Reduction of Cr(VI) by Bacillus toyonensis LBA36 and its effect on radish seedlings under Cr(VI) stress

Soil sample

The soil used for microbial isolation was obtained from the molybdenum and lead mining area in Luanchuan County, Henan Province (111°54′E, 33°82′N) (Fig. 1). The soil texture was sandy loam with a pH of 6.5, and the heavy metal content of the soil is shown in Table 1.

Isolation, screening and identification of Cr(VI)-resistant bacterial strain

One gram of soil was used to prepare a 100 mL soil suspension, followed by magnetic stirring and settling. Five milliliters of the supernatant were collected and added to Luria-Bertani (LB) broth containing 30 mg·L⁻¹ Cr(VI) (K₂Cr₂O₇), and incubated in shaking conditions at 30 °C and 150 r·min⁻¹ to obtain a bacterial suspension. Bacterial suspension (1 mL) was spread onto LB agar containing 30 mg·L⁻¹ Cr(VI) (30 °C, 24 h). Individual colonies that appeared were selected for cultivation. This process was repeated multiple times until the Cr(VI)-resistant bacterial strain was identified.

The DNA of the strain was extracted using the boiled template method, while the amplification of the 16S rDNA gene was conducted using polymerase chain reaction (PCR) with primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACG A-3′) (Zhang et al., 2022). The temperature cycling program was configured to preheat at 94 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 90 s. This cycling process was repeated 30 times, followed by a final extension at 72 °C for 10 min (Ji et al., 2018). The PCR amplification system is shown in Table 2. The PCR products were sequenced, and the results were compared with the data from the National Center for Biotechnology Information database and EZBioCloud database to construct a phylogenetic tree (Loong et al., 2016).

Minimum inhibitory concentration, chromium reduction, and adsorption experiments

The LBA36 strain was inoculated in LB medium at concentrations of 0, 30, 60, 90, 120, and 150 mg·L⁻¹ and incubated for 88 h. The OD_600nm value of the culture solution was measured at regular intervals to determine the minimum inhibitory Cr concentration (MIC) of the LBA36 strain. Three replications were set up for each set of experiments.

LBA36 strain was cultured in medium containing Cr(VI) 30, 60, and 90 mg·L⁻¹ to investigate the removal of heavy metals by LBA36 strain at different Cr(VI) concentrations (the minimum inhibitory concentration experiments showed that the LBA36 strain had different growth characteristics under Cr(VI) stress of 30, 60, and 90 mg·L⁻¹). Cr(VI)-containing medium without strain inoculation was used as a negative control. Three parallel groups were set up for each concentration. Samples were collected, incubated for 32 h (8,000 r·min⁻¹, 10 min) and then centrifuged, and 1 mL of supernatant was extracted for digestion. The LBA36 strain was cultured in medium containing Cr(VI) 30 mg·L⁻¹ to explore the effect of time on Cr(VI) removal by LBA36 strain. Cr(VI)-containing medium without strain inoculation was used as a negative control. The incubation period was 56 h and samples were taken every 8 h. Three replications were set up for each set of experiments.

Total chromium quantification can only be performed using a digested supernatant solution (Dong et al., 2022). The supernatant underwent digestion using the electrothermal plate method (Dong et al., 2022). One milliliter of the collected supernatant was transferred into a polytetrafluoroethylene crucible, to which 5 mL of HCl was added. Low-temperature (110 °C) digestion was conducted on an electric heating plate until the volume was reduced to 1–2 mL. Subsequently, 5 mL of HNO₃ was introduced for high-temperature (160 °C) digestion, and once the volume reached 1–2 mL, the temperature was lowered for acid evaporation until complete sample digestion was achieved. The digested sample was then dissolved in a 50 mL volumetric flask, supplemented with 1 mL of HNO₃ to ensure a 2% acidity level, and finally brought to volume with ultra-pure water (50 mL). Thus, total chromium content was measured in the digested solution using an inductively coupled plasma spectrometer (ICPS) (Astatkie, Ambelu & Beyene, 2021). The Cr(VI) content of the samples was determined spectrophotometrically using dibenzoyl dihydrazide (Ribas et al., 2022). The reduction rate of Cr(VI) by the strain was calculated with the following formula:

(1) $${\mathrm{q}}_1={\displaystyle \frac{\left({\mathrm{q}}_2-{\mathrm{q}}_3\right)}{{\mathrm{q}}_2}}$$where q₁ is the reduction efficiency of Cr(VI) by the strain; q₂ is the supernatant Cr(VI) content of the control group; and q₃ is the supernatant Cr(VI) content of the experimental group.

The adsorption rate of total chromium by the strain was calculated with the following equation:

(2) $${\mathrm{Q}}_1={\displaystyle \frac{\left({\mathrm{Q}}_2-{\mathrm{Q}}_3\right)}{{\mathrm{Q}}_2}}$$where Q₁ is the adsorption efficiency; Q₂ is the total chromium content in the supernatant of the control group; and Q₃ is the total chromium content in the supernatant of the experimental group.

Assessment of the reducing properties of various cellular components

Extracellular and intracellular materials and bacterial debris of the bacteria were extracted, and they were subsequently divided into resting and permeable cells based on treatment methods (Han et al., 2020). The isolated bacterial strain was cultured for 32 h. The culture fluid was then aspirated and centrifuged (6,000 r·min⁻¹, 4 °C, 20 min). The resulting supernatant was collected as the extracellular material. The untreated supernatant sample was termed the “resting sample”. The supernatant to which 1% toluene and 0.2% Triton-x100 was added was called the “permeabilized sample” (Megharaj, Avudainayagam & Naidu, 2003). The intracellular and cellular debris samples were generated as follows: the extracellular supernatant sample was broken by ultrasonication in an ice bath (20 s on, 30 s off, 200 W, 20 min duration), and the broken sample was centrifuged at 4 °C, 12,000 r·min⁻¹ for 30 min, and the supernatant was collected after centrifugation as the intracellular material (intracellular extract) along with cellular debris. The cell debris was suspended in 10 mL of 25 mM Tris-HCl buffer (pH = 7.0). Tris-HCl buffer (10 mL, 25 mMol, and pH = 7.0) was used as a blank instead of cell fractions. One milliliter each of the above-mentioned samples was added separately to culture media containing 10 mg·L⁻¹ Cr(VI), which was then inoculated with LBA36 strain and incubated for 6 h. The sample was then centrifuged and the Cr(VI) remaining in the supernatant was used to determine the reducing properties of the different cellular components.

Chemical analysis and characterization

The morphology of the bacterial strain was assessed using scanning electron microscopy (SEM). The bacterial solution was centrifuged (7,000 r·min⁻¹, 10 min), the bacterial body was collected and dehydrated using an ethanol gradient (Wang et al., 2019). The ethanol was allowed to evaporate, and the bacterial powder was coated on the sample stage and sprayed with gold. Bacterial cell images were observed using a scanning electron microscope.

LBA36 cells were cultured in media containing Cr(VI) 0 mg·L⁻¹, 30 mg·L⁻¹, and 60 mg·L⁻¹ for 32 h. The culture medium was taken and centrifuged to collect the cells, which were dried in a vacuum freeze dryer (An et al., 2022; Zeng et al., 2020). After drying, Fourier-transform infrared spectroscopy (FTIR), and XPS were used to investigate the changes in surface functional groups of the LBA36 strain and Cr(VI) valence before and after Cr(VI) stress.

Radish seedling hydroponic experiment

The radish seedling hydroponic experiment was performed to investigate the effects of LBA36 on the growth and development of radish seeds as well as their physiology and biochemistry in the presence of Cr(VI) ions. The radish used in the experiment was sweet and crispy white radish seeds (cultivated seeds) of Botou Yonghong Seed Co. The general-purpose hydroponic nutrient solution used in the experiment consisted of Ca(NO₃)₂, KNO₃, NH₄H₂PO₄, MgSO₄, Na₂B₄O₇, MnSO₄, (NH₄)₂MoO₄, CuSO₄ and ZnSO₄.

In vivo experiments: Sterilized radish seeds were soaked separately in a culture solution containing LBA36 and sterile water for 2 h. Sterile filter papers were soaked in Cr(VI) solution at graded concentrations (0, 30, 60, 90, 100, 200, and 400 mg·L⁻¹). The filter papers corresponding to different Cr(VI) concentrations were then placed in petri dishes, and 50 radish seeds were placed in each petri dish. To minimize variability in other conditions during incubation, petri dishes were incubated in the dark at 30 °C for 7 days. Sterile distilled water was added every 24 h. After the seeds germinated, the germination percentage was calculated, and the seedling diameter length, root length, seedling fresh weight, and dry weight were measured. The experiment was repeated thrice for each Cr(VI) concentration level.

In vitro experiment: White radish seeds were disinfected with a 10% sodium hypochlorite solution for 10 min, and then rinsed with sterile distilled water 3–4 times. Subsequently, the disinfected white radish seeds were soaked in sterile distilled water for 6 h. The soaked seeds were then placed into moistened seedling sponges and incubated at room temperature for a duration of 3 days. After this initial incubation period, the seedlings were transferred to containers for an additional 3-day incubation period, ensuring the intact growth of their roots. Finally, the viable seedlings were transplanted into transparent plastic containers for cultivation, with seven plants per pot. Each container was filled with diluted 900 mL of general-purpose hydroponic nutrient solution (general-purpose hydroponic nutrient solution: distilled water = 1:300). This experiment was set up with two test groups (heavy metal chromium + 1% inoculated strain treatment) and control group (adding heavy metal chromium + no inoculated strain). The results of the pre-test for normal growth of radish seedlings showed that radish seedlings could not grow under high concentration of Cr(VI) (30 mg·L⁻¹) (control and experimental groups). Therefore, a range of Cr(VI) concentrations in the growing environment of radish seedlings was screened. Radish seedlings were treated with 0, 1, 2, 3, 4, 5, 6, and 7 mg·L⁻¹ Cr(VI). Radish seedlings were placed in an artificial culture laboratory with a day/night time of 16/8 h, a day/night temperature of 26 ± 2.5 °C/18 ± 2 °C and a light intensity of 25,000 lux. During the cultivation of radish seedlings, nutrient solution was added every 3 days to ensure adequate nutrition. After 20 days of incubation, the radish seedlings were collected, and the plant height and fresh weight of the radish leaves were measured after draining the water from the surface.

Chlorophyll and biochemical analyses

Determination of chromium content in radish seedlings and hydroponic solutions

Radish seedlings were rinsed, their surfaces were wiped dry, and leaves and taproots were dried in an oven at 70 °C. Next, 0.1 g of leaves and 0.1 g of taproots were weighed and transferred to a crucible for elimination. Total chromium content in leaves and taproots were determined using an inductively coupled plasma spectrometer (ICPS) (Faisal & Hasnain, 2005). At the end of the incubation (20 days in vitro), the hydroponic solution was taken from the container and filtered through a 0.45 μm filter membrane, and the residual Cr(VI) ions were measured using the diphenyl carbamide method. One milliliter of the hydroponic solution was aspirated into a 50 mL crucible for digestion, and the total chromium content was determined using ICPS. The digestion of leaves, taproots, and hydroponic solutions was carried out using the electrothermal plate digestion method (HCl-HNO₃ system) as previously outlined.

Statistical analysis

Data were represented as mean values and standard error. All data were obtained from three replicate experiments. Data were processed using SPSS 26 and analyzed by ANOVA (P < 0.05). Data visualization was performed using Origin 2019b. FITR and XPS results were analyzed using Omnic and Avantage software, respectively.

Identification and developmental tree analysis of the LBA36 strain

The colony morphology of the LBA36 strain is shown in Fig. 2A, exhibiting a colony size of 2–4 mm. The colonies possessed neat edges and a uniform surface, and appeared white, non-sticky, and without odor. Under an optical microscope (Fig. 2B) the cells appear as a rod-shaped, gram-positive bacterium. SEM analysis revealed that LBA36 strain was long, rod-shaped bacterium measuring approximately 0.5 × 1.0 μm in size (Fig. 2C).

The phylogenetic tree of LBA36 strain is shown in Fig. 2D, and the degree of similarity between LBA36 strain and Bacillus toyonensis BCT-7112T (CP006863) was 99.5% (Data S1). 16S rDNA sequence homology revealed that LBA36 strain belongs to the genus Bacillus.

Chromium reduction capacity, adsorption efficiency, and growth curves of LBA36

The growth of LBA36 varied under different Cr(VI) concentrations. As shown in Fig. 3A, the logarithmic growth period of the LBA36 occurred between 3–18 h in the absence of Cr(VI) stress. The onset of the logarithmic growth phase of LBA36 was delayed with increasing Cr(VI) concentrations. The logarithmic growth phase of LBA36 occurred between 18–57 h when grown in media containing 60 mg·L⁻¹ Cr(VI). At 90 mg·L⁻¹ of Cr(VI), the growth of LBA36 strain was markedly inhibited. The MIC of Cr(VI) was 90 mg·L⁻¹.

The reduction and adsorption efficiencies of LBA36 decreased with an increase in the Cr(VI) concentration. The maximum reduction rate of LBA36 was greater than 90%, and the total Cr adsorption rate was about 9% (Fig. 3B). As shown in Fig. 3C, under stress of 30 mg·L⁻¹ Cr(VI), the reduction rate of Cr(VI) increased with time until it reached a certain limit, beyond which it stabilized at approximately 97%. This can be attributed to the inherent reduction characteristics of the strain itself. The Cr adsorption efficiency of the strain initially increased and then decreased with time, and the maximum adsorption rate was 8.8%, observed at 32 h. This may be because LBA36 reached its maximum Cr adsorption capacity, leading to a change in the surface or internal group of the strain. This may have triggered a change in the adsorption capacity, potentially resulting in desorption.

The Cr(VI) reduction ability of different components of the strain was as follows: extracellular material > resting cell > permeabilized cell > intracellular material, indicating that the reduction of Cr(VI) mainly took place in the extracellular area. The reduction rate of the extracellular material was 31.46% (Fig. 3D). The reduction capacity of permeabilized cells for Cr(VI) was lower than that of resting cells, which indicated that the cell membrane played an important role in the reduction process. The permeability of the cell membrane treated with toluene and Triton X-100 was noticeably increased, and Cr(VI) could penetrate the cell more easily, which altered the cells’ ability to reduce Cr(VI).

FTIR and XPS studies

The infrared spectra of LBA36 after 36 h of incubation at Cr(VI) concentrations of 0, 30, and 60 mg·L⁻¹ are shown in Fig. 4A. Owing to the different concentrations of Cr(VI), the FTIR spectra of LBA36 exhibited marked changes. The strong broad peak at 3,301.79 cm⁻¹ is the superposition of O-H and-NH telescopic vibration peaks; the C-H telescopic vibration peak of aliphatic CH₂ can be seen at 2,934.05 cm⁻¹; the C=O telescopic vibration peak of ester can be seen at 1,736.68 cm^-1; the C=O telescopic vibration peak of amide I band is observed at 1,655.02 cm⁻¹; the C-H asymmetric bending vibration peak of CH₃ group can be seen at 1,450.92 cm⁻¹; and the C-O stretching vibration peak of carboxylic acid can be seen at 1,389.41 cm⁻¹. These absorption peaks were shifted to high wave numbers, indicating that the numerous bacterial cell surface functional groups, including surface methyl-, amide I-and amide II groups may be involved in the reduction and adsorption of Cr(VI), preventing the entry of Cr(VI) into the cells, reducing the toxic effect of Cr(VI), and improving the tolerance of LBA36 to Cr(VI). The bending vibration peak of amide II with N-H at 1,546.21 cm⁻¹, the telescopic vibration peak of C-N at 1,242.14 cm⁻¹, the superposition of the C-O peak of ester and the C=S peak of sulfur carbonyl at 1,060.18 cm⁻¹, and the absorption peak of PO₄³⁻ at 628.66 cm⁻¹ were all shifted to lower wave numbers, indicating that they may have altered the toxicity of Cr(VI) to LBA36. These shifting of peaks to lower wave numbers indicates that Cr(VI) may have changed the chemical properties and concentration levels of carbohydrates, esters, proteins, and other biomolecules in LBA36. In LBA36, OH, CH, C-N, C=O, NH, C=S are usually part of proteins and sugars.

The valence states of Cr on the cell surface were analyzed by XPS (Figs. 4B, 4C). Compared with the LBA36 treated with 0 mg·L⁻¹ Cr(VI), the strain treated with 30 mg·L⁻¹ Cr(VI) exhibited relatively lower C-(O/N) content, relatively higher C-O, C=O and O-C-O content. The relative content of C=O in polysaccharides increased slightly and the relative content of C-O increased considerably. Combined with the infrared spectra, the characteristic peaks of polysaccharides and proteins in the LBA36 cells drifted when treated with 30 mg·L⁻¹ Cr(VI), indicating that the functional groups in polysaccharides and proteins interacted with Cr(VI).

As shown in Fig. 4D, the peaks appeared at 586.5–588 eV (Cr_2p1/2) and 576.88–578.76 eV (Cr_2p3/2), and LBA36 reduced Cr(VI) to Cr(III). The valence states of Cr present on the surface of LBA36 were +6 and +3, and the ratio of Cr(VI) to Cr(III) was 1:2. Thus, the ratio of adsorbed Cr(VI) to Cr(III) on the surface of LBA36 cells was 1:2.

Determination of fresh weight, dry weight, and length of radish seedlings

The height and weight of plant seedlings are important indicators of plant growth and development status. The degree of toxicity of Cr(VI) to radish seedlings was determined by measuring the germination rate, plant height, fresh weight, and root length of radish seedlings under Cr(VI) stress. As shown in Fig. 5A, in the control group (seeds + sterile distilled water), with increasing concentrations of Cr(VI), the germination rate of seeds decreased, reaching around 50%; in the experimental group (seeds + LBA36 bacterial solution), the concentration of Cr(VI) also showed a decreasing trend, but the overall germination rate of the experimental group was higher than that of the control group. The germination rate of the experimental group was higher than that of the control group. The germination rate of the experimental group increased by 17.74%, 16.39%, 13.33%, 13.56%, 15.52%, 9.4% and 10% at different Cr(VI) concentrations, respectively. Figure 5B shows that the weight of radish seedlings in both the control and the experimental groups initially increased and then decreased with increasing Cr(VI) concentrations. Figure 5C revealed that inhibition of root length of radish seedlings increased with increasing Cr(VI) concentrations in both control and experimental groups. Root length of radish seedlings in experimental groups eventually became larger than that of the control group due to the presence of LBA36. Figure 5D shows that with an increase in Cr(VI) concentration, the stem length of radish seedlings in the control group and experimental group initially increased and then decreased. When the Cr(VI) concentration was greater than 100 mg·L⁻¹, the stem length of radish seedlings in the experimental group was higher than that in the control group, and the inhibitory effect became considerably significant.

As shown in Figs. 5E and 5F, radish seedling growth and development decreased with an increase in Cr(VI) concentration. The fresh weight of the control group gradually decreased, and plant height initially increased and then decreased, indicating that small amounts of Cr(VI) or Cr(III) in the environment promote the growth of radish seedlings. In the experimental group, radish seedlings grown in 3 and 4 mg·L⁻¹ Cr(VI) exhibited the best growth, and the highest fresh weight and plant height values.

Figure 5G shows that the control and experimental groups grew well, and that the seedlings in the experimental group inoculated with LBA36 strain were taller, with wider leaves and longer taproots. In the control group, the size of radish seedlings, rootstocks, and leaves decreased with increasing Cr(VI) concentrations. Furthermore, the leaves became yellower with increasing Cr(VI) concentrations. In the experimental group, with increasing Cr(VI) concentrations, the taproots of radish seedlings gradually thickened, the diameter of the leaves increased, and the rootstock became shorter and smaller. This phenomenon indicated that LBA36 could promote the growth and development of radish seedlings, but after Cr(VI) exceeded 3 mg·L⁻¹, LBA36 was unable to prevent Cr(VI) from poisoning radish seedlings, and they suffered from stunted growth and development, yellowing of leaves, and impaired photosynthesis.

Under Cr(VI) stress, the presence of LBA36 could enhance fresh weight and promote rootstock growth. Compared with CG0-CG7, the fresh weight of EG0-EG7 radish seedlings increased by 16.75%, 66.81%, 73.29%, 85.17%, 87.73%, 81.4%, 85.47% and 87.81%, respectively; and the height of the plants increased by 30.45%, 35.96%, 32.65%, 37.5%, 45.47%, 32.58%, 28.09% and 37.07% respectively. Radish seedlings in the experimental group had longer taproots, more leaves, and wider and greener leaves.

Effect of LBA36 strain on chlorophyll and biochemical properties in radish seedlings

Figure 6A shows that with increasing Cr(VI) concentration, the chlorophyll content of leaves in the control group and experimental group decreased significantly. The effect of LBA36 on TP content in leaves of radish seedlings under Cr(VI) stress is shown in Fig 6B. As Cr(VI) levels increased, its inhibitory effect on the TP content in leaves became increasingly apparent. At the same Cr(VI) concentration, the TP content was higher in the experimental group. As shown in Fig. 6C, the MDA content in the leaves increased significantly with Cr(VI) concentration. The leaves in the control group gradually turned yellowed, indicating an increase in cell membrane lipidation in the leaves of radish seedlings, and the highest concentration of Cr(VI) caused serious damage to the cell membrane of the leaves. Figure 6D shows the effect of LBA36 on SOD enzyme activity in leaves of radish seedlings under Cr(VI) stress. As Cr(VI) concentration increased, SOD enzyme activity increased and then decreased. Lower concentrations of Cr(VI) enhanced SOD enzyme activity, and high concentration of Cr(VI) significantly inhibited SOD activity. Figure 6E shows the effect of LBA36 on CAT enzyme activity in the leaves of radish seedlings under Cr(VI) stress. As Cr(VI) concentrations increased, CAT activity was significantly inhibited in the leaves of radish seedlings. At similar Cr(VI) concentrations, CAT content in the experimental group markedly differed from that of the control group across the concentration gradient range of 1–6 mg·L⁻¹. In the control group, the CAT content decreased as Cr(VI) concentration increased, whereas, in the experimental group, CAT concentration increased with increasing Cr (VI) levels.

Changes in Cr(VI) content in radish seedlings in vivo and in hydroponic solution

As shown in Fig. 7A, under similar Cr(VI) concentrations, Cr accumulation in taproots was higher in the experimental group than that in the control group. In contrast, Cr accumulation was higher in leaves in the control group than that in the experimental group (Fig. 7B). This may be due to the fact that LBA36 reduces environmental Cr(VI) to Cr(III), which decreases Cr uptake and accumulation in the leaves of radish seedlings. Furthermore, at the same Cr(VI) concentration, Cr accumulation capacity varied in different parts of radish seedlings. Cr content in the roots of radish seedlings was much higher than that in their leaves, indicating that Cr primarily accumulated in the roots of radish seedlings and exhibited limited transference to the leaves. A related study demonstrated that certain plants, such as water hyacinth and lobelia, accumulated more Cr in their roots than their stems and leaves (Batool, Tabassum & Ali, 2017).

Cr(VI) was completely reduced in the control group within the concentration range of 1–2 mg·L⁻¹, possibly attributed to the presence of reducing substances in the hydroponic solution or the release of such substances through the plant’s own protective mechanisms (Fig. 7C). At a concentration of 1–5 mg·L⁻¹, Cr(VI) was completely reduced in the experimental group, and at 6- and 7 mg·L⁻¹, the Cr(VI) concentration was only 0.01 and 0.08 mg·L⁻¹, respectively. At the same Cr concentration, the total Cr adsorption rate of the experimental groups exhibited varying degrees of increase. Overall, LBA36 reduced a large portion of Cr(VI) in the environment and promoted the uptake of Cr ions from aqueous solutions by the roots of radish seedlings.