Fatty acid ethyl ester from Manilkara zapota seed oil: a completely renewable biofuel for sustainable development

This article reports the deliverables of the experimental study on the production of a completely renewable biofuel from Manilkara zapota fruit and seed oil. It was attempted to synthesis ethyl ester from Manilkara zapota seed oil using bioethanol synthesized from decayed Manilkara zapota fruit. Bioethanol was produced through fermentation of decayed Manilkara zapota fruit, waste skin, and pulp with Saccharomyces cerevisiae and then distilled at 72°C. The bioethanol yield was noted as 10.45% (v/w). The 95.09% pure bioethanol and 4.9% water molecules were present in the distilled sample. Mechanically extracted raw Manilkara zapota seed oil was used for ethyl ester conversion. The molar ratio of bioethanol to oil, the quantity of KOH, and process temperature were investigated for the maximum yield of Manilkara zapota ethyl ester. A 9:1 molar ratio of bioethanol to oil, 1.5% (w/w) KOH, and 70°C process temperature were identified as enhanced ethanolysis process parameters. The maximum yield of ethyl ester was identified as 93.1%. Physicochemical characteristics of Manilkara zapota oil, bioethanol, and ethyl ester were measured as per the corresponding ASTM standards. It was found that both Manilkara Zapota ethyl ester and bioethanol synthesized from decayed Manilkara zapota fruit could be promising substitutes for fossil diesel and gasoline.


Introduction
Biodiesel, a mono-alkyl ester, has been produced from many resources by many researchers. The major resources predominantly used for biodiesel production are edible and nonedible plant-based seed oils and some animal fats. Among the abovementioned raw materials, plant-based edible oils have more benefits such as low free fatty acid content, lower viscosity than that of nonedible oils, abundant availability, and straightforwardly cultivatable in a short period. The presence of a high amount of fatty acids with single carbon double bond and other suitable properties to readily convert into biodiesel are few key properties of first-generation edible oils than inedible vegetable oils (Karabas 2013;Wu and Leung 2011;Kafuku and Mbarawa 2010a). Biodiesel synthesized from edible vegetable oils is generally recognized as first-generation biodiesel. Few first-generation biodiesel resources are coconut oil, peanut oil, sunflower oil, gingelly oil, mustard oil, rice bran oil, and mahua oil (Mani et al. 2020;Supraja et al. 2020;Ghosh et al. 2020). The nonedible vegetable oils are biodiesel resources that belong to the second-generation group. They are preferable to edible oils because of the low cost (Melvin Jose et al. 2011;Kafuku and Mbarawa 2010b).
The popularly used alcohol for biodiesel production is methanol or methyl alcohol. It is highly reactive in the transesterification process. Though methanol is alcohol with lower molecular weight and highly reactive in the conversion of biodiesel, it is toxic and synthesized chemically. It is vulnerable to human lives (Arslan and Ulusoy 2018;Korkie et al. 2002). Unknowing inhalation causes suffocation problems for human beings. Consumption of 10-ml pure methanol affects the optic nerves and causes blindness; consuming beyond that is sometimes fatal also. Hence ethanol nontoxic alcohol could be a better substitute for methanol in biodiesel production (Neelakandan and Usharani 2009;Sharma et al. 2007;Taherzadeh and Karimi 2007).

Responsible Editor: Ta Yeong Wu
Ethanol is a colorless, renewable, volatile, and flammable liquid widely called as ethyl alcohol or EtOH. It consists of two single-bonded carbon atoms of the alkyl group and oxygen of a hydroxyl group (Talebnia et al. 2010;Tiwari et al. 2010). It is nontoxic than methanol; hence, inhalation or leakage will not cause any human health issues. It has lower autoignition temperature and higher energy density than methanol. It also has higher flame speed, higher octane number, and high heat of vaporization (Madani et al. 2020;Sargar et al. 2017). It has been used in many industrial applications such as antiseptic and disinfectant in drug industries, refrigerant and solvent in chemical industries, and biofuel in transportation and power industries. Ethanol can be produced by chemical synthesis technique through hydration of ethylene and biosynthesis technique through fermentation of sugar (Jahid et al. 2018;Asli 2010;Pramanik and Rao 2005). The ethanol synthesized by the fermentation method is recognized as bioethanol (Sharma et al. 2004;Suresh et al. 2020).
Based on the feedstocks used for biosynthesis of ethanol, the bioethanol produced can be classified in to three categories such as first, second, and third generation. Sucrose and starch rich feedstocks are used in first-generation bioethanol production, lignocellulosic biomass feedstocks are used for secondgeneration bioethanol production, and algal biomass feedstocks are used for third-generation bioethanol production (Zaky et al. 2020;Nazli 2020;Mikulski and Kłosowski 2020). The role of yeast (microorganism) in biosynthesis of ethanol is very significant which converts sugars into bioethanol during fermentation. Yeast is a low-cost microorganism which has high productivity and ethanol conversion rate (Nigam and Singh 2011;Kasavi et al. 2012). Simultaneous saccharification and fermentation (SSF) is one of the most preferred processes for batch type bioethanol production among other process such as separate hydrolysis and fermentation and simultaneous saccharification and cofermentation (SSCF) (Canilha et al. 2012).
Fermentation process temperature, time, pH, concentration of sugar, size of inoculum, and rate of agitation are few key parameters influencing the production of bioethanol, out of which temperature of fermentation process plays a vital role since it directly affects the microorganism growth rate. The optimal temperature band varies from 20 to 40°C (Zabed et al. 2014;Charoenchai et al. 1998). The higher temperature increases the stress factor on microorganism and severely affects the growth rate; on the other hand, the lower temperature will not be favorable to the growth of microorganism during fermentation. Thus selection of optimal temperature and maintain it constantly throughout the fermentation process becomes very important. Several researchers noticed a detrimental effect on ethanol concentration while employing Saccharomyces cerevisiae at temperatures over 37°C (MarelneCot et al. 2007;Liu and Shen 2008;Phisalaphong et al. 2006).
The cost of feedstock accounts for around 60% of the bioethanol production cost; hence, identification of low-cost feedstock for bioethanol production is highly needed. In this context, it is focused on producing bioethanol from low-cost feedstocks including cellulosic and agro-food wastes in this research work. Decomposed fruit wastes are one of the greatest feedstocks for bioethanol production. The pulp of the Manilkara Zapota, one of India's most widely picked fruits, has higher saccharinity. Due to its nature during harvesting, transit, and storage in the stockyard, this pulpy fruit is more prone to spoiling. Twenty to 25% of Manilkara zapota fruits harvested in India may be damaged, resulting in trash and disposal in a landfill. Fruit skins and waste pulps from Manilkara zapota pulp, jam, and juice industries are also thrown into garbage yard. These neglected rotting fruit wastes dumped into the garbage can be exploited as a possible growing substrate for yeast strains, which could be a better resource for bioethanol production. The seeds from the fruits which contain around 20% to 30% (w/w) oil content could be a potential oil resource for biodiesel production (Sathish Kumar and Sureshkumar 2016;Sathish Kumar et al. 2015.
Through an intensive literature review, it is observed that very little research efforts have been attempted to synthesize biodiesel using bioethanol synthesized from decayed fruits or waste vegetables. Also, no literature contribution has been found on ethyl ester production using zapota seed oil with bioethanol synthesized from Manilkara zapota fruit. This experimental research work aims to investigate the use of Manilkara zapota fruit wastes from the cultivation field and pulp industries to synthesis bioethanol. Further, the synthesised bioethanol has been used for ethyl ester production from Manilkara zapota seed oil. Characterization of bioethanol and biodiesel generated for quality control has been carried out and described in this research.

Materials
Decayed fruit wastes were collected from the local fruit market and cultivation fields in and around Chennai, India. Manilkara fruit skins, pulp wastes, and seeds were collected from the pulp, jam, and juice industries in Andhra Pradesh state, India. KOH pellets, potassium permanganate, urea, sucrose, and Saccharomyces cerevisiae were acquired from Pentagon chemicals manufacturer, Chennai, India. The transesterification process was carried out using a hot plate with a magnetic stirrer (Remi, India), a 200-ml conical flask equipped with a reflex condenser, a thermometer with a temperature range of 0 to 100°C, and a 200-ml separating funnel. A standardized digital balance of 0.001-g precision was employed for precise weighing of raw materials. A precise temperature controllable incubator of temperature range 0 to 100°C was used to preserve the fermentation process. A temperature-controllable heating mantle, a three-necked round bottom borosilicate glass flask with a 5-l capacity, a water-cooled condenser, and a distillate collecting flask were utilized in this work.

Preparation of substrate and inoculum for fermentation process
Fruit debris and skins were collected and rinsed in clean water to eliminate dust and other pollutants before being cleansed in a 5% potassium permanganate solution (KMnO 4 ) to eradicate any illnesses caused by other microorganisms. Then they were rinsed and cleaned in distilled water to remove potassium permanganate to avoid adverse reaction between KMnO 4 and yeast. The seeds of cleansed and disinfected Manilkara zapota fruits were collected for oil extraction. The seedless fruits, skins, and collected pulps were then smashed with a juicer to prepare the substrate. The inoculum was prepared by mixing 10 g of Saccharomyces cerevisiae (baker's yeast), 50 g of sucrose, and 1 g of urea, with 100 g of warm water in a distinct glass container. Prepared inoculum was mixed with 200g of the Manilkara zapota substrate, and required quantity of distilled water was added to bring the final volume to 1000 ml. The prepared material was preserved in the incubator and maintained at 37°C to facilitate the fermentation process. Calibrated hydrometer was used to measure the specific gravity of the fermentation sample at every 12 h. The achievement of a constant specific gravity value denotes the completion of the fermentation process.

Ethanol extraction by distillation process
When the fermentation process is complete, the conical flask is withdrawn from the incubator and the product is distilled. Distillation is the technique of extracting a distinct material from a liquid mixture by selective evaporation followed by condensation. The purity of the extracted components using distillation is very high. Fermented products are transported to the distillation setup, and the temperature of the mantle is kept precisely to the boiling point of the ethanol. The evaporated ethanol is condensed and collected in a collecting flask with the help of water-cooled condenser.

Infrared spectroscopy test for bioethanol
An infrared (IR) spectrometry test is conducted to confirm the presence of ethanol in a distillate product. Each molecule present in the bioethanol absorbs the infrared rays at certain wavelengths based on their structural characteristics. The infrared light beam emitted from the IR source is continuously passed to the bioethanol sample at different wavelengths to record the IR spectrum. The absorption of the IR beam occurs when the vibrational frequency of the molecule matches with source IR wavelength. The amount of energy absorbed at each wavelength of the IR beam is measured by a monochromator.
This research uses the JASCO 6300 model Fourier transform infrared spectrometer. Two NaCl salt plates are washed using dichloromethane and cyclohexane with full care, without any finger prints on the plates. In the middle of one plate, a drop of bioethanol sample is deposited, and another plate is placed on the top, then softly squeezed together, and rotated slightly using a soft tissue to create a thin film of bioethanol sample on the plates. The sample plates are then carefully placed in the holder of the sample chamber in the instrument. The scale is fixed to 4000-400cm −1 and the range is fixed to 0-100%T and spectrum is recorded. Once the spectrum is recorded, the sample plates are cleaned.

Gas chromatograph test for bioethanol, raw oil, and ethyl ester
The quantity of bioethanol generated and its purity was assessed using a gas chromatograph apparatus aided with the mass spectrum. The chemical composition and components present in any solution or oil or fat can be identified and quantified by gas chromatograph test. When the sample is injected in a stationary or source column, each component present in the mixture will move at a different rate based on their characteristics. The injected sample is sent to the transfer line, which uses the rate of migration to separate the constituent components in a mixture. The flow rate of the mobile or transfer phase is measured in ml/min or μl/min. The term GC-MS refers to a mass spectrometric detector (MSD) coupled to gas chromatography. A component in a combination is first transformed into ionic fragments in GC-MS, after which the total number of ions is identified and shown against time.
In the present investigation, Turbo EI mass spectrometer inbuilt Perkin-Elmer Clarus 500 gas chromatograph instrument is used to identify the chemical composition of raw oil and bioethanol. The specification of the capillary column is 30m × 0.25mm × 1μm. The carrier gas used in this instrument is helium with a flow rate of 1 ml/min. The initial temperature of the oven is set to 100°C and maintained for 10 min to attain stability, later raised to 200°C, and remained constant. The rate of increase in oven temperature is 10°C/min. The temperatures of the source, transfer, and injector line are set to 200°C, 200°C, and 220°C, respectively. NIST Version 2.1 MS data library is used to identify the components.

Manilkara zapota ethyl ester production using bioethanol
The Manilkara zapota seeds were collected and dried for 48 h in sunlight, the shells were taken away manually, and oil was squeezed using a mechanical expeller. The oil was characterized using ASTM standards. A total of 100 g of pure Manilkara zapota oil was taken in a conical flask and heated up to the required temperature and maintained constantly. At 500 rpm, the oil was constantly stirred using a hot plate with a magnetic stirrer. The ethoxide solution was prepared separately by thoroughly dissolving the required quantity of KOH in the required amount of bioethanol. The oil temperature, amount of KOH, and quantity of bioethanol were fixed with respect to the corresponding experimental conditions. Upon attaining the required temperature of the oil, the ethoxide solution was gradually transferred into the conical flask to initiate the transesterification reaction. All the experiments were conducted for 90 min, and then the products were shifted to the separating funnel and kept undisturbed for 24 h to facilitate the phase separation. The low viscous biodiesel settled on the top was separated and cleaned using distilled water for removing the unreacted chemicals.

Investigation of ethanolysis process parameters
Three highly influencing ethanolysis process parameters were taken for the investigation, namely bioethanol to oil molar ratio, catalyst quantity, and oil temperature. Other transesterification parameters such as stirring speed and reaction time were maintained at 90 min and 500 rpm, respectively, throughout the investigation. Initial investigations were carried out to assess the feasibility of using bioethanol to produce ethyl ester at random time intervals and discovered that there was no improvement in ethyl ester production beyond 80 min; hence, all tests in this work were carried out at 90 min. The best value of a given parameter was achieved by executing a series of tests with various values of that specific parameter while maintaining other parameters constant at some arbitrary value. The obtained optimal value of a parameter was held constant for the investigation of the other parameters until the best value was achieved for that particular parameter. Three replications were performed for each experiment and the average value was considered for the analysis.

Identification of completion of fermentation process
One method for determining the completion of the fermentation process is to measure the specific gravity of the fermentation sample. The initial specific gravity of the fermentable product will be greater, and after fermentation begins, the specific gravity value will continue to decrease with regard to the rate of fermentation. This decrease in specific gravity is a direct indication conversion of ethanol from sugar. When the fermentation process is finished, the specific gravity reaches a minimal value and remains stable. The fluctuation of the sample's specific gravity with respect to time is measured and displayed in Figure 1. The sample's specific gravity was initially 1.08, but at the end of the fourth day, it had dropped to 0.978 and stayed steady. The total time taken for the completion of fermentation process was found as approximately 96 h. This stable value of the sample's specific gravity guarantees that the fermentation process is completed. Figure 2 shows the infrared spectroscopy of the synthesized bioethanol. The presence of alcohol functional group (OH group) is evidenced from the IR spectroscopy of the sample at the peak value of 3358.43 cm −1 . At the same time, the broad OH vibration band in IR spectrum showed the existence of water molecules in the bioethanol sample. The IR spectroscopy cannot quantify the amount of ethanol generated; hence, a gas chromatography test utilizing the GC-MS system was performed to determine the purity and quantity of ethanol production.

Infrared spectroscopy results for bioethanol
Gas chromatograph results for bioethanol, raw oil, and ethyl ester Figure 3 shows the result of gas chromatograph for the synthesized bioethanol. The comparison of the chromatograph peak of the bioethanol sample and peak value of ethanol from NIST library are given in Figure 4. The percentage of bioethanol attained from the synthesized bioethanol sample is calculated using the area under the peak from chromatography graph using the formula: Percentage of Ethanol (%) = Individual peak area / total peak area *100 It was observed from the calculation that 95.09% pure bioethanol and 4.9% water molecules were present in the sample. The molecular peaks of water molecules are observed between m/z 17.9 and 18.9. The total bioethanol yield was calculated as 10.45% (w/v).
The chemical constituents of Manilkara zapota seed oil were identified and quantified as palmitic acid 12.98%, stearic acid 5.12%, oleic acid 61.81%, linoleic acid 16.26%, linolenic acid 3.21%, and other unidentified components were 0.62%. The chromatograph result of the raw oil sample was presented  Figure 5. Raw vegetable oil with high saturated fatty acid content will affect the cold flow characteristics of the biodiesel. The increased degree of saturation of the raw oil increases the viscosity of the biodiesel. Oil with increased poly unsaturated fatty acid will reduce the cetane value of the biodiesel. Hence, vegetable oils consist of low saturated and  polyunsaturated fatty acids, and high monounsaturated fatty acids will be best suitable for biodiesel production (Sathish Kumar and Sureshkumar 2016). Manilkara zapota seed oil includes a high concentration of monounsaturated fatty acids (61.81% oleic) and a low concentration of saturated and polyunsaturated fatty acids, making it ideal for biodiesel production.
The chromatograph sample for Manilkara zapota ethyl ester (MZEE) is shown in Figure 6

Effect of bioethanol to oil molar ratio
The effect of bioethanol to oil molar ratio on the yield of Manilkara zapota ethyl ester (MZEE) is shown in Figure 7. The molar ratio in this analysis ranged from 3:1 to 15:1 in the 3:1 interval. The other two parameters catalyst quantity and process temperature were kept constant as 1% and 60°C, respectively. It was observed clearly from Figure 6 that the maximum MZEE yield was attained at a 9:1 molar ratio as 91.6%. The transesterification process is highly reversible; hence, the stoichiometric molar ratio will not be good enough to result in higher biodiesel yield. Less reactivity of bioethanol in comparison with methanol was another reason for the higher amount of bioethanol consumption in this process. A noticeable reduction in biodiesel yield was recorded beyond the 9:1 molar ratio. This was due to the hydration caused by surplus alcohol present in the reaction which reduces the fatty acid ethyl ester conversion rate (Fernandez et al. 2010;Bolonio et al. 2019;Hanh et al. 2009;Saydut et al. 2008;Morshed et al. 2011).

Effect of catalyst quantity
The effect of catalyst quantity on the MZEE yield is presented in Figure 8. Investigations were performed by varying the quantity of the catalyst from 0.5 to 2.5% (w/w). The best value obtained from the previous segment was set at 9:1 for the bioethanol to oil molar ratio, and other parameters were kept constant as before. The maximum Manilkara zapota ethyl ester yield was noted as 91.8% for 1.5% catalyst quantity. At 0.5% catalyst quantity, only 63.6% biodiesel yield was recorded. This was because of the insufficient quantity of catalyst to aggravate the mechanism of the reaction. Due to the higher degree of saponification formation of excess KOH and water molecules present in the bioethanol with triglycerides, further, a rise in catalyst quantity adversely affects the MZEE yield, thus the ethyl ester production was significantly decreased, and even greater soap formation was observed. (Rashid et al. 2008;Nakpong and Wootthikanokkhan 2010).

Effect of process temperature
The temperature of the transesterification process is one among the most critical factors that influence the ethyl ester yield. Figure 9 depicts the fluctuation in Manilkara zapota ethyl ester yield as a function of process temperature. Experiments were conducted on five distinct temperatures ranging from 50 C to 90°C with 10°C interval. The best bioethanol to oil molar ratio values and the quantity of catalysts obtained from previous segments have been introduced, and other considerations have remained stable. From Figure 8, it was found that the rise in temperature increases the MZEE yield up to 70°C. This was due to the higher temperature that allows coherent bonding between triglyceride molecules to be quickly braked and made active in transesterification conversion (Sánchez et al. 2013;Schinas et al. 2009). A further rise in temperature had a reverse effect and reduced the biodiesel yield. This was due to the evaporation of bioethanol at 80°C and 90°C process temperature and would have not participated in the ethyl ester production. The optimal value of the MZEE yield was found to be 93.1% at 70°C.
Physicochemical properties of bioethanol, raw oil, and Manilkara zapota ethyl ester Table 1 shows key physicochemical properties of bioethanol determined using ASTM standards and compared to laboratory ethanol values. The higher density of bioethanol as 873 kg/ m 3 is noted due to the presence of small amount of water molecules in the bioethanol. The physicochemical properties of raw oil were estimated and presented as follows. The density of oil at 15°C was 0.895 g/cm 3 , the kinematic viscosity at 40°C was measured as 33.97 mm 2 /s, the estimated acid value of the oil was 3.92 mg KOH/g, and the free fatty acid value was determined as 1.96%, hence best suitable for single-step transesterification process. Free fatty acid content more than 2.5% in the oil leads to high saponification reaction with KOH during ethyl ester synthesis; hence, biodiesel synthesis is to be carried out in two steps. The iodine value of the oil was estimated as 64.3 g iodine/100g. The physical appearance of the oil was clear brownish-yellow, and the molecular weight was determined as 873.31 g/mol. The physicochemical properties of Manilkara zapota ethyl ester were assessed and compared with Manilkara zapota methyl ester properties (Sathish Kumar et al. 2015) and given in Table 2. From the results, it was found that all the property values are comparable with methyl ester produced from Manilkara zapota oil and biodiesel standards. The ester content in the MZEE is 93.1% which is 3.4% lesser than the minimum limit of EN14214 standard. This might be attributed to two factors: first, ethanol is less reactive than methanol in the transesterification process, and second, the presence of water molecules in the bioethanol synthesized. The density of MZEE is 0.889 g/cm 3 , which is within the limitations of biodiesel standards and slightly higher than MZEE owing to the effects of water content in bioethanol.
The kinematic viscosity is 4.96 mm 2 /s, which is within the maximum limit of biodiesel requirements and somewhat higher than MZME. The acid value is 0.28 mg KOH/g, which is considerably within the maximum limit of biodiesel standards and nearly double the MZEE. This is owing to the bioethanol's acidic nature. Other properties, such as heating value, pour point, flash point, and iodine value, are within biodiesel requirements and comparable to MZME. Mono-, di-, and triglyceride and total glycerol contents are well within the limits of the biodiesel requirements.

Conclusion
Decayed zapota fruits were collected from cultivation field, and waste pulp and skin were collected from jam, juice, and pulp industries and have been used for biosynthesis of ethanol. Saccharomyces cerevisiae yeast was used to facilitate the fermentation process. The produced bioethanol was utilized to transform Manilkara zapota seed oil into Manilkara zapota ethyl ester (biodiesel). Three key parameters of the ethanolysis process were investigated for maximum ethyl ester yield. Based on the findings of the investigation, the following conclusions were reached. Unused damaged zapota fruits could be a good substrate for bioethanol production. The addition of 50-gm sucrose and 10gm of Saccharomyces cerevisiae along with 1-gm urea in 200g of zapota fruit smash produced around 21ml (10.45% (w/v)) of bioethanol. According to the GC-MS data, the concentration of the ethanol acquired in the sample is 95.09% by volume.
Bioethanol to oil molar ratio of 9:1, KOH quantity of 1.5% (w/w), and oil temperature of 70°C were identified as enhanced ethanolysis reaction factors. The maximum yield of Manilkara zapota ethyl ester was identified as 93.1%. The physicochemical properties of Manilkara zapota-based  bioethanol and biodiesel show that they are a good substitute for fossil diesel and gasoline in overcoming the global alarming twin problems of environmental pollution and energy crisis. This research work can be further extended by considering the following points in the future. The quality of the bioethanol from the decayed Manilkara zapota fruit can be improved by optimizing the fermentation process. The biodiesel quality can be further improved by implementing ultrasonic-assisted transesterification process.
Availability of data and materials All data generated or analyzed during this study are included in this published article.
Author contribution SKR conceptualized, collected resources and investigated the experimental analysis, and validated the results. He was the major contributor in writing the manuscript. SK supervised the experimental investigation and validated the results. He has reviewed and edited the manuscript. All authors read and approved the final manuscript.

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Competing interests The authors declare no competing interests.