Di-(2-ethylhexyl) phthalate-degrading functional microorganisms were identified in black soil based on high throughput analysis

Highlights

• •Through high-throughput sequencing and analysis of multiple soil genomes, functional microorganisms involved in DEHP degradation were elucidated.
• •The combination of soil extract and inorganic salt medium was used to screen DEHP-degrading bacterial communities with good degradability, which was consistent with the bioinformatics data.
• •DEHP-degrading functional microorganisms in black soil have been identified.

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) has become an increasingly serious pollutant in soils. Microbial degradation represents a highly promising approach for its remediation. In this study, four black soils were used to simulate the natural degradation of DEHP over a 75-day microenvironmental experiment. High-throughput analysis was conducted to investigate the distribution and abundance of functional genes in soil microorganisms, aiming to explore functional microbial information. The degradation efficiency of DEHP in black soils was 76.37 %, 74.16 %, 92.21 %, and 75.35 %. The α-diversity of microbial community was positively correlated with the degradation rate. Actinobacteria and Proteobacteria exhibited sensitivity to DEHP contamination. Xanthomonaceae, Sphingomonadaceae, Hypomicrobiaceae, and Comamonadaceae contributed to the upstream metabolism of DEHP. The abundances of Rhodococcus, Sphingomonas, Nocardioides, and Arthrobacter were positively correlated with the abundance of functional genes enriched in the black soil for benzoate degradation. Concurrently, 10 DEHP-degrading bacterial communities were identified, and the taxonomic and functional profiles of certain members within these communities were consistent with the metagenomic data. Bacterial communities JQ104, JQ52, and JQ129 degraded >98 % of DEHP (400 mg/L) in 48 h, demonstrating remarkable degradation efficiency. This study demonstrated the dynamic impact of the indigenous microbiome on DEHP contamination and verified the degradation capabilities of key functional microorganisms.

Graphical abstract

Environmental implications.

DEHP is the most commonly detected contaminant in Chinese soil, often found at significantly high concentrations. In Northeast China, DEHP concentrations are predominate within the total concentration of phthalates (PAEs) in the soil. This study employed microcosm experiments to simulate the natural degradation of DEHP in black soil, integrating metagenomics to identify functional microorganisms capable of participating in DEHP degradation. Additionally, soil extract and minimal salt medium (MSM) were used to screen for microorganisms that can degrade DEHP. This study provided a theoretical foundation for the microbial remediation of DEHP in black soil by identifying functional microbial communities, offering valuable resources for its future in-situ degradation.

Alt-text: Unlabelled box

1. Introduction

Black soil is a valuable natural resource recognized for its high productivity and fertility (Ma et al., 2023a; Shen et al., 2025). It has the capacity to effectively adsorb heavy metal ions and organic pollutants (Nguyen et al., 2021). The use of plastic films is increasingly common in agricultural practices. However, these films may contain phthalates (PAEs), which can leach into the soil over time (Scopetani et al., 2023). PAEs are highly hydrophobic and readily adsorbed by soil organic matter, allowing their residues to persist in the soil for extended periods (Sharma and Kaur, 2020). PAEs, commonly employed as plasticizers, have emerged as widespread contaminants in agricultural soils (Zou et al., 2025). According to the environmental emission inventory of polycyclic aromatic hydrocarbons in China, the cumulative emissions of Di-(2-ethylhexyl) phthalate (DEHP) were 29.022 million tons by 2019 (Bi et al., 2021). In the investigation of representative agricultural soils, DEHP showed the highest concentration level (Zou et al., 2025). In northeast China, the concentration of DEHP accounted for 83.79 % of the total concentration of PAEs, indicating its dominant position in soil (Li et al., 2021). In black soil, the bioavailability of DEHP was high, with approximately 6.8 % of the initial application being absorbed by plants and microorganisms (Zhang et al., 2023b). DEHP contamination can alter the diversity of soil microorganisms and affect the stability and function of microbial communities. It has been demonstrated that DEHP significantly inhibits soil microbial biomass carbon (Shah et al., 2024). Due to the inhibitory effect of DEHP on microorganisms, the nitrogen cycling process in soil was disrupted, resulting in an imbalance between nitrogen transformation and release, and inhibiting transformation of nitrogen in soil (Tao et al., 2022; Wang et al., 2024a). DEHP competed negatively with other phthalates, such as dibutyl phthalate (DBP), during the nitrification process in black soil (Tao et al., 2022).

The degradation capacity of microorganisms is pivotal in influencing the fate of DEHP in soil. Under optimal conditions, specific microorganisms can effectively degrade DEHP, thereby reducing its environmental toxicity (Singh et al., 2023). Microbial remediation offeres distinct advantages over physicochemical methods, exhibiting greater controllability, broader applicability, and enhanced adaptability. Microorganisms consistently demonstrate rapid adaptive responses to environmental changes, which are attributed to their high metabolic and reproductive turnover rates (Sazykina et al., 2022). Bacteria-driven biodegradation was considered to be the most promising strategy, with outstanding advantages including complete degradation of pollutants, lower treatment costs, higher safety, and less environmental disruption (Hu et al., 2021). Additionally, bacterial communities were demonstrated to have superior environmental adaptability compared to single-strain degradation (Liu et al., 2023). Microbial interactions and regulation are essential in promoting soil health through microbial mediation, including the bioremediation of soil contaminants. This approach is regarded as an environmentally friendly strategy for sustaining healthy soil conditions (Wu et al., 2023). Microbial communities enhanced the biodegradation and biotransformation processes of PAEs through a combination of diverse metabolic genes and enzymes (Puri et al., 2023). Studies have demonstrated that alterations in microbial functional genes can serve as a crucial tool for predicting environmental pollution. By monitoring changes in specific functional genes, researchers can assess the adaptive capacity of microorganisms to environmental shifts, evaluate pollutant levels in soil and water, and gauge ecosystem responsiveness (Liao et al., 2023; Sveen et al., 2024). Microbial functional diversity significantly contributed to ecosystem robustness (Wang and Xue, 2021). Therefore, variations in functional genes can influence the structure and function of microbial communities, impacting the resistance and resilience of ecosystems.

DEHP-degrading bacterial communities are primarily sourced from natural environments and utilize DEHP as a substrates in selective medium. There are two main approaches for designing and constructing synthetic microbial communities: top-down and bottom-up (Hu et al., 2021). Metagenomics identifies genes and metabolic pathways, studies genes associated with pollution metabolism, and provides critical information on metabolic function (Xu et al., 2022). The changes in degradation genes directly affect the degradation ability of microorganisms, and thereby influence the removal efficiency of DEHP in soil. In this study, black soil was used as the material, with DEHP serving as the degradation substrate in microcosm simulation experiments aimed at identifying the microbial classes potentially involved in the natural degradation of DEHP in black soil. Although numerous studies have successfully isolated DEHP-degrading bacterial communities from various environmental matrices, reports on functional strain resources derived from black soil remain poorly documented. Furthermore, conventional screening methods have largely relied on nutrient-limited minimal salt medium (MSM), which often fails to support the growth of environmentally relevant but unculturable microorganisms, thereby limiting the discovery of novel degraders. In this study, a novel screening strategy was developed by integrating soil extract with MSM to balance “culturability” and “functionality”. The soil extract supplied essential growth-supporting factors and acted as a crucial buffer that facilitated the adaptation of indigenous microorganisms from their natural habitat to laboratory conditions. Meanwhile, MSM maintained a strong selective pressure for DEHP degradation. This combined approach achieved an optimal equilibrium between “enrichment” and “targeted selection”. This study explored the functional microorganisms for DEHP degradation by integrating bioinformatics analysis with in situ microbiota screening. Multiple bacterial communities, exhibiting diverse species richness and abundance as well as high DEHP degradation efficiency, were successfully isolated. The composition of the bacterial community aligned well with the metagenomic sequencing results. In summary, this study not only provides an effective strategy for obtaining highly efficient DEHP-degrading microbial consortia from black soil but also offers essential insights for constructing synthetic microbial communities aimed at the bioremediation of DEHP-contaminated environments.

2. Materials and methods

2.1. Construction of soil microenvironment for DEHP degradation

The experimental flowchart of this study is shown in Fig. 1. The black soils were collected from the Inner Mongolia Autonomous Region, China (Oroqen Autonomous Banner, 123.72°N, 50.59°E) and Heilongjiang Province (Hongxinglong, 130.93°N, 46.94°E; Hailun, 126.96°N, 47.46°E; Keshan, 125.86°N, 48.03°E). The soil was air-dried naturally, and then stones, roots, branches, and leaves were removed before being sieved through a 2-mm mesh. Sterilized water was added to the air-dried soil to achieve a moisture content of 20 %. The soil was packed into porcelain pots (20 cm in diameter × 25 cm in height) and subjected to a pre-incubation at 25 °C for 14 days to restore and stabilize microbial activity. The soils were contaminated with DEHP at 40 mg/kg using acetone as a co-solvent and thoroughly mixed to ensure homogeneity. To evaluate the effects of the co-solvent, an acetone treatment group was established, which received an equivalent amount of acetone. Additionally, a sterile water treatment group was created that contained neither DEHP nor acetone. The microcosm experiment was conducted in an incubator maintained at a constant temperature of 25 °C and shielded from light for a duration of 75 days to simulate the natural degradation of DEHP in the soil. Throughout the experimental period, sterilized water was added to the soil every two days to maintain the moisture content at 20 %, using the constant weight method.

Fig. 1.

Flow chart of high-throughput identification of DEHP degrading functional microorganisms. Black soil was collected from four regions in China: Hailun City (HL), Keshan County (KS), Nongken Hongxinglong Farm (HXL) and Oroqen Autonomous Banner (ELC) of Inner Mongolia Autonomous Region. A 75-day microcosmic experiment was conducted to simulate the natural degradation of DEHP in black soil. Initially, soil samples from the DEHP treatment group (DEHP) were collected on days 0, 30, 60, and 75 to assess the concentrations of DEHP, phthalic acid, and benzoic acid. Subsequently, soil samples from the DEHP treatment group (DEHP), acetone control group (BT), and sterile water control group (S) were collected at each time point for metagenomic sequencing. Finally, the screening, identification, and degradation capacity of DEHP-degrading bacteria were evaluated.

2.2. Determination of DEHP and its intermediate metabolites in black soil

On days 0, 30, 60, and 75, 10 g of black soil samples were collected from various DEHP treatment groups and transferred to glass centrifuge tubes. The collected supernatant was then transferred to a round-bottomed flask for concentration using a rotary evaporator. A volume of 1 mL of methanol was added for reconstitution, after which the solution was transferred to a brown wide-mouth glass bottle for subsequent LC–MS analysis (Wang et al., 2025).

2.3. Metagenomic sequencing workflow and data analyses

The PE150 sequencing strategy was used on the Illumina HiSeq 2500 platform. The average sequencing depth of the DNA samples was 12 Gb per sample. Total DNA was extracted and tested according to the instructions of the PF Mag-Bind Soil DNA Kit, and the purity and concentration of the DNA were assessed. The high-quality sequences obtained from each sample were spliced, assembled and clustered to construct a non-redundant gene set (Chang et al., 2024). Species and abundance information was obtained for each taxonomic level in each sample. The KEGG functional annotation information of genes at the Pathway, Enzyme, Module and other levels was obtained by comparing with KEGG database.

2.4. Screening and identification of DEHP-degrading bacterial communities in black soil

The test medium was modified soil extract and MSM (Wang et al., 2022a). Soil extract was prepared by adding 500 g of naturally dried soil to 1 L of sterilized water. The mixture was shaken at 120 rpm for 24 h at room temperature, followed by centrifugation at 12,000 rpm for 10 min. The supernatant was filtered through folded qualitative filter paper, and 0.05 g/L of yeast extract was subsequently added. The above can be used as the soil extract medium after sterilization.

A total of 0.01 g of DEHP-contaminated soil was inoculated into 500 mL of soil extract medium. The medium was supplemented with 20 mg of DEHP, resulting in a final concentration of 40 mg/L. After vortexing at 120 rpm and 25 °C for 30 min, 20 mL of the culture was transferred to a sterilized glass centrifuge tube and vortexed for an additional 30 min. From this, 160 μL aliquots were dispensed into each well of a 96-well plate. The cultures were incubated in the dark at 25 °C for 3 days. Following incubation, all cultures from the turbid wells were pooled and transferred into MSM containing 40 mg/L DEHP. The culture was shaken at 120 rpm and 25 °C. The culture was subcultured (1 % v/v inoculum) into medium containing DEHP at sequentially increasing concentrations: 100, 200, 300, and 400 mg/L. Each domestication cycle lasted for 7 days. To stabilize the microbial community, the final domesticated culture was successively subcultured four times in fresh medium containing 400 mg/L of DEHP. The growth curve of this stabilized bacterial community was then determined under the same conditions (400 mg/L of DEHP, 120 rpm, 25 °C).

Bacterial communities with an OD at 600 nm of 1.0 were inoculated with a 1 % inoculum and cultured in MSM for 72 h. The cultures were collected by centrifugation and used for microbial diversity sequencing to elucidate the taxonomic composition of the community members and their relative abundance. High-throughput sequencing using Illumina PE300/PE250 technology was conducted by Shanghai Meiji Bio-pharmaceutical Science and Technology. The total genomic DNA of the microbial community was extracted, and its quality was assessed to determine both concentration and purity. PCR amplification and sequencing library construction were then performed. The V3-V4 variable region of the 16S rRNA was amplified using the upstream primer 338F and the downstream primer 806R, which included the barcode sequence. Taxonomic analysis of amplicon sequence variants (ASVs) was conducted based on the SILVA 16S rRNA gene database (version 138) to determine the composition of the DEHP-degrading bacterial communities and their relative abundance.

2.5. Determination of the degradation capacity of the bacterial communities

Bacterial communities (OD ₆₀₀ = 1.0) were inoculated at 1 % (v/v) into conical flasks containing 50 mL of MSM and incubated at 25 °C with shaking at 120 rpm for 72 h. Samples were collected at 12 h intervals throughout the incubation period (12, 24, 36, 48, 60, and 72 h). An equal volume of ethyl acetate was then added to the cultures, which were shaken vigorously to ensure complete contact among the ethyl acetate, the bacteria, and the walls of the flask. The mixtures were transferred to glass centrifuge tubes for centrifugation. The aqueous phase was extracted three times, and all organic phases were combined and concentrated using a rotary evaporator. The residue was re-dissolved in 5 mL of hexane, filtered through a 0.22 μm sterile membrane, and collected in a brown wide-mouth vial. The filtrate was collected in a brown wide-mouth vial to determine the DEHP content in the bacterial communities during the 72 h incubation period using GC–MS (Zhang et al., 2021).

2.6. Statistical analyses

The original metagenomic sequencing data, 16S sequencing data, and GC–MS data have been stored in the NCBI SRA database with the accession number PRJNA1322269. Intergroup differences in the microbial α-diversity indices across different periods were tested using the Kruskal–Wallis rank-sum test. Data were visualized using the ggplot and ggpubr packages in R. The microbial β-diversity of the black soil microbial community accross various periods was characterized using a non-constrained dimensionality reduction analysis method, specifically Principal Coordinates Analysis (PCoA). Differences between groups were assessed using ANOSIM with the Bray-Curtis distance algorithm, and two-dimensional scatter plots were utilized to illustrate the similarities and differences between the control and treatment group communities. Correlation analyses involving soil microbial diversity, soil physicochemical factors, and the rate of DEHP degradation were performed using the Mantel test, with correlation coefficients calculated via the Pearson algorithm. Data visualization for these correlations was achieved using the ggcor package in R to create heatmaps. Additionally, the composition of microbial community structures during DEHP degradation in four black soil sample plots was visualized using stacked bar plots generated with the ggplot package in R, enabling comparison of genus-level composition. Differences between groups were assessed using Student’ s t-test. The differences in microbial functional abundance among the non-treated groups exposed to DEHP were analyzed using one-way analysis of variance (ANOVA). All statistical analyses were considered significant at the P < 0.05 level.

3. Results and discussion

3.1. Analysis of DEHP content and degradation efficiency in black soil

The DEHP content in the black soil under various treatments at each site was assessed on days 30, 60, and 75 of the microcosm experimental period. On day 30, the DEHP content in black soil at each site was reduced to <20 mg/kg. By day 75, the DEHP content in the contaminated black soil from ELC, HL, HXL, and KS sites was measured at 9.75 ± 0.16 mg/kg, 10.66 ± 0.44 mg/kg, 3.24 ± 0.05 mg/kg, and 10.15 ± 0.13 mg/kg, respectively. The corresponding DEHP degradation efficiency was 76.37 ± 0.38 %, 74.16 ± 1.06 %, 92.21 ± 0.12 %, and 75.35 ± 0.35 %, with HXL soil exhibiting the highest degradation rate (Fig. 2). It was observed that the effective phosphorus and potassium content in the soil significantly increased with the addition of 10 mg/kg DBP, but decreased as the DBP concentration further increased. Furthermore, the total nitrogen and potassium content was higher than in the control when PAEs were added at 40 mg/kg (Gao et al., 2020). In this study, the effective potassium content in KS and ELC soils was significantly elevated in black soils contaminated with DEHP, while the effective phosphorus content in KS soil also showed a significant increase (Fig. S1). Therefore, DEHP contamination of black soil led to notable alterations in certain physicochemical properties. The rate of DBP degradation varies across different soils, which is associated with degradation activity (Kong et al., 2022). In this study, the DEHP degradation rates varied among the four black soils due to differences in soil properties and microbial community composition, which influence degradation activities in black soils from different regions. Soils with high organic matter content generally demonstrate increased microbial activity, which in turn enhances the degradation rate of DEHP. Previous studies have indicated that elevated organic matter content can significantly enhance the desorption of DEHP, thereby facilitating its microbial utilization and promoting degradation (Ramanayaka et al., 2023; Shah et al., 2024). In this study, HXL soil exhibited the highest organic matter content and the highest DEHP degradation rate (Tab. S1).

Fig. 2.

Variation of DEHP and its metabolites phthalic acid and benzoic acid in black soil. (A) DEHP; (B) phthalic acid; (C) benzoic acid.

Some studies have demonstrated a significant positive correlation between the levels of benzoic acid (BA) and phthalic acid (PA). This correlation suggests a tendency for these two acids to co-accumulate in the soil, which could be attributed to their common source or similar degradation pathways (Li et al., 2024b). PAEs in soil may interact directly or indirectly with soil microorganisms, thereby influencing the metabolic cycles of the microbiome (Wu et al., 2022). Consequently, monitoring changes in DEHP metabolites in soil can serve as an indirect indicator of alterations in microbial metabolism. In this study, the content changes of PA and BA in soil were monitored. The accumulation of PA in HL, HXL, and KS soils was highest on the 30th day of DEHP contamination, with PA concentrations of 0.08 ± 0.007 μg/kg, 0.14 ± 0.006 μg/kg, and 0.06 ± 0.002 μg/kg, respectively. The accumulation of PA in ELC soils peaked on the 60th day of DEHP contamination, reaching 0.03 ± 0.005 μg/kg. BA was detected in the original soil samples from ELC, indicating its presence in black soil under natural conditions. Furthermore, the maximum accumulation of BA in the soils of ELC, HXL, and KS occurred on the 30th day of DEHP contamination, with concentrations of 2.33 ± 0.09 μg/kg, 4.42 ± 0.30 μg/kg, and 1.44 ± 0.05 μg/kg, respectively. The accumulation of PA in HL soil reached its maximum value of 3.00 ± 0.02 μg/kg on the 60th day of DEHP contamination. During the test period, the accumulation of PA and BA occurred in all four black soils samples. However, on the 75th day after DEHP contamination, the concentrations of PA and BA were both less than the peak. The entry of PAEs into the bacterial cell is a key process before microbial degradation of PAEs. This process was primarily driven by transporter systems, including ABC transporters and permease-type PAEs transporters (Hu et al., 2021). PAEs were initially converted to PA via side-chain β-oxidation, transesterification, and de-esterification. This conversion represented a critical step in PAEs biodegradation, thereby reducing their environmental accumulation (Li et al., 2022a; Wang et al., 2024b). Then, PA was metabolized into water and carbon dioxide. LC-MS analysis of samples collected from the DEHP-treated groups on days 0, 30, 60, and 75 revealed the accumulation of PA and BA in the black soil, indicating that the DEHP biodegradation pathway was activated.

3.2. Effects of DEHP on microbial communities in black soils

The high-throughput sequencing results revealed significant alterations in the Shannon index of microbial diversity across the three periods (Fig. 3A). The Shannon index significantly increased in ELC soil by day 75 of DEHP contamination compared to day 0. Significant increases were also observed in HL, HXL, and KS soils on day 30. To assess temporal shifts in microbial community structure, principal coordinate analysis (PCoA) was performed based on Bray-Curtis distances to evaluate compositional similarity across the four black soils. The matrices of different treatment groups were distinctly separated, revealing larger discrete ranges among black soil samples at various times of DEHP contamination and smaller within-group differences (Fig. 3B). In ELC soil, PC1 accounted for 62.34 % of the variation, while PC2 explained 32.27 %, together accounting for 94.61 % of the differences in sample composition (R = 1, P < 0.001). In HL soil, PC1 explained 72.28 % of the variation, with PC2 accounting for 19.12 %, cumulatively explaining 91.40 % of the variation in sample composition (R = 0.988, P < 0.001). HXL soil revealed that PC1 explained 75.35 % of the variation, while PC2 contributed 19.06 %, together explaining 91.41 % of the variation in sample composition (R = 1, P < 0.001). In KS soil, the principal axis PC1 accounted for 86.47 %, and PC2 explained 9.72 %, cumulatively accounting for 96.19 % of the differences in sample composition (R = 1, P < 0.001). In summary, DEHP contamination in black soil altered the microbial β-diversity and bacterial community structure. Previous studies have demonstrated that DEHP significantly reduces both the abundance and diversity of soil microorganisms, with this effect being more pronounced at higher concentrations (Kim and Sang, 2023; Zhang et al., 2020a). Interestingly, in this study, the Shannon index of DEHP-contaminated soil exhibited an opposite trend. This discrepancy may be attributed to the enhanced DEHP-degrading potential of the indigenous microbial communities in black soil.

Fig. 3.

Changes and correlation analysis of soil microbial community diversity in different periods of black soil (A) The Shannnon index for days 0, 30 and 75; (B) PCoA analysis at days 0, 30 and 75; (C) Mantel Test analysis of soil microbial diversity, soil physicochemical factors and DEHP degradation rate.The corresponding value of the heatmap is Pearson correlation coefficient (r), where r > 0 is positively correlated but r < 0 is negatively correlated. AK, soil available potassium content. AP, soil available phosphorus content. TN, soil total nitrogen content. EC is soil conductivity. *, **, *** respectively indicate significant differences at P < 0.05, P < 0.01, and P < 0.001 levels, the same below.

To identify the key environmental drivers of DEHP degradation, the relationships among soil physicochemical properties, microbial diversity indices, and the DEHP degradation rate were assessed using a Mantel test. The DEHP degradation rate in ELC soil was significantly and positively correlated with total nitrogen content, and microbial α-diversity. In HL soil, the DEHP degradation rate showed a significant and positive correlation with total nitrogen content, pH, and microbial α-diversity. In HXL soil, the DEHP degradation rate was significantly and positively correlated with available potassium content, available phosphorus content, total nitrogen content, pH, and microbial α-diversity. Similarly, the degradation rate of KS soil was significantly and positively correlated with available phosphorus content and microbial α-diversity. In summary, DEHP degradation rates were positively correlated with soil pH in HL and HXL soils, while a negative correlation was observed in ELC and KS soils. The biodegradation of PAEs in soil is mainly mediated by degrading enzymes secreted by functional microorganisms. Gordonia sp. GZ-YC7 had efficiently degraded DEHP by secreting esterases (Hu et al., 2022). Carboxylesterases and lipases have exhibited notable selectivity and specificity in the degradation process of PAEs, with particularly higher efficiency observed in degrading short-chain PAEs (Sun et al., 2022). The optimal pH of PAEs-degrading enzymes in soil varies depending on the type and source of the enzymes. The PAEs hydrolase screened from Rhodococcus sp. 2 G had an optimal pH range of 4.0–10.0, which demonstrated its broad pH adaptability and stability (Du et al., 2021). The carboxylesterase Dkca1 from Dietzia kunjamensis IITR165 had an optimal pH range of 7.0–9.0, suggesting effectiveness under mildly acidic to mildly alkaline conditions (Singh et al., 2024). Pseudarthrobacter defluvii E5 was more conducive to the activity of its enzyme at pH 5.3 (Pereyra-Camacho et al., 2024). In this study, the inherent microbial communities and dominant microbial populations differed across the four soil samples from distinct sources, which may have led to significantly different optimal pH values for the key enzyme systems involved in degradation and secreted by these communities. Soil microorganisms engage in a complex “reaction-change-adaptation-feedback” process in response to various stresses (Lü et al., 2023), and microbial communities exhibit high sensitivity to soil contamination. Physicochemical factors and microbial diversity contribute positively to the degradation of DEHP. Analysis revealed a significant positive correlation between DEHP degradation rate and microbial α-diversity across all four regions, demonstrating the predominant role of microbial communities as a key environmental factor driving DEHP degradation in the microcosm experiments.

The total sequence length of all sample genes prior to redundancy removal was 82,864,460,034 bp. The amino acid sequences of the non-redundant gene set were compared with the NR database, and the corresponding species annotations were obtained from the Taxonomic Information Database (TID). This data included a total of 254 phyla, 502 orders, 1048 families, 2157 genera, and 45,507 species. To identify microorganisms stimulated by DEHP or potentially involved in its degradation, the microbial composition and their abundance were analyzed at both the phylum and genus levels. At the microbial phylum level, the top 10 phyla in terms of relative abundance were annotated, while less abundant phyla were grouped into “others” category (Fig. S2). Actinobacteria and Proteobacteria were the dominant phyla in all four soils. The community structure at the phylum level was significantly altered by DEHP contamination in the black soil.

The relative abundance of microbial genera in the DEHP treatment group on day 75 was compared with that of the sterile water control group at baseline (day 0) (Fig. 4). The relative abundance of several genera was significantly increased in ELC soil, notably Nocardioides, Methylotenera, Sphingomonas, Rhodococcus, Lysobacter, and Streptomyces. In HL soil, the genera exhibiting significantly higher abundance included Nocardioides, Sphingomonas, Mycobacterium, Pseudonocardia, Kribbella, Mycolicibacterium, and Lysobacter. In HXL soil, the genera that showed a significantly higher abundance included Bradyrhizobium and Nocardioides. In KS soil, the genera that showed a significantly increased abundance included Sphingomonas, Nocardioides, and Lysobacter. DEHP contamination can result in the reorganization of soil microbial communities. DEHP pollution can alter the diversity, abundance, and community structure of soil microorganisms, leading to the reorganization of microbial communities (Jiang et al., 2022; Zhou et al., 2022a). Certain microbial taxa declined in response to DEHP exposure, whereas others proliferated owing to their adaptive capacity. This microbial restructuring resulted in shifts in soil ecological functions, ultimately affecting soil health and productivity (Kim et al., 2019).

Fig. 4.

Comparison of two groups of bacterial genus level community composition in four sample sites. (A) ELC; (B) HXL; (C) HL; (D) KS.

Biological communities exhibited a high degree of sensitivity to soil contamination. Bacteria in the soil were more responsive to DEHP contamination than fungi and archaea (Lü et al., 2023). The microbiome exhibited a strong potential for adaptation to polluted conditions (Su et al., 2022), resulting in a significant increase in the relative abundance of certain stress-tolerant microbial taxa (Gao et al., 2020). In this study, Actinobacteria and Proteobacteria responded most sensitively to DEHP contamination, exhibiting significant shifts in their relative abundance. In addition, some phyla (such as Proteobacteria, Chloroflexi, and Acidobacteria) and PAEs degrading genera (such as Sphingomonas and Lysobacter) are considered key bacterial species in the co-occurrence network under PAEs stress (Huang et al., 2023). The Sphingomonas strain degraded aromatic compounds and harbored the catabolic genes for protocatechuic acid (PCA) (Gao et al., 2020). The relative abundance of Sphingomonas was significantly higher at day 75 of DEHP contamination compared to day 0 in all four black soils. Similarly, the relative abundance of Nocardioides, which are known as “specialists” in degrading difficult-to-degrade pollutants in the environment (Ma et al., 2023b), increased in the four black soils. Nocardioides were found to harbor lipase genes contributing to DEHP degradation (Ningthoujam, 2022). Nocardioides demonstrated the capability to degrade PAEs via specialized enzyme systems, thereby mitigating the toxicity of these compounds to soil organisms and reducing environmental contamination (Zhang et al., 2023a). Nocardioides collaborated synergistically with other microorganisms to degrade PAEs, thereby enhancing the breakdown of DEHP (Li et al., 2024a; Lin et al., 2024). In brown soils, the relative abundance of Glutamicibacter, Paenibacillus, and Cohnella was significantly lower in DBP- and DEHP-treated soils compared to the control. In tidal soils, the relative abundance of Streptomyces, Nocardioides, and Methylobacillus was significantly higher in the DEHP-added group compared to the control (Gao et al., 2020). During the shift in microbial community composition, it is important to identify key members of the bacterial community that have significantly increased in abundance, as they may be involved in the biodegradation of PAEs at contaminated sites (Hu et al., 2021).

3.3. Identification of DEHP-degrading functional microorganisms

The upstream degradation pathway of PAEs initially activates pollutants and decomposes them into key intermediate metabolites. The downstream degradation pathway of PAEs further mineralizes the intermediate products generated by the upstream pathway, ultimately yielding harmless inorganic substances such as CO₂ and H₂O. In short, the upstream pathway involves decomposition and the downstream one involes mineralization. The two are connected in series to complete the entire degradation process from complex pollutants to inorganic substances. An analysis of the abundance changes in two PAEs degradation-related modules (M00623 and M00636) was conducted across different treatment groups using the original KEGG gene set, aiming to identify microorganisms involved in the upstream metabolism of PAEs in black soil (Fig. 5). The taxonomic identities of species harboring the degradation genes pht3, pht4, and pht5 was investigated. A gene set associated with BA metabolism was established in black soil and applied to species and functional annotation to identify functional microorganisms involved in the downstream metabolic processes of PAEs.

Fig. 5.

Change in abundance of M00623 and M00636 after DEHP contamination based on KEGG Moudel level, (A) M00623, (B) M00636.

Modules M00623 and M00636 are pathways for the degradation of PAEs to PCA. Following DEHP contamination, the abundance of both modules M00623 and M00636 increased significantly by day 75 in ELC soil. However, in HL soil, the abundance of M00623 decreased significantly, with no significant change in M00636. A significant increase in M00636 abundance was observed in HXL soil at day 75, while M00623 remained unchanged. In KS soil, the abundance of M00636 increased significantly over time, in direct contrast to M00623, which exhibited a significant decrease.

Investigating the expression changes of the pht gene under various environmental conditions enhances our understanding of how microorganisms respond to environmental fluctuations. Different microorganisms collaborated to facilitate DEHP degradation through their interactions, highlighting the significance of the complex relationships between genes and the synergies involved in the degradation process (Wei et al., 2021). The module M00623 pathway encodes the functional gene pht3 (K18068), which metabolizes PAEs to phthalic acid-4, 5-cis-dihydrodiol ester, this intermediate is then metabolized to 4, 5-dihydroxyphthalate by pht4 (K18067) and finally to PCA by pht5 (K04102). To investigate the relationship between functional genes and their host microorganisms, abundance data for both were employed in an association analysis (Fig. 6). At the microbial family level, the contribution of the top 20 families to the abundance of functional genes associated with DEHP degradation was analyzed. Microorganisms exhibiting high contributions to gene K18068 in ELC soils included unclassified_c_Deltaproteobacteria, unclassified_p_Candidatus_Rokubacteria, unclassified_p_Chloroflexi, and unclassified_c_Betaproteobacteria. On days 0, 30, and 75 of DEHP contamination, the Sphingomonadaceae associated with gene K18068 contributed 0.36 %, 4.19 % and 15.46 %, while unclassified_o_Sphingomonadales contributed 1.93 %, 5.21 %, and 8.79 %, respectively. In KS soil, the contribution rate of unclassified_c_Alphaproteobacteria of gene K18068 on day 0, day 30 and day 75 of DEHP contamination was 7.5 %, 12.11 % and 12.23 %, and unclassified_c_Betaproteobacteria contributed 4.15 %, 9.21 %, and 8.98 %, respectively. In HL and HXL soils, the families with higher contributions to the pair gene K18068 were unclassified_c_Deltaproteobacteria, unclassified_p_Candidatus_Rokubacteria, unclassified_c_ Alphaproteobacteria, and unclassified_c_Alphaproteobacteria. Xanthobacteraceae, Sphingomonadaceae, Hyphomicrobiaceae, and Comamonadaceae contribute to the upstream metabolism of PAEs with a high degree of contribution.

Fig. 6.

Analysis of microbial contribution to functional genes K18068, K18067, and K04102.

3.4. Changes in functional genes and functional microorganisms involved in benzoate degradation in black soil

BA is a common downstream metabolite of DEHP. In various microbial populations, alterations in benzoate degradation genes directly influence the degradation efficiency of DEHP. For instance, genomic analyses of Rhodococcus opacus 1CP have revealed the presence of multiple genes associated with DEHP degradation (Bhattacharyya et al., 2023). The expression levels and functional changes of these genes may influence the DEHP degradation capacity. Following the construction of a gene set associated with benzoate degradation and subsequent species and functional annotation, the results demonstrated that sequences involved in benzoate degradation were upregulated in black soil after DEHP contamination. On the 30th and 75th days following DEHP contamination in ELC and HL soils, the proportion of sequences associated with benzoate degradation significantly increased compared to day 0 (Fig. 7). Similarly, a significant increase was observed in KS and HXL soils on the 30th day relative to the initial time point. Meanwhile, the abundance of functional genes involved in BA degradation increased in black soil (Fig. S3). On the 75th day of DEHP contamination in ELC soil, the abundance of 17 functional genes related to BA metabolism, including K10220, K10218, and K04100, was significantly upregulated. The abundance of functional genes related to BA metabolism in HL soil was upregulated on the 30th and 75th days of DEHP contamination. The abundance of BA metabolic genes was upregulated in KS soil after 75 days of DEHP contamination. In summary, soil microorganisms promoted the downstream metabolism of DEHP by increasing the abundance of BA degradation functional genes.

Fig. 7.

Sequence proportions with different periods of benzoic acid metabolism in different plots.

Functional genes and microorganisms enriched in black soil were clustered separately and subjected to Spearman correlation analysis. The correlation coefficients were expressed as r, with values of r < 0.3 in the matrix of weak correlations being converted to 0 (Fig. 8). Bradyrhizobium, Rhodococcus, Sphingomonas, Lysobacter, Mycobacterium, Mycolicibacterium, Nocardioides, Arthrobacter, Streptomyces, and Pseudonocardia were positively correlated with the abundance of BA metabolism functional genes enriched in ELC soil. Nocardioides, Sphingomonas, Streptomyces, Bradyrhizobium, and Arthrobacter were positively correlated with the abundance of BA metabolism function genes enriched in KS soil. Nocardioides, Rhodococcus, Sphingomonas, and Lysobacter were positively correlated with the abundance of most of the BA metabolic function genes enriched in HL soil. In HXL soil, the abundance of Sphingomonas, Marmoricola, Nocardioides, Pseudonocardia, Rhodococcus, Solirubrobacter, and Mycolicibacterium exhibited a positive correlation with the abundance of functional genes K05783 and K04112.

Fig. 8.

Correlation analysis of benzoate degradation genes enriched in each sample site with microbial abundance.

Microbial communities in soil adapt to contaminated environments by selectively enriching contaminant-degrading taxa and associated functions, including specific genes (Huang et al., 2023). In this study, it was found that the abundance of modules involved in the degradation of PAEs changed significantly in black soil after DEHP contamination at the KEGG Module level, while the percentage of sequences involved in benzoate degradation increased significantly among black soil microorganisms. By constructing the benzoate degradation gene set, the changes in genes related to BA degradation in different black soils were analyzed separately, further highlighting the key categories involved in DEHP degradation. The abundance of functional genes involved in BA metabolism was significantly up-regulated throughout the experimental cycle in each of the four sampled black soils (Fig. S3). The observed increases in both microbial abundance and functional gene expression enhance the adaptive capacity of these communities to environmental stressors, such as climate change and soil pollution. This enhanced adaptability is essential for maintaining ecosystem stability and function (Li et al., 2022b). In this study, the abundance of Arthrobacter, Rhodococcus, and Sphingomonas showed a significant positive correlation with the abundance of several benzoate degradation-related functional genes, suggesting that these taxa may play key roles in the degradation of DEHP in black soil. The abundance of genes encoding hydrolases, esterases, cytochrome P450, lipases, carboxylesterases, laccases, and keratinases was increased in indigenous bacterial communities under DEHP (1000 mg/kg) contamination, and these elevated functional genes were derived from Gordonella, Rhodococcus, Nocardia, Mycobacterium, Arthrobacter, Nocardioides, and Lysobacter. Therefore, these bacteria may collectively be involved in the degradation of DEHP (Zhu et al., 2020). Arthrobacter sp. JQ-1 can completely degrade 500 mg/L of DEHP within 3 days (Zhang et al., 2020b). Twenty potential esterase genes were identified in the genome of Rhodococcus sp. AH-ZY2, four of which were expressed and demonstrated to be capable of degrading PAEs (Hou et al., 2024). Rhodococcus pyridinivorans DNHP-S2 efficiently degraded DEHP within a temperature range of 10 °C to 35 °C and maintained high degradation activity even under low-temperature conditions (Wang et al., 2022a). Sphingomonas species are environmentally important bacteria recognized for their broad metabolic versatility (Feller et al., 2021). They are capable of degrading polycyclic aromatic hydrocarbons, which are more complex than DEHP (Zhou et al., 2022b). Comamonadaceae grew fastest in the medium with DEHP as the sole carbon source, indicating that it showed strong adaptation and tolerance to DEHP during the 2-month domestication process (Ruiz et al., 2021). In this study, Comamonadaceae was shown to contribute functional genes associated with the upstream metabolism of PAEs. It is important to screen for the bacterial communities with a stable degradation effect and lower requirements for environmental conditions for practical remediation (Ding et al., 2021). The above provides guiding information for the subsequent screening of DEHP-degrading bacterial communities.

3.5. Identification of DEHP-degrading bacterial communities and determination of degradation capacity in black soil

A total of 10 bacterial communities with degradation capabilities were identified in this study, including JQ135 dominated by Klebsiella, JQ55 dominated by unclassified_f_Enterobacteriaceae, JQ112 dominated by Aquamicrobium, JQ104 dominated by Chryseobacterium, JQ94 dominated by Klebsiella, JQ36 dominated by Burkholderia, JQ52 dominated by Glutamacibacter, JQ74 dominated by Rhodococcus, JQ81 dominated by unclassifiedd_f_Enterobacteriaceae, and JQ129 dominated by Pseudomonas. The composition of the bacterial communities was presented in Figure S4. The degradation efficieny of DEHP by various bacterial communities was assessed over different cultivation periods (Fig. 9). Specifically, the degradation efficiency of DEHP by JQ135 after 60 h of cultivation was 98.03 ± 0.32 %. JQ55 achieved a degradation efficiency of 98.27 ± 0.87 % after 72 h. JQ112 demonstrated the highest degradation efficiency of 99.00 ± 0.27 % after 60 h. JQ104 exhibited a degradation efficiency of 98.63 ± 0.56 % after 48 h, while JQ94 achieved a rate of 96.26 ± 0.05 % in the same time. JQ36 reached a degradation efficiency of 98.81 ± 0.08 % after 72 h, and JQ52 achieved 98.02 ± 0.19 % after 48 h. Notably, JQ74 demonstrated a remarkable degradation efficiency of 99.79 ± 0.01 % after 72 h. Meanwhile, JQ81 recorded a degradation efficiency of 90.80 ± 3.62 % after 72 h, and JQ129 achieved 98.38 ± 0.77 % after 48 h. These bacterial communities were able to degrade over 90 % of DEHP within 72 h.

Fig. 9.

Growth and residual amount of DEHP of bacteria and within 72 h.

These bacterial communities were capable of utilizing diverse carbon sources and demonstrated a broad capacity to degrade various PAEs (Fig. S5-S14). Notably, JQ135 exhibited optimal growth when PCA served as the sole carbon source. It indicated that JQ135 had a pronounced preference for PCA metabolism over alternative carbon sources. In contrast, JQ55 grew more rapidly on PAEs than on their downstream metabolites. At concentrations below 200 mg/L, JQ112 and JQ94 showed no significant difference in substrate preference. JQ112 exhibited the strongest preference for PCA at a concentration of 400 mg/L. Additionally, JQ36, JQ52, JQ74, JQ81, and JQ129 exhibited strong growth on substrates other than BA when used as the sole carbon source at a concentration of 400 mg/L.

3.6. Identification of metabolites of DEHP-degrading bacterial communities

According to the mass spectrometry and scanning data obtained from GC–MS (Fig. 10), the following organic compounds were detected in all bacterial communities: 2-ethylhexyl benzoate (EHBA), DBP, dimethyl phthalate (DMP), and 2-ethylhexyl alcohol. The retention times recorded were 9.294 min for EHBA, 11.068 min for DBP, 5.918 min for DMP, and 4.555 min for 2-ethylhexyl alcohol. It indicated that the functional bacterial communities could simultaneously degrade DEHP into DBP and DMP, which have shorter alkyl side chains, via β-oxidation, and hydrolyze DEHP into EHBA through ester hydrolysis. Typically, the biodegradation of PAEs primarily involves the sequential hydrolysis of the ester side chains, resulting in the formation of monoesters. Under the influence of esterase, DEHP is hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) and 2-ethylhexyl alcohol, a critical and rate-limiting step in its metabolic pathway (Wei et al., 2021). In the present study, 2- ethylhexyl alcohol was detected and found to accumulate in the metabolites of bacterial communities. Furthermore, these communities were shown to reduce the carbon number in the side chain of DEHP via β-oxidation. The results from substrate preference experiments demonstrated that the DEHP-degrading bacteria screened in this study possess the capability to metabolize short-chain PAEs, PA, BA, and PCA, further confirming the functionality of these degrading bacteria in DEHP degradation.

Fig. 10.

Mass spectra of metabolites of degradation community.

During the degradation of organic compounds, certain strains specialize in catalyzing the initial steps, whereas others are responsible for subsequent metabolic reactions. This division of labor enhances the overall metabolic efficiency of the community (Wang et al., 2022b). Moreover, some strains break down complex substrates into simpler compounds that can be utilized by others, thereby reducing the individual metabolic burden and optimizing energy allocation within the consortium (Ioannou et al., 2024). In this study, in situ degrading bacterial communities were isolated from constructed soil microcosms, and their degradation capabilities were experimentally validated. The identified communities exhibited considerable diversity in both richness and abundance among their constituent members. It has been noted that Comamonadaceae contribute to the upstream metabolism of PAEs. Among these isolates, JQ135, JQ55, JQ112, JQ104, JQ94, JQ52, JQ74, and JQ81 were found to contain varying proportions of Comamonas. Additionally, the genus Rhodococcus was found to be positively correlated with the degradation of BA in the soils from ELC and HL. In this study, strain JQ74 was successfully isolated, with Rhodococcus identified as the dominant genus. Notably, JQ74 degraded 99.79 % of DEHP within 72 h, demonstrating high degradation efficiency.

4. Conclusion

In this study, black soils from four distinct regions were used as experimental materials, and exogenous DEHP was added to create a microenvironment favorable for the natural degradation of DEHP. Through metagenomic sequencing and bioinformatics analysis, the effects of DEHP on the microbial community structure of black soil were elucidated, revealing information about microorganisms that may be involved in DEHP degradation. In situ DEHP-degrading bacterial communities were successfully isolated and identified, and their degradation functionality was experimentally validated. The conclusions of this study are as follows: (1) DEHP contamination caused the change in physical and chemical properties of black soil, and the intermediate metabolites of DEHP, PA and BA, were accumulated in the soil. The DEHP degradation rate was found to be positively correlated with the α-diversity of the black soil microbial community. (2) Metagenomic sequencing results indicated that DEHP altered community diversity and composition, with significant enrichment of functional genes associated with DEHP degradation. Species classification information for functional microorganisms contributing these genes was traced, revealing a significant positive correlation between the functional microorganisms and the abundance of functional genes. (3) A total of 10 DEHP-degrading bacterial communities were isolated using soil extract and MSM. These communities degraded over 90 % of DEHP within 72 h, producing metabolic intermediates including EHBA, DBP, DMP, and 2- ethylhexyl alcohol during the degradation process.

CRediT authorship contribution statement

Wenli Zhang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. Heqi Guo: Formal analysis, Investigation, Visualization, Supervision. Weihui Xu: Resources, Writing – review & editing. Wenjing Chen: Validation, Formal analysis, Visualization. Yunlong Hu: Formal analysis, Supervision. Zhigang Wang: Conceptualization, Funding acquisition, Project administration, Resources, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Zhigang Wang reports financial support was provided by Natural Science Foundation for Distinguished Youth Scholars of Heilongjiang Province. Reports a relationship with that includes:. Has patent pending to. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.