Biodegradation Potential and Taxonomic Composition of Hydrocarbon-Degrading Bacterial Consortia in Diesel-Contaminated Agricultural Soils

Abstract

This study explored the potential of bacterial consortia to remediate real diesel-contaminated agricultural soils. Two consortia were tested: a native consortium isolated from contaminated soil and an exogenous consortium derived from vermicompost. Bacterial communities (consortia and soils) were characterized through high-throughput sequencing. Within 30 days, total petroleum hydrocarbons (TPH) were removed most efficiently by bioaugmentation with the native consortium (53.32%), followed by the exogenous vermicompost consortium (47.14%) and the indigenous microbiota (42.52%). Gas chromatography confirmed the reduction of polycyclic aromatic hydrocarbons (PAHs) with 2–5 rings; however, terphenyl, chrysene, and pyrene persisted. The highest TPH biodegradation rate was observed in the treatment inoculated with the native consortium (208.5 mg/kg per day), followed by the treatment with indigenous microbiota (181.8 mg/kg per day) and the exogenous consortium (161.9 mg/kg per day). Furthermore, hydrocarbon-degrading bacterial populations increased significantly during the first week but declined after day 21, showing a negative correlation with TPH concentrations across all treatments, indicating that the highest bacterial activity and degradation occurred during the first 14 days. Taxonomic analysis identified Actinobacteria as the most abundant phylum in the initial soil, whereas Proteobacteria dominated both the consortia and the bioremediated soils. Significant differences in community structure and composition were observed between the consortia according to their origin, influencing removal efficiency. Dominant genera shifted from Nocardioides and Streptomyces in untreated soil to Pseudomonas, Sphingobium, and Pseudoxanthomonas following biological treatments, while Nocardia, Rhodococcus, and Bacillus remained nearly constant. These findings underscore the effectiveness of adapted bacterial consortia in restoring real diesel-contaminated agricultural soils and highlight potential microbial succession patterns associated with biodegradation and soil ecological recovery.

1. Introduction

The high global consumption of crude oil and its derivatives has led to severe soil contamination, primarily resulting from leaks and spills during extraction and transportation processes [1,2]. In Mexico, fuel spills in agroecosystems are common due to fuel theft for black market [3,4]. The presence of petroleum-derived hydrocarbons in agricultural soils alters their physical and chemical properties, as well as their biological interactions [5,6]. These changes negatively impact the environment and the health of exposed organisms [7,8]. Moreover, the quality and productivity of agri-food systems are reduced [9,10], leading to socioeconomic issues such as poverty and marginalization, and ultimately compromising regional food security [11,12].

Fuels derived from petroleum, such as diesel, are composed of complex mixtures of aliphatic, aromatic, and polycyclic aromatic hydrocarbons (PAHs) [13,14]. Among these, sixteen PAHs have been identified as priority pollutants by both the scientific community and the United States Environmental Protection Agency (EPA), largely due to their hydrophobic nature and the stability of their molecular structures, which make them highly resistant to degradation [15,16]. Numerous studies have shown that exposure to PAHs can cause genetic and cellular damage in living organisms [17,18]. These compounds are recognized for their mutagenic and carcinogenic properties [19,20], highlighting the importance of developing sustainable mitigation strategies, such as bioremediation approaches, to reduce their impact on the environment [21,22].

Currently, biological decontamination methods are recognized as effective alternatives to physical, chemical, and thermal techniques for remediating hydrocarbon-contaminated soils, being generally faster and more cost-effective [23,24]. Bioremediation processes also support the restoration of agroecosystems without interrupting agricultural production [25]. In particular, bioaugmentation promotes the removal of contaminants through the introduction of microbial strains or consortia with degradative capacities, and in some cases, plant growth-promoting traits [26,27,28]. Several bacterial genera have been widely studied for their role in hydrocarbon biodegradation [29,30], due to their ability to use petrogenic hydrocarbons as the sole carbon or energy source, enabling complete mineralization while avoiding the accumulation of potentially toxic intermediates [31,32].

Hydrocarbon-degrading bacterial consortia are commonly used to enhance biodegradation through complementary metabolic pathways, while also modulating soil microbial composition and diversity [33,34,35]. The origin and specific composition of these consortia influence their performance, conferring adaptive advantages and functional stability that favor complete mineralization of organic pollutants [36,37,38]. Despite the extensive research on microbial consortia, their effectiveness can vary depending on soil conditions and community composition, and few studies systematically evaluate consortia that combine robust degradative capacity with ecosystem compatibility. In this context, the present work investigates native microbial consortia with the potential to provide stable and efficient hydrocarbon degradation, highlighting their advantages compared to previously reported systems.

However, studying soil microbiota in contaminated sites remains challenging, as less than 1% of these microorganisms can be cultured under laboratory conditions [39]. As a result, environmental biotechnology has increasingly relied on molecular and bioinformatic tools to gain a deeper understanding of microbial dynamics in polluted environments. These tools enable the genomic analysis of entire uncultured microbial communities, and their gene expression profiles [40], allowing the identification of key microbial groups, families, and genera actively involved in hydrocarbon biodegradation processes [41,42,43].

In this context, the aim of this study was to evaluate the effectiveness of two hydrocarbon-degrading bacterial consortia—one native, isolated from the contaminated site, and the other exogenous, obtained from vermicompost—in diesel removal from contaminated agricultural soils. Additionally, the bacterial community structure of both the consortia and the treated soils was analyzed.

2. Materials and Methods

2.1. Samples

Contaminated agricultural soil was sampled from a rural area affected by a diesel spill in 2017, located in the Acatzingo region, Puebla, Mexico (18°58′43.0″ N, 97°46′49.7″ W). The physical and chemical properties of the soil were determined according to the Mexican standard NOM-021-SEMARNAT-2000 [44]. The entire soil sample was stored at 4 °C for preservation and subsequent use.

A sample of Green Forest México^® (Puebla, Puebla, México) vermicompost was used, with the following characteristics provided by the manufacturer: total humic extract 9–10%, humic acids 6.5–7.0%, fulvic acids 2.5–3.0%, nitrogen (NH₃) 0.9–1.5%, phosphorus (P₂O₅) 0.4–1.0%, potassium (K₂O) 1.1–1.7%, calcium 2.3–2.9%, magnesium 1.6–1.9%, organic matter 40–45%, moisture 30–35%, pH 7.5–8.7, electrical conductivity 3.6–5.6 μS/cm, bulk density 0.65–0.75 g/cm³, and culturable bacteria 2 × 10¹² CFU/g.

2.2. Development of Hydrocarbon-Degrading Bacterial Consortia

The bacterial consortia were obtained through individual enrichment and successive culturing procedures [45,46]. The native consortium (NC) was isolated from diesel-contaminated agricultural soil, while the exogenous consortium (EC) was obtained from a vermicompost sample. Heterotrophic bacterial enrichment was performed by cultivating 1 g of each sample separately in nutrient broth (yeast extract 2.0 g/L, peptone 5.0 g/L, NaCl 5.0 g/L) at 37 °C and 150 rpm for 48 h (Culture 1). Subsequently, 1000 µL of Culture 1 from each sample was inoculated into minimal mineral medium supplemented with diesel as the primary carbon source and glucose as a co-substrate (NH₄SO₂ 7 g/L, K₂HPO₄ 5.7 g/L, MgSO₄ 7 g/L, glucose 2 g/L, diesel 2000 µL), and incubated at 37 °C and 150 rpm for 10 days (Culture 2). Finally, 1000 µL of Culture 2 was transferred into Bushnell Haas broth containing diesel as the sole carbon source (NH₄SO₂ 1 g/L, K₂HPO₄ 1 g/L, KH₂PO₄ 0.5 g/L, MgSO₄ 0.2 g/L, MnSO₄ 0.001 g/L, FeSO₄ 0.001 g/L, ZnSO₄ 0.001 g/L, CaCl₂ 0.001 g/L, diesel 2000 µL/L), and incubated at 37 °C and 150 rpm for 14 days.

The biomass from each consortium was recovered by centrifugation and preserved in Bushnell Haas broth supplemented with glycerol (80:20 v/v) at –80 °C. Prior to use, the hydrocarbon-degrading bacterial consortia were reactivated in Bushnell Haas broth at 37 °C and 150 rpm for 48 h (

Table 1. Summary of experimental design.

Bioremediation Treatment	Code	Description *
Native microbiota	NM	Soil
Bioaugmentation with native consortium	BNC	Soil + Native consortium **
Bioaugmentation with exogenous consortium	BEC	Soil + Exogenous consortium **
Abiotic control	AC	Soil + HgCl2 2%

* 100 g of contaminated agricultural soil was used ** 10% v/v (10⁸ CFU/mL) of each consortium.

).

2.3. Microcosm-Scale Bioremediation Assays

The bioremediation assay was carried out in sterile glass flasks containing 100 g of contaminated agricultural soil, with the C:N:P ratio adjusted to 100:10:1 using (NH₄)₂SO₄ and K₂HPO₄ solutions based on material balance calculations [47]. The experiment ran for 30 days, during which soil moisture was maintained at field capacity (30%) by weekly additions of sterile distilled water. In addition, manual aeration was applied for 60 min every 24 h under laminar flow conditions. The treatments evaluated included native microbiota (NM), bioaugmentation with a native consortium isolated from the contaminated soil (BNC), bioaugmentation with an exogenous consortium derived from vermicompost (BEC), and an abiotic control (AC) in which the soil was treated with a 2% HgCl₂ solution (Table 1).

Each treatment consisted of 12 independent experimental units, which were designed as sacrificial samples to ensure measurement independence. Including the three initial soil samples (time 0), a total of 51 experimental units were established in the study. At each sampling time (0, 7, 14, 21 and 30 days), three units per treatment were sacrificed to monitor hydrocarbon degradation dynamics and viable bacterial counts. Concentrations of PAHs, along with bacterial taxonomic composition, were determined at both the beginning and the end of the experiment using composite samples.

2.3.1. Evaluation of Biodegradation Potential

Total Petroleum Hydrocarbons (TPH)

Changes in diesel concentration were monitored weekly for each treatment by determining TPH (mg/kg) through gravimetric analysis. Three grams of each soil sample were mixed with 5 mL of a 1:1 (v/v) dichloromethane-acetone solution. Subsequently, the soil was subjected to three ultrasonic bath extractions of 15 min each. Samples were then centrifuged at 2000 rpm for 10 min. The supernatant from each extraction was collected by filtration into pre-weighed test tubes, and solvents were evaporated to dryness under a fume hood [48]. TPH concentration (mg/kg) was calculated using Equation (1):

$$\mathrm{T}\mathrm{P}\mathrm{H} (\mathrm{m}\mathrm{g}/\mathrm{k}\mathrm{g})={\displaystyle \frac{{\mathrm{W}}_{\mathrm{h}}\ast 1000}{{\mathrm{W}}_{\mathrm{s}}}}$$

(1)

where W_h = weight of assay tube with hydrocarbon extract (mg)—weight of empty assay tube at constant weight (mg), W_s = soil sample weight (g).

The percentage of diesel removal (%) was calculated using Equation (2) [49].

$$\mathrm{R}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)={\displaystyle \frac{\left[\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{T}\mathrm{P}\mathrm{H}\right]-\left[\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{T}\mathrm{P}\mathrm{H}\right]}{\left[\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{T}\mathrm{P}\mathrm{H}\right]}}\ast 100$$

(2)

Finally, the biodegradation rate was calculated using a simple linear regression model based on the TPH concentration throughout the bioremediation experiment [50].

Polycyclic Aromatic Hydrocarbons (PAHs)

The concentration of PAHs (mg/kg) in the initial and final soil samples of all treatments were quantified using composite samples. Solid-phase extraction was performed, followed by analysis with a PerkinElmer^® Clarus 680 gas chromatograph (Shelton, CT, USA) coupled to a PerkinElmer^® SQ8C mass spectrometer. A DB-U1 8270D Agilent^® (Santa Clara, CA, USA) capillary column (30 m length, 0.25 mm internal diameter, 0.25 µm film thickness) coated with stationary phase was used. A 1 µL aliquot of the sample was injected into the chromatograph. The injector temperature was set at 240 °C, the transfer line at 280 °C, and the carrier gas flow rate was maintained at 1.2 mL/min. The oven temperature program started at 40 °C for 30 s, followed by an increase of 10 °C/min until 100 °C. Subsequently, the temperature was ramped up by 22 °C/min to 260 °C, then increased by 8 °C/min to 280 °C and finally ramped at 25 °C/min until reaching 320 °C. PAHs identification was confirmed by comparing retention times and relative ion abundances with profiles obtained from certified standards (Agilent^®).

2.4. Analysis of Bacterial Community Composition

2.4.1. Dynamics of Cultivable Biodegrading Bacterial Populations

The dynamics of cultivable bacteria over time were evaluated using the base-10 serial dilution technique with 0.09% saline solution. Dilutions 10⁻⁵ to 10⁻⁷ were plated, and colony-forming units (CFU) were counted weekly [51]. Hydrocarbon-degrading bacteria were cultured on Bushnell-Haas agar, with filter paper impregnated with diesel as the sole carbon source and incubated at 37 °C for 48 h. Colony-forming units per gram of soil (CFU/g) were calculated according to Equation (3):

$$\mathrm{C}\mathrm{F}\mathrm{U}/\mathrm{g}={\displaystyle \frac{\mathrm{N}\mathrm{C}\ast \frac{1}{\mathrm{D}\mathrm{F}}\ast \frac{1}{\mathrm{V}}}{\mathrm{W}\ast \mathrm{H}\mathrm{F}}}$$

(3)

where NC = number of colonies counted, DF = inoculated dilution factor (10⁵–10⁷), V = inoculated volume (mL), W = wet soil sample weight (g), HF = humidity correction factor (1 − (% of humidity/100)).

2.4.2. Bacterial Community Taxonomic Profile

Genetic material was extracted from soil samples using the DNeasy PowerMax Soil^® (Hilden, Germany) kit according to the manufacturer’s instructions, employing 15 g of sample. Subsequently, genomic DNA extraction from the bacterial consortium biomass, previously washed with 10 mM MgSO₄, was performed in triplicate using the Zymo Research Quick-gDNA Miniprep^® kit (Irvine, CA, USA). The triplicates of each sample were then pooled into a single 1.5 mL vial. DNA extracts from all samples were concentrated at 60 °C for 30 min using a Thermo Scientific Savant DNA Concentrator^® (Walthman, MA, USA). DNA quality and quantity were assessed with a Thermo Scientific Nanodrop^® spectrophotometer and 1% agarose gel electrophoresis.

The DNA was then used to amplify the V3–V4 variable regions of the 16S rRNA gene using primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 [52]. End-point PCR amplification was performed using the NZYTech^® Master Mix kit (Lisbon, Portugal) for all samples and controls (reaction components are detailed in Table S1 of the Supplementary Material). The cycling program was as follows: initial denaturation at 95 °C for 3 min; 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 15 s; followed by a final extension at 72 °C for 10 min.

PCR products were purified by fragments cut from agarose gel under UV illumination using a UVP Visi-Blue^® (Upland, CA, USA) transilluminator and a scalpel (Figure S1, Supplementary Material). Finally, DNA was purified using the Zymoclean Gel DNA Recovery^® kit.

Amplicon libraries were constructed following the 16S Metagenomic Sequencing Library Preparation protocol from Illumina MiSeq^® (San Diego, CA, USA) and sequenced on the Illumina MiSeq platform^® (v 4.1.0) with a paired-end read configuration. Sequences are available at the SRA site under the ID PRJNA1310627. The reads were used to reconstruct the original amplicon region (450–490 bp in length) by overlapping them with FLASH v1.2.11 software [53].

Taxonomic annotation was performed using Parallel-Meta Suite v3.7.2 [54] with the Refseq database [55]. Graphs were generated using ggplot2 (v 3.5.2) and ggVennDiagram (v 1.5.6) [56,57].

2.5. Statistical Analysis

To assess significant differences between treatments, response variables were statistically analyzed using paired Student’s t-test, one-way ANOVA, and Tukey’s multiple comparison test, simple linear regression, and Pearson’s correlation, as appropriate (Normality and homogeneity of variance tests are presented in Table S2 of the Supplementary Material). All analyses were performed using GraphPad Prism 9^® (10.3.1) software with a 95% confidence level.

3. Results

3.1. Samples

The contaminated agricultural soil sample was classified as sandy soil, with a pH of 7.47, organic matter content of 9.25%, total nitrogen 0.39%, phosphorus 20.32 mg/kg, bulk density 1.29 g/cm³, electrical conductivity 0.214 dS/m, and field capacity of 30%.

3.2. Microcosm-Scale Bioremediation Assays

Evaluation of Biodegradation Potential

After the bioremediation assay in microcosms, a significant degradation was observed in all biological treatments (Figure 1a); however, the abiotic control showed no hydrocarbon removal (p = 0.059). The analysis of variance of the final TPH concentration (Figure 1b) revealed the formation of three statistical groups (p < 0.0001), indicating that bioaugmentation with the native consortium (BCN) achieved the highest removal rate, reaching 53.32% after 30 days. This was followed by the native microbiota (NM) and the bioaugmentation treatment with the exogenous consortium, which shared a statistically similar mean, removing between 42.52% and 47.14% of the TPH present in the soil.

Significant removal was observed from the first week of the experiment in the treated groups, with similar TPH removal efficiency trends maintained across bioremediation treatments throughout the assay (Figure 1b).

Similarly, the simple linear regression model facilitated the estimation of TPH removal rates for each treatment (

Table 2. Simple linear regression results for biodegradation rate prediction *.

Treatment	Linear Equation	R-Squared	Biodegradation Rate (mg/kg per Day)
Native microbiota (NM)	y = −181.8x + 10857	0.8829	181.8
Bioaugmentation with native consortium (BNC)	y = −208.5x + 10967	0.9133	208.5
Bioaugmentation with exogenous consortium (BEC)	y = −161.9x + 10854	0.8564	161.9
Abiotic control (AC)	y = −8.975x+ 11771	0.9263	8.975

* α = 0.05.

). The slope of the bioaugmentation treatment with the native consortium exhibited the highest value, corresponding to a TPH degradation rate of 208.5 mg/kg per day, followed by the native microbiota treatment with 181.8 mg/kg per day and the bioaugmentation with the exogenous consortium, which achieved a TPH removal rate of 161.9 mg/kg per day (Figure S2, Supplementary Material). Finally, the mathematical model estimated a daily removal of 8.975 mg/kg TPH due to abiotic factors, highlighting the critical role of soil microbiota in hydrocarbon biodegradation processes.

Moreover, gas chromatography results revealed a reduction of over 95% in the concentration of 2- to 5-ring polycyclic aromatic hydrocarbons in the biological treatments (Figure 2). However, concentration did not decrease in any treatment for compounds such as Terphenyl-d14, Chrysene-d14, and Pyrene-d14, highlighting their recalcitrant nature. Additionally, the abiotic control (AC) exhibited a PAHs composition remarkably similar to that of the initial soil (IS), underscoring the critical role of microbiota in the degradation of xenobiotics.

3.3. Analysis of Bacterial Community Composition

3.3.1. Dynamics of Cultivable Biodegrading Bacterial Populations

The viable population of hydrocarbon-degrading bacteria in the initial soil was 1.60 × 10⁵ CFU/g. This concentration increased significantly in all biological treatments from the first week of the experiment (Figure 3a), with the strongest effect observed under bioaugmentation with hydrocarbon-degrading consortia. In the following week, the highest population was recorded in the bioaugmentation treatment with the native consortium, reaching 9.60 × 10⁸ CFU/g, whereas the lowest population was observed in the native microbiota treatment, differing by one order of magnitude (p < 0.0001). Quantification on day 21 revealed a significant decline across all treatments. Furthermore, in the last two measurements (day 21 and day 30), two statistically distinct groups were formed: the first comprised the bioaugmentation treatment with the native consortium, which maintained the highest concentration (~10⁷ CFU/g), and the second consisted of the bioaugmentation with the exogenous consortium and the native microbiota treatment (~10⁶ CFU/g).

Additionally, viable degrading bacterial concentrations and TPH levels exhibited a negative correlation across all biological treatments (Figure 3b), underscoring the relevance of high hydrocarbon-degrading bacterial density in hydrocarbon biodegradation processes.

3.3.2. Bacterial Community Taxonomic Profile

The results of DNA extract concentration and quality, as well as amplicon yields, are presented in Table S3 of the Supplementary Material. PCR product integrity analysis revealed bands corresponding to the V3–V4 region of the 16S rRNA gene (Figure S2, Supplementary Material). Bioinformatic analysis yielded a total of 99,466 operational taxonomic units (OTUs). The initial soil samples contained 2527 OTUs, while the native and exogenous hydrocarbon-degrading consortia exhibited 26,606 and 24,992 OTUs, respectively. Finally, the OTUs detected in the final soils were 11,846 for the native microbiota treatment, 12,782 for bioaugmentation with the native consortium, and 20,713 for bioaugmentation with the exogenous consortium.

Bacterial taxonomic composition at the phylum level for all samples is shown in Figure 4a. Initial soil analysis revealed 10 groups dominated by Actinobacteria (77.52%), Proteobacteria (14.05%), and Firmicutes (7.52%). The hydrocarbon-degrading consortia contained only four groups, with Proteobacteria being predominant at 99.7% in the native consortium and 96.70% in the exogenous consortium; Actinobacteria, Bacteroidetes, and Firmicutes were also present. In treated soils, the number of groups increased to 14, with a similar composition across samples. Proteobacteria remained dominant, averaging 68% across the three treatments. However, the second most abundant group differed: Firmicutes accounted for 11.36% in the native microbiota treatment, while Actinobacteria comprised 15.56% and 29.05% in soils bioaugmented with the native and exogenous consortia, respectively. Taxonomic groups identified at the Order and Family levels are presented in Figures S3 and S4 of the Supplementary Material.

The most abundant genera identified in the samples are shown in Figure 4b. The initial soil community was mainly composed of Nocardioides (36.17%), Nocardia (16.02%), Streptomyces (13.34%), Rhodococcus (4.35%), and Pseudomonas (3.13%). In contrast, both the hydrocarbon-degrading consortia and the treated soils displayed Pseudomonas as one of the dominant genera. Although the native consortium (NC) was isolated from the contaminated soil, its composition decreased considerably to 19 genera, being dominated by Stenotrophomonas (50.01%) and Pseudomonas (45.27%). The exogenous consortium (EC) exhibited a distinct composition, mainly represented by Stenotrophomonas (45.13%), Ochrobactrum (30.76%), Pseudomonas (13.12%), Achromobacter (6.70%), and Mycobacterium (2.47%). The soil treated with native microbiota (NM) showed an increase in taxonomic richness to 167 genera, with Pseudomonas (51.54%), Sphingobium (5.97%), Nocardia (4.69%), Coprococcus (3.35%), and Caloramator (2.52%) as the most abundant groups. Additionally, 9.38% of the sequences were assigned to an unclassified genus. Similarly, the soil bioaugmented with the native consortium (BNC) exhibited greater diversity, with 188 genera detected. The dominant groups were Pseudomonas (33.93%), Nocardia (10.02%), Sphingobium (6.52%), Sedimentibacter (5.46%), and Pseudoxanthomonas (5.27%), along with an unclassified genus accounting for 4.12% of the community. Finally, the soil bioaugmented with the exogenous consortium (BEC) comprised 173 genera, being dominated by Pseudomonas (29.95%), Pseudoxanthomonas (20.27%), Dietzia (12.22%), Ramlibacter (4.93%), Gordonia (4.67%), Rhodococcus (3.65%), and Sphingobium (2.60%).

The taxonomic composition of the hydrocarbon-degrading bacterial consortia revealed that the genus Stenotrophomonas was the most abundant in both samples, accounting for 50.01% in the native consortium and 45.13% in the exogenous consortium (Figure 5a). In contrast, the genus Pseudomonas was also present in both consortia; however, its relative abundance was markedly higher in the native consortium (45.27%) compared to the exogenous consortium (13.12%). Similarly, the genera Achromobacter, Vibrio, Candidatus Hepatobacter, Frankia, and Shinella were identified in both consortia, with comparable relative abundances. Notable differences between the consortia were observed in the genus Ochrobactrum, which was considerably more abundant in the exogenous consortium (30.76%) than in the native consortium (0.004%). Additionally, the genera Mycobacterium (2.26%), Bacillus (0.68%), and Brucella (0.55%) were exclusively detected in the exogenous consortium at relevant concentrations, whereas Cupriavidus was detected only in the native consortium with a relative abundance of 1.98%.

These differences in the composition and structure of hydrocarbon-degrading consortia were not directly reflected in the overall taxonomic structure of the 100 most abundant genera in the bioaugmented soils (BNC and BEC); that is, the dominant genera in the consortia did not correspond to those in the bioaugmented soils, considering that Pseudomonas also increased significantly in the non-bioaugmented treatment (NM). Nevertheless, these differences, together with the biostimulation of the native microbiota, appeared to influence the composition and structure of the bacterial communities in the bioremediated soils (Figure 4b), potentially affecting hydrocarbon biodegradation efficiency (Figure 1).

Finally, Figure 5b illustrates the number of unique and shared bacterial genera among the 30 most abundant taxa identified in the soil samples. The relative abundance of each genus is provided in Supplementary Table S3. The initial soil exhibited 23 genera that were not shared with the other treatments, with Streptomyces (13.34%), Arthrobacter (2.61%), Limnobacter (2.26%), Sphingomonas (2.10%), Metabacillus (1.74%), and Lysobacter (1.58%) as the most abundant. These differences highlight a substantial reconfiguration of the taxonomic composition at the genus level following the application of biological treatments, as well as a significant enrichment of genera not detected in the initial soil or in the consortia. In the soil treated solely with native microbiota, unique genera included Coprococcus (3.53%), Gracilibacteraceae Group (0.61%), Caldicoprobacter (0.37%), Altererythrobacter (0.32%), Lutispora (0.32%), Arenimonas (0.27%), and Bradyrhizobiaceae Group (0.23%). Similarly, the bioaugmented soil with the native consortium harbored distinctive genera such as Pusillimonas (3.40%), Alkanindiges (1.31%), Puniceicoccaceae Group (0.64%), Corynebacteriaceae Group (0.56%), Ellin6075 Group (0.42%), Rhodanobacter (0.30%), Luteolibacter (0.27%), and Methylophilaceae Group (0.25%). In contrast, the soil bioaugmented with the exogenous consortium exhibited exclusive genera including Chryseoglobus (4.47%), Iamia (1.54%), Mycobacterium (0.76%), Proteiniclasticum (0.42%), Rhodobacter (0.32%), Georgenia (0.27%), Methyloversatilis (0.22%), Agrobacterium (0.14%), and Parachlamydia (0.12%).

Furthermore, the samples subjected to bioremediation treatments shared eight abundant genera: Sphingobium, Achromobacter, Paracoccus, Phenylobacterium, Xanthomonas, the Sphingomonadaceae group, and Devosia, all of which have been previously reported as hydrocarbon-degrading agents in various environments. Finally, the four soil samples shared five of their thirty most abundant genera: Pseudomonas, Pseudoxanthomonas, Rhodococcus, Bacillus, and Nocardia. In addition to being recognized hydrocarbon-degrading microorganisms, the latter three are associated with the restoration of ecological function in soil.

4. Discussion

4.1. Microcosm-Scale Bioremediation Assays

Evaluation of Biodegradation Potential

All treatments applied in this study achieved significant removal by the end of the bioremediation assay, highlighting the critical role of the soil microbiota in biodegradation processes, as well as the contribution of both biostimulation and bioaugmentation strategies. Several studies have reported that the biodegradation potential of bioaugmentation with hydrocarbon-degrading bacterial consortia increases when combined with nutrient supplementation and aeration. This effect is attributed to the enhancement of the metabolic activity of the indigenous microbiota and the concomitant improvement of soil biodiversity in contaminated environments [58].

Bioaugmentation using the native microbial consortium led to markedly higher biodegradation efficiency than the exogenous consortium, emphasizing the advantage of site-adapted communities in accelerating hydrocarbon breakdown under soil-specific conditions. For example, Eze et al. [59] reported a 91% contaminant removal after 60 days following inoculation with a native consortium of hydrocarbon-degrading and plant growth-promoting bacteria, they attributed this removal to the presence of hydrocarbon-degrading genes encoding enzymes such as monooxygenases and dioxygenases, as revealed by metagenomic analyses. In a similar approach, Curiel-Alegre et al. [60] reported that bioaugmentation with a native bacterial consortium immobilized on biochar, combined with rhamnolipid-enhanced biostimulation, resulted in a 30% decrease in TPH after 90 days. These studies further highlighted the importance of temporal shifts in soil physicochemical properties in promoting more effective biodegradation.

Exogenous hydrocarbon-degrading bacterial consortia have also been employed for hydrocarbon biodegradation in soils, achieving removal efficiencies similar to those reported in the present study. For example, Abena et al. [61] evaluated the metabolic potential of an exogenous consortium composed of five bacterial strains for TPH degradation. This consortium outperformed natural attenuation across various soil samples, reaching up to 48.1% contaminant removal after 40 days. The enhanced performance was attributed to the increased bioavailability of hydrocarbons, as the bacterial strains used are well-known biosurfactant producers. In a separate study, a hydrocarbon-degrading consortium assembled from non-native strains of Bacillus megaterium, Pseudomonas putida, and Bacillus subtilis achieved a 34% reduction in TPH after 100 days, reflecting an overall improvement in bioremediation rates [62].

Although uncontaminated soils generally offer favorable conditions for the survival and activity of edaphic microbiota, conventional isolation-based approaches for obtaining hydrocarbon-degrading consortia frequently lead to reduced microbial diversity [63], which may in turn constrain the range of available biodegradation pathways. These findings underscore the importance of selecting microbial strains not only based on their inherent degradative capabilities but also on their adaptability to stressful environmental conditions and their potential to participate in cooperative ecological interactions. Despite these conceptual advances, direct comparative studies assessing the performance of native versus exogenous hydrocarbon-degrading consortia remain limited, highlighting the need for further research to clarify their effectiveness and ecological dynamics in hydrocarbon-contaminated soils.

The linear regression analysis further revealed a pronounced increase in the biodegradation rate when native microbial consortia were employed. This modeling approach has been previously used to quantify both the biodegradation kinetics and the theoretical extent of hydrocarbon removal in bioaugmentation studies, identifying critical factors that govern hydrocarbon degradation, including contaminant concentration, the bacterial strain applied, and the incubation period [64]. Importantly, the present findings demonstrate that stimulation of native microbiota is more effective than the inoculation of exogenous consortia in enhancing biodegradation rates (Table 2). This superior performance can be attributed to the intrinsic adaptive capacity of indigenous microorganisms to local soil conditions, including nutrient availability, pH, moisture, and contaminant composition, which enables them to metabolize hydrocarbons efficiently [65]. In contrast, exogenous consortia may experience reduced survival, competition with established microbial populations, or suboptimal adaptation to the soil environment, which can limit their effectiveness [36,37,38]. These findings reinforce the ecological advantage of leveraging native microbial communities for in situ bioremediation and suggest that strategies aimed at stimulating indigenous populations, such as bio-stimulation with nutrients or environmental optimization, may achieve higher hydrocarbon degradation rates than the introduction of external microbial consortia.

In addition, the qualitative analysis of PAHs demonstrated a pronounced reduction in 4- and 5-ring compounds following the application of biological remediation treatments (NA, BCN, and BEC). Biodegradation of PAHs in soil is facilitated by the ability of indigenous microorganisms to produce catalytic enzymes, including laccase, lignin peroxidase, and cytochrome P450 in fungi, as well as dioxygenases and monooxygenases in bacteria [66]. Nevertheless, the persistence of certain compounds, such as chrysene, can be attributed to their extremely low solubility (0.0003 mg/L) and high thermal stability, which limit microbial utilization as a substrate and consequently retard degradation [67]. Similarly, pyrene exhibits strong adsorption to soil organic matter, reducing its mobility and bioavailability, which further constrains microbial degradation [68]. PHAs compounds like terphenyls, chrysene, and pyrene are of particular concern due to their high hydrophobicity, chemical stability, and known toxicological properties, including mutagenicity, carcinogenicity, and potential for bioaccumulation in soil and aquatic organisms [15,16,17,18,19,20]. Their persistence in the environment therefore poses potential ecological risks, which should be carefully evaluated in future studies. These findings underscore the complexity of PAH biodegradation in soils and highlight the critical role of microbial enzymatic versatility and substrate accessibility. Optimizing environmental conditions, enhancing bioavailability through soil amendments [69], and promoting mechanisms such as cometabolism and interspecific metabolic interactions could accelerate the degradation of these highly recalcitrant and potentially toxic compounds [70].

4.2. Analysis of Bacterial Community Composition

4.2.1. Dynamics of Cultivable Biodegrading Bacterial Populations

The biostimulation and bioaugmentation treatments applied in this study promoted a significant increase in the population of degradative bacteria from the first week of the experiment. These findings are consistent with those reported by Li et al. [71], who evaluated similar biotechnologies and observed an increase in the abundance of hydrocarbon-degrading bacteria. This result can be attributed to greater nutrient availability and aeration during the experiment, as well as to the ability of the microorganisms present to metabolize petrogenic compounds as a source of carbon and energy, promoting their growth and simultaneously contributing to the reduction in TPH concentrations in the substrate, as also demonstrated in this study (Figure 3). Moreover, the dynamics of cultivable degradative bacterial populations showed a significant decline from day 21 of the experiment across all treatments. Similar results have been reported in microcosm bioremediation studies, where a decrease in viable bacterial populations was observed after the fourth week [72]. The observed decrease in bacterial populations during the later stages of TPH degradation may be associated with multiple factors. Gradual depletion of available substrates, including TPH and PAHs, limits the carbon and energy sources required for microbial metabolism and subsequent growth. In addition, the accumulation of potentially toxic metabolites produced during hydrocarbon degradation may inhibit certain bacterial populations, thereby contributing to the observed decline [73]. Nutrient limitation, particularly of essential elements such as nitrogen and phosphorus, also plays a critical role; reduced nutrient availability constrains the growth of hydrocarbon-degrading bacteria while favoring microbial groups better adapted to low-nutrient conditions, resulting in lower bacterial abundance at this stage and reduced biodegradation efficiency [2,7,8,15] (Figure 3b). Overall, these observations suggest that fluctuations in viable bacterial populations are driven by substrate limitation, metabolite toxicity, and nutrient competition, all of which directly influence the efficiency and rate of TPH biodegradation. These results show that bacterial activity and hydrocarbon biodegradation peak within the first 14 days, highlighting that the strategies used in this study offer a fast and effective way to restore contaminated soils.

Nevertheless, the Mexican Official Standard NOM-138-SEMARNAT/SSA1-2012 [74] sets permissible hydrocarbon limits according to land use, with maximum thresholds of 1200 mg/kg for medium-fraction hydrocarbons and 3000 mg/kg for heavy-fraction hydrocarbons in agricultural, forestry, livestock, or conservation soils. Under the conditions of this study, meeting these limits would require a TPH removal of approximately 65%, which was not achieved by any of the treatments applied. These results underscore the importance of nutrient or substrate replenishment and periodic reinoculation of microbial consortia to sustain a stable and active population of biodegrading bacteria, thereby improving hydrocarbon removal and the overall performance of biological treatment systems.

4.2.2. Bacterial Community Taxonomic Profile

The most abundant phylum in the initial soil sample was Actinobacteria. Jia et al. [63] reported an increase in this group in soils slightly contaminated with hydrocarbons compared to uncontaminated soils, where Firmicutes predominated. Similarly, Galitskaya et al. [75] observed a significant and sustained increase in the Actinobacteria population up to 120 days following contamination with heavy hydrocarbons, reaching a relative abundance of 42%. In this context, the soil used in the present study can be classified as aged and weathered, having undergone stabilization processes over time and achieving a state of relative equilibrium in the physical, chemical, and biological properties. Such conditions may have favored the establishment of Actinobacteria as the predominant phylum, even in the presence of petrochemical hydrocarbons.

However, the application of biological remediation treatments in soils, such as biostimulation and bioaugmentation, induced significant changes in the structure of the microbial community structure, as has been observed in previous studies [76]. In the present study, an increase in the phylum Proteobacteria was observed following the bioremediation assay across all treatments (<65% in all treatments). These findings are consistent with those reported by Rahmeh et al. [77], who observed an increase of up to 65% in Proteobacteria after bioremediation using hydrocarbon-degrading bacterial consortia in a biopile system. Similarly, Cui et al. [78] reported an increase from 55.01% to 90.3% in this bacterial group when optimizing hydrocarbon biodegradation conditions in activated sludge bioreactors; this increase was significantly associated with contaminant degradation. Several bacteria within the phylum Proteobacteria have been described as hydrocarbon-degrading [79]. In general, their capacity to biodegrade hydrocarbons in diverse environments is attributed to their high abundance in contaminated sites, their heterotrophic metabolism, and the presence of genes encoding enzymes involved in catabolic degradation processes [80]. Proteobacteria have been identified as key bioindicators in PAHs biodegradation processes [81]. In line with this ecological role, the present study revealed a high representation of this group across all samples, comprising 99.7% of the native consortium and 96.7% of the exogenous consortium, and exceeding 65% in soils following the bioremediation assay, regardless of the treatment applied. These findings emphasize the ecological significance of Proteobacteria as dominant and functionally active taxa in hydrocarbon degradation and underscore their potential utility as bioindicators for evaluating the effectiveness of soil restoration strategies in hydrocarbon-contaminated environments.

Other taxa that were predominant in soils after bioremediation included Firmicutes and Bacteroidetes, both of which have been reported as major phyla in similar studies [82]. Although the presence of Firmicutes in the native hydrocarbon-degrading consortium was below 0.01%, soils bioaugmented with this consortium exhibited a significant increase, reaching 8.11% of this phylum. This enhancement may be attributed to the establishment of favorable soil conditions, including increased nutrient availability, shifts in microbial competition, and improved substrate accessibility, which collectively promoted TPH removal. In contrast, soils bioaugmented with the exogenous consortium contained only 2.76% Firmicutes, despite the consortium itself comprising 0.68% of this group, suggesting limitations in the ability of these bacteria to adapt to the contaminated environment. Moreover, Firmicutes is recognized as one of the dominant bacterial groups with high efficiency in hydrocarbon degradation in soils, partly due to its ability to produce biosurfactants [22]. Therefore, its relative abundance may be associated with the observed degradation efficiency.

On the other hand, Bacteroidetes was also positively correlated with alkane degradation, TPH, and PAHs [83]. In the present study, Bacteroidetes were significantly more abundant in soils treated with native microbiota (9.96%), whereas bioaugmented soils with the exogenous consortium contained only 0.12% of this phylum. Although the difference in relative abundance was pronounced, both treatments achieved statistically equivalent reductions in TPH by the end of the experiment. These results suggest that, under the conditions of this study, Bacteroidetes may contribute to hydrocarbon degradation, but their relative abundance alone may not fully determine the overall effectiveness of the biodegradation process. In addition, this phylum is known for its role in organic matter degradation and nutrient cycling in soils, as well as for producing biosurfactants in the presence of hydrocarbons [84], its presence in soils may therefore be linked to biodegradation through mechanisms of metabolic cooperation.

In the hydrocarbon-degrading consortia, Stenotrophomonas initially dominated, representing 50.01% of the native consortium and 44.12% of the exogenous consortium. However, this genus was not detected among the top 100 genera in soils after bioaugmentation. Instead, Pseudomonas emerged as the predominant genus across all treatments (Figure 4b). This shift likely reflects the competitiveness and metabolic versatility of Pseudomonas, traits that enable it to degrade xenobiotic substrates such as hydrocarbons [85]. Although Stenotrophomonas was initially abundant, the assay conditions may not have been favorable for its proliferation, thereby restricting its role in hydrocarbon biodegradation. This finding is particularly noteworthy, given the well-documented capacity of this genus to degrade hydrocarbons. Genomic analyses have shown that Stenotrophomonas harbors genes encoding oxygenases and dehydrogenases and can utilize metabolic pathways involving phthalic and salicylic acids for PAHs degradation [86].

In this context, the abundant presence of Pseudomonas in soil samples after the bioremediation assay may be associated with the significant removal of TPH observed across all treatments. The genus Pseudomonas is widely recognized for its ability to degrade hydrocarbons and is commonly found in high abundance at contaminated sites [87]. This bacterial group can catabolize xenobiotic compounds, including hydrocarbons, due to the high metabolic plasticity, which provides diverse adaptive strategies allowing it to tolerate and utilize such compounds [88]. Pseudomonas spp. play a crucial role in the aerobic biodegradation of PAHs, as they synthesize the enzyme family responsible for initiating the first ring-cleavage reaction, the oxygenases [89]. In addition, a positive correlation between genes encoding these enzymes and hydrocarbon biodegradation efficiency has also been reported in bioaugmentation or biostimulation studies involving Pseudomonas, supported by functional metagenomics [90].

Similarly, another notable difference in consortium composition was observed in the abundance of Ochrobactrum in the exogenous consortium (30.76%), whereas this genus was scarcely detected in the native consortium (0.0038%). Although Ochrobactrum has been previously reported as effective in hydrocarbon biodegradation in soils, achieving significant contaminant reductions [91], its proliferation in the present study may have been limited due to competitive interactions with the resident microbiota, as well as suboptimal adaptation to the environment, given its status as an exogenous agent [92].

Moreover, a notable shift in taxonomic composition at the genus level was observed following both biostimulation and bioaugmentation (Figure 4b), highlighting the impact of these bioremediation treatments on the bacterial community structure at the genus level. Such alterations in soil bacterial community composition after the application of bioremediation strategies have been previously reported [93]. This phenomenon is primarily attributed to adaptive strategies employed by dominant microbial groups [76]. In addition, bioremediation processes often modify soil conditions, including pH, salinity, aeration, and nutrient availability, thereby inducing changes in the resident microbiota not only at the individual level but also through the alteration of ecological interactions [94].

Furthermore, other bacterial genera identified among the most abundant groups in the soils included Rhodococcus, Bacillus, Nocardia, Xanthomonas, and Devosia across all samples, while Sphingobium, Achromobacter, Paracoccus, and Phenylobacterium were detected only in the treated samples (Table S4 of the Supplementary Material). Various species within these genera have been investigated for their hydrocarbon-degrading capabilities in soils, and their presence and relative abundance are therefore considered important in hydrocarbon biodegradation processes.

The genera Rhodococcus, Bacillus, and Nocardia were the most abundant both in the initial and final soil samples. Specifically, Rhodococcus accounted for 0.71–4.35% of the total taxonomic composition, while Bacillus ranged from 0.41 to 1.78%. Since the abundance ranges of these genera did not exhibit significant differences across treatments, it can be inferred that they represent stable components of the soil microbiota. This stability may be attributed to their metabolic versatility, including the ability to degrade a wide array of organic compounds, which allows them to maintain relatively constant populations even under varying environmental conditions [95]. The hydrocarbon biodegradation potential of these genera has been previously reported. Rhodococcus erythropolis KB1 achieved 95% TPH removal in an alfalfa-assisted rhizoremediation system and promoted plant growth by increasing nitrogen and phosphorus availability in contaminated soils [96]. Similarly, Bacillus marsiflavi reached 65% TPH removal within five days and exhibited both emulsifying properties and plant growth-promoting effects [97]. In contrast, the abundance of Nocardia varied markedly among samples: 16.03% in the initial soil, 10.02% in the soil bioaugmented with the native consortium, 4.69% in soil treated with native microbiota, and 0.51% in soil bioaugmented with the exogenous consortium. Nocardia species inhabit soils rich in organic matter, and their stable presence is likely due to their competitive ability, including the production of antimicrobial metabolites [98]. However, the low abundance of Nocardia in the soil bioaugmented with the exogenous consortium again highlights potential adaptation limitations to the local environment. Moreover, the hydrocarbon-degrading potential of this genus has been further evidenced in the genomic analysis of a novel strain, Nocardia canadensis, which revealed a high abundance of hydrocarbon degradation-related genes, notably alkB (alkane monooxygenase) and ndo (naphthalene dioxygenase) [99]. Furthermore, these genera are also recognized as plant growth-promoting bacteria, suggesting that their persistence in the soil may be linked to the restoration of soil ecological function [100,101,102].

Finally, the genera Sphingobium, Achromobacter, Paracoccus, Phenylobacterium, Xanthomonas, and Devosia exhibited a significant increase in relative abundance after the biodegradation process. Notably, these taxa were absent from the 100 most abundant genera in the untreated soils but ranked among the top 25 in the bioremediated soils. This shift suggests a microbial succession likely driven by the bioaugmentation and biostimulation strategies employed in this study, promoting the proliferation of these groups and reflecting an adaptive response linked to their capabilities in hydrocarbon degradation, potentially involving them in metabolic cooperative webs that facilitate hydrocarbon mineralization in the soil, through cross-feeding mechanisms where primary bacteria produce intermediates that are used by secondary bacteria as sources of carbon and energy, allowing complete contaminant degradation, as well as sequential biodegradation, where different microorganisms degrade a contaminant in successive stages [103]. The hydrocarbon-degrading capabilities of some strains belonging to these groups are described below. Liu et al. [104] reported that Sphingobium sp. SJ10-10 achieved 92.6% phenanthrene degradation within 15 days, attributed to the expression of iron-dependent ring-hydroxylating dioxygenases (RHDs). Achromobacter xylosoxidans, isolated from petrochemical-contaminated soils, demonstrated the ability to utilize anthracene as its sole carbon source [105]. Similarly, Paracoccus aminovorans degraded 47.7% of pyrene and 30.7% of benzo[a]pyrene under microcosm conditions, with bioaugmentation significantly reshaping bacterial community composition at the phylum level [106]. Phenylobacterium sp., isolated from petroleum-contaminated saline soil, was able to grow on 200 ppm of anthracene and phenanthrene as the sole carbon sources. When incorporated into a hydrocarbon-degrading consortium, this strain facilitated crude oil removal of up to 86% within 90 days [107]. The hydrocarbon-degrading potential of Xanthomonas campestris was evaluated using crude oil, kerosene, diesel, and gasoline in media supplemented with varying concentrations of monoammonium phosphate, achieving biodegradation efficiencies between 60% and 80% after 21 days [108]. Moreover, Devosia polycyclovorans sp. degraded 58% of pyrene and 48% of benzo[a]pyrene within 5 days; genomic analysis revealed genes associated with PAHs degradation pathways [109]. Collectively, these findings support the notion that microbial community dynamics not only reflect contaminant removal but also promote novel ecological interactions within soils. Similar observations were reported by Yang et al. [110], who described the coexistence of Phenylobacterium with other hydrocarbon-degrading genera, suggesting that trophic-level interactions enhance contaminant removal through cooperative biodegradation processes such as cometabolism.

5. Conclusions

The results of this study demonstrate that biostimulation of the native microbiota and bioaugmentation with hydrocarbon-degrading bacterial consortia constitute effective strategies for hydrocarbon removal in real agricultural soils. Although the exogenous consortium exhibited greater diversity than the native consortium, this versatility did not confer an advantage during the biodegradation process. Instead, competition among microbial groups already adapted to the environment appeared to prevail, resulting in higher contaminant removal efficiency. Moreover, biostimulation of the native microbiota also modified the bacterial taxonomic composition, promoting the proliferation of hydrocarbon-degrading bacteria that enabled significant removal. Nevertheless, the treatment bioaugmented with the native consortium achieved the highest degradation percentage and fastest degradation rate. This underscores the importance of increasing the population of environmentally adapted hydrocarbon-degrading bacteria in these processes. In this context, the observed correlation between the increase in the viable population of cultivable hydrocarbon-degrading bacteria and the reduction TPH concentration demonstrates that the population dynamics of these microorganisms can serve as an indicator of metabolic activity during bioremediation. Notably, the highest degradative activity was observed within the first 14 days, underscoring the importance of maintaining a stable degradative population through nutrient supplementation and reinoculation of strains or consortia. Nevertheless, although overall PAH concentrations decreased, some high-molecular-weight compounds remained in the soil, highlighting the need for further studies to assess potential environmental and health risks.

Following bioremediation, significant shifts were observed in the composition and structure of the bacterial community. At the phylum level, Actinobacteria was replaced by Proteobacteria in biologically treated soils, underscoring the latter’s role as a key taxon and potential bioindicator of hydrocarbon degradation. Similarly, Pseudomonas emerged as the dominant genus, outcompeting previously more abundant genera within the consortia, such as Stenotrophomonas, reflecting its metabolic versatility and competitive advantage. In contrast, Rhodococcus, Bacillus, and Nocardia maintained relatively stable abundances across all soil samples, suggesting that their resilience traits are critical in contaminated soils, contributing both to hydrocarbon degradation and the restoration of soil ecological functions. Finally, the relative abundances of Sphingobium, Achromobacter, Paracoccus, Phenylobacterium, Xanthomonas, and Devosia increased significantly in the bioremediated treatments, indicating a probable microbial succession linked to the adaptive and metabolic functions of these bacterial groups, potentially involving cooperative trophic networks that enhanced hydrocarbon biodegradation. These results underscore the importance of examining microbial functions and interactions, which represent a valuable perspective for future research aimed at optimizing bioremediation strategies, complementing the insights gained here from changes in taxonomic composition and biodegradation efficiency.

In conclusion, the findings of this study emphasize the importance of selecting bacterial consortia naturally adapted to contaminated environments and demonstrate the value of specific bacterial groups as bioindicators for assessing the effectiveness of soil restoration strategies in hydrocarbon-impacted agroecosystems. Moreover, the results reveal viable, rapid, and effective hydrocarbon biodegradation strategies, highlighting potential patterns of microbial succession linked to both degradation processes and the ecological recovery of contaminated soils. Collectively, these insights provide a robust foundation for designing targeted, high-performance bioremediation approaches in agricultural soils affected by hydrocarbon pollution.