Abstract Euglena gracilis is a photosynthetic Euglenophyte capable of carbon dioxide fixation and lipid synthesis, offering a sustainable approach to renewable energy production. This study investigates the effects of mono- and multi-light spectra on the growth and fatty acid methyl ester (FAME) production of E. gracilis. The cultures were subjected to various light spectra, including red, blue, purple, red-blue, and white (control), to analyze their impact on growth rates, lipid accumulation, and FAME profiles. Growth metrics, including cell density, biomass, specific growth rate, and doubling time, were recorded, and FAME analysis was performed using GC-FID. Results indicated that purple light significantly enhanced growth, achieving the highest saturated fatty acid (SFA) content (31.7%), while red-blue light yielded the highest lipid concentration (0.680 ± 0.028 g L⁻¹). In contrast, blue light promoted the production of unsaturated fatty acids (UFA), with a composition of 74.62%. This study contributes to expanding the FAME profile database and highlights the potential of E. gracilis in Indonesia as a biodiesel source, particularly in the context of ecological modifications such as light spectrum optimization. The findings underline the organism's viability as a sustainable biofuel source, supporting efforts toward renewable energy development.

Keywords: Euglena, FAME, Lipid, SFA, UFA

Funding: This work was funded by the Ministry of Education, Culture, Research, and Technology, Republic of Indonesia, under the Enhancing International Publication Program 2023 [Grant number 165.16/E4.4/KU/2023].

Citation: Maghfiroh, K. Q., Budiman, A., Retnoaji, B., Suyono, E. A. 2025.  Fatty acid methyl ester production from Euglena gracilis through mono and multi-light spectrum modification. Natural and Life Sciences Communications. 24(2): e2025027.

INTRODUCTION

Given the limited availability of fossil fuels, and the presence of issues of food security and energy balance in the 1^st & 2^nd generation biofuel, there is a growing interest in biodiesel production from microalgae (Patnaik and Mallick 2021). Traditionally, producing vegetable or animal oils for biodiesel affects food security by competing for land and agricultural resources (Ogbonna et al., 2024). In contrast, fatty acids derived from microalgae do not disrupt food systems or other resources. Efficient biodiesel production depends on identifying novel microalgae strains with high lipid output, desirable fatty acid composition, and rapid growth rates (Vichachucherd, 2019; Ogbonna et al. 2024). Microalgae also offer a cost-effective solution for capturing and storing CO₂, with the ability to produce 1 kg of dry biomass by capturing approximately 1.83 kg of carbon dioxide (CO₂) through fixation (Herold et al., 2021). This can improve overall environmental quality, even in contaminated areas. Euglena can use carbon dioxide and water to produce large organic molecules, such as lipids. The lipid content varies depending on the growth conditions. Euglena exhibits a relatively high specific growth rate (SGR) of 0.168 µ (Maghfiroh et al., 2023), resulting in rapid species growth, increased metabolite accumulation, and enhanced pollutant absorption.

Algae exhibit a high degree of metabolic flexibility, allowing them to adapt rapidly to changing environmental conditions. It is well-established that factors such as salinity, temperature, nutrient availability, light, and cultivation duration influence the biochemical composition and growth of microalgae (Guihéneuf et al., 2015). The accumulation of polyunsaturated fatty acids (PUFA) in algal cells can also be affected by light wavelength and illumination duration. Previous studies have shown that different wavelengths result in varying effects on biomass production and lipid content. For example, Nannochloropsis oculata, Nannochloropsis oceanica, and Nannochloropsis salina experience accelerated growth under blue light-emitting diodes (LEDs) and achieve maximum lipid concentrations under green LEDs. A two-phase culture approach, where blue LEDs are used first to promote growth and green LEDs in the second phase to enhance lipid content, resulted in lipid compositions of 52%, 53%, and 56% for N. oculata, N. oceanica, and N. salina, respectively (Ra et al., 2016). Additionally, research on Scenedesmus obliquus demonstrated optimal biomass productivity and lipid content under both blue and red LEDs, outperforming results obtained under white light. A combination of blue and red illumination in a 1:1 ratio has been found to produce better outcomes compared to using a single wavelength (Abomohra et al., 2019). This indicates that each species responds differently to light stress, particularly when exposed to specific light spectra.

Under stress conditions, lipids are one of the metabolites produced by microalgae. In normal conditions, microalgae create generate two types of lipids: structural lipids and storage lipids. Triacylglycerols (TAG), which contain both saturated fatty acids (SFA) and unsaturated fatty acids (UFA), undergo transesterification to produce biodiesel, a process that converts stored lipids into fuel. Microalgae cells produce minimal TAG during the exponential growth phase but accumulate large amounts of TAG during the stationary growth phase. Among the various lipid categories, fatty acid (FA) composition can vary. Monounsaturated fatty acids (MUFA) are the primary fatty acids stored as lipids, while structural lipids mainly consist of polyunsaturated fatty acids (PUFA). Triacylglycerols are neutral lipids comprising three esters, where a glycerol molecule is bound to three long-chain fatty acids. During transesterification, alcohols such as methanol replace glycerols in the process (Ogbonna et al., 2024). 

To enhance the production of secondary metabolites like lipids, it is essential to prioritize optimal conditions for Euglena growth during the initial phase. Once growth conditions are refined and substantial biomass is achieved, the culture can then be exposed to light-induced stress, triggering the synthesis of the desired secondary metabolites. The optimal light spectrum for the growth of plants and microalgae includes blue light within the 400–500 nm range, red light spanning 600–700 nm (Helena et al., 2016), and far-red light between 700–800 nm. Chlorophyll a and b are most efficient at absorbing photons in the blue range of 400–480 nm and the red range of 630–680 nm (Kamolrat et al. 2023). This study is grounded on analyzing how Euglena chlorophyll absorbs light across various spectra to evaluate its impact on growth and secondary metabolite production. The research focuses on red, blue, purple (a blend of red and blue), and their combinations as key light variables to explore.

Despite advancements in science, no research has yet examined how light spectrum modifications affect Euglena's ability to produce FAME profiles. This discovery serves as a critical indicator for assessing the potential of Euglena as a biodiesel source. This study aimed to identify FAME types from E. gracilis, considering ecological adaptations and habitat characteristics in Dieng Peatland. The FAME profiles and lipid productivity derived from this research can be used to evaluate E. gracilis potential as a biofuel source. Furthermore, these findings could contribute to the development of the FAME industry as a leading source of renewable energy, particularly from plant-based sources, including microalgae.

MATERIALS AND METHODS

Culture stock preparation

The strain used in this study is E. gracilis, which was purified during a previous research project (Maghfiroh et al., 2023). The organism was successfully isolated from peatlands in an extreme ecological location in Dieng, Indonesia (7°12'33.84"S, 109°54'43.2"E), characterized by a pH level of 2.5. After screening alongside other strains, E. gracilis was identified as the optimal strain for growth. The strain was subsequently cultivated in 3 liters of Cramer Myers (CM) medium (the composition of the medium is provided in Table S1), using continuous light, until the optical density (680 nm) reached approximately 0.7–0.9. Research indicates that Euglena prefers ammonia nitrogen over nitrate nitrogen as its nitrogen source. It has the capability to utilize diverse carbon sources to boost its growth. Additionally, Euglena requires vitamin B1 and B12 to support its proliferation. Over time, culture media have been extensively refined for Euglena, leading to the development of Cramer–Myers (CM) medium (pH 3.5) as a fundamental autotrophic growth medium for this organism (Suzuki et al., 2015).

Cultivation of E. gracilis

Aeration-based cultivation was performed in 1-liter bottles at a controlled temperature of 24 ± 2°C. The culture was supplemented with 700 mL of steril CM medium, resulting in a final volume of 900 mL. Light treatment was applied using continuous light and consistent intensity of 100 µmol photons m⁻²s⁻¹ from Avaro Smart Lamp LEDs (PT Avaro Inovasi Teknologi, Jakarta, Indonesia). The light spectra used included red LED (685 nm), blue LED (470 nm), purple LED (415 nm), red-blue LED (685 and 470 nm), and white light, which served as the control. Cultivation lasted for 12 days, extending through the death phase, based on preliminary exploratory studies on Euglena growth.

Determination of growth rate

The growth of E. gracilis was analyzed using specific growth rate (µ), doubling time (Td), daily cell counts, and biomass measurements taken every three days. Biomass analysis was carried out using a Rocker 300 Vacuum Filtration System (167311-22, Rocker Scientific Co., Ltd., New Taipei City, Taiwan) and 1822-047 Cytiva Whatman Glass Microfiber Filter Paper GF/C (Global Life Sciences Solutions, Marlborough, Massachusetts, USA). A 10 mL sample underwent filtration, followed by drying in an oven 100°C for 1 hour and mass measurement. The values for Td and SGR (µ) were calculated using Daneshvar’s formulas (1) and (2) (Suyono et al., 2024), as shown below:

N₀: Number of cells on day 0

N_t: Number of cells on day t

t: Time interval

Determination of lipid production

Lipid synthesis in E. gracilis was assessed at 3-day intervals using a modified methodology from Wardana et al. (2023). Specifically, 5 mL of biomass was centrifuged at 4,000×g for 5 minutes. The pellet was dissolved in a mixture of 1 mL chloroform and 1 mL methanol, then vortexed until a uniform consistency was achieved. Following this, 1 mL of distilled water was added, forming three distinct layers. The upper and middle layers were removed, and the lower layer was transferred to a container for evaporation, after which its weight was measured as the total lipid content as shown in the formula (3) (Agus Suyono et al. 2024), as shown below:

Fatty acid profiling using GC-FID

The analysis of saturated and unsaturated fatty acids was performed using a gas chromatograph equipped with a flame ionization detector (GC-FID) through two processes: hydrolysis and methylation. Hydrolysis involved adding 5 mL of HCl to the Euglena pellet and heating it to 80°C for 3 hours. After cooling, extraction was carried out using 10 mL of diethyl ether and petroleum ether (1:1), vortexed, and left to separate. The top oil layer was collected and evaporated in a water bath with nitrogen (N₂). During the methylation stage, 0.5 mL of oil from the hydrolysis process was mixed with 1.5 mL of sodium methanolic solution and heated at 60°C for 10 minutes with agitation. After cooling, 2 mL of boron trifluoride methanoate was added and heated again at 60°C for 10 minutes. The extraction was carried out with 1 mL of heptane and 1 mL of NaCl. The top layer was collected, placed in a GC vial, and 1 µl of the sample was injected into an Agilent Technologies 7890B GC (Santa Clara, California, USA). Details of the procedure are provided in Table 1.

Table 1. Detailed settings of GC-FID method for identifying fatty acids.

Data analysis

Quantitative data were evaluated using ANOVA (analysis of variance) with a 95% significance level to determine the effects of the treatments. Duncan's Multiple Range Test (DMRT) was conducted at the same significance level to explore variations in detail. Data analysis was performed using SPSS 25.

RESULTS

Growth rates of E. gracilis under different light spectra

The results of the purple light treatment showed significant differences (P < 0.05) compared to other treatments, including the control. Data indicate that cell number, biomass, specific growth rate, and doubling time all demonstrate that purple light significantly (P < 0.05) enhances Euglena growth (Table 2).

Table 2. Growth rate of E. gracilis under different light spectra.

Note: Lowercase letters indicate significant differences based on Duncan’s test (P < 0.05).

The cell count under purple light reached 212 ± 24 × 10⁴ cells mL⁻¹, which is 52% higher than the control. The biomass value was 0.99 ± 0.100 gL^-1, 30% greater than the control treatment. The specific growth rate (SGR) was 0.27 ± 0.02 µ, 29% higher than the control, resulting in the shortest doubling time. All these results were significantly different (P < 0.05) from other treatments. The blue light treatment produced a cell count comparable to the control, while its biomass was 13% higher, suggesting that the biomass per cell was greater under blue light conditions. Although the red and red-blue light treatments showed lower growth parameters compared to the control, other findings depicted in Figure 1 clearly demonstrate that Euglena experienced substantial population growth from the initial period (days 0-5) to the exponential phase (days 6-9) in all treatments except the control. The increase in cell count indicates that Euglena peaked on day 9, while the control peaked on day 10. Afterward, cell growth declined as they entered the death phase.

Figure 1. Cell density of E. gracilis from the lag phase to the death phase. Error bars represent standard deviation (n=3).

Lipid production rate from E. gracilis

The objective was to identify an optimal approach for enhancing the production of specific lipid molecules. Figure 2 shows the highest lipid content, with significant differences between treatments (P < 0.05), achieved under the red-blue light treatment, which utilized multiple wavelengths as the culture light source. This treatment produced a lipid content of 0.680 ± 0.028 gL^-1. The application of light at 685 and 470 nm wavelengths enabled E. gracilis to generate more lipids than the control and other treatments. The other treatments showed lower lipid levels than the control group (0.660 ± 0.061 gL⁻¹), with purple light yielding 0.580 ± 0.042 gL⁻¹, blue light at 0.480 ± 0.010 gL⁻¹, and red light producing the lowest lipid content at 0.320 ± 0.046 gL⁻¹.

Figure 2. Lipid content of E. gracilis under different light spectra. Lowercase letters in the same column indicate significant differences based on Duncan’s test (P < 0.05).

FAME profile of E. gracilis under different light spectra

The fatty acids profile derived from this study showed significant variations across treatments. The treatments, except for the control, produced the highest quantities of fatty acids, highlighting the importance of selecting the appropriate wavelength to specifically target desired fatty acids. Table 3 presents the profiles of saturated fatty acids (SFA) generated under different light spectrum treatments. The results indicate that the purple light treatment resulted in the highest percentage of SFA at 31.7%, followed by red light at 29.90%, red-blue light at 28.28%, white light (control) at 28.04%, and the lowest SFA level in blue light at 25.38%.

The red light treatment produced the highest amounts of methyl arachidate (14.89%) and methyl tricosanoate (1.33%). Blue light treatment resulted in the highest production of methyl myristate (8.95%) and methyl heptadecanoate (4.39%). Optimal production of other SFA, including methyl tridecanoate (0.76%), methyl pentadecanoate (2.92%), methyl palmitate (3%), and methyl docosanoate (2.42%), occurred under purple light treatment. The red-blue light treatment led to the highest production of methyl laurate (1.48%). The control treatment exhibited minimal or no SFA production. The purple light treatment resulted in the greatest diversity of fatty acid types compared to all other treatments.

Table 3. Saturated fatty acid profiles of E. gracilis under spectrum treatments.

While other methods, such as carbon source engineering, two-stage culture, and genetic engineering, have significantly improved the production of unsaturated fatty acids (UFA), including polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA), light variation offers a low-cost and effective alternative. This strategy is particularly advantageous for medium to industrial-scale production, where it can more efficiently achieve desired outcomes. The PUFA and MUFA values are inversely related to the SFA value. Table 4 presents the PUFA and MUFA profiles. The highest PUFA and MUFA value was observed under blue light treatment at 74.62%, followed by the control light treatment at 71.96%, the red-blue treatment at 71.73%, the red light treatment at 70.11%, and the lowest PUFA and MUFA value under purple light at 68.30%. PUFA and MUFA play a crucial role in regulating the fluidity of biofuels, which is vital for enhancing the potential of Euglena as a sustainable energy-producing organism using simple, cost-effective techniques.

The UFA profile in this study depended on the specific light spectrum applied. The red and purple light treatments exhibited the greatest diversity in UFA types, each comprising 14 distinct fatty acids. The red light spectrum produced the highest concentrations of myristoleic acid methyl ester (2.24%), methyl cis-10-heptadecenoate (5.87%), methyl linolenate (6.44%), and methyl cis-11,14,17-eicosatrienoate (1.65%). Under blue light, the highest concentrations were found for methyl palmitoleate (29.93%), methyl cis-8,11,14-eicosatrienoate (9.3%), methyl erucate (1.93%), methyl nervonate (3.23%), and methyl cis-4,7,10,13,16,19-docosahexaenoate (1.94%) compared to other treatments. The purple light spectrum induced the highest production of methyl linolelaidate (5.77%), methyl cis-5,8,11,14-eicosatetraenoate (5.36%), and methyl cis-13,16-docosadienoate (3.08%). The red-blue light treatment produced the highest concentration of methyl cis-13,16-docosadienoate (2.25%).

Table 4. Unsaturated fatty acid profiles of E. gracilis under different spectrum treatments.

Ratio of SFA to PUFA and MUFA in E. gracilis under different light spectra

The results of this study show that the SFA values were lower compared to UFA in all treatments, with UFA values being at least twice as high as SFA. This indicates that E. gracilis predominantly produces UFA (Figure 3). The differences between SFA and UFA are significant for various industries, including fuel, pharmaceuticals (e.g., supplements and medications), food additives, and cosmetics. Figure 4 illustrates a mapping plot that shows the distribution of FAME profiles resulting from mono- and multi-wavelength light treatments on E. gracilis. The graph highlights methyl palmitoleate as the FAME variant with the highest concentration.

Figure 3. Ratio of SFA to UFA in E. gracilis under different light spectra.

Figure 4. Percentage of fatty acid composition after treatment.

DISCUSSION

This study demonstrates that the purple and blue light spectra significantly enhance the growth of E. gracilis. This is evident from the increased (P < 0.05) cell count, biomass, specific growth rate (SGR), and shorter division time compared to the control and other treatments. In contrast, cultures exposed to red and red-blue light, as shown in Table 2, exhibited growth parameters lower than the control, suggesting that these light spectra may suppress the growth of E. gracilis. This phenomenon is related to the ability of light spectra to regulate physiological activities, including the photochemical reaction centers of Photosystem I (PSI) and Photosystem II (PSII), cellular metabolic pathways, and hormonal signal transmission (Tang et al., 2019). PSI and PSII contain reaction center pigments known as P700 and P680, respectively, named after their peak absorption wavelengths of 700 nm and 680 nm. Light-harvesting complexes (LHCs), consisting of carotenoid and chlorophyll molecules bound to photoreceptor proteins, play a crucial role in photosynthesis. These photoreceptor proteins are activated by different light wavelengths, affecting cellular functions (Allorent and Petroutsos, 2017).

We present a previous studies of the effects of light spectra on pigment synthesis in microalgae as follows: Activation of Blue-Light Photoreceptors: Blue light is perceived by specific photoreceptors, such as cryptochromes and aureochromes, which initiate signaling pathways that regulate gene expression related to pigment biosynthesis (Takahashi 2016). Activation of Photosystem II (PSII): Red light is primarily absorbed by chlorophyll a, which efficiently drives PSII activity. This activation enhances photosynthetic efficiency, leading to increased chlorophyll synthesis (Larkum et al., 2020). Synergistic Activation of Photosystems: Red and blue light together can enhance the activity of both photosystem I and photosystem II, leading to increased photosynthetic efficiency and, consequently, higher pigment synthesis (Lv et al. 2022). Last in this studies about purple/violet light, with wavelengths ranging from approximately 380 to 450 nm, can influence pigment production in microalgae through several mechanisms: Violet light is absorbed by specific photoreceptors in microalgae, such as cryptochromes and phototropins, which initiate signaling pathways that regulate gene expression related to pigment biosynthesis (Maltsev et al. 2021).

Previous research has shown that Phaeodactylum tricornutum exhibited increased biomass production when exposed to red light (Sharma et al., 2020). Scenedesmus sp. responded positively to both red and blue light and similar responses were observed in Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Auxenochlorella pyrenoidosa (Wagner et al., 2016; Mao and Guo, 2018; Guo and Fang, 2019). Red and yellow pigments have been noted in Nannochloropsis sp. (Shin et al., 2018). Okumura et al. (2015) found that using monochromatic blue and mixed red-green-blue LEDs maximized biomass production in Botryococcus braunii. In this study, the physiological activity of E. gracilis was limited under red and red-blue light, with the highest response observed under purple light compared to white light.

In relation to red and red-blue light treatment, Euglena cells appeared to enter a state of increased relative electron transport rate (rETR) through Photosystem II (PSII), quickly enhancing their short-term tolerance to high-light stress. These findings suggest that the increase in rETR is due to a higher proportion of open PSII, driven by an increase in Photosystem I (PSI) activity (Tanno et al., 2020). The variations in the peak log phase of Euglena growth are determined by the specific light spectrum used. In natural environments, the lighting conditions that affect microalgae growth are highly variable, influenced by factors such as latitude, daily and seasonal shifts in solar altitude, and the scattering of light by the atmosphere and water. At low concentrations, light serves as a signal, activating mechanisms to mitigate stress, while at high concentrations, it can cause cellular damage and alter physiological and biochemical processes (Maltsev et al., 2021).

Euglena is a microalga that exhibits phagocytosis and efficiently acquires nutrients at a rapid rate. Algal cells accumulate substantial amounts of NADPH and ATP through photosynthesis and cellular respiration to support cellular synthesis. To meet the electron and energy demands, algal cells increase electron transfer and improve energy utilization within the photosynthetic system (Chen et al., 2024). Photosynthesis begins with light absorption by the light-harvesting complex, which contains pigments that capture light at specific wavelengths. The pigments in microalgae vary in their light absorption properties, resulting in differential light absorption (Hakim et al., 2023). Several photoreceptors, including red/far-red light receptors phytochromes A-E (PHYA-E), ultraviolet-B (UV-B) light receptor UV RESISTANCE LOCUS8 (UVR8), FLAVIN-BINDING KELCH REPEAT-BOX1 (FKF1), light/oxygen/voltage (LOV) KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL), phototropin (PHOTs), and blue/ultraviolet-A (UV-A) light receptors cryptochromes (CRYs), have been identified (Zhang et al., 2024). The variations in physiological responses to different light spectra are believed to reflect the regulation of these photoreceptors. This study found that the purple light spectrum led to the highest growth rate in E. gracilis compared to other light sources. The optimal light spectrum for microalgae growth varies across species, depending on the specific pigments present in each species.

HPLC analysis revealed the presence of chlorophyll a (Chl a) and chlorophyll b (Chl b), as well as specific carotenoids, including neoxanthin, diadinoxanthin, cis-diatoxanthin, trans-diatoxanthin, β-carotene, and zeaxanthin in Euglena. These carotenoids exhibited absorption peaks at wavelengths of 450 and 478 nm (Tanno et al., 2020). Total carotenoids contribute significantly to blue light absorption (Ma et al., 2022), covering the 300–600 nm range (Begum et al., 2016). In contrast, chlorophyll a and b are primarily responsible for absorbing red and blue light (Ma et al., 2022). Purple light, having a shorter wavelength than red and blue light, possesses higher frequencies and photon energies, which chloroplasts can efficiently absorb, leading to optimal growth of E. gracilis. The results of this study are in accordance with previous studies on other organisms that the intense energy emitted by purple light increase the growth of C. vulgaris and S. obliquus (Ruiz-Marin et al., 2020).

In this study, lipids produced under high biomass conditions, such as in the purple light spectrum (0.580 ± 0.042 gL⁻¹), were lower compared to lipids produced under lower biomass conditions, such as in the red-blue light spectrum (0.680 ± 0.028 gL⁻¹). This finding supports the idea that lipid content in microalgae has a negative correlation with biomass productivity. Increasing lipid content in microalgae typically leads to lower growth rates, which in turn reduces overall lipid productivity (Ogbonna et al., 2024). Further research indicates that elevated reactive oxygen species (ROS) levels alter carbon metabolism in algal cells, resulting in the accumulation of additional carbon elements as lipids. Eukaryotic microalgae, such as E. gracilis in this study, possess a comprehensive lipid metabolic pathway, enabling them to efficiently store more carbon as lipids. In contrast, prokaryotic microalgae have a limited lipid metabolic pathway, which restricts their capacity to accumulate carbon as lipids (Azizullah et al., 2022). The primary pathways for triacylglycerol (TAG) production—a key lipid compound essential for biodiesel—include the fatty acid synthesis pathway and the Kennedy pathway. The synthesis of fatty acids, particularly C16 and C18 fatty acids, begins with the conversion of Acyl-CoA to malonyl-CoA by the enzyme Acyl-CoA carboxylase (ACCase) (Song and Pei, 2018). It is plausible that the red-blue light spectrum triggers the activation of ACCase or enhances the quantity of precursors in the lipid biosynthetic pathway, leading to increased fatty acid production. Therefore, specific light spectra can potentially improve the efficiency of photosynthesis by providing sufficient energy for metabolite synthesis. The accumulation of triglycerides involves the action of various enzymes and cellular components, including ribulose bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase, the latter of which regulates carbon dioxide levels in microalgae. Increased enzyme activity leads to greater triglyceride accumulation (Teo et al., 2014).

Microalgae cultivated under stressful conditions, such as in this study with red-blue light, tend to prioritize carbon utilization for the production of amino acids and other cellular components, including neutral lipids. An increased lipid composition is essential for the recovery of photosynthesis under stress (Preechaphonkul et al., 2024). However, prolonged exposure to stress can lead to cellular damage and negatively affect metabolite production. During periods of intense light stress, microalgae can accumulate large quantities of reactive oxygen species (ROS), such as superoxide (O₂⁻), singlet oxygen (O₂), hydrogen peroxide (H₂O₂), and hydroxyl radicals. This accumulation can result in the oxidative degradation of lipids, proteins, and nucleic acids (Gaignard et al., 2021), ultimately leading to cell death before lipids can be produced.

Regarding lipid content, the low amount of saturated fatty acids (SFA) in the lipid profile of E. gracilis is considered optimal for biodiesel production. While biodiesel made from SFA has favorable oxidation resistance, it exhibits poor fuel performance in cold temperatures, making it unsuitable for winter use (Cao et al., 2014). The blue light treatment resulted in the highest proportion of unsaturated fatty acids (UFA) compared to other treatments, indicating that blue light effectively enhances UFA composition. Previous research has shown that increases in UFA are driven by the activation of fatty acid desaturase, an enzyme that introduces double bonds into the hydrocarbon chains of fatty acids. These enzymes are crucial for maintaining the integrity and functionality of cellular membranes.

The process of fatty acid synthesis can be summarized as follows: malonyl-CoA serves as the immediate precursor for fatty acid production in biological systems. Acetyl-CoA carboxylase (ACCase) is the key enzyme responsible for the irreversible carboxylation of acetyl-CoA, producing malonyl-CoA. In microalgae, acetyl-CoA can follow two pathways: participation in the tricarboxylic acid (TCA) cycle or conversion into malonyl-CoA for fatty acid synthesis (Lu et al., 2021). Previous research has demonstrated that overexpression of ACCase from various sources increases the abundance of malonyl-CoA and accelerates fatty acid synthesis (Cao et al., 2014).

Further lipid constituents, specifically MUFA and PUFA, are critical for biodiesel. MUFA are the preferred component due to their superior low-temperature fluidity and oxidative stability. However, biodiesel produced from microbial oils or vegetable lipids invariably contains significant amounts of alkyl esters of both polyunsaturated and saturated fatty acids, which can hinder its practical use (Cao et al., 2014). The findings of this research show that the E. gracilis strain from Indonesia has the highest MUFA profile across all treatments, ranging from 39.56% to 46.40%. This is followed by PUFA, which ranged from 25.56% to 28.48%, while SFA ranged from 28.04% to 31.7%. These results suggest that fatty acids derived from E. gracilis have the potential to serve as a viable biodiesel feedstock. Research indicates that biodiesel containing a greater proportion of monounsaturated fatty acids (MUFA), generally demonstrates superior oxidative stability compared to biodiesel high in polyunsaturated fatty acids (PUFAs). Shaltout and El-Din (2015), emphasized the importance of balancing saturated and unsaturated fatty acids to optimize fuel properties, including viscosity and low-temperature fluidity (Shaltout and El-Din 2015). The oxidative stability of polyunsaturated fatty acids diminishes as the degree of unsaturation rises, potentially leading to unfavorable combustion properties when these acids are utilized as a fuel source. During combustion, the oxidative breakdown of polyunsaturated fatty acids (PUFAs) can generate nitrogen oxides, as the resulting degradation compounds influence the temperature and pressure conditions within the engine. This effect is emphasized by Marchetti, et al., (2017) who underscored the importance of oxidative stability in assessing the viability of PUFAs for diesel engine applications, noting that low stability can contribute to elevated emissions of harmful pollutants like NOx.

The reduction in PUFA levels in microalgae can be attributed to the formation of intracellular reactive oxygen species (ROS) caused by high-intensity light. The buildup of ROS in algal cells can result in the oxidation of PUFA, thereby reducing their proportion in total fatty acids (TFA) (Lu et al., 2021). Under stressful conditions, the fluidity of lipid bilayers may change, causing a transition from a liquid state to a crystalline state (Huang et al., 2019). Unsaturated fatty acids help maintain the flexibility of the lipid bilayer. Algae produce unsaturated fatty acids under stress, enhancing membrane function and flexibility. This accumulation protects against crystallization and oxidation (Lu et al., 2021; Preechaphonkul et al., 2024).

Microalgae hold significant potential as a valuable source of renewable energy, primarily due to their high triglyceride content, which can reach up to 60% (Nuhma et al., 2021). Previous study demonstrated that the fatty acid profile of Chlorella vulgaris, cultivated under mixed-out growth conditions, is predominantly composed of MUFA, accounting for approximately 60–68% of the total fatty acids. The main components of this profile include oleic acid (C18:1), palmitic acid (C16:0), palmitoleic acid (C16:1), and stearic acid (C18:0), which is considered ideal for biodiesel production. Conversely, when grown under conditions favorable for growth, the fatty acid profile indicates unsuitability for biodiesel production, as it contains higher levels of PUFA, such as linolenic acid (C18:2, C18:3) and eicosapentaenoic acid (C20:5), making it more suitable for food consumption (Nuhma et al., 2021).

The fatty acids composition of algal lipids plays a critical role in influencing the lubricity, cetane number, cold flow properties, kinematic viscosity, density, oxidative stability, and heat of combustion of biodiesel. Additionally, the specific microalgal strain and the physicochemical conditions under which it is cultured are key factors in determining its fatty acid content (Ogbonna et al., 2024). Giakoumis and Sarakatsanis (2019) found that higher concentrations of saturated fatty acids, such as C16:0 and C18:0, increase the cetane number, thereby improving biodiesel's ignition quality. Previous studies have indicated that saturated fatty acids are preferable for biodiesel production (Ogbonna et al., 2024). However, other research emphasizes the importance of MUFA in enhancing biodiesel's flow properties at low temperatures (Cao et al., 2014). Minimal quantities of PUFA are advantageous for biodiesel, particularly in cold environments, as they improve fuel fluidity (Costa et al., 2017). In this study, PUFA levels were consistently lower than MUFA and SFA, underscoring the potential suitability of the investigated substance for biodiesel production. Elevated concentrations of PUFA, which tend to oxidize rapidly, negatively impact the oxidative stability (OS) of biodiesel and compromise its storage stability, a crucial factor for fuel applications. Biodiesel produced from plant and microalgae lipids via transesterification consists of FAME, which are the chemical components of biodiesel. The versatility of FAME allows it to be reprocessed according to industry needs, either as a primary component or as a supplementary ingredient with improved quality.

Figure 4 highlights the FAME variant with the highest value, specifically observed in the methyl palmitoleate category. The use of E. gracilis as a biofactory for methyl palmitoleate production is considered a viable alternative to address shortages arising from growing demand. The extraction process is relatively simpler than that required for plants, especially higher plants. Moreover, the prominence of methyl palmitoleate, a component of UFA, positions Euglena as a promising potential biodiesel source. Previous research has suggested that optimal biodiesel should have lower levels of SFA and PUFA compared to MUFA to mitigate concerns related to oxidative stability and cold flow properties (Saber et al., 2024). The EN 14214 standard establishes specific limits for the levels of polyunsaturated fatty acids (PUFAs) and tri-unsaturated fatty acids (TUFA) in biodiesel, directly influencing the SFA/UFA ratio. According to the standard, TUFA content must not exceed 12%, and PUFAs are limited to a maximum of 1%, as these unsaturated fatty acids can compromise the oxidative stability of biodiesel (Ferreira et al., 2022). This regulation highlights the critical role of maintaining an optimal SFA/UFA ratio to preserve the stability and performance of the fuel.

To achieve sustainable energy goals, significant progress is needed in developing multi-functional technologies that can perform various tasks simultaneously. In this context, Euglena acts as a carbon-absorbing organism and functions as a biological machine that produces metabolites with minimal energy consumption. This is accomplished by optimizing environmental conditions or applying specific modifications. The use of different light sources as an external factor has demonstrated variations in growth rates, biomass, and metabolite accumulation. These findings hold great potential for further application in eco-friendly and sustainable energy sectors.

CONCLUSION

This study highlights the potential of E. gracilis as a renewable biodiesel source, with its ability to produce diverse FAME profiles under different light spectra. Among the treatments, purple light significantly enhanced the growth rate of E. gracilis, while red-blue light yielded the highest lipid content. Blue light resulted in the highest proportion of unsaturated fatty acids (UFA), which are critical for biodiesel quality. The findings demonstrate that specific light spectrum modifications can be employed to optimize the production of desired FAME profiles, aligning with the requirements for sustainable biofuel production. The predominance of monounsaturated fatty acids (MUFA) in the FAME profiles suggests the suitability of E. gracilis for biodiesel applications, offering a balance between oxidative stability and cold flow properties. This study underscores the importance of ecological modifications, such as light spectrum variation, to enhance the biofuel potential of microalgae while maintaining environmental sustainability. Future work should investigate the physicochemical properties of biodiesel derived from E. gracilis to further validate its commercial feasibility and adaptability to diverse conditions.

ACKNOWLEDGEMENTS

The authors express their gratitude to the staff at the University of Edinburgh and Universitas Gadjah Mada for their technical support.

AUTHOR CONTRIBUTIONS

Khusnul Qonita Maghfiroh assisted with the experiments, performed statistical analysis, created data visualizations, and wrote the manuscript. Arief Budiman and Bambang Retnoaji designed and conducted all experiments and contributed to manuscript writing. Eko Agus Suyono supervised the experiments, validated the data, and co-wrote the manuscript. All authors have read and approved the final version of the manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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Supplementary

Tabel S1. Composition medium of Cramer Myers.