The strain CZ-B1 has a significant removal effect on Pb while resisting Pb stress (Figure 4A). The removal rate of Pb²⁺ in the treatment of 500 mg/L Pb²⁺ and 1000 mg/L Pb²⁺ by CZ-B1 at 72 h was 90.38% and 39.3% respectively. With the inverse ratio of Pb stress concentration to Pb²⁺ removal, this may be due to the fact that increased Pb²⁺ toxicity severely inhibits bacterial reproduction and growth functions, thus interfering with their Pb²⁺ adsorption. In addition, the removal of Pb²⁺ by CZ-B1 under 500 mg/L Pb²⁺ stress showed three stages of increase (0–12 h), decrease (12–24 h) and increase (24–72 h). Under the Pb²⁺ stress of 1000 mg/L, CZ-B1 was able to adapt to the Pb²⁺ stress within 24 h at the earliest and continued to perform its ecological restoration function.

Organic acid secretion is a key process for PSM to dissolve phosphorus sources in the environment (Su et al., 2019). Phosphate dissolved/released by PSM can form stable phosphate minerals with HMs ions (Xiao et al., 2021). In addition, the chelation of organic acids with HMs can reduce the toxicity of HMs, which is also a common way for microorganisms to resist HMs stress on their own (Que et al., 2020). Malic acid, citric acid, oxalic acid, etc. are small molecule organic acids usually secreted by PSM (Rawat et al., 2020; Sarmah and Sarma, 2022). In order to explore the mechanism of Pb²⁺ removal by CZ-B1, we determined six organic acids in the solution (Figure 2D). In LB medium without Pb²⁺ stress, oxalic acid, citric acid, malic acid and succinic acid, formic acid and tartaric acid were detected in the solution. This result was different from the oxalic acid content in the two inorganic media (Figure 2D), which could be attributed to the different stress of CZ-B1 itself to inorganic and organic phosphorus sources. Among them, the content of malic acid (6902.0 mg/L) > succinic acid (2812.7 mg/L) > citric acid (96.4 mg/L) > oxalic acid (68.7 mg/L) at 72 h (Supplementary Table S1). The maximum level of organic acid release from CZ-B1 in organic medium was significantly higher than that in inorganic medium.

Formic acid, malic acid and tartaric acid were not detected in solution under 500 mg/L-1000 mg/L Pb²⁺ stress, which suggesting that Pb stress may directly inhibit the secretion of CZ-B1 for the three acids. Meanwhile, oxalic and succinic acids in solution kept decreasing with increasing concentration of Pb stress, while citric acid gradually increased. The content of oxalic acid and succinic acid in the solution under 1000 mg/L Pb²⁺ stress was 10.5% and 83.3% of that under 500 mg/L Pb²⁺ stress, respectively, which indicated that the effect of Pb stress on oxalic acid content was greater than that on succinic acid content. Also, the increase of citric acid content in the solution (by 32.2%) with increasing Pb²⁺ concentration indicates that Pb²⁺ coercion stimulates the secretion of citric acid by CZ-B1. In addition, the decrease in oxalic and succinic acid content in solution could both be caused by slowed cellular metabolism and reduced activity or inactivation due to HMs stress (Babu et al., 2015). Studies have shown that environmental stress significantly inhibits the rate of tricarboxylic acid cycle in microorganisms, thereby reducing the secretion of succinate (an intermediate of the tricarboxylic acid cycle) (Guarnieri et al., 2017; Teng et al., 2019). In addition, some studies have shown that oxalic acid secreted by Aspergillus niger can form PbC₂O₄ precipitation with Pb²⁺ (Ceci et al., 2015; Feng et al., 2022). Therefore, the reduction of oxalic acid in solution may also be transferred to the precipitate in the form of Pb oxalate. On the other hand, the results of organic acid content per unit cell of CZ-B1 showed that the oxalic acid concentration of unit cell under 1000 mg/L Pb²⁺ stress was higher than that under 500 mg/L Pb²⁺ stress, which further confirmed that CZ-B1 could still release oxalic acid under Pb stress (Table 2).

Figure 5 there are five characteristic peaks were mainly present in the 3D-EEM of CZ-B1, among which peak 1 (Ex/Em 370/450) and peak 2 (Ex/Em 330/400) belonged to humic acid (Baker, 2001; Guo et al., 2014) and fulvic acid (Wang et al., 2009; Lin and Guo, 2020), which were mainly derived from the nutrients in LB. Peak 3 (Ex/Em 200/450) and peak 4 (Ex/Em 200/400) may originate from the secretion of CZ-B1. In addition, there is a small peak 5 at Ex/Em 280/380. Studies have shown that Ex/Em 280/380 may be a by-products of microorganisms (Chen et al., 2003) or tryptophan (Baker, 2001).

When the Pb²⁺ concentration increased from 500 mg/L to 1000 mg/L, the fluorescence intensity of peek 1 and peek 2 increased by 44.8% and 47.1%, respectively. Meanwhile, peak 3, peak 4, and peak 5 from microbial secretions were decreased by 50.1%, 52.6% and 60.0%, respectively. It can be concluded that the metabolic activity of the microorganisms decreased and the consumption of nutrients diminished with increasing Pb stress concentration and that microbial by-products such as tryptophan might be produced simultaneously under stress conditions. Tryptophan as a precursor of indoleacetic acid (Shahid et al., 2012), both of which possess the property of promoting phosphorus solubilization (Ji et al., 2012; Alemneh et al., 2021), which undoubtedly provides a gaining effect for Pb²⁺ passivation. Therefore, the increase of tryptophan can indirectly prove the enhanced bioimmobilization of Pb²⁺ by CZ-B1. This phenomenon also proves that under low-concentration stress, CZ-B1 are more inclined to use their own cells for Pb²⁺ fixation.

XRD results showed that Pb-phosphate and Pb-oxalate were present in the sediment (Figure 6A). Many strong diffraction peaks were present at 29.8°–30.9°, 21.6°, 26.3°–27.2° and 43.9° in both concentrations of Pb²⁺ stress, which may be attributed to pyromorphite [Pb₅(PO₄)₃Cl/OH] (Meng et al., 2020). Meanwhile, the diffraction peaks at 20.78°, 23.6°, 26.4° and 35.5° indicate the formation of PbC₂O₄ (Choi et al., 1994; Ma et al., 2016). Compared with 1000 mg/L Pb²⁺ stress, the pyromorphite peak signal of CZ-B1 under 500 mg/L Pb²⁺ stress was more significant. The formation of pyromorphite indicates that CZ-B1 can fix Pb2+ by releasing phosphorus. However, the Pb oxalate signal under 1000 mg/L Pb²⁺ stress is lower than that under 500 mg/L Pb²⁺ stress. Therefore, according to the oxalic acid content in solution under different Pb stress (Figure 4A), the decrease of oxalic acid under high concentration Pb stress may be more attributed to the decrease of cell number of CZ-B1 than to Pb oxalate precipitation. However, the formation of Pb phosphate and Pb oxalate can reduce the Pb concentration in the environment, which is also one of the main mechanisms of CZ-B1’s resistance to Pb stress.

ATR-IR results showed (Figure 4B) that the cell sediment under Pb stress increased the peak position related to PO₄ ³⁻ at 565 cm⁻¹, 603 cm⁻¹, 965 cm⁻¹ (Botto et al., 1997; Wei et al., 2021). In addition, the O-C-O stretching vibration peak of 782 cm⁻¹ (Mancilla et al., 2009) and the C-O bond peak of 1289 cm⁻¹, 1312 cm⁻¹, 1386 cm⁻¹ were enhanced (Mancilla et al., 2009; Salavati-Niasari et al., 2009). O-C-O and C-O may be related to Pb oxalate (Mancilla et al., 2009). This result further proves that there are Pb phosphate compounds and Pb oxalate compounds in the cell sediments under Pb stress. In addition, the peaks representing amides and vibration peaks of saturated aliphatic (-CH) substances were observed at 1533 cm⁻¹, 1640 cm⁻¹ and 2930 cm⁻¹ (Doshi et al., 2007; Wang et al., 2020), indicating that the hydrocarbon functional groups and cellular proteins produced by CZ-B1 astress, which may be attributed topyromorphitelso participated in Pb²⁺ adsorption.

SEM images showed not only the presence of intact cells of CZ-B1 in the precipitate, but also the presence of obvious mineral grains (Figure 7). CZ-B1 cells are rod-shaped, about 2 μm, the surface is smooth (Figure 7A). Under Pb²⁺ stress, CZ-B1 cells appear deformation and shrinkage to varying degrees, and the cell morphology was significantly smaller, which indicates that Pb stress hinders the growth and development of CZ-B1 (Figures 7B, C). In addition, the individual CZ-B1 cell becomes smaller under the stress of 500 mg/L Pb²⁺, but the cell surface was smooth. The deformation of CZ-B1 under 1000 mg/L Pb²⁺ stress was more serious than that under 500 mg/L Pb²⁺ stress, and even broken cell remains were present in the precipitate (Figure 7C). In addition, EDX spectroscopy found that there were sharp particles with highly overlapped P and Pb signals around CZ-B1 cells, which may form pyromorphite. However, the pyromorphite signal was stronger under 1000 mg/L Pb²⁺, so more Pb phosphate mineral precipitation may be produced under the stress of high concentration of Pb. Combined with the OD values it can be confirmed that under high Pb²⁺ stress, the solubilization and utilization of P by CZ-B1 may be preferentially supplied to mineral precipitation rather than used by itself. The resistance mechanism of microorganisms under high stress may be dominant over the reproduction mechanism.

The CV curves of CZ-B1 cell precipitates showed no significant redox peak positions in the absence of stress (Figure 8A). The CV curves under 500 mg/L Pb²⁺ stress showed significant reduction peaks between −0.4 V and −0.2 V, but not significant oxidation peaks (Fig. B). When Pb²⁺ stress was increased to 1000 mg/L, the reduction peak position of the CV curve shifted to between −0.4 V and 0.0 V, and a clear oxidation peak appeared around −0.4 V (Figure 8C). The generation and loading of minerals can change the electron transfer capacity of cell (Zhang et al., 2021), so the shift in the redox peak position of the CV curve may originate from the Pb minerals generated by CZ-B1. In addition, there were no significant oxidation or reduction peaks on the CV curves of the three different treatments of EPS (Figure 8D, E, F). However, the EPS peak area of CZ-B1 increased significantly by 67.15%–73.16% under Pb stress, which suggests that EPS may assist CZ-B1 in the removal of Pb²⁺ through adsorption. In addition, the area of CV closure increased continuously with increasing Pb stress concentration. The enrichment of Pb²⁺ by EPS was beneficial to increase the extracellular electron transfer rate and accelerate the mineralization response of cells to Pb²⁺, which is consistent with Figure 8C and XRD results. Compared to the cells, the main change in the CV curve of EPS was the peak area rather than the redox peak position. Therefore, we speculate that EPS mainly acts as an enriched adsorption for Pb²⁺ rather than generating Pb minerals on it. It is worth mentioning that the mineralization and precipitation of cells to Pb²⁺ were improved precisely because Pb²⁺ was adsorbed around CZ-B1 cells by EPS.

In summary, the resistance and repair mechanism of CZ-B1 to Pb stress may be mainly from three aspects: 1) CZ-B1 can combine with Pb²⁺ to form phosphate, succinic acid and oxalate minerals via secreted citric, succinic and oxalic acids. 2) CZ-B1 can secrete tryptophan and other microbial products for complexation to enhance the absorption of Pb²⁺, thus weakening the stress of Pb²⁺ on itself. 3) CZ-B1 adsorbs and enriches Pb²⁺ through pericellular and surface EPS to increase the extracellular electron transfer rate, which can increase the reaction rate of CZ-B1 with Pb²⁺ mineralization and promote mineral precipitation.