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Article

Screening and Application of Pseudomonas protegens from Municipal Sludge for the Degradation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) in Contaminated Soil and Water

by
Yanting Wu
1,
Yuanping Li
1,*,
Tianyun Zhou
1,
Yaoning Chen
2,3,*,
Li Zhu
1,
Guowen He
4,
Nianping Chi
1,
Shunyao Jia
2,3,
Wenqiang Luo
1 and
Ganquan Zhang
1
1
School of Municipal and Geomatics Engineering, Hunan City University, Yiyang 413000, China
2
College of Environmental Science and Engineering, Hunan University, Changsha 410082, China
3
Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082, China
4
School of Materials and Chemical Engineering, Hunan City University, Yiyang 413000, China
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(9), 547; https://doi.org/10.3390/fermentation11090547
Submission received: 4 August 2025 / Revised: 17 September 2025 / Accepted: 19 September 2025 / Published: 22 September 2025
(This article belongs to the Section Industrial Fermentation)
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Abstract

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is a refractory organic pollutant that is characterized by its persistence, toxicity and potential for bioaccumulation. As a typical biocontrol bacteria, Pseudomonas protegens has not been reported to degrade organic pollutants in the environment. A single strain of Pseudomonas protegens was isolated and acclimated from municipal sludge, and its ability to degrade BDE-47 was investigated. The enhancing effects of different carbon sources and inducers on Pseudomonas protegens were also examined. Through the reinforcement of bacterial enhancers, Pseudomonas protegens was applied to remediate soil and water contaminated with BDE-47. Based on colony characteristics, physiological and biochemical properties, and 16S rDNA gene sequence homology analysis, the strain was identified as Pseudomonas protegens and named YP1. This marks the first discovery of Pseudomonas protegens being capable of degrading BDE-47. Strain YP1 utilized BDE-47 as a carbon source and achieved a degradation rate of 69.57% after 75 h of incubation under conditions of 37 °C, pH 7, and constant temperature in a dark shaking incubator. After comparing the actual enhancement effects, glucose was selected as the carbon source and 2,4-dichlorophenol as the inducer to improve the environmental remediation capability of Pseudomonas protegens. After 14 days of remediation, the degradation rates of BDE-47 in contaminated soil and water reached 48.26% and 52.60%, respectively. The Pseudomonas protegens strain obtained from municipal sludge through screening, acclimation, and enhancement processes exhibits excellent environmental remediation capabilities and promising practical application prospects.

1. Introduction

Polybrominated diphenyl ethers (PBDEs), a group of dioxin-like compounds (DLCs), have been subject to growing scientific concern in recent years as persistent environmental pollutants. Polybrominated diphenyl ethers (PBDEs), owing to their semi-volatility, high mobility, environmental persistence, and low degradation rates [1,2,3,4,5], can accumulate in organisms through dietary intake, inhalation exposure, and dermal absorption. This bioaccumulation leads to neurotoxicity, endocrine disruption, and adverse effects on immune system function and reproductive development in humans [6,7,8,9]. Notably, tetrabromodiphenyl ether (BDE-47) [4,10,11] is recognized as one of the most abundant and most toxic PBDE congeners in environmental media [12,13,14]. Research has confirmed the detection of BDE-47 in both wastewater and soil at e-waste dismantling facilities [15,16,17,18]. Consequently, the development of effective degradation strategies for BDE-47 [19,20,21] has emerged as a key research focus among environmental scientists in recent years. Current research on BDE-47 degradation primarily focuses on microbial degradation [22], photocatalytic debromination [23], and advanced oxidation processes (AOPs) [24,25]. Among these approaches, microbial degradation has attracted significant research interest due to its cost-effectiveness, high degradation efficiency, and minimal secondary pollution compared to alternative techniques [26,27,28].
To date, multiple microbial strains capable of BDE-47 degradation have been reported, including polychlorinated biphenyl-degrading bacterium LB400 [29,30], Pseudomonas aeruginosa YH [31], Pseudomonas stutzeri BFR 01 [32], Phanerochaete chrysosporium [33,34], and Stenotrophomonas sp. WZN-1 [35]. Zhang et al. [32] isolated a novel BDE-47-degrading bacterial strain BFR 01 from contaminated soil. This strain demonstrated the capability to utilize BDE-47 as its sole carbon and energy source, achieving 97.94% degradation efficiency within a two-week incubation period. The degradation process followed first-order kinetics with a rate constant (k) of 0.32 d−1. Qi et al. [36] isolated a novel BDE-47-degrading strain, Pseudomonas plecoglossicida, from an e-waste dismantling facility. The biodegradation of BDE-47 by this strain is primarily mediated through intracellular enzymatic activity within the cellular matrix. Zhang et al. [35] investigated the degradation kinetics of BDE-47 using the bacterial strain Stenotrophomonas sp. WZN-1, and the degradation mechanism likely involves debromination pathways coupled with the formation of hydroxylated metabolites. Ti et al. [29] employed B. xenovorans LB400 for BDE-47 degradation, revealing that the primary mechanism involves intracellular accumulation of BDE-47 followed by enzyme-mediated degradation. This process efficiency is collectively governed by initial substrate concentrations, dynamics of intracellular accumulation, and biosurfactant-modulated bioavailability. Qi et al. [37] investigated the degradation dynamics of BDE-47 in the rhizosphere of Medicago sativa (alfalfa) following inoculation with Pseudomonas plecoglossicida, with parallel analysis of associated microbial response mechanisms. The findings demonstrate that rhizospheric microbial degradation—rather than phytoextraction—constitutes the primary route for BDE-47 dissipation. Compared with the non-inoculated control, the inoculation treatment elicited a significant 40.8% reduction in rhizospheric soil BDE-47 concentrations. Tang et al. [31] established that the aerobic degradation of BDE-47 by Pseudomonas aeruginosa strain YH is primarily dependent on intracellular enzymatic catalysis. Cao et al. [34] extracted both intracellular and extracellular crude enzyme fractions from Phanerochaete chrysosporium, identifying lignin peroxidase (LiP) and manganese peroxidase (MnP) within the extracellular fraction as the predominant enzymes responsible for BDE-47 degradation.
Following the isolation of competent degrading strains, the efficacy of BDE-47 biodegradation can be enhanced through strategic supplementation of nitrogen sources, cofactor metal ions, and surfactants within microbial degradation systems. Chen et al. [38] found that nitrogen source amendment in BDE-47-contaminated soil microbial degradation systems elevated BDE-47 removal efficiency from 47.3% to 58.5%. This supplementation concurrently enhanced soil nutritional status, increased activities of urease and dehydrogenase enzymes, and amplified the abundance of both total bacteria and dehalogenating bacterial consortia, demonstrating that nitrogen source addition significantly stimulates microbial degradation of BDE-47 in soil matrices. Feng et al. [33] elucidated the stress response of Phanerochaete chrysosporium to cadmium (Cd) during BDE-47 biodegradation. Cd exposure modulated degradation efficiency indirectly through impacts on extracellular enzyme secretion, organic acid excretion, and cellular antioxidant capacity. Critically, low-concentration Cd (<5 mg/L) enhanced degradation via hormetic stimulation, while elevated concentrations (>20 mg/L) exerted inhibitory effects. Ti et al. [29] reported that exposure to a defined concentration of the biosurfactant sucrose fatty-acid ester significantly elevated the bioavailability of BDE-47, whereas extracellular adsorption remained essentially unchanged. Intracellular accumulation, however, increased monotonically with surfactant concentration. At elevated surfactant levels, although aqueous solubility of BDE-47 was enhanced, its effective bioavailability declined, triggering inhibitory effects on the degradation process—including oxidative stress, cellular morphological distortion, and bacterial membrane disruption.
In microbial degradation approaches, the differential tolerance thresholds and degradation efficiencies exhibited by diverse bacterial strains toward varying concentrations of BDE-47 arise from strain-specific variations in biochemical properties, environmental origins, and acclimation protocols. Consequently, strategic selection of BDE-47-adapted degraders for optimal remediation under specific conditions is imperative. However, the current repertoire of validated BDE-47-degrading strains remains limited, impeding the targeted deployment of specialized microbial agents to maximize their degradation potential. This constraint frequently results in incomplete mineralization and suboptimal degradation kinetics of BDE-47 within complex environmental matrices. Consequently, the acquisition of novel microbial strains exhibiting high degradative competence toward BDE-47 represents a research imperative with considerable practical implications for the effective depuration of this contaminant across diverse environmental compartments.
For the first time, this study screened, isolated, and acclimatized a Pseudomonas protegens strain, designated YP1, from municipal sewage sludge for the efficient degradation of BDE-47 in environmental matrices. To date, Pseudomonas protegens has predominantly been exploited in the biosynthetic production of antibiotics and related bioactive metabolites [39,40], and no documented evidence exists for its application in BDE-47 degradation. Building upon a comprehensive elucidation of the physiological and biochemical characteristics of Pseudomonas protegens strain YP1, this study optimized exogenous carbon sources and inducers to markedly enhance its BDE-47-degradative performance. Subsequently, the degradative competence of the fortified strain was systematically evaluated in authentic aqueous matrices and soils to expand the microbial arsenal, furnish theoretical insights, and provide practical guidance for the remediation of BDE-47-contaminated environments.

2. Materials and Methods

2.1. Reagents and Instruments

Municipal sludge was collected from the Tuanzhou Wastewater Treatment Plant in Yiyang City, Hunan Province, China. The collected sludge was preserved using paraffin sealing film and stored at 4 °C in laboratory refrigerators until processing.
Mineral Salt Medium (MSM): Na2HPO4 (5 g/L), KH2PO4 (2.5 g/L), NH4Cl (5 g/L), MgSO4·7H2O (0.5 g/L); pH adjusted to 7.0–7.2 with HCl/NaOH; Sterilized by autoclaving at 121 °C for 20 min.
Beef Extract-Peptone Medium: Beef extract (5 g/L), Peptone (10 g/L), NaCl (5 g/L); Sterilized by autoclaving at 121 °C for 20 min.
Agar powder, glucose, 2,4-dichlorophenol, isooctane, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47).
Autoclave (ZEALWAY, Wilmington, DE, USA); UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan); high-performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA); constant-temperature shaking incubator (Shanghai Yuzhuo Instruments Co., Ltd., Shanghai, China); laminar flow hood (Suzhou Purification Co., Ltd., Suzhou, China); centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China); Infrared CO2 Analyzer (LI-COR Inc., Lincoln, NE, USA).

2.2. Screening of Pseudomonas protegens

A 10 g aliquot of the obtained sludge sample was transferred into a 250 mL Erlenmeyer flask. Subsequently, 90 mL of sterile ultrapure water was added, and the mixture was agitated in a constant-temperature incubator shaker at 180 rpm for 1 h. Following agitation, a 30 mL subsample of the sludge-water mixture was centrifuged at 3000 rpm for 15 min in sterile conical tubes. The resulting supernatant constituted the microbial suspension for subsequent procedures. The microbial suspension was incubated in a constant-temperature orbital shaker at 37 °C under dark conditions for 5 days of enrichment culture. This enriched culture served as the source stock for the subsequent isolation of BDE-47-degrading bacterial strains.
Throughout this process, bacterial growth kinetics were monitored by measuring optical density at 600 nm (OD600) using a UV-Vis spectrophotometer. Measurements were performed on Pseudomonas protegens cultures at designated time intervals: 2, 4, 6, 10, 12, 24, 50, 75, and 100 h post-inoculation.

2.3. Acclimation of Pseudomonas protegens

Microbial cultures were acclimated to BDE-47 tolerance through three rounds of stepwise concentration escalation. Each round was conducted under sterile conditions: Minimal Salt Medium (MSM) was prepared in 250 mL Erlenmeyer flasks, sterilized by autoclaving at 120 °C for 30 min, and cooled to 60–70 °C. Subsequently, an isooctane solution of BDE-47 was aseptically added within a laminar flow hood. Following complete evaporation of isooctane, media containing target BDE-47 concentrations were obtained (25 µg/L for the first round, 75 µg/L for the second round, and 115 µg/L for the third round). For the first acclimation round, a 10% (v/v) inoculum of a 24 h enriched bacterial suspension was introduced. The culture was then incubated under dark, aerobic conditions at 37 °C with constant agitation for 5 days, yielding first-round tolerance-adapted bacteria. For the second round, a 10% (v/v) inoculum of first-round tolerance-adapted bacteria was transferred and incubated under identical conditions for 7 days, yielding second-round tolerance-adapted bacteria. For the third round, plates containing BDE-47 amended MSM agar medium were prepared. A 10% (v/v) inoculum of growth-vigorous second-round adapted strains was streaked onto the plates using the streak-plate technique and incubated at 37 °C for 7 days in a constant-temperature incubator.

2.4. Purification and Preservation of Pseudomonas protegens

Using strains grown on third-round domestication plates, five successive streak-plating cycles were performed until discrete single colonies were obtained. The purified strains underwent physiological and biochemical characterization and were concurrently submitted to the General Biol (Anhui) Co., Ltd., (Chuzhou, China) for molecular identification by 16S rDNA sequencing.
The purified strain was deposited at the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan 430072, P.R. China (Wuhan University campus). This bacterial strain, taxonomically designated as Pseudomonas protegens strain YP1, bears the accession number CCTCC No: M 2023338.

2.5. Enhancement of Pseudomonas protegens

Prepare 45 mL of MSM in a 250 mL Erlenmeyer flask and autoclave at 120 °C for 30 min. When the sterilized medium cools to 60–70 °C, add tetrabromodiphenyl ether (BDE-47) to achieve a final concentration of 115 μg/L in the medium. The culture medium was supplemented, respectively, with the following exogenous carbon sources: yeast extract, glucose, acetate, and ethanol; and inducers: 2,4-dichlorophenol, bisphenol A, toluene, and hydroquinone. The enhancement effects on Pseudomonas protegens YP1 were evaluated under these distinct carbon source and inducer conditions [28,36].

2.6. Determination of PBDE Degradation Efficiency by Pseudomonas protegens

On days 1, 3, 5, 7, and 14 of cultivation, 2 mL aliquots were withdrawn from the supplemented MSM (containing exogenous carbon sources and inducers) under aseptic conditions. Using a 10 mL pipette, 20 mL of n-hexane was added in two sequential additions to create a 20 mL medium/20 mL *n*-hexane biphasic system. The conical flask was sealed and agitated at 27 °C for 1 h in an incubator shaker (180 rpm). Phase separation was then performed using a separatory funnel after 30 min of stationary settling. Transfer the upper n-hexane phase containing BDE-47 (typically appearing as a milky suspension) to a beaker and dehydrate it with an appropriate amount of anhydrous sodium sulfate. The extraction solvent was then concentrated via rotary evaporation under reduced pressure (water bath temperature: 29–32 °C; moderate rotation speed) using a rotary evaporator equipped with a vacuum pump and temperature-controlled water bath. This concentration step ensured the BDE-47 level fell within the instrument’s detection range. Finally, the concentrated extract was analyzed by high-performance liquid chromatography (HPLC) to quantify residual BDE-47 concentrations in the medium.
In addition, each BDE-47 degradation experiment was repeated three times to ensure the accuracy of the experimental data.

2.7. Determination of Respiration Intensity of Pseudomonas protegens

The respiration intensity of Pseudomonas protegens was determined using a closed static method for CO2 measurement [41]. Two portions of the bacterial strain were placed into separate 250 mL conical flasks. Flask 1# contained beef extract-peptone liquid medium, while Flask 2# contained inorganic salt liquid medium with BDE-47 as the sole carbon source. The flask openings were sealed with punctured plastic film and incubated at 37 °C. Each conical flask was connected to an infrared CO2 analyzer to record the CO2 concentration and airflow rate at 1 d, 2 d, 3 d, 5 d, 7 d, and 9 d.

2.8. Performance of Pseudomonas protegens in Degrading BDE-47 in Water and Soil Samples

The purified Pseudomonas protegens was inoculated into a medium containing glucose, 2,4-dichlorophenol, and BDE-47 for enhanced cultivation to prepare a bacterial suspension with a concentration of 105–107 CFU mL−1. Water samples were collected from Zi River, Yiyang City, China. The real water was pretreated via the suction filtration method, and the standard addition of BDE-47 was 5 μg/L. A 50 mL aliquot of the spiked aqueous sample was inoculated with 10 mL of the prepared bacterial suspension and incubated aerobically at room temperature in the dark. Residual BDE-47 concentrations were quantified at 1, 3, 5, 7, and 14 days post-inoculation. Soil samples were also collected from Yuhu Mountain, Hunan City University. BDE-47 was spiked into the soil at a concentration of 2.3 μg/g. One milliliter of the bacterial suspension was added per gram of spiked soil, followed by aerobic incubation at room temperature in the dark. Residual BDE-47 levels were determined after 1, 3, 5, 7 and 14 days. Under identical conditions, additional abiotic controls were prepared for both the water and soil microcosms by omitting the bacterial suspension. These negative controls were incubated aerobically in the dark at room temperature, and the residual BDE-47 concentration was quantified on days 1, 3, 5, 7, and 14.

3. Results and Discussion

3.1. Molecular Identification and Physiological–Biochemical Characterization of Pseudomonas protegens

The single colony obtained through screening and acclimation was subjected to bacterial species identification via the 16S rDNA method (QB/TYJC 0001-2018). The identification results indicated that the 16S rDNA sequence of the single strain exhibited a 99.63% similarity to that of Pseudomonas protegens strain CHA0. The phylogenetic tree constructed is shown in Figure 1. The strain was identified as a Gram-negative bacterium. Microscopic examination revealed a rod-shaped morphology, while single colonies appeared pale yellow with smooth, entire margins, wrinkle-free surfaces, and moist, viscous textures, exhibiting a spherical form. The strain demonstrated robust growth under aerobic conditions, as illustrated in the photo in Figure 1.
Physiological and biochemical characterization of the single Pseudomonas protegens strain revealed negative results for the following tests: Gram staining, growth at 41 °C, Voges-Proskauer (V-P) test, salt tolerance (10% NaCl), acetate utilization, maltose fermentation, D-cellobiose fermentation, lactose fermentation, ethanol utilization, methyl red test, and pectin degradation; while positive results were observed for: growth at 4 °C, gelatin liquefaction, protease activity, salt tolerance (1% NaCl), salt tolerance (5% NaCl), hydrogen peroxide tolerance, citrate utilization, D-mannose fermentation, fructose fermentation, xylose fermentation, esterase activity, and the catalase (contact enzyme) test. The optimal growth temperature for the strain was 37 °C, and it exhibited normal growth in media with an initial pH range of 6.0–8.0. The physiological and biochemical characteristics of Pseudomonas protegens are summarized in Table 1.

3.2. Growth Characteristics of Pseudomonas protegens

Pseudomonas protegens was cultured under laboratory conditions at 37 °C, pH 7.0, and with nutrient-replete media. Its growth dynamics are shown in Figure 2. The data indicate that Pseudomonas protegens exhibits rapid growth and a significant increase in bacterial population within 6 h under conditions of sufficient nutrients, including carbon and nitrogen sources. Subsequently, after 6 h, the growth rate within the culture medium begins to decline and the population stabilizes due to diminishing nutrient availability and inter-species competition [42,43]. The decline in growth rate observed after 75 h implies that applying Pseudomonas protegens cultured for this duration offers dual advantages. Firstly, it allows for the harvest of a substantial population of the bacterium in a relatively short timeframe. Secondly, and more significantly, the resulting nutrient-deficient state of the bacteria better mirrors real-world environmental conditions. This facilitates their rapid adaptation and enables more efficient biodegradation of BDE-47 in the target environment.

3.3. BDE-47 Degradation Efficacy of Pseudomonas protegens

The degradation process of BDE-47 by Pseudomonas protegens is illustrated in Figure 3. Initially, due to the bacterium’s limited tolerance to BDE-47 at a concentration of 115 μg/L, the degradation rate was relatively slow. Over a 24 h degradation period by Pseudomonas protegens, the BDE-47 concentration decreased to 113 μg/L. After five days, Pseudomonas protegens developed tolerance to BDE-47, with the concentration decreasing to 92 μg/L. This tolerance manifested in an increased degradation rate of BDE-47 and a concurrent decline in the growth rate of Pseudomonas protegens, highlighting the trade-off between these processes [44]. The concentration of BDE-47 on the 14th day was 35 μg/L, and Pseudomonas protegens achieved a degradation rate of 69.57% for BDE-47 within 14 days. Meanwhile, as shown in Figure 4, under nutrient-rich conditions, the respiration of the bacterial strain increased sharply over time. In the medium with BDE-47 as the sole carbon source, the respiration slowed down but still maintained a certain intensity, indicating that Pseudomonas protegens possesses a certain mineralization capacity for BDE-47. The results demonstrate that after a period of environmental exposure, Pseudomonas protegens exhibits significant tolerance to BDE-47 and is capable of efficiently degrading it, highlighting its potential for practical application. In the field of researching the remediation of toxic and harmful organic pollutants, such as BDE-47, by microorganisms, the OECD 301 (ready biodegradability test), 302 (inherent biodegradability test) and the Microtox test system are widely accepted as the gold standard of biodegradation and microbial toxicity tests [45,46]. These tests are instrumental in enhancing the understanding of the mechanism of microbial degradation and in strengthening the reliability and scientific rigor of the biodegradation data. Consequently, they were incorporated into the subsequent research to enhance comprehension of the Pseudomonas protegens strain physiological resilience.
When Pseudomonas protegens cultivated for 75 h under optimal conditions was applied to degrade BDE-47 at 115 μg L−1, the degradation process conformed to first-order kinetics, yielding a rate constant k of 0.0888 d−1 and a half-life t1/2 of 7.80 d, as depicted in Figure 5.
The first-order kinetic linear equation is:
y = 0.088x − 0.1306
The aerobic microbial degradation pathway for PBDEs involves an initial debromination step, followed by ring cleavage of the debrominated products to achieve complete mineralization into harmless compounds without secondary pollution; this process also proceeds at a significantly faster rate than anaerobic degradation. The aerobic degradation products of BDE-47 by Pseudomonas aeruginosa YH mainly include two debrominated products (BDE-28 and BDE-7), four hydroxylated degradation products (6-OH-BDE-47, 5-OH-BDE-47, 2′-OH-BDE-28, and 4′-OH-BDE-17), and two bromophenol products (2,4-DBP and 4-BP) [31]. In existing biocontrol studies, Pseudomonas protegens is known to produce a range of antimicrobial metabolites, including 2,4-diacetylphloroglucinol (2,4-DAPG), pyoverdine (PLT), surfactants (such as rhamnolipids, sophorolipids, lipopolysaccharides, and carbohydrate–protein–lipid complexes), and hydrolytic enzymes [47]. Therefore, during the degradation of BDE-47, Pseudomonas protegens should be capable of secreting key extracellular substances. These include extracellular functional enzymes [34,35,48], surfactants (such as rhamnolipids [48], lipopolysaccharides), and cell surface hydrophobicity (CSH). The involvement of rhamnolipids in BDE-47 degradation is presumably because surfactants possess unique hydrophobic and hydrophilic structural domains. At higher concentrations, rhamnolipids increase CSH in Pseudomonas protegens, making altered CSH another factor influencing BDE-47 degradation. The combined effect of these extracellular substances enables the initial dehalogenation of BDE-47 by removing bromine from the benzene ring. Subsequent monooxygenation/dioxygenation hydroxylation or methylation facilitates ring-opening. The resulting ring-opened products are then taken up by aerobic degrading bacteria as substrates. These products enter the TCA cycle or are mineralized into CO2 and H2O, thereby completing the degradation process.

3.4. Enhancement of BDE-47 Degradation by Strain-Conditioning Amendments

To investigate the influence on the degradation efficiency of Pseudomonas protegens toward BDE-47 at an initial concentration of 115 μg/L, the culture medium was supplemented with different exogenous carbon sources: yeast extract, glucose, acetate, and ethanol. Compared with the blank control, the BDE-47 degradation efficiencies achieved by Pseudomonas protegens were 60.87% without any supplemental carbon source, and 68.70%, 84.35%, 81.74%, and 80.87% in the presence of yeast extract, glucose, acetate, and ethanol, respectively (Figure 6). These data indicate that the addition of carbon sources significantly enhances BDE-47 degradation, with glucose providing the strongest stimulatory effect on Pseudomonas protegens.
To investigate the effects on the degradation efficiency of BDE-47 at an initial concentration of 115 μg/L, the culture medium was supplemented with 50 mg/L of the following inducers: 2,4-dichlorophenol, bisphenol A, toluene, and hydroquinone. After 14 days of degradation, the degradation rates of BDE-47 by Pseudomonas protegens were 83.48%, 81.74%, 78.26% and 51.30% with the addition of 2,4-dichlorophenol, bisphenol A, toluene and hydroquinone, respectively, as shown in Figure 7. Compared with the control group, the addition of the inducers 2,4-dichlorophenol, bisphenol A and toluene enhanced BDE-47 degradation by Pseudomonas protegens, while hydroquinone had an inhibitory effect. Notably, 2,4-dichlorophenol significantly boosted the strain’s degradation capacity.

3.5. Practical Application of Pseudomonas protegens for BDE-47 Degradation in Contaminated Water and Soil

Pseudomonas protegens enhanced with glucose as a carbon source and 2,4-dichlorophenol as an inducer was applied to degrade BDE-47-contaminated water and soil samples. In this study, Pseudomonas protegens effectively degraded BDE-47 in real water samples; after a 14-day incubation the residual BDE-47 concentration declined from 5 µg/L to 2.37 µg/L, as shown in Figure 8a. Concurrently, Pseudomonas protegens exhibited notable tolerance toward BDE-47 at an initial concentration of 2.3 µg/g in soil and achieved an efficient degradation, with a removal rate of 48.26%, as illustrated in Figure 8b. Simultaneously, we monitored the BDE-47 concentration in the abiotic negative controls (without bacterial inoculum) for both the water and soil samples; no appreciable change in BDE-47 level was detected throughout the incubation period. These findings demonstrate that Pseudomonas protegens can gradually acclimate to varying BDE-47 concentrations and develop tolerance, maintaining viability even under elevated contamination levels. Moreover, the strain utilizes BDE-47 as a nutritional substrate, achieving its degradation without generating secondary pollutants—fully aligning with the principles of green and sustainable development.

4. Conclusions

(1)
A BDE-47 aerobic-degrading bacterial strain designated YP1 was isolated from activated sludge at Tuanzhou Wastewater Treatment Plant in Yiyang City, Hunan Province. Based on morphological characteristics, physiological–biochemical properties, and 16S rDNA sequence homology analysis, this strain was identified as Pseudomonas protegens. This study reports the first discovery of Pseudomonas protegens capable of degrading BDE-47.
(2)
Pseudomonas protegens strain YP1 exhibited excellent BDE-47 degradation performance: when inoculated into a mineral-salt medium containing 115 μg/L of BDE-47 and incubated at 37 °C, pH 7, in darkness with shaking, it achieved a 69.57% removal efficiency within 75 h.
(3)
All tested carbon sources—yeast extract, glucose, acetate, and ethanol—enhanced the BDE-47 degradation efficiency of Pseudomonas protegens. While inducers 2,4-dichlorophenol, bisphenol A, and toluene promoted BDE-47 degradation, hydroquinone inhibited the process. Among these, glucose and 2,4-dichlorophenol demonstrated the most pronounced enhancement on BDE-47 degradation by Pseudomonas protegens. Consequently, both compounds were selected as strain-enhancing agents to boost the degradation capacity of Pseudomonas protegens toward BDE-47.
(4)
Pseudomonas protegens strain YP1 demonstrates considerable practical potential. When introduced into BDE-47-contaminated real water and soil matrices, it maintains growth under suboptimal conditions and at elevated pollutant levels. Within 14 days, the strain achieved BDE-47 removal efficiencies of 52.60% in water and 48.26% in soil, evidencing its robust tolerance and efficacious performance under field-relevant scenarios.

Author Contributions

Conceptualization, Y.L. and Y.W.; methodology, Y.L.; soft-ware, T.Z., W.L. and L.Z.; validation, T.Z., N.C. and S.J.; formal analysis, G.H. and Y.W.; investigation, S.J., G.Z., W.L. and L.Z.; resources, Y.C.; data curation Y.C.; writing—original draft preparation, Y.W.; writing—review and editing, Y.C. and Y.L.; visualization, G.Z. and T.Z.; supervision, Y.L.; project administration, Y.C.; funding acquisition, Y.L. and Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (42477020), the Research Foundation of Education Department of Hunan Province (24A0578), the Innovation Training Program for College Students of Hunan Province (S202511527042), the Natural Science Foundation of Hunan Province, China (2023JJ30130), and the Engineering and Technology Centre for Safeguarding Drinking Water Quality in Hunan Villages and Towns Project (2019TP2079).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of Pseudomonas protegens and strain morphology.
Figure 1. Phylogenetic tree of Pseudomonas protegens and strain morphology.
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Figure 2. Growth curve of Pseudomonas protegens. (Medium: Beef extract peptone medium; Carbon: Beef extract, Glucose; Growth rate constant: 0.0888 d−1).
Figure 2. Growth curve of Pseudomonas protegens. (Medium: Beef extract peptone medium; Carbon: Beef extract, Glucose; Growth rate constant: 0.0888 d−1).
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Figure 3. Degradation of BDE-47 (initial concentration 115 μg L−1) by Pseudomonas protegens strain YP1.
Figure 3. Degradation of BDE-47 (initial concentration 115 μg L−1) by Pseudomonas protegens strain YP1.
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Figure 4. Respiration Intensity of Pseudomonas protegens.
Figure 4. Respiration Intensity of Pseudomonas protegens.
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Figure 5. First-order kinetic fitting curve for BDE-47 degradation by Pseudomonas protegens strain YP1.
Figure 5. First-order kinetic fitting curve for BDE-47 degradation by Pseudomonas protegens strain YP1.
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Figure 6. Effects of different carbon sources on BDE-47 degradation by Pseudomonas protegens strain YP1.
Figure 6. Effects of different carbon sources on BDE-47 degradation by Pseudomonas protegens strain YP1.
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Figure 7. Degradation of BDE-47 by different inducers on Pseudomonas protegens strain YP1.
Figure 7. Degradation of BDE-47 by different inducers on Pseudomonas protegens strain YP1.
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Figure 8. Pseudomonas protegens strain degradation of BDE-47 in contaminated water (a) and soil (b).
Figure 8. Pseudomonas protegens strain degradation of BDE-47 in contaminated water (a) and soil (b).
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Table 1. Physiological and biochemical characteristics of Pseudomonas protegens.
Table 1. Physiological and biochemical characteristics of Pseudomonas protegens.
Test ItemsResultsTest ItemsResults
Gram staining−D-mannose+
4 °C+Maltose−
41 °C−Fructose+
Gelatin liquefaction+Xylose+
Voges-Proskauer test−D-cellobiose−
Protease+Lactose−
1%+Esterase+
Halotolerance5%+Ethanol (C2H5OH)−
10%−Methyl red−
H2O2+Catalase+
Citrate+Pectin−
Acetate−pH = 6~8+
“−” indicates negative or no reaction; “+” indicates positive reaction.
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Wu, Y.; Li, Y.; Zhou, T.; Chen, Y.; Zhu, L.; He, G.; Chi, N.; Jia, S.; Luo, W.; Zhang, G. Screening and Application of Pseudomonas protegens from Municipal Sludge for the Degradation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) in Contaminated Soil and Water. Fermentation 2025, 11, 547. https://doi.org/10.3390/fermentation11090547

AMA Style

Wu Y, Li Y, Zhou T, Chen Y, Zhu L, He G, Chi N, Jia S, Luo W, Zhang G. Screening and Application of Pseudomonas protegens from Municipal Sludge for the Degradation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) in Contaminated Soil and Water. Fermentation. 2025; 11(9):547. https://doi.org/10.3390/fermentation11090547

Chicago/Turabian Style

Wu, Yanting, Yuanping Li, Tianyun Zhou, Yaoning Chen, Li Zhu, Guowen He, Nianping Chi, Shunyao Jia, Wenqiang Luo, and Ganquan Zhang. 2025. "Screening and Application of Pseudomonas protegens from Municipal Sludge for the Degradation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) in Contaminated Soil and Water" Fermentation 11, no. 9: 547. https://doi.org/10.3390/fermentation11090547

APA Style

Wu, Y., Li, Y., Zhou, T., Chen, Y., Zhu, L., He, G., Chi, N., Jia, S., Luo, W., & Zhang, G. (2025). Screening and Application of Pseudomonas protegens from Municipal Sludge for the Degradation of 2,2′,4,4′-Tetrabromodiphenyl Ether (BDE-47) in Contaminated Soil and Water. Fermentation, 11(9), 547. https://doi.org/10.3390/fermentation11090547

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