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Article

Harnessing Bacillus thuringiensis and Bacillus cereus for Effective Biodegradation of Endocrine Disruptor 4-Nonylphenol

by
Lian Yang
,
Fanglian Lu
,
Deqin Luo
and
Ranran Dong
*
College of Animal Science, Guizhou University, Guiyang 550025, China
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(12), 247; https://doi.org/10.3390/microbiolres16120247
Submission received: 13 October 2025 / Revised: 15 November 2025 / Accepted: 17 November 2025 / Published: 25 November 2025
(This article belongs to the Topic The Role of Microorganisms in Waste Treatment)
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Abstract

4-Nonylphenol (4-NP), an important fine chemical precursor, can cause endocrine disruption, resist natural degradation, and bioconcentrate. Biodegradation is an effective and environmentally sustainable approach to its remediation. This study employed a mixture comprising equal proportions of six non-pathogenic Bacillus strains to screen and identify strains capable of degrading 4-NP, and degradation rate was measured using an ELISA kit, and metabolomic analyses and whole-genome sequencing were used to investigate the response of Bacillus to 4-NP and elucidate pathways involved in 4-NP degradation. The results revealed DY and LY strains isolated at 500 μg/L 4-NP. The DY strain was identified as Bacillus thuringiensis, and the LY strain was identified as Bacillus cereus via physiological, biochemical and PCR analyses. The degradation efficiency of a DY and LY strain mixture was 79.45% after 7 days. At 1000 μg/L 4-NP, only the LY strain was successfully isolated. Whole-genome sequencing indicated that the LY strain (accession number: CRA021210) shares the highest homology with B. cereus strain FORC-047. Notably, it showed a degradation rate of 86.34% after 7 days. Metabolomics analysis indicates that 4-NP affects the degradation pathways of aromatic compounds and benzoic acid in B. cereus. Combined with genome data, it is hypothesized that the 4-NP degradation pathway involves its conversion to p-hydroxybenzoic acid, catalyzed by monooxygenases, dioxygenases and oxidases. Subsequently, p-hydroxybenzoic acid degrades via one of two potential pathways: it produces phenol through decarboxylase or is oxidized to benzoic acid by monooxygenase. In summary, the DY and LY strains are capable of degrading 4-NP. Furthermore, we postulate potential 4-NP degradation pathways, providing insights for the remediation of 4-NP in aquatic environments.

1. Introduction

4-Nonylphenol (4-NP) is an environmental endocrine disruptor, classified as an emerging pollutant [1]. It is widely used in the manufacturing of various pesticides, industrial chemicals, and additives due to its cost-effectiveness and high efficiency [2]. Its widespread presence in urban, industrial, and domestic wastewater systems has significant toxic effects on aquatic organisms and human health, including endocrine disruption, decreased fecundity and carcinogenesis [3,4]. Consequently, the remediation of 4-NP-contaminated environments is of critical importance.
Remediation strategies for 4-NP include physicochemical and biological techniques [5]. Physicochemical methods such as adsorption, membrane filtration, and advanced oxidation processes are effective but often face limitations, including high cost, the generation of toxic by-products, and limited recyclability [6,7,8,9,10,11]. In contrast, bioremediation, particularly microbial degradation, is efficient, economical, and sustainable [12]. To date, approximately 37 NP-degrading bacterial strains have been isolated from aquatic environments [5]. However, a significant limitation of the currently available strains is that many are either pathogenic (e.g., Shigella, Aeromonas and Shewanella) or conditionally pathogenic (e.g., Enterobacter, Acinetobacter and Pseudomonas), which raises safety concerns for their application in environmental remediation [13,14,15]. Furthermore, the degradation mechanisms of nonpathogenic strains remain insufficiently researched, and comprehensive studies integrating multi-omics approaches to elucidate these pathways are still lacking.
To address these gaps, we screened and identified non-pathogenic Bacillus strains with high 4-NP degradation efficiency and investigated their degradation mechanism. We evaluated six non-pathogenic Bacillus strains isolated from fish—B. cereus, Bacillus licheniformis, B. Thuringiensis, Bacillus tropicus, Bacillus wiedmannii, Bacillus amyloliquefaciens—for their capacity to degrade 4-NP via stepwise acclimation. Efficient degraders were identified through physiological, biochemical, and molecular assays. More importantly, the degradation pathway of 4-NP was elucidated for the first time in these non-pathogenic Bacillus strains through an integrative analysis combining whole-genome sequencing and metabolomics. Our findings provide novel insights into the use of safe bacterial agents and their mechanistic basis for the bioremediation of 4-NP in aquatic environments.

2. Materials and Methods

2.1. Preliminary Bioinformatics Screening of 4-NP-Degrading Bacteria

Genomic sequences of Bacillus were obtained from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) for analysis of the enzymes associated with degradation.

2.2. Screening of 4-NP-Degrading Bacillus

The B. Cereus, Bacillus licheniformis, B. Thuringiensis, Bacillus tropicus, Bacillus wiedmannii and Bacillus amyloliquefaciens used in this study were previously isolated from southern catfish. The analytical standard nonylphenol (NP, C15H24O; purity: 93.8%; molecular weight 220.35 kg/mol; CAS: 84852-15-3) was purchased from Sigma-Aldrich. Strains tolerant to 4-NP were screened on Tryptic Soy Agar (TSA) medium with a pH of 7.3, using a stepwise increase in the 4-NP concentration. Plates were coated 10 μg/L, 100 μg/L, 300 μg/L, 400 μg/L, 500 μg/L, 600 μg/L, 800 μg/L and 1000 μg/L 4-NP, with TSA medium without 4-NP as a control. A 100 μL logarithmic-phase bacterial culture was inoculated into TSA medium. Each group was established in triplicate. First, the bacteria were streaked and inoculated separately onto 4-NP tryptone soy agarose (TSA) plates, progressively increasing the concentration from 100 to 500 μg/L to select for tolerant strains (Steps 1–3). Then, 1 mL of selected isolates was cultivated in liquid Tryptic Soy Broth (TSB) with 4-NP to confirm growth (Steps 4 and 5). Subsequently, the strains’ ability to utilize 4-NP as a sole carbon source was tested on a minimal salt medium (MSM). As no growth occurred, glucose was added as an auxiliary carbon source. The isolates grew successfully on the amended medium with 4-NP concentrations of up to 500 μg/L (Steps 6–9). The domesticated and screened strains were preliminarily identified through 16S rDNA sequencing (universal bacterial primers: F:5′-AGAGTTTGATCATGGCTCAG-3′, R:5′-ACGGTTA CCTTGTTACGACTT-3′) [16]. The PCR products were purified and sequenced by Sanger sequencing on an ABI 3730XL DNA Analyzer (Thermo Fisher Scientific, Vacaville, CA, USA).

2.3. Effects of 4-NP on the Morphology of Bacillus

Well-established Bacillus cultures were collected and prepared for scanning electron microscopy by sequential processing: primary fixation with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2–7.4) for structural preservation, secondary fixation with 1% osmium tetroxide for membrane stabilization and contrast enhancement, and stepwise dehydration through a graded acetone series (30–100%). After critical point drying and gold-palladium sputter-coating, the samples were examined using a JEM-1400FLASH scanning electron microscope (Japan Electronics Co., Ltd., Tokyo, Japan) at an accelerating voltage of 5–15 kV to visualize morphological changes before and after 4-NP exposure.

2.4. Morphological, Physiological and Biochemical Identification

The selected strains were inoculated individually onto TSA and TSB (Tryptic Soy Broth) media, followed by incubation at 36 °C for 18 to 24 h. Initial identification was conducted by observing the colony size, color, and shape on solid TSA plates. Gram staining was used to observe the morphological features and staining characteristics. Subsequently, physiological and biochemical analyses included tests for carbohydrate fermentation (Xylose, Sorbitol, Adonitol, Raffinose, Glucose), enzyme activity and metabolic traits (Peptone water, Gluconate, Urea, Hydrogen sulfide, Phenylalanine deaminase, Ornithine decarboxylase, Lysine decarboxylase), and substrate utilization (Citrate, Semi-solid agar for motility). Analyses were performed in accordance with the instructions provided with the commercial biochemical identification tubes used, which were purchased from Hangzhou Microbial Reagent Co., Ltd. (Hangzhou, China). The antibiotic sensitivity discs were obtained from Changde Biocomma Biotechnology Co., Ltd. (Changde, China).

2.5. Whole-Genome Sequencing to Identify 4-NP-Degrading Bacteria

The 4-NP-degradation bacteria, harvested from the TSB culture medium during the logarithmic growth phase, were subjected to centrifugation at 8000 rpm, flash-frozen in liquid nitrogen, and subsequently transported to Guangdong Magigene Biotechnology Co., Ltd. (Zhaoqing, China). for sequencing analysis. A sequencing library was constructed using the SQK-LSK109 ligation kit, following the manufacturer’s protocol, including DNA damage repair, end repair and A-tailing, and adapter ligation. The resulting libraries were loaded onto R9.4.1 flow cells and sequenced on the Nanopore PromethION platform. Base calling was performed using Guppy (v3.2.6), with the high-accuracy model. Raw reads were filtered using NanoFilt (v2.8.0) to generate subreads, which comprised reads longer than 2000 bp and served as the input for genome assembly with Canu v1.5 [17] and wtdbg2 v2.2 [18]. Functional annotation of the genes was performed using NCBI (ftp.ncbi.nlm.nih.gov), KEGG (http://www.kegg.jp), and CARD (https://card.mcmaster.ca/).

2.6. Determination of 4-NP Concentration

A degradation experiment was conducted with a 1:1 mix of B. thuringiensis and B. cereus, as well as B. cereus alone, in an oscillating incubator at 30 °C and 150 rpm for 7 days. The experiment was carried out in a defined inorganic salt medium (Minimal Salt Medium, MSM) with the following composition (g/L): Na2HPO4 2.5, KH2PO4 1.5, (NH4)2SO4 0.5, MgSO4 0.1, CaCl2 0.04, EDTA-2Na 0.005, FeSO4·7H2O 0.002. The pH was adjusted to 7.0 and the medium was sterilized by autoclaving at 115 °C for 15 min. Inorganic salt cultures were collected on days 1, 3, and 7, and 4-NP concentration was measured using an ELISA kit (Shenzhen Rongjin Technology Co., Ltd., Shenzhen, China, Catalog No. RNC95011). The assay was performed strictly according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader, and the concentration of 4-NP in the samples was determined by interpolating from the standard curve.

2.7. Metabolomics Analysis

The experiment was divided into two groups: the 4-NP group, containing 1 mg/L 4-NP and 1 mg/L glucose, and the GL group, containing 1 mg/L glucose in the inorganic salt medium. Each group was incubated in a shaker at 150 rpm and 37 °C. After 72 h, the broth culture was collected and centrifuged at 3000 rpm and 4 °C for 10 min to obtain the supernatant. Six parallel samples from each group (n = 6) were subsequently sent to Beijing Omicson Gene Technology Co., Ltd. (Beijing, China). for non-targeted metabolomic analysis. The analysis was performed using ultra-high performance liquid chromatography (UHPLC) coupled to a Thermo Scientific Q-Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, California, USA).
Raw data were processed using Compound Discoverer software (Version 3.1, Thermo Fisher Scientific) for peak alignment, retention time correction, and peak area extraction. Metabolites were identified by querying the mzCloud and HMDB databases with a mass tolerance of 5 ppm. Multivariate statistical analysis, including orthogonal partial least squares-discriminant analysis (OPLS-DA), was performed using the ropls R package (Version 1.6.2). Differential metabolites were screened with variable importance in projection (VIP) > 1.0 and p-value < 0.05 from Student’s t-test. Pathway analysis of differential metabolites was conducted using KEGG (http://www.kegg.jp) and MetaboAnalyst (Version 6.0) (http://www.metaboanalyst.ca/).

2.8. Statistical Analysis

All experiments were conducted in at least three independent replicates. Data are presented as mean ± standard deviation (SD) value. Statistical significance between groups was assessed using an unpaired two-tailed Student’s t-test, and a p-value of less than 0.05 was considered statistically significant. All analyses were performed using IBM SPSS Statistics 27.0.1 software.

3. Results

3.1. Primary Screening of 4-NP-Degrading Bacteria

Analysis of the Bacillus strains’ genomes from the NCBI database revealed that they possess a variety of benzene ring degradation-related enzymes including PabB, YhbJ, YhcA, YhcB, MenC, DhbA, CatD and YhjG (Table 1), suggesting their potential for 4-NP degradation.

3.2. Screening of Bacillus Under 4-NP

By screening bacterial growth under adverse conditions, LY was ultimately determined to be the dominant bacterium (Table 2). With increase in 4-NP concentration, two dominant strains, DY and LY, were isolated at a 4-NP concentration of 500 μg/L. A dominant strain, LY, was isolated at a concentration of 1000 μg/L.

3.3. Effect of 4-NP on Bacillus Morphology

Morphological alterations in the bacteria pre- and post-screening were examined using scanning electron microscopy. The analysis revealed that the DY strain exhibited dimensions ranging from 2.8 to 4.5 μm in length and 0.42 to 0.56 μm in width prior to screening. The bacteria were rod-shaped, with a smooth surface and clear arrangement. (Figure 1A). Post-screening, they appeared clustered and adherent (Figure 1B,C). Before screening, the LY strain was 1.8 to 3.1 μm long, 0.77 to 1.1 μm wide, rod-shaped, with a smooth surface, and arranged in chains (Figure 1a). After screening, its length was 1.5 to 2.5 μm and its width was 0.52 to 0.84 μm, and it exhibited collapsed ends, a rough surface, and an irregular arrangement (Figure 1b,c).

3.4. Morphological Identification Results for 4-NP Degrading Bacteria

After 18 h of incubation at 37 °C, the DY colony had a diameter of 1 to 1.5 mm and appeared white and circular, with a rough, filamentous structure around the edge. In contrast, the LY colony, with a diameter of 1 to 2 mm, also appeared white and circular but had a relatively rough surface, low transparency, and a patchy texture. (Figure 2A,B). Both strains were identified as Gram-positive bacilli after Gram staining (Figure 2a,b).

3.5. Physiological and Biochemical Identification Results

The DY strain tested negative for xylose, sorbose, adonitol, raffinose, peptone water, Simmons citrate, urea, hydrogen sulfide, phenylalanine deaminase, gluconate, ornithine decarboxylase, and lysine decarboxylase but positive for gas production from glucose, hydrogen sulfide, MR and VP, and motility in semi-solid agar. The LY strain had positive MR, VP, and agar test results, but all other tests were negative (Table 3). According to the Manual of Common Bacterial Systematic Identification [19] and Bergey’s Manual of Determinative Bacteriology [20], these results are consistent with the biochemical characteristics of the Bacillus.

3.6. 16S rDNA Identification Results

The 16S rDNA sequencing results of the single colonies of DY and LY strains were compared using BLAST (Version 2.17.0) (Basic Local Alignment Search Tool). The results showed that the DY strain is 99.7% similar to B. thuringiensis strain IAM 12077, while the LY strain is 99.9% similar to B. cereus ATCC 14579.

3.7. Whole-Genome Sequencing Results for LY Bacteria

The LY Strain’s genome sequence (accession number: CRA021210) was analyzed using BLAST, revealing that it exhibited the highest homology with B. cereus FORC_047 (Table 4). Further comparative genomic analysis using MUMmer software (version 3.23) confirmed strong collinearity between the LY and FORC_047 genomes (Figure 3), identifying the LY strain as B. cereus.

3.8. 4-NP Degradation Rate

During co-cultivation of the isolated strains B. thuringiensis DY and the isolated strains B. cereus LY, the degradation rate reached 79.45% over seven days at 500 µg/L 4-NP. However, when the 4-NP concentration was increased to 1000 μg/L, the degradation rate of the mixed culture decreased to 46.35%. In contrast, a monoculture of B. cereus LY showed a degradation rate of 59.45% within 7 days at 500 µg/L 4-NP, which increased to 86.34% at 1000 µg/L 4-NP (Figure 4). These results suggest that compared to the isolated strains B. cereus LY monoculture, the isolated strains B. thuringiensis DY and the B. cereus LY mixed culture showed higher degradation efficiency at low concentrations. However, as B. cereus LY became the predominant degrading bacterium at high concentrations, it is possible that B. thuringiensis DY experienced inhibition, thereby impeding its growth and reducing the degradation rate.

3.9. Effect of 4-NP on Metabolites of B. cereus

Six hundred forty-five metabolites were detected from the sample metabolites (Table 5). By combining univariate analysis (Student’s t-test) and multivariate analysis (OPLS-DA), calculating variable importance in projection (VIP) values, and integrating p-values or fold change values from the univariate analysis, differential metabolites were further screened [21]. A total of 120 differential metabolites were identified. Notable examples include key intermediates in aromatic compound degradation such as p-hydroxybenzoic acid and catechol, as well as amino acids like L-glutamate and sugars such as D-ribose, which are indicative of broader metabolic shifts. Correlation analysis was performed on significant differential metabolites (Figure 5), revealing that the majority of differential metabolites showed strong positive correlations.

3.10. Degradation-Related Pathways in Metabolome

From the KEGG results, pathways associated with the degradation of 4-NP were screened, identifying two potential degradation pathways: the degradation of aromatic compounds and the degradation of benzoic acid. The primary metabolic products identified were p-hydroxybenzoic acid, benzoic acid, and phenol (Table 6). Moreover, p-hydroxybenzoic acid interconverts with benzoic acid and phenol ( Figure 6A,B), indicating that these three metabolites may serve as intermediate products in 4-NP degradation.

3.11. Possible Pathways for 4-NP Biodegradation

The prospective 4-NP degradation pathway of B. cereus was inferred from the metabolomic and whole-genome analyses (Figure 6C). It is hypothesized that the degradation pathway involves 4-NP’s conversion to p-hydroxybenzoic acid, catalyzed by monooxygenases, dioxygenases, and oxidases. Subsequently, p-hydroxybenzoic acid undergoes one of two potential degradation pathways: it produces phenol through decarboxylase, or is oxidized to benzoic acid by monooxygenase.

4. Discussion

4.1. 4-NP Is Degraded by B. thuringiensis and B. cereus

The bioremediation of 4-NP in aquatic environments has been demonstrated using bacteria such as Enterobacteriaceae, Pseudomonas and Aeromonas [22,23,24]. However, using these bacteria in aquatic environmental remediation poses a risk of toxic by-products [25]. Moreover, non-pathogenic bacteria exhibit limited competitiveness against indigenous pathogens for survival, resulting in a scarcity of suitable non-pathogenic degraders for bioremediation applications [5]. In contrast to these limitations, our study successfully isolated and identified the non-pathogenic bacteria B. thuringiensis and B. cereus through successive domestication, offering a safer and more robust alternative for bioremediation. Bacillus, comprising aerobic or facultative anaerobic Gram-positive bacteria, is ubiquitous in diverse environments including air, water, and soil [26,27]. Notably, Bacillus species demonstrate remarkable resilience under harsh and arid conditions [27] and are known for their ability to adsorb and reduce toxic substances [28]. Consequently, Bacillus are frequently utilized in probiotic development [29]. Furthermore, because B. cereus reduces pathogens in the intestine and enhances host immunity it is widely employed as a microecological agent and feed additive in aquaculture [24,30], underscoring its environmental compatibility. Our findings confirm the degradative prowess of these two strains. While B. cereus has been reported to degrade phenol efficiently [31], this study demonstrates its high efficacy against 4-NP, achieving a degradation efficiency of 86.34%. This performance surpasses literature values for other documented degraders, such as Enterobacter asburiae (>50%) and Pseudomonas putida (75.28%) [22,23]. Both B. thuringiensis and B. cereus are known to inhibit harmful microorganisms like Vibrio, Pseudomonas, and Aeromonas [32,33,34]. By integrating this high degradation efficiency with their inherent environmental hardiness and established safety profile, B. thuringiensis and B. cereus offer a comprehensive and promising solution for the bioremediation of 4-NP.

4.2. Possible Degradation Pathways of B. cereus

By combining metabolomics and whole-genome data, we propose a putative pathway for 4-NP degradation in B. cereus, involving its transformation into p-hydroxybenzoic acid, benzoic acid, and phenol, likely catalyzed by oxidases or oxygenases. This hypothesis is consistent with previous reports identifying p-hydroxybenzoic acid as a key intermediate during 4-NP degradation under electrical stimulation [35] and is analogous to the pathway of malachite green degradation by B. cereus [36]. While these similarities support the plausibility of our proposed pathway, a critical distinction in our work is the use of an integrated multi-omics approach to investigate this pathway specifically in a non-pathogenic Bacillus strain, as many prior mechanistic studies have relied on single-method analyses or focused on pathogenic genera. These findings suggest that B. cereus primarily degrades aromatic compounds via hydroxylation and oxidation reactions, involving the benzoic acid degradation pathway. The metabolite p-hydroxybenzoic acid is generally considered harmless and can even inhibit bacterial growth [37]. Benzoic acid, a common environmental pollutant, has been extensively studied, and its degradation is well-understood [38]. In comparison with 4-NP, phenol is a less toxic organic compound for which mature degradation technologies are available [39]. It has been hypothesized that in the Pseudomonas putida MT4, NP is first converted to a carboxylic acid via an alkane hydroxylase. This is followed by the cleavage of the aromatic ring, generating straight-chain carboxylic acids that enter the tricarboxylic acid (TCA) cycle and are completely oxidized to CO2 and H2O [40]. This proposed terminal step, wherein the intermediates enter the TCA cycle, is consistent with our own findings. In summary, the degradation of 4-NP by Bacillus is effective and can harm and improve polluted aquatic environments. However, it should be noted that the proposed pathway remains hypothetical. In the future, degradation experiments will be conducted using carbon-13-labeled 4-NP. Intermediate products will be tracked by mass spectrometry to determine the pathway of 4-NP degradation. In addition, we will use heat-killed bacteria as control to definitively confirm biodegradation over adsorption, which represents a limitation of the present study.

5. Conclusions

In this study, we successfully screened and isolated two non-pathogenic Bacillus strains, B. thuringiensis and B. cereus, capable of degrading 4-NP via stepwise acclimation. Integrated metabolomic and genomic analyses proposed that B. cereus degrades 4-NP through hydroxylation and oxidation reactions, situating the process within the benzoic acid degradation pathway. Our findings provide not only promising, environmentally safe microbial resources for 4-NP bioremediation but also offer novel mechanistic insights into the metabolic capabilities of non-pathogenic Bacillus strains. Future work should focus on the degradation of carbon-13-labeled 4-NP to track intermediate products and test the efficacy of these strains in contaminated aquatic environments. The use of non-pathogenic Bacillus strains, which are compatible with aquatic ecosystems and even beneficial as probiotics in aquaculture, addresses a key safety challenge that has hindered the practical application of many previously reported degrading bacteria, highlighting the potential of our findings for developing safe bioremediation agents.

Author Contributions

Conceptualization, R.D. and L.Y.; methodology, L.Y.; investigation, D.L.; resources, R.D.; data curation, D.L.; writing—original draft preparation, L.Y.; writing—review and editing, R.D.; visualization, F.L.; supervision, D.L.; project administration, R.D.; funding acquisition, R.D. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Funder: Ranran Dong, Funding number: 32460918) and the Science and Technology Fund of Guizhou Province (Funder: Ranran Dong, Funding number: 2020-1Y100).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw sequence data reported in this paper have been deposited in GSA and OMIX in the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, and the Chinese Academy of Sciences (CRA021210 and OMIX011960) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa and https:/ngdc.cncb.ac.cn/omix, accessed on 19 September 2025 and 12 September 2025.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microbiolres 16 00247 g001
Figure 1. Scanning electron microscope images of B. thuringiensis strain DY and B. cereus strain LY before and after screening with 4-NP. (A) B. thuringiensis DY before screening; (B,C) B. Thuringiensis DY after screening at 500 μg/L (B) and 1000 μg/L (C) 4-NP. (a) B. cereus LY before screening; (b,c) B. cereus LY after screening at 500 μg/L (b) and 1000 μg/L (c) 4-NP. The red border in (C) showed that DY strain appeared clustered and adherent; The red border in c showed that LY strain exhibited a rough surface. All images were taken at a magnification of 5000×.
Figure 1. Scanning electron microscope images of B. thuringiensis strain DY and B. cereus strain LY before and after screening with 4-NP. (A) B. thuringiensis DY before screening; (B,C) B. Thuringiensis DY after screening at 500 μg/L (B) and 1000 μg/L (C) 4-NP. (a) B. cereus LY before screening; (b,c) B. cereus LY after screening at 500 μg/L (b) and 1000 μg/L (c) 4-NP. The red border in (C) showed that DY strain appeared clustered and adherent; The red border in c showed that LY strain exhibited a rough surface. All images were taken at a magnification of 5000×.
Microbiolres 16 00247 g001
Microbiolres 16 00247 g002
Figure 2. Colony morphology of B. Thuringiensis strain DY (A) and B. cereus strain LY (B), Gram staining of B. Thuringiensis strain DY (a) and B. cereus strain LY (b).
Figure 2. Colony morphology of B. Thuringiensis strain DY (A) and B. cereus strain LY (B), Gram staining of B. Thuringiensis strain DY (a) and B. cereus strain LY (b).
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Microbiolres 16 00247 g003
Figure 3. The collinearity diagram of LY strain and B. cereus FORC_047. Genomes depicted on the top and bottom horizontal bars, respectively. The horizontal bars represent the genomic sequences, with the scale in megabases (Mb), indicating the physical position along the chromosome. The diagram reveals a largely collinear genome architecture, suggesting a close phylogenetic relationship.
Figure 3. The collinearity diagram of LY strain and B. cereus FORC_047. Genomes depicted on the top and bottom horizontal bars, respectively. The horizontal bars represent the genomic sequences, with the scale in megabases (Mb), indicating the physical position along the chromosome. The diagram reveals a largely collinear genome architecture, suggesting a close phylogenetic relationship.
Microbiolres 16 00247 g003
Microbiolres 16 00247 g004
Figure 4. Detection of 4-NP concentration. ■ and ● degradation curves of B. cereus strain LY and B. thuringiensis strain DY under 1000 µg/L 4-NP. ▲ and ▼ degradation curves of B. cereus strain LY and B. thuringiensis strain DY under 500 µg/L 4-NP.
Figure 4. Detection of 4-NP concentration. ■ and ● degradation curves of B. cereus strain LY and B. thuringiensis strain DY under 1000 µg/L 4-NP. ▲ and ▼ degradation curves of B. cereus strain LY and B. thuringiensis strain DY under 500 µg/L 4-NP.
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Microbiolres 16 00247 g005
Figure 5. Differential metabolite correlation heat map. The heatmap displays Pearson correlation coefficients among the identified differential metabolites (VIP > 1.0 and p-value < 0.05). Each row and column represent a single metabolite. The color scale ranges from blue (strong negative correlation, r = −1) to red (strong positive correlation, r = +1). Metabolites and clusters were grouped by hierarchical clustering. Distinct blocks of high positive correlation (red squares) suggest co-regulated metabolic pathways, while strong negative correlations (blue squares) indicate potential inverse relationships in their abundances.
Figure 5. Differential metabolite correlation heat map. The heatmap displays Pearson correlation coefficients among the identified differential metabolites (VIP > 1.0 and p-value < 0.05). Each row and column represent a single metabolite. The color scale ranges from blue (strong negative correlation, r = −1) to red (strong positive correlation, r = +1). Metabolites and clusters were grouped by hierarchical clustering. Distinct blocks of high positive correlation (red squares) suggest co-regulated metabolic pathways, while strong negative correlations (blue squares) indicate potential inverse relationships in their abundances.
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Microbiolres 16 00247 g006
Figure 6. (A) Conversion between benzoic acid and p-hydroxybenzoic acid by KEGG. C00180 is benzoic acid; C00156 is p-hydroxybenzoic acid. (B) Conversion between p-hydroxybenzoic acid and phenol by KEGG; C00146 is phenol. (C) Possible pathways for LY strain to degrade 4-NP. The NP degradation pathway in B. cereus involves an initial step where 4-NP is oxidized to p-hydroxybenzoic acid, likely catalyzed by dioxygenases or monoxygenases. Subsequently, p-hydroxybenzoic acid is transformed into phenol (via decarboxylation) or benzoic acid (through oxidative decarboxylation), reactions mediated by decarboxylases or monooxygenases, respectively.
Figure 6. (A) Conversion between benzoic acid and p-hydroxybenzoic acid by KEGG. C00180 is benzoic acid; C00156 is p-hydroxybenzoic acid. (B) Conversion between p-hydroxybenzoic acid and phenol by KEGG; C00146 is phenol. (C) Possible pathways for LY strain to degrade 4-NP. The NP degradation pathway in B. cereus involves an initial step where 4-NP is oxidized to p-hydroxybenzoic acid, likely catalyzed by dioxygenases or monoxygenases. Subsequently, p-hydroxybenzoic acid is transformed into phenol (via decarboxylation) or benzoic acid (through oxidative decarboxylation), reactions mediated by decarboxylases or monooxygenases, respectively.
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Table 1. Bioinformatics analysis of degradation-related enzymes in Bacillus.
Table 1. Bioinformatics analysis of degradation-related enzymes in Bacillus.
Protein AbbreviationAccessing NumberFunction
PabBNP_387955.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_387955.1%20Bacillus
accessed on 12 February 2021		p-aminobenzoic acid synthetase
YhbJNP_388781.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_388781.1+Bacillus
accessed on 12 February 2021		Enzymes associated with benzoic acid degradation
YhcANP_388782.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_388782.1+Bacillus
accessed on 12 February 2021		Enzymes associated with benzoic acid degradation
YhcBNP_388783.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_388783.1+Bacillus
accessed on 12 February 2021		Quinone oxidoreductase related to benzoic acid
MenCNP_390956.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_388783.1+Bacillus
accessed on 12 February 2021		O-Succinylbenzoate-CoA Synthase
DhbANP_391080.2
https://www.ncbi.nlm.nih.gov/gene/?term=NP_391080.2+Bacillus
accessed on 12 February 2021		Benzoate dehydrogenase
CatDNP_388704.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_388704.1+Bacillus
accessed on 12 February 2021		Oxygenase
YhjGNP_388931.1
https://www.ncbi.nlm.nih.gov/gene/?term=NP_388931.1+Bacillus
accessed on 12 February 2021		Aromatic compounds monooxygenase/hydroxylase
Table 2. Screening of degrading bacteria.
Table 2. Screening of degrading bacteria.
StepsScreening MediumScreening NP Concentration (μg/L)Inoculation RegimesResults
1TSA solid medium10, 100, 1000StreakingGrowth at 100
2TSA solid medium100, 300, 600Transferred all colonies from Step 1 plateGrowth at 300
3TSA solid medium300, 400, 500, 600, 700, 800Washed all colonies from Step 3 plate with salineGrowth at 500
4TSB liquid medium100Inoculated from Step 1; washed with salineTurbid, growth
5TSB liquid medium 500Inoculated from Step 4; washed with salineTurbid, growth
6Inorganic salt solid medium100, 300, 500Transferred all colonies from Step 4 plate; inoculated with broth from Step 5No growth
7Inorganic salt solid medium50, 100, 200, 300Spread plate (1 μL, 10 μL, 100 μL); washed with salineNo growth
8Inorganic salt solid medium10, 50, 100, 300Transferred all colonies from Step 4 plate; washed with salineGrowth at 300
9Inorganic salt solid medium300, 400, 500Transferred all colonies from Step 8 plateGrowth at 500
10Inorganic salt solid medium500, 600, 700, 800Transferred all colonies from Step 9 plateGrowth at 800
11Inorganic salt solid medium800, 900, 1000Transferred all colonies from Step 10 plateGrowth at 1000
Note: At the end of step 9, there are two dominant bacteria, DY and LY, in the medium. By the end of step 11, LY has become the dominant bacterium on the medium.
Table 3. Biochemical identification of DY and LY strains.
Table 3. Biochemical identification of DY and LY strains.
Test ItemTest ResultTest ItemTest Result
DYLYDYLY
Xylose−−Urea−−
Sorbitol−−Hydrogen sulfide+−
Adonitol−−Phenylalanine deaminase−−
Raffinose−−Gluconate−−
Glucose+−Ornithine decarboxylase−−
Peptone water−−Lysine decarboxylase−−
Glufosinate water++Semi-solid agar++
Citrate−−
Note: “+” is positive; “-” is negative.
Table 4. Average Nucleic Acid Similarity Analysis.
Table 4. Average Nucleic Acid Similarity Analysis.
Query IDReference IDANIMapped_FragmentQuery_FragmentTaxon
LYGCF_00222028596.922716361785B. cereus strain FORC_047
Table 5. Metabolite number statistics.
Table 5. Metabolite number statistics.
ModeTotal Ion NumberAfter Pre-ProcessingRatio (%)
merge645645100.00%
Note: Databases include KEGG Database, Human Metabolome Database (HMDB), and METLIN database.
Table 6. Possible degradation pathways of 4-NP by metabolites KEGG annotation.
Table 6. Possible degradation pathways of 4-NP by metabolites KEGG annotation.
MetaboliteStructural FormulaPathway
4-Hydroxybenzoic acidMicrobiolres 16 00247 i001Degradation of aromatic compounds Benzoate degradation
Benzoic acidMicrobiolres 16 00247 i002Degradation of aromatic compounds
Benzoate degradation
PhenolMicrobiolres 16 00247 i003Degradation of aromatic compounds
Benzoate degradation
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Yang, L.; Lu, F.; Luo, D.; Dong, R. Harnessing Bacillus thuringiensis and Bacillus cereus for Effective Biodegradation of Endocrine Disruptor 4-Nonylphenol. Microbiol. Res. 2025, 16, 247. https://doi.org/10.3390/microbiolres16120247

AMA Style

Yang L, Lu F, Luo D, Dong R. Harnessing Bacillus thuringiensis and Bacillus cereus for Effective Biodegradation of Endocrine Disruptor 4-Nonylphenol. Microbiology Research. 2025; 16(12):247. https://doi.org/10.3390/microbiolres16120247

Chicago/Turabian Style

Yang, Lian, Fanglian Lu, Deqin Luo, and Ranran Dong. 2025. "Harnessing Bacillus thuringiensis and Bacillus cereus for Effective Biodegradation of Endocrine Disruptor 4-Nonylphenol" Microbiology Research 16, no. 12: 247. https://doi.org/10.3390/microbiolres16120247

APA Style

Yang, L., Lu, F., Luo, D., & Dong, R. (2025). Harnessing Bacillus thuringiensis and Bacillus cereus for Effective Biodegradation of Endocrine Disruptor 4-Nonylphenol. Microbiology Research, 16(12), 247. https://doi.org/10.3390/microbiolres16120247

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