In recent years, with population growth and industrial development, the use of pesticides and fertilizers and the large-scale exploitation of mineral resources have led to an increase in emissions of heavy metal pollutants, which enter agricultural soil ecosystems through dust and precipitation (Huang et al., 2019; Hossain Bhuiyan et al., 2021; Qin et al., 2021). Most of the heavy metal ions entering the soil are not degraded by microorganisms, but are either enriched in the soil or concentrated by biological action and accumulated in the food chain, ultimately reducing vegetable yields and threatening human health (Nagajyoti et al., 2010; Saadani et al., 2016; Rouillon et al., 2017). Lead (Pb) is one of the main metal elements polluting arable land worldwide (Kushwaha et al., 2018). Vegetables can accumulate heavy metals through root uptake, and excessive Pb accumulation in vegetables offers a significant risk to human health (Pelfrêne et al., 2013). In China, enhanced heavy metal concentrations is an increasing problem associated with rapid industrialization and agricultural intensification, with lead concentrations in agricultural soils of China ranging from 7.6 to 77.27 mg kg⁻¹ (Bi et al., 2018; Shifaw, 2018; Huang et al., 2019). Irreversible damage to the human body caused by excessive Pb accumulation can cause respiratory, cardiovascular, neurologic, digestive, and urinary diseases (Boskabady et al., 2018). In order to minimize the accumulation of heavy metals in vegetables, eco-friendly, economical, and energy-efficient procedures are urgently required.

Physical and chemical approaches for remediating soils contaminated with metals have limitations, including the fact that they modify soil microbial communities, which in turn changes soil characteristics, is time-consuming and expensive, and creates secondary pollutants (Tangahu et al., 2011; Yao et al., 2012; Zubair et al., 2016). These methods can only change the form of the problem and cannot completely eliminate the contaminants (Gomes et al., 2016). As a result, the in situ application of heavy metal-immobilizing bacteria, a new soil immobilizer, has been widely employed to encourage plant growth and minimize metal absorption of plants by forming indole acetic acid (IAA), siderophores, and reducing metal efficacy in soil (Ma et al., 2011; Wang et al., 2016; Han et al., 2018, 2020; Wang et al., 2020). Bacteria can expel heavy metals through permeable barriers or active export, detoxify them through chemical modification, and physically sequester them with extracellular polymers that bind metals, such as liposaccharides and exopolysaccharides (EPS) (Rouch et al., 1995; Guibaud et al., 2003; Morcillo and Manzanera, 2021).

The absorption and immobilization of heavy metals by EPS are carried out through different mechanisms such as ion exchange, complexation, precipitation etc. (De Philippis et al., 2011; Gupta and Diwan, 2017). Previous studies have suggested that EPS-producing bacteria could effectively reduce the bioavailability of soil metals and alleviate stressed conditions, thereby promoting plant growth and health (Karthik et al., 2017; Mukherjee et al., 2019; Zainab et al., 2020). For example, B. xiamenensis PM14 and Bacillus gibsoni PM11, two EPS-producing bacteria, improved nutrient availability and development of Linum usitatissimum by reducing the stress conditions caused by metals (Zainab et al., 2020). According to Karthik et al. (2017), Cellulosimicrobium funkei AR6 produced more EPS when Cr (VI) concentration increased (from 0 to 250 μg/mL), demonstrating the function of EPS as a defensive mechanism for reducing heavy metal stress in rhizospheric soils of plants. Notably, Mukherjee et al. (2019) observed that Halomonas sp. Exo1 exopolysaccharide production alleviated heavy metal stress through EPS-mediated biosorption in rice exposed to arsenic (2 mM). In addition, Pseudomonas. sp. MHR6, an EPS-producing bacterium, has been shown to decrease the adverse effects of salinity stress on plant growth by enhancing seedling growth, antioxidant levels, the osmotic regulator, and chlorophyll accumulation (Liu et al., 2022).

EPS secretion is an efficient biosorbent material essential for many marine microbes (Baria et al., 2022). Because they have evolved unique protective mechanisms and metabolic pathways to survive in harsh conditions, marine and marine extremophilic bacteria (those that can survive in extreme conditions of temperature, pressure, or the presence of toxic elements like heavy metals) have a greater capacity than other bacteria to produce unique bioactive compounds like EPSs (Casillo et al., 2018; Parrilli et al., 2021). Because of their strong affinity to heavy metals, certain EPSs that are generated by marine bacteria have found widespread application in the treatment of wastewater to remove heavy metals (Siddharth et al., 2021; Baria et al., 2022). As an illustration, the EPS made by Cyanothece sp. ATCC 51142 is particularly efficient in removing several metals from industrial wastes (Shah et al., 2000). Despite the fact that rhizobacterial EPS has the potential to reduce the negative effects of heavy metal stress on plants, very little is known about the impact of heavy metals on the composition of EPS and, as a result, the effects of this on root colonization by high-yielding EPS-producing marine bacteria and their positive impact on plant growth and health (Nocelli et al., 2016; Morcillo and Manzanera, 2021). The impacts of bacteria that produce EPS on the plant rhizosphere have seldom been studied, including the direct effects of bacteria on roots and indirect effects on the composition of soil bacterial communities, which are susceptible to soil Pb contamination (Chen et al., 2018). Pseudoalteromonas agarivorans Hao 2018, a high-yielding EPS-producing marine bacterium, was generated from the microbial membrane that is found on the surface of abalone seedlings, and previous studies have shown that its EPS production exhibits antioxidant activity, hygroscopicity, moisture retention, and excellent removal of Cu²⁺ and Pb²⁺ (Wang et al., 2018; Hao et al., 2019).

Pakchoi (Brassica chinensis L.) is extensively cultivated in China and accounts for 19% of its total production of leafy vegetables (Liu et al., 2010). Pakchoi is also commonly cultivated throughout the world and is easily contaminated with heavy metals when planted on farms (Liu et al., 2019). In this investigation, the ability of the high EPS-producing bacteria P. agarivorans Hao 2018 to produce EPS and absorb Pb was assessed. Additionally, the impacts of the strains on the development, quality, and Pb uptake of edible pakchoi tissues in soil contaminated with Pb were examined. Finally, the bioavailable Pb, and the relative abundance of bacteria producing EPS, as well as the structure and function of bacterial communities in rhizospheric soils, were also evaluated. The results contributed to a better understanding of the functions of high-yielding EPS bacteria in the soil microbial community and Pb immobilization and offered a theoretical basis for the employment of EPS-producing bacteria in the safe growth of plants in soil contaminated with heavy metals.

Reducing the bioavailability or mobility of heavy metal ions is very important to reduce the harm caused by their absorption and accumulation by vegetables (Khan et al., 2015; He et al., 2019). Microbial immobilization of heavy metals in soil is an economical and environmentally friendly way to reduce plant absorption of heavy metals (Ma et al., 2011; Han et al., 2020). Here, we explored the effects of the EPS-producing bacterium Hao 2018 on the immobilization of Pb in soil filtrate, plant growth, and Pb accumulation in pakchoi. The results of this study suggest that Hao 2018 effectively reduced the concentration of Pb (16–75%) in soil filtrate, and its EPS production increased in the presence of Pb²⁺. Additionally, EPS-producing bacteria Hao 2018 affected Pb bioavailability in rhizospheric soil, the results showed that strain Hao 2018 could effectively reduce the content of DTPA-extractable Pb in the rhizospheric soil, resulting in the reduction of Pb²⁺ absorption by the roots and edible tissues of pakchoi. Furthermore, strain Hao 2018 could effectively increase the biomass, Vc, and soluble protein content of pakchoi (Figure 3). Studies have shown that high concentrations of Pb²⁺ can affect the rooting and germination of seeds, thereby inhibiting photosynthesis and mineral absorption of the aboveground parts (Shen et al., 2016). Therefore, inoculation with Hao 2018, which is highly effective at immobilizing Pb²⁺ in soil, had a positive effect on the growth and development of pakchoi.

Nitrogen is an essential macronutrient that is often a limiting factor for normal plant growth (Giansoldati et al., 2012). Plants mainly absorb N in two forms, nitrate (NO₃⁻) and ammonium (NH₄⁺), which can be easily absorbed, thus increasing their biomass (Taiz et al., 2015). Similarly, Hao 2018 inoculation improved the biomass and quality of pakchoi in the presence of Pb pollution, which may be related to the increase in NH₄⁺-N and NO₃⁻-N content in rhizospheric soil. Previous studies have also found that the As-tolerant strain Halomonas sp. Exo1 promoted rice growth under metal As stress primarily through EPS secretion (Mukherjee et al., 2019).

Nitrogen (N) management is an agronomic strategy designed to prevent crops against metal contamination (Luo et al., 2012; Mao et al., 2014). Some scholars believe that NO₃⁻ absorption by plants is accompanied by proton (H⁺) absorption, leading to an increase in rhizosphere pH (Thomson et al., 1993). The basic function of the nitrate transporter (NRTs) gene is NO₃⁻ absorption. Zhu et al. (2019) found that Pb²⁺ induced the expression of the NRT1.1 gene and its encoded protein under co-supply of NO₃⁻ and NH₄⁺, thereby promoting NO₃⁻ uptake, increasing rhizosphere pH, and reducing Pb availability, and thus reducing Pb uptake in Arabidopsis. Many studies have suggested that the inoculation of soil with heavy metal-immobilizing bacteria increases soil pH and thereby reduces the availability of heavy metals (Madhaiyan et al., 2007). According to our study, the EPS-producing bacterium Hao 2018 contributes to a substantial increase in the supply of NH₄⁺ and NO₃⁻ to the rhizospheric soil and its pH, thereby reducing the level of DTPA-extractable Pb in the rhizospheric soil and the uptake of Pb by pakchoi.

The activity of soil enzymes is an essential measure of the health of soil ecosystems, especially those that have been affected by heavy metal pollution (Masto et al., 2009). Urease and alkaline phosphatase activities in soil are linked to the conversion of soil nitrogen and phosphatase, whereas dehydrogenase activity are crucial to soil respiration. Pb pollution disturbed the biochemical balance of soils in our study as evidenced by the inhibition of soil dehydrogenases (SDH) and alkaline phosphatase (ALP) activity and the stimulation of soil urease (URE) activity. Previous studies have also reported complex effects of heavy metals on soil enzyme activity (Khan et al., 2010; Pan and Yu, 2011; Wang et al., 2020). The increased enzyme activity in Hao 2018 inoculated-rhizospheric soil might imply that it may contribute to improving the soil biochemical reaction and thus improve plant growth. Similar to our research, Wang et al. (2020) found that enzyme (ALP, URE and SDH) activity and plant biomass significantly increased in Cd-contaminated rhizospheric soils inoculated with the γ-PGA-producing bacteria Bacillus amyloliquefaciens W25 and Bacillus subtilis W7. PCA analysis further verified the positive effect of Hao 2018 on physical and biochemical responses, rhizospheric soil chemical properties, and the enzyme activity of pakchoi under non- and Pb contamination.

Rhizospheric bacteria can alter metal bioavailability by modifying the pH of the surrounding environment, creating chelating agents, and altering redox potentials, which increases the efficiency of microbial bioremediation (Giller et al., 1998). In this study, the composition of the rhizosphere microbiome was affected not only by metal contamination but also by soil microbial modification, and metal contamination resulted in a significant decrease in microbial diversity. Furthermore, the results showed that the α-diversity of the Hao 2018-inoculated metal-contaminated rhizospheric soil was significantly higher than that of the control under high Pb pollution (Supplementary Table S2), indicating the recovery of microbial communities after Hao 2018 inoculation and the effectiveness of the remediation measures we adopted. Previous studies have also found that bacterial phyla including Proteobacteria, Chloroflexi, Acidobacteria, and Bacteroidetes have consistently been found to dominate metal-contaminated soils, most likely because of their high tolerance to metals (Jiang et al., 2016; Chen et al., 2019). These microbial communities colonize the rhizosphere due to their functional properties and effects on plant nutrient uptake, growth, and disease suppression (Yadav et al., 2020). The three phyla with the highest abundance in natural soils are Proteobacteria, Bacteroidetes, and Acidobacteria (Roesch et al., 2007), and they are believed to be linked to soil carbon and/or nitrogen cycles (Tang et al., 2011; Rawat et al., 2012; Chen et al., 2018). Researchers further point out that the bacterial genera belonging to Proteobacteria and some Firmicutes are most capable of dissolving inorganic forms of phosphate into effective forms (Selvakumar et al., 2012; Rana et al., 2019). The results of this study showed that Pb contamination reduced the abundance of Proteobacteria, Firmicutes, Chloroflexi, Acidobacteria, and Bacteroidetes in rhizospheric soils. Similar to our results, previous studies have found that metal contamination reduced the abundance of Proteobacteria, Chloroflexi, and Bacteroidetes in soil (Berg et al., 2012; Narendrula-Kotha and Nkongolo, 2017). We also observed a general increase in the abundance of Proteobacteria, Firmicutes, Chloroflexi, Acidobacteria, and Bacteroidetes in the rhizospheric soils inoculated with Hao 2018 compared with control soils under metal-contaminated conditions (especially at 50 mg L⁻¹ Pb²⁺) (Figures 6B, 7A–E), which consistently indicate a reduction in metal toxicity in the rhizosphere of pakchoi, as discussed previously. These results suggest that soil inoculated with Hao 2018 caused changes in microbial communities in metal-contaminated soils, making them more similar to uncontaminated soils. When soils with high Pb contamination were inoculated with Hao 2018, the relative abundances of the phyla BRC1, Entotheonellaeota, Nitrospirae, and Gemmatimonadetes increased dramatically (Figure 6B). Heavy metal resistance has been attributed to these phyla because of their role in C/N cycling activities such as urea hydrolysis, denitrification, nitrification, calcite precipitation, and EPS production (Zhang et al., 2021).

More importantly, the relative abundance of Streptomyces, Sphingomonas, Nocardioides, Bacillus, Gaiella, and Solirubrobacter was high in the Pb-contaminated rhizospheric soils inoculated with Hao 2018. Members of those genera were usually found to be metal-tolerant species and/or plant growth-promoting bacteria (PGPB) (Tangaromsuk et al., 2002; Dimkpa et al., 2008; Rodríguez-Tirado et al., 2012; Khan et al., 2014; Mahbub et al., 2016; Tan et al., 2017; Kou et al., 2018; Wang et al., 2020; Geng et al., 2022). In particular, the genera Sphingomonas, Streptomyces, and Bacillus are both metal-immobilizing and plant growth-promoting (So et al., 2001; Tangaromsuk et al., 2002; Dimkpa et al., 2008; Kumar et al., 2012; Rodríguez-Tirado et al., 2012; Khan et al., 2014; Tan et al., 2017). In addition, the presence of Hao 2018 might be linked to an increase in antibiotic-producing microorganisms in plant roots, and an improved plant immune system as a result of Lysobacter enrichment (Puopolo et al., 2016). Moreover, Guo et al. (2020) found that soil alkaline phosphatase activity (ALP) was significantly correlated with the relative abundance of Lysobacter, suggesting that this genus may be involved in the response of soil alkaline phosphatase to Pb addition. These findings show the feasibility of employing this EPS-producing bacterium in soil metal remediation by demonstrating an increase in microbial diversity, a reduction in metal bioavailability, and a reorganization of microbial composition in rhizospheric soil following inoculation of Hao 2018.

This research demonstrated that the EPS-producing bacteria Hao 2018 immobilized Pb by increasing its production of EPS and raising the pH of soil filtrate. Hao 2018 inoculation reduced Pb content in rhizospheric soil and Pb accumulation in pakchoi and increased its biomass, Vc, and soluble protein content. It is worth noting that the Pb content of the edible tissue of pakchoi inoculated with strain Hao 2018 reached the permissible limits of leafy vegetables stipulated by FAO/WHO (2011). Based on the results of this study, it is clear that Hao 2018 has the potential to decrease soil Pb availability and alleviate Pb stress by elevating soil pH, soil enzyme (ALP, URE, and SDH) activity, nitrogen content (NH₄⁺-N and NO₃⁻-N), and the relative abundance of bacteria that promote metal immobilization and plant growth, such as Streptomyces and Sphingomonas. The results of the study highlighted that the EPS-producing bacteria Hao 2018 has great potential for microbial application in remediating Pb-contaminated soils and mitigating heavy metal content in plants. Further research including field trials at metal-contaminated sites is needed to directly assess the in situ remediation capability of Hao 2018.

The data presented in the study are deposited in the National Center for Biotechnology Information (NCBI) BioProject repository, accession number PRJNA906320.

RC and YiZ conducted the whole experiment, did the analysis, and wrote the manuscript. RC and LH designed the study and revised the manuscript. LH provided the research grants for this study and also supervised the work. YJ, WeiW, YaZ, NL, GZ, and XW participated in part of the experiment. XX, CD, YL, HY, KS, CH, WeiyW, LZ, and CJ contributed to the data analysis. All authors contributed to the article and approved the submitted version.

This research was supported by the Natural Science Foundation of Shandong Province (ZR2022MD097), Education and Industry Integration Innovation Pilot Project of Qilu University of Technology (Shandong Academy of Sciences) (2022JBZ01-06), the Foundation of State Key Laboratory of Biobased Material and Green Papermaking (No. ZZ20190302), the Foundation of Shandong Provincial Key Laboratory of Biosensors (SWCG 2018–01), the Natural Science Foundation of Shandong Province (ZR2012CM019), and the Foundation (No. 202002) of Qilu University of Technology of Cultivating Subject for Biology and Biochemistry, Science, Education and Industry Integration Innovation Pilot Project of Qilu University of Technology (Shandong Academy of Sciences) (2020KJC-ZD08).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.