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Article

Effect of Artificial Diet Modification with Dextrose on the Growth and Fatty Acid Composition of Tenebrio molitor Larvae for Biodiesel Production

by
Miguel Alejandro Flores-Sánchez
1,
Amanda Kim Rico-Chávez
1,
Marco Martín González-Chávez
2,
Rodolfo Figueroa-Brito
3,
Juan Campos-Guillen
1,
Carlos Eduardo Zavala-Gómez
1,
Aldo Amaro-Reyes
1,
Joel de Jesús Barba-Franco
4 and
Miguel Angel Ramos-López
1,*
1
Facultad de Química, Universidad Autónoma de Querétaro, Querétaro 76010, Mexico
2
Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí 78210, Mexico
3
Centro de Desarrollo de Productos Bióticos (CEPROBI-IPN), Instituto Politécnico Nacional, Yautepec, Morelos 62731, Mexico
4
Centro Universitario de los Lagos, Universidad de Guadalajara, Lagos de Moreno 47463, Mexico
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(19), 10777; https://doi.org/10.3390/app151910777
Submission received: 6 August 2025 / Revised: 3 October 2025 / Accepted: 4 October 2025 / Published: 7 October 2025
Versions Notes

Abstract

Tenebrio molitor larvae are a promising source for the next generation of liquid biofuels. However, the conditions and processes required for rearing this insect for biodiesel production need to be investigated. In this study, the effect of dextrose addition to an artificial diet in the oil and biodiesel yield was evaluated. Larvae were fed artificial diets modified with 3, 6, 9, and 15% dextrose. Survival rate, mean dry weight, and oil yield were registered. The 15% dextrose addition resulted in 75% survival, 25 mg individual dry weight, and 29% oil yield. The main components of this oil were palmitic acid (14%), oleic acid (37%), and linoleic acid (20%). With the addition of dextrose, the total saturated fatty acids increased 13% and the polyunsaturated fatty acids decreased 16% compared to the control. However, with the transesterification reaction, the fatty acid methyl esters remained similar for both treatments, with methyl oleate, methyl linoleate, and methyl linolenate as major components. This suggest that the transesterification reaction was incomplete, due to the oil/methanol ratio or the catalyst. Thus, even though a more balanced saturated/unsaturated fatty acid profile can be achieved through dextrose addition, another transesterification method should be tested to obtain a complete reaction.

1. Introduction

Diesel is the main energy source of the freight industry globally, and around 27 million of barrels are consumed each year [1]. The use of this fuel generates greenhouse emissions, such as CO, NOx, and CO2, along with PM2.5, that cause damage to the environment and to human health [2,3]. Thus, it is necessary to migrate to new fuels that use renewable technologies that reduce the contaminant emissions [4].
Biodiesel from different sources of biomass is an alternative to fossil fuels. To obtain this fuel, a transesterification process is used. The fatty acids present in different vegetal or animal fats and oils react with methanol in an alkaline medium, usually sodium hydroxide (NaOH), resulting in corresponding fatty acid methyl esters (FAMEs). These molecules can be blended at 20% with commercial diesel without compromising the properties of the fuel in an engine, thereby also reducing the contaminant emissions [5]. Oils that can be used for biodiesel production can be from algae or non-edible plants, such as Jatropha curcas, Ricinus communis, or Pongamia pinnata [6,7].
Another potential source of biomass are insects; these organisms have a fat body in which they accumulate lipids as energy storage. Some species that have been investigated include the larvae of lepidopteran Galleria mellonella, the dipteran Hermetia illucens, and the coleoptera Tenebrio molitor [8,9]. Due to their high fat content and biomass, T. molitor larvae are considered to be a promising lipid source for biofuel. Currently, this organism is used as live feed in farm animals for its protein content [10]; however, it also can be exploited for oil extraction for biodiesel production [9,11]. Some of the advantages from this insect include its short lifecycle, approximately 90 d, a reproduction rate of 500 eggs per female, and the feed can be from different sources of organic waste, such as potato, carrot, and bran [9,10]. During the larval stage, these insects accumulate lipids in their fat body through their development which can vary in content and composition depending on the feeding substrate. The main substrate used for the rearing of this insect is wheat bran; however, some researchers have included other ingredients, such as flaxseed, oats, sunflower seeds, brewer’s yeast, and potato flour [12,13], reporting the prepupae as the instar with the highest rate of oil extraction, reaching yields between 30% and 40% dry basis [14,15]. The main fatty acids present in T. molitor oil are 35–45% linoleic (C18:2), 30–40% oleic (C18:1), and 20–25% palmitic (C16:0), which can be modulated trough the feeding substrate [16,17,18,19,20]. For example, in the research of Kotsou et al. [21], the authors determined that the combination of Moringa oleifera (Moringaceae) leaves combined with wheat bran reduced the oil content by 35%, but increased the oleic acid by 14%. While Melis et al. [22] reached a yield of 6% with brewer’s yeast, Montalbán et al. [23] obtained an oil yield of 34% with a diet based on 60% starch and 18% protein, but the yield was reduced to 27% when the starch was changed to 40% and the protein to 26%.
For biodiesel obtention from T. molitor larval oil, the transesterification method is used to convert the free fatty acids (FFAs) in the oil to the corresponding FAMEs. However, in T. molitor, the acid value ranges from 7 to 10 mg KOH g−1 oil, meaning a high FFA content. Therefore, a two-step transesterification process is needed. These reactions include an initial acid-catalyzed esterification to reduce the FFAs to avoid saponification, followed by an alkaline reaction to convert triglycerides into FAMEs. Similar to the oil, the main FAMEs obtained after transesterification are methyl palmitate (15–18%), methyl linoleate (10–20%), and methyl linolenate (20–25%) [24,25,26]. Another pathway to obtain biodiesel is by supercritical oil transesterification. The main advantages of this reaction are that it does not require a catalyst and the reaction time is 30 min. However, a higher methanol/oil ratio is needed (18:1), along with a pressure of 8 MPa and a temperature of 290 °C to obtain yields higher than 95% [27,28].
The composition of polyunsaturated fatty acids in T. molitor larval oil allow us to meet international biodiesel quality standards, such as ASTM D6751 [29] and EN14214 [30]. For instance, Siow et al. [24] obtained a cinematic viscosity of 2 mm2 s−1 and an acid value of 0.03 mg KOH g−1, while Zheng et al. [25] obtained a cetane number of 58 when, according to EN 14214, the minimum is 51. Wang et al. [26] found a cinematic viscosity of 4.51 mm2 s−1 and a cetane number of 52. These results, demonstrate the potential of T. molitor oil as a viable resource for biodiesel production.
In the present work, T. molitor larvae were fed with an artificial diet modified in the dextrose content to evaluate the weight increase. Subsequently, the oil from the larvae was extracted to produce biodiesel through catalytic transesterification, followed by the analysis of its chemical composition.

2. Materials and Methods

2.1. Tenebrio molitor Rearing

Tenebrio molitor larvae were obtained from an established colony at the Laboratory of Natural Insecticidal Compounds from the Autonomous University of Queretaro. For rearing, the larvae of this insect were placed in wooden boxes (50 cm × 50 cm × 10 cm) at temperatures of 25–27 °C with a relative humidity of 40–50% and a photoperiod of 10:14 h L/D. Each box was filled with a mix of oat, wheat bran, and sawdust in a proportion of 1:3:16 as rearing substrate, and the larvae were fed every third day ad libitum with 0.5 g slices of fresh carrot, which also maintained the humidity [31]. Once the pupae formed, they were separated from the rearing boxes and transferred to a plastic container (25 cm × 10 cm × 5 cm) without substrate until adult emergence. Newly emerged adults were placed in groups of 50 in plastic boxes (50 cm × 30 cm × 20 cm) with the same substrate as the larvae for mating and oviposition. After 5 days, the adults were transferred to another plastic box with substrate, leaving the mating box with the eggs until hatching. Afterward, the boxes were monitored every week to separate newborn larvae [32].

2.2. Diet Modification

For the evaluation of the development of T. molitor larvae trough modifications in the carbohydrates content in the diet, a preliminary bioassay was performed to select the carbohydrate that increased the larval fresh weight and survival rate. For this purpose, 50 larvae from the sixth instar were randomly selected in triplicate and placed in plastic boxes (50 cm × 30 cm × 15 cm) filled with a mix of oat, wheat bran, and sawdust in a proportion of 1:3:16 as a substrate. Artificial diets were formulated by modifying the content with three selected carbohydrates: starch, cellulose, and dextrose. The diets were formulated using sodium alginate microcapsules that contained carrot and wheat bran as a control, while for the carbohydrate modification 5%, 9%, and 16% were added, identified as PS5, PS9, and PS16 for starch, PC5, PC9, and PC16 for cellulose, and PD6, PD9, and PD16 for dextrose.
For the preparation of each diet, the solid ingredients (Table 1) were mixed with the corresponding volume of water in a 500 mL flask with a magnetic stirrer at 3500 rpm, until a homogenous mixture (referred as to “HM”) was obtained. At the same time, in a separate 500 mL flask, a solution of 0.8% w/v sodium alginate (Maesacv, Jalisco, Mexico) was prepared by dissolving the polymer in distilled water with constant stirring until a particle-free solution was obtained. Once the sodium alginate solution was fully dissolved, the HM was gradually added while maintaining the stirring for additional 10 min. The resulting mix was transferred to a plastic syringe and then dispensed in droplets into a 0.2% w/v calcium chloride solution to form microcapsules, until all the solution was poured. The formed beads were maintained in the salt solution for 15 min, then separated by filtration through Whatman #1 paper and washed in triplicate with distilled water, followed by ambient air-drying (25–28 °C) for 1 h, and were then stored in refrigeration until use [33].
Larvae from each treatment were fed ad libitum every third day with 5 g of the respective microencapsulated diet. The rearing conditions of the boxes were 25–27 °C, 40% humidity, and a photoperiod of 10:14 h L/D [34]. Insects were reared for 30 d, measuring survival rate, formed pupae, and larval fresh weight.
After the preliminary bioassay, the carbohydrate with more obtained larval survival and fresh weight was selected to repeat the bioassay at the same conditions, with 150 larvae from the third instar over 130 d. Subsequently, the larvae were slaughtered by 12 h freezing at −18 °C, followed by drying in a convection oven (FELISA FE-292A, Jalisco, Mexico) at 65 °C for 24 h until all moisture was removed. Once dried, the larvae were weighed, milled in a universal grinder (IKA M20, Wilmington, NC, USA) to achieve a uniform particle size (0.5–1 mm), and preserved in 150 mL sealed flasks for subsequent oil extraction. The measured variables included larval survival rate, pupae formation, and individual weight.

2.3. Oil Extraction

After the bioassay of the feeding with different dextrose concentrations, the diet with the highest survival rate and individual weight was selected for oil extraction. For each treatment group, the total alive larvae were weighed, sacrificed, dried, and milled for lipid extraction. The drying occurred at a constant weight in a controlled environment of 60 °C for 48 h to minimize moisture content, which can affect solvent efficiency.
For the extraction process, Soxhlet apparatus was employed, using 300 mL of analytical grade hexane (≥95% purity) (Maesacv, Jalisco, Mexico) as the solvent. The conditions included 6 h of continuous reflux to ensure complete lipid recovery. Afterward, the hexane containing the dissolved lipids was collected and evaporated using a rotary evaporator (IKA RV10, Wilmington, NC, USA) at 40 °C under reduced pressure until complete solvent removal.
The oil yield (%) was estimated gravimetrically, weighing the extraction flask before and after the extraction, with the difference corresponding to the extracted mass. The resulting oil was stored in a sealed amber vial to further compositional analysis.

2.4. Chemical Characterization of Tenebrio molitor Oil

The fatty acid profile was analyzed in a GC-MS (Agilent) equipped with a capillary column and ionization flame detector (6890N GC, 5975 MS Agilent Technologies, Santa Clara, CA, USA) with a 30 m × 0.25 mm × 0.25 mm SLB-IL60 column. Helium was used as a carrier gas. Initial temperature was 40 °C and was heated to 280 °C at a rate of 5° min−1 with ionization energy of 70 eV. Running time was 55 min. The results were compared with the Agilent MassHunter Quantitative Analysis library [35].

2.5. Oil Transesterification

To obtain biodiesel from the extracted T. molitor oil, a two-step transesterification process was performed, due to the high acid value reported in the oil [36]. Initially, preliminary acid esterification was performed to reduce the free fatty acid content and to prevent saponification during the alkaline step.
For this reaction, 30 g of T. molitor oil was placed in a 500 mL round-bottom flask and preheated to 60 °C with constant agitation at 500 rpm to ensure homogeneity, once the temperature was reached, methanol was added at a 6:1 ratio, and 0.1% of H2SO4 was added as the acid catalyst. After 60 min of reaction, the product was poured in a separation funnel to allow phase separation, the upper part was distilled to eliminate the methanol, and the remaining oil was poured in a new flask for the alkaline transesterification.
For the second reaction, the product of the acid esterification was mixed with methanol in an 8:1 molar ratio along with 0.8% w/w NaOH as the alkaline catalyst, the temperature was set to 60 °C, and the reaction time was 60 min. The result of the reaction was poured into a separation funnel to remove the lower glycerol layer, to distil the upper part to remove excess methanol, and to obtain the purified biodiesel.
The yield was estimated gravimetrically by weighing the oil previous to the acid esterification and the biodiesel after the alkaline transesterification. The resulting biofuel was stored in a sealed amber vial for further compositional analysis.

2.6. Chemical Characterization of Biodiesel

For the obtained biodiesel, 1 μL aliquot of each sample was separated in an HP-88 capillary column (30 m × 0.25 mm × 0.25 μm) installed in an Agilent 7890 GC system (Wilmington, DE, USA) coupled with an Agilent 5975C EI-SQ MS. Helium was used as the carrier gas at a constant flow rate of 1.5 mL/min. The injector was in split mode at 250 °C. The oven temperature program was as follows: initial temperature of 50 °C (held for 1 min), increased to 175 °C at 15 °C min−1, then ramped to 210 °C at 1 °C min−1. Electron ionization (EI) was performed at 70 eV, and the mass scan range was m/z 50–1100. Fatty acid methyl esters (FAMEs) were identified in comparison with the Supelco 37-component FAME Mix standard and quantified by the external standard method. Data acquisition and processing were conducted using Agilent ChemStation software (version C.01.08).

2.7. Statistical Analysis

For the evaluation of the effect of the diets on the survival rate, pupae formation and mean individual weight using a one-way analysis of variance (ANOVA) was performed, followed by Tukey’s test at a 95% confidence level to identify statistically significant differences.
Due to the limited weight of the total alive larvae, a statistical analysis of the obtained volumes of oil and biodiesel was not performed.

3. Results

3.1. Effect of the Diet Modification

As a result of the preliminary bioassay, dextrose was the selected carbohydrate, since the accumulated survival was 95% compared to 45% in the control, while for the rest of the treatments, the accumulated survival was around 50% (Table 2). In the case of the larval weight, for all the treatments it was around 130 mg with no statistically significant difference; however, with the PD5, PD9, and PD16 treatments, a gradual increase of weight was registered from 114 to 133 mg.
After selecting dextrose as the carbohydrate, the bioassay was repeated with the control diet supplemented with 6, 9, 12, and 15% of dextrose, identified as D6, D9, D12, and D15. In this bioassay, the survival of T. molitor larvae increased with the content of dextrose in the diet, reaching 30% more alive larvae with D15 compared with the control (Table 3), while the formed pupae doubled with the increase of dextrose in the diet, reaching a maximum of 21% for D15. The accumulated survival, which is the sum of the total larvae and pupae, increased with the content of dextrose, finding significant differences from D6, which increased 20%, and reaching an increase of 44% compared to the control for D15. In the case of the individual weight of each larva, an increase of 60%, 87%, and 218% was registered with D9, D12, and D15, respectively, which indicates that the addition of dextrose to the artificial diet affects the growth of this insect.

3.2. Oil Yield of Tenebrio molitor

The results of feeding 500 T. molitor larvae with a control diet and a modification of 15% of dextrose are shown in Table 4. The survival rate was similar to the results in Section 3.1, except from the control group, in which 50% of the larvae survived. With the D15 treatment, 47% more fresh weight was obtained due to the existence of 14% more alive larvae than the control; however, the average individual weight was 15% higher. The humidity and yield were similar between treatments at 70% and 29%, respectively.
Even though the oil yield was the same for both treatments, the average larval weight was different, being 77.36 mg for the control group and 89.31 mg for the D15 treatment group.

3.3. Tenebrio molitor Oil Characterization

The analysis of the Tenebrio molitor oil is shown in Table 5. Feeding the larvae with a modified diet with 15% dextrose (D15) increased the content of undecylic acid by 19%; while lauric acid (C12:0) was present in this oil, the linoleic acid (C18:2) decreased by 10%, while α-linoleic and nonadecanoic acid were not present. In the case of the total content of saturated fatty acids (SFAs) and unsaturated fatty acids (UFA), the oil obtained from D15 increased the SFAs by 13% and decreased in the same proportion for the UFAs. In both cases, the oleic acid (C18:0) was the most abundant, totaling nearly 35%.

3.4. Oil Transesterification

For the control treatment and D15, the yield of the reaction was 80.4% and 85.7%, respectively. While the methyl ester profile changed for the larval oil in both cases. The relative content is summarized in Table 6. In this case, for both treatments, the methyl ester profile was similar. The main differences were found in methyl undecanoate, with the presence of 0.26% in D15, in methyl laurate in the control, with 0.47%, and in methyl linolenate, with 2.39% in the control. The total PUFAs/SFAs in both treatments is nearly 1.3.

4. Discussion

The addition of dextrose in the diet of T. molitor larvae, modified the growth and pupation, which suggests that the presence of this carbohydrate affects larval development. In this study, the larval weight increased from 69 mg in the control group to 150 mg with the D15 treatment, while overall survival increased from 30 to 75% with the addition of dextrose. Similar results were reported by Ruschioni et al. [37], where the authors observed a decrease in the development time from 137 d when the larvae were fed with wheat middlings for 109 d when wheat flour was the substrate as a control, demonstrating that a shortened larval development time can be achieved through modifications in the diet. This was also observed by Huang et al. [38], who evaluated different protein to carbohydrate (P/C) ratios (1:8, 1:5, 1:3.3, 1:2.5, and 1:1.9), reaching a higher larval weight value (54 mg) with P/C ratio of 1:5, meaning that a higher content of carbohydrates in an artificial diet can increase the larval weight. Moreover, the survival rate was 64% with the P/C ratio of 1:8, while with the other treatments the survival rate was around 80%. Other similar findings include those of Montalbán et al. [23], who fed T. molitor larvae with a broccoli byproduct which contained 28.7% starch and 16% neutral detergent fiber (NDF), gained 80 mg larval weight, reaching an average weight of 140 mg per larva in 30 d, while only 40 mg was gained with grape pomace byproduct, which contained no starch and 47% NDF. The increase of fatty acids observed with the addition of dextrose in the diet can be explained by the de novo biosynthesis of lipids from carbohydrates. This mechanism has been described in another study for Hermetia illucens [39], in which the enzyme acetyl-CoA carboxylase participates in the conversion of carbohydrates in fatty acids, such as C12:0 and C16:0. This metabolic pathway confirms that higher carbohydrates in a formulated diet have a positive impact on larval growth and survival.
Regarding the oil content, Huang et al. [38] found that the ether extract yield reduced from 43% to 25% when the P/C increased from 1:5 to 1:8. The authors concluded that the carbohydrates incorporated in the feeding substrate had a direct correlation with the oil yield from the larvae. Another finding was that the higher content of carbohydrates in the diet increased the polyunsaturated fatty acid (PUFA) content in the insect; when the P/C ratio in the feeding substrate increased, the PUFAs decreased, obtaining a maximum of 32% at a P/C ratio of 1:1.9 and a minimum of 19% at a P/C ratio of 1:8. Similar results were obtained by Rho and Lee [40], who fed T. molitor larvae with different P/C diets, using only sucrose as a carbohydrate source, finding that a P/C ratio of 1:4 increased the oil content to 28%, compared to the P/C ratio of 6:1 used as a control, with an oil yield of 18%. Kröncke and Benning [41] reported a correlation of the carbohydrate in the diet with the oil in the insect, reaching a maximum of 48.6% crude fat when feeding the T. molitor larvae with cassava flour (P/C 1:6), compared to a wheat bran-based diet used as a control (P/C 1:3), which reached 35% of the oil yield. In this study, although dextrose did not affect the larval oil content, the mean larval dry weight increased, suggesting that a higher content of oil can be achieved with a diet formulated with 15% dextrose.
The composition of the oil from the larvae fed with the control diet consisted mainly of 14% C16:0, 34% C18:1, and 30% C18:2. By contrast, in the larvae from the D15 treatment, the C11:0 increased from 8% to 27%, while C16:0 and C18:1 remained similar, and C18:2 decreased from 30% to 20%. Also, in this study the PUFA/SFA rate decreased with the diet modification with dextrose. These results were similar to Huang et al. [38], who reported a reduction in the PUFAs/SFAs from 1.33 to 0.81 with the increase of P/C ratio from 1:1.9 to 1:8, confirming that the addition of dextrose to an artificial diet can modulate the composition of the oil, mainly in the proportion of UFAs. Another study, by Mlček et al. [42], used wheat as a control (P/C 1:1) and potatoes (P/C 1:9.5), finding a survival of 75% with the potatoes and 55% with the control. Also, the oil content on a dry basis was 37% for the control and 31% for the potato diet, while in the composition of the oil, C18:1 increased in the control diet from 32% to 41% with the potato diet, while 16:0 and C18:2 remained similar for both treatments (12% and 18%, respectively). In this study, the T. molitor larvae were fed with dextrose, maintaining the C18:1 in the obtained oil around 35%, while C18:2 decreased from 30% to 20%, reducing the total UFAs from 70% to 47% and increasing total SFAs from 29% to 43%. In the study by Mlček et al. [42], the SFAs remained at 16% for both diets, and the UFAs increased from 51% to 60% with the increase in the content of carbohydrates. The decrease in UFAs from this study can be explained by increasing only one source of carbohydrate (dextrose) and maintaining the rest of the ingredients in the formulation without changes; however, both studies demonstrate that the oil composition can be modulated through diet. It should be noted that the fatty acid composition analysis reported in this study is based on a single measurement (n = 1), as shown by other authors [38,42] who have provided a representative profile which provides evidence of the modulation of fatty acids through the diet.
In the obtained biodiesel, the methyl esters profile suggested that part of the fatty acids from the T. molitor larval oil remained unreacted. For instance, the C11:0, which was 27% in the D15 oil, decreased to 0.26% in the biodiesel, indicating that SFAs were more affected during transesterification. This is reflected in the PUFA/SFA ratio, being in the oil 1.22 for the control group and 0.48 for the D15 treatment; in the biodiesel, however, the PUFA/SFA ratio was around 1.3 for both treatments. Similar to the case of the obtained oil, the fatty acid methyl esters profile reported in this study is based on a single measurement (n = 1), but providing evidence of the presence of different fatty acid methyl esters, which has also been reported in other studies [24,43]. Despite these limitations, the biodiesel results were consistent with the research by Siow et al. [24] in which the major components of the biodiesel were methyl oleate, methyl linoleate, and methyl palmitate, which correspond to the fatty acids C18:1, C18:2, and C16:0, respectively. Also, a similar proportion of saturated and unsaturated fatty acids was reported by Khizar et al. [43] using Hermetia illucens larvae, where after the transesterification a total of 40% saturated fatty methyl esters and 30% of unsaturated fatty acid methyl esters were obtained. According to Lee et al. [36], the T. molitor oil has organic and inorganic impurities that affect the oil transesterification yield, this can be addressed by increasing the temperature and time in the reaction. Charoenchaitrakool and Thienmethangkoon [44] used a two-step transesterification from waste frying oil, finding that the optimal yield (93%) was achieved with a methanol/oil molar ratio of 9.1, 0.5% wt KOH as a catalyst, and 55 °C for 60 min. Compared to the lowest yield of 80% obtained with a methanol/oil molar ratio of 5.2, and 1.5% wt KOH under the same reaction time, meaning that increasing the methanol ratio while reducing alkali catalyst concentration, can lead to a higher biodiesel yield. With the T. molitor oil, an 8:1 methanol/oil molar ratio was used with 0.8% wt of NaOH, resulted in the incomplete conversion of the FAMEs. This suggest that a higher methanol/oil ratio, and a lower NaOH concentration is needed to improve the overall conversion of the FAMEs. Similarly, Abid et al. [45] used the fat from chicken skin to produce biodiesel. This fat had a percentage of > 0.5% FFAs, meaning that a two-step transesterification was needed. The authors also mentioned that the FFAs must be eliminated to optimize the transesterification reaction, registering a FAME yield of 4.37% when the FFA conversion was incomplete and higher than a 90% FAME yield when the majority of the FFAs were reduced through the acid catalytic reaction. These authors also used a methanol/oil ratio of 6:1. Another explanation for the incomplete conversion of C11:0 in the D5 could be related to the content of SFAs. According to Pinzi et al. [46], oils with high SFA, such as coconut oil (85% SFA) with lauric acid (C12:0) as the major component or palm oil (48% SFA) with palmitic acid (C16:0) as the main component, exhibited lower transesterification conversion rates, 54% conversion compared to 83% with the sunflower oil which had >80% UFAs. These authors determined that the content of saturated acids affected the transesterification yields, detecting that monoglycerides and diglycerides remained unreacted after 60–120 min, while the total conversion of triglycerides in palm oil took 45 min to fully react compared to oils with more UFAs (5–10 min). In another report, Awad et al. [47] used waste cooking oil transesterification with methanol, using different parameters, such as methanol quantity, temperature, time, and catalyst. The study determined yields of 50–60% biodiesel using 0.5–0.8% of NaOH as the catalyst and a 1:6 oil/methanol molar ratio at 40 °C, while the highest yield (82%) was obtained with a 1:12 oil/methanol molar ratio and 1% NaOH catalyst at 40 °C, showing that increasing the oil/methanol molar ratio and catalyst concentration can achieve higher biodiesel yields. However, these authors also highlighted that an oil/methanol molar ratio >1:6 promoted emulsion formation between the FAMEs and glycerol, limiting the separation of phases at the end of the process. This phenomenon was also observed by Hájek et al. [48], who transesterified rapeseed oil and analyzed the distribution of the FAMEs between biodiesel and the glycerol phases, finding that part of the esters migrated to the glycerol phase, resulting in a reduction of the presence of FAMEs in the biodiesel phase. For example, palmitic acid (C16:0) decreased from 5.2 to 0% in the biodiesel phase, while linolenic acid (C18:3) increased from 11 to 76%, and linoleic acid (C18:2) decreased from 62 to 17%. The authors also observed that increasing the catalyst concentration promoted saponification, which contributed fatty acid migration toward the glycerol phase. Furthermore, their results indicated that the oil/methanol molar ratio had a linear effect on the FAME losses.
In the case of the D15 larval oil, the total SFA content was 13% higher than the control diet. This may have limited the complete conversion of triglycerides to FAMEs. In addition, the disappearance of specific fatty acids from the biodiesel phase, as observed in our study, can be attributed to a partial migration to the glycerol fraction. Moreover, the use of a homogeneous catalyst may also influence the yield of the reaction. In the present work, a two-step process was performed. The use of a different catalyst and methanol in the influence in the biodiesel yield and composition was reported by He et al. [49], where Hermetia illucens larvae were used as a source of oil and the transesterification was performed to obtain the corresponding biodiesel. In this study, it was found that using lipase as a catalyst and a molar ratio of methanol/oil of 3:1 increased the fatty acid yields from 67% to 98% to the corresponding methyl esters, which confirmed that reaction parameters contribute to the total conversion of the oil to biodiesel. The results mentioned above suggest that an incomplete FAME conversion was obtained in the present work, which could be attributed to an incomplete reduction of the FFAs in the acid catalyzed step, while the total yield was <90%. Therefore, our results emphasize that optimizing the reaction conditions, such as catalyst concentration, oil/methanol molar ratio, temperature, and time, is necessary to prevent losses of short and medium chain FAMEs to achieve a complete biodiesel conversion.
Regarding economic and environmental concerns, T. molitor is a promising source for biomass obtention. Although the larval development for this insect is relatively long (130 d), dietary adjustments can reduce the development time, observed in higher pupae formation, while higher biomass and survival rates were also observed, which could result in more biomass in less time. Also, T. molitor can be reared under continuous production systems, allowing for overlapping generations. For example, Kowalski [50] reported a schedule for the maintenance of larvae, in which a new batch was introduced in week 12 (days 78–84), while separating larvae into size fractions. This approach demonstrated the feasibility of an industrial scale operation. According to Safavi et al. [51], this insect has the potential to be reared at industrial-scale since it produces less greenhouse emissions than other traditional processes. Moreover, this insect can be fed industrial organic waste, which could reduce operational costs. The process of feeding T. molitor and H. illucens with crop residues to produce biodiesel has been explored by Wang et al. [26], who found that mealworm larvae were capable of reducing corn stover waste by 50%, while obtaining an oil yield of 23% and a biodiesel conversion efficiency of 90%, demonstrating that insect biorefineries can convert crop residues to an energy source. In another sustainability assessment, Salomone et al. [52] evaluated the feasibility of using H. illucens for 1 kg of oil compared to rapeseed oil, highlighting that 6.44 m2 less land area was needed for production. However, the global warming potential increased by 0.2 kg CO2 eq, and the authors mentioned remaining uncertainties regarding the energy inputs, such as renewable sources. Furthermore, insect-based biorefinery processes produce more valuable byproducts, such as frass, which can be used as fertilizer, and oil residues that can serve as protein sources. In the case of T. molitor, the insect shows the potential for mass-rearing at industrial scale for biodiesel production, although further investigations and environmental assessments are still required.

5. Conclusions

The diet with 15% dextrose added to an artificial diet significantly influenced the growth and development of T. molitor larvae, increasing survival rate and individual weight, meaning that the overall biomass of this insect can be achieved with carbohydrate supplementation in an artificial diet. The oil extracted from the larvae fed with 15% dextrose has more saturated fatty acids, such as undecylic acid, and a reduction in unsaturated fatty acids, such as linoleic acid. However, the biodiesel obtained through acid/alkali transesterification did not have the same profile of fatty acid methyl esters as the oil, particularly in the D15 treatment, suggesting that reaction conditions such as the methanol/oil ratio, the catalyst, and/or the reaction time must be explored to achieve a total conversion of the fatty acids in the larval oil.
While most studies on T. molitor larvae mainly focuses on its application in animal feed, the present work provides novel evidence of the challenges and opportunities of using larval oil as an alternative for biofuel feedstock. This study highlights the need for further optimized transesterification methods based on the fatty acid profile for the biodiesel production from this insect.

Author Contributions

Conceptualization, M.A.F.-S. and M.A.R.-L.; data curation, M.A.F.-S., A.K.R.-C., J.d.J.B.-F. and M.A.R.-L.; formal analysis, M.A.F.-S., A.K.R.-C., J.d.J.B.-F., C.E.Z.-G. and M.A.R.-L.; funding acquisition, M.A.R.-L.; investigation, M.A.F.-S., A.K.R.-C., J.d.J.B.-F., C.E.Z.-G. and M.A.R.-L.; methodology, M.A.F.-S., A.K.R.-C., M.M.G.-C., R.F.-B., J.C.-G., C.E.Z.-G., A.A.-R., J.d.J.B.-F. and M.A.R.-L.; resources, M.A.F.-S., A.K.R.-C., J.d.J.B.-F. and M.A.R.-L.; supervision, J.C.-G., C.E.Z.-G., J.d.J.B.-F. and M.A.R.-L.; writing—original draft, M.A.F.-S. and M.A.R.-L.; writing—review and editing., J.C.-G., C.E.Z.-G., A.A.-R. and M.A.R.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the “Secretaría de Ciencia, Humanidades, Tecnología e Innovación” (SECIHTI), grant number 982882.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors acknowledge the SECIHTI for the support in this work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Formulation of the artificial diets for Tenebrio molitor larvae.
Table 1. Formulation of the artificial diets for Tenebrio molitor larvae.
Ingredient% Weight
ControlPS5PS9PS16PC5PC9PC16PD5PD9PD16
Carrot7777777777
Wheat bran11.711.711.711.711.711.711.711.711.711.7
Brewer’s yeast1.61.61.61.61.61.61.61.61.61.6
Ascorbic acid0.20.20.20.20.20.20.20.20.20.2
Neomycin0.10.10.10.10.10.10.10.10.10.1
Distilled water78.473.469.462.463.459.255.65248.444.8
Sodium triphosphate0.50.50.50.50.50.50.50.50.50.5
Methylparaben0.50.50.50.50.50.50.50.50.50.5
Starch05916000000
Cellulose00005916000
Dextrose00000005916
Table 2. Survival rate and T. molitor larval weight obtained from the feeding with modified artificial diets over 30 d.
Table 2. Survival rate and T. molitor larval weight obtained from the feeding with modified artificial diets over 30 d.
Treatment% Larval Survival% Formed Pupae% Accumulated
Survival		Mean Individual Fresh Larval Weight (mg)
Control15.76 ± 2.93 F28.50 ± 4.46 A44.26 ± 5.33 A133.33 ± 5.24 A
PS531.40 ± 3.36 DEF22.89 ± 3.96 ABC54.29 ± 2.03 A128.78 ± 7.48 A
PS924.34 ± 2.87 EF27.11 ± 3.31 AB51.45 ± 1.31 A131.53 ± 5.03 A
PS1632.85 ± 1.87 DE12.86 ± 2.63 CD45.71 ± 4.29 A128.63 ± 7.67 A
PC535.75 ± 1.32 DE7.19 ± 1.44 D42.94 ± 3.64 A128.77 ± 3.81 A
PC921.44 ± 2.65 EF21.44 ± 2.65 ABC42.88 ± 1.32 A129.84 ± 8.27 A
PC1644.18 ± 3.54 CD7.13 ± 1.59 D48.49 ± 3.50 A131.58 ± 3.55 A
PD565.76 ± 3.95 AB27.11 ± 2.02 AB92.87 ± 1.44 C114.47 ± 5.93 A
PD954.35 ± 2.70 BC18.60 ± 1.39 ABCD72.95 ± 3.31 B123.1 ± 1.85 A
PD1678.68 ± 3.18 A15.70 ± 1.30 BCD94.38 ± 1.48 C132.94 ± 2.31 A
The means with statistically significative difference have different letters n = 50 p < 0.05.
Table 3. Survival rate and T. molitor larval weight obtained from the feeding with modified artificial diets over 130 d.
Table 3. Survival rate and T. molitor larval weight obtained from the feeding with modified artificial diets over 130 d.
Treatment%Larval Survival %Formed Pupae% Accumulated
Survival		Mean Individual Fresh Larval Weight (mg)
Control22.67 ± 1.76 B8 ± 1.15 D30.67 ± 1.33 A69.39 ± 2.23 D
D641.16 ± 1.33 A10 ± 1.15 CD51.16 ± 1.76 B82.74 ± 2.76 CD
D946.67 ± 1.76 A14.67 ± 1.76 BC61.34 ± 2.67 BC112.16 ± 0.98 BC
D1252.67 ± 1.76 A17.33 ± 1.33 AB70 ± 1.15 C128.93 ± 1.40 AB
D1553.33 ± 1.76 A21.33 ± 1.33 A74.66 ± 0.67 C150.76 ± 1.89 A
The means with statistically significative difference have different letters n = 150 p < 0.05.
Table 4. Total oil yield from 500 Tenebrio molitor larvae fed with artificial control diet and 15% added dextrose after 130 d.
Table 4. Total oil yield from 500 Tenebrio molitor larvae fed with artificial control diet and 15% added dextrose after 130 d.
TreatmentSurvival Rate (%)Total Fresh Weight (g)Total Dry Weight (g)Humidity (%)Mean Individual Dry Weight (mg)Oil Yield (%)
Control5019.344.9674.3519.8429.17
D156428.588.0471.8725.1229.07
Table 5. Relative fatty acid composition of oil from Tenebrio molitor larvae fed on artificial diet modified with dextrose.
Table 5. Relative fatty acid composition of oil from Tenebrio molitor larvae fed on artificial diet modified with dextrose.
Fatty Acid Content (%)
Carbon ChainCommon NameControl DietD15
C11:0Undecylic acid8.1527.49
C12:0Lauric acid-0.65
C16:0Palmitic acid14.5714.14
C18:1Oleic acid34.2637.42
C18:2Linoleic acid30.3720.30
C18:3α-Linoleic acid5.82-
C19:0Nonadecanoic acid6.83-
Saturated Fatty Acids (SFAs) 29.5542.28
Unsaturated Fatty Acids (UFAs) 70.4557.72
Monounsaturated Fatty Acids (MUFAs) 34.2637.42
Polyunsaturated Fatty Acids (PUFAs) 36.1920.30
PUFAs/SFAs 1.220.48
Oil yield (%) 29.1729.07
The values of the fatty acids content are reported from one sample of each oil (n = 1).
Table 6. Relative fatty acid methyl esters composition of biodiesel from Tenebrio molitor larvae fed on control diet and D15.
Table 6. Relative fatty acid methyl esters composition of biodiesel from Tenebrio molitor larvae fed on control diet and D15.
Carbon ChainCommon NameControl DietD15
C11:0Methyl undecanoate-0.26
C12:0Methyl laurate0.47-
C16:0Methyl palmitate21.2522.77
C18:1Methyl oleate49.4447.10
C18:2Methyl linoleate26.4529.87
C18:3Methyl linolenate2.39-
Saturated Fatty Acids (SFAs) 21.7123.03
Unsaturated Fatty Acids (UFAs) 78.2976.97
Monounsaturated Fatty Acids (MUFAs) 49.4447.70
Polyunsaturated Fatty Acids (PUFAs) 28.8529.27
PUFAs/SFAs 1.331.27
Biodiesel yield (%) 80.485.7
The values of the fatty acid methyl esters content are reported from one sample of each biodiesel (n = 1).
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Flores-Sánchez, M.A.; Rico-Chávez, A.K.; González-Chávez, M.M.; Figueroa-Brito, R.; Campos-Guillen, J.; Zavala-Gómez, C.E.; Amaro-Reyes, A.; Barba-Franco, J.d.J.; Ramos-López, M.A. Effect of Artificial Diet Modification with Dextrose on the Growth and Fatty Acid Composition of Tenebrio molitor Larvae for Biodiesel Production. Appl. Sci. 2025, 15, 10777. https://doi.org/10.3390/app151910777

AMA Style

Flores-Sánchez MA, Rico-Chávez AK, González-Chávez MM, Figueroa-Brito R, Campos-Guillen J, Zavala-Gómez CE, Amaro-Reyes A, Barba-Franco JdJ, Ramos-López MA. Effect of Artificial Diet Modification with Dextrose on the Growth and Fatty Acid Composition of Tenebrio molitor Larvae for Biodiesel Production. Applied Sciences. 2025; 15(19):10777. https://doi.org/10.3390/app151910777

Chicago/Turabian Style

Flores-Sánchez, Miguel Alejandro, Amanda Kim Rico-Chávez, Marco Martín González-Chávez, Rodolfo Figueroa-Brito, Juan Campos-Guillen, Carlos Eduardo Zavala-Gómez, Aldo Amaro-Reyes, Joel de Jesús Barba-Franco, and Miguel Angel Ramos-López. 2025. "Effect of Artificial Diet Modification with Dextrose on the Growth and Fatty Acid Composition of Tenebrio molitor Larvae for Biodiesel Production" Applied Sciences 15, no. 19: 10777. https://doi.org/10.3390/app151910777

APA Style

Flores-Sánchez, M. A., Rico-Chávez, A. K., González-Chávez, M. M., Figueroa-Brito, R., Campos-Guillen, J., Zavala-Gómez, C. E., Amaro-Reyes, A., Barba-Franco, J. d. J., & Ramos-López, M. A. (2025). Effect of Artificial Diet Modification with Dextrose on the Growth and Fatty Acid Composition of Tenebrio molitor Larvae for Biodiesel Production. Applied Sciences, 15(19), 10777. https://doi.org/10.3390/app151910777

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