Thymelaea hirsuta and Echinops spinosus : Xerophytic Plants with High Potential for First ‐ Generation Biodiesel Production

: The negative impacts of fossil fuel on the environment should be replaced by clean and sustainable energy sources worldwide. Therefore, the use of biodiesel as a clean energy source is crucial. Biodiesel is produced from various natural resources through a transesteri ﬁ cation process. Considering the importance of this topic, this study focuses on the assessment of oil properties of Thymelaea hirsuta and Echinops spinosus as primary sources for biodiesel production. The two investigated plants were collected from the Western Desert of Egypt. The results showed that the lignocellulosic content was about 57.3 and 79.8 g/100 g in E. spinosus and 59.1 and 82.8 g/100 g in T. hirsuta, respectively. The two investigated samples showed variable lipid contents (30.2%–76.1%). The GC ‐ MS fatty acid profile characterized seven FAs in E. spinosus and twelf FAs in T. hirsuta . The greatest CN was calculated in T. hirsuta (379.2) compared to the lowest in E. spinosus (229.9). Furthermore, the values of saponification number (SN) were 27.9 in E. spinosus and 16.07 in T. hirsuta . The value of higher heating value (HHV) was about 47.5 MJ/kg in E. spinosus and 48.3 MJ/kg in T. hirsuta . Meanwhile, T. hirsuta exhibited a higher induction period (IP) value (19.3 h) comparable to that of E. spinosus (4.3 h). The results revealed that both plants are potential sources for biodiesel production according to various international standards for biodiesel production, and this work appears to be one of the first reports regarding such wild xerophytic plants as promising new primary sources for biodiesel production in Egypt .


Introduction
Energy is a key requirement for every country's economic growth. As the world's oil supplies have slowly declined [1,2], suitable alternative fuel sources are urgently needed. Currently, research has focused on biofuel production from various sustainable bio-resources, in particular plants that are not edible [3]. Moreover, the effect of airborne pollutants on human health has become a pressing issue worldwide [4], and biodiesel is a promising solution to this problem. Biodiesel is one of the nonpolluting renewable fuels and has received considerable attention [5].
Biodiesel derived from plant-based materials presents several advantages [6]. The use of biodiesel significantly minimizes the emissions of harmful greenhouse gases, aromatic hydrocarbons, and sulfur particles, but it somewhat raises the fuel consumption and decreases the engine power. Even though nitrogen oxide (NOx) emissions are increased in such cases, this can be diminished using As an alternate and possible way to produce biofuel, the steady growth in the use of wild plant oil in Africa and Asia should be concentrated [25,38]. Fossil fuel resources for energy production are rare in Egypt, so alternative bio-resources, such as non-edible wild plants, should be used for biodiesel production to meet the increasing demand in energy.
The genus Echinops spinosus (L.) belongs to the family Asteraceae; it is a perennial herb, growing to about 1 m, with erect brownish to reddish stems. It has a few long leaves, 10 to 15 cm in length, is hairy, arachnoid, and has very long spines [39]. Echinops spinosus is distributed regionally in North Africa and throughout the Sahara, including the Red Sea region and Sinai. Such wild shrubs are naturally adapted to drought conditions and have various medicinal purposes [40].
In addition, Thymelaea hirsuta (L.) Endl. belongs to the family thymelaceae; it is a perennial Mediterranean, evergreen shrub. Moreover, T. hirsuta is a drought-tolerant shrub that grows up to 2 m high and is considered as one of the psammophytes, with rigid, fibrous stems [41]. It is one of the most common species inhabiting the north coast of Egypt and extends inland approximately 70 km from the coast [42]. The economic potential of T. hirsuta as a medicinal plant and fiber source was reported by Schmidt and Stavisky [43]. These two xerophytic species have never been reported elsewhere as primary feedstock for biodiesel production.
The two investigated xerophytic plants in this study have been chosen for the following reasons: T. hirsuta has rigid, fibrous stems containing a large amount of cellulose, lignin, and oils [44]. Moreover, T. hirsuta shoots are characterized by a fast ignition time and are used by the Bedouins as solid fuel for energy production [45]. Furthermore, E. spinosus contains an appropriate amount of fatty acids that qualify it to produce biodiesel [46]. Additionally, these two species are widely distributed in arid and semiarid areas in Egypt, grow in soil with low water and nutrient contents, and do not need agricultural fertilizers. Finally, T. hirsuta and E. spinosus are perennial species, which can be cultivated in a sustainable way on non-arable land, especially since there are huge areas of desert land in Egypt that could be used for the cultivation of drought-tolerant plants. Therefore, the production of large quantities of biomass from these plants as raw materials to produce biofuels needs no effort or high cost and is of great economic feasibility. Therefore, this work is aimed at the determination and assessment of energy-generating compounds of Thymelaea hirsuta and Echinops spinosus and their potentiality as primary plant feedstocks for biodiesel production.

Plant Material
The aerial portions (stems and leaves) of two xerophytic plants, Echinops spinosus ( Figure 1) and Thymelaea hirsuta ( Figure 2), with similar size and age, were collected from their natural habitats (Western Desert of Egypt) in 2019. Botanical identification of the above two plants was confirmed in Ain Shams University, Faculty of Science, Botany Department (the Herbarium section). For two weeks, aerial plant parts have been dried in air under the shade, powdered and then used for analysis.

Estimation of Carbohydrates
A commonly used method for the extraction of carbohydrates is to boil a known weight of airdried plant tissue in 10 mL of 80% (v/v) ethanol. The extract was filtered, and the filtrate was ovendried at 60 °C, then dissolved in a known volume of water to be ready for the determination of soluble sugar. The soluble sugar and insoluble sugar contents were measured by the anthrone reagent according to the method of Fairbairn [47]. The concentration of soluble and insoluble sugar was determined finally from the standard curve of glucose and calculated as mg/g plant tissue. Moreover, the cellulose content was determined by the method described by Reference [48].

Estimation of Pectin and Lignin
The fiber quality for pectin and lignin was determined following the methods described by References [49] and [50], respectively.

Extraction and Assay of Peroxidase Activity (POX)
The method adopted for peroxidase extraction was that described by Reference [51]. A dried plant tissue (2 g) was extracted in 100 mM phosphate buffer (pH 6.8). The homogenates were centrifuged at 20000× g for 20 min. The supernatant was made to a known volume with the same buffer and was used in enzyme preparation for assaying the activity of certain enzymes. Moreover, POX activity was assayed according to the method of reference [52].

Estimation of Total Phenols
Total phenols were extracted and measured according to the method adopted by Reference [53]. In this method, the Folin-Ciocalteau reagent was used to estimate total phenols. The phenolic content was calculated as gallic acid equivalents of gallic acid/g extract on the basis of gallic acid standard curve.

Lipid Extraction
The air-dried powder (10 g) was extracted with petroleum ether for 18 h by using a Soxhlet apparatus [54]. The extract was then transferred quantitatively to a weighed flask, and the solvent was evaporated using an electric fan. The flask was then reweighed, and the difference in weight was equivalent to the weight of total lipids.

Fatty Acid Composition of oil by GC-MS
After extraction, the oil underwent to the transesterification process, which was carried out by using methanol and base (KOH) as described by References [55,56]. Then, the methyl ester fatty acid composition of yielded oil was analyzed by gas chromatography mass spectrometry (GC-MS) ( Figure  3) in accordance with Jun [57] by using an Agilent 7890B GC system coupled to an Agilent 5977A MSD with a capillary column size of 0.6 m × 100 μm × 0 μm (Agilent-Technologies, Sant a Clara, CA, USA). The injector and detector temperatures were set at 250 °C, and the ion source temperature was set at 230 °C. Helium gas was used as carrier gas at a constant flow rate of 1.5 mL/min. The column temperature was set at 40 °C for 2 min, then 10 °C/min to 180 °C for 5 min, and then 10 °C/min to 250 °C for 10 min. The total GC running time was 38 min. The GC/MS was run in Scan/SIM mode, and the identification of sample components was performed using Agilent Mass Hunter software (NIST14 L). The fatty acids composition was reported as the relative percentage of the total peak area by direct comparison of the retention times and mass spectral data reported in the literature.

The Fuel Properties of Biodiesel
Potential biodiesel fuel properties such as saponification number (SN), iodine value (IV), degree of unsaturation (DU), cetane number (CN), higher heating value (HHV), cold filter plugging point (CFPP), and induction period (IP) were empirically determined according to the method of Reference [58].

Saponification number (SN) and iodine value (IV)
The saponification number and iodine value were calculated according to Equations (1) and (2), respectively, from the fatty acid methyl ester composition of oils: where N is the percentage of each fatty acid component, M is the molecular mass of each fatty acid, and D is the number of double bonds in each fatty acid [59].

Degree of unsaturation (DU)
The degree of unsaturation was calculated from Equation (3): where MUFA is the percentage of monounsaturated fatty acids and PUFA is the polyunsaturated fatty acids [60].

Cetane number (CN)
The cetane number was calculated from the following empirical Equation (4) [61]:

Induction period (IP)
This was calculated from the empirical Equation (8)  where X is the content of PUFA (wt%).

Statistical Analysis
All results were expressed as mean value ± standard error by using two-way analysis of variance (ANOVA) using IBM Statistical Product and Service Solutions, SPSS Statistics for Windows, Version 21.

Results and Discussion
Nowadays, there is a great demand for new renewable, sustainable energy sources. Biodiesel production is considered as a potential alternative fuel to reduce the carbon footprint [65].
The major polymer constituents of biomass are cellulose, pectin, and lignin. The variability in lignocellulosic material depends upon the type of biomass species, plant parts, and the abiotic factors [66,67]. The present study found the cellulose content was about 57.3 g/100 g in E. spinosus, while it reached about 59.1 g/100 g in T. hirsuta (Figure 4). The high cellulose content of both investigated plants favors their efficiencies for biofuel production. On the other hand, the lignin content was about 79.8 g/100 g in E. spinosus and 82.8 g/100 g in T. hirsuta. Lignin can inhibit cellulase activity [68]; therefore, plant species with less lignin in their cell walls are desirable for biofuel production [69]. The pectin contents reached about 37.9 and 33.5 g/100 g in E. spinosus and T. hirsuta, respectively ( Figure 4). Although lignin is not used for conversion to ethanol, it can be converted to other biofuels or used for biopower generation [70]. Moreover, the proper proportion of lignocellulosic constituents in the cell wall is essential in the fermentation and thermo-chemical conversion processes of biofuels [71], and it helps increase oil yield [72]. In this study, the lignin content in E. spinosus was less than that estimated in T. hirsuta. It is interesting to note here that the peroxidase enzyme showed a variable activity among the two investigated plants E. spinosus and T. hirsuta ( Figure 5). The peroxidase activity was higher in E. spinosus (2.62) than that of T. hirsuta (1.91). In the present study, lignin content was negatively related to POX activity. Such relation indicated that this enzyme was not involved in lignin synthesis and/or exhibited high oxidative activity in the degradation of lignin dimer [73]. Moreover, the increase in peroxidase activity might be due to the accumulation of certain phenylpropanoids (monomeric units of lignin) resulting in the reduction of structural lignin [74]. Indeed, biofuel production is influenced by sugar content [75]. The amount of soluble and insoluble sugars reached about 12.6 mg/g and 46.9 mg/g in E. spinosus, while it amounted to be 8.81 mg/g and 39.5 mg/g in T. hirsuta, respectively ( Figure 6). Such parameters can also be utilized as a source of fermentable sugars used in the production of biofuels [75]. The assayed phenol contents were 6.89 mg/g in E. spinosus, while they reached about 3.66 mg/g in T. hirsuta ( Figure 6). Natural antioxidants, such as phenolic compounds, protect oils against the oxidation of unsaturated fatty acids [23].
The present data revealed that E. spinosus was one of the best oil producers ( Table 1). The oil content expressed as g/100 g dry weight reached about 76.1% in E. spinosus and 30.2% in T. hirsuta (Table 1). In fact, for species selection, the appropriate oil content was about 15% for biodiesel production [76]. Additionally, our results agree with previous findings on Jatropha spp. [77]. Meanwhile, each plant was characterized by its specific fatty acid profile [78]. The GC-MS fatty acids analysis showed the profile and the percentage of E. spinosus and T. hirsuta extracted crude oil. The oil was characterized seven FAs in E. spinosus and twelve FAs in T. hirsuta (Tables 2 and 3). It is interesting to note here that E. spinosus was a rich source of polyunsaturated fatty acids (PUFAs) such as linolelaidic acid (C18:2, about 70.31%), followed by margaric acid (C17:0, 13.97%) and palmitic acid (C16:0, 11.11%). At the same time, monounsaturated fatty acids MUFAs such as palmitoleic acid (C16:1, 0.47%) and gadoleic acid (C20:1, 1.00%) were abundantly found in E. spinosus, along with palmitic acid. Additionally, behenic acid (C22:0, 28.08%), arachidic acid (C20:0, 22.68%), palmitic acid (C16:0, 18.9%), and stearic acid (C18:0, 5.86) were the four major components of FA detected in T. hirsuta oil (Table 3). Other FAs such as lignoceric acid (C24:0), undecanoic acid (C11:0), and melissic acid (C30:0) were also differentially detected in trace amounts in T. hirsuta oil. Other MUFAs such as oleic acid (C18:1, 1.67%) and erucic acid (C22:1, 0.85%) were also detected. On the other hand, the PUFA linolelaidic unsaturated fatty acid (C18:2, about 6.85%) also was detected in the oil extracted from T. hirsuta (Table 3). The changes in fatty acid composition in plant membranes are critical for the plant to adapt to desert conditions [79,80]. The oil composition of the investigated plants inhabiting an arid habitat is greatly influenced by the environmental conditions and genetic factors [81]. In addition, the profiles of the fatty acid methyl ester determine the properties of biofuel [82]. The composition of fatty acids in the vegetable oil determines the cetane number of the produced biodiesel [82]. Indeed, the cetane number (CN) of biodiesel, which measures the ignition and combustion quality of the fuel, is the primary factor affecting the fuel properties. Higher CN improves the ignition and delays the combustion of fuel. In this study, CN values were about 229.9 in E. spinosus, which was lower than that in T. hirsuta (379.2; Table 1). These results were relevant to the United States (ASTM D 6751) and Europe (EN 14214:2008) biodiesel standards, who point out that the appropriate values are >51 and >47 respectively [83]. In addition, Iodine value (IV) is the most common indicator used to determine the quality of fuel [84]. The values of IV reached about 50.75 in E. spinosus and 29.16 in T. hirsuta (Table 1). Generally, standards of biodiesel by EN14214 indicate the value of iodine <115 g I2/100 g [83], while iodine < 120 is mentioned for European standards of biodiesel. It was recorded that lower values of iodine will encompass solidification of FAME at ambient temperature. However, in biodiesel production the fatty acid methyl ester formula desires unsaturated FAMEs, as they prevent biodiesel solidify [85]. Consequently, E. spinosus oil is better than T. hirsuta as a source of raw material for biodiesel production because of its higher iodine content, concomitant with the increase in the number of unsaturated double bonds, thereby reducing the oil viscosity [86]. The relatively observed low iodine values might be attributed to the high values of temperature in a desert habitat, which stimulate the occurrence of saturated fats instead of unsaturated ones [87]. The IV of FAMEs exhibited a negative relation with CN (Table 1), which reached about 50.75 g I2/100 g and 29.16 g I2/100 g in E. spinosus and T. hirsuta, respectively. These xerophytic oils exhibit values that are competitive with some traditional vegetable oils used for biodiesel production such as sunflower, soybean, and corn [88]. The high CN value attained in T. hirsuta matched the low IV as compared with those in E. spinosus ( Table 1). The results in this study were aligned with the results reported by [89] in palm oil. The current results follow the biodiesel standards in Europe (EN 14214), which indicate a value of iodine < 120. Meanwhile, low IVs in unsaturated FAMEs [90] prevent oxygen peroxide activity and solidification at ambient temperature, which is desirable for biodiesel quality [59,83]. Furthermore, the degree of unsaturation (DU) amounted to be 142.1 and 16.2 in E. spinosus and T. hirsuta, respectively ( Table 1). The unsaturated FAMEs are oxidatively unstable because fatty acids react quickly with oxygen double bonds. Moreover, DU significantly affects long-term storage capabilities [91]. The values of the saponification number (SN) in both investigated plants were 27.9 in E. spinosus and 16.07 in T. hirsuta ( Table 1). The SNs in this study were lower than those recorded in Jatropha curcas [92]. This biodiesel had highly saturated FAMEs (at most C14:0 and C16:0) and a high saponification value [93]. It was reported that the SN is related to IV; thus, a higher SN value improves the stability of biodiesel [16].
The value of the cold filter plugging point (CFPP) in T. hirsuta (261.5) was higher than the value recorded in E. spinosus (2.93) ( Table 1). Our results follow Park et al. [64], who reported that CFPP values increased concomitantly with the percentage of oil saturation. However, it is arduous to set up enough ranges for CFPP values because of the seasonal temperature variability worldwide [24].
Furthermore, higher heating value (HHV) is a parameter that plays an essential role in determining the quality of biofuels. The presented data showed that HHV reached about 47.5 MJ/kg in E. spinosus and 48.3 MJ/kg in T. hirsuta (Table 1). Such data indicated that the values of both tested plants exceeded the limit set (EN14213 (>35.0) standard) for this criterion (Table 1). Such results are parallel to other ones in tropical plants [90,94]. Additionally, the results in this study had higher values of HHV than those obtained from corn, cotton seed, and Jatropha curcas [88]. Moreover, such results are desirable properties for biodiesel energy, since the lower heating value of biodiesel is known to decrease the engine power.
The induction period (IP) evaluates the instabilities of physicochemical and thermal biofuel properties, which, in turn, increases its oxidation [95]. In this study, T. hirsuta exhibited a higher IP value (19.3 h) compared to that of E. spinosus (4.3 h) ( Table 1). The USA ASTMD6751 and European standards of biodiesel expressed induction periods (at 110 °C) of >3 h and >6 h, respectively [24]. Furthermore, the induction period improves linearly for natural antioxidants such as vitamins as well as phenolic compounds. These antioxidants slow down the rate of oxidation of biofuel [96].

Conclusions
We must face the energy demands of the growing population in our countries. Therefore, providing renewable resources is essential. It was shown in this study that two drought-tolerant plants, subjected to many abiotic stresses (i.e., drought and salinity) and biotic stresses (i.e., overgrazing and over collection), could be cultivated in a sustainable way on marginal and nonarable lands of Egypt. The yields will be not as high as when grown on arable land, but arable land should be reserved for the cultivation of food.
In conclusion, the data on biodiesel properties obtained using empirical equations for both Echinops spinosus and Thymelaea hirsuta species based on their FAMEs profile exhibit that both investigated species meet the norms required to be a diesel substitute, and they have enough oil content of moderately long-chain fatty acids with a high degree of unsaturation (DU). Oil of these species characterized by a high cetan number (CN), that improves the ignition and delays the combustion of fuel. In addition, the higher saponification values (SN) along with higher iodine values (IV) will improves the stability of biodiesel.
To summarize, the findings from the preliminary study carried out in this research show that the two xerophytic species investigated are a hopeful, sustainable and potential primary feedstock for future production of biodiesel.