Influence of Biosurfactants on the Efficiency of Petroleum Hydrocarbons Biodegradation in Soil

Abstract

Soil contamination with petroleum hydrocarbons is a serious environmental issue, necessitating the development of effective and environmentally friendly remediation methods that align with the principles of sustainable development. This study investigated the impact of selected biosurfactants on the efficiency of the biodegradation of total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAHs) in contaminated soil. Six biosurfactants—poly-γ-glutamic acid (γ-PGA), rhamnolipid, surfactin, a mixture of γ-PGA, rhamnolipids, and surfactin (PSR), as well as two commercial formulations (JBR 425 and JBR 320)—were evaluated in combination with a bacterial consortium. Biodegradation experiments were conducted under laboratory conditions for a 90-day period. The effectiveness of the tested biosurfactants was assessed using respirometric analysis, the chromatographic determination of the residual hydrocarbon content, and toxicity assays. The results showed that the application of a bacterial consortium enriched with a mixture of biosurfactants PSR (a biosurfactant concentration in the inoculating mixture: 5 g/dm³) was the most effective approach, resulting in an oxygen uptake of 5164.8 mgO₂/dm³ after 90 days, with TPH and PAH degradation rates of 77.3% and 70.32%, respectively. Phytotoxicity values decreased significantly, with TU values ranging from 6.32 to 4.62 (growth inhibition) and 3.77 to 4.13 (germination). Toxicity also decreased in the ostracodtoxkit test (TU = 4.35) and the Microtox SPT test (TU = 4.91). Among the tested biosurfactants, surfactin showed the least improvement in its bioremediation efficiency. Under the same concentration as in the PSR mixture, the oxygen uptake was 3446.7 mgO₂/dm³, with TPH and PAH degradation rates of 60.64% and 52.64%, respectively. In the system inoculated with the bacterial consortium alone (without biosurfactants), the biodegradation efficiency reached 44.35% for TPH and 36.97% for PAHs. The results demonstrate that biosurfactants can significantly enhance the biodegradation of petroleum hydrocarbons in soil, supporting their potential application in sustainable bioremediation strategies.

1. Introduction

The contamination of the environment by petroleum hydrocarbons represents a significant environmental concern in the contemporary world [1]. The primary sources of these pollutants are the activities of the petrochemical industry, oil spills, and the improper storage and transportation of fuels [2]. Consequently, substantial areas of soil and water have been contaminated with toxic compounds that exhibit a high degree of persistence in the environment [3]. These pollutants disrupt the structure and functioning of ecosystems, leading to a decline in biodiversity, alteration of biogeochemical cycles, and toxicity to soil organisms, plants, and animals [4,5].

Petroleum hydrocarbons, such as alkanes, cycloalkanes, monocyclic aromatic hydrocarbons (BTEX), and polycyclic aromatic hydrocarbons (PAHs), are hydrophobic, poorly biodegradable, and toxic compounds [6]. PAHs are of particular concern due to their mutagenic and carcinogenic properties [7,8]. The presence of these substances in the soil has been shown to result in a reduced oxygen availability, the inhibition of microbial activity, and changes in the physicochemical properties of the soil, thereby rendering it less capable of self-cleaning [9,10]. The remediation of petroleum hydrocarbon-contaminated soils is a multifaceted process that employs a range of physicochemical techniques, including thermal treatment [11], solvent extraction [12], solidification/stabilization (S/S) [13], photocatalysis [14], and oxidation processes [15]. While these approaches can be effective, they are often associated with high operational costs, require specialized equipment, and demand a stringent control of reaction conditions, which limits their feasibility for large-scale applications [16]. Consequently, biological methods such as bioremediation have gained increasing attention due to their environmental compatibility, cost-effectiveness, and efficiency under natural field conditions, aligning well with the principles of sustainability and environmentally responsible practices.

Bioremediation, defined as the treatment of the environment with microorganisms capable of breaking down toxic substances, is a highly effective method for the removal of oil pollution [17]. The process is based on the utilization of naturally occurring or introduced microorganisms (bioaugmentation) that facilitate the conversion of hydrocarbons into less harmful products, primarily carbon dioxide, water, and biomass [18]. The efficiency of this process can be significantly increased by the use of biosurfactants, defined as natural surfactant compounds produced by microorganisms [19]. It is evident that, as of 2022, the majority of microorganisms capable of producing biosurfactants are classified into a limited number of well-defined taxonomic groups [20]. Bacteria of the genus Pseudomonas constitute the majority of this group, accounting for up to 37% of all identified biosurfactant producers. Of particular note is Pseudomonas aeruginosa, which is distinguished by its efficient synthesis of rhamnolipids [21]. These biosurfactants are among the most extensively studied, exhibiting potent surfactant and emulsifying properties. The second most significant group is the Bacillus genus, comprising 34% of known biosurfactant producers [20]. It has been demonstrated that species such as Bacillus subtilis and Bacillus licheniformis are capable of producing biosurfactants [22], which have been shown to be efficacious in the remediation of hydrocarbon-contaminated environments. Among yeasts, a notable proportion (12%) is attributed to representatives of the Candida genus, including Candida bombicola [23]. Other groups of microorganisms are of lesser quantitative importance, although they are, nevertheless, important from the perspective of biological and metabolic diversity. Bacteria from the Serratia genus [24] and the filamentous fungus Rhizopus arrhizus [25] each account for 3% of the total number of known biosurfactant producers. The remaining 11% comprise a variety of strains, including consortia of microorganisms that can act synergistically in biosynthesis and biodegradation processes, rendering them a promising area for further biotechnological research [20]. Biosurfactant molecules are composed of both hydrophilic and lipophilic parts [26]. The hydrophilic groups are typically mono- or polysaccharides, amino acids, or peptides, while the hydrophobic part is constituted by long-chain fatty acids, fatty hydroxy acids, or α-alkyl-β-hydroxy acids. The structure of biosurfactants facilitates their localization at the phase boundary (e.g., oil/water), a process which has been demonstrated to reduce interfacial tensions and increase the contact surface between contaminants and microorganisms [27,28]. In contrast to synthetic surfactants, biosurfactants are characterized by their biodegradability [29], non-toxicity, and stability over a broad spectrum of pH, temperature, and salinity, thereby enhancing their environmental compatibility [30,31,32]. Biosurfactants can be classified on the basis of their chemical structure, adopting division into five main groups: glycolipids, lipoproteins and lipopeptides, phospholipids and fatty acids, biopolymers, and biological structures [33]. Glycolipids include compounds such as rhamnolipids, mannolipids, sophorolipids, trehalose lipids, and diglucosyl diglycerides [33,34,35]. Lipoproteins and lipopeptides include compounds like surfactin [36], viscosin, lichenysin, and gramicidin [35]. Phospholipids and fatty acids are represented by compounds such as spiculisporic acid and corynomycolic acids, which differ in the length of their carbon chains. Biopolymers include emulsan, biodispersan, allasan, liposan, polysaccharide–protein complexes, mannoproteins, and carbohydrate–protein complexes [33,35]. Additionally, biosurfactants may occur in the form of biological structures such as vesicles or entire cells that exhibit surface-active properties. An alternative classification of biosurfactants, commonly found in the literature, is based on their molecular weight [37]. This approach distinguishes low-molecular-weight biosurfactants, up to approximately 1500 Da, which mainly include glycolipids (such as rhamnolipids, sophorolipids, and trehalose lipids), lipopeptides (e.g., surfactin, gramicidin S, and polymyxin), as well as certain small proteins [37,38]. In contrast, high-molecular-weight biosurfactants (up to approximately 100 kDa) comprise polysaccharides, lipoproteins, and other complex polymeric molecules [37,39]. This classification reflects not only structural differences, but also the diverse physicochemical and functional properties of these compounds. Biosurfactants have been demonstrated to enhance the biodegradation efficiency by means of several mechanisms [40]. Firstly, biosurfactants can improve the solubility of hydrocarbons in water [41,42]. Secondly, they can mobilize contaminants from the surface of soil particles [43,44,45]. Thirdly, they can stimulate microbial growth and activity by serving as a carbon source and supplying essential nutrients necessary for proper microbial development [20,45,46]. Moreover, biosurfactants can reduce the toxic effects of hydrocarbons on microbial cells by altering cell surface hydrophobicity and membrane permeability, thereby increasing the cell’s tolerance to hydrophobic compounds [47]. Furthermore, biosurfactants have been shown to facilitate the emulsification of hydrophobic organic compounds, thereby significantly enhancing the rate of their biological decomposition [48,49,50,51].

2. Materials and Methods

2.1. Materials

The soil in this area has been heavily contaminated with petroleum hydrocarbons, with a TPH fraction concentration of 6062.59 mg/kg d.m. and a polycyclic aromatic hydrocarbon (PAH) content of 12.05 mg/kg d.m. The study material was sourced from the site of a former waste pit located in southeastern Poland. Soil samples were collected from two depth intervals of the near-surface layer: the samples were collected at depths ranging from 0 to 0.25 m and from 0.25 to 1.00 m. Subsequent to collection, the samples were meticulously placed in hermetically sealed, appropriately labeled containers and expedited to the laboratory for immediate analysis. In the context of laboratory experimentation, the samples were subjected to rigorous mixing and homogenization procedures to ensure the generation of a representative test material for subsequent analysis [52].

The bioremediation study utilized a bacterial consortium (MC), which was developed on the basis of indigenous microorganisms isolated from oil hydrocarbon-contaminated waste pits. The MC consortium comprised the following bacterial strains: Dietzia sp. IN118, Gordonia sp. IN101, Mycolicibacterium frederiksbergense IN53 (formerly Mycobacterium frederiksbergense IN53), Rhodococcus erythropolis IN119, Rhodococcus globerulus IN113, and Raoultella sp. IN109. The composition and method of construction of the consortium has been described in detail in earlier works [52,53].

Five biosurfactants of commercial availability and one mixture developed in the laboratory were utilized in the ongoing study of surfactant-assisted bioremediation. Each formulation was tested at two concentrations: 1 g/dm³ and 5 g/dm³. The following biosurfactants were included in the analysis: γ-polyglutamate (γ-PGA) (Ambioteco, Polska), rhamnolipids (Sigma-Aldrich, Burlington, MA, USA), surfactin (Sigma-Aldrich, Burlington, MA, USA), and commercial formulations JBR 425 (Janeil Biotechnology, Saukville, WI, USA) and JBR 320 (Janeil Biotechnology, Saukville, WI, USA). Furthermore, a mixture designated PSR was applied comprising γ-PGA, rhamnolipids, and surfactin, prepared at a volume ratio of 1:1:1.

2.2. Methods

Experiment Description

A series of studies were conducted to explore the potential of bioremediation in addressing soil contamination by petroleum hydrocarbons. This research was conducted under controlled laboratory conditions, utilizing the Oxi-Top (WTW, Weilheim, Germany) kit as a primary experimental apparatus. Experiments were conducted on soil samples of 30 g, contaminated with petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAH) under strictly controlled conditions. The temperature was maintained at 20 °C, the soil moisture content at 20%, and the air access and duration of the experiment was 90 days.

In the course of this study, a total of 26 experimental variants were prepared, the distinguishing features of which were the presence of the MC bacterial consortium, the type of biosurfactant utilized, and its respective concentration. The biosurfactants which were the focus of the present study included poly-γ-glutamic acid (γ-PGA), surfactin, rhamnolipids, biosurfactant preparations, JBR 425 and JBR 320, as well as a proprietary mixture designated PSR, consisting of γ-PGA, surfactin, and rhamnolipids in a 1:1:1 ratio. Variants 1–12 comprised samples containing both the bacterial consortium MC and biosurfactant formulations. For each biosurfactant type, measurements were taken at two concentrations (1 g/dm³ and 5 g/dm³). The remaining control variants (13–24) contained only the respective biosurfactants at concentrations of 1 g/dm³ or 5 g/dm³, without the consortium. Variant 25 was a control sample that was not inoculated, while variant 26 was inoculated with the bacterial consortium MC. Figure 1 shows the experimental design.

The experiment was performed in five replicates. During the course of this study, measurements of oxygen consumption were carried out in the analyzed sample enrichment variants (respirometric analyses), with the aim of reflecting microbial activity in the variants. The detailed procedure of the respirometric analyses is described in the Supplementary Materials. Subsequent to the completion of the measurements, the soil samples were subjected to a process of mixing and homogenization in order to obtain a sample that would be representative of the soil in question for the purposes of this study as a whole. This process included chromatographic analyses and ecotoxicological assessments.

The quantification of the TPH content in soil samples was conducted by gas chromatography, which was performed using a Clarus 500 chromatograph (PerkinElmer, Waltham, MA, USA) [52,53]. The analysis of the PAHs content was conducted by high-performance liquid chromatography (HPLC) on a Vanquish Core Series instrument (Thermo Scientific, Waltham, MA, USA) [54]. Concurrently, an ecotoxicological assessment was conducted employing standardized Phytotoxkit, Ostracodtoxkit, and Microtox SPT toxicological assays. The detailed procedures for the respirometric and chromatographic analyses and ecotoxicological tests are described in the authors’ previous publications [52] and in the Supplementary Materials (Tables S1 and S2).

2.3. Data Analysis and Statistical Information

The statistical analysis was conducted using Statistica 14.0 (TIBCO Software Inc., Palo Alto, CA, USA). Prior to further analysis, the data were tested for normality. Subsequently, one-way analysis of variance (ANOVA) was performed and in instances where significant differences were identified, Tukey’s post hoc test was employed for multiple comparisons. Standard deviation (SD), relative standard deviation (RSD, %), and Pearson correlation coefficients were also calculated. Statistical significance was considered at p < 0.05 [52,53].

3. Results

3.1. Respirometric Tests

In the 90-day respirometric tests conducted, the microbial activity in the soil samples was evaluated on the basis of oxygen consumption, expressed in mgO₂/dm³. The results demonstrated significant variations between the analyzed experimental variants (Figure 2).

In the blank sample (non-inoculated sample), minimal oxygen consumption (15.51 mgO₂/dm³) was recorded, confirming the absence of biological activity in this sample. Conversely, in the control sample, comprising soil inoculated with the bacterial consortium, a considerably elevated level of oxygen consumption (1944.8 ± 71.95 mgO₂/dm³) was detected, signifying an augmentation in microbial activity, associated with the biodegradation of petroleum pollutants by the bacterial strains present in the consortium. The incorporation of selected biosurfactants at a concentration of 1 g/dm³ into the bacterial consortium resulted in a substantial augmentation in the oxygen consumption within the examined samples. The highest values were recorded for the biosurfactant mixture of PSR (4131.9 ± 148.24 mgO₂/dm³), JBR425 (3885.3 ± 142.12 mgO₂/dm³), and JBR320 (3752.8 ± 133.33 mgO₂/dm³). The bacterial consortium exhibited suboptimal results when enriched with γ-PGA and rhamnolipids, exhibiting oxygen consumption levels of 3486.6 ± 128.31 mgO₂/dm³ and 3467.6 ± 125.39 mgO₂/dm³, respectively, after a 90-day period. Conversely, the inoculating mixture that was enriched with surfactin resulted in a variant oxygen consumption of 3332.3 ± 121.08 mgO₂/dm3. Research undertaken in analogous conditions on soil samples enrichment only with biosurfactants (i.e., without consortium) at a concentration of 1 g/dm³ demonstrated significantly diminished oxygen consumption during the experiment. The results of the experiment revealed that the presence of microorganisms is crucial for efficient biodegradation processes, as evidenced by the following measurements: 1081.6 ± 40.69 mgO₂/dm³ (surfactin), 1052.2 ± 38.95 mgO₂/dm³ (PSR mixture), 1446.2 ± 53.95 mgO₂/dm³ (JBR425), and 1281.3 ± 47.40 mgO₂/dm³ (JBR320) (Figure 2a). The results obtained from samples inoculated with a bacterial consortium enriched in biosurfactants and biosurfactants alone indicate a synergitic effect between microorganisms and biosurfactants, resulting in an intensification of the biological activity (Table S2 in Supplementary Materials).

It was established that an increase in the concentration of biosurfactants in the bacterial consortium to 5 g/dm³ resulted in a further increase in microbial activity. In a manner analogous to the studies conducted on lower surfactant concentrations, the highest oxygen consumption values were observed in the variant inoculated with the bacterial consortium enriched with a mixture of PSR (5164.8 ± 188.85 mgO₂/dm³), JBR425 (5087.2 ± 182.10 mgO₂/dm³), and JBR320 (4956.2 ± 179.25 mgO₂/dm³). The incorporation of γ-PGA, surfactin, and rhamnolipids into the inoculation mixture resulted in oxygen consumption levels of 4440.2 ± 161.36 mgO₂/dm³, 3446.7 ± 125.22 mgO₂/dm³ and 4258.3 ± 155.47 mgO₂/dm³, respectively, in the tested variants after a 90-day period. The values of oxygen consumption in the control samples (following the introduction of biosurfactants alone into the soil at a concentration of 5 g/dm³) ranged from 1273.2 ± 41.59 to 1749.8 ± 65.77 mgO₂/dm³ (Figure 2b).

3.2. Chromatographic Analysis

Chromatographic analyses demonstrated that the incorporation of biosurfactants within the inoculation mixture resulted in a substantial enhancement of the biodegradation efficiency of petroleum hydrocarbons within the soil environment. The findings unequivocally demonstrate that the type of surfactant employed, as well as its concentration, exert a substantial influence on the extent of degradation of contaminants. Figure 3 provides a summary of the biodegradation levels of TPH and PAH in the examined variants.

Tests conducted on a blank sample (non-inoculated sample) showed marginal degradation of the compounds (0.2% TPH and 0.05% PAH), confirming that the spontaneous treatment of soil contaminated with TPH and PAH under these conditions is minimal and requires the support of bioremediation processes. The inoculation of the soil with the bacterial consortium resulted in a decrease in the concentration of TPH from 6062.59 mg/kg dry matter (DM) to 3375.26 mg/kg DM (44.33%) and in the concentration of polycyclic aromatic hydrocarbons (PAH) from 12.05 mg/kg DM to 7.60 mg/kg DM (36.97%). These results indicate the ability of microorganisms to degrade contaminants independently, albeit with less efficiency than in variants enriched with biosurfactants.

The addition of biosurfactants to the bacterial consortium resulted in a substantial enhancement in the efficiency of petroleum substance removal from soil. The highest level of TPH and PAH degradation was achieved for the sample inoculated with the consortium MC and the PSR biosurfactant mixture. At a PSR concentration of 5 g/dm³, a decline in the concentration of TPH to 1376.34 mg/kg s.m. (77.30%) and PAH to 3.58 mg/kg DM (70.32%) was observed, representing the most optimal outcome among all the variants evaluated. Variants inoculated with the bacterial consortium enriched with JBR425 and JBR320 at a concentration of 5 g/dm³ demonstrated equivalent levels of efficiency. The final concentrations for variant 11 were as follows: the TPH concentration was found to be 1554.68 mg/kg, DM (74.36%), while the PAH concentration was determined to be 4.14 mg/kg, DM (65.68%). In variant 12, the TPH concentration was found to be 1641.94 mg/kg DM (72.92%), while the PAH concentration was determined to be 4.37 mg/kg DM (63.71%). The utilization of reduced concentrations of biosurfactants within the inoculating mixture yielded diminished biodegradation efficiency; nevertheless, the outcomes attained following a three-month treatment period remained satisfactory. In soil inoculated with the bacterial consortium enriched with a PSR mixture at a concentration of 1 g/dm³, the concentrations of TPH and PAH were 2067.09 mg/kg DM and 4.64 mg/kg DM, respectively, corresponding to degradation rates: the mean values for TPH and PAH were found to be 65.90% and 61.53%, respectively. In samples enriched with the bacterial consortium, along with the addition of JBR425 and JBR320 at a concentration of 1 g/dm³, the degradation efficiency of TPH was observed to range from 61.29% to 69.45%, while that of PAH was found to be between 56.20% and 58.87%.

Tests conducted on samples to which only biosurfactants were added demonstrated a significantly diminished biodegradation efficiency. For samples containing biosurfactants at a concentration of 5 g/dm³, the range of TPH degradation was from 12.13% to 19.32%, and the range of PAH degradation was from 6.84% to 11.74%. These findings suggest that the use of biosurfactants alone is not as effective as might have been expected. Analogous trends were observed at lower concentrations (1 g/dm³), where TPH degradation did not exceed 16.32%, and PAH degradation was 10.15%.

A comprehensive analysis of data pertaining to the degradation of TPH across a range of carbon chain lengths (n-C6-n-C9, n-C10-n-C21, n-C22-n-C30, and n-C31-n-C36) has been conducted. The analysis revealed a discernible correlation between the efficiency of biodegradation and the chemical structure of the pollutants. It has been demonstrated that hydrocarbons with shorter carbon chains (n-C6-n-C9 and n-C10-n-C21) are subject to biodegradation at a faster rate than heavier fractions containing longer carbon chains (n-C22-n-C30 and n-C31-n-C36). Figure 4 presents the average degradation percentage of n-alkanes with different carbon chain lengths across various experimental variants.

The highest degradation values were recorded for the n-C10-n-C21 fraction, in which the level of reduction in samples inoculated with the bacterial consortium with the addition of biosurfactants at a concentration of 5 g/dm³ usually exceeded 90%. The n-C6-n-C9 fraction, which contains light hydrocarbons, also exhibited a notably elevated level of biodegradation (in the range of 75.28–92.62%), despite its share of the total TPH mass typically being less than that of the medium alkane fractions. For the n-C22-n-C30 fraction, degradation values were typically in the range of 61.94–79.71% in variants inoculated with the bacterial consortium, with the addition of biosurfactants (5 g/dm³). Conversely, for the n-C31-n-C36 fraction, the degradation level rarely exceeded 50%, even in the most efficient variants.

The findings obtained from the experiments involving samples inoculated with biosurfactant-enriched samples at reduced concentrations (1 g/dm³) and the samples enriched exclusively with biosurfactants substantiate this correlation. However, it is noteworthy that the biodegradation rates in the various fractions were considerably diminished in these instances.

In a manner analogous to aliphatic hydrocarbons, a clear relationship was also observed for PAHs between the biodegradation efficiency and the structure of the molecule or, more specifically, the number of aromatic rings. Low-molecular-weight PAHs (containing two-to-three aromatic rings per molecule) have been shown to biodegrade more efficiently than high-molecular-weight PAHs (containing four or more aromatic rings per molecule). Variants incorporating the bacterial consortium and biosurfactants at a concentration of 5 g/dm³ exhibited the highest degradation efficiencies for low-ring PAHs, with naphthalene demonstrating the greatest degree of degradation (66.11–88.32%). For three-ring PAHs, the values ranged from 49.81 to 66.16%. For the same variants, the degree of degradation of five- and six-ring PAHs was noticeably lower, ranging from 25.84 to 38.22%. In the variants with a lower concentration of biosurfactants (1 g/dm³), the values of average biodegradation degrees were correspondingly lower, and for low-molecular-weight PAHs, they were in the range of 41.33–77.28%, while for high-molecular-weight PAHs, they were in the range of 21.44–36.60%. In control variants enriched exclusively with biosurfactants, the level of PAH biodegradation was found to be significantly lower for both low- and high-molecular-weight PAHs. However, it was also observed that the biodegradation spheres exhibited a dependence on the number of aromatic rings present in the hydrocarbon molecule. Figure 5 shows the distribution of the average PAH degradation values across different experimental variants (X-axis), categorized by the number of aromatic rings in the hydrocarbon molecules.

In evaluating the efficacy of the implemented remediation treatments, the values of the biodegradation coefficients (n-C17/Pr and n-C18/Ph) constitute a pivotal component of the analysis. It was observed that the enrichment of the bacterial consortium with biosurfactants led to a significant reduction in the values of these coefficients compared to the samples inoculated with the bacterial consortium only. This finding suggests that biosurfactants have the capacity to enhance the bioavailability of hydrocarbon contaminants to microorganisms, thereby increasing biodegradation processes. In comparison, the samples containing only biosurfactants exhibited higher values of biodegradation coefficients, which may indicate their limited effectiveness in the absence of microbial activity. The values of the biodegradation coefficients in the soil after the bioremediation process in the tested variants are presented in

Table 1. Summary of biodegradation rates of n-C17/Pr and n-C18/Ph in soil after bioremediation in the tested variants at biosurfactant concentrations of 1 g/dm³ and 5 g/dm³ in the inoculation solution (repetition number n = 7–10, p < 0.05).

Sample	n-C17/Pr	n-C18/Ph
Initial soil G6 (control sample)	2.716 ± 0.076	2.575 ± 0.075
Soil G6 + bacterial consortium	1.247 ± 0.027	1.303 ± 0.023
The concentration of biosurfactant in the enriching mixture is 1 g/dm3
Soil G6 + MC + γ-PGA	0.781 ± 0.009	0.818 ± 0.010
Soil G6 + MC + surfactin	0.959 ± 0.011	1.004 ± 0.016
Soil G6 + MC + rhamnolipids	0.845 ± 0.015	0.936 ± 0.026
Soil G6 + MC + mixture PSR	0.604 ± 0.016	0.665 ± 0.015
Soil G6 + MC + JBR425	0.802 ± 0.012	0.808 ± 0.022
Soil G6 + MC + JBR320	0.871 ± 0.011	0.925 ± 0.025
Soil G6 + γ-PGA	2.286 ± 0.056	2.350 ± 0.060
Soil G6 + surfactin	2.241 ± 0.059	2.282 ± 0.058
Soil G6 + rhamnolipids	2.273 ± 0.053	2.247 ± 0.053
Soil G6 + mixture PSR	2.192 ± 0.052	2.227 ± 0.057
Soil G6 + JBR425	2.125 ± 0.055	2.232 ± 0.052
Soil G6 + JBR320	2.172 ± 0.048	2.199 ± 0.049
The concentration of biosurfactant in the enriching mixture is 5 g/dm3
Soil G6 + MC + γ-PGA	0.257 ± 0.007	0.267 ± 0.003
Soil G6 + MC + surfactin	0.688 ± 0.012	0.719 ± 0.011
Soil G6 + MC + rhamnolipids	0.364 ± 0.006	0.408 ± 0.008
Soil G6 + MC + mixture PSR	0.164 ± 0.004	0.173 ± 0.003
Soil G6 + MC + JBR425	0.243 ± 0.007	0.252 ± 0.008
Soil G6 + MC + JBR320	0.273 ± 0.003	0.284 ± 0.006
Soil G6 + γ-PGA	2.286 ± 0.056	2.225 ± 0.055
Soil G6 + surfactin	2.251 ± 0.059	2.392 ± 0.052
Soil G6 + rhamnolipids	2.184 ± 0.046	2.235 ± 0.045
Soil G6 + mixture PSR	2.108 ± 0.042	2.109 ± 0.041
Soil G6 + JBR425	2.233 ± 0.043	2.211 ± 0.043
Soil G6 + JBR320	2.266 ± 0.056	2.193 ± 0.057

.

3.3. Ecotoxicological Assessment

The evaluation of the effectiveness of the applied remediation treatments was supplemented by an analysis of soil ecotoxicity after the treatment process in the tested variants. For the purpose of this study, the Phytotoxkit test was employed and the resulting data were converted into toxicity index (TU) values. A summary of the TU values in the Phytotoxkit test obtained for soil samples after bioremediation was completed in the tested variants of the process conduct is provided in Figure 6.

The analyses demonstrated that the enrichment of the MC bacterial consortium with biosurfactants had a clear effect on reducing the soil toxicity, as evidenced by the analysis of key test parameters, including seed germination and root length inhibition, in test organisms (Sinapis alba, Sorghum saccharatum, and Lepidium sativum). For the initial (contaminated) soil sample, prior to remediation, the toxicity coefficient values for germination ranged from 9.02 to 11.24, while for root length inhibition, the values ranged from 8.70 to 12.56. The lowest G6 soil TU values were observed in the variants inoculated with the MC bacterial consortium enriched with the PSR mixture and JBR425 and JBR320 preparations, with a biosurfactant concentration in the inoculating mixture of 5 g/dm³. In these variants, the post-experiment soil TU values for plant germination were 3.77 for Sinapis alba in the variant with PSR, 4.05 with JBR425, and 4.19 with JBR320, respectively. For Sorghum saccharatum, the values were 4.13, 4.49, and 4.65, respectively, while for Lepidium sativum, they were 4.58, 4.92, and 5.08. In the case of root growth inhibition, reductions in soil TU were observed for Sinapis alba, with values of 3.62 (MC + PSR), 3.89 (MC + JBR425), and 4.02 (MC + JBR320). For Sorghum saccharatum, the corresponding values were 4.19, 4.52, and 4.67, while for Lepidium sativum they were 4.62, 5.01, and 5.20, respectively (Figure 7b). For the lower concentration of biosurfactants in the mixture with the bacterial consortium of 1 g/dm³, slightly higher toxicity coefficient values were observed. For germination, the G6 soil TU values against Sinapis alba were 4.79 in the PSR variant, 5.16 with JBR425, and 5.31 with JBR320. For Sorghum saccharatum, the corresponding values were 5.44, 5.92, and 6.11, respectively. For Lepidium sativum, the values were marginally higher at 5.83, 6.29, and 6.46, respectively. For root growth inhibition, G6 soil TU values against Sinapis alba were 4.61 (MC + PSR), 4.97 (MC + JBR425), and 5.11 (MC + JBR320), respectively, for Sorghum saccharatum 5.39, 5.83, and 6.00, and for Lepidium sativum, 6.07, 6.62, and 6.83, respectively. Surfactin was found to be the least effective biosurfactant in this study. Its presence in the inoculating mixture, at a concentration of 1 g/dm³, resulted in a decline in G6 soil TU, ranging from 5.75 to 7.01 (germination) and 5.53 to 7.48 (root length inhibition). An increase in the surfactin concentration to 5 g/dm³ yielded only marginally superior outcomes; TU values decreased by 5.20–6.35 (germination) and 5.01–6.69 (root length inhibition) (Figure 7a).

Tests conducted on control samples enriched only in biosurfactants, without the MC bacterial consortium, showed only a slight decrease in soil toxicity to test organisms. The toxicity coefficient values for these samples for germination ranged between 7.94 and 10.29, and for root growth inhibition, between 7.66 and 11.57. The findings of this study demonstrate that the variants incorporating a blend of bacterial consortium and biosurfactants emerged as the most efficacious in mitigating soil toxicity. The efficacy of this mitigation was found to be contingent upon both the nature of the biosurfactant employed and its concentration.

Another test employed to evaluate the ecotoxicity of the soil following the treatment process in the variants examined was the Ostracodtoxkit test, in which the test organism is Heterocypris incongruens. In a manner analogous to the Phytotoxkit test, the results of the test, including the mortality and growth inhibition of bivalve mollusks, were converted into toxicity index (TU) values. A summary of the TU values in the Ostracodtoxkit test obtained for soil samples after bioremediation was completed in the tested variants is provided in Figure 7a.

The initial soil (G6) exhibited the highest toxicity value (TU) of 10.5, signifying the most pronounced toxic effect against the bioindicators employed among all the samples examined. The inoculation of the soil with the bacterial consortium (MC) alone resulted in a decrease in toxicity to TU = 8.05, suggesting partial detoxification by microbial action. The lowest TU values (and, therefore, the lowest toxicity) were obtained after the simultaneous application of the bacterial consortium MC and biosurfactants. When the soil was inoculated with the consortium enriched with biosurfactants at a concentration of 5 g/dm³, a range of TU values was observed, ranging from 4.35 to 6.42. The lowest toxicity was found for the combination of the bacterial consortium MC + PSR mixture, and the highest for bacterial consortium MC + surfactin. Slightly higher TU values were obtained with a lower concentration of biosurfactants (1 g/dm³), whose values ranged from 5.81 to 7.18. In soil samples inoculated with biosurfactants only, total toxicity values (TU) remained high, ranging from 9.79 to 10.15, indicating that biosurfactants used in isolation did not significantly reduce the soil toxicity.

In order to complement the ecotoxicological analysis, an evaluation of the toxicity of soil samples was conducted using the Microtox SPT test. In this test, the indicator organism is the bioluminescent bacterium Vibrio fischeri.

As with the other tests, the initial soil sample (G6) exhibited the highest level of toxicity (TU = 13.05), indicating the presence of substances that are harmful to aquatic organisms. Inoculating the soil with the MC bacterial consortium alone resulted in a marked decrease in toxicity (TU = 8.87) due to the bacteria breaking down xenobiotics. Significantly lower TU values were recorded in samples inoculated with both the MC bacterial consortium and biosurfactants at a concentration of 5 g/dm³. The TU values in this group ranged from 4.91 to 7.05. The soil sample with the lowest toxicity level was inoculated with the MC bacterial consortium enriched with the PSR mixture (TU = 4.91), and for the G6 soil sample, inoculated with the bacterial consortium with the addition of surfactin (TU = 7.05). At a lower concentration of biosurfactants (1 g/dm³) in the inoculating mixture, the TU values of the soil samples ranged from 6.42 to 7.87. Tests conducted on control samples enriched only with biosurfactants at concentrations of 1 and 5 g/dm³ showed a slight decrease in TU values relative to the initial sample. These values were in the ranges of 11.6–12.05 and 11.33–11.98, respectively, confirming the low effectiveness of biosurfactants alone in treating soils contaminated with petroleum hydrocarbons. A summary of the Microtox SPT test results for all soil samples after the experiment is provided in Figure 7b.

4. Discussion

Contamination of the soil and groundwater environment with petroleum hydrocarbons, including fractions of TPH and PAHs, is one of the key challenges of modern environmental protection [55]. Due to their persistence, low solubility, and high affinity for organic matter, these compounds exhibit limited bioavailability, making their biodegradation much more difficult [56,57]. In response to these challenges, combinatorial methods, such as, among others, biosurfactant-assisted bioaugmentation, are receiving increasing attention [58]. The employment of meticulously selected consortia of microorganisms, endowed with the capacity to degrade pollutants, in conjunction with biosurfactants that augment the bioavailability of hydrophobic compounds, has the potential to markedly enhance the efficacy of bioremediation processes [31,59]. These methods have been proven to enhance the efficiency of contaminant elimination whilst concurrently fostering sustainable development by minimizing the environmental impact, eschewing the use of deleterious chemicals, curtailing secondary pollution, and preserving ecosystem integrity. Biosurfactant-assisted techniques exemplify the balance between technological efficiency and environmental responsibility, rendering them particularly relevant in modern, sustainability-oriented environmental management.

The experimental results obtained unequivocally confirm the effectiveness of using a biosurfactant-assisted bioaugmentation strategy in the bioremediation of soils historically contaminated with petroleum hydrocarbons. The application of the selected bacterial consortium, comprising strains from the genera Rhodococcus, Dietzia, Mycolicibacterium, Gordonia, and Raoultella, to soil through inoculation resulted in a substantial enhancement of the degradation parameters, particularly in the presence of biosurfactants (γ-PGA, surfactin, rhamnolipids, the PSR mixture, and the commercially available preparations JBR425 and JBR320). This process led to an increase in the bioavailability of the contaminants.

Respirometric and chromatographic analyses indicated that biosurfactants played a key role in enhancing the efficiency of the biodegradation of oil-derived pollutants by microorganisms present in the MC consortium. In the course of respirometric analyses, the highest oxygen consumption, which is an indicator of intensive microbial activity, was recorded in samples enriched with the MC consortium and PSR mixture, which contain three biosurfactants (γ-PGA, surfactin, and rhamnolipids), and MC with the commercial preparations JBR425 and JBR320. Conversely, the lowest level of activity was exhibited by the variants containing the MC consortium and surfactin. The findings suggest a clear relationship between the capacity of TPH and PAH biodegradation and the type of biosurfactant employed. It can be assumed that mixtures of different surfactants, characterized by a more complex composition, interact more effectively with the diverse microorganisms present in the bacterial consortium, providing a broader spectrum of nutrients necessary for their growth and activity. Such conditions promote the intensification of metabolic processes of microflora in complex systems, which in turn may result in the increased bioavailability of contaminants and accelerated biodegradation [60,61]. This observation is corroborated by the finding that, in variants inoculated with the bacterial consortium alone or with bio-surfactants alone, significantly lower oxygen consumption was observed during the experiment. The observations made during respirometric analyses were corroborated by the results of chromatographic analyses, which were used to determine the concentrations of petroleum hydrocarbons (TPH, aliphatic hydrocarbons, and PAHs) in the initial soil sample and in the soil samples after bioremediation in the tested treatment variants. The optimal conditions for achieving maximum hydrocarbon biodegradation were determined to be variants augmented with the bacterial consortium and biosurfactants. The application of biosurfactants resulted in a substantial decrease in the concentrations of TPH and PAHs in the soil, in comparison to the baseline soil sample and the control sample inoculated solely with the biosurfactant. The highest efficiency was obtained with the PSR biosurfactant mixture at a higher concentration (5 g/dm³) in combination with the MC consortium, with the reduction of contaminants exceeding 70%, indicating a strong synergistic effect of these components. It was also observed that equivalent results were attained with reduced concentrations of PSR, though the efficacy was marginally diminished. Conversely, samples with surfactin exhibited reduced biodegradation efficiency, particularly at lower concentrations, indicating the limited efficacy of this biosurfactant under the examined conditions. For the purpose of comparison, the use of the bacterial consortium alone allowed for a moderate reduction in contaminants. Conversely, experiments with biosurfactants alone, without the participation of microorganisms, showed the lowest efficiency, especially with regard to TPH and PAHs, where reductions did not exceed 20% and 12%, respectively. The findings of the present research were consistent with those reported by others. For instance, in a study conducted by Zhang, the application of rhamnolipids in PAH-contaminated soil increased the degradation efficiency by more than 50% compared to the control sample [62]. A further study demonstrated that the incorporation of surfactin resulted in the acceleration of the degradation of phenanthrene and pyrene in PAH-contaminated soils [63]. Owsianiak et al. noted that the use of rhamnolipids in the biodegradation of hydrocarbons increased the degradation efficiency to 77% after 7 days, compared to 58% in a sample without biosurfactants [64]. In turn, Zeng et al. showed that the use of monoramnolipids significantly improved hexadecane biodegradation in a Candida yeast monoculture, achieving 93% degradation after 4 days, while only 78% was achieved in the sample without biosurfactant [65]. Husein’s observations confirmed the conjecture that biosurfactant mixtures allow better pyrene degradation efficiency. In their study, they showed that the use of a mixture of rhamnolipids, emulsane, and indigenous biosurfactants resulted in 98% pyrene degradation, while the absence of emulsane in the biosurfactant mixture resulted in a reduction in pyrene biodegradation to 91% [66,67]. These results are in line with our observations, suggesting the advantage of biosurfactant mixtures over their individual forms, due to a possible synergistic effect. In their study, Lai et al. compared the effectiveness of various biosurfactants, including surfactin and rhamnolipids, in removing total petroleum hydrocarbons (TPH) from soils contaminated with petroleum hydrocarbons. At the same concentrations of biosurfactants, the efficiency of TPH removal from soil for rhamnolipids was in the range of 23–63%, while for surfactin, it was in the range of 14–62%, indicating that surfactin was less effective [68]. The present study investigates the relationship between the intensity of biodegradation processes and the concentration of the biosurfactant in the inoculating mixture. This relationship was investigated through the conducting of tests with two variants of biosurfactant concentrations (1 g/dm³ and 5 g/dm³). However, it is important to consider whether the achieved biodegradation effect is commensurate with the increased financial outlay associated with a higher biosurfactant concentration. It is important to note that high efficiency does not necessarily equate to the optimization of the process from the point of view of operating costs. This is a particularly salient consideration in the context of industrial-scale implementations of bioremediation technologies. The experimental findings suggest that the optimal equilibrated dosage of biosurfactants can significantly impact the efficiency and cost-effectiveness of the overall bioremediation process by biosurfactant-enhanced bioaugmentation [69].

Furthermore, the findings of chromatographic analyses demonstrated a correlation between the chemical structure of organic pollutants and their biodegradability [56,70]. This relationship was observed for aliphatic carbon donors, where fractions with shorter carbon chains (n-C6-n-C9 and n-C10-n-C21) were degraded much faster than long-chain fractions (n-C22-n-C29 and n-C30-n-C36) [71,72,73]. A comparable phenomenon was observed in the context of polycyclic aromatic hydrocarbons, wherein compounds comprising fewer aromatic rings per molecule (<four aromatic rings per molecule) exhibited significantly higher biodegradability in comparison to compounds containing four or more aromatic rings [4,74,75,76]. The underlying mechanisms responsible for this trend can be elucidated by examining the physicochemical properties of the constituent compounds. Shorter-chain aliphatic hydrocarbons and low-molecular-weight PAHs exhibit higher volatility, enhanced water solubility, and greater bioavailability to microorganisms [77]. Consequently, these compounds are more rapidly transported into microbial cells and are more easily metabolized. In contrast, higher-molecular-weight hydrocarbons exhibit reduced mobility, diminished solubility, and an increased propensity for a strong affinity with the organic fraction of the soil or sediment. This, in turn, limits their bioavailability and consequently slows their rate of biodegradation [56,57,78,79]. The employment of biosurfactants, which enhance the solubility and bioavailability of contaminants, in conjunction with the introduction of selected consortia of microorganisms with high metabolic activity to a given type of compound [31,80], thus ensures optimal treatment outcomes. The effectiveness of biodegradation was also confirmed by an analysis of the n-C17/Pr and n-C18/Ph indices [80,81,82]. In the variants containing biosurfactants at a concentration of 5 g/dm³, the values of these indices fell into the ranges of 0.16–0.70 and 0.17–0.74, respectively, compared to the values in soil inoculated only with the consortium (1.22 and 1.28). This finding suggests that the presence of biosurfactants can significantly enhance the rate of biodegradation. In the control samples, the values remained close to the baseline, confirming their limited effectiveness without the participation of microorganisms.

Ecotoxicological analyses, conducted utilizing Phytotoxkit, Ostracodtoxkit, and Microtox SPT tests, constituted a significant component in the comprehensive evaluation of the efficacy of the bioremediation process. The lowest toxicity (TU) values were obtained in variants in which the bacterial consortium and biosurfactants (especially PSR, JBR425, and JBR320 at a concentration of 5 g/dm³) were applied simultaneously. TU values for phytotoxicity parameters (germination and root growth) fell into the range of 3.62–4.61, in the Ostracod-toxkit test to 4.35, while in the Mcrotx SPT test to 4.91, which is a significant reduction from the baseline values (8.70–12.56, 10.5, and 13.05, respectively). A further significant finding is that no secondary toxic by-products were identified in the tested variants following the remediation process. Concurrently, the findings substantiated the notion of a direct correlation between the concentration of TPH and PAHs and soil toxicity [69,83,84]. The outcomes of both chromatographic analyses and toxicological tests indicated that a decrease in the pollutant concentration was proportionate to a decrease in the TU values. Furthermore, the variants comprising of solely biosurfactants, devoid of microbial involvement, did not attain substantial enhancement in the soil quality parameters. This underscores the imperativeness for a synergistic interplay between biosurfactants and microorganisms in achieving efficacious environmental purification.

5. Conclusions

The accumulated data clearly indicate that the use of a composite method such as biosurfactant-assisted biodegradation is an effective strategy in the remediation of soils contaminated with petroleum hydrocarbons. The findings from the respirometric, chromatographic, and toxicological analyses substantiate that this integrated approach considerably enhances the biodegradation efficiency of both total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAHs). Furthermore, the toxicity unit (TU) values obtained in Phytotoxkit, Ostracodtoxkit, and Microtox tests indicate an enhancement in the overall quality of the soil environment and a reduction in potential ecological risks. The findings substantiate the assertion that the judicious selection of the bacterial consortium, in congruence with a biosurfactant, is paramount to the efficacy of remediation processes. This approach is a sustainable and economically viable solution for the remediation of industrial sites, supporting long-term environmental recovery while minimizing harmful side effects, which is in line with the core objectives of sustainable development.