The Application of a New Microbial Biosurfactant to Remove Residual Oil from Electric Power Plant and to Inhibit Metal Corrosion in a Salty Environment

Abstract

Human development has led to increased production of oil and gas, mainly as energy sources, which, however, are responsible for contamination and metal corrosion in industrial, marine, and terrestrial environments. Lubricating oil, in particular, is widely used in generators and industrial machines in the electric sector and is responsible for contamination not only in industrial environments but also in many terrestrial and aquatic ecosystems. In this context, this study aimed to apply the Starmerella bombicola ATCC 222214 biosurfactant to inhibit metal corrosion in seawater and in an Accelerated Corrosion Chamber (ACC). For this purpose, its toxicity against the microcrustacean Artemia salina, its dispersion capacity, and its ability to promote oil biodegradation in a saline environment were investigated. The biosurfactant, when applied at twice its Critical Micellar Concentration (CMC), caused low mortality (30.0%) of microcrustaceans in a saline environment, and, in its crude form, the biosurfactant ensured the dispersion of no less than 77.56% of residual engine oil in seawater. Oil biodegradation by autochthonous microorganisms reached 94.39% in the presence of the biosurfactant in seawater. Furthermore, the biosurfactant, when used at twice its CMC, acted satisfactorily as a corrosion inhibitor by reducing the mass loss of galvanized iron specimens (plates) in seawater in a static system to only 0.36%. On the other hand, when the biosurfactant was added at the CMC as an atmospheric corrosion inhibitor, the reduction in mass loss of carbon steel plates treated in the ACC was 17.38% compared to the control containing only a biodegradable matrix based on vegetable resin. When the biosurfactant was incorporated into different paints applied to galvanized iron plates placed in contact with the salt spray produced in the ACC, the best result was obtained using the biomolecule at a concentration of 3% in the satin paint, ensuring a plate mass loss (29.236 g/m²) that was almost half that obtained without surfactant (52.967 g/m²). The study indicated the use of yeast biosurfactant as a sustainable alternative in combating the contamination of marine environments and metal corrosion, with the aim of preserving the environment and improving the quality of life in aquatic and terrestrial ecosystems.

1. Introduction

The main source of energy comes from oil or oil derivatives; however, the transportation and use of these resources lead to contamination of the atmosphere as well as the aquatic and terrestrial environment by hydrocarbons [1,2,3,4]. Other pollutants present in ecosystems are heavy metals, also from industrial activities. Thus, the presence of these contaminants increases toxicity and bioaccumulation processes [5,6,7,8].

The transportation of oil in maritime environments, which amounts to 35 million barrels/year, is the main source of contamination in these sites. This causes an imbalance in ecosystems through the phenomena of bioaccumulation and biomagnification. In addition, petroleum derivatives are carcinogenic and recalcitrant compounds, which cause harm to the health of living beings affected by the pollution. Several studies describe the use of chemical surfactants, which are oil derivatives, to treat oil spilled into the environment and in industries themselves. These compounds are, in fact, able to make hydrophobic pollutants bioavailable so that microorganisms can degrade them, consequently increasing their biodegradation rate. On the other hand, the synthetic nature of these surfactants also has a negative impact on the treated environments, since they are toxic and non-biodegradable. The process of obtaining these agents also releases greenhouse gases [9,10,11].

Thus, in recent decades, several studies have been conducted to develop so-called green surfactants, a category of natural and biodegradable surfactants that primarily include microbial surfactants, also known as biosurfactants, and plant surfactants [12,13,14,15]. These compounds are amphipathic and, in the case of biosurfactants produced by bacteria, yeasts, and some fungi, they are able to reduce surface and interfacial tensions between phases with distinct polarities at lower concentrations than chemical surfactants [16,17,18,19,20,21,22].

The measurement of a surfactant’s functionality is based on the Critical Micellar Concentration (CMC), which is the lowest concentration at which its molecules begin to self-aggregate. The resulting micelles are rounded structures with hydrophilic heads facing outward and hydrophobic tails facing inward, which allow the interactivity of nonpolar liquids, such as oils, with polar liquids, such as water [23,24]. The most extensively studied chemical structures of the biosurfactant class are glycolipids and lipopeptides [25,26].

Micelle formation occurs when the water surface becomes saturated with biosurfactant molecules. However, this process is not directly related to the optimal concentration for inhibiting metal corrosion, as certain surfactants exhibit maximum inhibition efficiency at concentrations superior to the critical micelle concentration (CMC). It is known that the polar portion of biosurfactant molecules has a strong affinity for metal surfaces, promoting adsorption onto the metallic substrate. As more biosurfactant is added, a continuous protective layer is formed until the surface is completely covered and adsorption reaches equilibrium [27]. However, micelle formation plays a crucial role in aqueous systems, resulting in anisotropic distribution that alters the physical and mechanical properties of water, such as the flow direction and surface tension. These spherical structures are also essential for solubilizing nonpolar compounds, encapsulating oil droplets within their hydrophobic cores, thereby facilitating the dispersion and removal of contaminants [28]. Thus, the biosurfactant serves not only as a corrosion inhibitor but also as a highly efficient multifunctional agent in both chemical and environmental processes.

Energy industries, such as thermoelectric and oil plants, can apply biosurfactants as corrosion inhibitors in their metal equipment, since, through adsorption, their molecules form a protective layer on the metal surface against these corrosive processes [5,29,30,31].

Corrosion is also responsible for damage to equipment used in the transportation of oil and gases. Paints are usually applied, which form a barrier to the corrosion process in metal equipment [32,33,34]. The transportation of gas and oil presents problems of corrosion and formation of hydrated gas agglomerates in the pipelines, which are commonly treated with anti-agglomerants and corrosion inhibitors [35,36,37]. In recent years, the study of corrosion has gained increasing prominence in the environmental field, where biosurfactants are being explored as inhibitors of atmospheric oxidation [38,39,40]. Unlike their chemical counterparts, biosurfactants have the advantage of being biodegradable and environmentally compatible, in addition to exhibiting high specificity and stability in different temperature, salinity, and pH ranges. An advance in the area of these biomolecules is the use of low-cost substrate sources such as industrial waste, which makes the production of natural surfactants economically viable [41,42,43].

In the energy industry, such as the oil industry, biosurfactants, such as glycolipids or lipopeptides, are injected into oil reservoirs, contributing to the reduction in interfacial tensions between oil and water and the breaking of capillary forces that prevent oil from escaping through rock pores in Microbial Enhanced Oil Recovery (MEOR) processes. Biosurfactants such as Emulsan, Liposan, or Alasan are described in the literature for their ability to form stable emulsions with oils, reducing their viscosity and enabling their transportation in pipelines [5,44,45].

The specificity and sustainable nature of biosurfactants have driven research in the environmental field, especially in marine environments [41,46,47]. Seawater is widely used in desalination plants and for cooling equipment, and is often in contact with equipment, commonly causing corrosion. Studies have shown that increasing the concentration of biosurfactants in contact with steel structures reduces the mass loss of these materials submerged in saline water [48,49,50,51,52]. In this context, Starmerella bombica is one of the most studied yeast species in the world and is known for producing large amounts of sophorolipids, a type of biosurfactant, as secondary metabolites. This molecule, a glycolipid, contains sophorose and a fatty acid in its structure and is widely used in various industrial fields [53].

In light of this background, the present work aimed to produce a biosurfactant from the yeast strain Starmerella bombicola ATCC 222214, evaluate its capacity to decontaminate marine environments polluted by oil, and investigate its application as an inhibitor of metal corrosion caused by physical (air and marine) factors.

2. Materials and Methods

2.1. Microorganism

Starmerella bombicola ATCC 222214 yeast cells from the American Type Culture Collection (ATCC^®) were activated at 28 °C for 48 h in an incubator in Yeast Mold Agar (YMA) medium containing 2% agar, 1% D-glucose, 0.5% peptone, and 0.3% yeast extract. The microorganism was then transferred to Erlenmeyer flasks previously autoclaved at 121 °C for 20 min containing Yeast Mold Broth (YMB) medium, pH 7.0, which had the same composition as YMA but without agar. Yeast growth occurred for 24 h at 28 °C under orbital shaking at 150 rpm. Then, dilutions of the culture medium were made until reaching a pre-inoculum of 5% with 10⁶ cells/mL to be transferred to the biosurfactant production medium.

2.2. Biosurfactant Production

For biosurfactant production, yeast was grown in mineral medium containing 0.3% NaNO₃, 0.05% KH₂PO₃, 0.005% CaCl₂∙H₂O, 0.04% MgSO₄∙7H₂O, and 0.005% FeCl₃ and supplemented with 5% residual canola oil from frying. This oil, used as an insoluble substrate, was obtained from a commercial establishment in the city of Recife, Brazil. Peel flour of potato (Solanum tuberosum L.) acquired from local commercial waste was added as a soluble substrate to the production medium at concentration of 2%. The peels were dried and ground until a powder was obtained. Urea at concentration of 0.2% was used as the nitrogen source. Following the methodology described by Selva Filho et al. [54], fermentations were carried out at 28 °C and pH 6.0 in 1000 mL Erlenmeyer flasks containing 500 mL of the medium, under agitation at 200 rpm, for 180 h, with 5% inoculum.

2.3. Biosurfactant Extraction

Biosurfactant extraction was performed according to Farias et al. [55]. After centrifugation of the metabolic liquid resulting from microbial fermentation at 4400 rpm and 4 °C for 15 min, filtration was performed on Whatmann No. 1 filter paper. Then, the cell-free metabolic liquid was transferred to a separatory funnel, ethyl acetate was added up to a 1:1 (v/v) ratio, the mixture was stirred for 15 min, and the organic phase was extracted. The residue was subjected to extraction with the solvent again. Then, the solvent was evaporated at 40 °C, and the retained portion was washed twice with hexane, which in turn was evaporated. The resulting isolated biosurfactant was quantified by gravimetric analysis to determine the surfactant production yield.

2.4. Toxicity Test with Artemia Salina

Larvae of the microcrustacean Artemia salina were acquired from a commercial fishing establishment and incubated for 24 h. Then, 10 larvae were transferred to 10 mL of seawater placed in 15 mL vials containing 1% and 2% solutions of the isolated biosurfactant at half the CMC, the CMC, and twice the CMC [56]. Distilled water was used as a control. After 24 h, the mortality of the microcrustaceans was checked. The toxic concentration was assumed to be the lowest concentration capable of causing the death of the larvae. The CMC of the biosurfactant (2.0 g/L) was previously established by Selva Filho et al. [54].

2.5. Dispersion of Engine Oil in Seawater

The dispersion test was performed by contaminating 5% of the seawater in a Petri dish with engine oil collected from an electric power plant. Biosurfactant solutions were then added in volumetric ratios of 1:2, 1:8, and 1:25 (v/v) of biosurfactant to oil. These biosurfactant solutions were previously prepared in deionized water at concentrations corresponding to half the critical micelle concentration (½ CMC), the CMC, and twice the CMC (2× CMC), as well as biosurfactant at crude form. After preparation, these solutions were applied to the system at final proportions of 1% and 2% (v/v). The effectiveness of dispersion was visually evaluated by measuring the diameter of the clear zone formed in the oil droplet [57].

2.6. Tests of Bioremediation of Oil Spill in Seawater

Microbiological determinations to evaluate the potential of the biosurfactant as a bioremediation agent were performed according to the official Standard Methods for the Examination of Water and Wastewater [58]. Initially, seawater samples (100 mL) were collected. Then, 1% motor oil was added to the flasks together with solutions of the solated biosurfactant at half the CMC, the CMC and twice the CMC. The solutions were incubated at 27 °C and 150 rpm for 30 days, and samples were taken after 1, 7, 14, 21 and 30 days to count microorganisms using the Most Probable Number (MPN) technique. Decimal dilutions of samples (1/10, 1/1000, and 1/10,000,000) were performed using sterile saline solution (0.85% NaCl, pH 7.2). In parallel, the residual oil was also quantified through gravimetric analysis.

Standard bacterial counts were performed using the pour plate technique. For this purpose, decimal dilutions were performed with buffered water by adding 1 mL of each dilution in duplicate in Petri dishes to the Nutrient Agar culture medium. Colonies were enumerated after 2 days of incubation at 35 °C, and the results were expressed in CFU/mL.

Filamentous fungi and yeasts were counted using Sabouraud Agar culture medium with chloramphenicol distributed in Petri dishes. Dilutions of the samples were added (1 mL) to the culture medium and dispersed with a glass loop (Drigalski). The results obtained after incubation at 25 °C for 5 to 7 days were expressed in CFU/mL of filamentous colonies (filamentous fungi) or milky colonies (yeasts) [59].

2.7. Inhibition of Metal Corrosion in Seawater Using the Isolated Biosurfactant

To test the ability of the biosurfactant to inhibit metal corrosion, the method described by Faccioli et al. [60] was used. Briefly, seawater from Boa Viagem Beach (Recife, Brazil) (1 L); deionized water; and solutions of the isolated biosurfactant at half the CMC, the CMC, and twice the CMC were added to plastic bottles made from PET (polyethylene terephthalate). Distilled water was used as a control instead of seawater.

Metal specimens (plates) made of galvanized iron and carbon steel were initially weighed, tied, and submerged in the fluid. After 90 days, they were removed from the fluid, cleaned, and weighed again. The dynamic process followed the static method, but with the addition of aeration pumps in each fluid contained in each bottle.

The percentage of inhibition of metal corrosion (I_c) was calculated according to the equation

$$I_c={\displaystyle \frac{M_d}{M_a}}\times 100\%$$

(1)

where M_d and M_a are the masses of specimens after and before the corrosion process, respectively, both expressed in grams.

Furthermore, the corrosion rate (T_c) was calculated as the mass loss of each specimen per unit of time, according to the formula

$${\mathit{T}}_{\mathit{C}}={\displaystyle \frac{\mathit{∆}\mathit{M}}{\mathit{∆}\mathit{t}}}$$

(2)

where ∆M and ∆t are the changes in mass and time, expressed in grams and days, respectively.

2.8. Evaluation of Biosurfactant Incorporated into Biodegradable Matrix as an Inhibitor of Corrosion Due to Atmospheric Physical Factors on Metal Surfaces

Carbon steel test specimens (Ts) were manufactured with size of 100.0 × 70.0 × 1.0 mm for mass loss tests in the Accelerated Corrosion Chamber (ACC) (Figure 1) developed by Soares da Silva et al. [61]. The Ts were previously cleaned and weighed to determine the initial mass before the test [62]. The test consisted of exposing the Ts for 30 days to a mixture of 5.0% (w/v) sodium chloride in water at a constant temperature of 40 °C and relative humidity close to 100% (simulating the reality of a maritime atmosphere). It was decided to reproduce these atmospheric conditions in an ACC because it allows for (a) obtaining results in much shorter periods of time compared to tests performed in the field, (b) controlling the factors responsible for corrosion attacks, and (c) separately studying these agents with satisfactory repeatability.

The samples were prepared as described below. The Ts were subjected to the application of a biodegradable matrix, described by Soares da Silva et al. [61], containing rosin resin, oleic acid, ethanol, calcium carbonate (CaCO₃), and the isolated biosurfactant at concentrations of half the CMC, the CMC, and twice the CMC. The biosurfactant under study was incorporated into the matrix to act as an adhesion element on the surface of the Ts (Figure 2). The Ts were cleaned and dried to perform a careful evaluation of the extent of corrosion and other visible effects.

The Ts were then subjected to the following chemical cleaning process: immersion in 26% HCl, washing in running water, neutralization with 10% NaOH solution, washing again in running water, drying, sanding, immersion in isopropyl alcohol and acetone, drying with hot air and, finally, cooling to room temperature, as described by Dantas [63] and Oliveira [64]. The objective of this treatment was to remove corrosion products. The mass loss (L), expressed in g/m², was obtained by the equation

$$L={\displaystyle \frac{M_0-M_f}{A}}$$

(3)

where M_f and M₀ are the final and initial masses after and before the corrosion process, respectively, both expressed in grams, and A is the area of the plates in m². An analytical balance was used to determine the Ts final mass, after the test, to calculate the mass loss.

2.9. Biosurfactant’s Incorporation into Synthetic Enamels

Synthetic enamels, obtained from three different companies, were applied as coating matrix incorporating the biosurfactant from S. bombicola ATCC 222214. The Ts used in this kind of test were plates (61.0 × 31.0 × 5.0 mm) made of galvanized iron, a material commonly subjected to paint applications in everyday life (Figure 3). These paints, which followed their respective Material Safety Data Sheets, presented the characteristics and compositions listed in

Table 1. Characteristics and compositions of the paints used to coat the test specimens.

Paint	Characteristics	Composition	Color	Application
Type A	Easy-to-apply alkyd resin-based enamel with quick drying and good coverage, 900 mL	Resin consisting of polyalcohols, polyacids, aliphatic solvent, active pigments, additives, drying oils, and turpentine	Ice white	Covering of external and internal surfaces of wood, metal, and masonry
Type B	Quick-drying satin synthetic enamel, 900 mL	Resin containing polyacids, polyalcohols, pigments, solvents, additives, and oils	Bright ivory	External covering of wood, metal, galvanized steel, and aluminum
Type C	Premium quick-drying synthetic enamel, 900 mL	Hydrated light petroleum distillates, xylene, toluene, cobalt octoate, manganese octoate, and methyl ethyl ketoxime	Snow white	Covering of wood, aluminum, ferrous metals, galvanized steel, and masonry surfaces

.

2.10. Application of Biosurfactant as an Additive in Synthetic Enamels to Inhibit Metal Corrosion Due to Atmospheric Physical Factors

The S. bombicola ATCC 222214 biosurfactant was added at a concentration of 3% to each type of selected synthetic enamel in order to (a) reduce its toxicity, being a natural component, and (b) inhibit metal corrosion in ACC simulating a critical marine atmosphere. Particularly, the paints were applied, as described by Pontes et al. [65], by covering the galvanized iron plates with two thin and homogeneous layers, waiting 24 h for drying, and working in ACC for 15 days.

At the end of the process, the Ts, which had been sanded and weighed before the paint was applied, were subjected to chemical cleaning, as described by Dantas [63] and Oliveira [64]. The plates were then weighed again to quantify the corrosion extent using the mass loss formula (Equation (2)). Therefore, the tested Ts plus the plates coated with paints without biosurfactant (controls) included 6 systems.

2.11. Statistical Analysis of Results

The results of experiments were obtained in quintuplicate and expressed as mean ± standard deviation. In addition, Analysis of Variance (ANOVA) was performed in order to determine the significance levels at which p-values < 0.05 were considered statistically significant.

3. Results and Discussion

3.1. Toxicity of the Biosurfactant to Artemia Salina

The test of Starmerella bombicola ATCC 222214 biosurfactant toxicity to larvae of the microcrustacean Artemia salina revealed a slightly progressive mortality rate of the bioindicator with an increasing surfactant concentration. The highest mortality rate (30%) was observed using the biosurfactant at twice the CMC (

Table 2. Mortality rate of Artemia salina, expressed as a percentage, caused by the Starmerella bombicola ATCC 222214 biosurfactant.

Biosurfactant Concentration with Deionized Water as Solvent	Artemia salina Mortality Rate
	1% Biosurfactant Solution in Sea Water	2% Biosurfactant Solution in Sea Water
½ × CMC	3.0% ± 0.8	10.0% ± 0.3
CMC	13.0% ± 0.2	17.0% ± 0.2
2 × CMC	17.0% ± 0.2	30.0% ± 0.4

), which indicates its low toxicity for the microcrustacean, enabling its application in the environment (Table 2).

The corresponding percentage of live microcrustaceans (70%) can be considered satisfactory when compared to the 100% value observed in the presence of the lipopeptide produced by the bacterium Bacillus cereus [66] and the glycolipid produced by the fungus Trichoderma sp. MK116452 [67], both at a concentration of 1.0 g/L.

Furthermore, when comparing the 70% microcrustacean survival rate observed in the present study using 0.08 g/L S. bombicola ATCC 222214 biosurfactant with the value obtained by Deivakumari et al. (80%) for the same target organism using 0.10 g/L Pseudomonas aeruginosa DKB1 surfactant [68], it is evident that the efficiency of these biosurfactants is similar.

This type of toxicity test is crucial to prove that biosurfactants are less toxic and sustainable when compared to chemical surfactants. In fact, the work of Deivakumari et al. [68] demonstrated that the chemical surfactant sodium dodecyl sulfate was capable of causing 100% mortality in this marine organism at a concentration of only 0.05 g/L. Furthermore, it should be taken into account that the concentrations tested in this study for the S. bombicola ATCC 222214 biosurfactant led to mortality values always well below 50%, that is, below the median lethal concentration (LC50), thereby exhibiting no acute toxicity.

3.2. Dispersion of Engine Oil in Seawater

The dispersion experiment demonstrated that the metabolic liquid of S. bombicola ATCC 222214 containing the crude biosurfactant dispersed the engine oil more than the isolated biosurfactant at half the CMC, the CMC, and twice the CMC (Figure 4). In the case of the isolated biosurfactant, the higher its concentration, the greater its dispersion in engine oil (

Table 3. Results of the test of engine oil dispersion in seawater due to the action of the biosurfactant from Starmerella bombicola ATCC 222214.

Biosurfactant/Oil Ratio (v/v)	Oil Dispersion Rate (%)
	1/2 × CMC	CMC	2 × CMC	Crude Biosurfactant
1:2	23.3 ± 0.9	25.6 ± 0.6	28.9 ± 0.8	73.0 ± 0.7
1:8	25.6 ± 0.9	45.0 ± 0.7	52.3 ± 0.8	77.6 ± 0.5
1:25	22.2 ± 0.6	37.5 ± 0.6	46.7 ± 0.6	70.1 ± 0.9

). It is likely, therefore, that other residual metabolites present in the metabolic liquid contributed to the dispersion. This result can be considered satisfactory if we consider that there will be no costs involved in the extraction process when using the crude preparation. Another important fact is the use of all the metabolic liquid, avoiding medium disposal, in addition to increasing the feasibility of biosurfactant application, considering the volume necessary for treatment in the event of a spill at sea.

The best ratio using the metabolic liquid was 1:8 (v/v) biosurfactant:oil, at which the engine oil dispersion reached 77.6% (Table 3). This result is not so far from that observed by Santos et al. [69] (90%) using the biosurfactant from the yeast Candida sphaerica in the same biosurfactant:oil ratio. Continuing the comparison with values reported in the literature, the crude biosurfactant from the bacterium Serratia marcescens UCP 1549, later investigated by the same authors [70], was able to disperse 88.9% of engine oil in distilled water at a ratio of 1:2 (v/v) surfactant:oil, a value very close to that obtained in this study (73.0%) in the same oil proportion but in seawater.

In conclusion, it is worth remembering that the dispersion of hydrocarbons by biosurfactants is very important, as it aids in the microbial degradation of oil spilled in seawater [71]. The greater dispersion presented by the metabolic broth and the isolated biosurfactant at twice the critical micelle concentration (CMC) demonstrates enhanced hydrophobicity and, consequently, greater dispersion of the engine oil [72]. Similarly, a mixture of the lactonic sophorolipid produced from the same yeast with a synthetic surfactant, choline oleate, achieved 81.15% dispersion in a 1:25 proportion with petroleum oil in synthetic seawater [72]. In this case, the same proportion of the metabolic liquid containing the nonanedioic biosurfactant of Starmerella bombicola reached 70.1% with the engine oil in seawater [54].

3.3. Bioremediation of Oil Spills in Seawater

The potential of the biosurfactant as an adjuvant in degradation processes was evaluated in experiments simulating an oil spill in seawater. The behavior of the autochthonous microorganisms in seawater revealed that the bacteria grew until the 21st day, reaching a count of 1.0 × 10⁷ CFU/mL, before decreasing, while the yeasts grew until reaching 2.7 × 10² CFU/mL on the 14th day. Therefore, in this sample, the bacterial population was much greater than that of the other microorganisms (Figure 5A).

In the case of seawater contaminated with engine oil, the bacteria grew up to 3.0 × 10⁷ CFU/mL on the 7th day and maintained their count stable until the end of the test (30th day). The yeasts remained stable until the 14th day; grew until the 21st day, reaching 2.1 × 10² CFU/mL; and then their population began to decline. The filamentous fungi peaked on the 1st day with a count of 3.2 × 10¹ CFU/mL, from which point onwards there was a decline. The oil biodegradation rate reached its highest value (73.57%) at the peak of yeast growth (Figure 5B).

On the other hand, when the biosurfactant was added at half the CMC, the bacterial population, which initially totaled 2.0 × 10⁷ CFU/mL, progressively decreased until the end of the test. Unlike the bacteria, the yeasts grew until the 21st day, reaching 2.1 × 10² CFU/mL, and then started to decline, while the filamentous fungi, initially totaling 8.9 × 10¹ CFU/mL, showed a progressive decrease until the 14th day. The biodegradation accompanied the bacterial growth, starting with a 70.64% engine oil removal yield, followed by a slight decrease to 68.25% on the 30th day (Figure 5C).

The addition of the biosurfactant at the CMC caused the bacteria to grow exponentially up to 6.5 × 10⁵ CFU/mL on the 14th day, reaching the stationary phase on the 21st day and then declining until the end of the run. The yeast population remained approximately stable throughout the run, with the highest count (1.3 × 10² CFU/mL) occurring on the 14th day. Filamentous fungi grew from the 7th to the 14th day, reaching 4.0 × 10¹ CFU/mL, and then their population began to decline until the end of the run. Biodegradation was stable in a similar way to yeast growth, reaching an engine oil removal yield as high as 90.76% on the 1st day (Figure 5D).

The last run was carried out using the biosurfactant at twice the CMC. The bacteria and yeasts showed qualitatively the same behavior, growing sharply until the 7th day and progressively until the 21st day, when both populations began to decrease. The largest yeast population (7.8 × 10² CFU/mL) was found precisely in this system, while the bacteria reached only 9.1 × 10⁵ CFU/mL. The filamentous fungi began to grow on the 14th day, reached a count of 2.0 × 10¹ CFU/mL on the 21st day, and then began to decline. Biodegradation increased until the 7th day and then decreased progressively. It was precisely in this system that the highest oil removal yield (94.39%) was obtained (Figure 5E).

In summary, the experiment of petroleum derivative bioremediation demonstrated that bacteria grew the most in the control, reaching 3.0 × 10⁷ CFU/mL on the 7th day, while the highest counts of yeasts (7.8 × 10² CFU/mL) and filamentous fungi (8.9 × 10¹ CFU/mL) were observed using the biosurfactant at twice the CMC on the 21st day and at half the CMC on the 1st day, respectively. The highest biodegradation yield was obtained using the highest biosurfactant concentration after 7 days (Figure 5E). Furthermore, the pH in all systems remained between 6.0 and 7.7 throughout the process, a range that is commonly found in the presence of microorganisms, being ideal for microbial growth (Figure 6).

The present study identified an optimal pH of approximately 6.0 for bacterial growth, around 7.0 for yeasts, and near 7.5 for filamentous fungi. The highest rate of oil biodegradation was observed at a pH of approximately 6.5, aligning with the findings reported by Kebede et al. [73], who previously reported and reinforced that slightly acidic to neutral conditions are optimal for enhanced microbial activity in hydrocarbon degradation. Residual products of microorganisms alter the pH, which consequently influences the availability of nutrients and the solubility of the contaminant [73]. In a study carried out by Soares da Silva et al. [74], bacteria showed greater growth up to the 14th day and fungi after the 14th day, while the highest biodegradation rate (75%) was observed using the Pseudomonas cepacia biosurfactant at concentrations equal to three and five times the CMC. As in the study carried out by Almeida et al. [75] with the biosurfactant from Candida tropicalis UCP0996, the surfactant tested in the present study provided greater growth of autochthonous microorganisms and consequently greater degradation of engine oil in seawater when compared to the control, demonstrating its ability as a bioremediation additive. The yield of petroleum-derived hydrocarbon degradation in the presence of the bacterial biosurfactant produced in situ by Enterobacter hormaechei STP-3 reached a value close to 80% in seawater within 7 days [76]. Finally, the engine oil removal yield (94.39%) obtained using the S. bombicola ATCC 222214 biosurfactant ex situ was higher than that (85%) reported for the bacterial biosurfactant from Enterobacter hormaechei by Muneeswari et al. [76].

3.4. Inhibition of Metal Corrosion by the Isolated Biosurfactant in Seawater

The experiment on metal corrosion inhibition in seawater demonstrated that in the dynamic system, greater mass loss was observed when compared to the static system, due to aeration, which made the Ts more susceptible to oxidation (Figure 7). This was verified by the values of mass loss over time illustrated in Figure 8. In addition, the biosurfactant at a concentration equal to half the CMC reduced the metal corrosion of the carbon steel specimens by no less than 99.75%. In general, there was greater inhibition in the carbon steel Ts than in the galvanized iron ones (Figure 8).

The mass loss in galvanized iron Ts using deionized water as a control reached 13.41% in the dynamic system, a value close to that reported by Faccioli et al. [60] (18%) in a similar system. Furthermore, in that study, the isolated biosurfactant from P. cepacia led to a nearly 2% mass loss decrease in galvanized iron specimens in the static system, while in the present study, the addition of S. bombicola ATCC 222214 biosurfactant at twice the CMC in a similar system ensured a mass loss as low as 0.36% (Figure 9).

In the study performed by Li et al. [77] simulating seawater conditions, the P. aeruginosa biosurfactant reduced the mass loss in carbon steel Ts by 1.96 and 0.79 mg/cm² compared to the control and the initial mass, respectively. In the present study, the S. bombicola ATCC 222214 biosurfactant at twice the CMC in a static system had a reduction that was an order of magnitude higher (16.44 mg/cm²) for galvanized iron Ts in seawater compared to the control, thereby demonstrating its great effectiveness in this regard.

In the study developed by Tang et al. [36], oleic acid-based biosurfactants were physically and chemically adsorbed on the surface of solid steel in an almost parallel direction, effectively protecting the surface, leading to a surface exposed to corrosion of only 1%. Similarly, the yeast biosurfactant resulted in a minimal mass loss of 1.01% in carbon steel in a static system in seawater. It is essential to note that the salts present in seawater render the medium highly corrosive due to electrochemical reactions, similar to the water simulated to contain H₂S and CO₂ in an oil field, which was also highly corrosive due to electrochemical reactions.

3.5. Biosurfactant in Biodegradable Matrix as an Inhibitor of Corrosion Due to Atmospheric Physical Factors

It can be seen in Figure 10 that, in the biodegradable matrix, the isolated S. bombicola ATCC 222214 biosurfactant significantly reduced corrosion by atmospheric physical factors. Particularly, the Ts mass losses per unit area in the control and in the presence of the biosurfactant at half the CMC, the CMC, and twice the CMC were, at the end of the 30-day experiment in the ACC (30 days), 123.60, 25.22, 18.93, and 22.12 g/m², respectively.

The treatment with the biosurfactant, compared to the control, led to a mass loss due to corrosion by atmospheric physical factors (17.38%) close to that due to biological factors (15.7%) reported by Faccioli et al. [60] using the P. cepacia biosurfactant. Furthermore, Soares da Silva et al. [61] reported a mass loss per unit area of 90.1 g/m² using the same biodegradable matrix under the same conditions, but incorporating the P. cepacia surfactant at half of the CMC. This proves that biosurfactants are promising in reducing mass loss on metal surfaces.

When carbon steel specimens containing the biodegradable matrix incorporating the S. bombicola ATCC 222214 biosurfactant at the CMC were exposed to salt spray in ACC at 40 °C, the mass loss (18.93 g/m²) was much lower than that extrapolated to 30 days for copper foils submerged in 1.0 M HNO₃ at 24.85 °C (10,800 g/m²) using the biosurfactant sodium N-hexadecyl valine [78] at a concentration of 80 mg/L.

3.6. Use of Synthetic Enamels with Biosurfactant to Inhibit Corrosion Due to Atmospheric Physical Factors

The galvanized iron Ts, after being subjected to the corrosion process in ACC for 15 days, were cleaned and weighed in order to measure the mass loss per unit area. This time was selected so that the corrosion process would become visually evident in specimens coated with the three selected enamels from different brands, as well as in the controls without biosurfactant.

The control specimens suffered a higher level of corrosion than those coated with paints containing the S. bombicola ATCC 222214 biosurfactant. This was confirmed by the higher values of mass loss per unit area in the control specimens coated with ice white (type A) (58.032 g/m²), bright ivory (type B) (52.967 g/m²), and snow white (type C) (82.208 g/m²) paints compared to those coated with biosurfactant-incorporating paints (36.595, 29.236 and 41.099 g/m², respectively) (Figure 11).

In the work carried out by Soares da Silva et al. [61], the same enamels were used on galvanized iron plates treated in the same ACC, but incorporating the P. cepacia biosurfactant. The loss of mass per unit area in the Ts coated with paint type A (46.010 g/m²) was approximately 25% higher than that obtained in the present study using the biosurfactant from S. bombicola ATCC 222214. The mass loss values for the coating with type B paint, in turn, were similar for both biosurfactants.

Nao-ionic gemini surfactants synthesized by Deyab et al. [79] using natural sources, including polyethenoxy di-dodecanoate, led to much higher mass loss (2916 g/m² corresponding to 0.81 mg/cm²∙h) in carbon steel Ts after 15 days of exposure to 1.0 M HCl solution at 54.85 °C than the maximum value detected in this study using the biosurfactant from S. bombicola ATCC 222214 (41.099 g/m²).

On the other hand, the study developed by Semeniuk et al. [32], who worked with rhamnolipid-incorporated paint to coat steel plates in a 0.1% NaCl solution environment at 20 °C, indicated values for corrosion rate and degree of protection of the plates against corrosion of 1.85 10⁻⁶ g/cm²∙h and 55%, respectively. Thus, the corrosion rate obtained by Semeniuk et al. [32] was lower than that of the present study, incorporating the yeast biosurfactant, which was estimated to be 8.1211∙10⁻⁶ g/cm²∙h. However, the degree of protection of the plates was similar, being 45% for the yeast biomolecule added to type B paint, even in a more critical environment (5% NaCl at 30 °C), than the rhamnolipid.

In summary, the present mass loss results demonstrated that the addition of the biosurfactant from S. bombicola ATCC 222214 to enamels improved their deposition on the metal surface of galvanized iron plates and increased the Ts resistance against corrosion resulting from the simulation of a maritime atmosphere. Thus, the biomolecule acted effectively in inhibiting metal corrosion.

As is known, the mechanism by which a biosurfactant acts to protect a metal surface against electrochemical corrosion reactions occurs through its physical adsorption on a metal surface. Furthermore, the presence of an oxygen atom in the polar moiety of a biosurfactant molecule provides a pair of electrons to the metal surface, strengthening the intermolecular bond, in addition to its nonpolar moiety forming a barrier capable of repelling the electrochemical reactions responsible for corrosion [32]. The presence of oxygen in the hydrophilic portion and of hydrocarbons in the hydrophobic portion of the biosurfactant investigated in this study was previously proven through chemical characterization by gas chromatography with mass spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance, which allowed the entify of 6,6-dimethoxyoctanoic acid and nonanedioic acid [54].

4. Conclusions

The biosurfactant from the yeast Starmerella bombicola ATCC 222214 was shown to be harmless to larvae of the microcrustacean Artemia salina and demonstrated dispersion power in seawater. The biomolecule acted as a stimulant of the growth of autochthonous microorganisms in seawater, which suggests its use as an agent to promote bioremediation processes in marine environments contaminated by oil and derivatives.

Corrosion tests in an Accelerated Corrosion Chamber also demonstrated the ability of the yeast biosurfactant to protect metal surfaces subjected to atmospheric oxidation and in seawater. This study also revealed that the biosurfactant can act incorporated into a biodegradable coating matrix and as a paint additive, contributing to the better protection of metal surfaces against corrosion. This was proven by low values of mass loss in carbon steel and galvanized iron specimens.

In conclusion, the microbial surfactant investigated in this study can be considered promising since bioremediation and corrosion processes are quite common in tropical regions with high temperatures and salinities, especially in Brazil, causing damage to both the environment and industries. Visionary developed countries are already investing in ecological corrosion inhibitors, which, in turn, are biodegradable and do not cause environmental damage. As a result, ecological products are gaining ground in the market.