Application Potential of Lysinibacillus sp. UA7 for the Remediation of Cadmium Pollution

Abstract

Background: Cadmium (Cd) pollution poses a significant environmental challenge. Microbially induced carbonate precipitation (MICP), an advanced bioremediation approach, relies on the co-precipitation of soluble metals with the microbial hydrolysate from urea. This study isolated a urease-producing strain and evaluated its Cd remediation potential. Methods: The isolated strain UA7 was identified through 16S rDNA gene sequencing. Urease production was enhanced by optimizing the culture conditions, including temperature, dissolved oxygen levels—which were affected by the rotational speed and the design of the Erlenmeyer flask, and the concentration of urea added. Its Cd remediation efficacy was assessed both in water and soil. Results: UA7 was identified as Lysinibacillus sp., achieving peak urease activity of 188 U/mL. The immobilization rates of soluble Cd reached as high as 99.61% and 63.37%, respectively, at initial concentrations of 2000 mg/L in water and 50 mg/kg in soil. The mechanism of Cd immobilization by strain UA7 via MICP was confirmed by the microstructure of the immobilized products with attached bacteria, characteristic absorption peaks, and the formed compound Ca_0.67Cd_0.33CO₃, which were analyzed using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The Cd-remediation effect of strain UA7, which reduces lodging in wheat plants, prevents the thinning and yellowing of stems and leaves, and hinders the transition of soluble Cd to the above-ground parts of the plant, was also demonstrated in a pot experiment. Conclusions: Therefore, Lysinibacillus sp. UA7 exhibited high potential for efficiently remediating contaminated Cd.

1. Introduction

Cadmium (Cd), a widely used industrial metal, is extensively employed in batteries, pigments, plastic stabilizers, and solar panels [1]. However, it poses significant environmental and health risks as a highly toxic metal [1,2,3,4]. Due to its exceptional solubility and prolonged half-life (17–30 years), Cd adversely affects aquatic and terrestrial organisms, reduces agricultural productivity, and endangers human health, with its presence documented across nearly all global ecosystems [1,2,3,4,5,6]. In China, approximately 33.54% of farmland and 44.65% of urban soil sites are contaminated with Cd, with an average concentration of 0.19 mg/kg. The world average concentration, as well as those in Iran and Africa, are even higher at 1.10 mg/kg, 1.53 mg/kg, and 5.00 mg/kg, respectively [7]. The soil with the highest reported Cd concentration is in France, at 16.7 mg/kg, followed by Belgium at 7.61 mg/kg, and China at 7.43 mg/kg [2]. Consequently, Cd pollution remediation has gained increasing attention, and among the proposed methods, a biomineralization technique known as microbially induced carbonate precipitation (MICP) has demonstrated promising effectiveness [8,9,10].

The basic principle of MICP is that bacteria produce urease to hydrolyze the uncharged urea molecule (CO(NH₂)₂) into two charged ions (ammonium NH₄⁺ and carbonate CO₃²⁻). Under alkaline conditions caused by the released NH₄⁺, metal ions could combine with CO₃²⁻ to precipitate in the presence of Ca²⁺ [8]. Consequently, soluble heavy metals are transformed into insoluble carbonate mineral crystals, which decreases their mobility and bioavailability [10,11,12]. Generally, high urease activity is beneficial for the immobilization of Cd [13], and microbial urease production is typically influenced by culture conditions [14].

Numerous studies have been conducted on urease-producing bacteria to reduce the levels of soluble Cd. The majority of these studies concentrate on the genera Bacillus, Citrobacter, Enterobacter, Pseudomonas, Serratia, and Yersinia [9,11,13,15,16,17]. In the study of Peng et al. [9], it was reported that Enterobacter sp. exhibited the highest Cd removal rate (99.50%) after 7 days at an initial Cd concentration of 20 mg/L in water, and demonstrated the highest Cd tolerance in soil (20 mg/kg) achieving a 56.10% immobilization rate after 40 days of treatment. Nevertheless, the cadmium tolerance, immobilization rates, and treatment duration of these documented strains were generally inadequate for use in environments characterized by high pollution levels. Thus, researchers are still actively searching for promising strains that could more effectively immobilize Cd. Specifically, the use of Lysinibacillus, a type of Gram-positive bacterium, has been of particular interest because of its excellent metal resistance [18]. Chen et al. [19] reported that Lysinibacillus fusiformis S01 reduced water treatment duration to 2 days (achieving 80% removal of initial 5 mg/L Cd) and increased soil Cd immobilization to 66.43% after 7 days at an initial concentration of 9.324 mg/kg. In this study, we also isolated a Lysinibacillus strain and achieve a higher immobilization rate, even at elevated initial concentrations and within a reduced treatment duration.

2. Materials and Methods

2.1. Isolation and Identification of Urease-Producing Strains

Urease-producing strains were hypothesized to be efficiently isolated from Cd-contaminated soil. The Cd-contaminated soil was artificially prepared by mixing a CdCl₂ solution into the original soil to achieve a uniformly distributed Cd concentration of 10 mg/kg. The original soil sample was collected from the urban farmland located at Beijing University of Agriculture (E 116.291964, N 40.0978690), a site selected for its close resemblance to actual application environments.

Firstly, 1 g of Cd-contaminated soil and 99 mL of sterile water with glass beads were shaken at 180 rpm and at 30 °C for 30 min, and stood for 1 min. The supernatant was collected and decimal dilutions until 10⁻⁶ were prepared with sterile water. Then, the three dilutions from 10⁻⁴ to 10⁻⁶ were transferred onto nine plates, marked from UA1 to UA9, containing urea agar medium (1 g/L peptone, 2 g/L potassium dihydrogen phosphate, 5 g/L sodium chloride, 1 g/L dextrose, 20 g/L urea, 15 g/L agar, and 0.012 g/L phenol red; pH 6.8 ± 0.2), and incubated at 30 °C for 48 h. Color transition from pale yellow to pink-red on the agar medium served as an indicator of the presence of urease-producing bacteria [16]. Finally, the colonies that changed the medium color were isolated and further purified on urea agar medium. The one with a completely pink-red color was chosen as the candidate strain (named UA7) for identification. Genomic DNA was extracted from the bacterial cells using the Bacterial Genomic DNA Extraction Kit (purchased from Beijing Biomed Technology Co., Ltd., Beijing, China). The gene 16S rDNA was amplified by polymerase chain reaction (PCR) with primers 27F and 1492R [20], and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The resulting sequence was uploaded to the GenBank database of the National Center for Biotechnology Information (NCBI), assigned GenBank Accession No. OQ977014, and analyzed using the Basic Local Alignment Search Tool (BLAST). The phylogenetic tree was constructed using MEGA7.0 software.

2.2. Determination of Urease Activity

The obtained strain UA7 was grown overnight in lysogeny broth (LB) medium until OD₆₀₀ reached ~0.6. The culture was then inoculated (1%, v/v) into 100 mL of urease-producing medium (3 g/L beef extract powder, 5 g/L sodium chloride, 10 g/L peptone, 20 g/L urea; pH 7.0 ± 0.2) in 250 mL unbaffled flasks. The flasks were incubated for 48 h at 180 rpm and at 30 °C. During the incubation, the growth was followed by OD₆₀₀. Urease activity was quantified using a modified version of the phenol-hypochlorite method, which is based on the Berthelot reaction [21]. In this method, the culture broth was thoroughly mixed with an equal volume of phosphate buffer (pH 7.0, containing 30 g/L urea) and allowed to react at 37 °C for 20 min. One unit of urease activity (U) was defined as the quantity of enzyme that hydrolyzes 1 μmol of urea per minute. The specific urease activity was calculated by dividing the urease activity by the bacterial biomass OD₆₀₀ [16].

2.3. Optimization of Cultural Conditions

Urease production of strain UA7 was further investigated under various cultural conditions, including incubation temperatures (25 °C, 30 °C, 35 °C, and 40 °C), dissolved oxygen levels influenced by the rotational speed and the structure of the Erlenmeyer flask (180 rpm and unbaffled, 180 rpm and baffled, 200 rpm and baffled, and 220 rpm and baffled), urea concentrations (1%, 2%, 3%, and 4%), and incubation periods (12 h, 24 h, and 36 h). The bacterial biomass OD₆₀₀ was measured and the urease activity were determined at the end of the incubation period.

2.4. Determination of the Cd Immobilization Rate in Water

Strain UA7 was cultivated in urease-producing medium (with a urea concentration of 3%) at the optimal conditions (35 °C, 200 rpm, in a baffled Erlenmeyer flask). The culture broth, incubated for varying duration (12–36 h), was supplemented with CaCl₂ and CdCl₂, yielding final concentrations of Ca²⁺ 25 mM and Cd²⁺ 5 mg/L, respectively. Following supplementation, the mixture was further incubated for 4–6 h and then centrifuged at 12,000× g for 10 min.

The supernatant was collected to detect the soluble Cd concentration using inductively coupled plasma optical emission spectrometry (ICP-OES, ICP6300, Thermo Fisher Scientific, Waltham, MA, USA). Meanwhile, a culture broth supplemented with varying concentrations of Cd (5–2000 mg/L) was also tested to investigate the effect of UA7 on the immobilization of Cd in water. The Cd immobilization rate was calculated using the equation Y(%) = (C₀ – C_t)/C₀ × 100%, where Y represents the immobilization rate (%), C₀ is the initial concentration of soluble Cd (mg/L), and C_t is the concentration of soluble Cd (mg/L) following treatment.

2.5. Characterization of Immobilized Products

Immobilized products, the precipitates after centrifugation, were gathered to be characterized by a scanning electron microscope (SEM, 5136 SB, TESCAN, Brno, Czech Republic), a Fourier transform infrared spectrometer (FTIR, Agilent Cary 630, Agilent, Santa Clara, CA, USA), and X-ray diffraction (XRD, Utima IV, Rigaku, Tokyo, Japan). The acid-resistance test was conducted as follows: the immobilized products were placed on coverslips and exposed to hydrochloric acid (HCl) solutions at various pH levels, from 6.0 to 2.0 with an interval value of 0.5 pH units. The generation of bubbles was observed using a microscope (BM2100, Novel Optics, Ningbo, China). The acid resistance was defined as the pH value at which before the first bubble was generated.

2.6. Determination of the Cd Immobilization Rate in Soil

Different levels of Cd-contaminated soil (1–50 mg/kg) were prepared by mixing CdCl₂ to investigate the immobilization effect of strain UA7. Fifteen grams of soil were mixed with 1 mL of solution comprising 3% of urea and 25 mM CaCl₂, and 4 mL of UA7 culture broth in a 50 mL tube. Samples were taken on the 5th and 10th day to measure the soluble Cd content and calculate the immobilization rate. A diethylene–triamine–pentaacetic (DTPA) solution was used to extract the soluble Cd from the soil environment [22] and detected using ICP-OES.

2.7. Planting Test

Soil polluted with soluble Cd at concentrations of 1 and 5 mg/kg was transferred to square flower pots (7 cm in height, 10 cm in upper side length, and 7 cm in bottom side length), sprayed with UA7 culture broth (27 mL/kg), and stayed in a fume hood at room temperature (~25 °C) for 5 days before planting. A blank control group was prepared with equal volume of deionized water. Each group includes five pots. Wheat seeds were planted, and after 12 days, the leaves and roots of the wheat seedlings were separated, dried, and digested with HNO₃ [23]. The concentration of Cd in the digestion solution was measured using ICP-OES.

2.8. Data Analysis

The mean and standard deviation values were calculated for three replicates (with five replicates in the planting test). The data were analyzed using analysis of variance (ANOVA), and the means were compared using Duncan’s test at the 0.05 significance level with SPSS 21.0 software. The graphs were plotted using Origin 2021 software. The XRD patterns of the precipitated products were analyzed using MDI Jade 6 software.

3. Results

3.1. Isolation and Identification of Urease-Producing Strain

The farmland soil mixed with soluble Cd and the urea agar medium were used to isolate strains efficiently. Seven strains with both Cd tolerance and urease production were isolated. As depicted in Figure 1A, the first three strains with obvious color changes was UA6, UA7 and UA9. The presence of a pink-red color across the entire agar medium indicated that strain UA7 had the highest capacity for urease production. The 16S rDNA sequencing result of strain UA7 was submitted to NCBI database (GenBank Accession No. OQ977014) and compared with the related sequences in GenBank. The phylogenetic tree (Figure 1B) suggested that UA7 was closely related to Lysinibacillus sphaericus CHP05 (GenBank Accession No. MT341799), hence being identified as Lysinibacillus sp.

3.2. Urease Production of Strain UA7

Figure 2A shows the growth and enzyme production characteristics of strain UA7. The biomass increased rapidly, entering the logarithmic growth phase (0–4 h), reached the plateau phase (4–36 h), and then entered a decline phase. The urease activity reached a higher level after 4 h with a highest value at 24 h and maintained about 80–100 U/mL within 12–36 h.

Optimization of cultural conditions has been proven to be beneficial for further improving microbial urease production [14]. Here, cultural conditions including temperature, dissolved oxygen level, and urea concentration in the cultural medium, were optimized for improving the urease production of strain UA7.

The effect of incubation temperatures on the urease activity of UA7 is shown in Figure 2B. Urease activity was higher when cultured at 25–35 °C and decreased at 40 °C. At 35 °C, urease activity was maintained at 100–120 U/mL, and the maximum specific urease activity was observed (Table S1). Additionally, controlling large-scale fermentation at this temperature was more economically viable than at 25 °C. Thus, 35 °C is recommended as the optimal temperature for cultivating UA7.

The distribution regularity of urease activity was reported to be similar to that of dissolved oxygen [24]. Here the effect of different dissolved oxygen levels influenced by the rotational speed and the structure of the Erlenmeyer flask (unbaffled or baffled) was investigated on urease production of strain UA7 (Figure 2C). The urease activity of strain UA7 was maintained at approximately 140–160 U/mL within 12–36 h when cultivated in a baffled Erlenmeyer flask at a rotation speed of 200 rpm.

A relatively higher urea concentration is favorable for urease production [14] while excessive urea application in heavy metals-polluted soil can cause soil acidification, salinization, and microbial biomass reduction [25]. As shown in Figure 2D, the addition of 3% urea was recommended as urease activity reached as high as 188 U/mL.

Based on the results above, UA7 was recommended to be cultivated in a baffled Erlenmeyer flask for 12–36 h at 35 °C, 200 rpm, using 3% urea, for further application.

3.3. Immobilization of Cd by Strain UA7 in Water

The culture broth of strain UA7, within different cell growth time intervals (12–36 h) and initial Cd concentrations (5–2000 mg/L), was tested to study its effectiveness in immobilizing Cd in water. As shown in Figure 3A, it demonstrated exceptional performance in immobilizing Cd at a concentration of 5 mg/L in water, with no significant differences observed among the various cell growth time intervals. The trend of Cd immobilization by UA7 was consistent with its urease production in the period of 12–36 h. The results, originating from varying initial Cd concentrations, further indicated that UA7 exhibited a strong tolerance to Cd. Even if the initial soluble Cd concentration in water is as high as 2000 mg/L, it can be reduced to 7.8 mg/L after being remediated by UA7, with the immobilization rate reaching 99.61% (Figure 3B).

To confirm the mechanism of MICP using strain UA7, the microstructure of the immobilized products was observed by SEM at various magnification. In the low-magnification image (1.99k×, Figure 3C), spherical particles with surface-attached rod-shaped structures were predominantly observed, and some particles were aggregated. High-magnification imaging (10.01k×, Figure 3D) revealed densely interwoven rod-like structures with crystallographic axis alignment, along with branched growth patterns indicative of hierarchical crystallization. The observed rod–particle composite structure implies microbial involvement in nucleation and crystal growth regulation.

The characteristic peaks associated with the immobilized products were obtained by FTIR analysis. As shown in Figure 3E, the functional groups of the bacterial precipitation varied when MICP process was employed by strain UA7. The absorption peaks were analyzed referring to the similar research reports [11,17]. The absorption peaks around 3280 cm⁻¹ (represented stretching vibration peak of O-H and N-H bonds), 2960 cm⁻¹ (caused by the vibration of C-H in polysaccharides, amino acids or alkyl compounds which were mainly components of various membrane and cell wall), and 1085 cm⁻¹ (corresponding to symmetric stretching vibration peak of CO₃²⁻) were slightly weaker in the MICP group. The characteristic absorption peaks at 1388 cm⁻¹ (corresponding to internal antisymmetric stretching vibration of CO₃²⁻), 870 cm⁻¹ and 711 cm⁻¹ (corresponding to internal bending vibration of CO₃²⁻) were much stronger or newly emerged in the MICP group. Changes in the absorption peaks represented the MICP process involved in the treatment of Cd in water by UA7.

The immobilized products were further analyzed using XRD, and the results were confirmed with a standard PDF (Powder Diffraction File) card, a standardized diffraction data file published by the International Centre for Diffraction Data (ICDD). According to the characteristic absorption peak of the XRD pattern in Figure 3F, calcite (CaCO₃ PDF-#05-0586) was clearly identified. The presence of calcite confirmed that UA7 hydrolyzed urea to produce CO₃²⁻, then formed CaCO₃. The comparison between the standard PDF card and the main diffraction peaks indicated that the co-precipitation of Cd²⁺ immobilized products by UA7 predominantly formed stable Ca_0.67Cd_0.33CO₃ (PDF-#72-1938) co-crystallization, effectively reducing the mobility of soluble Cd.

Based on the characteristics of precipitation, the exceptional ability of strain UA7 to immobilize Cd is attributed to the formation of the stable compound and the strength of cell attachment. The immobilized products also demonstrated acid resistance, remaining stable even at a pH of 4 (Table S2).

3.4. Immobilization of Cd by Strain UA7 in Soil

Samples with different initial Cd concentrations were used to investigate the ability of strain UA7 to remediate Cd-polluted soil. As shown in Figure 4A, the immobilization rate of soluble Cd treated with the culture broth of UA7 was more than 70% after 10 days, with no significant differences observed when the initial concentration was within 20 mg/kg. Even at an initial concentration as high as 50 mg/kg, the immobilization rate remained above 60% after 10 days.

To further test the remediation effect of strain UA7 on Cd pollution in soil, the growth of wheat in the contaminated soil was investigated through a pot experiment. The growth of wheat planted in Cd-contaminated soil, treated with or without UA7, did not show a difference at a low concentration of 1 mg/kg (Figure 4B). However, a higher concentration of Cd pollution (5 mg/kg) seriously affected the growth of wheat, resulting in the lodging of wheat plants and a gradually thinning and yellowing of stems and leaves. In contrast, these harms were remediated if the soil was treated with strain UA7 (Figure 4C).

The results of soluble Cd detected in the leaves (Figure 4D) and roots (Figure 4E) of the wheat seedlings indicated that the Cd in the soil was scarcely transferred to the wheat plant at the low concentration (1 mg/kg), even without treatment. In contrast, the control group exposed to 5 mg/kg of soil Cd exhibited significantly higher Cd translocation: 3.444 mg/kg in roots and 0.525 mg/kg in leaves. Notably, treatment with strain UA7 reduced these concentrations to 1.205 mg/kg in roots and 0.090 mg/kg in leaves.

4. Discussion

In the absence of Cd pressure in soil samples, numerous ureolytic bacterial species would also be isolated, requiring further secondary screening [16]. Accordingly, researchers tended to collect Cd-contaminated soil samples to enhance the efficiency of isolating target strains capable of Cd immobilization. Zhou et al. [23] successfully isolated the Comamonas testosteroni ZG2 strain from a soil sample with a Cd content of 2.5 mg/kg, achieving 42.86% immobilization of 0.448 mg/kg Cd. Similarly, Zhao et al. [11] isolated the strain Cupriavidus sp. CZW-2 from a soil sample containing 5.27 mg/kg Cd, resulting in 53.30% immobilization of 5.10 mg/kg Cd. To enhance Cd-tolerance, immobilization, and real-world applicability, this study employed artificially contaminated soil composed of urban farmland soil spiked with Cd up to 10 mg/kg for isolating ureolytic bacteria. The isolated strain of Lysinibacillus sp. UA7 exhibited notable Cd-tolerance and an immobilization rate of 50 mg/kg and 63.37%, respectively.

Research has demonstrated that urease has been investigated in multiple bacterial genera, including Bacillus, Citrobacter, Enterobacter, Pseudomonas, Serratia, and Yersinia [16]. Many urease-producing microorganisms are capable of immobilizing Cd, and most studies indicate that different bacterial strains, even those belonging to the same genus, have varying abilities to immobilize Cd. Generally, high urease activity is beneficial for the immobilization of Cd [13]. However, evaluating the potential of a microbial strain for application in MICP solely based on urease activity data is unreliable. Methodological variations, including conductivity [14], phenol-hypochlorite [20], and Nessler’s reagent methods [23], complicate direct cross-study comparisons. Differences in unit definitions and measurement techniques also affect activity readings. Therefore, a comprehensive evaluation was conducted based on the initial concentration of soluble Cd, the immobilization rate, and the treatment duration (

Table 1. Comparison of urease-producing strains on cadmium immobilization in water.

Bacterial Strains	Urease Activity (U/mL)	Initial Cd Concentration, Immobilization Rate, and Treatment Duration	Reference
Enterobacter bugandensis TJ6	~35.6 ①	5 mg/L, 84.9%, 24 h	Wang et al., 2020 [15]
Bacillus megaterium HD8	~54.2 ①	5 mg/L, 93.2%, 24 h
Serratia marcescens C5-6	804.51 ③	5 mg/L, 69.83%, 48 h	Yang et al., 2024 [26]
Lysinibacillus fusiformis S01	NA	5 mg/L, 80%, 2 d	Chen et al., 2025 [19]
Enterobacter sp. TJ6	36.1 ①	10 mg/L, 61.3%, 5 d	Su et al., 2024 [27]
Enterobacter sp.	NA	20 mg/L, 99.50%, 7 d	Peng et al., 2020 [9]
Paenarthrobactor nitroguajacolicus	NA	100 mg/L, 46%, 144 h	Ma et al., 2023 [17]
Cupriavidus sp. CZW-2	51.6 ①	2 mM, 80.10%, 120 h	Zhao et al., 2019 [11]
Stenotrophomonas rhizophila A323	1.65 ①	2 mM, 71.3%, 72 h	Jalilvand et al., 2019 [13]
Variovorax boronicumulans C113	1.46 ①	2 mM, 73.45%, 72 h
Stenotrophomonas pasteurii	11.08 ①	2 mM, 97.15%, 72 h
Bacillus sp. UR21	55.2 ①	2 mM, 65.0%, 72 h	Wei et al., 2022 [28]
Lysinibacillus sp. UA7	188 ②	2000 mg/L (7.57 mM), 99.61%, 36 h	This study
Comamonas testosteroni ZG2	~200 ②	~11.9 mM, 98.4%, 48 h	Zhou et al., 2021 [23]

Note: ① Urease activity determined by the conductivity method [14,29], and one unit of urease activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 mM urea per minute. The conductivity changes of 1 mS cm⁻¹ min⁻¹ equals to hydrolysis of 11.1 mM urea min⁻¹ [13]. ② Urease activity determined by the phenol-hypochlorite assay method [21,30], and one unit of urease is defined as the amount of enzyme that hydrolyzes 1 µmol of urea per minute. ③ Urease activity determined by the Nessler’s reagent assay method [26], and one unit of urease is defined as the amount of enzyme catalyzed the production of 1 µmol of ammonia per minute. Data with “~” was estimated or calculated based on related information in the corresponding reference. NA means data not available.

and

Table 2. Comparison of urease-producing strains on cadmium immobilization in soil.

Bacterial Strains	Urease Activity (U/mL)	Initial Cd Concentration, Immobilization Rate and Treatment Duration	Reference
Comamonas testosteroni ZG2	~200 ②	0.448 mg/kg, 42.86%, 1 week + 30 days	Zhou et al., 2021 [23]
Enterobacter sp. TJ6	36.1 ①	3.14 mg/kg, 49.1%, 56 days	Su et al., 2024 [27]
Lysinibacillus fusiformis S01	NA	3.504 mg/kg, 76.96%, 7 days	Chen et al. 2025 [19]
Cupriavidus sp. CZW-2	51.6 ①	5.10 mg/kg, 53.30%, 2 weeks + 1 month	Zhao et al., 2019 [11]
Lysinibacillus fusiformis S01	NA	9.324 mg/kg, 66.43%, 7 days	Chen et al. 2025 [19]
Bacillus sp. UR21	55.2 ①	10 mg/kg, 25.2%, 40 days	Wei et al., 2022 [28]
Enterobacter sp.	NA	20 mg/kg, 56.10%, 40 days	Peng et al., 2020 [9]
Lysinibacillus sp. UA7	188 ②	20 mg/kg, 70.25%, 10 days	This study
		50 mg/kg, 63.37%, 10 days

Note: ① Urease activity determined by the conductivity method [14,29], and one unit of urease activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 mM urea per minute. The conductivity changes of 1 mS cm⁻¹ min⁻¹ equals to hydrolysis of 11.1 mM urea min⁻¹ [13]. ② Urease activity determined by the phenol-hypochlorite assay method [21,30], and one unit of urease is defined as the amount of enzyme that hydrolyzes 1 µmol of urea per minute. Data with “~” was estimated or calculated based on related information in the corresponding reference. NA means data not available.

) according to the literature. The results indicated that the immobilization of soluble Cd in water is easier than in soil.

Numerous urease-producing strains are capable of immobilizing soluble Cd in water (Table 1). Strains such as Stenotrophomonas pasteurii [13], Bacillus megaterium HD8 [15], Enterobacter sp. [9], Comamonas testosteroni ZG2 [23], and Lysinibacillus sp. UA7 (this study) have achieved immobilization rates exceeding 90%. Notably, Comamonas testosteroni ZG2 and Lysinibacillus sp. UA7 demonstrated exceptional performance, immobilizing over 98% of Cd even under high stress (up to 11.9 mM and 2000 mg/L, respectively) and a shorter treatment (down to 48 h and 36 h, respectively). These findings underscore their strong potential for treating heavily contaminated water.

The reported strains capable of immobilizing soluble Cd in soil (Table 2) are not as numerous as those that can immobilize Cd in water (Table 1). Moreover, only Lysinibacillus sp. has demonstrated superior capabilities in the remediation of Cd, with immobilization rates exceeding 60%. Chen et al. [19] reported a high immobilization rate of Cd at a low initial concentration (76.96% at 3.504 mg/kg), which decreased to 66.43% as the concentration increased to 9.324 mg/kg, using the Lysinibacillus fusiformis S01 strain. In this study, the rates of Cd immobilization followed the same pattern of variation with changes in initial concentrations. However, the Lysinibacillus sp. UA7 strain maintained high immobilization rates (70.25% and 63.37%) of Cd even at higher concentrations (20 mg/kg and 50 mg/kg).

The genus of Lysinibacillus has attracted considerable attention and warrants further exploration of its application potential [18,31]. Recent advancements have primarily concentrated on the species Lysinibacillus fusiformes. Zhou et al. [32] revealed humic acid significantly inhibits the MICP process by reducing urease activity in Cd remediation using L. fusiformis. Chen et al. [19] also reported the immobilization of Cd in a sequencing batch reactor using the strain L. fusiformis S01. In this study, a bacterial strain identified as Lysinibacillus sp. UA7 demonstrated high urease activity and effectively promoted the co-precipitation of Ca_0.67Cd_0.33CO₃. This process resulted in a remarkable 99.61% immobilization of 2000 mg/L Cd in water. Additionally, it significantly reduced the Cd content by 83% in leaves and 65% in roots when the wheat plants were grown in soil containing 5 mg/kg Cd. To our knowledge, this is the first comprehensive remediation of high-concentration Cd pollution using Lysinibacillus sp.

5. Conclusions

In this study, a urease-producing microbial strain was isolated from simulated Cd-contaminated soil and identified as Lysinibacillus sp. UA7. The strain effectively decreased the mobility of toxic Cd through co-precipitation with Ca, predominantly forming the stable compound Ca_0.67Cd_0.33CO₃. Its exceptional Cd resistance and significant immobilization rate over a short treatment duration highlight its substantial potential for Cd remediation, particularly in immobilizing Cd in water and soil with high contamination levels. Reduced Cd uptake in seedlings suggests it could improve crop safety in contaminated soils. Future studies should prioritize whole-genome sequencing of the isolated strain UA7 to enhance its characterization and facilitate further exploitation. This would enable strategies to reduce the reliance on calcium and urea during treatment processes, uncover its multifunctional potential, and support the translation of laboratory findings into practical applications under natural conditions.