isotopic composition is expressed as δ¹³C values, which denote the ratio of ¹³C–¹²C in a sample relative to a standard (Mašalaitė et al., 2012). Indeed, δ¹³C-based metabolic tracing is a prevailing approach used to investigate lipids’ biosynthesis in microalgae through following the stream of ¹³C-labeled carbon in metabolic pathways (Twining et al., 2020; Remize et al., 2022). Through analysis of δ¹³C values of diverse lipids, researchers can identify the active metabolic pathways in lipid biosynthesis (Zhang and Liu, 2021) and how fast carbon flows via these pathways (Xin et al., 2024; Meier-Augenstein, 2002; Rhee et al., 1997).

Despite the application of CSIA in microalgal systems, no fundamental research was conducted till day on the application of Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) for CSIA at natural abundance levels mainly in relation to DHA biosynthesis and metabolic flux changes between growth phases in A. limacinum. Hence, the objective of the present study was to first optimize the culture of A. limacinum and investigate the FAs profiling through GC-FID. Then, we developed a novel CSIA approach to estimate δ¹³C values at natural abundance level in FAs produced using GC-C-IRMS.

The life cycles of thraustochytrids are extremely reliant on nutrition. When cultivated in artificial media comprising yeast extract or peptone, thraustochytrids can accumulate great quantities of biomass and lipids, making them very attractive for biotechnological applications (Dellero et al., 2018). It is well recognised that lipid production is influenced by the composition of the culture media and differs with the life cycle. Such fluctuations are associated with significant environmental/cultivation restrictions including the C:N ratio. Certainly, a carbon source is mandatory for these heterotrophic cells to produce FAs and lipids. This carbon source can be assumed through glucose or glycerol addition to the culture media. The FA compositions of several thraustochytrid types have been investigated in order to evaluate their content in terms of several PUFAs mainly DHA. Diverse culture environments (changing the source of carbon, oxygen concentration, salinity, temperature, nitrogen accessibility, etc.) were tested to determine the effect of the culture medium on FAs’ production and unsaturation (Zhu et al., 2007). Previous researchers have showed that thraustochytrid types produced mainly C16:0 and DHA, the latter varying from one species to another. The genera Aurantiochytrium, and Schizochytrium were the most productive of DHA (Fossier Marchan et al., 2018). Certainly, the medium composition is an important parameter, the presence of glucose or glycerol as a carbon source and yeast extract or peptone as a nitrogen source allowing the highest yield (Aasen et al., 2016).

In the present study, A. limacinum cells were grown on glucose with a natural carbon isotope abundance in the presence of peptone, yeast extract as a nitrogen source allowing the highest yield and sea water collected from Persian Gulf. To avoid bacterial proliferation, three different antibiotics were added to the culture medium. All experiments were conducted in biological triplicates. Within 2 days of cultivation, the cell count was 5.53 × 10⁶ per ml of the culture, whereas, after 7 days, the cell count increased by a factor of 10⁴ and reached 5.35 × 10¹⁰ per ml of the culture. No bacterial growth or contamination were observed during the full culture’s period.

Glucose decreased in the culture medium to 51% ± 18% on day 7. We could have increased the cultivation duration to 9 or 10 days, but it could be risky mainly related to bacterial proliferation and death of cells. The carbon conversion yield, defined as the amount of biomass produced per Gram of substrate consumed, was 0.08 g biomass/g glucose. This fairly low conversion yield is steady with literature values for Aurantiochytrium strains with comparable heterotrophic cultivation, where an important fraction of carbon is oriented toward lipid accumulation (especially PUFAs) rather than biomass. Moreover, some carbon may be missed as CO₂ as a result of respiratory metabolism or diverted into non-biomass cellular components including extracellular polysaccharides or secondary metabolites. Furthermore, the high standard deviation observed in glucose consumption might represent natural biological variability among different cultures. During the stationary phase, this could reflect metabolic heterogeneity in response to nutrients in A. limacinum influencing glucose consumption and lipid biosynthesis.

A good quantity of biomass was collected with 72 ± 8 mg and 197 ± 36 mg on day 2 and day 7 of cultivation, respectively. Total lipids produced were around 16% and 52% on day 2 and day 7 of cultivation, respectively and these results aligned well with other researchers who reported that A. limacinum typically accumulates lipids up 30%–60% of its dry weight lipids at stationary phase (Hoshino et al., 2016). From the FAs profiling, we observed that at an exponential phase, DHA was the most copious FAs with 65.6% of total FAs followed by C16:0 with 34.4%. However, at the stationary phase, DPA and DHA were the most abundant FAs with 45.4% and 33.9%, respectively. Furthermore, C14:0, C14:1n-9, C16:0 were 4.6%, 6.2% and 9.9%, respectively. Indeed, the pragmatic decline in DHA abundance from 65.6% in the exponential phase to 33.9% in the stationary phase might be an indicative of a metabolic flux redirection, probably related to the physiological transition of A. limacinum from active growth to lipids’ accumulation. Throughout the exponential phase, cells prioritize membrane lipid biosynthesis and speedy proliferation (Soto-Sánchez et al., 2023), with high DHA level playing a fundamental structural role in PL-rich cellular membranes. Contrarily, during the stationary phase, the metabolic focus moves toward the accretion of storage lipids mainly triacylglycerols (TAGs), less enhanced in DHA and more connected to saturated and monounsaturated FAs. This metabolic rerouting might decrease the flux toward PUFA biosynthesis, counting DHA, thus dropping its proportion in total FAs pool. These findings are aligned with those in the literature showing that, when thraustochytrids including Aurantiochytrium and Schizochytrium are grown up in suitable environments, they can accumulate DHA with almost around half of the total FAs (Coplen et al., 2006; Brand and Coplen, 2012).

When investigating the CSIA using GC-C-IRMS at ¹³C natural abundance level in FAs, we found that δ¹³C signature of all FAME were negative suggesting that FAME were depleted in ¹³C since the cells were grown on ¹²C-glucose and not ¹³C. Indeed, for negative values of δ¹³C, the carbon isotope fractionation often happens in the process of fatty acids biosynthesis, and more ¹²C is used. Thus, providing a lower δ¹³C (Kelleway et al., 2018). The δ¹³C (‰) of C16:0 and DHA were −16.8 ± 0.2 and −18.5‰ ± 0.1‰, respectively, at the exponential phase while at the stationary phase, δ¹³C (‰) of C14:0, C14:1n-9, C16:0, C22:5n-3 and DHA were −10.6 ± 1.1, −11.3 ± 0.1, −11.1 ± 0.2, −8.3 ± 0.2 and −10.6‰ ± 0.1‰. respectively.

Notably, the exponential phase displays more negative δ¹³C values for DHA at −18.5‰ compared to the isotopic enrichment of DHA in the stationary phase with −10.6‰, reflecting a further direct and effective accommodation of ¹²C-rich glucose via central carbon metabolism and suggesting a shift in carbon source utilization or carbon allocation efficiency between growth phases. As glucose concentration decrease in the culture medium, cells might use internal carbon reserves or alternatives, which have a tendency to be comparatively ¹³C-enriched. Moreover, the recycling of intracellular carbon via anaplerotic paths or the improved involvement of endogenous CO₂ fixation may decrease isotope fractionation, ensuing less negative δ¹³C values (Bowling et al., 2008). Together, these results support the hypothesis that carbon flux redirection and nutrient-driven metabolic remodeling meaningfully impact both FAs profiles and their isotopic signatures throughout diverse growth phases (Kobayashi et al., 2020; Mang Gao, 2013; D.H. et al., 1998).

During the stationary phase, the δ¹³C value of DPA was remarkably less negative (−8.3‰) compared to DHA (−10.6‰). This isotopic enrichment in DPA may reflect underlying variances in metabolic pathways and enzyme activity during FA biosynthesis. Additionally, DPA and DHA biosynthesis in A. limacinum likely proceeds via the polyketide synthase (PKS)-like pathway or the elongase/desaturase pathway. In the latter case, DHA is naturally derived from DPA via a Δ4 desaturase enzyme, and DPA in turn is synthesized from eicosatetraenoic acid (ETA) or arachidonic acid through elongation and Δ6 desaturation. The less negative δ¹³C of DPA may consequently indicate that it serves as an intermediate pool which accumulates during the stationary phase, possibly due to reduced Δ4 desaturase activity or altered enzyme kinetics under nutrient-depleted conditions. Furthermore, this isotopic pattern might be linked to carbon source allocation preferences and fractionation effects. The carbon flux during the stationary phase is likely redirected toward energy storage and structural lipid synthesis, with selective incorporation of isotopically heavier carbon (¹³C-enriched) into certain intermediates like DPA. This could occur due to kinetic isotope effects in elongase or desaturase enzymes or due to precursor channeling dynamics where isotopically lighter carbon is preferentially routed to DHA synthesis or other metabolic sinks.

Furthermore, at stationary phase, the higher standard deviation detected for the δ¹³C value of C14:0 (±1.1‰) is steady with its lower relative abundance and better sensitivity to slight shifts in precursor accessibility or enzymatic turnover. Since C14:0 is a trivial FA in the stationary phase, minor variations in its biosynthesis might lead to amplified isotopic variance. Thus, these differential δ¹³C signatures provide insight into the dynamic regulation of PUFA biosynthesis under varying physiological conditions and supports the utility of GC-C-IRMS in elucidating pathway-level isotope fractionation.

The newly developed CSIA method can be applied in the context of several studies focusing on targeting the brain with PUFAs, especially DHA to estimate the ¹³C natural abundance level in FAs. To purify different FAs including DHA produced from A. limacinum, several approaches could be applied including solid phase extraction (SPE) as well as HPLC. The purified PUFAs including DPA and DHA can have several beneficial applications mainly in nutrition and health sectors.

Aurantiochytrium limacinum can be considered as a sustainable and biotechnological source for DHA production for medical applications. The newly developed CSIA at natural abundance levels in microalgal systems could allow a better understanding of the dynamic regulation of PUFA biosynthesis as well as the metabolic flux discrepancies between growth phases supporting the significance of GC-C-IRMS in explicating pathway-level isotope fractionation.