Eco-Friendly Biosurfactant: Tackling Oil Pollution in Terrestrial and Aquatic Ecosystems

Abstract

Spills involving fuels and lubricating oils in industrial environments caused by the fueling of machines, inadequate storage and the washing of equipment are significant sources of environmental pollution, impacting soil and water bodies. Such incidents alter the microbiological, chemical and physical properties of affected environments. The use of biosurfactants is an effective option for the cleaning of storage tanks and the remediation of contaminated soils and effluents. The scope of this work was to assess the production and application of a Starmerella bombicola ATCC 22214 biosurfactant to remediate marine and terrestrial environment polluted by oil. The production of the biosurfactant was optimized in terms of carbon/nitrogen sources and culture conditions using flasks. The performance of the biosurfactant was tested in clayey soil, silty soil, and standard sand, as well as smooth surfaces and industrial effluents contaminated with oils (fuel oils B1 for thermal power generation, diesel, and motor oil). The ideal culture medium for the production of the biosurfactant contained 2% glucose and 5% glycerol, with agitation at 200 rpm, fermentation for 180 h and a 5% inoculum, resulting in a yield of 1.5 g/L. The biosurfactant had high emulsification indices (86.6% for motor oil and 51.7% for diesel) and exhibited good stability under different pH values, temperatures and concentrations of NaCl. The critical micelle concentration was 0.4 g/L, with a surface tension of 26.85 mN/m. In remediation tests, the biosurfactant enabled the removal of no less than 99% of motor oil from different types of soil. The results showed that the biosurfactant produced by Starmerella bombicola is a promising agent for the remediation of environments contaminated by oil derivatives, especially in industrial environments and for the treatment of oily effluents.

1. Introduction

Petroleum, also called crude oil, is an essential feedstock in the chemical industry, which plays a key role in world economic development [1]. Petroleum is a primary fuel for transport, electricity production and a vast gamut of industrial and domestic activities. The estimated consumption of petroleum is about 90 million barrels/day, with a projected increase of 1.7% annually by the year 2030 [2,3].

With the continual development of industrialization on the global scale, the demand for crude oil as an energy source is also continually increasing. However, oil spills due to exploration and transportation often occur throughout the world [4]. As a consequence, large quantities of oil have been released into soils and oceans, with devastating effects on the environment, which are even greater due to the toxic potential of the chemical dispersants commonly used for the remediation of such spills [5,6,7].

Environmental concerns related to the burning of fossil fuels, especially gasoline and coal, have led to a greater social awareness that has driven the search for sustainable processes [8,9]. Several countries have begun to adopt specific measures in line with the concept of sustainable development in an attempt to minimize the climate and environmental crises that place the entire planet at risk [10,11].

The petroleum sector includes offshore activities performed on the ocean, some of which involve accidental spills of the transported crude oil into the sea; therefore, dispersants and containment measures are required [11,12].

In the environmental field, chemical dispersants used for the treatment of areas contaminated with oil derivatives are mainly composed of surfactants [13,14]. As amphiphilic molecules, surfactants are characterized by the capacity to reduce surface tension between immiscible fluids, such as oil and water, providing detergency, dispersion and emulsification, which are essential to the remediation of environments contaminated with hydrocarbons [15,16,17].

Oil-derived surfactants are synthetically obtained from finite sources, including petroleum (paraffin oil, aromatic compounds, etc.) and natural gas (propene, ethene, etc.) [18,19]. Such compounds lead the surfactant market in developed world, corresponding to around 90% of sales because of their high yields and low costs [20,21,22]. Nonetheless, present concerns about health, environmental contamination, global warming and the exhaustion of petroleum resources need measures to overcome the widespread utilization of synthetic surfactants [23,24].

Green surfactants—known as biosurfactants or biobased surfactants—emerge as promising candidates due to their unique properties and versatility [17,25,26]. Biosurfactants have applications in a set of industrial processes, such as the cleaning, food, agricultural, environmental, petrochemical and cosmetic industries [27]. Due to the broad gamut of chemical compositions, biosurfactants have more favorable structural diversity than their conventional counterparts. Their hydrophilic moiety, called head, is made up of carbohydrates, amino acids, peptides or polar functionalities, including carboxylic acids, whereas the lipophilic portion is formed by fatty acids or saturated, unsaturated and/or hydroxylated fatty acids [28,29,30].

Biosurfactants are less toxic, more biodegradable and more stable under adverse environmental conditions compared to chemical surfactants [31,32]. Properties such as detergency, emulsification, dispersion and solubilization confer considerable versatility to these biomolecules, making them a promising commercial alternative [33,34]. Moreover, biosurfactants have a considerable diversity of molecular structures, including glycolipid, lipopeptide, protein–sugar, phospholipid complexes, fatty acids and neutral lipids when cultivated on insoluble substrates (oils, wastes and hydrocarbons) or soluble substrates (sugars) [35,36]. The surface tension reduction capacity and the emulsification activity of biosurfactants may be attributed to the molecular mass, as those with a greater molecular mass generally achieve better emulsification, whereas those with a lower molecular mass have better surface tension activity, although other factors, such as the viscosity of the medium and the interaction capacity between the molecular structures of the biomolecule and substrate should also be considered [34].

Yeasts of the genus Starmerella have been widely studied for the potential production of glycolipids, especially Starmerella bombicola, which is known for the production of sophorolipids, which are biosurfactants formed by a glucose disaccharide and a chain of fatty acids with 16 or 18 carbon atoms [36,37,38]. Therefore, this work aimed to obtain a biosurfactant from Starmerella bombicola ATCC 22214 to be applied in the remediation of oil-polluted marine and terrestrial environments. The biosurfactant was assessed for its capacity as a dispersing agent of complex hydrocarbons on surfaces and in soils and oily industrial effluent as a sustainable, nontoxic, low-cost solution.

2. Materials and Methods

2.1. Materials

All reagents used for the composition of the mineral medium were analytical grade. Soluble substrates were used as the carbon sources, such as analytical-grade glucose and sucrose, glycerol and sugarcane molasses (obtained from a sugar plant located in the city of Vitória de Santo Antão, Brazil). The insoluble substances were analytical-grade N-hexadecane and waste soybean fry oil collected in car workshops located in Recife, Brazil. The nitrogen sources were yeast extract and corn steep liquor (a bioproduct of corn production) kindly provided by Corn Products S.A. do Brasil (Cabo de Santo Agostinho, Brazil).

The hydrophobic pollutant used in this study was residual motor oil, containing residues of fuel, metallic particles and other contaminants generated by the functioning of engines. Heavy oil, supplied by a thermoelectrical device and categorized as special B1 fuel oil (OCB1—fuel oil for thermal power generation) (PETROBRAS, Rio de Janeiro, Brazil), was a tangled hydrocarbon blend, with kinematic viscosity at 60 °C of 620 cSt, a flashpoint of 66 °C and density at 20 °C of 0.968 g/mL.

Oily effluent was obtained from ULTRACARGO, which is a storage and distribution company for fuels and chemical products located at the Suape Port in the state of Pernambuco, Brazil. The effluent has a high content of petroleum derivatives and is composed of a mixture of biodiesel—B100, diesel oil—S10, gasoline, anhydrous and hydrated ethyl alcohol and heavy oil (OCB1), resulting from routine activities of the company.

2.2. Microorganism and Preparation of Inoculum

The yeast Starmerella bombicola ATCC 22214, purchased from the American Type Culture Collection, was employed to produce the biosurfactant. The cells were activated by transfer to tubes with sterilized Yeast Mold Agar (YMA) medium containing 0.3% yeast extract, 1% D-glucose, 0.5% peptone, 2% agar and 100 mL of distilled water at pH 7. The culture was incubated for 24 h at 28 °C, 150 rpm and pH 7.0 in flasks containing 50 mL of autoclaved Yeast Mold Broth (YMB), which had the same composition as the YMA medium but without agar. After that, dilutions of the medium were carried out until a 10⁶ cells/mL concentration was obtained. Cell counts were performed in a Neubauer chamber. Autoclaving was performed at 121 °C for 20 min.

2.3. The Production of the Biosurfactant

The yeast was cultivated in mineral medium with the following composition (g/L): KH₂PO₄: 0.5 g/L; NaNO₃: 3 g/L; CaCl₂.H₂O: 0.05 g/L; FeCl₃: 0.05 g/L MgSO₄.7H₂O: 0.4 g/L; and 0.05 g/L of yeast extract. Mixtures of carbon and nitrogen sources were added to this medium. The culture conditions for the selection of the best pre-production medium for the yeast are displayed in

Table 1. Media and culture conditions adopted to produce the Starmerella bombicola ATCC 22214 biosurfactant.

Media Tested	Culture Conditions
5.0% glycerol + 2.0% glucose	pH 6.0
1.0% N-hexadecane + 2.0% glucose	200 rpm
3.0% sucrose + 0.1% yeast extract (*)	96, 120 and 144 h
2.0% sugarcane molasses + 3.0% corn steep liquor (*)	28 °C

* With and without waste soybean fry oil (2.0%).

. After medium preparation, its pH was adjusted to 6.0 ± 0.2 by adding 5.0 M NaOH solution, and sterilization was performed at 121 °C for 20 min. To produce the biosurfactant, cultivations were carried out in 250 mL Erlenmeyers containing 100 mL of the broth, in which the inoculum at a concentration of 5.0% was incubated. Samples were finally taken to determine the surface tension and the yield of the isolated biosurfactant.

2.4. Determination of Surface Tension

The surface tension of the biosurfactant was determined using an automatic tensiometer (KSV Sigma 700, Helsinki, Finland) with a Du Noüy ring. After immersing the platinum ring in the broth, the tensiometer recorded the force needed to pull it through the air–liquid interface.

2.5. Emulsification Activity Assessment

To assess the emulsification activity, cell-free broth specimens were tested according to Cooper and Goldenberg [39]. In total, 2 mL of the cell-free broth (crude biosurfactant) was added to graduated tubes containing 2.0 mL of the hydrophobic substrates (motor oil, diesel, kerosene, hexane and vegetable oils) and the mixture was agitated in a vortex for 2 min. The stability of the emulsion was assessed as a percentage after 24 h, and the emulsification index (EI) was defined as the ratio of the emulsion height to the total mixture height, multiplied by 100. The emulsification rate was calculated using Equation (1):

$$EI (\%)={\displaystyle \frac{Height of emulsion }{Total height of mixture}}\times 100$$

(1)

2.6. Assessment of Biosurfactant Stability

The stability of the biosurfactant was analyzed by varying one parameter at a time while keeping the others constant. The conditions tested were as follows: temperature (5 to 120 °C) with pH fixed at 7.0; NaCl concentration (2.0 to 10.0%) with temperature fixed at 28 °C and pH at 7.0; pH (2.0 to 12.0) with temperature at 28 °C; and heat exposure at 90 °C for 10 to 120 min, maintaining pH at 7.0. Stability was evaluated using cell-free broth to determine surface tension [39].

2.7. Extraction and Isolation of Biosurfactant

The biosurfactant was isolated according to Samak et al. [40]. After centrifugation at 4400 rpm and 4 °C for 15 min, the cell-free broth was transferred to a decantation funnel containing the same volume of ethyl acetate, and the mixture shaken intensely for 15 min. After separating the phases, the organic one was extracted again using the same solvent amount. The solvent was evaporated from the organic phases at 40 °C, and the resulting residue was washed twice with hexane to remove possible trace lipophilic compounds and then removed by evaporation. The yield of the isolated biosurfactant was assessed gravimetrically, considering the sample volume of the fermentation medium, enabling the precise determination of the efficiency of the process.

2.8. Determination of Critical Micelle Concentration

The critical micelle concentration (CMC) of the biosurfactant was determined automatically in the tensiometer (KSV Sigma 700, Finland) with the Du Noüy ring. The platinum ring was immersed in different samples of ultrapure water to which the biosurfactant was step-by-step added up to a constant surface tension.

2.9. Composition of Biosurfactant

The protein concentration of the isolated biosurfactant was determined using a LabTest Kit (São Paulo, Brazil). Carbohydrates were assessed using the phenol-sulfuric acid method with D-glucose as a standard [41]. Lipids were quantified based on Manocha et al. [42].

2.10. Analysis of Ecotoxicity

The toxicity of the biosurfactant was determined using terrestrial and aquatic indicators. Cabbage seeds (Brassica oleracea) and tomato seeds (Solanum lycopersicum) were used as suggested by Tiquia et al. [43]. For the aquatic indicator, larvae of the microcrustacean Artemia salina were placed to 15 mL penicillin tubes containing 10 mL of seawater, with 1% and 2% of the biosurfactant diluted in distilled water and the surfactant at half its CMC, the CMC and twice the CMC, based on Meyer et al. [44].

2.11. Dispersion of Hydrophobic Compounds in Seawater

The capacity to disperse or aggregate oil slicks was determined by polluting seawater samples with weathered engine oil. Experiments were carried out by adding the crude biosurfactant to the weathered oil in 1:2, 1:8 and 1:25 (v/v) proportions. The results were evaluated visually [45].

2.12. Removal of Petroleum Derivative Adsorbed to Sand and Soils Using Biosurfactant in Flasks—Kinetic Assay

The contaminant was removed following the method described by Bezza et al. [46]. Samples of 50 g of standard sand, clayey soil and silty soil were contaminated with a 10% v/w motor oil solution. The samples were placed in 500 mL Erlenmeyers, and 100 mL of the isolated biosurfactant solution were added at half the CMC, the CMC and twice the CMC. The same volume of cell-free broth was also assessed as crude surfactant and water without surfactant as the control. All flasks were kept under stirring at 150 rpm and 28 °C. Samples were withdrawn after 30, 60 and 120 min and 24 h. After treatment, the liquid was removed and the sand and soil samples were washed with the solvent (n-hexane) for two hours with stirring at 150 rpm and 28 °C. After solvent evaporation, the mass of the residual oil was recorded.

2.13. Removal of Petroleum Derivative Adsorbed to Sand and Soils Using Biosurfactant in Packed Columns—Static Assay

About 200 g of standard sand, clayey soil or silty soil previously contaminated with a 10% solution of exhausted motor oil was loaded into glass columns (55 cm × 4 cm), whose surfaces were then flooded with 200 mL of the biosurfactant solution at half the CMC, the CMC and twice the CMC. The same volume of cell-free medium was also tested as a raw surfactant source, while a column containing contaminated soil and 200 mL of fresh water without surfactant was employed as the control. Solution percolation was tracked for 24 h. The soil was then washed with hexane and, after evaporating the solvent, the residual oil was evaluated by gravimetry [47].

2.14. Cleaning of Oily Surface

The cleaning capacity of the biosurfactant was investigated by coating the internal walls of beakers borosilicate (capacity 50 mL) with used motor oil and heavy oil (OCB1). A total of 50.0 mL of the washing solution containing the aqueous solution of surfactant at various concentrations (half the CMC, the CMC, twice the CMC and five times the CMC) was added to each beaker. The beakers were shaken for 1.0 min and left to rest for 6 h [48].

2.15. Destabilization of OCB1 and Diesel Oil on Smooth Surface

The isolated biosurfactant was diluted to obtain different concentrations (half the CMC, the CMC, twice the CMC and five times the CMC), which were used to assess the capacity to destabilize heavy oil and diesel oil spread on a smooth surface as a function of time. Borosilicate Petri dishes (diameter: 12 cm) were previously weighed to determine their initial mass and the surfaces were then evenly spread with the oil derivatives. The dishes with OCB1 oil were left to rest for two consecutive days to ensure impregnation. For the more fluid pollutant (diesel), a hydrophobic dye (Scarlet Red) was used to facilitate visualization. For these types of oils, the dishes were submitted to heating in a laboratory oven at 100 °C for 48 h for the volatilization of the lighter fractions of the hydrocarbon and better oil fixation. Dishes soaked in the petroleum derivative were then reweighed to determine the oil amount. Established portions of the test solutions (15 and 20 mL) were added to the layer of spread oil, spread without manual action and left to rest for different periods of time (30 and 60 min). After the destabilization of the oil as a function of the established volume and time, the dishes were washed in distilled water without pressure to remove the excess test solution and dispersed petroleum derivatives. After drying in a laboratory oven at 50 °C for 30 min, the dishes were weighed after returning to room temperature. The removal was accompanied by photographic documentation after each assay. The removal efficiency (I) was calculated by the following equation:

I = [(M_c − M_W)/(M_c − M_i)] × 100

(2)

in which M_c is the mass of the polluted dish, M_W is the mass of the washed dish, and M_i is the starting dish mass.

2.16. Application of Biosurfactant in Treatment of Oily Industrial Effluent

The “swirling flask” or “cylindrical bottle” test is often performed to assess the effectiveness of biosurfactants as spilled oil dispersing agents [49]. In this work, such a test was conducted to assess the efficacy of the biosurfactant produced by Starmerella bombicola ATCC 22214 in the treatment of an oily effluent from a fuel storage and distribution company.

Oily effluent obtained from the company ULTRACARGO, which specializes in the storage and distribution of fuels, was used to assess the effectiveness of the biosurfactant in the removal of petroleum derivatives in a real oily effluent. Open 1 L cylindrical glass vessels (ϕ = 10 cm) with a drain valve beneath used to take samples were employed in the removal test. The methodology followed in this study was based on the procedures described by Sobrinho et al. [49] and Soares da Silva et al. [50].

After the addition of oil effluent (200 mL) in the vessels, we added the crude surfactant at 1:1 (v/v) and 1:2 (v/v) surfactant/effluent proportions or the isolated surfactant at concentrations of half the CMC, the CMC and twice the CMC at 1:1 (v/v) surfactant/effluent proportion. The vessels were put on an orbital shaker at 28 °C to ensure the vortical movement of the contents. Agitation was performed at 150 rpm for 10 min, followed by 1 to 2 min of stabilization of the system. Samples were collected soon afterward. The first 2 mL of samples were taken using the outlet valve and thrown away, while the next 50 mL samples were retained for analysis. The samples were extracted three times with hexane. The oil removal percentage (δ) was calculated using Equation (3) based on sample absorbance (determined by the analysis of the hexane extract) compared with the control (with no surfactant):

δ = [(C_B − C_A)/C_B] × 100

(3)

in which C_B and C_A are the concentrations of oil before and after treatment with the biosurfactant, respectively. The wavelength was set at 320 nm, and triplicate analyses were performed.

2.17. Statistical Analysis of Results

The results were expressed as means ± standard deviations in tests carried out in triplicate. Comparisons were performed using the analysis of variance (ANOVA). A p-value < 0.05 was taken as indicative of statistical significance.

3. Results and Discussion

3.1. Selection of Biosurfactant Production Medium

The purpose of this step was to identify the most adequate culture media for the production of the biosurfactant by the yeast Starmerella bombicola ATCC 22214. The selection of the most favorable fermentation variables was based on the determination of surface tension. No technical–economic viability study was performed in this work. However, such a study needs to be considered in future steps of the research to determine the viability of large-scale application and achieve the yield optimization and the reduction in downstream steps. Therefore, culture media with “cleaner” substrates and others composed of industrial waste products were used, assessing the production of the microbial surfactant with a focus on its potential practical and economic viability for the future.

The results of the fermentations in different media and with different culture conditions are presented in Figure 1, which displays the surface tension values. Combinations of substrates were added to the mineral medium to ensure a soluble and insoluble carbon source, totaling six different culture media. Temperature, agitation and pH were maintained at constant values, whereas three different culture times were used (96, 120 and 144 h).

Media containing industrial waste products achieved different results, especially in conditions containing waste fry oil, in which a greater reduction in surface tension occurred. This effect was more evident in the medium composed of molasses and corn steep liquor. Culture time also exerted a significant influence, with a more accentuated reduction in surface tension as the culture time increased.

The mineral medium formulated with glycerol and glucose as the carbon sources was the most efficient among all media tested at all culture times. The most promising performance was achieved with the use of 5% glucose and 2% glycerol after 144 h of fermentation, resulting in a significant reduction in surface tension to 27.24 mN/m. Moreover, the use of more soluble substrates free of solid particles favors the economy of the process, reducing costs associated with materials and reagents in downstream steps. This approach also contributes to the maintenance of a consistent standard in the productive process, which, in turn, enhances the market competitiveness of biosurfactants. It should be pointed out that glycerol, which is a byproduct of the transesterification of plant- and animal-based oils to produce biodiesel, is an option that has generated considerable interest, as the annual quantity generated throughout the world has been increasing due to the growing demand for biodiesel and other derivatives [51].

The literature offers various studies on the selection of substates for the production of microbial surfactants with similar properties to those obtained in the present investigation. In a study conducted by Farias et al. [52], biosurfactants obtained from bacteria of the genus Pseudomonas were assessed in different culture media with the same substrates used in the present study. The most promising result was achieved with a medium composed of 5.0% glycerol and 2.0% glucose fermented for 96 h at 28 °C, reaching excellent surface tension values ranging from 25.0 to 29.0 mN/m.

In a study conducted with the yeast Starmerella bombicola ATCC 22214, Selva Filho et al. [53] identified potato peel powder at a concentration of 2.0% and waste canola fry oil at a concentration of 5.0% as the most promising soluble and insoluble carbon sources. The combination of these sources led to a surface tension of 29.60 mN/m. Moreover, these raw materials are easily accessible and economically viable.

The reduction in surface tension (ST) is extremely important to assess the efficacy of a surfactant. This property differs considerably from one chemical surfactant to another. Some examples include Triton X (33.0 mN/m), sodium dodecyl sulfate—SDS (40.5 mN/m), sodium octyl sulfate—SOS (40.7 mN/m), sodium 2-ethylhexyl sulfate—SEHS (29.5 mN/m), linear alkylbenzene sulfonate—LAS (36 mN/m) and sorbitan oleate—Span 80 (44.9 mN/m). Biosurfactants capable of reducing water ST from 72 to less than 35 mN/m are regarded as very efficient [11].

The ST of the biosurfactant produced by Starmerella bombicola ATCC 22214 was 27.24 mN/m, which is considered very promising in terms of tensioactive activity. As pointed out by Shaji et al. [54], biosurfactants are capable of reducing water ST from 72 to 27 mN/m, confirming the efficiency of that produced in the present investigation. The literature offers various studies on biosurfactants with similar properties to those found in the biosurfactant studied herein. The fungus Curvularia lunata UFPEDA885 achieved an ST value of 32.9 mN/m [55]. Yuan et al. [56] investigated Chitinophaga eiseniae 4H for biosurfactant production and found a decrease in water ST to 32.35 mN/m. The biosurfactant produced by Saccharomyces cerevisiae URM 6670 cultured in a broth based on waste soybean fry oil and corn steep liquor lowered the medium ST to as little as 6 mN/m [57], and that produced by Candia mogii UFPEDA 3968 in a medium containing licuri palm oil and glucose reduced ST from 71.04 to 28.66 mN/m [58].

3.2. Emulsification Activity

One way to assess the properties of a surfactant is to analyze its emulsifying activity with regard to hydrocarbons and/or insoluble compounds in water. The emulsification index is a rapidly determinable parameter that allows for the evaluation of the emulsifying power of a surfactant. Biosurfactants have emulsifying capacity, forming stable oil-in-water or water-in-oil emulsions [59].

Figure 2 shows the emulsification index of the biosurfactant for the hydrophobic substrates (motor oil, diesel, kerosene, hexane and soybean oil).

The emulsification index was higher than 50% for all hydrophobic substrates, with the exception of hexane (29.4%). The biosurfactant demonstrated a very satisfactory interaction with motor oil, achieving an emulsification rate of 86.6 ± 0.5%, revealing greater affinity for more complex hydrocarbons. These results indicate that the biosurfactant produced has good emulsifying properties. More complex oils, such as motor oil, are composed of both nonpolar and polar molecules, which makes them interact more with the biosurfactant. As hexane is a pure nonpolar compound, it will not interact as much with the hydrophobic part of the biosurfactant, making the layer between the hydrophobic part of the biosurfactant and the hexane brittle. The high volatility of hexane is another factor that prevents it from remaining in the mixture, making emulsification difficult. According to Ferreira et al. [60], emulsification rates higher than 50% indicate efficacy in emulsion formation.

Jaysree et al. [61] and Hassanshahian [62] found very similar rates (15–54% and 10–65%, respectively) for biosurfactant-producing microorganisms isolated from locations contaminated with oil. Barakat et al. [63] reported emulsification rates of 56% and 57% for two different strains of Bacillus. The emulsification rate for kerosene in the present investigation (52.1%) was much higher than that reported by Da Silva et al. [58] for the biosurfactant synthesized by the yeast C. moggi (3.28%).

3.3. Physicochemical Properties of the Biosurfactant

The applicability of a biosurfactant in different fields depends on its stability when exposed to various values of temperature, pH and salt concentration as well as its resistance to heat for a given length of time. Many biosurfactants are considered hopeful because of their tolerance to stress conditions found in food treatment and bioremediation activities [22].

Figure 3 depicts the change in the ST of the biosurfactant synthesized by Starmerella bombicola ATCC 22214 when submitted to different temperatures, pH values, NaCl concentrations and heating times at 90 °C. Little variation in surface tension (26.89 to 27.55 mN/m) was found when the biosurfactant was submitted to a temperature of 90 °C for 10, 20, 30, 40, 50, 60 and 120 min (Figure 3a).

When submitted to different pH values (2, 4, 6, 8, 10 and 12), a slight increase in ST was observed in acidic media, reaching 32.30 mN/m at pH 4, whereas it ranged from 27.45 to 28.63 mN/m in alkaline media (Figure 3b). Souza et al. [64] investigated the stability of the biosurfactant of Wickerhamomyces anomalus in the same pH range and found a greater variation in ST (30.5–34.7 mN/m). Such findings confirm the effectiveness of the biosurfactant produced by S. bombicola ATCC 22214 within the pH range investigated.

Satisfactory results were found for the tests in which the biosurfactant was submitted to different temperatures, with a trend of increasing ST with increasing temperature (Figure 3c). Purwasena et al. [65] analyzed the thermostability of a biosurfactant produced by Pseudoxanthomonas taiwanensis when submitted to high temperatures (120 °C), reporting that the performance of the biomolecule diminished only slightly, as occurred in the present investigation.

The biosurfactant also maintained its efficiency with the increase in salinity, reducing the surface tension to 25.82 mN/m when exposed to a 10% concentration of NaCl, as shown in Figure 3d. Studies show that microorganisms generally benefit in isotonic solutions. High salinity can slow the growth of many microorganisms, suggesting that a mildly saline solution would exert a positive effect on biosurfactant production [66]. Thus, the present findings are consistent with data from previous reports.

3.4. Critical Micelle Concentration of Biosurfactant

Once they reach the CMC, amphipathic molecules cluster together with polar moieties facing the micelle exterior and the lipophilic one facing the interior. Adding further surfactants does not lead to a greater ST decrease. Surfactants with low CMC are more efficient and, therefore, economically advantageous compared to those with a high CMC [36].

The biosurfactant produced by Starmerella bombicola ATCC 22214 lowered water ST from 72 to 26.85 mN/m, with a CMC of 0.4 g/L (Figure 4) and a yield of the isolated biosurfactant of 1.5 g/L.

Although this yield is lower than that described in the literature for the majority of biosurfactants produced by the yeast Starmerella bombicola, many bacterial and yeast surfactants have similar yields, as described by Gaur et al. [67] for C. albicans and C. glabrata cultivated in media composed of glucose and a yeast nitrogen base, with yields of 1.320 g/L and 1.6 g/L, respectively. Xia et al. [68] produced a biosurfactant from Pseudomonas mosselii, describing a CMC of 0.93 g/L, with an ST decline from 73.20 to 30.61 mN/m. In a study involving Bacillus subtillis, Sharma and Singh [69] determined a CMC of 0.5 g/L and an isolated biosurfactant yield of 3.5 g/L. Silva et al. [70] found a CMC ranging from 7 to 16 mg/mL for a biosurfactant from the yeast Pichia pseudolambica, with an ST reduction from 70.82 to 36.47 mN/m.

3.5. Ecotoxicity with Seeds

Toxicity regards the negative impact of a compound on a living organism, and the toxic effect depends on compound concentration and features in addition to exposure time [71]. In the present study, cabbage (Brassica oleracea) and tomato (Solanum lycopersicum) seeds were exposed to various biosurfactant levels (half the CMC, the CMC and twice the CMC), and toxicity was assessed based on the germination index (GI), a parameter that depends on the relative germination of seeds and growth of roots. The findings of these runs, displayed in

Table 2. Germination index of tomato (Solanum lycopersicum) and cabbage (Brassica oleracea) seeds for assessment of phytotoxicity of biosurfactant from Starmerella bombicola ATCC 22214 cultivated in mineral oil supplemented with 5.0% glycerol and 2.0% glucose at 28 °C and 200 rpm for 144 h. Results expressed as mean ± SD (n = 3).

Concentration of Isolated Biosurfactant	Germination Index (%)
	Tomato (Solanum lycopersicum)	Cabbage (Brassica oleracea)
½ CMC	90.5 ± 0.18	80.5 ± 0.11
CMC	86.0 ± 0.13	72.2 ± 0.14
2 CMC	80.8 ± 0.15	65.0 ± 0.12

, show that none of the examined solutions exerted a significant inhibition on seed germination or root growth, thus pointing to low toxicity. However, a rise in biosurfactant concentration resulted in a slight reduction in the GI of the seeds of Brassica oleracea and Solanum lycopersicum.

Umar et al. [72] investigated the optimization of the production of a biosurfactant from Bacillus subtilis SNW3 and found a greater germination rate for Solanum lycopersicum seeds at a concentration of 0.7 g/100 mL (68.75%) compared to water (control), for which the germination rate was 56.25%. Lima et al. [73] studied a biosurfactant produced by the yeast C. lipolytica UCP 0988, reporting no inhibitory effects on seed germination or root growth, with germination rates of 99, 98 and 95% for solutions at ½ the CMC (0.25 g/L), the CMC (0.5 g/L) and twice the CMC (1.0 g/L), respectively.

3.6. Toxicity with Artemia salina

Artemia salina, a microcrustacean capable of developing in highly salty niches, is often employed as a model to determine the acute toxicity of different organic and inorganic compounds. In addition to being easily cultivated, A. salina is widely available and can be easily found in markets, making it a desirable option in toxicity tests [74].

In the present study, A. salinas larvae were exposed to various biosurfactant concentrations (half the CMC, the CMC and twice the CMC) and 1.0 and 2.0% biosurfactant solutions. Survival rates ranged from 95 to 100%. The highest mortality rate (5%) was found when the biosurfactant was used at twice its CMC (0.04%) and at a concentration of 2.0%. These findings are similar to the ones reported by Lima et al. [73], who found a 95% survival rate for twice the CMC and a solution with a 2% concentration of the biosurfactant produced by C. lipolytica UCP 0988.

3.7. Chemical Composition of Biosurfactant

Known for the production of glycolipids, yeasts of the species Starmerella bombicola have been studied for their biosurfactant production potential and have considerable market prospects [75,76]. The biosurfactant in the present study was composed of 42.03% lipids and 55.90% carbohydrates, which suggests a glycolipid nature. This classification is based on the chemical composition of the biosurfactant, which includes a glucidic portion, indicating the presence of the polar fraction, and a lipid portion with an apolar nature, which confers the amphipathic characteristic essential to the reduction in surface/interfacial tension [22]. The interaction of these components gives rise to a multifunctional biosurfactant with potential for various applications, such as bioremediation, the stabilization of emulsions and the development of sustainable industrial products [22]. The biosurfactant produced by C. lunata UFPEDA885 in the study conducted by Maciel et al. [55] had 0.02% proteins, 6.5% sugars and 87% total lipids.

3.8. Dispersion of Hydrophobic Compounds in Seawater

The oil dispersion test is a fast, simple method for detecting the dispersant capacity of a biosurfactant. It consists of the reduction in water/oil interfacial tension and the enlargement of a clear zone in the presence of an efficient biosurfactant [77]. The cell-free broth (crude biosurfactant) was added to motor oil at different biosurfactant/oil ratios (1:2, 1:8 and 1:25 vol/vol) to test its dispersion power. Oil dispersion ranged from 50 to 80%, with the highest dispersion rate achieved at the 1:25 ratio, as shown in Figure 5.

Lima et al. [73] described similar results in a study involving the production of a biosurfactant from C. lipolytica UCP 0988. At concentrations of 0.5 g/L (CMC) and 0.25 g/L (½ CMC), the biosurfactant was able to disperse 70% and 40% of the motor oil, respectively, demonstrating efficiency in the dispersion of hydrocarbons.

3.9. Removal of Petroleum Derivative Adsorbed to Sand and Soils by Biosurfactant—Kinetic Assay

Engine oil removal from the reference sand in the kinetic test increased progressively with the addition of the surfactant up to the CMC. However, no additional improvement was observed utilizing it at twice its CMC. The increase in time also exerted a positive influence on the removal of the hydrophobic compound, as shown in

Table 3. Removal of engine oil (kinetic assay) from sand and soils using biosurfactant from Starmerella bombicola ATCC 22214 cultivated in mineral oil supplemented with 5.0% glycerol and 2.0% glucose at 28 °C and 200 rpm for 144 h. Results expressed as mean ± SD (n = 3).

Type of Soil	Biosurfactant	Contact Time (h)	Removal Rate (%)
Standard sand	Crude biosurfactant	0.5	16.96 ± 0.21
		1.0	27.39 ± 0.05
		2.0	53.51 ± 0.81
		24.0	55.11 ± 0.22
	Isolated biosurfactant at ½ CMC	0.5	48.89 ± 0.16
		1.0	65.66 ± 0.92
		2.0	72.26 ± 0.11
		24.0	75.75 ± 0.14
	Isolated biosurfactant at CMC	0.5	56.05 ± 0.16
		1.0	69.51 ± 0.18
		2.0	73.62 ± 0.23
		24.0	82.48 ± 0.12
	Isolated biosurfactant at 2 × CMC	0.5	55.85 ± 0.17
		1.0	69.82 ± 1.14
		2.0	74.02 ± 0.27
		24.0	82.01 ± 0.22
Clayey soil	Crude biosurfactant	0.5	62.40 ± 2.65
		1.0	77.43 ± 0.05
		2.0	77.92 ± 2.75
		24.0	88.00 ± 0.24
	Isolated biosurfactant at ½ CMC	0.5	76.87 ± 0.48
		1.0	91.00 ± 0.08
		2.0	91.40 ± 1.08
		24.0	98.68 ± 0.01
	Isolated biosurfactant at CMC	0.5	81.07 ± 0.06
		1.0	91.45 ± 0.17
		2.0	91.72 ± 2.23
		24.0	98.81 ± 0.01
	Isolated biosurfactant at 2 × CMC	0.5	85.79 ± 1.58
		1.0	92.66 ± 0.17
		2.0	93.87 ± 0.63
		24.0	99.01 ± 0.02
Silty soil	Crude biosurfactant	0.5	50.31 ± 0.45
		1.0	82.94 ± 0.05
		2.0	85.38 ± 0.04
		24.0	89.80 ± 0.01
	Isolated biosurfactant at ½ CMC	0.5	62.57 ± 1.57
		1.0	96.00 ± 0.00
		2.0	96.20 ± 0.78
		24.0	97.01 ± 0.03
	Isolated biosurfactant at CMC	0.5	68.50 ± 0.85
		1.0	96.25 ± 0.01
		2.0	97.00 ± 0.11
		24.0	97.21 ± 0.01
	Isolated biosurfactant at 2 × CMC	0.5	71.00 ± 2.18
		1.0	97.04 ± 0.08
		2.0	97.22 ± 0.63
		24.0	97.61 ± 0.00

.

The use of silty soil as the sorbent and the surfactant at half its CMC enabled the removal of up to 97.21 ± 0.01% of the contaminant. However, the increase in concentration to the CMC and twice the CMC did not exert a significant additional effect in the 24 h period. Similar behavior was found when silty soil was employed, with a removal rate of 98.81 ± 0.01% for the biosurfactant at the CMC and 99.01 ± 0.02% at twice the CMC.

The crude biosurfactant proved to be efficient in the removal of motor oil, with a somewhat better performance on the two types of soil compared to standard sand. Comparing the concentrations of the isolated biosurfactant (½ CMC, CMC and 2 CMC), no significant differences were found. Thus, the utilization of the raw biosurfactant may be a low-cost option, especially in applications that require large volumes of biosurfactant.

Studies conducted by Sharma et al. [69] revealed the effect of biosurfactants produced by Bacillus amyloliquefaciens SAS-1 and Bacillus subtilis BR-15 in the removal of motor oil from soil, demonstrating that the lipopeptide biosurfactants were able to remove between 56.91 ± 1.52 and 66.31 ± 2.32% of the oil from the soil. In a study involving a biosurfactant produced with the same yeast used in the present investigation cultivated in industrial waste products, Selva Filho et al. [53] reported removal rates of 82.30 ± 0.07%, 94.20 ± 0.07% and 96.65 ± 0.09% for standard sand, silty soil and clayey soil, respectively. Therefore, the present results demonstrate that the biosurfactant produced by Starmerella bombicola is highly efficient.

3.10. Removal of Petroleum Derivative Adsorbed to Sand and Soils by Biosurfactant—Static Assay

Biosurfactants modify the wetting ability of soil particles through the sorption of lipophilic components on the surface of particles and interactions with them in the aqueous phase. Moreover, the removal of the contaminant is optimized due to the repulsion between the polar portion of the biosurfactant and the soil particles [78,79].

The biosurfactant was tested in the removal of motor oil by its percolation through clayey and silty soils as well as standard sand. The results displayed in

Table 4. Motor oil removal (static assay) from sand and soils by biosurfactant from Starmerella bombicola ATCC 22214 cultivated in mineral oil supplemented with 5.0% glycerol and 2.0% glucose at 28 °C and 200 rpm for 144 h. Results expressed as mean ± SD (n = 3).

Soil	Removal Rate (%)
	Water (Control)	Crude Biosurfactant	Isolated Biosurfactant
			½ CMC	CMC	2 × CMC
Standard sand	10.52 ± 2.3%	49.35 ± 1.08%	52.00 ± 0.60%	63.70 ± 0.59%	72.05 ± 0.80%
Silty soil	5.67 ± 0.64%	85.48 ± 0.34%	92. 81 ± 0.21%	93.95 ± 0.43%	96.87 ± 0.30%
Clayey soil	8.12 ± 1.59%	81.40 ± 0.27%	97.15 ± 0.11%	99.20 ± 0.08%	99. 50 ± 0.03%

demonstrate the efficiency of the biomolecule for the cleaning of soils, reaching a removal rate of 99.50 ± 0.03% in the clayey soil at twice the CMC.

The use of the crude biosurfactant achieved satisfactory results, especially with silty and clayey soils, achieving removal rates of 85 ± 0.34% and 81.40 ± 0.27%, respectively. The cell-free broth also demonstrated a good performance in standard sand, with a contaminant removal rate of 49.35 ± 1.08%, as the increase in oil removal by the biosurfactant at the CMC was only 14.35% higher, enabling the application of the crude biomolecule under static conditions at a lower cost. According to Galitskaya et al. [80], the expensive separation and purification steps can be eliminated, as cell-free broths of isolates are directly usable under field conditions to improve heavy oil recovery.

Figure 6 shows the capacity of the removal of the hydrophobic compound through the percolation of the biosurfactant, demonstrating the efficiency of the biomolecule.

3.11. Cleaning of Petroleum Derivative on Oily Surface by Biosurfactant

Satisfactory removal rates were found for motor oil on the smooth surfaces of the glass recipients.

Table 5. Removal of hydrophobic compounds (motor oil and OCB1 oil) from oily surfaces due to action of biosurfactant from Starmerella bombicola ATCC 22214 cultivated in mineral oil supplemented with 5.0% glycerol and 2.0% glucose at 28 °C and 200 rpm for 144 h.

Hydrophobic Compound	Concentration of Biosurfactant
	Water (Control)	Isolated Biosurfactant
		½ CMC	CMC	2 × CMC	5 × CMC
Motor oil	25.38 ± 4.21	72.15 ± 0.69	80.83 ± 8.10	83.34 ± 2.54	85.58 ± 0.71
OCB1 oil	5.94 ± 2.21	58.93 ± 8.93	72.76 ± 0.57	86.87 ± 1.36	88.49 ± 3.49

shows that removal rate increased with raising surfactant concentration. The removal rates for the motor oil and OCB1 oil were, respectively, 85.58 ± 0.71% and 88.49 ± 3.49% using the biosurfactant at 5 CMC, demonstrating the effectiveness of the microbial surfactant in cleaning oily surfaces.

OCB1 heavy oil is a petroleum derivative with greater removal difficulty due to its high viscosity. Figure 7 shows a significant increase in the removal of the OCB1 oil from the walls of the beakers with raising surfactant concentration, demonstrating that the biomolecule effectively removes heavy oil from smooth surfaces. Barata et al. [81] performed an oil removal test on glass slides contaminated with motor oil to determine the removal power of a detergent formulated with a biosurfactant produced by Bacillus invictae UCP1617, reporting a 98.42 ± 1.02% removal rate.

3.12. Destabilization of Petroleum Derivative

The biosurfactant was tested at concentrations of half the CMC, the CMC, twice the CMC and five times the CMC for its capacity to remove OCB1 oil and diesel oil spread on Petri dishes. The graphs in Figure 8 show the removal rate of each hydrophobic substance used in the experiment. The diesel oil removal rate reached 99.87 ± 0.13%, with the lowest rate (94.44 ± 0.04%) found using the biosurfactant at half its CMC. The OCB1 removal rate was much lower than that found for diesel oil, which was expected due to the high viscosity of the heavy oil, with the highest rates around 22.22 ± 1.58% and 28.88 ± 1.40%. The difference in the volume of the biosurfactant and exposure time did not exert a direct influence on the removal rate of either petroleum derivative.

Figure 9 shows the removal of the hydrophobic pollutants by different concentrations of the biosurfactant. For OCB1, a rise in biosurfactant concentration promoted the removal process. In contrast, the biosurfactant at even low concentrations, such as half the CMC, was extremely effective at removing diesel oil.

Soares da Silva et al. [82] utilized the same test to evaluate the efficacy of a nontoxic biodetergent and a commercial detergent for the destabilization of OCB1 fuel oil. The biodetergent achieved the complete destabilization of the petroleum derivative in only two minutes, whereas the commercial product required twice the amount of time to achieve the partial destabilization of the oil.

3.13. Treatment of Oily Industrial Effluent by Biosurfactant

The isolated biosurfactant achieved an oil removal rate of 70.73% when used at half its CMC, 83.76 ± 1.53% when used at the CMC and 92.52 ± 1.18% when used at twice the CMC, highlighting high efficacy and suggesting that a higher biosurfactant concentration favors the removal of the oil. Distilled water—used as the control condition—only removed 0.75 ± 1.32% of the contaminant. Figure 10 illustrates the removal power of the microbial surfactant.

In a similar study involving the use of a biosurfactant produced by Pseudomonas cepacia for the removal of oil from a thermoelectric industrial effluent, Soares Silva et al. [50] reported similar rates to those found in the present investigation, with 40%, 60% and 100% at concentrations of ½ CMC, CMC and 2 × CMC, respectively.

Considering the highly practical nature of this study, an economic evaluation of the process for obtaining the biosurfactant produced by Starmerella bombicola ATCC 22214 was carried out. The results of this evaluation suggest the biosurfactant’s viability for industrial and environmental applications. In the present study, the production cost was estimated considering a culture medium composed of 2% glucose (USD 0.40/kg) and 5% glycerol (USD 0.25/kg), in addition to mineral salts and yeast extract. The fermentation process, conducted for 144 h, presented an average consumption of 4.2 kWh/L produced, resulting in an approximate energy cost of USD 0.63/L, considering an industrial tariff of USD 0.15/kWh. The final average production cost was estimated at USD 0.11/L based on these factors. Additionally, the cost associated with the downstream process, including centrifugation, solvent extraction, and drying, was estimated at USD 3.50 per kg of the isolated biosurfactant. In comparison, synthetic surfactants have an average cost of USD 2 per kg, while biosurfactants can range from USD 5 to USD 20 per kg [83]. However, using industrial by-products, such as glycerol, can significantly reduce these costs, making production more competitive and sustainable [84].

According to the literature, Noll et al. [83] obtained total costs for producing di-rhamnolipids between USD 12 and 56/kg, using glucose and stearic acid from the strain Pseudomonas putida KT2440. Freitas et al. [85] evaluated the commercial application of a cell-free broth of a biosurfactant produced by Candida bombicola formulated with potassium sorbate at the industrial level and estimated a cost of only USD 0.1–0.22/L. Like the production cost described in this study, Soares da Silva et al. [82] reported a cost of USD 0.11/L for producing a surfactant obtained from Pseudomonas cepacia. In their research, production in a 50 L bioreactor using industrial waste reached 40.5 g/L, demonstrating economic viability. The estimated cost was USD 0.14–0.15/L for the biosurfactant formulated with a preservative and USD 0.02/g for the isolate. Given the potential shown in this study, it is recommended that future investigations explore strategies for scaling up the production of the biosurfactant, evaluating the optimization of fermentation parameters and cost reduction in downstream processes. Additional studies on using agro-industrial waste as a substrate and implementing purification technologies can contribute to large-scale production and increase viability and competitiveness in the biosurfactant market.

4. Conclusions

Starmerella bombicola ATCC 22214 demonstrated the capacity to produce a highly efficient biosurfactant in media formulated with low-cost substrates, highlighting its significant biotechnological potential. The yeast produced compounds with surface and emulsifying activities using commercially viable raw materials. The biosurfactant exhibited an excellent performance as a tensioactive agent, achieving surface tension compatible with values described in the literature for microorganisms recognized as super-biosurfactant producers. The biosurfactant proved effective as an emulsifier of oils and hydrocarbons derived from petroleum, demonstrating its potential for the treatment of environments contaminated with heavy oils. The crude biosurfactant proved viable for direct use in the environmental field, with the advantage of significantly reducing production costs. Moreover, the biomolecule exhibited stability under extreme environmental conditions, suggesting its applicability in adverse scenarios, such as oil spills at sea and in oil recovery and industrial processes that involve high temperatures. The absence of toxicity at the concentrations tested underscores the safety of the biosurfactant for environmental and industrial purposes.

The use of the biosurfactant in the treatment of an oily industrial effluent confirmed its potential for environmental decontamination processes. Additionally, the economic analysis performed in this study suggests that biosurfactant production is feasible using low-cost raw materials, considering fermentation, energy consumption, and downstream processing. These findings align with previously reported biosurfactant data and reinforce their potential for commercial application. Lastly, the possible commercial application of the biosurfactant as a biotechnological additive aligns with environmental preservation policies and the reduction in impacts on ecosystems, consolidating it as an innovative, sustainable solution.