Genomic Insights into Dechloromonas sp. TW-R-39-2: A Dual-Function Bacterium for Heavy Metal Sequestration and Chlorinated Organic Degradation

TL;DR

A new Dechloromonas bacterium can both remove heavy metals and break down toxic organic pollutants, making it useful for cleaning contaminated water.

Contribution

Discovery of a novel Dechloromonas strain with dual capabilities for heavy metal sequestration and chlorinated compound degradation.

Findings

Strain TW-R-39-2 showed 78% cadmium and 75% zinc adsorption efficiency.

The bacterium degraded 83.6% trichloroethylene and 81% chlorophenol in 7 days.

Chloride ion release confirmed complete dechlorination of pollutants.

Abstract

Environmental contamination caused by various types of heavy metals and chlorinated organic compounds poses a significant threat to global ecosystems. While bioremediation offers a sustainable solution, identifying microbial strains that possess the metabolic versatility to withstand metal toxicity and degrade persistent organic pollutants remains a major challenge. In this study, we characterized strain TW-R-39-2, a novel bacterium isolated from a wastewater treatment plant. Phylogenomic analysis based on complete genome sequencing revealed that strain TW-R-39-2 represents a novel species within the genus Dechloromonas, showing Average Nucleotide Identity (ANI) and digital DNA–DNA hybridization (dDDH) values of 80.17% and 23.4%, respectively, with its closest relative, Dechloromonas denitrificans. Genomic insights revealed a 3.46 Mb circular chromosome containing a diverse array of genes associated with heavy metal resistance (e.g., predicted czc and cadA clusters) and chlorinated compound degradation (e.g., dehalogenases). Phenotypic assays validated these genomic predictions, demonstrating the strain’s dual functionality: it exhibited high adsorption efficiencies for cadmium (Cd2+, 78.0%) and zinc (Zn2+, 75.1%), alongside significant degradative capacity for trichloroethylene (TCE, 83.6%) and chlorophenol (81.0%) within a 7-day incubation period, corresponding to the strain’s active growth and early stationary phases. The stoichiometric release of chloride ions confirmed the complete dechlorination of the organic pollutants. These findings demonstrate that strain TW-R-39-2 possesses versatile capacities for both the sequestration of toxic metals and the biodegradation of persistent organic compounds. This study highlights the potential of strain TW-R-39-2 as a promising candidate for the efficient bioremediation of diverse industrial wastewaters.

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1. Introduction

Environmental contamination caused by the co-occurrence of heavy metals and chlorinated organic compounds has emerged as a critical global challenge [1]. Industrial activities, including electroplating, mining, and chemical manufacturing, frequently discharge complex effluents containing both inorganic toxic ions (e.g., Cd^2+^, Zn^2+^, and Pb^2+^) and persistent organic pollutants such as trichloroethylene (TCE) [2]. Unlike isolated contaminants, these mixed pollutants exert synergistic toxicity; heavy metals often inhibit the enzymatic activities of microbes responsible for organic degradation, thereby severely limiting the efficiency of conventional bioremediation strategies [3,4].

The genus Dechloromonas, belonging to the family Rhodocyclaceae, is well-recognized for its metabolic diversity, particularly in perchlorate reduction, aromatic hydrocarbon degradation, and nitrate-dependent oxidation [5,6]. While previous studies have extensively explored the degradative potential of Dechloromonas species, their resilience and sequestration mechanisms against high concentrations of heavy metals remain less understood [7]. Given the necessity for significant microbial candidates capable of functioning in multi-stressor environments, identifying novel Dechloromonas strains with dual functionality—effectively managing metal toxicity while performing organic pollutant breakdown—is of significant environmental and industrial importance [8].

In the present study, we characterized strain TW-R-39-2, a novel member of the genus Dechloromonas isolated from a wastewater treatment plant, which exhibits the dual-functional potential for the mitigation of heavy metal toxicity and the degradation of chlorinated organic compounds. To elucidate the molecular basis of its resilience, we performed complete genome sequencing and comparative phylogenomic analysis [9,10]. Furthermore, we experimentally validated its adsorption efficiency for multiple divalent heavy metals and its degradative performance against TCE and chlorophenol. This study provides a comprehensive understanding of the genomic and phenotypic features of strain TW-R-39-2, highlighting its potential as a versatile tool for the integrated bioremediation of complex industrial wastewaters [11,12].

Notably, the unique dual-functional properties of Dechloromonas sp. TW-R-39-2 for both heavy metal sequestration and organic pollutant biodegradation have been recognized for their industrial novelty, leading to its official registration as a patent in the Republic of Korea (Patent No. 10-2845955, titled ‘Novel Dechloromonas sp. TW-R-39-2 strain with heavy metal adsorption and chlorinated compound degradation activities and environmental remediation method using the same’).

2. Material and Methods

2.1. Strain Isolation, Culture Conditions, and Morphological Characterization

A sewage sample was collected in 2019 from the Tongbok wastewater treatment plant (Pyeongtaek, Republic of Korea; 36°59′34.6″ N, 127°04′00.5″ E). At the time of sampling, the physicochemical properties of the wastewater were as follows: temperature, 15.3 °C; pH, 7.3; dissolved oxygen (DO), 8.12 mg/L; and electrical conductivity, 8.12 µS/cm. The sample was serially diluted in sterile saline (0.85% NaCl, w/v) and spread onto Reasoner’s 2A (R2A) agar (MBcell, Seoul, Republic of Korea). Plates were incubated at 28 °C for 3 days. Single colonies were selected and subcultured on R2A agar to ensure purity. The isolated strain, designated TW-R-39-2, was deposited at the Korean Collection for Type Cultures (KCTC) under the accession number KCTC 18799P.

The growth conditions of strain TW-R-39-2 were evaluated over a range of temperatures (10–45 °C), pH values (4.0–10.0), and salinities (0–5% NaCl, w/v). Optimal growth was observed at 28 °C and pH 7.0 in the absence of NaCl. Notably, the strain exhibited the ability to grow under both aerobic and anaerobic conditions, reflecting its metabolic versatility. Working cultures were maintained on R2A agar at 28 °C, and for long-term preservation, the strain was stored at −80 °C in 20% (v/v) glycerol.

The Gram reaction was performed using a standard Gram staining kit (BD Difco, Sparks, MD, USA) according to the manufacturer’s instructions to determine the cell wall type [13]. Cellular morphology was examined using phase-contrast microscopy (Nikon 80i, Tokyo, Japan) and transmission electron microscopy (TEM) (CM200; Philips, Amsterdam, The Netherlands) [14]. For TEM analysis, bacterial cells were harvested from R2A agar, resuspended in sterile saline, and negatively stained with 2.0% (w/v) uranyl acetate on carbon-coated copper grids to observe fine cellular details, including surface structures and the presence of flagella [15].

2.2. 16S rRNA Gene Sequencing and Phylogenetic Analysis

Genomic DNA was extracted from strain TW-R-39-2, cultured on R2A agar at 28 °C for 3 days, using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quality and quantity of the extracted DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis [16]. The 16S rRNA gene was amplified by PCR using universal bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) [17].

The resulting sequence was compared with type strain sequences available in the EzBioCloud database (https://www.ezbiocloud.net, accessed on 15 January 2026) to identify its taxonomic position [18]. Reference sequences of closely related taxa were retrieved from the NCBI GenBank database. Multiple sequence alignments were performed using ClustalW v. 2.1 and further refined using EzEditor2. Phylogenetic trees were reconstructed using neighbor-joining (NJ) [19], maximum-likelihood (ML) [20], and maximum-parsimony (MP) [21] methods implemented in the MEGA version 7.0 software [22]. The evolutionary distances for the NJ tree were computed using the Kimura 2-parameter model [23]. The robustness of the tree topologies was evaluated by bootstrap analysis with 1000 replicates [24].

2.3. Complete Genome Sequencing and Annotation

Whole-genome sequencing of strain TW-R-39-2 was performed using a hybrid approach combining the PacBio RS II and Illumina HiSeq platforms at Macrogen (Seoul, Republic of Korea) [25]. For PacBio sequencing, a 20-kb SMRTbell library was constructed and sequenced, yielding 1.26 Gb of data of filtered subreads. Additionally, Illumina HiSeq raw data (1.85 Gb) were generated to provide high-quality short reads for sequence error correction [26].

De novo assembly was conducted using RS HGAP Assembly (v3.0) within the SMRT Portal (v2.3) [27], and the assembled contigs were further polished using Pilon (v1.21) to ensure maximum sequence accuracy [28]. Genome annotation was performed using Prokka (v1.13) [29]. Functional genes were identified and classified using Glimmer (v3.02) [30] and searched against the Kyoto Encyclopedia of Genes and Genomes (KEGG) [31] and eggNOG [32] databases. The complete genome sequence and its associated data have been deposited in GenBank under the accession number CP045202.

2.4. Comparative Genomic and Phylogenomic Analysis

To evaluate the taxonomic position and genomic distinctiveness of strain TW-R-39-2, comparative genomic analyses were performed against closely related Dechloromonas species, including D. denitrificans ATCC BAA-841^T^, D. hortensis MA-1^T^, and D. agitata DSM 13637. Average Nucleotide Identity (ANI) and digital DNA–DNA hybridization (dDDH) values were calculated using the OrthoANI tool [33] and the Genome-to-Genome Distance Calculator (GGDC) version 3.0 [34], respectively. Phylogenomic relationships were further analyzed using the Type (Strain) Genome Server (TYGS; https://tygs.dsmz.de (accessed on 16 January 2026)) [35]. Intergenomic distances were calculated based on the Genome BLAST Distance Phylogeny (GBDP) method using GGDC 3.0 (Genome-to-Genome Distance Calculator [34], and a phylogenomic tree was constructed to confirm the evolutionary placement of strain TW-R-39-2.

Pan-genome analysis was conducted to identify the core genome shared among Dechloromonas species and the accessory and strain-specific genes unique to strain TW-R-39-2. The pan-genome was constructed using the Comparative Genomics tool on the EzBioCloud server, which applies the OrthoMCL algorithm to cluster orthologous gene groups based on reciprocal best-hit criteria [36]. Genes were classified into core, accessory, and singleton categories. The distribution of orthologous clusters was visualized using jvenn to provide a comprehensive comparison of shared and unique gene repertoires [37]. This analysis focused on identifying unique metabolic pathways potentially contributing to the strain’s specialized bioremediation capabilities toward heavy metals and chlorinated compounds.

2.5. Functional Gene Prediction and Putative Operon Visualization

The genomic organization of heavy metal resistance genes was analyzed to predict putative operon structures based on intergenic distances and functional co-localization. Putative functional gene clusters, including the czc (cobalt–zinc–cadmium), cnr (nickel–cobalt), ncc (nickel–cobalt–cadmium), and ars (arsenic) systems, were identified using the Center for Genomic Epidemiology database [38] and mapped onto the circular chromosome of strain TW-R-39-2.

The spatial arrangement, orientation, and transcriptional direction of these genes were visualized using Geneious Prime v. 2026.0 (Biomatters, Auckland, New Zealand) [39] and the R package v. 0.4.1 gggenes [40] to illustrate the genetic architecture underlying the strain’s multi-metal resistance phenotype. In addition, comparative locus maps were generated to examine synteny with corresponding gene clusters in closely related Dechloromonas species, such as D. denitrificans ATCC BAA-841^T^ [41]. This analysis provided insights into the evolutionary conservation and potential horizontal gene transfer of these functional determinants.

2.6. Remediation Performance Assays

The resistance of strain TW-R-39-2 to five divalent heavy metal ions (Cd^2+^, Co^2+^, Cu^2+^, Pb^2+^, and Zn^2+^) was evaluated in Nutrient Broth (NB) supplemented with 100 mg/L of each metal using analytical grade reagents (CdCl_2_, CoCl_2_ · 6H_2_O, CuCl_2_, Pb(NO_3_)2, and ZnCl_2_; Sigma Aldrich, St. Louis, MO, USA). Cultures were inoculated at an initial density of 10^7^ CFU/mL and incubated statically at 28 °C for up to 10 days. Bacterial growth was monitored daily by measuring the optical density at 600 nm (OD_600_) using a spectrophotometer. After incubation, cultures were centrifuged at 8000 rpm for 10 min at 4 °C to minimize protein denaturation. Metal adsorption efficiency was calculated based on the difference between initial and residual metal concentrations measured by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES; Agilent 5110, Agilent Technologies, Santa Clara, CA, USA) [42,43].

The biodegradation of TCE and chlorophenol (≥99% purity, Sigma-Aldrich, St. Louis, MO, USA) was assessed in Mineral Salt Medium (MSM). Strain TW-R-39-2 was inoculated at an initial density of 10^7^ CFU/mL into sealed serum bottles containing 50 mg/L of each compound as the sole carbon and energy source. The cultures were incubated statically at 28 °C for 7 days. Residual concentrations were monitored every 24 h using gas chromatography–mass spectrometry (GC–MS; Agilent 7890B GC/5977B MSD; Agilent Technologies, Santa Clara, CA, USA) [44] equipped with a DB-624 column (30 m × 0.25 mm × 1.4 μm) and a headspace sampler. Dechlorination activity was further validated by quantifying the release of chloride ions (Cl^−^) [45].

The growth inhibition rate was calculated according to the following Equation (1):

$$[eqn]$$

where OD_sample_ is the maximum optical density of the heavy metal-treated group and OD_control_ is the maximum optical density of the control group.

The removal efficiency (R) for both heavy metals and chlorinated organic compounds was determined using the following Equation (2) [46]:

$$[eqn]$$

where C_i_ is the initial concentration of the pollutant and C_f_ is the final (residual) concentration after the incubation period. All phenotypic assays, including heavy metal removal and organic compound biodegradation, were performed in independent biological triplicates. Data are presented as the mean ± standard deviation (SD) of these biological replicates.

2.7. Statistical Analysis

All experiments were performed in triplicate to ensure statistical reliability. Data are presented as the mean ± standard deviation. Statistical significance was analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test (p < 0.05). All statistical analyses and visualizations were conducted using GraphPad Prism (version 9.0; GraphPad Software, San Diego, CA, USA) [47,48].

3. Results

3.1. Morphological and Phylogenetic Identification Based on 16S rRNA Gene

Strain TW-R-39-2 was isolated from the sewage of the Tongbok wastewater treatment plant. On R2A agar, the colonies appeared circular, smooth, and cream-colored after 3 days of incubation at 28 °C (Figure S1). Cellular morphology, observed via phase-contrast and TEM, revealed that the strain is a Gram-negative, rod-shaped bacterium, approximately 1.2–1.8 µm in length and 0.5–0.7 µm in width. Notably, the TEM analysis confirmed the presence of a single polar flagellum, providing motility to the cells (Figure 1). Physiological tests showed that the strain is facultatively anaerobic and grows optimally at 28 °C and pH 7.0 in the absence of NaCl.

Phylogenetic analysis based on the nearly full-length 16S rRNA gene sequence (1458 bp) showed that strain TW-R-39-2 belongs to the genus Dechloromonas within the family Rhodocyclaceae. A comparative analysis using the EzBioCloud database indicated that the strain shared the highest 16S rRNA gene sequence similarity with Dechloromonas denitrificans ATCC BAA-841^T^ (97.7%), followed by Dechloromonas hortensis MA-1^T^ (97.4%), Dechloromonas agitata CKB^T^ (96.3%) and Dechloromonas aquae ZY10^T^ (95.5%) [18]. In the neighbor-joining phylogenetic tree, strain TW-R-39-2 formed a distinct and robust monophyletic cluster with these species, supported by high bootstrap values (Figure 2). Consistent topologies were also confirmed in the maximum-likelihood and maximum-parsimony trees (Figures S2 and S3), further validating the taxonomic placement. Since the 16S rRNA gene sequence similarities to its closest relatives were significantly below the recognized species delineation threshold of 98.7% [49], strain TW-R-39-2 was strongly suggested to represent a novel species within the genus Dechloromonas, a finding that was further subjected to comprehensive genomic-level validation.

3.2. General Genomic Features of Strain TW-R-39-2

The complete genome of Dechloromonas sp. TW-R-39-2 consists of a single circular chromosome of 3,462,267 bp with an average sequencing depth of 136.5×. The genomic G+C content was determined to be 58.4%, which is slightly lower than that of its closest relatives, such as D. denitrificans ATCC BAA-841^T^ (61.7%) and D. hortensis MA-1^T^ (61.4%). Genome annotation using Prokka v1.14.6 and Glimmer v3.02 identified a total of 3267 protein-coding sequences (CDS). In addition, the genome contains 12 rRNA genes (comprising four sets of 5S, 16S, and 23S rRNA operons) and 61 tRNA genes, highlighting a robust protein synthesis machinery.

Functional classification based on the KEGG and eggNOG databases revealed a diverse metabolic repertoire, including pathways for nitrogen metabolism and environmental stress response. A circular representation of the genome illustrates the distribution of CDS on forward and reverse strands, along with GC content and GC skew (Figure 3), confirming the structural integrity and typical genomic organization of the genus Dechloromonas. The presence of numerous genes associated with secondary metabolite biosynthesis, such as bacitracin synthase, further suggests the strain’s competitive advantage in complex microbial ecosystems.

3.3. Phylogenomic Analysis and Species Delineation

To further resolve the taxonomic position of strain TW-R-39-2, genome-based comparative analyses were conducted against its closest relatives identified in the 16S rRNA analysis. ANI values between strain TW-R-39-2 and the type strains of the genus Dechloromonas were significantly low. The highest ANI value was observed with Dechloromonas denitrificans ATCC BAA-841^T^ (80.17%), followed by D. hortensis MA-1^T^ (80.11%), D. agitata DSM 13637^T^ (78.05%), and D. aquae ZY10^T^ (76.45%) (Figure S4; Table 1). These values are far below the widely accepted species-level threshold of 95–96%, supporting its status as a distinct species [50].

Similarly, the dDDH values ranged from 21.4% to 23.4%, which fall significantly below the 70.0% threshold required for species delineation (Table 1) [10,51]. Phylogenomic analysis based on concatenated whole-genome sequences using the TYGS further demonstrated that strain TW-R-39-2 forms a distinct and well-supported lineage within the genus Dechloromonas (Figure S5). Collectively, these genomic metrics provide robust evidence that strain TW-R-39-2 represents a taxonomically distinct lineage, corresponding to a novel species within the genus Dechloromonas.

3.4. Comparative Genomics and Pan-Genome Analysis

To evaluate the genomic divergence and environmental adaptation of strain TW-R-39-2, a comparative pan-genome analysis was performed with its closest relatives: D. denitrificans ATCC BAA-841^T^, D. hortensis MA-1^T^, and D. agitata DSM 13637^T^. The pan-genome consisted of 5842 orthologous clusters, reflecting substantial genetic diversity within the genus [52]. Among these, a core genome of 2294 gene clusters was shared across all four strains, representing the essential metabolic and structural backbone of the genus Dechloromonas (Figure 4).

Notably, strain TW-R-39-2 possessed 499 strain-specific genes (singletons) that were not present in the other three species. Functional annotation of these unique genes revealed a significant enrichment in pathways associated with environmental stress response, signal transduction, and pollutant sensing (Table 2). Unlike its closest relatives, TW-R-39-2 harbors specialized chemoreceptors such as NahY (TW-R-39-2_01696) and PctC (TW-R-39-2_01305), which are known to facilitate chemotaxis toward naphthalene and other aromatic pollutants [53]. This suggests that the strain can actively migrate toward high concentrations of contaminants, enhancing its degradation efficiency in complex environments.

Furthermore, the unique presence of the metal response regulator czcI (TW-R-39-2_02842) and a tributyltin chloride resistance protein (TW-R-39-2_02393) provides a specialized genetic toolkit for surviving in multi-metal-contaminated industrial effluents [54]. These strain-specific determinants are complemented by a significant nitrogen metabolism, including a unique nitrite reductase (NO-forming; TW-R-39-2_02443) and a nitrate/nitrite response regulator (TW-R-39-2_02391). These genomic features suggest an enhanced capacity for denitrification under fluctuating nitrogen loads, a common characteristic of industrial effluents [55]. Collectively, these unique functional determinants underscore the ecological versatility of strain TW-R-39-2 and its evolved superiority for the integrated bioremediation of complex wastewater systems.

3.5. Identification of Functional Genes for Heavy Metal Resistance

To elucidate the genetic basis of the metabolic versatility in strain TW-R-39-2, we performed an in-depth analysis of its Putative functional gene clusters. The genome harbors a diverse array of heavy metal resistance determinants, primarily organized into putative operons based on genomic proximity (Table 3). A major feature of its resistance machinery is the presence of the RND (Resistance-Nodulation-Division) family efflux systems, which are known to mediate the expulsion of divalent metal ions [54].

In addition to the systems for divalent metals, the genome contains putative gene clusters for arsenic resistance (e.g., putative ars operon), indicating a broad genomic potential for environmental resilience. While these findings suggest a wide-ranging capacity for metal resistance, the current study focused on the experimental validation of the most prevalent divalent heavy metals found in the source environment.

Specifically, a high-identity predicted Czc/Ncc cluster (TW-R-39-2_00730–00732) was identified, comprising czcC, czcB, and nccA. This cluster showed remarkably high amino acid sequence identities (98.8–99.5%) to known RND-type transporters, suggesting it is the primary mechanism for cobalt, zinc, and cadmium resistance. In addition, a putative nickel/cobalt system (cnrA; TW-R-39-2_01524) and an arsenic system (arsCB; TW-R-39-2_02332–02333) were mapped, indicating that the strain possesses the genomic potential for a broad resistance spectrum. While these genomic signatures suggest a wide-ranging capacity for metal mitigation, further phenotypic studies are required to confirm the functional efficacy of these pathways.

Interestingly, the strain also possesses a lead/cadmium P-type ATPase transporter (cadA; TW-R-39-2_02651) and a divergent predicted czcBC cluster (TW-R-39-2_02186), which may offer supplementary detoxification routes [56]. Regulatory genes, such as czcD and the metal response regulator czcI, were found at distal loci, indicating a complex and multi-layered transcriptional response to metal stress. Comparative locus analysis revealed high synteny with D. denitrificans ATCC BAA-841T, yet the unique spatial arrangement of these core resistance genes underscores the specialized genomic adaptation of strain TW-R-39-2 to heavy metal-rich environments.

3.6. Performance Evaluation of Heavy Metal Resistance and Adsorption

To validate the functional predictions from genomic analysis, the resistance and adsorption capacities of strain TW-R-39-2 were evaluated against five divalent heavy metals (Cd^2+^, Co^2+^, Cu^2+^, Pb^2+^, and Zn^2+^). The strain exhibited high tolerance to all tested metals at a concentration of 100 mg/L. Growth kinetic analysis showed that, although heavy metal stress induced a prolonged lag phase compared to the control, strain TW-R-39-2 reached a stable stationary phase within 4–6 days of incubation, demonstrating significant metabolic resilience.

The metal sequestration efficiency was quantified using ICP-OES after a 10-day incubation period. To distinguish biological sequestration from non-biological factors, abiotic controls (cell-free medium) were monitored under identical conditions. As shown in Figure 5, strain TW-R-39-2 exhibited significant removal efficiencies for multiple metals, whereas the abiotic controls showed negligible changes (<3.0%) for all tested ions. The highest removal was observed for cadmium (78.0%) and zinc (75.1%), followed by lead (68.6%) and cobalt (66.5%). In contrast, the removal efficiency for copper (Cu^2+^) was notably lower at 7.7%, despite the strain’s growth tolerance to this metal. These results indicate that while the strain possesses broad-spectrum resistance, its sequestration efficiency varies depending on the specific metal ion and its associated efflux or binding mechanisms.

These experimental results correlate strongly with the genomic identification of high-identity efflux systems and P-type ATPases, such as the predicted czc cluster and cadA. The ability of strain TW-R-39-2 to maintain growth while effectively sequestering toxic ions, particularly Cd^2+^ and Zn^2+^, highlights its specialized genetic architecture for mitigating metal toxicity [57]. These findings suggest that the strain likely employs putative mechanisms involving both active efflux and surface adsorption to remediate wastewaters contaminated with diverse heavy metals.

3.7. Biodegradation of Chlorinated Organic Compounds

The metabolic versatility of strain TW-R-39-2 was further evaluated through its ability to degrade chlorinated organic compounds, specifically TCE and chlorophenol. In MSM supplemented with 50 mg/L of each compound, the strain exhibited substantial degradative activity within a 7-day incubation period, aligning with the completion of its active growth and early stationary phases. The degradation efficiency for TCE reached 83.6%, while chlorophenol was degraded by 81.0% (Figure 6). Notably, the stoichiometric release of chloride ions (83.6% for TCE and 81.0% for chlorophenol) indicated that the majority of the parent compounds had undergone near-complete dechlorination by the end of the incubation period. This chemical evidence suggests that a 7-day duration is sufficient to evaluate the primary degradative potential of the strain.

The biodegradation process was accompanied by a stoichiometric release of chloride ions (Cl^−^), confirming the complete dechlorination of the target pollutants rather than simple adsorption or partial transformation [45]. These findings are consistent with the identification of various reductive dehalogenase-related genes and specialized metabolic pathways in the TW-R-39-2 genome. The dual-functional potential for heavy metal sequestration and organic pollutant degradation identifies strain TW-R-39-2 as a promising candidate for the comprehensive bioremediation of complex industrial wastewaters containing mixed inorganic and organic contaminants.

4. Discussion

The phylogenomic and phenotypic characterization of strain TW-R-39-2 provides comprehensive insights into its classification as a novel member of the genus Dechloromonas and its specialized adaptations for the remediation of diverse pollutants. Comparative genomic analysis confirms the designation of strain TW-R-39-2 as a novel species, showing ANI and dDDH values (80.17% and 23.4%, respectively) significantly below the recognized species-delineation thresholds [58]. Although the observed ANI value (~80.17%) is notably lower than the typical species-level cutoff (95–96%), it unequivocally confirms the status of TW-R-39-2 as a taxonomically distinct novel species within the Dechloromonas lineage. This pronounced genomic divergence reflects the strain’s specialized evolutionary trajectory, which has likely driven the acquisition of its unique functional repertoire for environmental resilience—a feature that distinguishes it from other characterized members of the genus. Based on these phylogenomic results, strain TW-R-39-2 is considered a novel member of the genus Dechloromonas; however, further comprehensive polyphasic taxonomic studies, including chemotaxonomic and comparative phenotypic analyses, are required for its formal species description and nomenclature.

4.1. Specialized Genomic Architecture for Heavy Metal Resilience

A defining characteristic of strain TW-R-39-2 that distinguishes it from other reported Dechloromonas species is its extraordinary resilience to divalent heavy metals. Historically, members of the genus Dechloromonas, such as D. agitata and D. aromatica, have been predominantly studied for their roles in perchlorate reduction and anaerobic degradation of aromatic hydrocarbons [5]. While these strains possess significant pathways for organic pollutant transformation, their genetic capacity for mitigating high-concentration heavy metal stress has been relatively limited or undocumented.

In stark contrast, the genome of strain TW-R-39-2 harbors a specialized and expanded repertoire of heavy metal resistance determinants. The identification of a complete putative core czc (czcCBA) operon (TW-R-39-2_00730–00732) and multiple P-type ATPases (e.g., cadA, TW-R-39-2_02651) suggests a potential molecular basis for its superior tolerance, meriting further investigation into the specific partitioning of metal ions. Notably, the presence of the strain-specific metal response regulator czcI (TW-R-39-2_02842), identified as a singleton in our pan-genome analysis, suggests a highly evolved and regulated mechanism for the active extrusion of toxic ions such as Cd^2+^, Zn^2+^, and Pb^2+^ [59]. This organized clustering of RND family efflux systems likely reflects an evolutionary adaptation to the multi-stressor environments of industrial wastewater treatment plants, allowing TW-R-39-2 to maintain metabolic integrity where other members of the genus might suffer from metal-induced enzyme inhibition [54]. Interestingly, while strain TW-R-39-2 exhibited high removal efficiencies for Cd^2+^ and Zn^2+^, its sequestration capacity for Cu^2+^ was notably lower (7.7%), which aligns with the substrate specificity of the identified RND family efflux systems. The putative czc operon is primarily optimized for the trans-periplasmic export of Cd^2+^, Zn^2+^, and Co^2+^, whereas it typically shows lower affinity toward Cu^2+^ [54,60]. Furthermore, the inherent proteotoxicity of copper at high concentrations (100 mg/L) might have prioritized cellular survival over active adsorption or uptake, reflecting a specialized rather than universal metal response strategy. In addition to divalent metals, the identification of the putative arsCB operon suggests a genomic potential for arsenic resistance, although further phenotypic studies are required to confirm its functional efficacy.

4.2. Dual-Functionality in Metal Resistance and Biodegradation

The experimental validation of its dual functionality—encompassing both heavy metal sequestration and chlorinated compound degradation—further underscores the unique value of strain TW-R-39-2 as a versatile bioremediation agent. The strain demonstrated high removal efficiencies for cadmium (78.0%) and zinc (75.1%), correlating strongly with the genomic presence of high-identity efflux systems. While the current study focuses on characterizing these putative functional clusters and their corresponding phenotypic resistance, the high correlation between the identified genetic repertoire (e.g., predicted czc and cadA clusters) and the observed high MIC values provides strong circumstantial evidence of their functional involvement. Most notably, this metal tolerance does not compromise its degradative activity; the strain achieved significant degradation of TCE (83.6%) and chlorophenol (81.0%) within the monitored 7-day period.

This versatility is rare in bioremediation, as heavy metals often act as a “metabolic brake” by binding to the sulfhydryl groups of essential enzymes or displacing cofactors, thereby inhibiting the breakdown of organic pollutants [46]. However, the diverse array of dehalogenases and the significant energy metabolism identified in TW-R-39-2 enable it to bypass these constraints. Although definitive confirmation of these pathways would require future transcriptomic or proteomic validation to observe differential gene expression under stress, the alignment between our genomic predictions and experimental phenotypes suggests that these systems are the primary drivers of the strain’s resilience. The observed 7-day incubation, aligned with the strain’s active metabolic phase, was sufficient to demonstrate its dual-functional potential, as evidenced by the high degradation efficiencies and stoichiometric chloride release. Furthermore, the discovery of unique singletons such as the chemoreceptors NahY (TW-R-39-2_01696) and PctC (TW-R-39-2_01305) suggests an enhanced capability for “active remediation” [61]. These proteins likely facilitate chemotaxis toward aromatic pollutants, allowing the strain to actively sense and migrate toward contaminants in complex wastewater matrices, a feature not typically emphasized in other Dechloromonas type strains.

4.3. Implications for Integrated Bioremediation

Collectively, these findings position strain TW-R-39-2 as a superior candidate for the treatment of diverse industrial effluents. Its dual-functional capacity for ‘multi-target remediation’—neutralizing toxic metals as well as degrading persistent organic compounds—offers a significant advantage over conventional strains optimized for single-pollutant degradation [62]. The stoichiometric release of chloride ions confirms that the organic pollutants are completely dechlorinated rather than merely adsorbed, ensuring the safe detoxification of the environment [63]. While the current study evaluated heavy metal sequestration and organic compound biodegradation independently, this characterization provides a fundamental basis for the strain’s application. Future research employing co-exposure scenarios will further elucidate the potential interactions between these diverse pollutants during the remediation process. Future studies focusing on the optimization of TW-R-39-2 in large-scale systems will further validate its practical application in restoring multi-pollutant contaminated environments.

5. Conclusions

In this study, we characterized strain TW-R-39-2 as a novel species within the genus Dechloromonas through a polyphasic approach combining high-quality whole-genome sequencing and phenotypic assays [64]. Phylogenomic analysis confirmed its distinct taxonomic position, while functional annotation revealed a specialized genetic repertoire for environmental resilience. Experimentally, the strain demonstrated notable dual functionality: high adsorption efficiency for heavy metals, particularly cadmium (78.0%) and zinc (75.1%), and significant degradative capacity for chlorinated organic pollutants, including TCE (83.6%) and chlorophenol (81.0%), within a 7-day incubation period.

The correlation between the identified resistance genes (e.g., predicted czc and cadA clusters) and the observed phenotypes suggests a potential molecular basis for its environmental resilience. Given its ability to maintain metabolic activity under diverse heavy metal stress while effectively neutralizing organic contaminants, strain TW-R-39-2 represents a versatile biological tool with potential for the integrated bioremediation of complex industrial wastewaters. Future studies focusing on co-exposure scenarios and the optimization of its performance in large-scale pilot systems will further validate its practical application in restoring multi-pollutant contaminated environments.