The sample was prepared by sieving pine powder through a 100-mesh, washed with deionized water to remove impurities, and subsequently dried. Under the protection of nitrogen gas, it underwent thermal decomposition at a heating rate of 10°C/min until reaching 600°C for a duration of 2 h, resulting in the sample referred to as C. A solution containing 5.40 g FeCl₃·H₂O dissolved in 100 mL oxygen-free water with 30% anhydrous ethanol was prepared. Different loading materials and concentrations of nano zero-valent iron (nZVI) were synthesized by adding 1% carboxymethyl cellulose (CMC), varying quantities of biochar or bentonite. The solute mixture was thoroughly mixed by shaking at room temperature and at a speed of 150 r/min for 24 h. Subsequently, under a nitrogen atmosphere, sodium borohydride (NaBH₄) weighing 3.04 g was slowly added dropwise into the above mixture to form nZVI stabilized by CMC, biochar or bentonite (Supplementary Text S1 and Figure S1), named as nZVI/CMC, nZVI/C, nZVI/B, respectively.

To assess the removal efficiency of stabilized nZVI for Cr(VI) in aqueous solution, a concentration of 2 g/L of the aforementioned material was added to a solution containing Cr(VI) with an initial concentration of 200 mg/L. The reaction was conducted in a reactor operating at a rotation speed of 150 rpm and a temperature of 25°C for a duration of 72 h. Supernatant was regularly collected and filtered through a 0.22 µm filter. The concentration of Cr(VI) in the filtrate was determined by employing the diphenyl-carbohydrazide spectrophotometric method. Subsequently, several stable materials with the best effect were selected to establish isothermal adsorption experiments with initial concentration gradients of 10, 20, 50, 100, 200, 400, and 800 mg/L for Cr(VI). Various kinetic models, including the pseudo-first-order model, pseudo-second-order model, Avrami model and intraparticle diffusion model were employed to fit the adsorption process of Cr(VI), along with adsorption isotherm models such as Langmuir, Freundlich and Sips models.

The microstructure of synthesized materials above was imaged by scanning electron microscopy (SEM, Zeiss Sigma500). Also, the products were analyzed by X-ray diffraction (XRD, D8 Advance, Bruker, U.S.A) and data were collected the 2θ range from 10 to 90°. The functional groups on the surface of the materials were characterized by Fourier Transform Infrared Spectroscopy (FTIR, Thermo Scientific Nicolet iS20). In addition, the species of Cr and Fe on the surface of reaction products were analyzed by X-ray photoelectron spectroscopy (XPS, Escalab 250Xi, UK).

To assess the oxidative stress response of SL2 to stable nZVI, 1% spore SL2 suspension (10⁷ CFU/mL) and 1 g/L different materials (nZVI/B was not measured because of the poor growth of SL2) were introduced into Cr(VI) solution. The mixture was incubated in a shaker at 30°C and 150 rpm for 2 days. After centrifugation, the collected bacterial slurry was analyzed for intracellular ATP, reactive oxygen species (ROS), and glutathione (GSH) levels (Supplementary Text S2) as indicators of the oxidative stress reaction.

The morphology and size of the newly prepared stabilized nZVI were characterized by scanning electron microscopy (SEM). As depicted in Figure 2A, individual particles of nZVI appeared irregularly spherical and had a diameter of approximately 50 nm. After stabilization, the nZVI was uniformly dispersed on the surface of the carriers. The CMC stabilized nZVI exhibited a reticular dendritic structure and smaller particle size, due to the significant influence of high-concentration CMC solution on the nucleation of nZVI. Throughout the nanoscale particle growth process, CMC molecules adhere to the particle surface, impeding further growth through electrostatic repulsion and steric hindrance (He and Zhao, 2007). This preservation of smaller particle sizes for nZVI results in higher reactivity in reactions. In the EDS maps, each material exhibited elevated levels of Fe element, along with characteristic elements of the loaded material, such as C, Si and Al, indicating successful loading of iron onto the material. It is noteworthy that the iron element content on the surface of nZVI/CMC (89.4%) is significantly higher than that of biochar (53.28%) and bentonite (20.21%). This characteristic enables better preservation of the nZVI properties and facilitates the reactivity towards Cr(VI) reduction. To elucidate the stabilization mechanisms and gain further insight into the various functional groups of the nanoparticles, XRD, FTIR and XPS measurements were carried out on stabilized nanoparticles. From XRD pattern, a distinctive peak corresponding to Fe (0) is observed at 2θ = 45° (Bian et al., 2021; Zhou et al., 2022). The broad peak signified the body-centered cubic (bcc) Fe (0) crystal lattice plane (110) (Lin et al., 2010) with a relatively poor crystallinity (Gong et al., 2017), providing further evidence of the presence of iron in the material in the form of zero-valent iron. The types of surface functional groups on the nanoparticles were qualitatively analyzed using FTIR spectroscopy, the characteristic broad peaks near 3,400 cm⁻¹ related to the stretching vibration of -OH (Xu et al., 2020; Ji et al., 2022) were observed in all materials, potentially associated with iron hydroxides (Bian et al., 2021). Additionally, bands corresponding to C=O were identified in the range of 1,610–1,660 cm⁻¹, those acidic O-containing functional groups were reported to have a great effect on absorption and reduction of Cr(VI) (Wang K. et al., 2020). Particularly, nZVI/CMC revealed a carboxyl group (-COO) at 1,420 cm⁻¹, suggesting its role as one of the binding sites for Fe. To identify the carboxylate-metal complexation mechanism, the separation of the symmetric and asymmetric stretches [Δν = Δ(asym) − Δ(sym)] of the carboxylate group was calculated (Deng et al., 2021). In the present work, Δν was determined to be 189 cm⁻¹ (1,615–1,426 cm⁻¹), proved that bidentate bridging was the primary mechanism for binding CMC molecules to Fe nanoparticles, same as the previous research (He et al., 2007; Lin et al., 2010). Simultaneously, polydentate ligands were commonly reported to possess stronger chelating affinities compared to monodentate ligands (Su and Puls, 2004), this structural characteristic enhanced the stability of nZVI/CMC, underlining the increased robustness conferred by the multidentate coordination in this complex. It is noteworthy that oxygen-containing functional groups in the materials, such as C=O, -OH and -COO may concurrently contribute electrons, thereby facilitating the reduction of Cr(VI) (Zhao et al., 2022). The implication is that the O functional groups may play a crucial role as electron transfer stations in the Cr-Fe reaction.

Further, XPS was performed for CMC-nZVI before and after Cr(VI) uptake (Figure 3). The XPS spectra before the reaction confirmed the presence of zero-valent iron in the material by a characteristic peak of Fe2p with a binding energy of 706.23 eV (Ren et al., 2018), accounting for 30.21%. Another portion of Fe, likely oxidized upon exposure to air during transportation and storage, formed Fe(II), constituting 69.79% (Yamashita and Hayes, 2008). After reaction, the peaks corresponding to Fe (0) and Fe(II) disappeared, and a characteristic satellite peak of Fe(III) emerged (Liu et al., 2020), indicating the complete oxidation of iron in the material to trivalent state. Additionally, the results of the Cr elemental partition spectra revealed that the Cr in the reaction product existed mainly in the valence state of Cr(III), including chromium oxides and hydroxides. Therefore, both Fe (0) and Fe(II) in the material directly serve as electron donors for the reduction of Cr(VI) to Cr(III). Subsequently, these Cr(III) species co-precipitate with Fe³⁺, forming Cr_nFe_1−n (OH)₃ (Pan et al., 2016; Ye et al., 2021). Moreover, upon contact with water, zero-valent iron generated H*, which was confirmed to be the predominant reactive species for Cr(VI) reduction (Qian et al., 2014). Besides, the abundant -OH and -C=O groups on CMC play a crucial role as electron donors for the reduction of Cr(VI) (Rajapaksha et al., 2018; Xu et al., 2019). In conclusion, the analysis confirmed that the removal of Cr(VI) by nZVI/CMC involved both adsorption and reduction processes, consistent with previous study (Fang et al., 2011).

In summary, the potential electrostatic repulsion and spatial hindrance capabilities of CMC contributed to maintaining the small size of nZVI particles, preventing its easy aggregation. Additionally, the combination of nZVI with CMC was achieved through a more stable bidentate bridging mechanism. Furthermore, it revealed that both Fe (0) and Fe(II) contributed to the reduction of Cr(VI), and the presence of oxygen-containing acidic functional groups suggested the potential for electron donation in the reduction of Cr(VI).

Through the assessment of intracellular ATP, GSH, and ROS concentrations, we compared the toxicity of nZVI with different stabilization methods on SL2. ATP, which is known to play a crucial role in cellular metabolism, and its concentration reflects the cellular activity (Tsitonaki et al., 2010; Wu et al., 2013). In the experimental group where only SL2 was added, the intracellular ATP content was highest, while the ATP levels significantly decreased in the groups with added materials, indicating a certain toxicity of the materials to SL2, affecting its metabolism. In comparison, the ATP content in the nZVI/CMC experimental group (0.5981 μmol/g) was higher than that in the nZVI (0.1655 μmol/g) and nZVI/C (0.2532 μmol/g) groups, suggesting that the encapsulation of CMC reduced the toxicity of nZVI to SL2.

nZVI exhibits considerable oxidative power in oxygen-containing water (Cheng et al., 2016). It can be oxidated to generate Fe(II) and H₂O₂ (Kim et al., 2011), followed by Fenton reaction to produce highly toxic ROS, such as ·OH and ·O₂ ⁻ (Keenan and Sedlak, 2008; Xu et al., 2022). The reaction equations are as follows (Eqs 8-11): F e 0 + O 2 + 2 H + → F e I I + H 2 O 2 (8) F e 0 + H 2 O 2 + 2 H + → F e I I + 2 H 2 O (9) F e I I + H 2 O 2 → F e I I I + · O H + 2 O H − (10) F e I I + O 2 → F e I I I + · O 2 − (11)

Typically, ROS produced at low frequencies are easily neutralized by antioxidant defenses (Nel et al., 2006). However, excessive ROS can overwhelm cellular antioxidant defenses, causing membrane damage (Xia et al., 2020), DNA fragmentation (Xia et al., 2019), and even cellular inactivation (Nel, 2005). Therefore, cellular oxidative stress responses were further investigated by assessing intracellular levels of ROS and GSH to elucidate the molecular mechanisms. As depicted in the Figure 4, the ROS content in the nZVI-treated group exhibited a dramatic increase (5376 u/s/g), surpassing that of other groups (819, 387 and 361 u/s/g respectively). This elevation suggested a potential dual effect: an increase in endogenous ROS production within the cells and a possible disruption of cell structures (Liu et al., 2011; Mao et al., 2019) allowing enhanced penetration of exogenous ROS (Li et al., 2022), leading to a substantial concentration surge. Simultaneously, the ROS content in the nZVI/CMC and nZVI/C remained comparatively lower, indicating a certain degree of mitigation of cellular oxidative stress reactions. From the perspective of GSH content, CMC-stabilized nZVI generated a significant amount of antioxidant enzymes to shield itself from external oxidation, thereby maintaining lower ROS levels. Although the nZVI-treated group also exhibited elevated GSH levels, it proved insufficient to eliminate the excessive intracellular ROS, resulting in an imbalance in the cellular redox state and compromising the cell’s self-protective capacity.

Taken together, the results presented herein indicate a significant enhancement in surface reactivity after CMC-coating, accompanied by a concurrent reduction in the nanomaterial’s toxicity towards SL2. This aligns with findings from previous studies conducted on Escherichia coli (Zhou et al., 2014). We hypothesize that the detoxifying effect of this CMC-coating can be attributed to three key factors: Firstly, its role as a scavenger of free radicals, leading to the substantial elimination of ·OH radicals (·OH + CMC → H₂O+ CMC^*) (Joo and Zhao, 2008); Secondly, its ability to prevent direct contact between SL2 and nZVI particles (Dong et al., 2016), thereby mitigating oxidative stress reactions in cells and ensuring their viability; Thirdly, CMC can provide an organic carbon source for the growth of SL2 (Wang ZY. et al., 2020).

In order to investigate the application potential of nZVI/CMC@SL2 in reducing Cr(VI) in soil, we conducted remediation experiments using soils from different types of contaminated sites. The TCLP method was employed to assess the leaching risk of Cr, and the changes in the total Cr(VI) content in the soil were also measured. As shown in Figure 5, after a 3-day remediation period, a significant reduction in TCLP-Cr(VI) was observed in soils amended with 0.4% nZVI/CMC. For NM, ZH, and ZL soils, the Cr(VI) concentration decreased from 28.6, 7.05, and 3.21 mg/L to less than 0.5 mg/L. However, a slight rebound was observed after 15 days, potentially attributed to the oxidation of ferrous species (Wang et al., 2023), leading to a loss of remediation capacity. Notably, the rebound was less pronounced in the presence of nZVI/CMC@SL2, indicating that the addition of SL2 could inhibit the oxidation of Cr(III). Furthermore, after 30-days remediation, the Fe(II) concentration in the nZVI/CMC@SL2 treatment group was significantly higher than in the group that only received nZVI/CMC (Figure 6). This observation indicated that SL2 could effectively facilitate the reduction of Fe(III) to Fe(II), thereby sustaining the reduction of Cr(VI). According to previous studies, oxalic acid was the most abundant LMWOA produced by strain SL2, and the affinity of oxalic acid with Cr(VI) leads to the expansion of Cr(VI) coordination from tetrahedron to hexahedron, which was preferable for hexahedral species of Cr(III). Oxalic acid was used as a substitute electron donor for Cr(VI) reduction through intramolecular electron transfer reaction, which significantly improved the Cr(VI) removal efficiency (Jiang et al., 2017). Furthermore, nZVI promoted oxalic acid secretion by SL2, and its generated iron ions could accelerate the reduction of Cr(VI) by oxalic acid (Hug et al., 1997). Studies have detected the formation of compounds containing Fe(II), Cr(V), and oxalate salts (HCrFeC₄O₉)(Luo et al., 2023), providing crucial evidence for the involvement of Fe(II)/Fe(III) cycling in organic acid reduction of Cr(VI). In the case of the reaction group with only SL2 added, the removal efficiency for Cr(VI) increased with prolonged incubation time within the 30-day period, suggesting that SL2 successfully colonized and persisted in the soil, continuously fixing Cr(VI) through biological processes, in accordance with SL2 biomass change (Supplementary Figure S4). For all nZVI/CMC@SL2 composite systems, the treatment groups achieved a removal efficiency of over 99.5% for TCLP-Cr(VI) after 30 days, and the TCLP-Cr(VI) was lower than the level IV standard of groundwater quality criterion in China (0.1 mg/L, GB/T 14848–2017). In addition, the total Cr(VI) in the NM soil decreased from over 600 mg/kg to 16 mg/kg, which was below the regulatory limit for Class I construction land specified in GB36600-2018 (30 mg/kg). In heavily contaminated soil (ZH, Figure 5E) with elevated concentrations of Cr(VI), the growth of SL2 was suboptimal due to the toxic effects of Cr(VI), therefore basically did not demonstrate any removal effectiveness against Cr(VI). However, with the addition of nZVI/CMC, the pollutant concentration was reduced to a level conducive to growth. This combination successfully decreased the total Cr(VI) content from around 1,200 mg/kg to approximately 300 mg/kg within a 30-day period.

This study evaluated sequential extraction procedures for identifying the relative availability of soil-bound heavy metal by partitioning the particulate trace metals into five fractions. The five species have been defined as exchangeable (F1), bound to carbonates (F2), Fe-Mn oxides-bound (F3), organic matter-bound (F4) and residual (F5). Figure 7 showed the transformation in chromium components in different treatment group which aligned with the earlier leaching results. After a 30-day treatment period, the exchangeable Cr content in all soils significantly decreased, undergoing transformation into Fe-Mn oxide-bound and the organic-bound forms. According to previous investigations, the elevated Fe-Mn oxides-bound fraction might be largely attributed to the precipitation of Cr(OH)₃ or Cr(III)/Fe(III) oxides/hydroxides (Cr_nFe_1−nOOH and Cr_nFe_1−n (OH)₃) during the CMC-nZVI remediation (Manning et al., 2007). Specifically, 17.82% of exchangeable chromium in the NM soil was completely transformed into other more stable forms of Cr. According to Supplementary Figure S5, after 7 days of treatment, noticeable changes in pH were observed in the experimental groups. The group with the addition of nZVI/CMC exhibited the highest pH increase, rising from 8.70, 8.55, and 8.45 to 8.93, 8.72, and 8.70, respectively. This phenomenon may be attributed to the alkaline nature of the material and the reduction of Cr(VI) acting in concert. In the experimental group with the addition of SL2, the pH initially showed a decline followed by an upward trend, gradually stabilizing. This suggested that SL2 proliferated extensively in the early stages, producing acidic substances, and later reached a stable state. Due to the natural buffering capacity of the complex soil system, the fluctuation range of pH during the remediation process remained relatively small.

In short, the results indicated the excellent stabilization and environmental adaptability of the nZVI/CMC@SL2 composite remediation system towards chromium. It promoted the conversion of more easily available Cr (EX and CB) to the less available (OX and OM) and thus reduce the toxic effect of Cr. Furthermore, SL2 possessed the ability to regenerate Fe(II), facilitating the sustained reduction of Cr(VI).

After 30 days of treatment, the quantity of SL2 in the nZVI/CMC@SL2 treatment group gradually stabilized and maintained at a high level (4.23 × 10⁴ CFU/mL, Supplementary Figure S4). Microbial community analysis of the ZL soil revealed an average of 81,938 quality sequences and 740 OUTs generated from the ITS1 gene. The fungal community’s similarities and differences were analyzed based on OTUs using a Venn diagram (Figure 8A). There were 353 shared OTUs, constituting 47.7% of the total observed OTUs (740), with 296 and 91 unique OUTs in the CK and treatment groups, respectively. This indicates that different treatments cultivated distinct microbial populations. Comparing with the CK, the treatment group exhibited a slight reduction in OUT levels, suggesting that this composite system could influence the fungal abundance in the soil. In terms of alpha diversity, both fungi and bacteria were significantly affected (Supplementary Table S6). In the CK group, the three most abundant fungi were Ascomycota (68.66%), Basidiomycota (6.65%), and Mortierellomycota (4.03%). In the experimental group, the relative abundance of Ascomycota surged to 98.09%. As SL2 belongs to Ascomycota, its relative abundance increased from 4.75% to 87.18% at genus level, further confirming the successful colonization of SL2 in the soil and its competition with indigenous microorganisms to become the dominant strain (Figure 8). Looking at the bacterial community (Supplementary Figure S6), there was a significant increase in the relative abundance of Proteobacteria and Firmicutes, rising from 38.70% to 10.68%–55.84% and 35.47%, respectively. Meanwhile, Actinobacteria decreased substantially (38.66%–7.35%). Proteobacteria was often found as a dominant species in chromium-contaminated soil (Desai et al., 2009), with Bacillus within this phylum known to possess Cr(VI) reductase activity, capable of resisting and reducing high concentrations of Cr(VI) (Elangovan et al., 2006; Desai et al., 2008). Firmicutes harbored various heavy metal resistance genes, exhibiting stronger adaptability to heavy metal-polluted environments. Also, they are reported typically to be Fe(III)-reducing bacteria (FeRB)(Jin et al., 2023), thus regenerating Fe(II) and reduce Cr(VI).

In conclusion, the nZVI/CMC@SL2 composite system enabled the successful colonization of SL2 in soils highly contaminated with Cr(VI). It demonstrates the ability to enrich chromium-tolerant and chromium-removing microorganisms, thereby promoting the reduction of Cr(VI).