Exploring the potential of biosurfactants produced by fungi found in soil contaminated with petrochemical wastes

Yehia A-G Mahmoud; Yasser H El-Halmouch; Ebtsam E Nasr; Yassin M Al-Sodany; Baher A El-Nogoumy; Sameh S Ali

doi:10.1038/s41598-024-75865-5

. 2024 Oct 28;14:25733. doi: 10.1038/s41598-024-75865-5

Exploring the potential of biosurfactants produced by fungi found in soil contaminated with petrochemical wastes

Yehia A-G Mahmoud ¹, Yasser H El-Halmouch ^2,^✉, Ebtsam E Nasr ², Yassin M Al-Sodany ², Baher A El-Nogoumy ², Sameh S Ali ^1,^✉

PMCID: PMC11519656 PMID: 39468200

Abstract

Biosurfactants are a diverse group of compounds derived from microorganisms, possessing various structures and applications. The current study was seeking to isolate and identify a new biosurfactant-producing fungus from soil contaminated with petrochemical waste. The bioprocess conditions were optimized to maximize biosurfactant production for Aspergillus carneus OQ152507 using a glucose peptone culture medium with a pH of 7.0 and a temperature of 35 °C. The carbon source was glucose (3%), and ammonium sulfate (0.25%) was utilized as the nitrogen source. For Aspergillus niger OQ195934, the optimized conditions involved a starch nitrate culture medium with a pH of 7.0 and a temperature of 30 °C. The carbon source used was sucrose (3.5%), and ammonium sulfate (0.25%) served as the nitrogen source. The phenol-H₂SO₄ and phosphate tests showed that the biosurfactants that were extracted did contain glycolipid and/or phospholipid molecules. They showed considerable antimicrobial activity against certain microbes. The obtained biosurfactants increased the solubility of tested polyaromatic hydrocarbons, including fluoranthene, pyrene, anthracene, and fluorine, and successfully removed the lubricating oil from contaminated soil and aqueous media surface tension reduction. Based on the obtained results, A. carneus and A. niger biosurfactants could be potential candidates for environmental oil remediation processes.

Keywords: Biosurfactant, Bioremediation, Hydrocarbon oils, Bioprocess, Petrochemical wastes, Environmental safety

Subject terms: Biotechnology, Microbiology, Environmental sciences

Introduction

Biosurfactants, as amphiphilic surface-active substances, specialize in accumulating at the interface of immiscible fluids to decrease surface and interfacial tensions, thereby augmenting the surface areas of insoluble substances for enhanced mobility. The amphiphilic structure of a surfactant generally comprises a hydrophobic moiety, such as a long-chain fatty acid, α-alkyl-β-hydroxy fatty acid, or hydroxyl fatty acid, in conjunction with a hydrophilic moiety that may include carbohydrates, peptides, amino acids, phosphate groups, carboxylic acids, alcohols, or other compounds¹. They are unique secondary metabolites synthesized non-ribosomally by specific bacteria and fungi². Bacteria such as Bacillus sp., Pseudomonas sp., and Acinetobacter sp. are the predominant producers of biosurfactants, alongside fungi including Trichoderma atroviride, Trichoderma citrinoviride, Trichoderma reesei, Trichoderma harzianum, Fusarium fujikuroi, Aspergillus niger, and Penicillium chrysogenum^3,4. The process of biosurfactants emulsifying hydrophobic substrates plays a crucial role in enhancing the availability of essential nutrients required for the development of organisms⁵.

Several recent studies have highlighted various strategies and advancements aimed at improving biosurfactant production. These strategies include isolating and identifying new efficient biosurfactant producers⁶; manipulating the genetic makeup of microorganisms⁷; optimizing nutrients for carbon sources, nitrogen sources, and other nutrients⁸; adjusting process parameters by optimizing conditions like pH, temperature, and agitation speed; and cost-effective methods for extracting and purifying biosurfactants⁸, and ensuring the sustainability of biosurfactant production⁹. Research is ongoing to tailor the properties of biosurfactants for various applications¹⁰. For biosurfactants to be useful in a wide range of biological situations, they need to have certain physical and chemical properties. These include the ability to lower surface tension, biodegrade oil, keep emulsions stable, recover oil efficiently, inhibit microbes, and dissolve in hydrocarbons. Biosurfactants are better than chemical surfactants in many ways, including being less harmful, breaking down more quickly, making more foam, being safe for the environment, and working well in tough conditions¹¹. They are a desired class of chemicals due to their diversity, which makes them useful for a variety of industrial and biotechnological applications, such as chemistry, pharmaceutics, cosmetics, and food production^12,13.

The objective of this study was to isolate, characterize, and identify biosurfactants produced by fungi found in soil contaminated with petrochemical wastes. It also aimed to optimize the productivity of these biosurfactants and evaluate their potential applications in areas such as oil recovery, microbial enhanced oil recovery, polyaromatic hydrocarbon solubility, and antibacterial activity. Additionally, the study aimed to provide a detailed description of the chemical structure of the identified biosurfactants. Hence, this study’s findings advance our understanding of biosurfactants, their potential applications, and provide insights into their production and chemical structure. The knowledge gained from this research has the potential to drive innovation and contribute to the development of sustainable and environmentally friendly technologies.

Material and method

Isolation of biosurfactant producer fungal strains

Fungi were isolated from crude oil-contaminated soil and water samples taken from Kafr El-Sheikh governorate, Egypt, and stored in sterile glass bottles and sterilized plastic bags in PDA as pure cultures. A serial dilution was prepared to 10^− 4 using one gram of a petrochemical soil sample in a sterile tube with nine milliliters of sterilized distilled water, followed by plating 100 µl of each dilution onto Sabouraud agar medium¹⁴. The colonies that developed were isolated in sterile Petri dishes using the identical culture following 72 h of incubation at 29 °C. The identical culture media was employed to preserve fungal colonies in slants, which were subsequently identified by examining their macroscopic and microscopic features¹⁵. In this study, a total of 89 morphological different fungal isolates were obtained.

Biosurfactant-producing fungi cultivation

The fungal isolates that were effectively carried out using potato dextrose broth culture medium supplemented with 10 ml/l sunflower seed oil were selected. The emulsification index and oil spreading test were used to evaluate the potential of biosurfactant-producing fungi. Using morphological and microscopical features, A. carneus and A. niger have been identified as the most promising fungi for biosurfactant production among all tested isolates. Then A. carneus and A. niger were cultured in Erlenmeyer flasks containing 100 ml of the liquid medium with 10 ml/l of sunflower oil. The culture medium underwent inoculation with spores of the isolates, reaching a final concentration of 1 × 10⁴ spores/ml. Subsequently, the flasks were incubated for 7 days at 29 ± 2 °C with vigorous shaking at 130 rpm. Post-incubation, the mixture was subjected to filtration using a quantitative 12.5 cm filter paper with a pore size of 28 m to eliminate fungal cells. The resulting filtrate was isolated for subsequent utilization in the evaluation of biosurfactants through oil spreading tests and determination of the emulsification index¹⁶.

Fungal identification and biosurfactant production optimization

The isolates were described by their morphological characteristics (color, colony diameter, and texture), microscopic characteristics (mycelium morphology, spore color and shape, phialide structure, and sterigmata form), and molecular analysis utilizing 28 S-rRNA. The molecular evolutionary genetics analysis version 7.0 software (MEGA 7.0) was employed to align sequences from NCBI GenBank data to ascertain the taxonomic position of strains and construct the phylogenetic tree. The nucleotide sequences of fungal strains were submitted to GenBank to acquire their entry numbers. On the other hand, various cultural conditions like culture media type, time course of fermentation, initial medium pH, and carbon and nitrogen sources were optimized by one factor at a time for the biosurfactant production by A. carneus and A. niger.

Emulsification index and oil spreading

To evaluate the emulsification index (E₂₄), a 4 ml aliquot of the cell-free culture filtrate was mixed with 6 ml of toluene in a test tube. The mixture was agitated strongly for 2 min utilizing a tube shaker vortex. The ratio of emulsified toluene was assessed relative to the total volume after 24 h. The emulsification index of a 1% (w/v) sodium dodecyl sulfate (SDS) solution was also assessed for comparative analysis. E₂₄ was calculated utilizing the formula¹⁷.

The oil spreading test was performed according to the procedures described previously^17,18, with minor modifications. Fifty milliliters of sterilized distilled water were initially dispensed into a 9 cm glass Petri dish. A thin layer of sunflower seed oil (2 ml) was thereafter uniformly placed on the water’s surface. Subsequently, 100 µl of the cell-free supernatant containing the biosurfactant was carefully introduced to the center of the Petri dish. The area of oil displacement was subsequently measured.

Biosurfactant extraction and characterization

For the extraction of biosurfactants, each flask was supplied with 100 ml of sterilized distilled water and subjected to agitation at 150 rpm for 1 h at 29 °C¹⁹. The suspension was centrifuged at 4 ºC at 5,000 rpm for 15 min. After centrifugation, the pellet underwent treatment with a 10 ml mixture of chloroform and methanol (2:1 volume ratio) and was then incubated at 30 °C in a shaking incubator at 200 rpm for 20 min. Following a 30min centrifugation at 5,000 rpm and 4 °C, the supernatant was let to evaporate through air drying²⁰.

To do the phenol-H₂SO₄ test, 1 ml of cell-free supernatant was mixed with 1 ml of 5% phenol. Then, 3–5 ml of concentrated H₂SO₄ was added dropwise until an orange color appeared, which showed that the glycolipids were present in the crude biosurfactant²¹. For the Biuret test, a volume of two ml of cell-free supernatant was heated to 70 ºC and mixed with 1 M NaOH solution, and then CuSO₄ solution was added to it drop-wise. Formation of a violet or pink color ring indicates the presence of lipopeptides²². Additionally, a phosphate test was carried out by mixing two ml of cell-free supernatant with 6–10 drops of 5 M HNO₃ and heated to 70 °C, then 5% ammonium molybdate was added dropwise until the appearance of a yellow-colored precipitate, which indicates the phospholipid incidence²³.

The biosurfactant composition was analyzed by comparing it to a methylated fatty acid ester mix standard using gas chromatography-mass spectrometry (GC-MS) with a QP2010 Ultra model detector. Each component was individually recognized according to its relative retention indices and cross-referenced with the Wiley Registry of Mass Spectral Data²⁴. The biosurfactant was also characterized by FTIR analysis²⁵. The column-purified biosurfactant was examined with a TENSOZ 27 (series no. 2887), covering the range of 500 to 4000 cm^− 1, to identify the functional groups and bond types²⁶.

Polyaromatic hydrocarbons solubilization

A polyaromatic hydrocarbons (PAHs) solubilization assay was performed following the previously described methods^27,28. Glass test tubes containing stock solutions of PAHs (anthracene, fluoranthene, fluorine, and pyrene) were filled with 60 µg of PAH each, and then the tubes were maintained open on a chemical fume hood to eliminate the solvent. The biosurfactant being tested was combined with 3.0 ml of assay buffer (comprising 20 mM Tris-HCl, pH 7.0) in concentrations ranging from 0 to 50 mg/ml. Assay buffer with solitary biosurfactant was utilized as a control. The tubes were sealed and left in a dark, 30 °C incubator with 200 rpm shaking. Filters measuring 1.2 μm were used to filter the samples (Whatman, No. 1). This filtrate was extracted in 2.0 ml using an equivalent volume of hexane. The aqueous solution and hexane phases of this emulsion were separated by centrifuging it for 10 min at 8,500 rpm. PAH concentration was measured spectrophotometrically at specific wavelengths of each compound: 350, 360, 239, and 263 nm wavelengths for anthracene, fluoranthene, pyrene, and fluorine, respectively. Hexane was used to extract the assay buffer, which included biosurfactant but no PAH, serving as a blank²⁸.

Applications of fungal biosurfactant

Antimicrobial activity

The antimicrobial activity of the biosurfactant extracted from fungi was achieved by using the agar well diffusion method^29,30. Previously extracted compounds were examined against various actively growing standard target reference strains: Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, Escherichia coli ATCC8739, Salmonella typhi ATCC6539, K. pneumoniae ATCC13883, Candida albicans ATCC10221, Candida tropicalis ATCC 750, and Geotrichum candidum. The crude biosurfactant was dissolved in distilled sterilized water to give 30 mg/ml in concentration. To generate a microbial lawn, a volume of 100 µl overnight culture of the test microbial isolates grown in Luria-Bertani broth was plated onto the previously described solid media. Then, 50 µl of the biosurfactant (30 mg/ml) was pipetted directly into the well. The antimicrobial tests were carried out with a positive control of two different antibiotics. Positive control was gentamicin (30 mg/ml) for bacteria and fluconazole (30 mg/ml) for fungi. All microbial strain tests were carried out in triplicate. All the plates were then incubated at 37 °C for 24–48 h. The diameter of the inhibition zone around the inoculated well was measured in mm³⁰.

A half-maximal inhibitory concentration (IC₅₀) test has been implemented. To complete 100 ml of nutrient broth in 250 ml Erlenmeyer flasks, we added varying concentrations of A. carneus or A. niger crude biosurfactants (30 mg/ml). The flasks were incubated at 35 °C for 48 h after being inoculated with 10 µl (3 × 10⁵ CFU/ml) of B. subtilis ATCC 6633. The bacterial pellets were extracted by centrifugation at 5000 rpm and dried at 80 °C for 2 h, following the measurement of the optical density at 600 nm.

Estimation of critical micelle concentration

The biosurfactants from A. carneus and A. niger cultures that were growing in the best conditions possible before show great ability to lower surface tension. A Du Nouy ring type tensiometer was used to measure the surface tension of diluted biosurfactants in various doses (2–120 mg/l)³¹. Pure water surface tension was measured as a control.

Elimination of lubricating oil from contaminated soils

The capacity of biosurfactants to extract oil from soil particles was assessed using 800 g of soil (1–2 mm in diameter) that had been acid-washed and combined with 50 ml of used lubricating oil. Samples comprising 20 g of contaminated soil were placed into 250 ml flasks and underwent the following treatments: 60 ml of distilled water (control), 60 ml of an aqueous solution of the biosurfactant Tween 80, and SDS. The studies were performed at the critical micelle concentration (CMC) of each crude biosurfactant, as well as at the CMC of each chemical, determined to be 18 mg/ml for SDS and 2 mg/ml for Tween 80, respectively. The samples were centrifuged for 20 min at 5,000 rpm to separate the dirt particles from the washing solution, after a 24-h incubation at 30 °C on a rotary shaker set to 200 rpm. Gravimetric analysis was employed to determine the quantity of material extracted from the soil using hexane. This enabled us to quantify the oil content in the soil particles subsequent to exposure to the biosurfactant³². The experiment was performed at different temperatures to assess the effect of temperature on oil recovery enhanced by biosurfactants.

Statistical analysis

SPSS software (version 16 for Windows) was used for all statistical analyses. The obtained date was analyzed using one way ANOVA test. Finally, the means were compared using the Student–Newman–Keuls multiple range test at p ≤ 0.05.

Results and discussion

Screening for best biosurfactant producer fungal strains

Microorganisms synthesize a class of active compounds, which exhibit structural diversity^33–35. Various industries, such as oil recovery, pollution bioremediation, healthcare, and food processing, have delineated their applications and prospective commercial uses¹. Microbial surfactants demonstrate significant specificity, as evidenced by numerous studies on biosurfactant applications across various industries in the past decade, particularly in environmental remediation, including the extraction of heavy metals and organic compounds, as well as the bioremediation of hydrophobic substances^1,4.

The results of this study revealed the existence of many physiologically active fungus sources in oil-contaminated soil samples. A total of eighty-nine morphologically different fungi were recovered from crude oil-contaminated soil and water samples in Kafr El-Sheikh governorate, Egypt, using PDA as a pure culture medium. The microbial isolation was successfully conducted utilizing potato dextrose broth culture medium augmented with 10 ml/l of sunflower seed oil. The emulsification index and oil spreading assay were employed to assess the capability of fungi to produce biosurfactants. Three species of Aspergillus had the highest efficacy in biosurfactant synthesis. The most promising fungi for biosurfactant synthesis were identified as A. carneus and A. niger. Aspergillus is among the most prevalent hydrocarbon degraders within fungal genera^36,37. Aspergillus spp. has been widely employed to synthesize biosurfactants from several synthetic and biobased substrates¹⁷. The production of biosurfactants from the sea sponge A. ustus has been also reported³⁸.

As for the phenotypic traits of the most potent isolates, A. carneus colonies are white at first but turn buff (brownish) over time, have a diameter of 1.2 cm, and have droplets that range in color from pale yellow to pale brown that come out of the cells. Microscopically, the conidial head is composed of large globose biserrate, brown conidia, thick-walled hyphae, and a smooth, brownish conidiophore that carries a globose vesicle. A. niger undergoes a notable transformation during its growth cycle. Initially, it appears as white colonies, but over time, it transitions into a distinct black color due to the production of conidial spores. Microscopic examination of A. niger reveals characteristic features that aid in its identification. A. niger conidia are smooth in texture and exhibit a range of colors. These conidia have radial heads and align in two rows, forming a biseriate pattern. This arrangement is a distinguishing feature of A. niger under microscopic observation. The A. niger conidiophores are also hyaline, which means they are clear or glass-like, and septate, which means they have cross-walls between the hyphae. The 28 S-rRNA genes of the selected fungi, A. carneus and A. niger, have been identified and entered into GenBank with accession numbers OQ152507 and OQ195934, respectively. After multiple sequence alignment between the obtained sequences, the result shown in Fig. 1 indicated that the tested sequences had 100% similarity PCR sequences with their respective species, A. carneus and A. niger.

Fig. 1 — Phylogenetic analysis of the selected fungal isolates based on the results of PCR amplification of the 28 S-rRNA gene.

Optimization of biosurfactant production

Effect of culture media

Important characteristics of the culture medium should include strong selectivity towards the intended products over the unwanted ones. The availability of nutrient supplies for microorganisms and other environmental conditions has a significant impact on the production of biosurfactant³⁹. The required data has shown that A. carneus OQ152507 and A. niger OQ195934 have a potential ability for bio-surfactant production as analyzed using common screening tests, such as E₂₄ percentage, oil spreading, and surface tension tests^17,40. The cultivation of A. carneus and A. niger revealed that these isolates possess the capability to efficiently produce biosurfactants under submerged culture conditions. The type of culture medium used significantly influenced both the emulsification index and the oil spreading test of A. carneus and A. niger biosurfactants (Table 1). A. niger surfactant, using starch nitrate medium, obtained the highest value (62.7%) for the emulsification index. A. carneus favored the glucose peptone medium for biosurfactant production, achieving an emulsification index of 57.27%. The oil spreading test conferred that the starch nitrate and glucose peptone media were the best for A. carneus and A. niger, respectively, to produce their biosurfactant. A. carneus showed the highest oil displacement (3.47 cm) when using the glucose peptone medium. A. niger biosurfactant’s oil displacement varied insignificantly, maintaining a mean of 3.30 cm. Candida lipolytica UCP0988 was capable of producing glycolipids in a medium of 5% animal fat and 2.5% corn steep liquor⁴¹. This resulted in the most significant reduction in surface tension, decreasing from 50 mN/m to 28 mN/m. Patil et al.⁴² employed 2% fresh coconut oil in an optimized mineral salt medium to synthesize biosurfactants from Pseudomonas aeruginosa F23, achieving a decrease in the surface tension of the culture medium from 45 mN/m to 31 mN/m. Substantial biosurfactant synthesis has been demonstrated by Trichoderma citrinoviride C1, HL, and B3 when cultured in Saunders medium and mineral-glucose-peptone medium⁴. They additionally demonstrated that a Saunders medium enriched with yeast biomass was more efficacious.

Table 1.

Effect of different culture media on the fungal biosurfactant production.

Culture media	Emulsification index (%)		Oil spreading test (cm)
Culture media	A. carneus	A. niger	A. carneus	A. niger
Glucose liquid	44.79^b	32.50^d	3.20^b	2.10^c
Glucose peptone broth	57.27^a	50.40^b	3.47^a	2.50^b
Yeast-malt extract broth	40.19^c	45.53^c	2.00^d	2.13^c
Bennett´s	41.26^c	52.26^b	2.00^d	3.30^a
Starch nitrate	42.11^c	62.7^a	2.53^c	3.30^a
Potato dextrose	42.17^c	31.2^d	2.00^d	1.92^c

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Means with different letters in the same column differ significantly at p ≤0.05.

Effect of hydrocarbon oil

To study the effect of different hydrocarbon oils on the biosurfactant activity, the identified isolates, A. carneus and A. niger, were cultivated separately in an optimal free carbon source media (glucose peptone and starch nitrate) supplemented with 10 ml/l of certain hydrocarbon oils. All of the tested fungi’s supernatant showed emulsification activity, with an emulsification index ranging from 22.22 to 65.42% and an oil displacement ranging from 0.6 to 4.27 cm (Table 2). The emulsification index for each fungus varied depending on the hydrocarbon oil used. Kerosene had the highest emulsification index of A. carneus(65.42%), followed by petrol 80 (62.33%) for A. niger. According to the oil spreading test, kerosene was the most favorable substrate for A. carneus biosurfactant activity (4.27 cm), while A. niger had the highest oil spreading (4.13 cm) when using petrol 80 as a substrate. In conclusion, the presence of kerosene in the culture medium induced the highest emulsification index of A. carneus surfactant, followed by petrol 80 for A. niger surfactant. The addition of plant oils such as soybean, peanut, and olive oil to the culture medium led to the highest biosurfactant yields from Pseudomonas⁴³. Pseudomonas aeruginosa achieved an emulsification index ranging from 55 to 60% against diesel when sunflower oil served as the carbon source⁴⁴. Also, B. subtilis strain Al-Dhabi-130 made a crude biosurfactant that could remove 89% of the crude oil (which contained kerosene) from soil samples⁴⁵. Wu et al.⁴⁶ conducted a study that corroborated these findings, demonstrating that the B. subtilis SL biosurfactant exhibited a notable emulsification index of 67% with kerosene.

Table 2.

Effect of different hydrocarbon oils on the fungal biosurfactant production.

Hydrocarbon oils	Emulsification index (%)		Oil spreading test (cm)
Hydrocarbon oils	A. carneus	A. niger	A. carneus	A. niger
Sun flower seed	53.33^c	42.33^c	2.00^d	2.60^d
Paraffin	55.67^bc	31.67^d	2.60^c	1.50^e
Olive	29.18^d	45.45^c	1.93^d	3.00^c
Diesel	59.06^b	28.00^d	3.33^b	1.00^f
Petrol 80	24.36^d	62.33^a	1.50^e	4.13^a
Kerosene	65.42^a	57.33^b	4.27^a	3.70^b
Soy bean	24.33^d	22.22^e	1.00^f	0.60^g

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Means with different letters in the same column differ significantly at p ≤0.05.

Effect of incubation period

It became clear that as the incubation period increases, the biosurfactant potential expressed by emulsification and oil spreading increases to some extent, reaching the optimum period. Any increase in time leads to a reduction in activity. The highest emulsification index was obtained at 10 and 9 days of incubation for A. carneus and A. niger, respectively. They were 69, 74.70, and 68.33%, respectively (Fig. 2a). The oil displacement of the tested fungi had relatively the same pattern as the emulsification index, with the maximum value at 10 and 9 days for A. carneus and A. niger, respectively (Fig. 2b). In terms of microbial growth, the optimum dry weight was 3.57 g for A. carneus at the 10th day and 5.77 g for A. niger at the 9th day (Fig. 2c). Based on an increase in E₂₄ and oil spreading of the culture filtrate, our data showed that the fungal strains began to excrete their biosurfactant following the lag phase. While the maximum biosurfactant activity was observed in the stationary phase from 9 to 11 days for A. carneus and from 8 to 11 days for A. niger. A lot of research has shown that some types of fungi, like A. flavus⁴⁷, A. ustus³⁸, A. versicolor⁴⁸, A. niger⁴⁷, and Piper hispidum⁴⁹, produce biosurfactants during both their stationary phase and logarithmic phase. More precisely, Penicillium sp. 8CC2 reached its maximum biosurfactant production on the ninth day of development⁵⁰. Similarly, the production of biosurfactant by F. fujikuroi UFSM-BAS-01, Virgibacillus salarius and Bacillus licheniformis has been documented^51–53. Several researchers have also reported high biosurfactant production by yeasts during their stationary phase⁵⁴. The production of Bacillus subtilis ANR 88 biosurfactant did not appear to be growth-associated¹².

Fig. 2 — Effect of incubation period on emulsification index (A), oil spreading (B), and fungal biomass (C) of *A. carneus* and *A. niger* in submerged culture filtrate.

Effect of temperature and initial pH

Current data showed that the emulsification index, oil spreading test, and biomass of the tested fungi varied significantly with the temperature. Growing A. carneus and A. niger at 35 °C resulted in the highest emulsification index (E₂₄ = 77.78% for A. carneus and 71.78% for A. niger). Additionally, the spread of oil varied depending on the temperature. Both A. carneus and A. niger yielded the highest value at 35 °C (Table 3). At 40 °C and 35 °C, A. carneus and A. niger, respectively, displayed the highest biomass. In conclusion, it was observed that the growth at 35 °C and 30 °C was favorable for A. carneus and A. niger to give the maximal productivity of biosurfactant and biomass. Several studies have demonstrated the positive influence of temperatures on the production of biosurfactants by bacteria and fungi⁴⁰. For example, trehalose lipids with a lower surface tension (20.08 mN/m) are produced by Fusarium fujikuroi at 47 °C⁵⁵ However, psychotolerant yeasts produce biosurfactants at 20 °C⁵⁶. At submerged fermentation temperatures of 35 °C, the results show that the biosurfactant with the highest E₂₄ and oil spreading was made. Aspergillus strains produced the maximum biosurfactant at 35 °C¹⁷. High moisture and moderate temperatures favor fungal growth. For example, field studies have demonstrated that fungi grow best in environments with high moisture and moderate temperatures. Temperature directly affects fungal growth and protein dynamics, which ultimately impact crop performance⁵⁷. The optimum temperature for fungal growth varies among species, with some species growing best at temperatures around 25 °C, while others may require conditions up to 35 °C⁵⁸. Other fungal species report that the ideal temperature for biosurfactant production is 30 °C⁵⁹. Therefore, temperature plays a crucial role in both biosurfactant activity and fungal growth. Specific temperatures generally favor biosurfactant production, while moisture and temperature influence fungal growth, with moderate temperatures typically being the most favorable.

Table 3.

Effect of temperature on biosurfactant activity and growth of A. carneus and A. niger.

Temperature (°C)	Emulsification index (%)		Oil spreading test (cm)		Dry weight (gm/l)
Temperature (°C)	A. carneus	A. niger	A. carneus	A. niger	A. carneus	A. niger
15	0.00^g	16.67^f	0.00^e	0.00^e	0.09^e	1.90^e
20	16.33^f	23.23^e	1.00^d	0.43^d	0.53^d	2.03^e
25	32.78^e	42.22^c	0.20^e	0.76^d	1.27^cd	2.99^c
30	50.00^c	61.44^b	1.33^c	3.80^b	2.06^b	4.09^b
35	77.78^a	71.78^a	5.40^a	4.33^a	4.48^a	5.21^a
40	62.89^b	38.18^d	2.83^b	1.67^c	4.96^a	2.29^de
45	37.89^d	21.42^e	1.00^d	0.50^d	1.77^b	2.5^cd

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Means with different letters in the same column differ significantly at p ≤0.05.

In submerged culture media, the initial pH values showed a significant effect on the biosurfactant activity and biomass of A. carneus and A. niger. All the variables (emulsification index, oil spreading test, and biomass) varied significantly according to the pH values (Table 4). A. carneus and A. niger recorded the highest and best emulsification activity at pH 7. It was 61.11% and 69.0% for A. carneus and A. niger, respectively. The results indicated that the optimum pH was 7 for the emulsification activity, oil spreading, and biomass productivity for A. carneus and A.niger(Table 4). The obtained data aligned with the findings of Alyousif et al.⁶⁰, who observed that Pseudomonas sp. and B.subtilis produced their maximum biosurfactant at pH 7. Changing in the pH to acidity or alkaline regions decreased the biosurfactant activity of A. carneus and A. nige. These results almost align with previous findings by Saimmai et al.⁶¹, which confirmed a decrease in the emulsification activity of Oleomonas sagaranensis AT18 surfactant with increasing acidity. Fouda et al.⁶² reported that raising the pH from 8 to 10 resulted in lower biosurfactant productivity in P. aeruginosa and B. cereus.

Table 4.

Effect of initial pH on biosurfactant activity and growth of A. carneus and A. niger.

pH	Emulsification index (%)		Oil spreading test (cm)		Dry weight (gm/l)
pH	A. carneus	A. niger	A. carneus	A. niger	A. carneus	A. niger
4	35.85^c	30.00^e	1.30^c	0.50^e	4.24^bc	1.81^d
5	37.28^c	36.33^d	1.50^c	0.70^e	4.15^bc	2.82^c
6	54.20^b	64.12^b	1.83^b	4.57^b	4.96^b	7.54^a
7	61.11^a	69.00^a	4.47^a	5.37^a	6.52^a	8.08^a
8	53.00^b	45.33^c	1.80^b	3.13^c	3.36^cd	7.64^a
9	28.67^d	43.52^c	0.50^d	1.83^d	1.93^d	5.62^b

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Means with different letters in the same column differ significantly at p ≤0.05.

Effect of carbon and nitrogen sources

The carbon consumption rate of microorganisms during cultivation often regulates growth and secondary biochemical production, including biosurfactants⁶³. Microorganisms can produce biosurfactants in aqueous media by adding carbon sources such as glucose, fructose, glycerol, mannitol, different oils, and feedstock wastes. Different renewable feedstocks as substrates in fermentation bioprocesses have been studied. Most of the carbon used to make biosurfactants comes from carbohydrates, such as simple sugar, starch, and plant sugar-based carbohydrates^64,65. Various biosurfactant producers can benefit from using pure carbohydrates as carbon sources, such as glucose, sucrose, and glycerol⁶⁶. Data shown in Fig. 3 indicated that the organic carbon sources were much better than inorganic ones. To make the most biosurfactant activity (E₂₄ = 64.39%; Fig. 3a), oil displacement (4.60 cm; Fig. 3b); and biomass production (4.95 g/ml; Fig. 3c), A. carneus used glucose optimally. P. aeruginosa MTCC 7815 can use glucose better than other carbon sources to make an emulsification index of 76.77% and a surface tension of 34.53 mN/m⁶⁵. These results support their findings. On the other hand, sucrose was the best carbon source for A. niger growth and biosurfactant productivity, with an emulsification index of 73.33% (Fig. 3a), oil spreading of 5.5 cm (Fig. 3b), and biomass of 6.70 g/ml (Fig. 3c). In the same context, P. aeruginosa UMTKB-5 produced high amounts of rhamnolipid (above 250 mg/l) when grown in either glucose or sucrose medium⁶⁷.

Fig. 3 — Effect of different carbon sources on emulsification index (A), oil spreading (B), and fungal biomass (C) of *A. carneus* and *A. nige*r in submerged culture filtrate.

Several inorganic and organic nitrogen sources can influence biosurfactant production. Commonly utilized inorganic sources include nitrate and ammonium salts, while organic sources include corn-steep liquor, yeast extract, and urea¹³. In this study, eight nitrogen compounds were used to study their effect on biosurfactant activity and biomass production of the tested fungi (Fig. 4). Ammonium sulfate and potassium nitrate were the best nitrogen sources for the emulsification index of A. carneus and A. niger, respectively (Fig. 4a). For A. carneus and A. niger, it was 75.54 and 64.00%, respectively. These results are similar to those found by Adamczak and Bednarski⁶⁸, who found that ammonium salts are the best nitrogen sources for Candida antartica to make biosurfactants. Additionally, Banat et al.⁶⁹ demonstrated that ammonium sulfate was similarly the most effective nitrogen source for biosurfactant production from Serratia marcescens. However, potassium nitrate yielded the highest biosurfactant activity for A. niger. Alyousif et al.⁶⁰ identified NaNO₃ as the ideal nitrogen source for P. aeruginosa to produce biosurfactants. Some B. subtilis strains were unable to produce biosurfactants using (NH₄)₂SO₄ or KNO₃, but they could use NaNO₃, NH₄NO₃, or KNO₃^70,71. Ma et al.⁷² validated that inorganic nitrogen sources were more suitable for sophorolipid production than organic sources. Peptone also reduced the emulsification index of Aspergillus favus biosurfactants⁷³. The optimized A. carneus OQ152507 and A. niger OQ195934 biosurfactants resulted in a significant decrease in water surface tension. These results were in accordance with the data obtained from biosurfactant of Fusarium fujikuroi, Leucobacter komagatae, and mutated strain of B. subtilis^17,28. While, among the three nitrogen sources, Lysinibacillus fusiformis biosurfactants had the highest emulsification index (78.31 ± 0.87%) using urea followed by yeast extract⁵⁸.

Fig. 4 — Effect of different nitrogen sources on emulsification index (A), oil spreading (B), and fungal biomass (C) of *A. carneus* and *A. niger* in submerged culture filtrate.

Effect of sugar, nitrogen, and critical micelle concentrations

Glucose was the best carbon source for A. carneus, while sucrose was favored by A. niger to produce the highest biosurfactant activity and the fungal biomass. A. carneus recorded the highest emulsification index (61.07%) with a concentration of thirty grams of glucose, while A. niger’s supernatant demonstrated the highest emulsification index (64.33%) with a concentration of 30 g/l of sucrose. Concerning the oil spreading test, 30 and 35 g/l of the preferred sugar were the best carbon source concentrations, giving the highest oil displacement: 3.5 and 4.73 cm for A. carneus and A. niger, respectively. The highest biomass for A. carneus and A. niger was obtained using 35 g/l of the favorable carbon source for each microbial isolate (Table 5). For biosurfactant production, several microorganisms evaluated various raw materials containing different concentrations of carbon sources. For example, a medium containing pure cashew apple juice supplemented with 10 g/l glucose and 8.7 g/l fructose resulted in a twofold increase in B. subtilis LAMI005 biosurfactant production compared to a mineral medium containing sugar at a concentration of 12.05 g/l⁷⁴. Roberta et al.⁷⁵ achieved the best biosurfactant production of Coprinus sp. FS-4.1 using potato dextrose broth containing both 1% glucose and 2% glycerol. Using glucose (10%) as the sole carbon source for Rhodotorula babjevae YS3 growth increased the production of biocurfactants. This decreased the surface tension from 70 to 32.6 mN/m⁵⁰. Adding glucose (2%) to the paneer whey medium increased the yield of pseudomonas aeruginosa SR17 rhamnolipids to 4.8 g/l⁷⁶.

Table 5.

Effect of different sugar concentrations on fungal biosurfactant activity and growth.

Sugar conc. (gm/l)	Emulsification index (%)		Oil spreading test (cm)		Dry weight (gm/l)
Sugar conc. (gm/l)	A. carneus	A. niger	A. carneus	A. niger	A. carneus	A. niger
5	14.45^e	12.61^d	0.63^e	0.13^e	1.43^e	1.87^c
10	18.36^d	13.84^d	0.7^e	1.20^d	2.93^d	2.30^bc
15	33.33^c	34.89^c	1.53^d	2.53^c	3.00^cd	2.93^b
20	57.90^a	58.82^b	3.07^c	3.20^bc	3.93^bc	4.60^a
25	59.63^a	60.23^b	3.17^bc	3.40^b	4.37^ab	4.47^a
30	61.07^a	64.49^a	3.50^a	4.13^a	4.73^ab	4.83^a
35	40.97^b	67.73^a	1.37^d	4.73^a	5.00^a	5.10^a
40	31.87^c	38.70^c	0.47^e	1.77^d	3.30^cd	3.07^b

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Means with different letters in the same column differ significantly at p ≤0.05.

The activity of A. carneus and A. niger to produce their surfactant depending upon the different nitrogen source concentrations was evaluated. The emulsification index, oil spreading, and fungal biomass of the two fungi were used to express the biosurfactant activity, revealing significant variations between nitrogen concentrations ranging between 1 and 4 g/l. In this experiment, A. carneus preferred ammonium sulfate as a nitrogen source, while A. niger preferred potassium nitrate. A dramatic increase was observed in emulsification index and oil spreading with increasing the nitrogen concentration, with a peak at a concentration of 2.5 g/l for A. carneus and A. niger. The highest biomass was obtained for A. carneus and A. niger at 2.5 g of the favorable nitrogen source for each microbial fungus (Table 6). Ustilago maydis produced different concentrations of cellobiose and mannosylerythritol lipids under nitrogen starvation⁷⁷. The biosurfactants from A. carneus and A. niger cultures that were growing in the best conditions possible before have great properties for lowering surface tension. The water tension surface (71.57 mN/m) decreased to 33.3 and 30.86 mN/m, respectively, when 120 mg/l of A. carneus and A. niger biosurfactant were used (Table 7). This indicates that the crude biosurfactant of A. carneus and A. niger reduced the surface tension by 53.5 and 56.9%, respectively. The critical micelle concentration of biosurfactant produced from A. carneus was 30 mg/l, which decreased the water surface tension to 36.7 mN/m, whereas in the case of A. niger, it was 40 mg/l, which decreased the water surface tension to 32.73 mN/m. On the other hand, Bacillus megaterium pL6 had a CMC of 100 mg/l⁷⁸, while Agrobacterium fabrum SLAJ731 had a CMC of 650 mg/l⁷⁹.

Table 6.

Effect of different nitrogen concentrations on fungal biosurfactant activity and growth.

Nitrogen source concentration (gm/l)	Emulsification index (%)		Oil spreading test (cm)		Dry weight (gm/l)
Nitrogen source concentration (gm/l)	A. carneus	A. niger	A. carneus	A. niger	A. carneus	A. niger
1	19.02^e	26.15^e	0.40^e	0.50^f	1.23^d	2.20^d
1.5	43.07^c	34.92^d	1.27^cd	1.20^e	2.63^bc	3.00^c
2	49.82^b	40.23^c	2.17^b	1.40^de	3.13^ab	3.10^c
2.5	58.70^a	58.95^a	3.33^a	3.37^a	3.67^a	5.10^a
3	49.30^b	54.21^ab	2.07^b	2.60^bc	2.93^b	4.13^b
3.5	41.33^c	55.44^ab	1.43^c	2.70^b	2.17^c	3.13^c
4	31.45^d	52.58^b	0.93^d	1.97^cd	1.97^c	2.70^cd

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Means with different letters in the same column differ significantly at p ≤0.05.

Table 7.

Critical micelle concentration of A. carneus and A. niger biosurfactant.

Biosurfactant concentration (mg/l)	Surface tension (mN/m)
Biosurfactant concentration (mg/l)	A. carneus	A. niger
0	71.57^a	71.57^a
2	62.81^b	50.02^b
4	58.28^c	46.25^c
6	55.34^d	43.79^d
8	50.47^e	40.48^e
10	46.65^f	37.53^f
20	39.25^g	34.04^g
30	36.70^h	33.24^h
40	36.67^hi	32.73^h
50	36.62^hi	32.10ⁱ
60	36.60^ij	32.08ⁱ
70	36.53^j	31.36^j
80	35.50^k	31.28^j
90	35.37^l	31.18^j
100	34.74^m	31.08^j
110	33.41ⁿ	31.00^j
120	33.33ⁿ	30.86^j

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Means with different letters in the same column differ significantly at p ≤0.05.

Biosurfactant application

Antimicrobial activity

The biosurfactant antimicrobial activity was carried out against nine selected pathogenic microbes (Table 8). The biosurfactant from A. carneus and A. niger demonstrated significant antimicrobial activity against all tested microbes. It varied significantly according to the tested microbes (Table 8). A. niger biosurfactant exhibited the highest inhibitory effect against S. aureus ATCCG538 (40 mm), followed by B. subtilis ATCC6633 (39.07 mm), K. pneumoniae ATCC13883 (35 mm), and E. coli ATCC8739 (33 mm), while A. carneus biosurfactant demonstrated the highest effect against B. subtilis ATCC6633 (34.03 mm) in comparison to the control. The effect of the extracted crude biosurfactant of A. carneus and A. niger on the growth of (B) subtilis ATCC 6633 was studied by determining its optical density and biomass. Regarding the optical density, the results indicated that A. carneus and A. niger biosurfactant caused a relatively complete growth inhibition at 420 and 300 mg/100 ml, respectively (Fig. 5A). The highest concentration of (A) niger biosurfactant that stopped the growth of B. subtilis ATCC 6633 was 300 mg/100 ml (Fig. 5B). Conversely, using A. carneus biosurfactant yielded results between 420 and 480 mg/100 mL (Fig. 5B). The IC₅₀ was 300 mg/100 ml for A. carneus biosurfactant and 220 mg/100 ml for A. niger biosurfactant (Fig. 5A). The biomass measurement revealed that it was approximately 205 and 170 mg/100 ml, respectively (Fig. 5B). Many studies proved the antimicrobial activity of various biosurfactants. Serrawettin W1, a biosurfactant found in Serratia marcescens, can inhibit Gram-positive bacteria⁸⁰. A lot of different types of bacteria, including Shigella dysenteriae, P. aeruginosa, S. aureus, and Micrococcus luteus, can be inhibited by Serrawettin W2, a biosurfactant found in Serratia surfactantfaciens YD25T⁸¹. Crude biosurfactant extracts from Lactobacillus jensenii and Lactobacillus rhamnosus could inhibit clinical multidrug-resistant strains of S. aureus, E. coli EC433, and Acinetobacter baumannii⁸². The broad antimicrobial action of sophorolipids of Candida bombicola ATCC 22,214 against several microorganisms, including E. coli, P. aeruginosa, and S. aureus has been reported⁸³. Morais et al.⁸⁴ demonstrated the biosurfactant using L. jensenii P6A and L. gasseri P65. Liu et al.⁸⁵ demonstrated that the biosurfactant surfactin C15 effectively inhibited C. albicans SC5314. Another investigation found that biosurfactants made by Lactobacillus spp. had antibacterial effects on oral streptococci⁸⁶.

Table 8.

Antimicrobial activity of A. carneus and A. niger biosurfactant.

Microorganisms	Inhibition zone (mm)
Microorganisms	A. carneus	A. niger	Control*
B. subtilis ATCC 6633	34.03^a	39.07^b	25.07^b
S. aureus ATCC 6538	27.00^e	40.00^a	22.03^d
E. faecalis ATCC 10,541	32.93^b	31.03^e	30.13^a
E. coli ATCC 8739	30.97^d	32.97^d	22.93^c
S. typhi ATCC 6539	24.03^c	29.90^f	25.17^b
K. pneumoniae ATCC13883	32.03^c	35.03^c	19.13^g
C. albicans ATCC 10,221	24.03^f	27.90^g	20.97^e
C. Tropicalis ATCC 750	23.97^f	30.00^f	22.17^d
G. candidum	23.10^g	21.90^h	19.90^f

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*Positive control was Gentamicin at 30 mg/ml for bacterial isolates and Fluconazole at 30 mg/ml for yeasts and filamentous fungi.

Means with different letters in the same column differ significantly at p ≤0.05.

Fig. 5 — Effect of *A. carneus* and *A. niger* biosurfactant concentration on bacterial growth of *B. subtilis* ATCC 6633 optical density (A) and biomass (B).

Biosurfactant effect

Solubilization tests with biosurfactant concentrations between 5 and 30 mg/ml were done to evaluate the effect of the biosurfactant on the apparent water solubility of PAHs. An increase in biosurfactant concentration typically leads to a rise in the apparent solubility of PAHs. The incorporation of A. carneus or A. niger biosurfactant enhances the solubility of fluoranthene, pyrene, anthracene, and fluorine in aqueous solutions (Table 9). The maximum degree of solubilization was observed at a concentration of 30 mg/ml for both biosurfactants tested. Treatment with 30 mg/ml of A. carneus biosurfactant led to a substantial enhancement in the solubility of pyrene, fluoranthene, anthracene, and fluorine by over 17, 27, 26, and 3 times, respectively, in comparison to pure water. Conversely, a 27-fold enhancement in the solubility of PAHs for anthracene has been obtained when treated with A. niger biosurfactant, relative to its control (Table 9). Results have proven that the A. carneus or A. niger biosurfactant improved the solubility of PHAs, fluoranthene, pyrene, anthracene, and fluorine in water. The solubility of PHAs was directly proportional to the biosurfactant concentration. Aafter 16 days of incubation, Phanerochaete sordida YK-624 totally destroyed pyrene at the low concentration⁸⁷. Biosurfactants from A. carneus and A. niger can break up hydrocarbons, which lowers the surface tension and makes it easier for oil to move from soil particles⁸⁸. Naphthalene, phenanthrene, and pyrene became more soluble as the levels of the biosurfactant rhamnolipid biosurfactant in Bacillus Lz-2 increased⁸⁹. The use of biosurfactants may increase PAH biodegradation efficiency more than chemical surfactants. for example, soil that was contaminated with PAHs and treated with rhamnolipid, a biosurfactant, had a biodegradation rate that was 95%, which was higher than the biodegradation rates of SDS (90%) and Tween 80 (92%)⁹⁰. Also, a basic biosurfactant from Bacillus stratosphericus, B. subtilis, B. megaterium, and P. aeruginosa improved the breakdown of PAHs in soil that had been contaminated with creosote⁹¹. Zhou et al.⁹² observed a high degrading efficiency of an aqueous system containing pyrene, phenanthrene, benzo[b]fluoranthene, and benzo[a]pyrene in Mycobacterium gilvum MI, Mycobacterium sp. ZL7, and Rhodococcus rhodochrous Q3. In the same context, a number of fungi can breakdown PAHs. The white rot fungus P. ostreatus can break down pyrene, phenanthrene, fluorene, anthracene, and benzo[a]pyrene in water⁹³. A combination of four PAHs (chrysene, phenanthrene, naphthalene, and benzo(a)pyrene) could be broken down by the Fusarium solani strain HESHAM-1 isolated from soil contaminated with oil⁹⁴.

Table 9.

Effect of different concentration of crude biosurfactant of A. carneus and A. niger on the solubility of polyaromatic hydrocarbons.

Biosurfactant conc. (mg/ml)	Solubility of PAHs* (mg/l)
	A. carneus				A. niger
	Fluoranthene	Pyrene	Anthracene	Fluorene	Fluoranthene	Pyrene	Anthracene	Fluorene
0	0.26^g	0.17^f	0.08^g	2.01^g	0.24^g	0.17^g	0.06^g	1.76^g
5	0.99^f	0.89^e	0.23^f	2.13^f	0.81^f	1.53^f	0.17^f	2.08^f
10	1.86^e	1.08^e	0.54^e	3.15^e	1.34^e	1.86^e	0.40^e	2.90^e
15	2.87^d	1.41^d	0.84^d	3.91^d	1.77^d	2.34^d	0.82^d	3.69^d
20	3.61^c	2.24^c	1.13^c	4.63^c	2.70^c	2.70^c	0.94^c	4.54^c
25	5.00^b	2.59^b	1.52^b	5.35^b	3.73^b	2.91^b	1.12^b	5.80^b
30	7.12^a	2.96^a	2.14^a	6.92^a	4.27^a	3.08^a	1.63^a	7.09^a

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Means with different letters in the same column differ significantly at p ≤0.05.

Removal of lubricating oil from the contaminated soil

The ability of the tested biosurfactants of A. carneus and A. niger to remove lubricating oil from the contaminated soil was examined compared to certain synthetic surfactants, that is, a nonionic surfactant tween 80 and an ionic surfactant SDS. The recovery percentage of lubricating oil by both synthetic and biosurfactant varied significantly depending on the temperature. Table 10 illustrates the varying effectiveness of different surfactants in recovering oil from contaminated soil under different temperature conditions. Tween 80 demonstrated significant recovery rates, retrieving 22% of the oil at 25 °C, 38% at ambient temperature, 67% at 45 °C, and 75% at 60 °C. In contrast, the synthetic surfactant SDS showed lower efficacy, with recovery rates ranging from 7 to 12% across the temperature range. The control group that received distilled water showed minimal recovery, ranging from 10 to 27%. Additionally, A. carneus biosurfactant recovered 25% of the oil at 25 °C, 37% at ambient temperature, 64% at 45 °C, and 76% at 60 °C, while A. niger biosurfactant retrieved 32% of the oil at 25 °C, 40% at room temperature (30 °C), 63% at 45 °C, and 80% at 60 °C. Generally, biosurfactants offer a promising alternative for enhancing oil recovery due to their unique properties and environmental benefits. Two of the most popular forms of biosurfactants, rhamnolipid and cyclohexanone, have successfully removed oil molecules from soil⁹⁵. People have widely used them to remediate oil pollutants in contaminated soil. These results showed that biosurfactants are better than synthetic surfactants like SDS and Tween at getting oil pollutants out of contaminated soil. In this work, we investigated biosurfactants that could serve as biostimulator agents for oil-contaminated soil bioremediation.

Table 10.

Removal of lubricating oil from the contaminated soil.

Temperature (°C)	Chemical surfactant			Biosurfactant
Temperature (°C)	Control	SDS	Tween 80	A. carneus	A. niger
25	0.90^a	0.93^a	0.78^a	0.75^a	0.68^a
30	0.85^b	0.91^a	0.62^b	0.63^b	0.60^b
45	0.78^c	0.89^b	0.43^c	0.36^c	0.37^c
60	0.73^d	0.88^b	0.25^d	0.24^d	0.20^d

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Means with different letters in the same column differ significantly at p ≤0.05.

Biosurfactant characterization

The chemical nature of a biosurfactant can be determined by the phenol-H₂SO₄ test to detect glycolipids, the biuret test to detect lipopeptides, and the phosphate test to detect phospholipids. First, it was found that the biosurfactants from A. carneus and A. niger are made up of glycolipid and lipopeptide molecules. The orange color showed that glycolipids were present in the biosurfactant crude of A. carneus and A. niger biosurfactant by phenol-H₂SO₄ test. Also, phospholipids were detected in the tested biosurfactant (Table 11). The negative results of the Biuret test indicated the absence of lipopeptides in A. carneus and A. niger biosurfactants.

Table 11.

Detection of chemical compounds in A. carneus and A. niger biosurfactant.

Compound	Biosurfactant
Compound	A. carneus	A. niger
Glucolipids	+	+
Lipopeptides	−	−
Phospholipids	+	+

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A GC-MS analysis revealed the presence of 50 different chemical compounds. They have different retention times, ranging from 3.429 to 36.057 min. The highest percentage content (area %) of the compounds are as follows: DL-Ethionine (12.48%), 1,2,3-Propanetriol, monoacetate (12.382%), Glycerin (6.353%), Methanamine, N-methoxy (4.76%), Octadecanoic acid, 2,3-dihydroxypropyl ester (2.787%), Hexadecanoic acid, 2-hydroxy-1- 5(hydroxymethyl) ethyl ester (2.453%), 9,12-octadecadienoic acid, methyl ester, (E, E)- (2.143%), n-Hexadecanoic acid (1.942%), 9-Octadecenoic acid (Z)-, methyl ester (1.114%), and trans-13-octadecenoic acid (0.467%). Out of these compounds, the GC-MS analysis identified ten hydrophobic compounds listed in Table 12 with retention times ranging from 26.278 to 36.057 min (Fig. 6). These hydrophobic tails may represent a part of certain biosurfactant(s). In terms of area percentages, 9,12-Octadecadienoic acid, methyl ester, (E, E)- has the most content (2.923%). It is followed by Octadecanoic acid, 2,3-dihydroxypropyl ester (2.787%), Palmitic acid ME P1297 (2.774%), and Hexadecanoic acid, 2-hydroxy-1(hydroxymethyl)ethyl ester (2.453%). These results suggest that the sample contains a diverse range of chemical compounds, with varying retention times and percentage contents. The presence of hydrophobic compounds within the biosurfactant(s) indicates that they may have a significant impact on the overall properties and effectiveness of the biosurfactants. More research into these compounds and how they work can help us understand what biosurfactants are and how they might be used in different fields, like food, medicine, cleaning up the environment, and oil and gas.

Table 12.

Hydrophobic tail compounds detected by GC-MS analysis.

Structure	Compound name	RT	Area %
A. carneus
C₁₆₃₂O₂	Palmitic acid ME P1297	26.278 – 26.633	2.774
C₁₉H₃₄O₂	9,12-Octadecadienoic acid, methyl ester, (E, E)-	27.984 – 28.354	2.923
C₁₉H₃₆O₂	9-Octadecenoic acid (Z)-, methyl ester	28.054	1.114
C₁₈H₃₄O₂	Trans-13-Octadecenoic acid	28.414	0.467
C₁₈H₃₆O₂	Octadecanoic acid	28.654	1.635
C₂₀H₃₈O₂	Ethyl Oleate	28.709	0.54
C₁₆H₃₄O	2-Hexadecanol	30.019	0.275
C₂₆H₅₄	Octadecane, 3-ethyl-5-(2-ethylbutyl)-	32.355	0.296
C₁₉H₃₈O₄	Hexadecanoic acid, 2-hydroxy-1(hydroxymethyl)ethyl ester	32.405	2.453
C₂₁H₄₂O₄	Octadecanoic acid, 2,3-dihydroxypropyl ester	36.057	2.787
A. niger
C₁₈H₃₄O	5-Octadecenal-	23.002	0.881
C₁₆H₃₂O₂	Palmitic acid P1210	26.623	1.508
C₁₅H₃₀O₃	Methyl 2-hydroxy-tetradecanoate	27.418	3.74
C₁₈H₃₆O₂	Octadecanoic acid	28.659	0.643
C₁₉H₃₈O₄	Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl)ethyl ester	32.4	1.57
C₂₁H₄₂O₄	Octadecanoic acid, 2,3-dihydroxypropyl ester	36.032	1.162

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Fig. 6 — GC-MS analysis of *A. carneus* and *A. niger* culture filtrate.

FT-IR evaluated the molecular composition of biosurfactants of A. carneus OQ152507 and OQ195934 produced under optimal conditions (Fig. 7). The FT-IR spectrum of A. carneus surfactants may show that all the compounds are present in the crude A. carneus surfactant (Figs. 6 and 7). The characteristic peaks at 3310, 1738, and 1465 cm^− 1 correspond to the OH, COO, and C = C stretching vibrations, respectively. The IR spectrum of A. niger OQ195934 showed the spectrum exhibited characteristic peaks at 3350, 1658, and 1455 cm^− 1 assigned for OH, COOH, COO, and C = C stretching vibration, respectively (Figs. 6 and 7). Generally, these spectra perfectly match the compound identified through GC-MS analysis. Varjani and Upasani⁹⁶ found that the main classifications for the biosurfactants are glycolipids, fatty acids, lipopeptides, lipoproteins, phospholipids, and polymeric compounds. The current study confirmed that the biosurfactants from A. carneus and A. niger are classified as glycolipids, which aligns with Bhardwaj et al.⁹⁷ who found that the majority of fungal biosurfactants are lipid derivatives. In the A. carneus supernatant, GC-MS showed that palmitic acid ME P1297 (16:0), 9,12-octadecadienoic acid, methyl ester, (E, E)-(19:2), hexadecanoic acid, 2-hydroxy1(hydroxymethyl) ethyl ester (19:0), and octadecanoic acid, 2,3-dihydroxypropyl ester (21:0) were all present. In the A. niger filtrate, methyl 2-hydroxy-tetradecanoate (15:0) was the most common acid. In glycolipid biosurfactants, these fatty acid moieties may act as hydrophobic tails. Likewise, the FTIR analysis confirmed that these biosurfactants are glycolipids. Similar results were detected concerning Mucor hiemalis, Mucor indicus, Candida glabrata surfactants, and Gordonia sp^88,98.

Conclusion

The fungi A. carneus OQ152507 and A. niger OQ195934, extracted from soil samples tainted with hydrocarbons, exhibited proficient biosurfactant synthesis capacities. The biosurfactant synthesis by these fungi was markedly improved when cultivated under optimum circumstances. The biosurfactants produced by A. carneus and A. niger demonstrated several advantageous characteristics. These biosurfactants exhibited enhanced emulsification capacity, significantly lowering the surface tension of aqueous solutions. Moreover, they exhibited the capability to augment the extraction of oil from polluted sand and raise the apparent solubility of PAHs. Furthermore, the biosurfactants demonstrated diverse biological activities, including antimicrobial capabilities. Chemical research indicated that the biosurfactants consisted of glycolipid and phospholipid molecules. This study’s findings underscore the potential applications of A. carneus and A. niger biosurfactants in environmental remediation and industrial approaches.

Author contributions

YAM, Writing, reviewing, and editing; investigation; YHE, Conceptualization, methodology, writing-original draft, preparation, writing, reviewing, and editing; EEN, Validation, conceptualization, writing, reviewing, and editing; YMA, writing, reviewing, and editing; BAE, Writing, reviewing, and editing; investigation. SSA, Writing, reviewing, and editing; investigation, software. All authors gave final approval for publication.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

Sequence data that support the findings of this study have been deposited in the NCBI with the accession codes of QQ152507 (https://www.ncbi.nlm.nih.gov/nuccore/OQ152507.1/) and QQ195934 (https://www.ncbi.nlm.nih.gov/nuccore/OQ195934).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Yasser H. El-Halmouch, Email: yasser.halmouch1970@sci.kfs.edu.eg

Sameh S. Ali, Email: samh_samir@science.tanta.edu.eg

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Data Availability Statement

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PERMALINK

Exploring the potential of biosurfactants produced by fungi found in soil contaminated with petrochemical wastes

Yehia A-G Mahmoud

Yasser H El-Halmouch

Ebtsam E Nasr

Yassin M Al-Sodany

Baher A El-Nogoumy

Sameh S Ali

Abstract

Introduction

Material and method

Isolation of biosurfactant producer fungal strains

Biosurfactant-producing fungi cultivation

Fungal identification and biosurfactant production optimization

Emulsification index and oil spreading

Biosurfactant extraction and characterization

Polyaromatic hydrocarbons solubilization

Applications of fungal biosurfactant

Antimicrobial activity

Estimation of critical micelle concentration

Elimination of lubricating oil from contaminated soils

Statistical analysis

Results and discussion

Screening for best biosurfactant producer fungal strains

Fig. 1.

Optimization of biosurfactant production

Effect of culture media

Table 1.

Effect of hydrocarbon oil

Table 2.

Effect of incubation period

Fig. 2.

Effect of temperature and initial pH

Table 3.

Table 4.

Effect of carbon and nitrogen sources

Fig. 3.

Fig. 4.

Effect of sugar, nitrogen, and critical micelle concentrations

Table 5.

Table 6.

Table 7.

Biosurfactant application

Antimicrobial activity

Table 8.

Fig. 5.

Biosurfactant effect

Table 9.

Removal of lubricating oil from the contaminated soil

Table 10.

Biosurfactant characterization

Table 11.

Table 12.

Fig. 6.

Fig. 7.

Conclusion

Author contributions

Funding

Data availability

Declarations

Competing interests

Footnotes

Contributor Information

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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