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Research Article
2026
:38;
4262024
doi:
10.25259/JKSUS_426_2024

Evaluation of biodiesel production for some Penicillium sp. isolates from Aramco oil field, Saudi Arabia, Western region

aApplied College, Shaqra University, Shaqra, 15526, Saudi Arabia

*Corresponding author: E-mail address: fahadaldhbaan@su.edu.sa or fahad.aldhabaan@gmail.com (F Al-Dhabaan)

Received: , Accepted: ,
© 2026 Journal of King Saud University – Science - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

In this investigation, we isolated fifteen fungal isolates from three sites belonging to the Saudi Aramco Oil Company. The first site showed the highest number of isolates (9), compared with four and two isolates for the third and second sites, respectively. Three isolates (A4, A8, and B5) reflected a distinguishably superior biodiesel yield compared with other fungal isolates. Based on the 18S ribosomal RNA gene sequence, three isolates were identified as Penicillium commune (for A4 and A8 isolates) and Penicillium expansum (for isolate B5) and submitted to the gene bank with PP330078, PP330079, and PP330080 accession numbers, with 100% identity. Gas chromatography was used to compare the biodiesel profiles of the three isolates and showed that An A4 isolate is considered the best biodiesel production isolate as it contains an elevated percentage of mono- and polyunsaturated fats, primarily 15.4% O.C., 34.8% L.I., 1.1% α-linoleic acid, and 20.3% arachidonic acid, and a small proportion of saturated fats, mainly 22.7% palmitic acid and 3.4% stearic acid.

Keywords

Biodeseil production
Fungal isolates
Polymerase chain reaction (PCR)
Saudia Arabia

1. Introduction

The global biodiesel production is anticipated to be ∼41.4 billion liters by 2025, with a compound annual growth rate (CAGR) between 8 and 10% from 2022 to 2032 (Sales et al. 2022). Relaying the fact that biodiesel is considered an effective renewable for conventional diesel fuel (Mahlia et al., 2020), using vegetable oils was familiar for biofuel production (Abdulrazak & Ahmed, 2020). Moreover, it exposed Penicillium commune NRC 2016 to ethyl methane sulfonate (EMS)-induced mutants, which exhibited lipid yields of 1.71-2.55 g L⁻1 (Abdelhamid et al. 2024). Differentiable sources of energy for the buildup of lipids were used by oleaginous fungi, like glycerol (Papanikolaou & Aggelis, 2002), agricultural-industrial waste, and lignocellulose dry mass (Ekas et al., 2019; Campos et al., 2020; Marwa et al., 2020).

During fermentation, various microorganisms can be adversely affected in the presence of many inhibitors. The detoxification process can be removed from the hydrolysates, which causes the loss of time and cost (Yan et al., 2022).

These carbon sources include agro-industrial residues, lignocellulosic material, wastewater, monosugars, cereals, corncobs, sweet sorghum, and crude glycerol (Youssef et al., 2021; Pajares et al., 2024). Many optimized steps were performed to improve biodiesel production through fungi. Improving phase solubility and reducing mass transfer limitations were obtained via catalyst-free alcoholysis reactions at high pressure. Furthermore, higher processing rates facilitate the separation and purification of the products (Supang et al., 2024). Many oleaginous filamentous fungi distinguished with biodiesel production, such as Aspergillus terreus and A. niger, Fusarium oxysporum, and Mucor circinelloides, are capable of producing a significant amount of lipids. Identifying fungal species capable of producing a significant amount of lipids will be highly beneficial for enhancing process economics (Al-Zaban et al., 2022).

In this study, our goal was to isolate and identify fungal isolates with promising biodiesel production. Furthermore, biodiesel compounds for each promising isolate were profiled.

2. Materials and Methods

2.1 Samples of soil

Fifteen soil samples were isolated at locations from the Saudi Aramco Oil Company (Photograph 1, https://www.google.com/maps/place/Aramco). After storing samples in sterile polyethylene bags below −20°C, one gram of soil was liquefied to get soil suspensions. For isolating fungal isolates, 2 mL of gentamicin was added to potato dextrose agar (PDA) to culture diluted samples and incubated at 28°C for 2 to 3 days (Kumar et al., 2011). Then, PDA slants were applied for subculturing pure colonies, which were stored at 4°C for further analysis (Photograph 1).

 Location of Aramco oil field in Western Region Saudi Arabia (https://www.google.com/maps/place/Aramco).
Photograph 1.
 Location of Aramco oil field in Western Region Saudi Arabia (https://www.google.com/maps/place/Aramco).

2.2 Evaluating lipid production for fungal isolates

To estimate and evaluate superior lipid-producing fungal isolates, a basal medium was used for regular culturing. Also, 30% glycerol was applied to the assay consumption capability of low-carbon sources for the nominated isolates. After incubation, the liquid culture was centrifuged, and the collected growth was washed in triplicate with distilled water and used to evaluate lipid content. Then, biomass was extracted and frozen at -80°C. To evaluate lipid production for fungal isolates, direct transformation (Lewis et al., 2000) was used. 300 mg of dried biomass was sonicated for 7 min at 30 kHz after adding 1:1:10 of chloroform, hydrochloric acid, and methanol. After suspending and vortexing pre-sonicated cells, after 8 h of stirring, pre-sonicated cells were suspended at 90°C. Then, distilled water was used to dilute the mixture. Ethyl acetate was applied to extract samples. Finally, weighting was performed on the leftover fatty acid methyl esters (FAMEs).

2.3 Gas chromatography profile analysis

Agilent Technologies 6890 (Net Work GC System, USA) was used to estimate and evaluate the (FAMEs) production of fungal isolates and detect a record-composed profile. 

FAME analysis was carried out in the Central Laboratories at the National Research Centre (NRC). Rates of 2, 7, 4, and 20 min and a run time of 60.10 min were used. 250°C was applied as the injector temperature. Then, injector temperature was used at 280°C as the temperature for the flame ionization detector with a flow-specific rate (1.5 mL/min) by using mobile phase (nitrogen) at a flow rate of 30 mL/min.

2.4 Identification using the 18S rRNA marker

18S rRNA (5’-CCTGGT TGATCCTGCCAGTA-3’, 5’-GCTTGATCCTTCTGCA GGTT-3’) was used as a molecular marker to identify fungal isolates with high biodiesel production potentials. After detecting amplicons on agarose gel (White et al., 1990), eluted fragments were sequenced, aligned, analyzed, and identified via the Basic Local Alignment Search Tool. A phylogenetic tree was constructed by Clustal W through the neighbor-joining method.

2.5 Statistical analysis

One-way analysis of variance (ANOVA) was used for the collected data and followed by Tukey’s Pairwise Comparisons with p < 0.05 using the software Minitab Statistics version 18.

3. Results

3.1 Tracking and evaluating microbial isolates to produce lipids

Fifteen fungal isolates were isolated from three soil samples that were taken from three locations. The standard serial dilution technique was used for obtaining samples. Our findings reflected that the first site showed the highest isolates (9 isolates) compared with four and two isolates for the third and second sites, respectively (Fig. 1).

Number of isolates from three soil sample sites.
Fig. 1.
Number of isolates from three soil sample sites.

3.2 Biomass and biodiesel production

As shown in Table 1, isolates A4, A8, and B5 reflected a distinguishably superior biodiesel yield. Thus, all three isolates were nominated as promising biodiesel production isolates.

Table 1. Biodiesel (g/L) production and biodiesel for fungal isolates from three isolated locations.
Isolates Biomass (g/L) Biodiesel (g/L) Biodiesel (%)
First site A 0.812 0.321 7.277
A 1.747 0.098 8.288
A 0.458 0.261 13.282
A 0.637 0.312 44.298
A 4.377 0.177 3.287
A 1.738 0.166 11.381
A 0.977 0.028 11.280
A 1.226 0.266 17.287
A 2.777 0.250 6.280
Second site B 0.277 0.281 4.893
B 0.388 0.046 1.629
B 0.922 0.381 9.287
B 0.826 0.177 28.387
Third site C 2.096 0.026 3.992
C 1.367 0.732 5.782

3.3 Molecular identification of fungal isolates

Our three superior biodiesel production isolates were identified via sequencing 18S ribosomal RNA gene fragments via the GeneBank database. Selected amplicons (approximately 1500 bp) have been sequenced (Fig. 2), aligned (Fig. 3), and identified as Penicillium commune (for A4 and A8 isolates) and Penicillium expansum (for isolate B5) and submitted to the gene bank with PP330078, PP330079, and PP330080 accession numbers with 100% identity. Based on the 18S ribosomal RNA gene sequence, phylogenetic trees were constructed with the closest homology isolates from the database (Fig. 4).

18S ribosomal RNA (a) gene fragments, (b) detecting, and (c) fragment length for three nominated isolates, A4, A8, and B5, respectively.
Fig. 2.
18S ribosomal RNA (a) gene fragments, (b) detecting, and (c) fragment length for three nominated isolates, A4, A8, and B5, respectively.
Alignments result for three nominated isolates (a) A4, (b) A8, and (c) B5 respectively.
Fig. 3.
Alignments result for three nominated isolates (a) A4, (b) A8, and (c) B5 respectively.
Constructed phyllogenetic tree for (a) A4, (b) A8, and (c) B5 fungal isolates according to 18S ribosomal RNA gene sequences.
Fig. 4.
Constructed phyllogenetic tree for (a) A4, (b) A8, and (c) B5 fungal isolates according to 18S ribosomal RNA gene sequences.

3.4 Biodiesel profile by gas chromatography

Our obtained results reflected superior fungal isolates with promising fatty acid methyl ester profiles for superior isolates for each location (A4, A8, and B5). According to the data represented in Table 2 and Fig. 5, the A4 isolate is considered the best biodiesel production isolate as a result of highly mono- and polyunsaturated fatty acid compounds, such as 15.4% O.C., 34.8% L.I., 1.1% α-linoleic acid, and 20.3% arachidonic acid, containing only a small amount of saturated fat, mainly 22.7% palmitic acid and 3.4% stearic acid. Our findings agreed with those of Farias et al. (2018), who provided comparable data. Compared to plant oils, the total saturated FA content (60.86%) was higher than that of Palm oil (44%) and Jatropha oil (21.52%) (Vyas & Chhabra, 2017), indicative of high-quality biodiesel. Furthermore, the lipids produced by promising fungal isolates and their FAME profile displayed characteristics akin to those of biodiesel. (Gadallah & Abd-El-Haleem, 2014; Babakura et al., 2019). Moreover, in agreement with Çiçek & Yalçın’s (2013) results, long-chain FAs exist; it has been reported that the FAME profile shows both high fuel efficiency and improved biodiesel properties (Zheng et al., 2012; Çiçek & Yalç, 2013).

Table 2. Identification results for A4, A8, and B5 fungal isolates.
Accession number Identification isolates Identity %
PP330078 Penicillium commune isolate A4 99.36
PP330079 Penicillium commune isolate A8 99.25
PP330080 Penicillium expansum isolate B5 99.90
Biodiesel production profiles for three fungal isolates (A4, A8, and B5).
Fig. 5.
Biodiesel production profiles for three fungal isolates (A4, A8, and B5).

4. Discussion

According to our findings recorded in Table 1, three isolates were characterized as potential biodiesel production isolates. More support was added by Qiao et al. (2018). They indicated that M. circinelloides demonstrated the maximum lipid content and the highest yield in static conditions. Our findings were in agreement with Ali and El-Ghonemy (2014), who discovered that there was more lipid buildup in static settings than in shaking conditions. Furthermore, varied species of oleaginous fungi reflected the accumulation activity of substantial intracellular lipid content (Somasekhar et al., 2003).

Varied biodiesel production capability was noticed for three fungal isolates. A4 isolate was superior for biodiesel production due to highly mono- and polyunsaturated fatty acid compounds such as 15.4% O.C., 34.8% L.I., 1.1% α-linoleic acid, and 20.3% arachidonic acid, containing only a small amount of saturated fat, mainly 22.7% palmitic acid and 3.4% stearic acid. Our findings were in agreement with those of Farias et al. (2018), who provided comparable data. Compared to plant oils, the total saturated FA content (60.86%) was higher than that of Palm oil (44%) and Jatropha oil (21.52%) (Vyas & Chhabra, 2017), indicative of high-quality biodiesel. Furthermore, the lipids produced by promising fungal isolates and their FAME profile displayed characteristics akin to those of biodiesel. (Gadallah & Abd-El-Haleem, 2014; Babakura et al., 2019). Moreover, in agreement with Çiçek & Yalçın’s (2013) results, long-chain FAs exist; it has been reported that the FAME profile shows both high fuel efficiency and improved biodiesel properties (Zheng et al., 2012; Çiçek & Yalç, 2013) (Table 3).

Table 3. Biodiesel production profiles for three fungal isolates Penicillium commune, Penicillium commune and Penicillium expansum isolates.
Isolates Biodiesel fractions
αLinolenic Palmitoleic Palmitic Linoleic Oleic Arachidonic Stearic
A4 1.1929 0.0000 22.7991 34.8820 15.4113 20.3178 3.4018
A8 2.2899 0.0000 7.1998 13.1993 7.2919 11.9373 2.1993
B5 0.7784 0.0000 2.5673 8.3016 2.4799 9.8826 0.2928

5. Conclusions

Our findings could be summarized as follows: we identified three fungal isolates, Penicillium commune (for A4 and A8 isolates) and Penicillium expansum (for isolate B5), and submitted them to the gene bank with PP330078, PP330079, and PP330080 accession numbers, with 100% identity, that reflected superior biodiesel production. An A4 isolate is considered the best biodiesel production isolate as it contains an elevated percentage of mono- and polyunsaturated fats, primarily 15.4% O.C., 34.8% L.I., 1.1% α-linoleic acid, and 20.3% arachidonic acid, and a small proportion of saturated fats, mainly 22.7% palmitic acid and 3.4% stearic acid.

Acknowledgement

I’m extremely grateful to Dr. Ahmed I. Marzouk (executive manager of the Biovision Scientific Consultancy Center); it would not have been possible without the support and nurturing.

CRediT authorship contribution statement

Fahad A. Al-Dhabaan, Conceptualization, methodology, software, investigation, writing - original draft, writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of Artificial Intelligence (AI)-Assisted Technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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