Biodiesel Production Catalyzed by Lipase Extract Powder of Leonotis nepetifolia (Christmas Candlestick) Seed

Abstract

This work aimed to evaluate the ability of lipase extract powder obtained from Leonotis nepetifolia seed for enzyme-catalyzed biodiesel production using Leonotis nepetifolia oil, commercial olive oil, and waste cooking oil as substrates. The lipase extract powder showed an enzymatic activity and hydrolysis percentage of 24.7 U/g and 21.31%, respectively, using commercial olive oil as a reference. Transesterification reaction conditions were 40 g of substrate, 34 °C, molar ratio oil: methanol of 1:3, lipase extract powder 20 wt%, phosphates buffer (pH 4.8) 20 wt%, and a reaction time of 8 h. Transesterification yields of 74.5%, 71.5%, and 69.3% for commercial olive oil, waste cooking oil, and Leonotis nepetifolia oil were obtained, respectively. Biodiesel physicochemical parameters were analyzed and compared with the international standards: EN 14214 (European Union) and ASTM D6751 (American Society for Testing and Materials). The biodiesel’s moisture and volatile matter percentages, iodine index, cooper strip corrosion, and methyl esters content conformed to the standards’ specifications. The fatty acid methyl ester content of the vegetable oils showed the presence of methyl oleate after enzyme-catalyzed transesterification. This study reveals that biodiesel production catalyzed by lipase extract powder from Leonotis nepetifolia could be a viable alternative, showing that transesterification yields competitive results.

1. Introduction

The development of alternative energy has increased due to the need to change the energy matrix based on fossil fuels due to the gradual depletion of the world’s petroleum reserves and the impact of fuel combustion exhaust emissions on the environment and human health [1]. Biodiesel is a sustainable, eco-friendly, and biodegradable biofuel used to supplant petroleum diesel due to lower emissions (SO₂, CO, and halogens) during its combustion process [2,3]. Biodiesel can be derived from various sources, including vegetable oils, animal fats, waste cooking oil, and microalgae oils [3]. Biodiesel is usually classified as the first, second, and third generation based on its origin or source used to produce it. First-generation biodiesel is produced from edible oils, second generation is from non-edible oils, and third- generation is from microalgae oils [4]. The use of non-edible oils for biodiesel production is increasing nowadays compared with edible and microalgae oils. This is because they are not used for food applications and do not require a large amount of investment or the necessity of sunlight. Furthermore, there are fewer difficulties in oil extraction and less complications in the production of non-edible oils at a larger scale [4,5].

The method applied for biodiesel production is transesterification based on non-catalyzed, chemical-catalyzed, and enzymatic-catalyzed reactions. The enzymatic reaction is preferred over the other two reactions because of the following advantages: mild reaction conditions, less energy-intensive, easy product recovery (glycerol), high-quality products, no saponification, and no wastewater generation [6,7]. However, it has some limitations, including the high cost of enzyme and reaction time, low yield, and the amount of water and organic solvents in the reaction mixture [6]. The main components in this reaction are the enzymes (lipases), which can catalyze a wide variety of substrates including free fatty acids, oil, and acyl acceptor (alcohol) [7].

Biodiesel catalyzed by lipases is affected by many factors, such as enzyme specificity and immobilization, oil composition and purity, acyl acceptors, oil-to-acyl acceptor molar ratio, water content, and temperature. Various methods have been examined to control these factors and enhance the enzymatic reaction for biodiesel production [7]. Different lipases have been applied to other substrates, obtaining the following yields: Corn oil 81% [8], jatropha 37–89% [9,10], rapeseed oil 10–85% [11], chicken feather meal oil 98% [12], and waste cooking oil 90% [13].

Leonotis nepetifolia belongs to the family Lamiaceae and has flowering stems bearing dense vertices with orange or, infrequently, yellowish cream flowers [14]. This plant has distinct features such as long internodes, quadrangular stems, orange spines, scarlet flowers with densely wooly upper lips, and black seeds [15]. Furthermore, it can survive during drought and produce a high seed amount during the rainy season. Its propagation is fast since it does not need specific agro-ecological conditions for its reproduction [16,17].

Leonotis nepetifolia is native to Tropical Africa and is a tall annual herb growing in plains, along roadsides, and complete wastelands. It often grows throughout India, Latin America, and the West Indies [15]. In Mexico, this plant is considered an exotic invasive species and a frequent weed that stands out for its showy flowers introduced for ornamental, medicinal, and melliferous purposes [16]. Benni et al. [15] reported that its seeds contain 37% fatty acids, such as oleic, labalenic, and linoleic. These characteristics make Leonotis nepetifolia an attractive species to produce biodiesel from its oil. Biodiesel production from Leonotis nepetifolia oil through the in situ chemical-catalyzed transesterification method has been reported. The results showed a maximum yield (about 96%) [15]. The biodiesel production from vegetable oils catalyzed by lipases is well documented. However, using plant lipase from Leonotis nepetifolia seeds is still scarce in the literature.

This work aimed to evaluate a lipase extract powder obtained from Leonotis nepetifolia seeds to catalyze the transesterification reaction of vegetable oils such as Leonotis nepetifolia oil, commercial olive oil (as reference), and waste cooking oil. The catalytic and hydrolytic activity of the lipase extract powder was evaluated, and the obtained biodiesel was physicochemically characterized by international standards to determine the biofuel quality.

2. Materials and Methods

2.1. Raw Materials

Leonotis nepetifolia samples were collected in the wastelands of Zacatecas (Zacatecas, Mexico). The clusters were cut during the last ripening stage and close to the dormancy stage. Then, they were sun-dried for one week, obtaining the seeds by shelling. The seeds were refrigerated for preservation (4 °C) until use.

2.2. Obtainment of Lipase Extract Powder

The lipase extract powder was obtained according to the methodology described by Avelar et al. [18] with the following modifications. An amount of 40 g of seeds was triturated using a commercial blender for 10 min with an addition of 10 mL of acetone. The acetone was pre-chilled at a temperature of 4 °C. Then, the obtained paste was remixed with pre-chilled acetone with a ratio of 1:5 w/v at a temperature of 4 °C for 16 h under agitation (700 rpm). Later, the suspension was filtered under a vacuum via a Buchner funnel and washed with pre-chilled acetone in excess. The obtained product was placed in aluminum trays at room temperature for 24 h. This step was performed to evaporate the solvent completely. Finally, it was sieved to a particle size <1 mm. Likewise, it was stored and kept sterile at 4 °C until use. This product was defined as lipase extract powder. The yield of enzyme extract/seed was 61 ± 0.24 wt%. This yield value is high since extract purification and lipase classification were not performed. Therefore, the lipase extract powder shows impurities associated with the shells of the seeds, which were not removed due to their size.

2.3. Determination of the Catalytic and Hydrolytic Activity of Lipase Extract Powder

The catalytic activity of the lipase extract powder from Leonotis nepetifolia seed was determined on the hydrolysis of an emulsified vegetable oil in triplicate based on the methodology described by Avelar et al. [18] with slight modifications. Commercial olive oil was used as a reference substrate since Avelar et al. [18] and Santos et al. [19] found the highest hydrolysis degree for this substrate. An emulsion of 50 g of commercial olive oil and 150 g of gum Arabic solution at 30 g/L was used as the substrate. Then, 5 g emulsion, 0.1 g of lipase extract powder, and 5 mL phosphate buffer (pH 4.8) were used as the reaction mixture. This mixture was incubated at 34 °C for 5 min under agitation (1000 rpm). An amount of 10 mL of ethanol was added to the sample to stop the reaction and denaturalize the enzyme. Then, it was titrated against a standard 20 mmol/L sodium hydroxide solution in the presence of phenolphthalein as an indicator. The enzyme activity was calculated by Equation (1). One unit of enzymatic activity (U) was defined as the amount of enzyme that releases one μmol of free fatty acid per minute under the assay conditions [20]:

$$A=\frac{{10}^6\times \left(V-V_1\right)\times M_{NaOH}}{t\mathrm{\ast }m}$$

(1)

where A: enzyme catalytic activity (U/g), V: volume of sodium hydroxide solution used in the titration of the sample (L), V₁: volume of sodium hydroxide solution used in the titration of the blank (L), M_NaOH: molarity of sodium hydroxide solution (mol/L), t: time of reaction (min), and m: mass of the lipase extract powder used in the assay (g).

Hydrolysis reactions were performed in triplicate using a mixture of 5 g of commercial olive oil, 45 mL buffer acetate (pH 4.8), and 1 g of lipase extract powder. This mixture was agitated for 240 min using a mechanical stirrer at 1000 rpm, atmospheric pressure, and 25 °C. Samples of 1 g were periodically removed at intervals of 30 min. An amount of 10 mL of commercial ethanol was added to stop the reaction. Then, it was titrated against a standard 20 mmol/L sodium hydroxide solution in the presence of phenolphthalein as an indicator [18]. The hydrolysis degree was defined as the percentage weight of free fatty acids in the sample divided by the maximum theoretical amount using Equation (2) [18]:

$$Hydrolysis\left(\%\right)=\left(\frac{V_{NaOH}\times {10}^{-3}\times M_{NaOH}\times MM}{wt\times f}\right)\times 100$$

(2)

where V_NaOH: volume of sodium hydroxide solution used in the titration of the sample (mL); M_NaOH: concentration of sodium hydroxide solution (mmol/L); MM: average molecular mass of fatty acids in the commercial olive oil (g/mol); wt: weight of the sample (g); and f: fraction of oil at the start of the reaction.

2.4. Leonotis Nepetifolia Oil Extraction

An amount of 30 g of seeds was triturated using a commercial blender for 5 min. Then, the blended material was placed in a convective flow stove FELISA FE-291AD (Feligneo, Zapopan, Mexico) at 60 °C for 24 h. The oil was obtained from seeds using the Soxhlet extraction method with 150 mL of hexane as solvent. The reflux was made for 240 min, controlling the temperature (~180 °C) to keep a steady drip of 3 or 4 drops per second. The obtained oil was placed in a convective flow stove FELISA FE-291AD (Feligneo, Zapopan, Mexico) at 105 °C for 24 h to remove the moisture and the remaining solvent. The oil was purified, degummed, and neutralized following the methodology reported by Ávila Vázquez et al. [21].

2.5. Transesterification Reactions Using Enzyme Catalysis

Enzyme-catalyzed transesterification was performed as follows. An amount of 40 g of commercial olive oil, 40 g of waste cooking oil, and 40 g of Leonotis nepetifolia oil were used as the substrates. An amount of 20 wt% lipase extract powder was used as the biocatalyst, and 20 wt% phosphates buffer (pH 4.8), regarding the substrate amount, was added using a molar ratio oil: methanol of 1:3. The reactors were incubated in a water bath JeioTech BS-11 (JeioTech, Daejeon, Republic of Korea) at 34 °C and 185 rpm for 480 min. The reactions were performed in triplicate. Later, the reaction liquid components (mixture of biodiesel–glycerin–methanol–buffer–water) were separated from the solid components (biocatalyst). This process was performed by centrifugation using a centrifuge SOLBAT-J40 (Solbat, Puebla, Mexico) at 4500 rpm for 20 min. Then, the recuperated liquid was mixed with 5 mL hexane and 5 mL deionized water. The mixture was stirred manually for 1 min and then centrifuged at 4500 rpm for 20 min, recuperating the organic phase (biodiesel) from the aqueous phase (water and glycerin). Later, the biodiesel was purified by drying in a convective flow oven FELISA, FE-291AD (Feligneo, Zapopan, Mexico) at 120 °C for 180 min to remove the remaining water and alcohol [22]. Finally, the biodiesel was stored at room temperature and in the dark until its use in the physicochemical characterization. The biodiesel yield and free fatty acid conversion into biodiesel were calculated by Equation (3) [23].

$$Biodieselyield\left(\%\right)=100\times \frac{weightofbiodiesel}{amountofsubstrateused}$$

(3)

2.6. Biodiesel Physicochemical Characterization

Biodiesel physicochemical characterization was performed based on the guidelines of the American Society for Testing and Materials (ASTM) of the United States of America (USA) and the Mexican standards of the Ministry of Commerce and Industrial Development (MCID), as shown in

Table 1. Parameters and analysis method guidelines used in the biodiesel physicochemical characterization.

Physicochemical Parameter	Analysis Method Guidelines	Reference
Density	ASTM D7042	[24]
Moisture and volatile matter	NMX-F-211-SCFI-2012	[25]
Saponification value	NMX-F-174-SCFI-2014	[26]
Acid value	ASTM D974	[27]
Iodine value	NMX-F-152-SCFI-2011	[28]
Peroxide value	NMX-F-154-SCFI-2010	[29]
Copper strip corrosion	ASTM D130	[30]

. Each parameter was evaluated in triplicate, reporting the mean and deviation standard. The results were compared with EN 14214 (European Union) and ASTM D6751 (USA) international standards.

2.7. Fatty Acid Methyl Ester Content in Biodiesel Analysis

Fatty acid methyl ester (FAMEs) content in biodiesel was analyzed in a gas chromatograph Varian (model 3800, Varian Inc., Walnut Creek, CA, USA). This analysis was performed with a Stabilwax (Restek, Bellefonte, PA, USA) capillary column (60 m × 0.25 mm × 0.25 mm) and split injection at 250 °C (split ratio 50:50). The oven temperature was set at 150 to 200 °C at 10 °C/min. Then, it increased to 250 °C at 3 °C/min. Finally, the temperature was held to 250 °C for 20 min. The injection volume was 3 µL, and the injection and FID detector temperatures were 300 °C with high-purity-grade hydrogen as the carrier gas. The peaks corresponding to fatty acid methyl esters in the biodiesel samples were confirmed by comparing their retention times with those obtained in the GC chromatogram of the individual compounds, using the Supelco37 standard, Sigma Aldrich Inc., St. Louis, MI, USA. They were quantified as the percentage of the corresponding peak area concerning the area sum of all the peaks.

3. Results and Discussion

3.1. Catalytic and Hydrolytic Activity of Lipase Extract Powder

Figure 1 shows the catalytic activity of the lipase extract powder from Leonotis nepetifolia seeds obtained in this study and its comparison with other studies and lipase sources. It can be observed that the catalytic activity of the lipase extract powder from Leonotis nepetifolia (24.7 ± 0.4 U/g) is less than other lipase sources. Avelar et al. [18], Santos et al. [19], and De Sousa et al. [31] reported that germination is fundamental to producing plant lipases since the lipase activity is absent in ungerminated (or dormant) seeds, increasing when germination starts. In this study, Leonotis nepetifolia seeds were used in an ungerminated state, so the seed germination study could be performed to improve the catalytic activity of the lipase extract powder from Leonotis nepetifolia. However, Santos et al. [19] reported that germination time reduces lipase catalytic activity from the castor bean, passion fruit, and sunflower seeds. Therefore, the catalytic activity of the lipase extract powder from Leonotis nepetifolia could be reduced with germination.

Figure 2 shows the hydrolytic activity of the lipase extract powder obtained from Leonotis nepetifolia seeds. The highest hydrolysis percentage was 21.31 ± 0.43% at a reaction time of 240 min. This value is low compared with other sources of lipase (Figure 1). The lipase extract powder of Leonotis nepetifolia seeds was obtained in the mature stage in this study. Thus, it favors producing a high oil content and minimum lipase amount. Gu et al. [32] and Tavares Cavalcante et al. [33] reported that lipase presents the maximum hydrolytic activity of vegetable oils in the early stages of seed germination. However, the temperature and pH are significant parameters to optimize the process since both determine the reaction rate and help to obtain a higher yield in the transesterification reaction. Moreover, these parameters guarantee adequate lipase activity and stability. Pooja et al. [34] and Madhu et al. [35] reported a high biodiesel yield at 35 °C. However, when the temperature incremented to 40 °C, the biodiesel yield fell 20%. This situation was associated with the enzyme’s denaturation. Thus, it is necessary to determine the optimal conditions for hydrolysis. Ranges of temperature between 30 and 55 °C and pH between 4 and 8 have been reported. However, this study used a temperature of 34 °C and a pH of 4.8.

3.2. Oil and Transesterification Reaction Yields

The oil yield obtained from Leonotis nepetifolia seed was 25.6 ± 0.34 wt%.

Table 2. Percentage of seed non-edible oil content from different plant species.

Source	Seed Non-Edible Oil Content (wt%)	Reference
Leonotis nepetifolia seeds	25.6	This study
Castor bean seeds	36–51	[21,36,37,38]
Rubber seeds	53–68	[39]
Jatropha seeds	27–40	[1,36]
Neem seeds	20–30	[40]
Karanja seeds	27–39	[36,41]

shows the percentage of oil content from different plant species with non-edible oil used as the substrate in the transesterification, including the value obtained in this study for Leonotis nepetifolia. It can be noted that Leonotis nepetifolia shows a low oil content compared with the other species, which may be associated with the collection source or maturation stage.

The reaction mechanism for the transesterification catalyzed by lipases is represented by ping-pong models, where each product is released between additions of the substrates [42,43]. This transesterification involves a two-step mechanism, looking at a single ester bond. The first step is the hydrolysis of the ester bond and the alcohol moiety release (Equation (4)). The second step is the esterification of the second substrate (Equation (5)) [44].

$$E+E{s}_s\leftrightarrow E·E{s}_s\leftrightarrow F·B_p\leftrightarrow F+B_p$$

(4)

$$F+A_s\leftrightarrow F·A_s\leftrightarrow E·{Es}_p\leftrightarrow E+{Es}_p$$

(5)

Subscripts s and p indicate the substrate and product, respectively. For biodiesel, A_s = alcohol substrate (methanol, ethanol, or others), B_p = product with alcohol moiety (glycerol or di- or monoglyceride), E = free enzyme, Es_s = ester substrate (tri-, di- or monoglyceride), Es_p = fatty acid alkyl esters, and F = fatty acid.

For this work, the transesterification was performed in triplicate using commercial olive oil, waste cooking oil, and Leonotis nepetifolia oil as substrates.

Table 3. Comparison of the transesterification yields using lipase sources as biocatalysts.

Physicochemical Parameter	Lipase Form	Vegetable Oils	Alcohol	Yields (wt%)	Reaction Conditions	Reference
Leonotis nepetifolia seeds	Free	Commercial olive	Methanol	74.5 ± 1.2	8 h, 34 °C, oil: alcohol molar ratio 1:3, 20 wt% lipase	This study
		Waste cooking		71.5 ± 2.3
		Leonotis nepetifolia		69.3 ± 1.4
Lipozyme-TL IM	Immobilized	Waste cooking	Methanol	95.0	105 h, 24 °C, oil: alcohol molar ratio 1:4, 4 wt% lipase	[45]
Pseudomonas fluoresces	Free	Soybean	Methanol	90.0	90 h, 35 °C, oil: alcohol molar ratio 1:3, 5 wt% lipase	[46]
Pseudomonas cepacian	Immobilized	Jatropha curcas	Ethanol	98.0	8 h, 40 °C, oil: alcohol molar ratio 1:4, 10 wt% lipase	[47]
Chromobacterium viscosum	Free	Jatropha	Ethanol	73.0	8 h, 40 °C, oil: alcohol molar ratio 1:4, 1 wt% lipase	[48]
Candida rugosa	Immobilized	Cotton seed	Methanol	98.3	48 h, 40 °C, oil: alcohol molar ratio 1:12, 5 wt% lipase	[49]
Burkholderia cepacian	Immobilized	Jatropha curcas	Ethanol	99.9	24 h, 35 °C, oil: alcohol molar ratio 1:10, 52.5 wt% lipase	[50]
Candida antarctica	Immobilized	Olive	2-propanol	30.3	72 h, 40 °C, oil: alcohol molar ratio 1:6, 1 wt% lipase	[51]
Mucor miehei	Free	Soybean	Methanol	75.4	5 h, 45 °C, oil: alcohol molar ratio 1:3, 12.5 wt% lipase	[52]

shows the average value of the transesterification yields with a standard deviation of ±0.5 for the three substrates. Likewise, Table 3 shows data for other lipase sources used as biocatalysts reported by other authors.

An enzyme catalyst for transesterification seems to be a promising alternative. Compared with microbial lipases, plant lipases can be extracted from low-cost substrates (such as plant biomass) without requiring genetic manipulation to be synthesized [33]. The advantages of enzymatic processes are that they can avoid product contamination, favor an easier glycerol recovery, and are more resistant to free fatty acid and water interferences caused by chemical catalysis. However, the enzyme cost limits the commercialization of lipase-catalyzed biodiesel production [53]. In this work, the capacity of the lipase extract powder was evaluated without immobilizing it since, although immobilized enzymes show a significant improvement in its stability, increasing the transesterification yield, this issue would also increase biodiesel production cost. Therefore, the possibility of future work remains open to study the transesterification yield using this enzymatic extract in an immobilized form.

Transesterification yields between 69.3% and 74.5% were obtained in this study. The lowest value was obtained using Leonotis nepetifolia oil as the substrate, followed by waste cooking oil, and, finally, the highest one was obtained using commercial olive oil. However, the industrial production of biodiesel from commercial olive oil is not feasible in terms of cost. This work shows that the lipase extract powder obtained from Leonotis nepetifolia seeds can favorably catalyze the transesterification reaction using more feasible substrates for its scaling, such as waste cooking oil. Some authors reported efficiencies between 30.3% and 99.9%. This variation depends on the conditions used in the transesterification reaction. Some factors are temperature, reaction time, oil: alcohol molar ratio (inactivation of the lipase exposed to higher concentrations of methanol), and lipase amount (wt%) [34,54]. For this reason, the optimal conditions of the factors to achieve a high yield in the transesterification reaction must be studied.

3.3. Biodiesel Quality

The obtained biodiesel was physiochemically characterized under USA and European Union standards.

Table 4. Physicochemical characterization of the biodiesel obtained from commercial olive oil, waste cooking oil, and Leonotis nepetifolia oil using the lipase extract powder as the biocatalyst.

Parameter	Commercial Olive Oil	Waste Cooking Oil	Leonotis nepetifolia Oil	Unit	Recommended Value from ASTM D6751	Recommended Value from EN 14214	Reference Value from Other Authors	Reference
Density	0.91 ± 0.02	0.91 ± 0.06	0.94 ± 0.04 (40 °C)	g/mL	0.86	0.86–0.90	0.92 (15 °C)	[2]
							0.91 (40 °C)	[37]
Moisture and volatile matter	0.05 ± 0.006	0.05 ± 0.007	0.04 ± 0.004	%	≤0.05%	≤500 mg/kg	0.8	[45]
							0.1
Saponification value	162.8 ± 3.47	167.9 ± 3.09	180.8 ± 0.56	mg of KOH/g of oil	-	-	181.4–199	[38]
							176.3	[47]
Acid index	0.74 ± 0.03	0.53 ± 0.007	0.96 ± 0.03	mg of KOH/g of oil	<0.5	<0.5	0.92–1.87	[39]
							0.99	[49]
Iodine value	74.7 ± 3.24	63.4 ± 1.88	61.9 ± 4.1	g of I2/100 g of oil	-	≤120	87.0	[50]
							86.0	[45]
Copper strip corrosion	1a	1a	1a	Dimensionless	<N° 3	1	1a	[40]
							1b	[39]

shows the results obtained from the characterization and reference values.

Different biodiesels must comply with international standards before being marketed to be comparable to other biodiesels and petroleum diesel. These standards are employed to analyze B100 biodiesel and its blends (B6 and B20) [33].

In this work, the average density value of biodiesel obtained was higher than the limits stipulated by the USA and European Union standards. This value influences fuel atomization efficiency (ignition quality) for air-less combustion systems. The density is a critical parameter because it indicates the delay between injection and fuel combustion in a diesel engine and the energy specifications (energy per unit mass) [55]. The value obtained is similar to that reported by other authors [2,37]. However, a high density is caused by the presence of unsaturated fatty acids, which cause a reduction in the viscosity and affect the flashpoint, resulting in a loss of power in the engine. Therefore, it is recommended to blend biodiesel with petroleum diesel so that biofuel pulverization in the motor is finer and proper combustion is achieved [21,33].

The percentage of moisture and the volatile matter is within the limit stipulated by the American standard. Moisture reacts with the catalyst during the transesterification reaction, causing soap formation and emulsions [55]. High moisture content in biodiesel can cause problems such as water accumulation and microbial growth in fuel handling and storage. He et al. [56] reported a moisture absorption rate of 22.2 ppm/°C for biodiesel, which was 9 times higher than diesel (about 2.4 ppm/°C) due to the oxygen content and polar chemical structure of carboxyl groups.

The moisture content of biodiesel reduces combustion heat and causes corrosion of vital fuel systems components such as injector pumps, fuel pumps, and fuel tubes [55]. An excess of water in biofuel can cause triglycerides hydrolysis, producing free fatty acids, especially at elevated temperatures. The excessive formation of soap occurs at average temperatures in the presence of alkaline catalysts (such as potassium hydroxide) [14]. For this reason, the saponification value must be evaluated, although this parameter is not specified in both quality standards.

The saponification value obtained was comparable to the values reported by other authors [38,47]. This value indicates that the formation of saponified products will not occur since the amount of catalyst was adequate in the transesterification reaction. In the same way, the acid value of biodiesel obtained for Leonotis nepetifolia oil was the highest (0.96 mg of KOH/g of oil), and waste cooking oil (0.53 mg of KOH/g of oil) was the lowest. The values obtained for the three cases were higher than the limits stipulated by both standards (<0.5 mg of KOH/g of oil).

Likewise, the acid index (presence of free fatty acids) was also higher than the limits stipulated by both standards and was comparable to the values reported by other authors [38,49]. Moisture in the transesterification reaction causes a high-acid index and saponifiable product formation [15]. Furthermore, it could cause corrosion in the engine’s fuel channel and indicate fuel deterioration and lubricant degradation while the fuel is in service [37,55].

The American standards do not provide a limit of iodine value for biodiesel. However, the values obtained in this study for different substrates are within the limit stipulated by the European standard (≤120 g of I₂/100 g of oil), and they are comparable to those reported by authors [45,50]. This parameter indicates the degree of biodiesel unsaturation through the existing number of double bonds in its composition. This property can significantly influence the oxidation stability and polymerization of glycerides and lead to deposit formation in diesel engine injectors, causing clogging. The iodine value is related to the cetane number, biodiesel viscosity, and the cold flow characteristics, i.e., the “cold filter plugging point” [55].

One of the advantages of biodiesel usage compared with petroleum diesel is its anti-corrosion capacity. This parameter is evaluated by a copper plate subjected to high temperatures and immersed in biodiesel. The value of copper strip corrosion was classified as 1b and is within limits stipulated by the American standard (<N° 3) and similar to the values reported by other authors [39,40], which means that although there are acids in the biofuel, they are not sufficient to cause corrosion in copper materials [37].

The fatty acid profile of the three oils used in this study is shown in

Table 5. Fatty acids profile in vegetable oils: commercial olive, waste cooking, and Leonotis nepetifolia.

Oil	Fatty Acid	Lipid Number	Retention Time (min)	GC-FID (%)
Commercial olive	Palmitic acid	C16:0	15.30	13.02
	Stearic acid	C18:0	17.50	5.28
	Oleic acid	C18:1	19.56	80.38
	Linoleic acid	C18:3 n6	20.70	1.32
Waste cooking	Caprilic acid	C8:0	4.86	2.95
	Palmitic acid	C16:0	15.30	10.05
	Stearic acid	C18:0	17.50	5.62
	Oleic acid	C18:1	19.56	73.96
	Linoleic acid	C18:2	20.70	7.42
Leonotis nepetifolia	Palmitic acid	C16:0	15.36	11.03
	Stearic acid	C18:0	17.50	3.35
	Oleic acid	C18:1	19.56	54.00
	Linoleic acid	C18:2 n6	19.70	19.01
	Linolelaidic acid	C18:2 n9	20.70	12.60

. Oleic acid (C18:1) had the highest percentage in all three oils: 73.96% in waste cooking oil, 80.38% in commercial olive oil, and 54% in Leonotis nepetifolia seed oil. Consult the Supplementary Materials for more information.

Although the fatty acid profile of cooked residual oils changes depending on the cooking type, temperature, and duration, oleic acid is the primary fatty acid present in the residual oil for all the food-cooked types [57]. In olive oil, the fatty acid composition may vary depending on zone meteorological conditions, the variety, and the maturation stage of the fruits [58]. However, oleic acid is reported as the primary component of olive oil (53–83%) [59,60].

The results of the fatty acid profile of Leonotis nepetifolia agree with those reported by Benni et al. [15], who reported 54.96% oleic acid (C18:1) and 25% linoleic acid (C18:2 n6).

On the other hand, in the analysis of the FAMEs of the transesterified vegetable oils (waste cooking, commercial olive, and Leonotis nepetifolia), it was possible to estimate the potential of Leonotis nepetifolia lipases for biodiesel production.

Table 6. Retention time and percentage of methyl oleate in enzymatic biodiesel samples.

Oil Biodiesel	Retention Time	GC-FID (%)
Commercial olive	27.08	48.28
Waste cooking	24.35	12.46
Leonotis nepetifolia	26.91	34.51

shows the retention times of methyl oleate and the central peak obtained after enzymatic catalysis (consult Supplementary Materials for more information).

The percentage of methyl oleate from olive oil was about 1.5-fold higher than Leonotis nepetifolia ester (Table 6). These results can be attributed to the high amount of oleic acid found in the olive oil sample (Table 5) and not to the catalytic efficiency of the lipases on the Leonotis nepetifolia oil. It has been reported that plant lipases have a catalytic preference over certain fatty acids [18]. For example, oleic acid esterification was 2.5 times faster than palmitic acid [61]. Therefore, it is likely that in Leonotis nepetifolia oil, containing a low proportion of oleic acid and a considerable amount of palmitic acid C16:0 (10–20%), the catalytic rate of lipases is decreased compared with the other substrates that are rich in oleic acid. Canet et al. [43] also reported that the transesterification rate increased with the amount of oleic acid used in the reaction.

Low biodiesel conversion was obtained using waste cooking oil as the substrate (Table 6). A peak (41.04%) with a retention time (rt) of 19.66 min was detected and attributed to the non-transesterified oil. 2,3-dihydroxy propyl elaidate, 1-hexanol, i-propyl-14-methyl-pentadecanoate, 1-heptene, and cis-9-hexadecenal are some hazardous chemical compounds in waste cooking oil that can interfere with the lipase catalytic activity [62]. Moreover, the high water content accelerates the hydrolysis reaction and reduces the amount of ester formation. Therefore, transesterification methodology modifications can be made to increase the biodiesel conversion rates from waste cooking oil, such as the stepwise addition of methanol to the system to reduce enzyme inhibition [63], in situ glycerol removal [64], and enzyme immobilization [65]. Likewise, the lipase-catalysis time could be increased since a time range of 7–48 h has been applied for a biodiesel yield of 90–99% [66].

4. Conclusions

Leonotis nepetifolia lipase extract powder is an environment-friendly biocatalyst to produce biodiesel enzymatically. Enzyme-catalyzed transesterification yields of 74.5%, 71.5%, and 69.3% for commercial olive oil, waste cooking oil, and Leonotis nepetifolia oil, respectively, were obtained under the following reaction conditions: temperature of 34 °C, oil to methanol molar ratio of 1:3, lipase extract powder 20 wt%, phosphates buffer (pH 4.8) 20 wt%, and a reaction time of 8 h. Biodiesel physicochemical properties (moisture and volatile matter, acid and iodine index, copper strip corrosion, and methyl esters content) satisfied the reference values stipulated by international standards. The search for other plant lipases and optimized techniques are opportunity areas for the transesterification reaction. In addition, the selection of plants with non-edible oils, which are adaptable to local climatic conditions, will allow biodiesel to be more viable as an alternative liquid fuel, can solve the global problem of food versus fuels, and is techno-economically feasible. Biodiesel production from proposed substrates using a lipase extract powder of Leonotis nepetifolia seed can be a promising alternative for biodiesel production catalysis.