Assessing the Bioenergy Potential of Novel Non-Edible Biomass Resources via Ultrastructural Analysis of Seed Sculpturing Using Microscopic Imaging Visualization

Abstract

Recently, intensifying energy crises accompanying ecological crises due to the decline in fossil-fuel reserves and extensive greenhouse gas emissions have triggered the exploration of renewable substitutes for petro-diesel. In this scenario, biodiesel is the best alternative to non-renewable finite conventional fuels due to its cost-effectiveness, sustainability, renewability, biodegradability, and eco-friendly nature. Hence, the current research was designed to utilize scanning electron microscopy to investigate and identify the micro-morphological characteristics of selected seed-bearing crops. Light-microscopy (LM) indicated discrete variations in macro-morphological characters such as seed shape (ovoid, ovate, oblong, semi-spheroid, or discoid), seed size (3.5–14 mm in length and 2.25 to 6.5 mm in width), seed color (yellow to black), and number of seeds per kilogram (from 6000 to 260,000). Chemical extraction via Soxhlet apparatus resulted in the estimation of oil content within the range of 20.3–48.0% (wt./wt.), FFA content (0.63–6.91 mg KOH/g), and maximum product, i.e., 98% biodiesel yield was achieved. Multivariate analysis via principal component analysis (PCA) was done using PAST 3 software to investigate similarities and differences among factors/variables. SEM examination exhibited ultra-morphological characters and distinct variation in cell-wall ornamentation; hilum occurrence, position, and level; wall-sculpturing variations such as ruminate, verrucate, wrinkled, or striate; cell arrangement (anticlinal or periclinal); and cell shape and margins. To conclude, SEM could be an advanced technique to disclose the ultra-micromorphological characteristics of oil-bearing energy crops providing a convenient way for scientists to determine correct identification, authentication, and classification.

1. Introduction

The energy that directly streamlines industry, power generation, and the transportation sector determines the economic success, development, and sustainability of a country [1]. In the past few decades, the population explosion has resulted in an upsurge in urbanization and industrialization. Therefore, finite fossil-fuel reservoirs have been exploited to meet the rising demand for energy [2]. Moreover, fossil-fuel combustion has intensified local air pollution and magnified global warming due to increased greenhouse effect, acid rain, ozone depletion, climatic changes, seasonal shift, and eventually, biodiversity declination. This scenario of declining fossil-fuel reserves and resulting petroleum price hikes has directed researchers worldwide to investigate alternate renewable fuels [3]. Rapid changes in the harvesting and use of renewable energy have lead to a transition in energy provision [4]. Due to geo-political, commercial, and environmental issues, diverse forms of renewable energy, e.g., wind, solar, hydrothermal, and biomass energy, have been explored [5]. Due to their diversity, sustainability, abundance, and eco-friendly qualities, all biomass-energy components, or biofuels, such as bioethanol, biogas, and biodiesel, are regarded as the most practical and best replacement [6,7]. Due to diminishing and finite petro-diesel reserves, sharp changes in petroleum prices, harmful gas emissions, and environmental concerns, the demand for biodiesel has rapidly increased. Biodiesel is preferred due to its renewability, high flash-point, non-toxicity, and biodegradability, so it can abate ecological crises by direct utilization in diesel engines. Biodiesel is a fatty acid methyl ester synthesized from triglycerides via transesterification. Alternatively, biodiesel can be synthesized by alcoholysis of different raw materials, e.g., animal fats, agronomic plants (edible/non-edible), microalgae, and waste biomass [8,9,10]. The selection of readily available, cost-effective, feedstock contributes to the budget of the biodiesel trade. The contradiction between fuel and food has been created by edible feedstock such as animal fat and vegetable oils, which has limited their use [10]. Therefore, non-edible raw materials, especially non-edible seed oils, is preferred to avoid fuel–food competition and for the sustainability of the biodiesel industry. Non-edible seed oils offer low cultivation costs due to their ability to grow on barren land. Moreover, toxins present in these seeds make them inappropriate for human use. Hence, the best option is for them to be used as feedstock and this resolves the issue of food insecurity, production cost, and environmental pollution. Examples of popular non-edible oils are those obtained from Capparis spinosa [2], Raphanus raphanistrum [9], Celastrus paniculatus [10], Brachychiton populneus [11], Elaeagnus angustifolia [12], Zanthoxylum armatum [13], and Cucumis melo var. agrestis [3]. Hence, the exploration and identification of non-edible seed oils as feedstock has become indispensable for accelerating large-scale biodiesel production. Due to the stability of the seed testa, scanning electron microscopy (SEM) is an advanced technique has been determined to be the best method of detection and authentication of various oil-bearing seed plants. High magnification SEM is used to quantitatively examine fine details of seed sculpturing, surface, and ornamentation, and seed ultrastructure. SEM has made revolutionary advancement in the field of microscopy for detailed examination that is not feasible via light microscopy. Ornithogalum species, seed-born fungi, and taxonomic variation in lapinus and onion seeds can be examined with SEM [14,15].

SEM has been used as a tool to identify various plant taxa based on anatomy and environmental significance. Micro-morphological characteristics of seed coats have been identified, followed by the identification of related genera. Furthermore, seed testa investigation via SEM has aided in tracking phylogenetic relationships, solving taxonomic and systematic problems, classification, and in investigating phenetic linkage to variation.

The deficiency in gross morphological data has been overcome by the ability to carry out detailed analysis of seed coat sculpturing and texture, which is important in solving taxonomic problems [16]. SEM has recently offered a new possibility to identify and authenticate oil-bearing seeds which are used as adulterants due to their morphological similarities. Moreover, high-liquid-pressure chromatography, gas chromatography, and Fourier transform infrared spectroscopy (for olive oil) are advanced analytical techniques to determine seed-oil adulterants. These techniques involve sample preparation that takes time and the use of costly chemicals and complex procedures, hence making the process expensive. Therefore, this has shifted the researcher’s attention towards a fast and reliable technique, i.e., SEM for the purpose of resolving taxonomy-related issues of close taxa and species as well as to authenticate oil seeds [17]. According to the best of our knowledge, SEM has not yet been employed in order to study the ultra-micro morphology of oil-producing seeds. The current work was focused on detailed investigation of micro-morphological characters of six novel oil seeds via SEM. Relatively similar seeds are often mixed and mishandled, so this research focused on reliable characterization techniques that are helpful in seed identification and authentication. The work also aimed to provide details that help in understanding related species in terms of their systematic relationships by the assessment of micro morphological features of their seeds. Additionally, along with a number of additional plant features that are important in accurate identification of closely related species, this study will operate as an additional reference for scientists.

2. Materials and Methods

2.1. Sample Collection

The selected plants’ seeds, including Bischofia javanica, Praecitrulus fistulosus, Luffa acutangula, Diospyros lotus, Solanum surattense, and Cucumis melo var. agrestis, were collected from different places in Pakistan during frequent field trips in respective seed seasons. The seeds were collected to examine the micro-morphological characteristics of the seed coat and to assess their potential in biodiesel synthesis. In addition, the samples were dried under shade to decrease moisture content and stored for future utilization.

2.2. Identification and Morphology

A binocular light microscope (Bosch and Lomb model, New York) was used to carry out the morphological description of six novel non-edible, oil-yielding, seeds. Seeds were examined with various magnifications, including 10×, 20×, 30×, and 40×, followed by 3–5 concordant readings and recording of the mean value of seed dimensions, where seeds’ width and length were measured using a ruler scale [6]. The author citation with plant species was authenticated via The Plant List and International Plant Name Index websites. In addition, validation of macro-morphological characteristics was made from the Flora of Pakistan. An expert taxonomist conducted the name verification of the selected seed samples, and then the names were compared with the plant specimens preserved in the Herbarium of Pakistan [18].

2.3. Seed Surface Ornamentation

Scanning electron microscopy was employed to examine seed ornamentation and micro-morphological features in detail. This was carried out at CRL, University of Peshawar, Pakistan, using scanning electron (SEM) model JEOL JSM 5910. Dry ripened seeds were washed for 1–2 min in ethyl alcohol (70%) to remove dust particles. Dried seeds were mounted on stubs via sticky carbon tape and processed in a gold sputter machine for gold coating. SEM micrographs of gold-coated seeds were taken to investigate various micro-morphological features such as seed dimension, structure, surface shape, and texture. Significant structural features such as the anticlinal wall pattern, cell arrangement, cell form, surface sculpturing, and margins, as well as the protuberance of the periclinal wall, were also investigated [16].

2.4. Seed Oil Content

Seed samples were kept at 60 °C in an oven overnight to remove moisture content until well dried. A pestle and mortar were then used to make a fine powder of dried seeds for oil extraction. A known amount of sample was placed in a thimble in the Soxhlet apparatus at 60 °C. Petroleum ether (250 mL) was used as a solvent for chemical extraction. The mean weight value (before and after extraction) was used to estimate the amount of oil content followed by solvent recovery (55 °C) via the rotatory evaporator upon reaction completion. Oil content was finally calculated by Equation (1):

$$\mathrm{Oil} \mathrm{Content} =\frac{Weight of extracted oil}{Weight of Sample used}\times 100$$

(1)

2.5. Free Fatty Acid Content Determination

Determining FFA content is a vital step to be conducted prior to the transesterification reaction. Current research involves FFA content determination via the acid–base titration method. Two steps involving blank and sample titrations were carried out with isopropanol in the flask and KOH (0.025 M) solution in the burette and phenolphthalein as indicator. While sample titration was performed by using a known amount of seed oil, FFA content was determined using the following equation:

$$\begin{array}{c}FFA content=\\ \frac{\left(Vol. used in sample titration-Vol. used in blank titration\right)\times mass of catalyst \left(\mathrm{g}/\mathrm{L}\right)}{Total volume of oil used}\end{array}$$

(2)

2.6. Statistical Analysis

Principal component analysis (PCA), a statistical technique, was used to examine the similarities and differences between the groups of the components. Using SPSS 16.0 and PAST 3, multivariate analysis via PCA was carried out to determine the variation in a set of parameters, including oil content, FFA content, and biodiesel production.

3. Results and Discussion

This work involved the detailed investigation of micro-morphological seed characteristics of six novel non-edible feedstocks that bear oil-yielding seeds. These included Cucumis melo var. agrestis, Bischofia javanica, Praecitrulus fistulosus, Luffa acutangula, Diospyros lotus, and Solanum surattense, which were found to be sustainable sources with high potential of bioenergy. Micro-morphological characteristics, oil content (%), FFA content (mg KOH/g), and the plants’ biodiesel potential were determined. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6 show photographs and SEM micrographs of the selected non-edible oil seeds. The micro-morphological characteristics included margins, cell arrangement, anticlinal and periclinal walls, cell shapes, and protuberances. There was a strikingly diversity in seed size and form as well as in micro-morphological traits, which serve as reliable identifiers to identify particular species. SEM has recently become the most sophisticated and reliable method for resolving many taxonomical issues related to seed categorization and identification. [18]. Hence, micro-morphological study and ultrastructural examination of seeds forms the basis of investigation of angiosperms’ evolution and the modern artificial classification system [14] and this ultrastructural and micro-morphological study provides a valid source of data. Furthermore, this research involved the evaluation of different inedible novel seeds via SEM. The obtained results will assist in determining the potential and promising sources of biodiesel for dealing with energy crises. A low free-fatty-acid content and high seed-oil content (>20%) are essential criteria for selecting seeds as a biodiesel feedstock [3].

Table 1. Qualitative characteristics of seed-bearing energy crops.

Plant Name	Family	Seed Color	Inner Color	Seed Shape	Surface Sculpturing	Wall Ornamentation	Hilum			Compression
							Occurrence	Position	Level
Cucumis melo var. agrestis Naudin	Cucurbitaceae	Dark brown	Off white	Ovoid	Ruminate	Thick and raised	Visible	Terminal	Raised	Lateral
Bischofia javanica blume	Phyllanthaceae	Light brown	Brown/black	Ovoid-oblong	Verrucate	Very thick and raised	Not visible	Sub-terminal	Depressed	Lateral
Praecitrullus fistulosus (Stocks) Pangalo	Cucurbitaceae	Dark brown	Pale yellow	Ovate-oblong	Wrinkled	Thick and raised	Not visible	Terminal	Raised	Lateral
Luffa acutangula (L.) Roxb	Cucurbitaceae	Black	Yellow	Ovate-Obovate	Verrucate	Very thick and raised	Visible	Terminal	Raised	Dorso-ventral
Diospyros lotus L.	Ebenaceae	Dark brown	Grey	Semi-spheroid	Striate	Thin and raised	Visible	Depressed	Depressed	Lateral
Solanum surattense Burm. f.	Solanaceae	Orange to yellow	Yellow	Discoid-obovate	Ruminate	Thick and raised	Visible	Terminal	Depressed	Lateral

shows several qualitative characteristics of seed-bearing energy crops, while

Table 2. Quantitative characteristics of seed-bearing energy crops.

Plant Name	Seed Length (mm)	Mean ± Standard Deviation (Length)	Seed Width (mm)	Mean ± Standard Deviation (Width)	Length/Width Ratio	No. of Seeds/kg	Seed Oil Content (%)	Mean ± Standard Deviation (Seed Oil Content)	FFA Content (mg KOH/g)	Mean ± Standard Deviation (FFA Content)	Biodiesel Potential (%)	Mean ± Standard Deviation (Biodiesel Potential)
Cucumis melo var. agrestis	10–12 (11)	11.0 ± 1.0	5–6 (5.5)	5.2 ± 0.26	2	1000–11,000	30	30.0 ± 1.0	0.63	0.63 ± 0.01	96	95.0 ± 1.45
Bischofia javanica	3–4 (3.5)	3.5 ± 0.50	2–3 (2.5)	2.3 ± 0.47	1.4	205,000–207,000	29.7	29.7 ± 0.01	1.95	1.95 ± 0.01	98	98.5 ± 0.707
Praecitrullus fistulosus	9–11 (10)	10 ± 1.0	5–6 (5.5)	5.3 ± 0.47	1.81	1000–11,000	34	34 ± 1.00	2.01	2.01 ± 0.01	91	91 ± 1.00
Luffa acutangula	11–13 (12)	12.0 ± 1.0	6–7 (6.5)	6.3 ± 0.47	1.84	5000–6000	48	48 ± 1.0	3.91	3.91 ± 0.01	93	93 ± 1.00
Diospyros lotus	13–15 (14)	14.0 ± 1.0	5–6 (5.5)	5.3 ± 0.47	2.54	7000–9000	20.3	20.4 ± 0.10	6.91	6.92 ± 0.01	87	88 ± 1.00
Solanum surattense	3–4 (3.5)	3.5 ± 0.50	2–2.5 (2.25)	2.1 ± 0.23	1.5	25,000–260,000	21	21 ± 1.00	3.91	3.92 ± 0.01	89	89 ±1.00

shows each seed’s oil content, FFA content, and biodiesel potential along with mean ± standard deviation.

3.1. Cucumis melo var. agrestis

Cucumis melo var. agrestis is a perennial or annual branched climbing herb and invasive weed, commonly known as wild musk melon, small gourd, or wild melon throughout the Old World tropics [19]. It is distributed in arid zones; pastures; fields of soybean, alfalfa, and sugarcane; railroad banks; marsh landscapes; rubbish dumps; vacant lots; disturbed areas; abandoned home sites; creek beds; and waste marshy lands where other plants seedlings are unable to germinate. It infests maize, cotton, pearl millet, cotton, and sorghum, as well as cyprus heads. The scabrid stem is prostrate with hairs (scabrous hairy stem) with 3–5 (–7) lobed, ovate, triangular leaves; the calyx is subulate (1.5 mm); the plant is petiolate (1–6 cm); the corolla is yellowish, ovate-oblong, and 6–8 mm in length; flowers are solitary or sometimes in pairs or threes, and the peduncle is 5–10 mm and densely hipsid. Cucumis melo var. agrestis has a rounded (2.5–5 cm diameter) ellipsoidal fruit consumed as a vegetable in Pakistan, while poisonous seeds remain unused and wasted.

Memon et al., 2018, demonstrated the nutritional profile of fruit (3.8% ash content, 31.2% carbohydrate content, 18.9% protein content, 80.9% moisture content) of this non-conventional vegetable [20]. Our experimental work showed that seeds are dark brown, 12 mm in length, and 6 mm wide, with a mean weight of 11–13 g (Table 1). The SEM micrograph shows that the seeds are broad ovoid in shape with irregular epidermal cell arrangement. Moreover, it can be seen that the anticlinal wall is irregularly thickened. The wall ornamentation is thick and raised with ruminate surface sculpturing. The seed hilum is visible, terminal, raised and has lateral compression, as shown in Figure 1.

The seeds had high oil content, i.e., 30% wt./wt. and FFA content with 0.63 mg KOH/g and were found to be a promising feedstock (96%) for biodiesel production. The phyto-pharmacological aspects of the seeds have been demonstrated in different studies that depict its antidiabetic [21], anti-inflammatory [22], antioxidant, analgesic, anti-adipogenic, anti-dyslipidemic, and antifungal activities due to its phyto-constituents [23]. A previous study by Ameen et al., 2018, showed that wild melon meets the international standards (ASTM) as a source of bioenergy and fuel properties [3].

3.2. Bischofia javanica

Bischofia javanica blume belongs to the Phyllanthaceae family and is widely distributed in Southeast Asia, India, Southern China, Malesia, Southern Japan, Taiwan, and Myanmar. Bischofia javanica is an evergreen tree with fissured, shallow, narrow bark, heartwood in the dark brown and reddish stem, and a straight cylindrical trunk. It grows fast, even up to 35 m in height, with smooth, dense, glabrous, round-head branches with milky sap. Leaves are shiny, bright green, membranous, palmate, 3 or 5 foliate, stipulate, petiolate, lanceolate, pulvinate, leaflets 7–15 × 4–8 cm, broad base obtuse to cuneate having lateral nerves (12–16), papery, glabrescent, obovate, elliptic, oblong, ovate. Its inflorescence is dioecious with separate female and male flowers. It grows paniculate, green, and raceme with many floral clusters, without petal, axillary, and greenish-yellow. The female flower has ovate-oblong sepals, and membranous margins, with a 15–27 cm long peduncle, having an exerted, glabrous, and smooth, tri-locular ovary with three entire, linear styles. The male flower (2.5 mm diameter) is also without petals having sepals that are semi-orbicular, membranous, outside pubescent abaxially, concave adaxially, and ladle shaped. It has five stamen, short filaments, is peltate, pistillode, with lengthwise dehiscing large anthers, 13 cm long, glabrous, pubescent, with a peduncle. Its fruit is a reddish brown, smooth berry (1.2–1.5 cm diameter), subglobose or globose with fleshy mesocarp and leathery to horny pericarp and shining surface, and 3–4 shiny non-edible seeds (4–5 mm in length and 2–3 mm in width) are embedded in colorless fruit pulp (edible) [24]. The seeds are light brown from the outside with brown to black on the inner surface. They are smooth, have two flat and one rounded sides, and are ovoid to oblong in shape, with approximately 60,000–100,000 seeds per kilogram, as depicted in Figure 2. SEM micrographs shows that surface sculpturing is verrucate with very thick and raised wall ornamentation. Cell arrangement is irregular with buttressed anticlinal wall pattern, and periclinal walls have thick and raised elevation. The hilum is not visible, sub-terminal, and depressed whereas a lateral compression is present. The seeds have high oil content, i.e., 29.7% wt./wt. Furthermore, the FFA content of 1.95 mg KOH/g makes them a promising potential feedstock (98%) for biodiesel production. The phytochemical and physicochemical properties of Bischofia javanica oil-yielding seeds included 5.93% moisture content, 6.83% ash content, 18.9% carbohydrate content, 18.6% protein content, and 5.32% fiber content. Total phenolics were 0.59%, total tannins 9.65%, and total alkaloids 0.22% [25].

Bischofia javanica is reported as an anti-inflammatory for ulcer and tonsillitis treatment in Asia, including the Philippines, China, and Indonesia [26]. It is also used to treat tuberculosis, sore throat, diarrhea, dislocation, and neuro-disorders. It is anti-diabetic due to phytochemical constituents such as gallic acid, beta-amyrine, betulinic acid, cynaroside, beta-sitosterol, friedelan-3-one, and quercetin, and has considerable therapeutic and nutraceutical properties [27].

3.3. Praecitrullus fistulosus

Praecitrullus fistulosus belongs to the Cucurbitaceae family, is commonly known as Indian squash, round melon, apple gourd, or squash melon, and is cultivated in two forms (light green and dark green) for its fruit to be used as a vegetable. It is grown in Asia, especially in the northwest of India and in Pakistan, Afghanistan, and Africa. It is also considered an export commodity in Kenya and Ghana (for the UK and US markets and cultivated at a small scale). The plant is a trailer or climbing herb that grows annually with a hispid to villous robust stem and slender stem. Leaves are entire with meticulously denticulate margins. It has veins and veinlets on the lower surface that are dense hispid and villous, lightly hispid lamina, hirsute petiole, and sparingly pinnatifid leaves. Praecitrullus fistulosus is a monoecious plant having yellowish, solitary, and smaller flowers. The female flower has a villous, hairy, and soft ovary with campanulate lobed calyx and lanceolate and is 5 mm long. The male flowers are pedicellate (1 mm in length) and have three stamens; two connate, and free obconic lobes; and a campanulate and hairy calyx. Flowering period is from March to September, and its fruit is spherical (6 cm diameter). The seeds are dark brown, whereas the inner surface is smooth, pale yellow, or whitish, oblong to ovate. It is 8–9 mm in length and 4–5 mm in width. SEM micrographs show that seeds have wrinkled wall sculpturing, whereas wall ornamentation is thick and raised with irregular epidermal cell arrangement and irregular thickened anticlinal wall pattern, whereas the periclinal walls have depressed elevation. The seed’s hilum does not have a visible terminal, while lateral compression is present (Figure 3).

The seeds have high oil content, i.e., 34% wt./wt. and FFA content with 2.01 mg KOH/g so have good potential as feedstock (91%) for biodiesel production. Our current results follow Tyagi et al., 2012, who reported a considerable percentage of fatty oil in seeds (52.8%) with the following composition: linoleic 50.8, oleic 21.2, stearic 10.7, palmitic 11.8, and myristic 1.74% with saponification and iodine values 192.5 and 126.5, respectively [28].

3.4. Luffa acutangula

Luffa acutangula is commonly known as angled loofah, ridged gourd, or Chinese okra. It belongs to the Cucurbitaceae family and is widely distributed in tropical South Asia, India, Pakistan, China, Russia, tropical Africa, the West Indies, Mexico, Oceania, and tropical America. The Luffa acutangula is an annual climber and creeper vine with a herbaceous, green, five-angled scabrous (along ribs) stem (grows up to 5–10 m), glabrous, having trifid axillary sub-scabrous, and 2–3 inch tendrils. The leaves are scabrous, pale green, palmatilobed (5–7 lobed that are more or less deep), glabrous, chartaceous, alternate, acuminate, or acute apex, 15–20 cm across the blade, have a hastate or cordiform base and denticulate or sinuate-dentate margins, and are petiolate (8–12 cm in length). Luffa acutangula is monoecious or unisexual, pedicellate, with pale yellow flowers, 4.5 cm across, and actinomorphic. Its calyx is slightly hairy with a pentagonal tube and slightly longer lobes compared to the tube. Staminate or male flowers are elongated, with racemes, deep lobes, pale yellow corolla, obtuse, 10–20 flowered, axillary, and erect, with three stamens with 4 mm long free filaments, villous, 10–15 cm in length. Petals are 2 cm long, 2.5 cm wide, obcordate, pale yellow, and may be emarginate. Pistillate or female flowers are found in the same axil as staminate flowers, have less than 1 cm hypanthium and are solitary, having globose stigma, a short style, tri-carpellate, 10-angled, inferior, and claviform, and the ovary has many horizontal ovules. The fruit is dehiscent from apical pores and has a crustose pericarp, 10-angled, longitudinal ribs, and acute. It is 15–30 cm long and 6–10 cm in diameter. The seeds of Luffa acutangula are soft, pale white, and lighter yellow when immature and dark brown or blackish and hard when mature. The inner seed surface is still yellowish, elliptic (1/16th of inch thick and ¼ to 3/8th of inch long, 1/8th to ¼ of inch wide), 11–12 mm in length and 6–8 mm in width, emarginated, smooth, and slightly rugose. The seeds are ovate to obovate and numerous with more than 30 seeds per mature fruit (Figure 4).

Our SEM micrographs show verrucate surface sculpturing and very thick and raised wall ornamentation. Cell arrangement is irregular with a buttressed anticlinal wall pattern, and periclinal walls have thick and raised elevations. Luffa acutangula seeds have a visible terminal with raised hilum, whereas dorsoventral compression is present. The seeds have high oil content, i.e., 48% wt./wt. and FFA content with 3.91 mg KOH/g, and hence, have good potential as feedstock (93%) for biodiesel production. Previous studies reported the pharmacological properties of Luffa acutangula, such as antioxidant, immunomodulatory, anti-cancer, anti-inflammatory, gastro-protective, and behavior-changing effects [29]. In general, the seed morphological patterns were supportive in distinguishing the different species of Cucurbitaceae and helpful in confirming tribes and subtribes classification. Based on these characteristics, Luffa was placed in a different tribe (Sicyoeae) [30].

3.5. Diospyros lotus

Diospyros lotus belongs to the Ebenaceae family, commonly known as date-plum, Caucasian persimmon, lilac persimmon, and Japanese persimmon and is distributed in Iran, Japan, India, Pakistan, Afghanistan, China, the Mediterranean region, Caucasia, the Balkans, and Anatolia. Diospyros lotus is 2–20 m tall with a glabrous or pubescent stem and petiolate (4–10 mm petiole length). Its leaves are elliptic, acuminate, lanceolate, with a rounded base, undulate at margins, and pubescent on lower or both sides. Diospyros lotus flowers are sessile and sometimes pedicellate with dense pubescent and short pedicel. Corolla are reflexed, 7 mm long, with yellowish-brown petals with rounded lobes and the inside is hairy or ciliated. The female flower is solitary and larger than the male. It has persistent calyx, a single row of eight staminodes, and 4–6 styles. The ovary is persistent and glabrous below and hairy at the apex. The male flowers have a 2.5 mm long calyx, cymes (2- or 3-flowered), two opposite rows of sixteen stamens, 3 mm long anthers, connective pilose, short filaments, 4–5 triangular lobes, pubescent, and subacute to the obtuse and rudimentary ovary. The fruit is a glaucous berry ovoid to globose when unripe and mealy white. When it ripens, it becomes dark purple to blackish and can grow up to 13–22 mm in diameter. The seeds of Diospyros lotus are dark brown on the outer surface and greyish on the inner side. It is semi-spheroid, 11–15 mm long, and 6–8 mm in width. The SEM micrographs show striate wall sculpturing, thin and raised wall ornamentation, undulate and rough anticlinal wall pattern, and asymmetric slightly depressed periclinal wall pattern. The hilum is visible and depressed and laterally compressed, as shown in Figure 5. The seeds have 20.3% oil content and a high FFA content of 6.91 mg KOH/g. However, it is worth noting that too high FFA makes the oil unfit for human consumption; therefore, it can be considered a non-edible seed oil with good potential (87%) for biodiesel production.

Our SEM results follow to evaluated the seeds’ surface via SEM images that revealed an uneven rough appearance with uniformly spread oil layers and interlacing in both non-extracted solid portion and oil. However, these layers appeared cracked on oil extraction, and the surface became deflated and highly porous, indicating oil depletion depicting higher extraction efficacy [31]. Lee et al. [32] quantitatively evaluated the nephroprotective, anti-hemolytic, and antioxidant activity of the seed extract of Diospyros lotus.

3.6. Solanum surattense

Solanum surattense belongs to the Solanaceae family, commonly known as yellow-fruit nightshade, Thai striped eggplant (from the unripe fruit), or Indian nightshade and is distributed as a weed or wild plant in South and South-East Asia, especially Pakistan, India, North Africa, Polynesia, and Australia. The plant is herbaceous, perennial, and prostrate with approximately 15 mm long prickles. The prickly herb diffuses with pubescent stems and branches that are stellate to glabrous. The leaves are dark green, elliptic to oblong, lobulated with unequal lobes, deeply lobed to sinuate, often toothed, acute or obtuse and are 30–80 × 25–50 mm in size. The flowers are purple, with peduncle cymes, a 10–20 mm long peduncle, 2–4 in number, corolla limb of 10–12 mm long, 2–2.8 cm broad, triangular to ovate and a stamen that has 7.5 mm long anthers. The calyx is also prickly and acute with 5 mm long lobes. The fruit is fleshy, 15–20 mm in diameter, and a globose berry. However, the seeds are orange to yellow on the outer surface and yellow on the inner side. They are 3 mm in length and 2 mm in width, smooth, obovate to discoid, and faintly reticulate.

The SEM micrographs depict the seeds’ ruminate surface sculpturing with thick and raised wall ornamentation, irregular and asymmetric cell arrangement, buttressed anticlinal wall, and rough and thickened periclinal walls. The seed’s hilum has a visible terminal and is depressed and laterally compressed, as shown in Figure 6. The seeds have considerable oil content, i.e., 21% wt./wt. and FFA content with 3.91 mg KOH/g. Hence, they have good potential to be used as feedstock (89%) for biodiesel production.

Solanum surattense has steroidal compounds such as diosgenin, solasonoine, campesterol, and solasonoine and alkaloids showing cardio-protective, anti-asthmatic, and hepato-protective properties [33,34]. In addition, Al-Snafi et al. [35] reviewed the presence of active phytochemicals in Solanum surattense, such as glycosides, ascorbic acid, steroids, flavonoids, phenols, gums, and sterols.

Figure 7 depicts the comparative analysis of seed-oil content and biodiesel potential of the six energy crops. Luffa acutangula (48%) possessed the highest oil content in its seeds, followed by Praecitrullus fistulosus (34%), Cucumis melo var. agrestis (30%), and Bischofia javanica (29.7%) which showed high potential for biofuel synthesis (>90%). Wang et al. [6] reported seed oil content in Luffa cylindrica of about 40%. However, Luffa acutangula seed oil content was found to be around 48% in our study. Hence, its oil-yielding seeds is more suitable for use as a novel non-edible feedstock for biodiesel production. Furthermore, Indra et al., 2013, reported high oil content (>20%) in Bischofia javanica. Therefore it can be regarded as a promising energy crop [36]. Finally, Cucumis melo var. agrestis contained a considerable amount of seed oil (30%), just like Luffa acutangula (48%) and Praecitrullus fistulosus (34%). Hence, it can be assessed that the three selected members of the Cucurbitaceae family possess seeds with high oil content and produce >90% biodiesel yield.

3.7. Multivariate Analysis

Variations among the six energy crops, which belong to four different families, were assessed by multivariate analysis for their oil content (%), FFA content (mg KOH/g), and biodiesel potential (%) using PCA. Additionally, key variables were carefully selected in the form of a biplot for both visual inspection of the data set and to increase the accuracy of the statistical categorization. The PCA factor loading is shown in

Table 3. PCA variables/factors loading for first three components.

Factors/Variable	PC1	PC2	PC3
Oil content (%)	0.97733	−0.21135	−0.012511
FFA content (mg KOH/g)	−0.079398	−0.42065	0.90374
Biodiesel potential (%)	0.19627	0.88226	0.42789
Eigen value	106.771	16.8027	1.49917
Variablity (%)	85.367	13.434	1.1986

.

Factor loadings are projections of dependent variables (green lines in Figure 8) on PC1 and PC2. PC1 and PC2 are the most significant variables as their eigenvalues appeared to be greater than 1, with 85.36% and 13.43% variance shown by PC1 and PC2 in total data variation, respectively. Cucumis melo var. agrestis and Bischofia javanica were positioned on the positive side of axis 1. Luffa acutangula and Praecitrulus fistulosus were located on the positive side of axis 2, whereas Solanum surattense and Diospyros lotus were found on negative side of axis 2, as shown in Figure 8. The PCA bi-plot (Figure 8) indicates the following aspects:

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Luffa acutangula had a higher seed oil content than Solanum surattense and Diospyros lotus species (discrimination on PC1);

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Cucumis melo and Bischofia javanica had a higher biodiesel potential and a lower FFA content than the other species (discrimination on PC2).

4. Conclusions

This study attempted to explore the detailed micro-morphological characteristics of the selected oil-bearing seeds from six energy crops, i.e., Cucumis melo var. agrestis Naudin, Bischofia javanica blume, Praecitrullus fistulosus (Stocks) Pangalo, Luffa acutangula (L.) Roxb, Diospyros lotus L., and Solanum surattense Burm. f. The seeds are similar in shape and color, and their recognition is challenging for indigenous people. Hence, LM and SEM could reveal topographic features that contribute to authenticating the identification of closely related counterparts. The ultra-structures of the selected seeds, such as seed ornamentation; seed sculpturing; hilum occurrence, position, and level; cell arrangement; and wall features (anticlinal and periclinal), exhibited distinct variations in understudy plant species. Therefore, SEM characteristics and light microscopy seem to be the most suitable, convenient, and versatile diagnostic techniques to identify oil seeds accurately and precisely. In addition, high oil-potential and ready accessibility are promising attributes that mark a seed as an exclusive candidate for biofuel synthesis to overcome power crises and ecological concerns. However, it is recommended to scrutinize the comprehensive history of such oil-bearing seeds by using modern practices such as scanning and transmission electron microscopy, owing to their significance in pharmaceutical, nutraceutical, and cosmeceutical industries and future energy crops. The physical properties of these oil-producing seeds should be determined to design a technology for enhancing oil-yielding capacity at a large scale. Furthermore, the improvement of oil-extraction techniques mean that data mining is a promising tools to analyze seed morphology and classification via bay networks and multinomial and logistic regression, and eventually configure peeling techniques for oil extraction to get a higher oil fraction that would efficiently run the biofuel industry.