Sustainable biodiesel synthesis via non-catalytic transesterification of biomass waste-derived oil and ethanol

Abstract

Ethanol (EtOH)-based biodiesel, known as fatty acid ethyl esters (FAEE), offers better fuel properties than methanol-based biodiesel. Given that EtOH can be produced via fermentation of lignocellulosic waste, the use of waste-derived ethanol represents an environmentally sustainable strategy for biodiesel production. In this approach, EtOH and oil are derived from waste materials, such as silkworm litter and discarded silkworm pupae, reinforcing the potential for an integrated waste-to-biofuel platform. This study demonstrated that EtOH production from silkworm litter hydrolysed using yeast was comparable to that from synthetic media, highlighting a green and cost-effective method for EtOH synthesis. EtOH was used in the transesterification process to convert oil extracted from silkworm pupae (which had a high oil content of 32.0 wt%) into biodiesel. Given the presence of impurities in pupal oil, a non-catalytic transesterification process was selected as it remained unaffected by such contaminants. Based on the availability of silkworm litter and discarded silkworm pupae, it was estimated that 50% of the required energy could be supplied by energy production from in situ waste. This innovative waste-to-energy strategy provides a sustainable method for FAEE production and contributes to the environmental and economic sustainability of the textile industry.

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Green foundation

1. This study aimed to contribute to achieving SDG 7 (affordable and clean energy) and SDG 12 (responsible consumption and production) through an in situ waste-to-energy approach, specifically targeting waste from the textile industry. We used silkworm litter to produce ethanol, which was subsequently used to generate ethanol-based biodiesel (fatty acid ethyl ester). The oil for the biodiesel was derived from another textile industry byproduct: discarded silkworm pupae.

2. Our research findings indicate that 776 MJ of energy can be obtained from the valorisation of 2 kg of silkworm pupae and 100 kg of silkworm litter, which are byproducts from the production of 1 kg of silk. These results suggest that the energy produced from sericulture waste can supply half of the energy required for the sericulture process itself.

3. Future work could focus on evaluating the economic feasibility of this in situ waste-to-energy approach.

1. Introduction

The exploitation of fossil resources has been a cornerstone in the rapid advancement and upkeep of modern society.^1,2 Although fossil fuel use has facilitated economic growth and technological progress, it poses environmental risks. The combustion of fossil fuels is a primary contributor to the greenhouse gas (GHG) emissions that drive global warming.³ This dependence on fossil fuels for energy production presents a critical challenge: should we continue to support the existing economic frameworks at the expense of the environment, or should we curtail economic activities to preserve our ecological resources?^4,5 This predicament highlights the fundamental conflict between economic development and environmental conservation, meaning that it is difficult to achieve a sustainable balance under current paradigms.⁶ To address these intertwined challenges (sustaining societal functions while minimising environmental damage), the United Nations introduced Sustainable Development Goal 7. This goal emphasises increasing access to clean and sustainable energy sources, thereby reducing the dependence on fossil fuels.⁷

In the pursuit of sustainable energy solutions, biofuels have gained considerable attention because of their carbon-neutral potential.⁸ Biomass can be converted into platform molecules through biorefinery processes.⁹ These platform molecules can then be further upgraded into biofuels via chemical methods such as catalytic transfer hydrogenation.¹⁰ While these processes hold potential for sustainable fuel production, they have not yet been commercialised. In contrast, biodiesel production from biomass is commercially available and is considered a cleaner alternative to petroleum-based diesel for engine applications.¹¹ Biodiesel, chemically known as fatty acid alkyl ester, is synthesised through transesterification, in which biomass-derived oils react with primary alcohols to form esters and glycerol.¹² Methanol (MeOH) has traditionally been the preferred alcohol for producing fatty acid methyl esters (FAMEs) because of its affordability and high reactivity.¹³ However, MeOH presents several challenges, including safety risks associated with its toxicity and flammability.¹⁴ The sustainability of biodiesel is also compromised because >90% of MeOH is derived from fossil fuels, particularly natural gas.¹⁵ This reliance on fossil-fuel-based MeOH undermines the environmental benefits of biodiesel, necessitating the exploration of a more sustainable feedstock for its production.

In recent years, ethanol (EtOH) has emerged as a promising “green” alcohol in biodiesel production, primarily due to its lower toxicity and the potential for being sourced from biomass through fermentation processes.^14,16 Biodiesel produced with EtOH, known as fatty acid ethyl esters (FAEEs), exhibits distinct fuel characteristics compared with MeOH-based biodiesel (FAMEs). The presence of an additional carbon atom in EtOH compared with MeOH imparts several advantages to FAEEs.¹⁷ FAEEs possess lower cloud and pour points and higher heat of combustion than FAMEs.¹⁸ These facts can contribute to superior fuel quality, making FAEEs a potentially more efficient and versatile alternative to FAMEs and traditional petro-diesel.¹⁶ Moreover, the combustion of FAEEs, compared to FAMEs, results in lower pollutant emissions (including nitrogen oxides, carbon dioxide, and particulate matter).¹⁹ This reduced emission profile indicates the potential of FAEEs as an environmentally friendly option for biodiesel production.

Based on this rationale, this study introduces an in situ waste-to-energy approach for the sustainable production of FAEEs using waste from the textile industry, with a focus on the silk production sector. In this strategy, the oil and alcohol (particularly EtOH) required for FAEE production were sourced from waste materials generated during silk manufacturing. Specifically, oil is extracted from waste silkworm pupae, and EtOH is synthesised by fermenting silkworm litter (a lignocellulose-based waste) after acid pretreatment and saccharification. To enhance bioethanol production, the lignocellulosic waste was characterised, and acid pretreatment and saccharification were optimised using response surface methodology (RSM). The production of 1 kg of silk generates 2 kg of pupal waste and 100 kg of litter.²⁰ Converting these substantial waste streams into biodiesel addresses waste disposal challenges and enhances economic and environmental sustainability. This study employed a non-catalytic transesterification process to convert oil and EtOH into FAEEs, thus eliminating the need for expensive catalysts and simplifying the production process. This approach leverages the vast scale of the textile industry,^21,22 offering global applicability and promising a contribution to sustainability in the sector by repurposing waste into valuable biofuel.

2. Materials and methods

2.1. Silkworm litter characterisation

Silkworm litter and mulberry leaves were obtained from a farm in the Republic of Korea. The lignocellulosic components (cellulose, hemicellulose, and lignin) were analysed following previously reported analytical protocols.^23,24 Crude fat was extracted using a Soxhlet apparatus, and its content was determined using the gravimetric method. Crude protein content was determined using the Kjeldahl method.²⁵ The ash content was also measured using the gravimetric method after combustion of the litter at 550 °C for 3 h.

2.2. Acid pretreatment and saccharification

Response surface methodology (RSM) was used to determine the optimal conditions for the acid treatment of silkworm litter. A 3-level-3-factor using a Box–Behnken design was employed with the following variables: (i) temperature (110, 120, and 130 °C), (ii) reaction time (1.5, 2, and 2.5 h), and (iii) sulfuric acid concentration (3, 4, and 5 wt%). Design-Expert 12 software (Stat-Ease, Inc., USA) was used for prediction.²⁶ The model equation was derived using multiple regression analysis, and its accuracy was assessed using the coefficients of determination and analysis of variance (ANOVA).

Saccharification using CTec2 (Novozymes, Denmark) was conducted on the acid hydrolysate produced under optimised conditions. Specifically, 4 vol% of the enzyme was added to the acid hydrolysate and incubated for 72 h at 50 °C while shaking at 120 rpm. The sugar content (glucose, xylose, cellobiose, and arabinose) was quantified using a high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific, USA) equipped with an Aminex HPX-87H column (length: 300 mm, internal diameter: 7.8 mm). Sulfuric acid (5 mM) was used as the mobile phase at a flow rate of 0.6 mL min⁻¹. A refractive index detector, maintained at 35 °C, was used to detect the sugar content. Standard chemicals (glucose (purity: 98%, Junsei, Japan), xylose (purity: 98%, Junsei, Japan), D(+)-cellobiose (purity: >99%, Fluka, Switzerland), DL-arabinose (purity: >98%, Fluka, Switzerland), and pure EtOH (Sigma-Aldrich, USA)) were dissolved in distilled water and the prepared standard solutions were used to quantify the concentration of each sugar compound. The standard calibration curves of each compound are shown in Fig. S1.†

2.3. Fermentation

The yeast strain Pichia stipitis KCTC 7222 was obtained from the Korean Collection for Type Cultures (Republic of Korea). For cell maintenance, the YPD medium was used, containing 20 g L⁻¹ xylose, 5 g L⁻¹ yeast extract, 5 g L⁻¹ peptone, 5 g L⁻¹ glucose, 1 g L⁻¹ magnesium sulphate, and 1 g L⁻¹ of monopotassium phosphate. Cells were subcultured for 24 h using a 1 : 1 mixture of YPD medium and silkworm litter hydrolysate. Twenty millilitres of the subculture (optical density at 600 nm: 3.0) was inoculated into 180 mL of fresh medium, either modified YPD medium or silkworm litter hydrolysate. The modified YPD medium contained high amounts of glucose (30 g L⁻¹) and xylose (10 g L⁻¹), which matched the glucose and xylose concentrations in silkworm litter hydrolysate. Fermentation was conducted in a shaking incubator at 90 rpm, maintained at 30 °C. The resulting EtOH was separated from the cell culture solution via distillation. The EtOH content was quantified using an HPLC system following the same method used for sugar content quantification.

2.4. Oil extraction from wasted silkworm pupae

Silkworm pupae were obtained from a farm in the Republic of Korea. Silkworm pupae were dried in an oven (100 °C) and ground before oil extraction. The oil was extracted using a Soxhlet apparatus with n-hexane as the solvent. Thermogravimetric analysis (TGA) was conducted (STA 449 F5 Jupiter, NETZSCH, Germany) to confirm the thermal degradation behaviour of the extracted oil. At a heating rate of 10 °C min⁻¹, 10 mg of the oil was heated from 50 to 900 °C. TGA was performed under nitrogen (N₂) conditions at a flow rate of 100 mL min⁻¹.

2.5. Non-catalytic transesterification

Non-catalytic transesterification has been conducted to convert silkworm pupal oil into biodiesel.²⁷ Silica (320 mg), the oil (8 μL), and alcohol (MeOH, EtOH, or a mixture; total 5 mmol) were added to a bulkhead union (SS-400-61, Swagelok, USA) and the union was sealed with two stoppers (SS-400-P, Swagelok). The sample-loaded reactor was placed in a furnace (450 °C). When the internal temperature of the reactor reached the target value, the reactor was removed from the furnace and cooled to room temperature. The internal temperature of the reactor was measured by using a K-type thermocouple (OMEGA, USA).

2.6. FAME/FAEE quantification

After non-catalytic transesterification, the FAMEs and/or FAEEs were collected by dissolving the products in dichloromethane. The diluted products were analysed using a gas chromatography (GC)/flame ionisation detector (8890, Agilent, USA) and a GC/mass spectrometer (5977 B, Agilent), equipped with a DB-WAX column (length: 30 m, internal diameter: 0.25 mm, and film thickness: 0.25 μm). The operational parameters of the GC were as follows: initial hold at 50 °C for 3 min, ramp to 180 °C (10 °C min⁻¹) and hold for 5 min, ramp to 190 °C (5 °C min⁻¹) and hold for 5 min, ramp to 220 °C (2 °C min⁻¹) and hold for 5 min, and final ramp to 230 °C (2 °C min⁻¹) and hold for 7 min. External standard mixtures (FAME and FAEE mixtures purchased from Sigma Aldrich, USA) were diluted with dichloromethane. The prepared standard solutions were used for FAME/FAEE quantification (the standard calibration curves of each compound are shown in Fig. S2 and S3†). The compounds that were not included in the FAEE standard mixtures were estimated using a relative response factor based on the effective-carbon-number method.²⁸

3. Results and discussion

3.1. EtOH production from silkworm litter

The biochemical compositions of the mulberry leaves and silkworm litter are listed in Table 1. Given that the primary feed for silkworms is mulberry leaves,²⁹ the biochemical compositions of mulberry leaves and silkworm litter are similar, particularly the lignocellulosic components (cellulose, hemicellulose, and lignin). The major component of the silkworm litter is lignocellulosic material, followed by protein and ash. Lipid content was negligible (1.2 wt% of silkworm litter). Because silkworm litter contains carbon-rich lignocellulosic materials, it was assumed that saccharification could be efficiently realised for sugar production. In addition, it was expected that the proteins in silkworm litter could serve as nitrogen sources for yeast.

Table 1 Biochemical analysis of silkworm litter and mulberry leaves (unit: wt%)

	Lignocellulosic components			Crude fat	Crude protein	Ash	Moisture
	Cellulose	Hemicellulose	Lignin
Silkworm litter	22.8	3.0	3.5	1.2	12.9	14.7	12.2
Mulberry leaves	24.4	2.2	4.2	2.9	16.3	13.9	7.3

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Prior to the enzymatic saccharification of silkworm litter, sulfuric acid pretreatment was conducted to enhance the saccharification efficiency. Dilute sulfuric acid is effective for recovering hemicellulose sugars and enhancing enzymatic convertibility towards cellulose degradation.³⁰ The efficiency of the acid pretreatment depends on the reaction conditions, including the acid concentration, reaction time, and temperature.³¹ Therefore, the optimal sulfuric acid pretreatment conditions were determined using RSM. Specifically, the Box–Behnken method (3-level-3-factor) was employed to predict the optimal temperature (X₁), reaction time (X₂), and sulfuric acid concentration (X₃).²⁶ Sugar recovery yields under various conditions are listed in Table 2. The interactions between the parameters and sugar recovery yield are illustrated in 3D plots (Fig. 1).

Fig. 1 Response surface and contour plots for optimised pretreatment conditions of silkworm litter. Interaction between sugar recovery yield and the parameters: (A) temperature and reaction time, (B) reaction time and sulfuric acid concentration, and (C) temperature and sulfuric acid concentration.

Table 2 RSM with a 3-level-3-factor design with the employment of a Box–Behnken method

Temperature (°C)	Reaction time (h)	Sulfuric acid concentration (wt%)	Sugar recovery yield (wt%)
120	2	4	87.9
110	2.5	4	68.0
130	2	5	86.7
120	2	4	88.4
130	2.5	4	89.7
120	2	4	90.2
120	2	4	90.3
130	1.5	4	80.0
110	2	3	73.9
120	2.5	3	81.2
130	2	3	88.3
120	2.5	5	74.0
120	2	4	89.6
110	1.5	4	83.6
110	2	5	70.2
120	1.5	3	61.4
120	1.5	5	76.3

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ANOVA was conducted to evaluate the accuracy of the model. As shown in Table 3, the quadratic model has a high R² value (0.9487), suggesting high reliability. This model was statistically significant (p < 0.01) (Table 4). Based on the ANOVA results, the sugar recovery yield was influenced by five model terms (X₁, X₁X₂, X₂X₃, X₂², and X₃²), all of which were significant at the 1% level (p < 0.01). The coded factors detailed in Table 3 are used to forecast the responses for each factor level. This coded equation facilitates the determination of the relative influence of the factors by allowing a comparison of the magnitudes of the coefficients corresponding to each factor.³²

Table 3 Model equations for sugar recovery yield. X₁, X₂, and X₃ indicate the temperature, reaction time, and sulfuric acid concentration, respectively

Equation in terms of actual factors	Model	R ^2
Sugar recovery yield (wt%) = 12.29173–2.38979 X1 − 18.58883 X2 + 96.66639 X3 + 1.53610 X1X2 − 11.02630 X2X3 − 29.34322 X2^2 − 9.29057 X3^2	Quadratic	0.9487
Equation in terms of coded factors	Sugar recovery yield (wt%) = 89.44 + 6.82 X1 + 2.13 X2 − 0.2892 X3 + 7.68 X1X2 − 5.51 X2X3 − 7.34 X2^2 − 9.29 X3^2

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Table 4 Analysis of variance (ANOVA) for the sugar recovery models. X₁, X₂, and X₃ indicate temperature, reaction time, and sulfuric acid concentration, respectively

Source	Sum of squares	Degrees of freedom	Mean square	F-Value	p-Value
Model	1392.71	7	198.96	23.79	<0.01
Residual	75.26	9	8.36
Lack of fit	70.52	5	14.10	11.90	0.0163
Pure error	4.74	4	1.19
Corrected total	1467.98	16
X 1	372.56	1	372.56	44.55	<0.01
X 1 X 2	235.96	1	235.96	28.22	<0.01
X 2 X 3	121.58	1	121.58	14.54	<0.01
X 2 ^2	227.21	1	227.21	27.17	<0.01
X 3 ^2	364.44	1	364.44	43.58	<0.01

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Among the single-factor effects on sugar recovery yield, one linear coefficient (X₁) and two quadratic coefficients (X₂² and X₃²) were the most crucial. The order of single-factor effects on sugar recovery yields was X₃² (sulfuric acid concentration) > X₂² (reaction time) > X₁ (temperature). For the combined factors, the two linear coefficients were significant in the sequence X₁X₂ > X₂X₃. Overall, the significance of each factor was ranked as follows: X₁X₂ > X₃² > X₂² > X₁ > X₂X₃. This ranking indicates that the squared terms of the factors are significant influencers, with the combination of temperature and reaction time being the most critical. Considering the RSM results, the optimal conditions for silkworm litter pretreatment were the addition of 4 wt% of sulfuric acid and incubation for 2 h at 120 °C. Under the optimal pretreatment conditions, the sugar recovery yield was 90.3 wt%.

The silkworm litter hydrolysate produced after saccharification contained four types of sugar compounds: cellobiose, glucose, xylose, and arabinose. Glucose and xylose were the most dominant, accounting for 57 and 20 wt%, respectively, of the total sugary compounds. The yeast species P. stipitis was used to produce EtOH from the silkworm litter hydrolysate. Given that P. stipitis can co-metabolise glucose and xylose,³³ this species is suitable for cultivation in silkworm litter hydrolysate. The profile of EtOH production by P. stipitis grown in silkworm litter hydrolysate is shown in Fig. 2. As a reference, a synthetic medium (modified YPD) was used for the cultivation of P. stipitis.

Fig. 2 Profiles of ethanol (EtOH) production by P. stipitis grown in (A) modified YPD medium and (B) silkworm litter hydrolysate.

The EtOH yield from the synthetic medium after 96 h was 13.2 g L⁻¹ (Fig. 2A). Although P. stipitis can metabolise both glucose and xylose, it shows greater preference for glucose. The glucose concentration in the medium decreased starting from 6 h of fermentation and was completely depleted in 36 h. However, xylose consumption by P. stipitis began after 30 h and decreased after glucose was depleted. This consumption pattern suggests repression of xylose uptake in the presence of glucose.³⁴

The growth pattern of P. stipitis in the silkworm litter hydrolysate differed from that in the synthetic medium. Simultaneous consumption of glucose and xylose was observed starting at 48 h (Fig. 2B). These results suggest that sugar metabolism in P. stipitis is likely affected by unknown compounds in the hydrolysate. Silkworm litter contains various (in)organic compounds, including amino acids.²⁰ Given that calcium, magnesium, amino acids, and other compounds are likely to influence the growth and EtOH production of P. stipitis, it is plausible that these compounds in silkworm litter affect sugar transport systems.³⁵

Nevertheless, EtOH production from silkworm litter hydrolysate was comparable to that from the synthetic medium, yielding 12.2 g L⁻¹. No additional resources were added to the silkworm litter hydrolysates. These results indicate that silkworm litter provides nitrogen sources, as hypothesised from the results of the biochemical analysis (Table 1). Considering the cost of chemicals such as peptone and yeast extract for synthetic medium preparation, the lack of additional substrates can contribute to the economic viability of the process. In summary, using silkworm litter as a feedstock for EtOH production offers environmental (by minimising waste through valorisation) and economic (by reducing production costs) advantages.

3.2. Biodiesel production from wasted silkworm pupae using EtOH derived from silkworm litter

To produce biodiesel from waste silkworm pupae, pupal oil was extracted using a Soxhlet extraction system. The extracted pupal oil had a dark brown colour. The oil content was 32 wt%. Given that non-catalytic transesterification is realised using thermal energy, it is necessary to scrutinise the thermal mass loss pattern of the oil.³⁶ TGA was conducted to characterise the mass loss behaviour of the pupal oil (Fig. 3). Moreover, the TGA test indicated the presence of impurities.³⁷ As shown in Fig. 3, the mass loss of pupal oil was initiated at 200 °C. The mass loss rate, represented in a differential thermogram (DTG), increased from 200 °C and slightly decreased between 300 and 314 °C. At >314 °C, the mass loss rate rapidly increased again, peaking at 416 °C. The residual mass of oil was <3 wt% at >480 °C. Refined oils containing >99 wt% triglycerides (TGs) typically have one DTG peak; the mass loss pattern of pupal oil between 200 and 300 °C is attributed to the volatilisation of impurities rather than TGs.³⁸ Indeed, the impurities such as tricosane, pentacosane, and heptacosane were detected in silkworm pupal oil. A previous study reported that the impurities accounted for 6.2 wt% of silkworm pupal oil.³⁹

Fig. 3 Thermogram and DTG curve of silkworm pupal oil.

It is worth noting that the initiation of mass loss of TGs at 200 °C does not signify the initiation of thermal decomposition (thermolysis) of TGs. Rather, this early mass loss could be attributed to the volatilisation of TG molecules. The thermal cracking of TGs proceeds through a sequence of reactions, beginning with ester bond cleavage, leading to the formation of fatty acids and glycerides, followed by decarboxylation and β-scission of fatty acids to generate hydrocarbons and aromatics.⁴⁰ In particular, while ester bond cleavage may occur at relatively lower temperatures, the thermal cracking of the liberated fatty acids generally requires temperatures ≥400 °C.⁴¹ Thus, in the temperature range of 200 to 400 °C, the observed mass loss might be due to the volatilisation of TGs rather than their chemical decomposition. It is plausible that although the scission of ester bonds in volatilised TGs may result in the formation of fatty acids and glycerides, the subsequent cracking of fatty acids is unlikely to proceed at these lower temperatures.

Conventional transesterification methods using acidic or alkaline catalysts are sensitive to the presence of impurities in oils, which likely induce side reactions such as saponification.⁴² In contrast, non-catalytic transesterification exhibits an extremely high tolerance to impurities.^43,44 In non-catalytic transesterification, the chemical bonds between fatty acids and the glyceride backbone are initially cleaved by thermal energy.²⁷ The liberated fatty acids then react with alcohols, such as MeOH and EtOH, resulting in the formation of FAMEs/FAEEs. This reaction is completed in <1 min because of the different phases of the reactants (alcohols in the gas phase and TGs in the liquid phase), which are attributed to the low boiling points of the alcohols.⁴⁵ According to collision theory, the heterogeneous phases of the reactants facilitate increased reaction kinetics, particularly in nano-sized pores.^36,46 Silica, which has 6–8 nm pores, was used as the porous material.⁴⁷

EtOH derived from silkworm litter was used to convert pupal oil into biodiesel (FAEEs) through non-catalytic transesterification. Chemically synthesised MeOH was used as a reference to produce FAMEs. The FAME yield from the pupal oil was <20 wt% at temperatures of ≤300 °C (Fig. 4A). These results indicate that the ester bonds were not cleaved completely under 300 °C. As the reaction temperature increased, FAME yield increased, reaching 91.3 wt% at 380 °C. The FAME yield via the acid-catalysed transesterification of silkworm pupal oil was 73.0 wt%.⁴⁸ Halada Nandakrishnan et al. reported a high FAME yield (90 wt%) from silkworm pupal oil; they conducted the two-step biodiesel conversion process (acid-catalysed esterification followed by base-catalysed transesterification); however, this method required more time and resources.⁴⁹

Fig. 4 Production of (A) FAME and (B) FAEE using silkworm pupal oil through non-catalytic transesterification.

In contrast, the non-catalytic transesterification of silkworm pupal oil afforded a high yield (91.3 wt%) without any pretreatment processes. This high yield further supports the hypothesis that thermal cracking of fatty acids did not occur at the reaction temperature of 380 °C, as such degradation would have reduced the FAME yield. Instead, the reaction conditions appear to have favoured transesterification over thermal decomposition, maintaining the integrity of the fatty acid chains necessary for efficient FAME production. Although thermal decomposition of fatty acids can also occur at temperatures below 400 °C, depending on factors such as reaction duration and pressure,⁵⁰ it was unlikely to be a concern in this study. This is because the non-catalytic transesterification was conducted in less than one minute under ambient pressure, conditions that are not conducive to significant thermal cracking. Simultaneously, thermal cracking of the products did not occur, as thermal cracking of FAMEs occurs at ≥530 °C.⁵¹

Fig. 4B shows the FAEE yield of pupal oil obtained through non-catalytic transesterification. Similar to the FAME conversion, the FAEE yield increased with the reaction temperature and showed a maximal yield at 380 °C. Because the boiling point of EtOH is 78 °C,⁵² EtOH exists in a gaseous state in the reactor. Therefore, the reaction mechanism for FAME conversion via non-catalytic transesterification (a heterogeneous reaction between gaseous alcohols and liquid TGs) can be applied identically to that for FAEE conversion. However, the FAEE yield at 380 °C (81.8 wt%) was lower than the FAME yield at the same reaction temperature (91.3 wt%). Because the FAEE yield at 390 °C did not increase compared with that at 380 °C, it is reasonable to conclude that a reaction temperature of 380 °C is sufficient for FAEE conversion. In other words, the FAEE yield through the non-catalytic transesterification of pupal oil is fully saturated.

There are two possible reasons for this. First, the lower FAEE yield compared to the FAME yield may be attributed to the different reactivities of the alcohols.⁵³ The lower reactivity of EtOH in transesterification reactions compared to that of MeOH was confirmed by conducting non-catalytic transesterification at various molar ratios of MeOH/EtOH. The compositional changes of the four major fatty acid alkyl esters (C_16:0, C_18:0, C_18:1, and C_18:3) at various MeOH/EtOH molar ratios are shown in Fig. 5A. The FAEE content increased with increasing EtOH content. Nevertheless, as hypothesised, the reactivity of EtOH in non-catalytic transesterification was lower than that of MeOH (Fig. 5B). Notably, the FAME content (63 wt%) was higher than the FAEE content (37 wt%), even at an equal molar ratio (5 : 5).

Fig. 5 (A) FAME and FAEE compositions of silkworm pupal oil under diverse molar ratios of MeOH/EtOH and (B) yield ratios.

Another explanation could be the purity of the EtOH derived from the silkworm litter. Distillation was employed to recover EtOH from the fermentation broth, and the purity was determined to be 93.8%. Although this technique is well developed and commercially available, it has limitations in separating azeotropic mixtures such as water and EtOH.⁵⁴ In general, the purity of EtOH obtained by distillation is <95%.⁵⁵ Given that the conversion efficiency of non-catalytic transesterification is not affected by impurities such as water,⁵⁶ it is plausible that the lower purity of EtOH resulted in fewer moles of EtOH than calculated, which may have contributed to the lower observed yield. The FAEE yield increased to over 85.2 wt% when high-purity (≥99%) EtOH is used. This suggests that the FAEE yield can be enhanced using high-purity EtOH. An increase in the purity of EtOH can be achieved by integrating the distillation technique with an additional separation process, such as membrane techniques.⁵⁵

Indeed, FAEEs can be advantageous over FAMEs in terms of fuel performance owing to their longer carbon chains.⁵⁷ Parameters such as gross heat of combustion, lubricity, and viscosity are improved in FAEEs compared to FAMEs.¹⁸ The high lubricity of FAEEs can reduce the wear of engine components,⁵⁸ thereby lowering engine maintenance costs. FAEEs also exhibit higher oxidative stability than FAMEs.¹⁴ A comparison of the fuel properties of FAEEs and FAMEs derived from silkworm pupal oil should be a subject for future studies.

One of the major limitations of FAEEs is the higher cost of EtOH compared with MeOH.⁵⁹ Indeed, the price of FAME starts at $0.28 L⁻¹, whereas the price of FAEE starts at $0.92 L⁻¹.¹⁶ Nevertheless, this study produced EtOH from textile industry waste (silkworm litter) in an environmentally friendly manner. Because the feedstock cost is negligible (as it is waste) and no additional compounds are used to produce EtOH, the production cost is expected to decrease. Comprehensive economic and environmental analyses must be conducted to thoroughly evaluate the viability of this strategy. In particular, scale-up experiments will be necessary to validate the feasibility and performance of the proposed processes under industrial conditions. Despite this, the waste valorisation approach might contribute to enhancing the sustainability of the industry.

A schematic of the entire process (EtOH production from silkworm litter, FAEE production from silkworm pupae, and EtOH derived from the former process) is shown in Fig. 6. To present the results clearly, calculations were based on the amount of waste generated during the production of 1 kg of silk, using the results obtained from this study as the basis. As previously mentioned, 1 kg of silk generates 2 kg of pupal waste and 100 kg of litter.²⁰ Given that the oil content in the wasted silkworm pupae was 32.0 wt%, 0.6 kg of pupal oil could be obtained from 2 kg of waste pupae. To convert 0.6 kg of pupal oil into FAEEs via non-catalytic transesterification, 88.4 g of EtOH (purity: 93.8% basis) was required. Considering the sugar yield (90.3 wt%) and total sugar-to-EtOH conversion yield (28.3 wt%), 25.6 kg of EtOH could be obtained from 100 kg of silkworm litter. This calculation indicates that FAEEs from silkworm pupae could be fully produced using EtOH from the fermentation of silkworm litter. In short, 0.5 kg of FAEEs and 25.5 kg of EtOH (remaining after transesterification) were obtained from the valorisation of 2 kg of pupae and 100 kg of litter. Given that diesel and gasoline engines differ,⁶⁰ the dual production of biodiesel and EtOH could benefit practical applications of the energy produced.

Fig. 6 Conceptual schematic of the proposed cascading valorisation pathway for sericulture waste based on the experimental results of this study.

Sericulture is an energy-intensive industry, requiring substantial energy for irrigation, cocoon drying, stifling, and reeling.⁶¹ In particular, 1539 MJ of energy is required to produce 1 kg of silk.⁶² Given that the higher heating values of biodiesel and EtOH were 38.6 and 29.7 MJ kg⁻¹,^63,64 it was calculated that 776 MJ of energy could be obtained from the valorisation of 2 kg of pupae and 100 kg of litter (generated from the production of 1 kg of silk). These results indicate that the energy produced from sericulture waste supplies half the energy required for sericulture waste. Overall, this strategy enhances the sustainability of the textile industry (particularly sericulture) by using textile waste as a feedstock for renewable energy production.

4. Conclusions

This study introduced a strategic approach for converting textile industry waste (sericulture waste) into FAEEs (biodiesel). Acid pretreatment was conducted on silkworm litter to enhance the saccharification efficiency. Under optimised conditions, the sugar recovery yield was 90.3 wt%. The silkworm litter was converted to EtOH via yeast fermentation, yielding 13.2 g L⁻¹. The resulting EtOH was used for FAEE production. Another sericulture waste product, silkworm pupae, was used as a lipid feedstock because of its high oil content (32.0 wt%). The extracted pupal oil contained impurities; therefore, non-catalytic transesterification, which shows a high tolerance towards impurities, was employed to convert the pupal oil into biodiesel. FAEEs were produced using pupal oil and EtOH derived from silkworm litter, although the yield was lower than the FAME yield, owing to the lower reactivity of EtOH. Nevertheless, the use of waste-derived EtOH for FAEE production offers environmental and economic advantages.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgements

This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean Government (MSIT) (NRF-2023R1A2C3003011 and NRF-2021R1F1A1052782).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5gc02712h

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