Introduction

Heavy metal pollution has the characteristics of non-degradability, bioaccumulation, and toxicity sustainability (Weber et al. 2001). Once ingested, they are hardly removed by excretion, which makes them easily enriched in organisms continuously through the food chain (Ayangbenro et al. 2019). Hence, even a tiny dose of heavy metals poses a great threat to human body and ecological environment (Chowdhury et al. 2016). Copper is one of the most common heavy metals with extensive biological toxicity. It was reported that exposing microorganisms to solid copper surface would increase the mutation rate or DNA damage (Espirito Santo et al. 2011). Therefore, discharging copper-containing wastewater into water bodies greatly reduces the self-purification capacity of the water and brings great pressure to the balance of the aquatic ecosystem (Vale et al. 2016). At present, the treatment technologies for heavy metal-containing wastewater include reverse osmosis, ion exchange, chemical deposition, electrochemical treatment, electrodialysis, membrane filtration, and flocculation adsorption (Vasudevan and Oturan 2013; Jamshidifard et al. 2019). Among them, flocculation adsorption is widely used, but the added chemical reagent can become a secondary pollutant (Xu et al. 2018). To solve this dilemma, bioflocculation, which does not incur the risk of secondary pollution, was investigated (Hashim et al. 2011). The core of bioflocculation is bioflocculants such as chitosan extracted from shellfish or fish by-products (Ola et al. 2023) and extracellular polymeric substances (EPS) secreted by microorganisms (bacteria, fungi, algae) (Mohd Nasir et al. 2019).

EPS could be secreted in situ, which means less cost and excellent solubility compared to chitosan, making it more suitable for application in wastewater treatment. EPS are natural organic compounds secreted by cells, and the main components are polysaccharides, proteins, nucleic acid, and humus (Li et al. 2021). EPS were investigated as a potential alternative to chemical flocculation due to their environment-friendly advantages (Hua et al. 2021). Despite these merits, heavy metals can be universally toxic to living cells, leading to cell death (Lian et al. 2022). Additionally, the preparation of EPS before use increases the cost in engineering applications. The solution to this predicament is to promote in situ EPS secretion, which can serve as a bioflocculant in wastewater treatment. It has been noted that when cells are exposed to adverse environments, i.e., heavy metals, EPS secretion is stimulated, which can serve as a self-defense mechanisms (Van Acker et al. 2014; Holmes et al. 2016) (Yu 2020). This defense mechanism could solve the dilemma that EPS must be preprepared before use, making it possible to apply in situ produced EPS directly to heavy metal wastewater treatment. Moreover, the stimulation of more EPS can act as a bioflocculant to improve heavy metal removal efficiency.

Research on the EPS self-defense mechanism can be summarized as follows. (1) Reasons why microorganisms tend to produce EPS under adverse conditions: EPS can serve as a permeation barrier and reducing reactant. For example, EPS wrapping around E. coil cells was secreted to decrease the toxicity of Ag+ (Kang et al. 2017), and EPS of Acidithiobacillus ferrooxidans strain serve as an electron donor in iron tailings treatment (Yi et al. 2021). (2) Quantity analysis of EPS secretion response: in general, it is well known that EPS secretion increases under low concentrations of heavy metal stress. EPS production increased by 8% when dosing 0.75 mg L−1 nZnO (Ma et al. 2022) (3) Evaluation of functional genes for EPS secretion: EPS secretion was stimulated under the stress of triclosan for the increased expression of four functional genes IntI1, sul1, mexB, and tnpA (Zhao et al. 2022). In conclusion, current studies about the self-defense mechanisms are limited to the mechanisms of how heavy metals affect EPS secretion. It is unclear whether it can initiate the self-protection mechanism of EPS secretion in situ when treating heavy metal wastewater. If it does exist, further exploration of the EPS-regulated functional resistance genes under copper stress is expected.

The main objective of this study is to compare the differences in EPS secretion previously studied for Bacillus (Zheng et al. 2008; Fang et al. 2011) and to investigate the changes in functional resistance gene expression and metabolic levels under Cu(II) stress. This work aims to provide more insights into the expression of unknown genes and broaden the possible mechanisms of EPS regulation. Bacillus sp. (CICC 23870) was used as the model strain to study the response of EPS secretion under different stress of simulated copper-containing wastewater. The cell growth, Cu(II) removal efficiency, and the EPS secretion would be analyzed through batch experiments and chemical assays. An array of spectroscopic technologies including FTIR, 3D-EEM, and XPS was used to analyze the variations in functional groups, active components, and elements involved in the reactions. Transcriptome gene sequencing was employed to determine the regulation of resistance genes under Cu(II) stress, and GO and KEGG pathway enrichment analyses were used to analyze the specific metabolic level changes. This study provided valuable insight into the possible self-defense mechanism of EPS regulation under Cu(II) stress, providing technical guidance and theoretical support for the biological treatment of heavy metal wastewater.

Methods and materials

Chemicals

The polysaccharide fluorescent dye, Calcofluor white stain, was purchased from Sigma-Aldrich. LIVE/DEAD™ BacLight™ bacterial viability kit (L13152) was bought from Thermo Fisher. Tryptone and yeast extract were obtained from OXOID. All other analytical chemicals including CuSO4 were purchased from Sinopharm Chemical Reagent, China.

Strain and culture medium

In this study, strain Bacillus sp. (CICC 23870) was used as the model EPS-producing strain. Liquid Luria-Bertani (LB) medium (g L−1) (Tryptone, 10; yeast extract, 5; NaCl, 10; pH,7.2) was used for aerobic cultivation of Bacillus sp. cells. The main constituents of EPS fermentation medium contain the following (g L−1): glucose, 20; yeast extract, 2.0; urea, 1.0; KH2PO4, 2.0; K2HPO4, 2.0; MgSO4·7H2O, 0.1 and 1% of SL-6 trace element solution. The SL-6 solution includes (mg L−1) ZnSO4·7H2O, 100; MnCl2·4H2O, 30; boric acid, 300; CoCl2·6H2O, 200; CuCl2·2H2O, 10; NiCl2·6H2O, 20; and Na2MoO4·2H2O, 30. The culture medium must be sterilized before using, and the carbon source and MgSO4 solutions were autoclaved separately then combined aseptically before using.

EPS extraction

The Bacillus sp. cells were aerobically cultured in 100 mL of LB medium at 37°C for 12 h to reach the stationary phase. The cells (OD660 ≈ 1.5) were separated from the medium by centrifugation (8000g at 4 °C for 10 min) and then resuspended in phosphate buffer solutions (PBS). The mixture was transferred into 98 mL of fresh, sterilized EPS fermentation medium with a 2% inoculation rate under different initial Cu(II) concentrations (0, 10, and 30 mg L−1). The cells were recultured to reach the stationary phase and were used for EPS extraction using the sonication and alkali treatment method (Hua et al. 2021). Briefly, the pH value of the broth was adjusted to 12.5 and then subjected to ultrasonic treatment for 10 min. Then, the broth was centrifuged (8000g, 4°C, 15min) and the supernatant was poured into twice the volume of ice-cold alcohol. The mixture was settled at 4 °C for 48 hours to obtain the precipitation, which was dissolved in deionized water and then dialyzed for 24 h at 4 °C (Zhang et al. 2021b). Finally, the cotton-like white cluster EPS were obtained after lyophilization.

Analytical methods

Cell growth was monitored by measuring the optical density (OD) at 660 nm using a UV-vis spectrophotometer (UV-2550, Shimadzu, Japan). Liquid samples were collected from the fermentation broth at predetermined time intervals to determine the Cu(II) removal efficiency. The samples were then centrifuged at 8000g for 10 min, and the supernatant was filtered through a 0.22 μm hydrophilic polytetrafluoroethylene membrane. The obtained filtrate was used to determine the Cu(II) concentration using atomic absorption spectrometry (TAS-990, Puxi, China).

The EPS surrounding the cell surface before and after EPS extraction were stained by Calcofluor white stain (Wang et al. 2021). In short, the cell pellets before and after EPS extraction were transferred (1.5 μL) to a slide. Then, a drop of Calcofluor white stain and KOH solution (10%, w/v) were added and settled for 20 min; afterwards, excess dyes were washed with sterile water. The stained samples were then observed using a fluorescence microscope (BX53, Olympus) with a fluorescent CCD camera (DP73, Olympus). The bacterial viability before and after EPS extraction was detected by the LIVE/DEAD™ BacLight™ bacterial viability kit according to the manufacturer’s instructions. The ratios of live and dead cells were estimated using ImageJ software. The protein and polysaccharide contents in EPS were measured using the Bradford assay method and Anthrone reagent method, respectively. The dried EPS powder was mixed and grinded with KBr (1:150) and then treated using tablet pressing method (Hua et al. 2021). The functional groups in EPS were detected in a FTIR spectrometer (Bruker VERTEX 80 V) with a spectral range of 4000−400 cm−1. The element composition of EPS was determined using XPS spectrometer (AXIS Ultra DLD, Shimadzu, Japan). The fluorescence intensity of the EPS samples was analyzed using a 3D-EEM fluorescence spectrometry (LS-55, PerkinElmer, USA) with an excitation wavelength range of 200−450 nm and an emission wavelength range of 250−550 nm.

Functional gene change detection

Transcriptome sequencing was used to analyze changes in related resistance genes and metabolic levels among Bacillus sp. cultured under 10 mg L−1 Cu(II) in contrast to the normal culture conditions. Initially, total RNA was extracted and agar gel electrophoresis was used to detect the integrity and purity. The qualified RNA (RIN > 7) was then isolated using a rRNA removal kit and broken into 200–300 bp fragments by ion disruption (Ola et al. 2023). Random primers and reverse transcriptase were used to synthesize cDNA, and a library was constructed with a size of 300–400 bp (Meis and Khanna 2009). After library construction, quality inspection was carried out using an Agilent 2100 Bioanalyzer and the sequencing was performed on the Illumina platform. The sequencing results were compared and annotated with reference genes. Finally, differential gene cluster analysis and GO and KEGG pathway enrichment analysis were used to analyze differentially expressed genes and changes in metabolic levels.

Results and discussion

Adsorption of Cu(II) onto cell and its toxic effect on cell growth

The cells obtained after aerobic cultivation in LB medium were inoculated into a copper containing (0, 10, and 30 mg L−1 of Cu(II)) fermentation medium. As shown in Fig. 1A, the maximum OD660 value (3.56 ± 0.23) for cell growth was reached in the control experiment after 48 h, while the OD660 values for batch experiments with Cu(II) were significantly lower, especially at 30 mg L−1 of Cu(II). And the cell growth was completely inhibited under 50 mg L−1 of Cu(II), as shown in Fig. S2. These results suggested that the strain Bacillus sp. can only adapt to stress of Cu(II) lower than 30 mg L−1 and then 10 and 30 mg L−1 chosen as appropriate mild and high stress levels for further research on EPS secretion. However, 30 mg L−1 of Cu(II) resulted in cell death and severe RNA degradation, making it unsuitable for gene sequencing. Therefore, 10 mg L−1 of Cu(II) was chosen as the suitable stress level for investigating the regulation of functional genes and metabolic levels. The cell growth curve under Cu(II) was similar to that in the presence of n-BuOH (Xia et al. 2016), indicating that higher stress concentration resulted in stronger inhibition of cell growth. Interestingly, Cu(II) ions were gradually removed as the cells grew (Fig. 1B), with removal efficiencies of 38.5 ± 1.14% and 34.04 ± 1.46% for 10 mg L−1 and 30 mg L−1 of Cu(II), respectively. This suggested that higher concentrations of Cu(II) resulted in better removal effects, possibly because copper ions can enter the cells and be removed from the medium. Moreover, the dead cells, along with the secreted EPS, could serve as an adsorbent for copper ions (He et al. 2021). Based on these findings, EPS secretion under Cu(II) stress would be compared as follows.

Fig. 1
figure 1

Aerobic growth of Bacillus sp. under difference Cu(II) concentration (A) and Cu(II) concentration versus incubation time (B)

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EPS variation in the presence of Cu(II)

As showed in Fig. 2A, more EPS were secreted with a significant increase in Cu(II) concentration. EPS concentrations (g CDW−1) were measured to evaluate the changes in EPS secreted by a single dry cell, and the exchange formula between cell dry weight (CDW) and the OD660 value was 1.0 OD660 ≈ 170.43 μg CDW ml−1 (Yu et al. 2020). The total extracted EPS concentration in the control (0 mg L−1) was 3.48 ± 0.19 g CDW−1, while that extracted under 10 and 30 mg L−1 of Cu(II) was 11.38 ± 0.57 g CDW−1 and 12.95 ± 0.72 g CDW−1, respectively. EPS secretion was stimulated and increased the most (9.47 ± 0.65 g CDW−1) despite severe inhibition of cell growth under 30 mg L−1 of Cu(II) (Fig. 1A). This illustrated that heavy metals, i.e., copper, can trigger bacteria to produce more EPS as a self-defense mechanism (Fu et al. 2022). Interestingly, EPS production was the highest at 30 mg L−1 Cu(II) stress in Fig. 2A, and the adsorption effect was the best at the same concentration in Fig. 2B. Furthermore, Cu(II) removal efficiency showed a positive correlation with initial Cu(II) concentrations, which may be because the EPS secreted under 30 mg L−1 of Cu(II) has stronger adsorption capacity and can absorb more copper ions efficiently compared to that secreted under 10 mg L−1 of Cu(II) (Li et al. 2020; Li et al. 2022). This demonstrated that more EPS secretion stimulated by copper ions can further increase the removal capacity for copper ions (Fang et al. 2022b).

Fig. 2
figure 2

Effect of Cu(II) with different initial concentration (0, 10, and 30 mg L-1) on the production of EPS (A), and the variations of protein and polysaccharide component concentrations in EPS (B)

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As exhibited in Fig. 2B, the main components in extracted EPS (PS and PN) were analyzed (Bai et al. 2022). The increased secretion of EPS was mainly caused by the rising PS content, which increased from 2.20 ± 0.16 g CDW−1 to 4.55 ± 0.43 g CDW−1 as the Cu(II) concentration increased from 0 to 30 mg L−1. Cu(II) stress overwhelmingly stimulated the secretion of PS over PN, indicating that the bacteria could secrete more PS as a resistance response mechanism, probably due to the fact that PS can act as a stable permeation barrier due to its thermodynamic stability and superior dispersibility compared with PN (Dong et al. 2022). The PN/PS ratio values showed a step-by-step increase with the increase in Cu(II) concentration from 0 to 30 mg L−1. The increased PN/PS ratio suggested an improvement in the hydrophobicity of anammox granules (Chen et al. 2019), thereby improving cell aggregation and acting as a coping strategy to relieve copper stress (Tang et al. 2021; Fu et al. 2022). Additionally, it was shown more EPS secretion but lower PN and PS total contents under 30 mg L−1 of Cu(II), as contrast in Fig. 2A, B. This verified that more cell lysis occurred, leading to more cell lysate, such as nucleic acid and humus, being extracted as EPS (Yu 2020), which was consistent with the 3D-EEM results in Fig. 4. In short, the coping strategy employed by Bacillus sp. to alleviate copper stress was as follows: increased Cu(II) stress triggered greater secretion of EPS with a higher PN/PS ratio, resulting in increased cell aggregation and binding with Cu(II) to prevent its entry into cells. The overwhelmingly stimulated secretion of PS over PN indicated a more stable effect of the EPS permeation barrier. However, higher concentrations, i.e., 30 mg L−1 of Cu(II) can break through the resistance threshold, causing cell lysis and decreasing the secretion of PS and PN contents.

FTIR and XPS analysis

As shown in Fig. 3A, the peaks at approximately 3300 cm−1, 1070 cm−1, and 540 cm−1 displayed the greatest changes under Cu(II) stress. The broad peaks near 3300 cm−1 and 1400 cm−1 were assigned to the stretching vibration of hydroxyl group (−OH), which may be due to the vibration of amines and/or alcohols in the sugar ring (Yu et al. 2020). However, the overlapping series of vibration in this region made it difficult to accurately determine the changes in functional groups (Fang et al. 2022b). With an increase in Cu(II) concentration from 0 to 30 mg L−1, the two peaks near 1070 cm−1 and 540 cm−1 were most obviously strengthened in Fig. 3A. This finding indicated an increase in stretching vibration of C−O/C−O−C bond (1070 cm−1) in polysaccharide rings (Zhang et al. 2022) and the in-plane bending vibration of C−C=O bond (540 cm−1) in ketones (Zhang et al. 2021a). These two peaks were recognized as glycosidic linkage (Kang et al. 2014), and the significant enhancement of these two peaks confirmed the results in Fig. 2B, demonstrating that Cu(II) stress could stimulate the secretion of more PS contents to strengthen the EPS effect of permeation barrier.

figure 3

Fig. 3 Fourier-transform infrared (FTIR) spectra of EPS (A) and the XPS analysis of high-resolution peak spectra of C1s (B–D) and O1s (E–G) for EPS extracted from Bacillus sp. cultured under initial Cu(II) concentration of 0, 10, and 30 mg L−1

The high-resolution scans of the C1s and O1s provided in Fig. 3B–G were obtained using EPS. It was observed that the peak intensity at 286.23 eV for C1s and at 533.1 eV and 532.28 eV for O1s changed most obviously with increasing Cu(II) concentrations. After reacting with copper, the intensity of the peak near 286.23 eV showed the most improvement, which was considered as the −C=O or −C−O−C (An et al. 2022). This indicated an increase in carboxylic acid, carboxylate, or amide, which was consistent with the FTIR results as shown in Fig. 3A. This increase may be due to copper stress triggering the secretion of more EPS contents as a protective barrier (Zhang et al. 2021b). Regarding the O1s for EPS, three peak intensities at 533.1 eV, 532.28 eV, and 531.44 were observed and were ascribed to the −C−O−C, −C−O, and −C=O bonds, respectively, which showed different trends with increasing Cu(II) stress. This is consistent with the C1s results, which showed an enhancement of the −C−O−C bond. The variations in the other two peaks may be ascribed to the hydroxyl, carboxyl, and amide groups in PN contents and amino acids (Sha et al. 2022). These observations indicated that the electronic density in the EPS was disturbed by Cu(II) since the three peak intensities showed complementary. In conclusion, the XPS results were consistent well with the FTIR observations, demonstrating an increase in hydroxyl, carboxyl, and amide groups of the EPS under copper stress. The enhancement of these hydrophobic bonds further verified the results in Fig. 2B, which showed an increase in the hydrophobicity of the cell surface under Cu(II) stress. This suggested that the increase in hydrophobicity served as a coping strategy to relieve copper stress.

3D-EEM spectroscopy analysis

Figure 4A–C shows a sharp decrease in peak intensity in four regions (Flu I–III and Flu V) in the EPS. Peak A (Ex/Em 285/350) was assigned to soluble microbial by-product-like substances (SMPs) that were associated with aromatic protein-like substances, such as tyrosine and tryptophan, and could be summarized as PN contents (Zhu et al. 2015) (Qi et al. 2021). Peak A (Ex/Em 285/350) intensity in Flu IV also decreased sharply in the EPS, which may have been due to the combination of tryptophan-like substances with Cu(II) that led to quenching of its fluorescence intensity (Zhang et al. 2021a). Peak B (Ex/Em 350/450) intensity in Flu V weakened the most, indicating the formation of complex substances of copper ions and HS (Zhang et al. 2021b). This is because peak B (Ex/Em 350/450) was associated with fulvic acid-like substances (Bai et al. 2022). Quenching of HS was shown to be attributed to electrostatic interactions between toxic substances and humus through the NH3+ and COO− groups (Yin and Chen 2022), which was consistent with the FTIR results in Fig. 3A that showed an increase in hydroxyl, carboxyl, and amide groups of the EPS under copper stress. This could result in the enhancement of electrostatic interaction between EPS, as demonstrated by Yu (2020) and He et al. (2021).

Fig. 4
figure 4

Three-dimensional excitation-emission matrix (3D-EEM) spectra of EPS extracted under 0, 10, and 30 mg L−1 Cu(II) stress (A–C). And the 3D-EEM spectra were collected with subsequent emission (Em) spectra from 250 to 550 nm at 5 nm increment by varying the excitation (Ex) wavelength from 200 to 500 nm at 5 nm increments. Ex and Em slits were maintained at 5 nm, and the scanning speed was set at 600 nm/min for all the measurements

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The differential expression of functional genes

Transcriptome sequencing was used to analyze functional gene changes in Bacillus sp. without copper stress (RIN = 9.3) and after exposure to 10 mg L−1 of Cu(II) (RIN = 7.2). The gene contrast was carried out using DESeq with the reference gene of Bacillus altitudinis (NCBI 66324737), and the contrast rate was high as 94.22%. As shown in Fig. 5A, the circlize package of R language was used to mark differential expression genes, and the volcano map was drawn using the ggplot2 software package (Fig. 5B). The left side of the volcano map showed the downregulated genes, while the right side showed the upregulated genes compared to the control. The results demonstrated that 26 genes were upregulated, and 93 genes were downregulated in Bacillus sp. cultured under 10 mg L−1 Cu(II) compared to the blank. The greater number of downregulated genes indicated that the strain activity was inhibited but also initiated the copper resistance mechanism under Cu(II) stress. As shown in the heatmap in Fig. 6A, the two-way clustering analysis using Euclidean method (Guo et al. 2022) clearly divided the differential genes into 9 clusters in Fig. 6B, of which three showed an upward trend, and six showed a downward trend. It demonstrated that the Bacillus strain exhibited certain resistance against Cu(II) stress, despite its severe toxicity.

Fig. 5
figure 5

Comparison of differentially expressed gene amounts for Bacillus sp. cultivated under 0 and 10 mg L−1 Cu(II) stress. The genome circle map (A) and volcano plot of differentially expressed genes (B)

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Fig. 6
figure 6

The heatmap of differentially expressed genes (A) and the gene expression trend analysis in each corresponding cluster (B) for Bacillus sp. cultivated under 0 and 10 mg L−1 Cu(II) stress

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GO and KEGG pathway enrichment analysis

Enrichment analysis was performed on the differentially expressed genes (Xiao et al. 2022). The bubble diagram and histogram of GO enrichment analysis are shown in Fig. 7A, B, respectively. This finding indicated that the biosynthesis of uridine-5′-monophosphate synthase (UMPs) was significantly enhanced, followed by the pyrimidine biosynthesis pathway. The UMP biosynthesis pathway had been shown to enable bacterial cells to sense starvation, nucleic acid degradation, and adapt the production of extracellular matrix to changing environmental conditions (Garavaglia et al. 2012). Interestingly, it has been demonstrated that the adenosine regulatory enzyme, cyclic dimeric guanosine monophosphate (c-di-GMP), played an important role in regulating EPS synthesis (Yu 2020). Therefore, the enhanced UMP biosynthesis pathway served as one of the adenosine regulatory pathways. This pathway was indicated to play a role in EPS regulation resistance mechanisms for cells to adapt to external Cu(II) stress (Wang et al. 2022). The resistance genes enriched in different GO terms could be seen in Table 1, with genes concerning copper ion binding and transport (GO:0005507 and GO0006826) being upregulated. This finding confirmed that copper ions entered the cells and triggered the Bacillus sp. strain to improve copper ion binding and transportation, thus verifying that the removal efficiency of 30 mg L−1 Cu(II)-containing wastewater was the best, as shown in Fig. 1B.

Fig. 7
figure 7

GO pathway enrichment analysis result comparison of differentially expressed genes for Bacillus sp. cultivated under 0 and 10 mg L−1 Cu(II) stress. Histogram of GO enrichment analysis (A) and the bubble diagram of GO enrichment analysis (B)

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Table 1 Function enrichment analysis results of upregulated genes
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The upregulation and downregulation of genes enriched in the KEGG pathway are shown in Fig. 8A, B. Three main pathways were found to be enriched for downregulated genes, namely, the secondary metabolic pathway, thiamine metabolic pathway, and C5-branched dibasic acid metabolic pathway. The secondary metabolic pathway was associated with the production of microbial by-products (Fang et al. 2022a). This indicated that the toxicity of Cu(II) suppressed strain activity, which is consistent with the results shown in Fig. 4, where the SMPs in EPS decreased significantly under Cu(II) stress. The thiamine metabolic pathway played a key role in carbohydrate metabolism and the generation of pyruvate and other α-ketoacid coenzyme (Jacobs 2014). The significant downregulation of this pathway indicated that the microbial glucose metabolism was disordered under Cu(II) stress, leading to an increased PN/PS ratio in EPS, as shown in Fig. 2B. The C5-branched dibasic acid metabolic pathway, an essential pathway for microbial electron transport, played an important role in microbial functional metabolism. The downregulation of this pathway indicated that the electron transport capacity of Bacillus sp. decreased under Cu(II) toxicity, which was confirmed by the three genes downregulated in Table 2. It was worth noting that cytochromes were recognized as electron transfer substances (Yu 2020), and the downregulation of genes related to cytochrome-mediating enzyme (NM232_RS08855, NM232_RS08860, and NM232_RS18130) indicated a decreased electron transport capacity and oxidative phosphorylation metabolism level of Bacillus sp. under Cu(II) stress.

Fig. 8
figure 8

The KEGG pathway enrichment analysis for of downregulated (C) and upregulated (D) genes

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Table 2 Changes in the expression of copper ion resistance genes
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Meanwhile, three KEGG enrichment pathways were significantly upregulated in Fig. 8B: the two-component system (TCS) pathway, the pyrimidine metabolism pathway, and the alanine aspartate and glutamate pathway. The TCS pathway could regulate microbial response to changing environments and facilitate adaptation (Kowluru 2002; Liu et al. 2021). Its significant upregulation indicated that Bacillus sp. had initiated an obvious defense mechanism in response to Cu(II) stress. The pyrimidine metabolism played an essential role in the synthesizing polysaccharides, phospholipids, and glycosylating proteins and lipids, leading to the variation of the PS/PN ratio in EPS (Löffler and Zameitat 2013). The alanine aspartate and glutamate pathway was an important pathway in microbial amino acid metabolism, positively correlated with the total antioxidant capacity of cells (Xiao et al. 2022). Its significant upregulation indicated that the redox of amino acid played an important role in the defense mechanism of Bacillus sp. under Cu(II) stress. It also confirmed that the PN contents in EPS played an important role in Cu(II) resistance (Wang et al. 2022). Additionally, seven genes related to heavy metal resistance were recognized as upregulated in Table 2, confirming that the strain had established a heavy metal resistance capacity.

Conclusions

In conclusion, this study demonstrated that Bacillus sp. could adapt to Cu(II) stress by increasing EPS secretion as a biological self-defense mechanism. The PN/PS ratio of EPS increased with higher Cu(II) stress, and FTIR, XPS, and 3D-EEM spectra showed changes in EPS groups. Cu(II) stress disturbed functional resistance gene expression and metabolic levels, with downregulation of genes related to electron transport and upregulation of genes related to copper ion binding and transport, UMP biosynthesis, pyrimidine metabolism, and TCS pathways. The study suggests that Bacillus sp. initiates self-defense mechanisms under Cu(II) stress despite severe cell toxicity, reflected in increased EPS secretion and upregulation of EPS-related genes and metabolic levels.