Ecological Shifts and Functional Adaptations of Soil Microbial Communities Under Petroleum Hydrocarbon Contamination

Abstract

Petroleum hydrocarbon contamination has emerged as a significant global environmental issue, severely impacting soil microbial communities and their functions. This study employed high-throughput sequencing to systematically analyze the bacterial community structure and functional genes in soils with varying levels of petroleum hydrocarbon contamination. The results demonstrated that petroleum contamination led to a significant decline in microbial diversity, while enhancing the abundance of specific functional genes, such as those involved in polycyclic aromatic hydrocarbon (PAH) degradation, methane production, and denitrification. Phylogenetic analysis further revealed that microbial communities in highly contaminated soils tended to form highly clustered and specialized groups, while simultaneously promoting the coexistence of phylogenetically distant microorganisms. The Mantel test identified significant correlations between ammonium ion concentration, soil moisture content, and microbial metabolic pathways, particularly those related to petroleum hydrocarbon degradation and denitrification. These findings suggest that petroleum contamination not only disrupts the carbon and nitrogen metabolism balance but also has profound implications for greenhouse gas emissions and nitrogen cycling, potentially destabilizing the ecosystem. This study provides novel insights into the ecological functions of microbial communities in petroleum-contaminated soils and highlights potential key factors for pollution management and ecological restoration.

1. Introduction

Soil ecosystems play a crucial role in maintaining global biogeochemical cycles and environmental regulation [1]. Among these processes, the carbon and nitrogen cycles are key pathways that directly influence the nutrient supply, pollutant degradation, and ecosystem stability [2]. Soil microbial communities are central drivers of these cycles, facilitating organic matter decomposition, nitrogen transformation, and energy flow through diverse metabolic processes [3]. In addition to these core functions, soil microbial communities also regulate the dynamic balance of carbon and nitrogen cycles by forming complex metabolic networks. These networks facilitate microbial cooperation, enabling efficient resource utilization and improving resilience against environmental disturbances [4,5]. However, soil ecosystems are increasingly threatened by various anthropogenic activities, with petroleum hydrocarbon pollution emerging as a significant global environmental concern [6]. Petroleum hydrocarbons, major constituents of petroleum and its derivatives, frequently enter the soil environment due to accidental spills during oil exploration, transportation, and storage, posing a serious threat to ecosystem functions [7]. Due to the complex chemical structure and persistence of petroleum hydrocarbons, this type of pollution has difficulty degrading naturally in the short term, further aggravating its impact on the soil environment [8]. The hydrophobic nature of these compounds further limits their bioavailability, reducing microbial access and slowing degradation rates [9].

The adverse effects of petroleum hydrocarbon pollution extend beyond alterations in soil physicochemical properties, significantly impacting microbial community structure and ecological functions [10]. Upon entering the soil, petroleum hydrocarbons modify parameters such as the pH, moisture content, organic carbon levels, and nitrogen content, thereby altering the microbial growth conditions, community composition, and metabolic activities [11,12]. These changes not only impair the original microbial community functions but may also promote the enrichment of certain pollution-resistant microbial populations, further altering the soil ecological balance [13]. Such enriched populations may develop specialized metabolic pathways that enable them to degrade specific hydrocarbon fractions, demonstrating adaptive evolution in polluted environments [14]. As a crucial determinant of microbial community structure and function, soil characteristics may exert a gradient effect on microbial communities at different pollution levels [15].

Previous studies have indicated that petroleum hydrocarbon pollution can significantly reduce microbial diversity while shifting metabolic functionality [16,17]. In petroleum hydrocarbon-contaminated soils, microbial communities play a pivotal role in pollutant degradation and ecological restoration through diverse metabolic pathways, with notable contributions in alkane degradation, polycyclic aromatic hydrocarbon (PAH) degradation, methanogenesis, and denitrification [12,18]. Alkane-degrading microbes utilize alkanes as carbon sources, transforming them into intermediate metabolites through key enzyme systems such as monooxygenases and dioxygenases, ultimately achieving mineralization [19,20]. Moreover, certain microbial populations exhibit co-metabolism during degradation, providing a potential advantage in breaking down complex pollutants [21]. This cooperative metabolism expands the spectrum of degradable hydrocarbons, allowing microbial consortia to target structurally diverse pollutants [22]. PAHs, as structurally complex and recalcitrant petroleum hydrocarbon components, undergo degradation via intricate biochemical pathways involving ring cleavage and aromatic oxidation [23,24]. In anaerobic conditions, methanogenic archaea convert organic intermediates into methane, representing a critical step in carbon turnover [25,26]. Denitrifying bacteria facilitate nitrogen cycling by reducing nitrate to nitrogen gas, mitigating the risk of soil eutrophication during petroleum hydrocarbon degradation [27,28]. The occurrence of these metabolic processes depends not only on microbial community composition but also on pollution levels and the resulting changes in soil physicochemical properties [29,30]. Although there has been an increase in research on petroleum hydrocarbon pollution, three critical knowledge gaps persist: (1) the continuum of microbial functional adaptation across different pollution levels (e.g., moderate contamination) remains underexplored, with most studies classifying environments as either polluted or pristine; (2) the systematic link between pollution-induced shifts in physicochemical properties (such as organic carbon and nitrogen changes) and the conservation of phylogenetic traits has rarely been quantified; and (3) the trade-offs between hydrocarbon degradation priorities and essential biogeochemical processes across pollution gradients lack mechanistic explanation. Previous studies have mostly relied on single-pollution-level snapshots or focus on isolated metabolic pathways, failing to capture how microbial communities allocate metabolic resources across contamination gradients while maintaining core ecosystem functions.

Therefore, this study aims to investigate the effects of different pollution levels (highly, lightly, and non-contaminated) on soil physicochemical properties, microbial community structure, and metabolic functions associated with alkane degradation, PAH degradation, methanogenesis, and denitrification. By analyzing the phylogenetic characteristics and metabolic potential of microbial communities in contaminated soils, this study seeks to elucidate the complex regulatory mechanisms governing carbon and nitrogen metabolism networks in petroleum hydrocarbon-polluted environments, ultimately providing theoretical support for ecological remediation strategies and microbial regulation approaches.

2. Materials and Methods

2.1. Site Description and Sample Collection

The data for this study were derived from a previous sample collection conducted in an abandoned coking plant industrial park in Beijing, China (39°53′~39°59′ N, 116°07′~116°14′ E) [31]. Based on the historical pollution levels in the area, the study region was categorized into highly contaminated (H), lightly contaminated (L), and non-contaminated zones (N), representing different levels of soil contamination. During the sample collection, 10 soil samples were randomly collected from the highly contaminated zone, 8 from the lightly contaminated zone, and 5 control soil samples were obtained from the unpolluted zone. To minimize the impact of soil heterogeneity on the experimental results, three parallel samples were taken from each sampling point, thoroughly mixed, and analyzed as a single composite sample. The data used in this study are based on the aforementioned sample collection, ensuring both the representativeness of the samples and the consistency of the data.

2.2. Soil Properties Analysis

This study measured several physicochemical parameters of the soil, including the pH, moisture content, total carbon (TC), total nitrogen (TN), nitrate (NO₃⁻), nitrite (NO₂⁻), and ammonium nitrogen (NH₄⁺). The pH was determined using a 1:5 soil-to-water mixture and measured with a pH analyzer (HI2221, Hanna Instruments, Italy). The soil moisture content was determined by drying 2 g of fresh soil at 105 °C until a constant weight was achieved. The TC and TN contents were quantified using an elemental analyzer (Elementar, Elementar Analysensysteme GmbH, Germany). NH₄⁺, NO₂⁻, and NO₃⁻ were extracted from the soil using a 1:5 mixture of fresh soil and 2 M potassium chloride (KCl), and measured with a flow injection analyzer (AACE, AACE GmbH, Germany). The concentration of total petroleum hydrocarbons (TPHs, C10–C40) was determined following the standard method HJ 1021-2019 issued by the Ministry of Ecology and Environment of China. All physicochemical parameters of the soil samples were measured in triplicate to ensure the reliability and accuracy of the data.

2.3. DNA Extraction and Sequencing

Microbial community DNA was extracted using the E.Z.N.A.^® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s protocol. The quality of the extracted DNA was assessed using 1% agarose gel electrophoresis. Amplification of the bacterial 16S rRNA gene V3-V4 region was performed using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 10 min. After quality verification of the amplification products via 2% agarose gel electrophoresis, high-throughput sequencing was conducted using the Illumina MiSeq PE300 platform (Majorbio Bio-Pharm Technology, Shanghai, China). The raw sequences obtained were quality controlled using fastp (v0.20.0) and assembled using FLASH (v1.2.7). Sequence clustering was performed using USEARCH (v7-uparse) with a 97% similarity threshold for OTU classification. Species annotation was based on alignment with the SILVA 138/16S_bacteria database, with a confidence threshold of 0.7.

2.4. Data Analyses

Phylogenetic diversity analysis included the phylogenetic diversity (PD), phylogenetic species variability (PSV), phylogenetic species richness (PSR), phylogenetic species evenness (PSE), and phylogenetic species clustering (PSC). Correlation analysis was performed using the “picante” package in R, and the phylogenetic tree was visualized using iTOL (https://itol.embl.de/) (accessed on 20 August 2024). The microbial community metabolic function prediction was performed using the PICRUSt2 tool (https://github.com/picrust/picrust2) (accessed on 10 October 2024).

A one-way ANOVA was conducted to assess the effects of petroleum hydrocarbon pollution levels on the measured variables. SIMPER analysis was performed using PAST3 software. Comparison of metabolic pathways at level 2 was carried out using STAMP. The Mantel test was performed with the “linkET” function in the R package to evaluate the relationships between microbial communities and environmental variables.

3. Results and Discussion

3.1. Geochemical Conditions in Petroleum-Contaminated Soils

The geochemical characteristics of soils contaminated with varying levels of petroleum were compared (Figure 1). The results revealed significant differences in the concentrations of total carbon (TC), total nitrogen (TN), and total petroleum hydrocarbons (TPHs) across the different contamination levels. Specifically, the average TC content in soils with light petroleum contamination was significantly higher than that in heavily contaminated and non-contaminated soils (H: 1.62%; L: 3.68%; N: 2.38%). The TC content observed in lightly polluted soils could be a result of increased organic matter inputs, possibly from petroleum degradation products or natural vegetation contributions [32]. As petroleum compounds undergo microbial degradation, they can release various organic intermediates, which may contribute additional carbon to the soil [6]. Additionally, the presence of vegetation, particularly plants that grow in contaminated environments, may increase the input of organic matter through root exudates, decaying plant material, and microbial interactions [33]. This organic matter can provide a richer substrate for microbial communities, fostering greater microbial activity and potentially enhancing the soil’s ability to assimilate and degrade contaminants [34]. Such changes in organic inputs might initially increase the carbon content in contaminated soils, promoting microbial diversity and activity [35]. The TN content in heavily contaminated soils was significantly lower compared to both L and N. The decrease in TN in heavily contaminated soils could be attributed to microbial immobilization or the alteration of nitrogen cycling processes, potentially due to petroleum hydrocarbons interfering with nitrogen-fixing bacteria or nitrifying microorganisms [36,37]. In addition, TPH concentrations in the heavily contaminated soils were significantly elevated compared to the lightly contaminated and non-contaminated soils, reaching 1412.00 mg/kg, which was 10.16 times higher than that in lightly polluted soils. The elevated TPH concentrations in H further highlight the significant impact of petroleum contamination, which could disrupt microbial communities, inhibit the breakdown of organic matter, and alter the natural biogeochemical cycling of nutrients in the soil [38,39]. No significant differences were observed in the soil moisture content, pH, or other nutrient levels, including NH₄⁺, NO₂⁻, and NO₃⁻, across the different sampling locations (p > 0.05). These findings suggest that varying levels of petroleum contamination can alter the physicochemical properties of the soil, which in turn may influence the assembly of soil microbial communities, potentially leading to distinct geochemical ecological niche effects.

3.2. Impact of Petroleum Contamination on Soil Bacterial Phylogenetic Structure

The phylogenetic diversity characteristics of the bacterial community in all the petroleum-contaminated soil samples revealed significant differences (Figure 2). The SR, PD, and PSR were significantly higher in both the lightly contaminated and uncontaminated soils compared to the heavily contaminated soils (p < 0.05), with no significant differences between the former two groups. This suggests that increased petroleum contamination may lead to a decline in bacterial community richness and phylogenetic diversity. This decline in richness and diversity can be attributed to the inhibitory effects of high petroleum contamination, which may hinder microbial growth and reproduction, creating a less favorable environment for diverse microbial species [10,40]. The PSV was highest in heavily contaminated soils, followed by lightly contaminated and non-contaminated soils (p < 0.05), indicating that contamination may promote the coexistence of phylogenetically distant microorganisms. The higher PSV in H suggested that contamination stress may push microbial communities to adapt through the coexistence of diverse, phylogenetically distant species, potentially as an adaptive survival strategy in the harsh contaminated environment [41]. The PSE was significantly lower in H compared to the other two groups (p < 0.05), suggesting that high contamination levels may result in community dominance by a few specialized taxa, reducing phylogenetic evenness. This reduction in evenness indicates that, in response to pollution stress, only a few specialized species may thrive, leading to an imbalanced microbial community where dominant taxa suppress others [42]. The PSC was highest in H, significantly exceeding that of the other groups (p < 0.05), implying that contamination may favor the enrichment of phylogenetically related microorganisms, possibly those with shared resistance mechanisms or metabolic pathways suited for degrading hydrocarbons [20].

Overall, petroleum contamination substantially alters the phylogenetic structure of soil bacterial communities, with heavy contamination reducing diversity and evenness while increasing phylogenetic variability and clustering. These findings suggest that pollution stress exerts strong selective pressure on microbial communities, potentially leading to the dominance of specific resistant taxa while promoting the coexistence of phylogenetically distant microorganisms as a means to adapt to and survive in the contaminated environment.

The bacterial genera identified through SIMPER analysis, such as Tepidiphilus (OTU160), Pseudomonas (OTU171, OTU5612), Paenisporosarcina (OTU153), and Arthrobacter (OTU2583), exhibit distinct metabolic characteristics that reflect their adaptation to varying levels of petroleum hydrocarbon pollution. Tepidiphilus, often associated with hydrocarbon-degrading environments, possess the metabolic pathways necessary for the breakdown of complex hydrocarbons, making them ideal candidates for petroleum degradation in highly contaminated soils [43]. Similarly, Pseudomonas, known for its ability to degrade a wide range of organic pollutants, including hydrocarbons, likely plays a crucial role in petroleum hydrocarbon breakdown in heavily contaminated soils [44]. In contrast, Paenisporosarcina, which was enriched in lightly contaminated soils, may specialize in the degradation of specific hydrocarbons or serve as part of the microbial community that balances hydrocarbon degradation with other ecological functions at intermediate contamination levels [45]. On the other hand, Arthrobacter, which was primarily found in non-contaminated soils, is known for its ability to metabolize simpler carbon compounds and may have a preference for low-pollution environments, where it likely contributes to basic soil functions, such as organic matter decomposition and nutrient cycling [46,47]. These differences in metabolic abilities highlight how microbial communities in polluted soils adapt to varying environmental conditions, with distinct genera playing pivotal roles in contaminant degradation and ecosystem balance.

These findings highlight clear microbial shifts along the pollution gradient, shedding light on how microbial communities adapt to varying levels of petroleum contamination. The differential enrichment of specific bacterial genera underscores their potential roles in pollutant degradation and their ability to thrive under different contamination levels. For instance, genera such as Tepidiphilus and Pseudomonas are particularly enriched in highly contaminated soils, suggesting their active involvement in hydrocarbon degradation. Meanwhile, Paenisporosarcina appears to thrive in lightly contaminated soils, potentially playing a key role in balancing degradation and ecological stability. In contrast, Arthrobacter is more prevalent in non-contaminated soils, indicating its preference for less disturbed environments. These shifts in microbial community structure not only reveal how specific genera adapt to environmental stressors but also emphasize the dynamic nature of soil ecosystems in response to contamination gradients.

3.3. Overall Microbial Functional Patterns

Functional prediction based on high-throughput sequencing data identified a total of 408 KEGG metabolic pathways. Among the primary metabolic processes, metabolism dominated microbial functional activity, accounting for 74–78% of the total (Figure S2). However, the contribution of primary metabolic processes varied across different levels of petroleum hydrocarbon contamination. Under high contamination levels, the relative abundance of metabolism decreased from 77% in non-contaminated soils to 74%, while the proportions of environmental information processing and cellular processes increased to 7% and 6%, respectively. This shift suggests that microbial communities are adjusting their metabolic focus in response to the stress induced by high petroleum contamination, prioritizing processes related to environmental adaptation and cellular survival over basic metabolic functions [48].

Further analysis of secondary metabolic pathways revealed that in highly contaminated soils, cellular processes exhibited a higher relative abundance compared to N and L. Specifically, cell growth and death, cell motility, cellular community with prokaryotes, and transport and catabolism were more enriched under high contamination levels (Figure S3). This enrichment indicates that petroleum contamination may stimulate mechanisms related to microbial survival and community dynamics, such as motility and cellular communication, which are essential for microbes to navigate and thrive in a contaminated environment [49]. Similarly, within environmental information processing, membrane transport and signal transduction showed increased abundance in H (Figure S3). These findings further highlight the importance of cellular adaptation to environmental stress, with microbial communities enhancing their ability to sense and respond to pollution through more efficient transport and signaling mechanisms [50]. These findings suggest that microbial communities in petroleum hydrocarbon-contaminated environments undergo significant metabolic shifts, with a reduced emphasis on primary metabolism and an enhanced capacity for cellular adaptation and environmental response. This shift reflects a microbial survival strategy, where the community adapts to high contamination stress by reprogramming its metabolic processes to focus more on cellular maintenance, communication, and the detoxification of harmful pollutants, thereby optimizing its chances of survival in the polluted environment [51].

Compared to non-contaminated soil, xenobiotics biodegradation and metabolism were significantly enhanced in contaminated soil (Figure 3), indicating an increased microbial capacity for degrading exogenous organic pollutants under polluted conditions. This suggests that petroleum contamination serves as a selective pressure, favoring the growth and activity of microbes capable of breaking down hydrocarbons and other xenobiotic compounds [52]. This metabolic process primarily involves the transformation, degradation, and mineralization of toxic organic compounds, facilitating their breakdown into non-toxic or less toxic small molecules, such as carbon dioxide and water, through a series of enzymatic reactions [53]. Moreover, this pathway plays a crucial role in pollutant tolerance, environmental adaptation, and microbial community regulation, enabling microbial populations to optimize their functional potential and ecological niche adaptation under pollution stress, thereby enhancing community resilience and stability [54]. The increased activity of this pathway under contamination conditions further underscores the microbial community’s capacity to mitigate pollutant effects and restore environmental balance, suggesting that microbial communities may be playing a pivotal role in the bioremediation of contaminated soils [55].

3.4. Key Microbial Functional Genes

Figure 4 illustrates the variations in microbial functional gene abundance across soils with different levels of petroleum hydrocarbon contamination and their roles in carbon and nitrogen metabolism, highlighting the profound impact of petroleum hydrocarbons on microbial community functions. Despite their hydrophobicity and toxicity, petroleum hydrocarbons can serve as potential carbon sources, promoting the enrichment of genes associated with alkane and PAH degradation (e.g., alkB, dhaA, bph, nah) in contaminated environments. These genes were significantly upregulated in the H and L groups, whereas their abundance was lower in the N group. This suggests that petroleum contamination not only introduces toxic compounds but also provides selective pressure, favoring microbes capable of metabolizing these hydrocarbons as a carbon source [56]. Additionally, genes involved in aromatic ring cleavage (e.g., pca, cat, lig) were more abundant in petroleum-contaminated soils, indicating enhanced PAH degradation potential. The increased abundance of these genes in polluted environments reflects the microbial community’s adaptation to the unique carbon substrates present in the soil, highlighting the role of these microbial communities in breaking down potentially harmful compounds into simpler, more manageable forms [57].

The degradation of hydrocarbons generates fatty acids and acetyl-CoA, which can enter the TCA cycle, providing energy for microbial metabolism and partially contributing to methane production. In contaminated soils, genes related to hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis (e.g., mcr, fwd, ackA) exhibited higher abundance in the H and L groups than in the N group, suggesting that contamination may facilitate methane emissions. The increased methanogenesis potential in contaminated soils could be indicative of a shift in microbial community composition towards those that are more capable of utilizing the by-products of hydrocarbon degradation, further emphasizing the complex interplay between hydrocarbon degradation and methane production [58]. Furthermore, denitrification-related genes (e.g., nir, nar, nap) were enriched in polluted environments, potentially enhancing nitrate reduction and leading to increased emissions of N₂O, a potent greenhouse gas. This shift in microbial function towards denitrification could exacerbate global warming effects by increasing N₂O emissions, further complicating the environmental impacts of petroleum contamination [59].

In contrast, nitrogen fixation genes (nif) were more abundant in the N group but significantly reduced in the H and L groups, suggesting that petroleum hydrocarbons may inhibit biological nitrogen fixation. This reduction in nitrogen fixation gene abundance in contaminated soils highlights the negative impact of petroleum hydrocarbons on microbial nitrogen fixation, a crucial process for maintaining soil fertility and ecosystem health [60]. Although petroleum contamination creates extreme environmental conditions, studies indicate that contaminated soils still support complex microbial ecosystems. To adapt to pollution-induced stress, microbes have evolved a suite of functional mechanisms, including carbon metabolism, methane metabolism, stress responses, signaling, and transport systems, enabling their survival and ecological functionality in contaminated environments [61,62]. These functional adaptations underscore the resilience of microbial communities, which can evolve diverse strategies to cope with pollutants and maintain ecosystem processes [4]. However, these metabolic alterations not only influence carbon and nitrogen cycling but may also exacerbate greenhouse gas emissions and disrupt soil nitrogen dynamics, thereby affecting ecosystem stability on a broader scale [63]. This dual impact on greenhouse gas emissions and nitrogen cycling emphasizes the need to consider both microbial adaptations and the long-term ecological consequences of petroleum contamination [64]. It suggests that while microbes may adapt to the presence of petroleum hydrocarbons, their functional shifts could have broader implications for soil fertility, greenhouse gas fluxes, and overall ecosystem health.

3.5. Factors Controlling Soil Bacterial Metabolism Under Petroleum Contamination

The Mantel test helps analyze the relationship between the abundance of petroleum hydrocarbon degradation-related functional genes (including alkane degradation, PAHs degradation, denitrification, nitrogen fixation, and methane production genes) and soil physicochemical properties (Figure 5). The results indicated that the TPH content was significantly correlated with the initial degradation of PAHs (PAHs-initial degradation), fatty acid-acetoclastic metabolism, and denitrification. Moreover, NH₄⁺ concentration showed significant correlations with the initial degradation of PAHs, PAH-ring cleavage, fatty acid-acetoclastic metabolism, and denitrification. The soil moisture content was primarily associated with the initial degradation of PAHs and denitrification. These correlations reflect the close coupling of carbon and nitrogen metabolism under soil contamination conditions and the underlying mechanisms. Firstly, TPH, as the main component of petroleum contamination, provides a carbon source for microorganisms, thus promoting PAH degradation and acetate-utilizing methane production processes. The presence of TPH may serve as a selective pressure for microbial communities, favoring those capable of hydrocarbon degradation, and consequently shaping the microbial composition in contaminated soils. This selective enrichment of hydrocarbon-degrading microbes suggests that petroleum hydrocarbons act as both an environmental stressor and a resource for microbial communities [65]. Additionally, organic acids and other intermediate metabolites produced during petroleum hydrocarbon degradation may serve as additional electron donors for denitrifying microorganisms, enhancing nitrate reduction [66]. The interplay between petroleum degradation and denitrification highlights the possibility of synergistic interactions between different microbial groups in the soil, where the products of one metabolic process can support another, promoting overall ecosystem function under contamination stress [67]. The accumulation of ammonium ions likely results from organic nitrogen degradation or ammonification, and both PAH degradation and denitrification processes are involved in nitrogen metabolism. Consequently, elevated NH₄⁺ concentrations may stimulate these metabolic pathways. This suggests that high ammonium concentrations may act as a driver for nitrogen cycling processes in contaminated soils, enhancing the functional capacity of microbial communities involved in nitrogen transformations.

The PAH-ring cleavage process, which involves the further breakdown of aromatic compounds, may also be regulated by NH₄⁺. The regulation of PAH degradation by ammonium may point to a feedback loop, where nitrogen-rich metabolites influence the efficiency of aromatic compound breakdown, demonstrating the interconnected nature of nitrogen and carbon cycles in contaminated soils [68]. The soil moisture content affects the diffusion of dissolved oxygen and determines the microbial metabolic activity [69]. Under high moisture conditions, anaerobic microorganisms, such as denitrifiers and anaerobic PAH-degrading bacteria, are more likely to be active, thereby enhancing PAH degradation and denitrification [70]. This indicates that the moisture content not only affects the physical properties of the soil but also plays a critical role in the regulation of microbial metabolic activity, particularly influencing the activities of anaerobic bacteria involved in the degradation of hydrocarbons and the nitrogen cycle. Collectively, these results reveal the complex regulatory mechanisms of the soil microbial metabolic network under petroleum hydrocarbon contamination. The findings underscore the importance of understanding the multifaceted interactions between soil physicochemical factors and microbial communities in polluted environments. The regulation of microbial functions by both carbon and nitrogen compounds suggests that managing soil contamination may require a holistic approach that considers not only the direct effects of pollutants but also the broader impacts on nutrient cycling and microbial community dynamics.

3.6. Comparative Ecological Implications and Future Perspectives

The decline in microbial diversity with increasing contamination levels, as observed in this study, may have significant implications for soil health and ecosystem services. Microbial diversity plays a crucial role in nutrient cycling, organic matter decomposition, and pollutant degradation. As microbial diversity decreases, these ecosystem services are at risk, potentially leading to reduced soil fertility, impaired carbon sequestration, and weakened pollutant degradation capacity. Furthermore, the enrichment of specific microbial populations, such as those involved in methane production, could have long-term consequences for greenhouse gas emissions. Increased methane production in contaminated soils may contribute to higher emissions, exacerbating climate change. The long-term impacts of microbial community shifts may also affect the overall ecosystem stability. As microbial communities adapt to contamination, changes in functional groups could either support or hinder ecosystem recovery. Understanding how these shifts influence ecosystem services, including soil health, nutrient cycling, and greenhouse gas emissions, is critical for developing effective management strategies for contaminated environments.

One of the primary limitations of this study is its cross-sectional design. Given that microbial communities are dynamic and may exhibit time-dependent responses to petroleum hydrocarbon contamination, the lack of temporal sampling restricts our ability to observe how microbial communities evolve and adapt over time in response to contamination. Microbial dynamics, such as shifts in community composition, metabolic activity, and functional adaptation, may not be fully captured at a single time point. For example, microbial populations might undergo initial stress responses, followed by longer-term adaptation processes that influence their functionality and degradation capacities. Future research incorporating temporal sampling through longitudinal studies would be necessary to better understand the time-dependent nature of microbial responses to contamination. Such studies would provide insights into how microbial communities shift across different stages of pollution exposure and how their functional profiles evolve over time. Furthermore, periodic monitoring would help identify key time points where microbial adaptation or community collapse occurs, which could offer valuable information for improving ecological restoration strategies and microbial management in contaminated environments. The integration of such studies would enhance our understanding of microbial resilience and adaptation, allowing for more effective management of contaminated sites and better prediction of long-term ecological outcomes.

4. Conclusions

Our study revealed that as contamination levels rise, the phylogenetic diversity of soil bacterial communities significantly declines, while species variability and clustering increase. Microorganisms in contaminated soils show enhanced petroleum hydrocarbon degradation potential, particularly in PAH degradation, methane production, and denitrification, with significant increases in gene abundance. Additionally, the NH₄⁺ concentration and soil moisture content are crucial for microbial metabolic pathways, promoting petroleum hydrocarbon degradation and enhancing the environmental adaptability of microbial communities. These findings highlight the potential for targeted microbial management in pollution remediation. Understanding how soil microbial communities respond to petroleum contamination can inform strategies for ecological restoration, emphasizing the role of microbial diversity and environmental factors in enhancing soil health. Such insights could contribute to more effective pollution control and the restoration of ecosystems impacted by petroleum hydrocarbons.