Synthesis of bio-oil from waste Trichosanthes cucumerina seeds: a substitute for conventional fuel

The present study explores the methodology for the synthesis of bio-oil from waste trichosanthes cucumerina seeds by the solvent extraction method. It investigates the yield percentage, concentration of free fatty acids and acid contents in the extracted bio-oil. Effects of size of the crushed seeds, moisture content, extraction time, solvent to seed ratio and extraction temperatures were examined. The non-polar hexane solvent resulted in a higher percentage of oil yield (28.4 ± 0.4%) for the crushed seed size of 0.21 mm, 6% moisture content, 270 min extraction time, 68 °C temperature and 6:1(ml/g) of solvent to seed ratio. The synthesized bio-oil was characterized using Fourier Transform Infra-Red spectrum and Gas Chromatography–Mass Spectroscopy analysis. The properties of the bio-oil and biodiesel were assessed according to the American Society for Testing and Materials and the Association of Official Analytical Chemists standards. The obtained methyl-ester by trans-esterification process results in the fuel properties closer to the conventional fuel. Thus, Trichosanthes cucumerina bio-diesel can be used as a potential substitute.


Materials and methods
Trichosanthes cucumerina seed and solvents. The trichosanthes cucumerina is an oil-rich seed plant that comes under the family of curcubitaceae. It originated in Asian countries and referred as snake gourd, viper gourd and snake tomato. Figure 1 shows the trichosanthes cucumerina which are 40-120 cm long, pale-green in colour, 0.5-1 kg weight (single fruit) and contains 40-70 seeds. Seeds are received from the local market and segregated for the bio-oil production. Five solvents namely polar protic (methanol, ethanol and isopropanol), dipolar aprotic (acetone) and non-polar (hexane) purchased from SRL Chemicals Pvt Ltd, Mumbai, India. The physical properties of the different solvents are listed in Table 1.  www.nature.com/scientificreports/ Bio-oil extraction process. The segregated seeds were washed with distilled water (in-house) for the removal of impurities. The seeds outer layer were removed and dried in an oven at 65 °C temperature. The dried seeds were crushed in the oilseed crushing machine (SASTRA Deemed University, Tanjore, Tamil Nadu, India) and obtained with 7% of the bio-oil yield from the taken 200 g of trichosanthes cucumerina seeds. From the literature, the soxhlet extractor was selected to extract the bio-oil yield 8,9 . The round bottom flask of 500 ml capacity, filled with 280 ml of hexane and kept in the heating mantle. The powdered cucumerina seeds of 200 g were packed with a satin cloth which was held inside the thimble. A reflux condenser was attached at the top with inlet and outlet ports for cooling water circulation using aquarium motor. The heated solvent vapors were passed over the bio-mass through distillation path and cooled using a condenser. The cold vapor drips back into the chamber, which was emptied by siphoning action. It results in an extracted trichosanthes cucumerina biooil with solvent and the oil retrieved seed waste is depicted in Fig. 2. In batch distillation process, the obtained bio-oil and solvent mixtures were heated (25 to 85 °C) to evaporate the solvents to separate the bio-oil from the solvent as shown in Fig. 3. The experimental work was performed for three times with each solvent and the average was considered.
Trans-esterification process. The bio-oil leached from trichosanthes cucumerina seeds found with FFA content via GC-MS test which needs the trans-esterification process. In the process, the triglycerides of trichosanthes cucumerina bio-oil converted into its mono-esters by reacting with alcohols in the presence of NaOH or KOH 18,22 . The bio-oil prepared from the seeds of trichosanthes cucumerina fruit was subjected to the transesterification process using acid-catalyzed esterification, alkali catalyzed esterification and purification stages. Initially, the bio-oil was mixed with methyl alcohol in 16:1 molar ratio then added with 1% of H 2 SO 4 . The mixture was heated at 60 °C for 45 min which reduced the acid value of bio-oil to less than 4 mg of KOH/g 23 . Next, the bio-oil (750 ml) and methyl alcohol (400 ml) were mixed with alkali catalyst (NaOH) and subjected to stirring at a constant speed of 1500 rpm for 30 min duration. The end of the process observed with fatty acid methyl ester, glycerol and the traces of NaOH. Further processing with the addition of HCL and H 2 O and followed by the purification process, the traces were removed and obtained with 93.4 ± 0.2% Trichosanthes Cucumerina Biodiesel (TCB). The trans-esterification process, as shown in Fig. 4, was carried out at SASTRA Deemed University, Tanjore, Tamil Nadu, India.
Bio-oil yield. The percentage of bio-oil yield was calculated with the formula (1) for all the five different solvents considered in the research (methanol, ethanol, isopropanol, acetone and hexane). www.nature.com/scientificreports/ The corresponding percentage differences between the different solvents is shown in Fig. 5. It was evident that hexane resulted in a high percentage of yield was about 28.4 ± 0.4%. It may be attributed due to its low latent heat of vaporization, the minimum value of dielectric constant and surface tension, lower density and viscosity as compared with other solvents are listed in Table 1. The least percentage of 20.54 ± 0.2% was found with ethanol due to their higher polarity and solubility in water which declined the bio-oil yield. The results obtained in this research were in line with the results of the other researchers 8,17,20,[24][25][26][27] , are reported in Table 2.

Results and Discussion
Effect of moisture content. The weight of the seeds with moisture and without moisture was measured using infrared moisture analyzer. For the first set of experiments, trichosanthes cucumerina seeds with the moisture content of 1 ± 0.02% were considered to check the yield of bio-oil with hexane as the solvent and resulted in 20.5 ± 0.3%. The increase in bio-oil yield was observed for the moisture content of seed between 1 ± 0.02% to   www.nature.com/scientificreports/ 6 ± 0.02%, as shown in Fig. 6. Further increase in moisture content reduced the yield since the penetration of hexane into the trichosanthes cucumerina seed, and the higher moisture content functioned as the barrier for bio-oil extraction [28][29][30] . The higher percentage of yield (28.01 ± 0.3%) was observed at 6% of moisture content, by keeping other parameters as constant (size of seed as 0.21 mm, extraction time of 270 min, at 68 °C and the solvent-to-seed ratio of 6:1 ml/g hexane). The further increase in the percentage of moisture content shows in the reduction of bio-oil yield. Farsie and Singh 31 reported that the maximum percentage of bio-oil yield was obtained from sunflower seeds expressed at 6% of moisture content. Muhammad Muhammad Fadhlullah et al. 32 determined the effect of moisture content in the bio-oil generation using Calophyllum inophyllum L. seeds. It was observed that the yield was increased from 28.87% to 33.39% for the moisture content of the seeds of 0% and 1.2% respectively. In contrast, the increase in moisture content to 20% the yield gets reduced to 15.56%. Orhevba et al. 33  Effect of seed size. The extraction of bio-oil was carried out with different sizes of the seed in the range 0.6 ± 0.02 mm to 0.15 ± 0.02 mm were present in Fig. 7. Initially, the seed of size 0.6 ± 0.02 mm was considered for the experimentation with hexane as a solvent which resulted in 20.54 ± 0.3% of bio-oil yield. Further experiments have seen with an increasing trend of yield till 0.21 ± 0.02 mm size of the seed (bio-oil yield as 28.30 ± 0.4%) after  www.nature.com/scientificreports/ which for the seed size of 0.15 ± 0.02 mm, the bio-oil yield get declined. Thereby the seed size seems to be the next critical parameter for the bio-oil yield extraction. The increase in contact between the solvent and seed, and mass transfer of seed (solid-state) to solvent (liquid state) shows that the decrease in the size of the seeds increased the bio-oil yield 34 . Qian et al. 35 reported that yield from cottonseed gets increased with the reduction of particle size; in contrast, the further reduction has not shown the improvement in the yield extraction. The decrease in seed size would not always increase the yield due to the range limit, which helps in optimizing the yield 32    www.nature.com/scientificreports/ bio-oil yield from 90 to 270 min, a further increase in extraction time decreased the yield (say at 330 min). The bio-oil extraction time increased gradually, which also increases the yield percentage up to a certain extent, after that the yield reaches a plateau at longer time duration 36 . Hence the extraction time 270 min was remarked to be the optimum time for further examinations. From the literature, it was observed that the Senna occidentalis seeds produced the maximum oil yield 23.46% with the optimum extraction time of 210 min 37 . Theresa et al. 38 investigated the extraction time between 30 to 300 min using Indigofera colutea seeds with hexane as the solvent and seen with maximum yield (38.45%) obtained at 210 min, further extension of time decreased the bio-oil yield.
Effect of solvent-to-seed ratio. The effect of solvent-to-seed ratio was also explored by varying the ratio between 2:1 to 7:1, as given in Table 3. The increase in the bio-oil yield percentage was observed until 6:1, a further increase in ratio reduced the yield percentage. The increase in solvent volume significantly improved the yield until the value of the equilibrium reached, a further increase in solvent volume not seen with any improvement 29 . Suganya and Renganathan 24 experimented on marine macroalgae Ulva Lactuca with the solvent-to-seed ratio varied from 3:1 to 6:1, which increased the bio-oil yield from 9% to 10.88%. The yield gets reduced for the further increase of solvent volume.
Effect of extraction temperature. The variation of extraction temperature on the bio-oil yield obtained from Trichosanthes cucumerina seed with all solvents was conducted over the range of 28 to 78 °C. The increase in bio-oil yield (hexane as solvent) from 18.34 ± 0.15% to 25.3 ± 0.3 was obtained as given in Table 3. The rise in extraction temperature increases the bio-oil yield, due to the mass transfer rate and solubilization of hexane. The lower viscosity (0.32 mPa s) and surface tension (18.43 mN/m) of hexane which improves the diffusivity and solubilization inside the solid matrix at higher temperature resulted in maximum extraction rate. The solvent dissolution capacity also would increase the bio-oil yield. On the further rise in temperature beyond 68 °C (boiling point of hexane), the bio-oil yield content decreased 39 . This rise in temperature increased the solvent boil off and reduced the active contact area between solid and liquid phases 40 . The hexane provided the maximum  Table 3. Effects of solvent-to-seed (ml/g) ration and temperature (°C) on bio-oil yield extraction. www.nature.com/scientificreports/ amount of oil extraction (27.81 ± 0.4%) with the optimum temperature of 68 °C. It was detected that the solubility of the solvent increased with an increase in the diffusion rate 41 . Sayyar et al. 16 considered the role of extraction temperature, the higher percentage of bio-oil yield of 47.3% from Jatropha curcas using hexane as the solvent at 68 °C. Milan D. Kostic et al. 42 optimized the extraction of bio-oil from hemp seeds using n-hexane solvent. The results show that the extraction temperature of n-hexane (at 70 °C) increased the hemp seed oil yield because of improved oil solubility in the solvent. Karthikeyan Murugesan and Renganathan Sahadevan 27 optimized the non-edible bio-oil extraction from Cassia javanica seeds using different solvents. The hexane was the best extractor to extract the maximum percentage of bio-oil yield was about 24.4% with the optimum conditions (extraction temperature at 68 °C and extraction time at 3.5 h). Table 4) was performed to measure the various factors of bio-oil, presence of FFA composition and different types of hydrocarbons 43,44 . The trichosanthes cucumerina bio-oil organic compounds were measured by the test method of gas chromatography (Fig. 9), model Perki-nElmer Clarus 500 coupled with a mass spectrometer. The Capillary Column Elite-5MS was used for the separation of components in bio-oil fraction for the length of 30 m. The flow rate of carrier gas fixed at 1 mL/min and the GC was operated at 58.   45 . This analytical method was used to analyze the chemical bonds and its nature, based on its stretching or bending on exposure to infrared radiation. In this study, the Deuterated Tri Glycine Sulphate (DTGS) detector was used, which works on the variation in the temperature and IR radiation intensity. The FTIR spectra were recorded between 500 and 4000 cm −1 in the transmission mode for the trichosanthes cucumerina bio-oil, as shown in Fig. 10. The various functional groups which were identified are tabulated in Table 5  www.nature.com/scientificreports/  Table 6.   www.nature.com/scientificreports/ Fuel density (ρ) and kinematic viscosity (ν). The fuel density was measured at the reference temperature of 15 °C using hydrometer and found to be 8.33% (TCO) and 6.14% (TCB) higher than diesel. The higher number of unsaturated fatty acid contents in the bio-oil increases the molecular weight. It results in higher fuel density which leads to increased compression ratio, specific fuel consumption and the rate of oxidation. The TCB density was very closer to the value of diesel fuel caused more thermal stability of the biodiesel 53,54 . The Redwood viscometer was used to measure the viscosity. The TCO obtained 42.75 cSt of kinematic viscosity which was 14.82 times higher than that of diesel which attributed to an increase in the free fatty acid chain, the breakdown of intermolecular forces and adhesion between biofuel molecules 7 . The TCB resulted in decreased kinematic viscosity and 9.05 times lesser than TCO, which improves the fuel characteristics of atomization and vaporization and provides better engine performance 55 .

GC-MS Analysis. The GC-MS test analysis (refer
Other properties. The other properties like fuel heating value, iodine number, saponification value, cetane number can also be referred in Table 6. The fuel heating values were numerically calculated using Dulong's formula 56 . The higher proportion of oxygen content in the bio-oil resulted in lower heat energy (32.45 MJ/kg) as compared to diesel. The heating value of TCB moderately increased with the trans-esterification to 38.50 MJ/kg. It can release a higher amount of heat energy during fuel combustion, which improves engine performance 57,58 .
The percentage of unsaturated FFA present in the bio-oil was measured as the iodine number. The Trichosanthes cucumerina bio-oil has an iodine number of 82.4. The degree of unsaturation affects the thermal stability of the fuel and results in carbon deposits 59 . The saponification values were calculated as the amount of potassium hydroxide required for the complete hydrolysis per gram of bio-oil. The saponification value of the Trichosanthes cucumerina bio-oil obtained as 121.37 mg KOH/g. In the other reported works, the saponification values of 192 and 212 mg KOH/g were obtained for Calophyllum inophyllum L. and Ulva lactuca seeds respectively 24,60 which are higher than the value obtained in this research. The higher saponification values may result in corrosion problem in the diesel engine 61 . The cetane number of 51, 35 and 44 was obtained for diesel, TCO and TCB respectively. Higher cetane number of TCB entails shorter ignition delay leads to improved diffusion part of combustion equated with premixed phase 39,62 .

Conclusion
Five different solvents were used to synthesis the bio-oil from Trichosanthes cucumerina seeds. The hexane was found to be the better solvent as compared with other solvents. The maximum bio-oil yield was achieved of 28.4 ± 0.4% at a temperature of 68 °C, 0.21 mm crushed seed size, 6% moisture content, 270 min extraction time, and 6:1(ml/g) of solvent to seed ratio. The FTIR analysis shows the higher percentage of oxygen content and less sulphur content in the trichosanthes cucumerina bio-oil. The GC-MS results are seen with the saturated (43.27%) and unsaturated fatty acids (61.14%). The biodiesel was produced from trichosanthes cucumerina bio-oil through the three stages of the trans-esterification process, which produced 93.4 ± 0.2% of biodiesel. The physicochemical properties of the trichosanthes cucumerina bio-oil and biodiesel were analyzed using AOAC and ASTM standards. The biodiesel properties obtained (heating value-38.74 MJ/kg, kinematic viscosity-4.26 cSt and cetane number-44) were closer to diesel fuel, and it can be considered in the diesel engine as an efficient alternative source.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.