Heavy Metal Immobilization by Phosphate-Solubilizing Fungus and Phosphogypsum Under the Co-Existence of Pb(II) and Cd(II)

Abstract

Globally, phosphogypsum (PG) is the primary by-product of the phosphorus industry. Aspergillus niger (A. niger), one of the most powerful types of phosphate-solubilizing fungi (PSF), can secrete organic acids to dissolve the phosphates in PG. This study investigated heavy metal (HM) remediation by PG and A. niger under the co-existence of Pb and Cd. It demonstrated that 1 mmol/L Pb²⁺ stimulated the bioactivity of A. niger during incubation, based on the CO₂ emission rate. PG successfully functioned as P source for the fungus, and promoted the growth of the fungal cells. Meanwhile, it also provided sulfates to immobilize Pb in the solution. The subsequently generated anglesite was confirmed using SEM imaging. The immobilization rate of Pb reached over 95%. Under co-existence, Pb²⁺ and 0.01 mmol/L Cd²⁺ maximized the stimulating effect of A. niger. However, the biotoxicity of Pb²⁺ and elevated Cd²⁺ (0.1 mmol/L) counterbalanced the stimulating effect. Finally, 1 mmol/L Cd²⁺ dramatically reduced the fungal activity. In addition, organic matters from the debris of A. niger could still bind Pb²⁺ and Cd²⁺ according to the significantly lowered water-soluble Pb and Cd concentrations. In all treatments with the addition of Cd²⁺, the relatively high biotoxicity of Cd²⁺ induced A. niger to absorb more Pb²⁺ to minimize the sorption of Cd²⁺ based on the XRD results. The functional group analysis of ATR-IR also confirmed the phenomenon. This pathway maintained the stability of Pb²⁺ immobilization using the fungus and PG. This study, hence, shed light on the application of A. niger and solid waste PG to remediate the pollution of Pb and Cd.

1. Introduction

Lead (Pb) is a persistent, bio-accumulative, and toxic heavy metal (HM) in the environment [1,2]. It might cause cardiovascular disease and mild mental retardation to humans [3]. Pb will bind to carboxyl and amino groups on the microbial cell walls, thus disrupting the integrity of the cell wall [4]. Although Cd is not as abundant as Pb on Earth, it usually exhibits a higher environmental toxicity [5,6]. It can disrupt microbial cellular mechanisms, such as damaging mitochondrial function [7]. In addition, Cd²⁺ can replace the functional cations in microbial cells, thus disrupting the normal physiological metabolism of microorganisms [8]. Pb and Cd pollution is particularly serious in agricultural soil. Approximately 82.8% of the contaminated agricultural soils were contaminated by inorganic pollutants, among which Pb and Cd accounted for 1.5% and 7.0%, respectively [9,10,11]. Approximately 200,000 square kilometers of rice fields have been contaminated by Cd [12].

Cd contamination is often associated with Pb contamination [13,14]. Pb and Cd can also block functional groups of biologically important molecules and denature/inactivate enzymes in fungi [15]. The adsorption of Pb and Cd by organisms showed significant differences. For example, when Pb and Cd were jointly exposed to the soil planted with Solanum nigrum L., the uptake of Cd was significantly reduced [16]. Additionally, co-exposure of Pb and Cd weakened toxicity with respect to single Pb or Cd exposure [17]. This is because Pb and Cd co-exposure induced antagonistic interactions at potential binding sites of plant roots [18]. Similarly, the biotoxicity of Pb and Cd together towards zebrafish larvae was significantly less than that of single Pb or Cd [19]. Furthermore, co-exposure can induce novel physiological effects that were not manifested under single-element pollution [20,21]. For example, it disrupts the mRNA expression rhythm of the melatonin-rate-limiting enzyme in zebrafish larvae [22].

The remediation of HM contamination includes physical remediation, chemical remediation, and bioremediation. Physical remediation often disrupts native ecosystems and requires extended remediation periods [23]. Chemical remediation is prone to cause secondary environmental pollution [24]. Bioremediation utilizes environmentally friendly and cost-effective techniques [25]. The introduction of Bacillus coagulans and olivine strengthened the immobilization of Pb and Cd within the sediment matrix [26]. The maximum tolerance of the fungus Aspergillus niger (A. niger) to Pb²⁺ was over 1500 mg/L [15], while the tolerance to Cd²⁺ was as low as <100 mg/L [27,28]. An appropriate concentration of HMs can induce excitability of microorganisms within a short time [29]. For example, the bioactivity of A. niger was stimulated in a short time at Pb of <1000 mg/L [15,30]. The strong reproductive capacity of microorganisms such as fungi and bacteria ensures the sustainability of the HM bioremediation process, which is an advantage that physical and chemical bioremediation do not possess [31].

Phosphogypsum (PG) is the by-product of the wet chemical attack process of phosphate ores, most of which is geological apatite [32,33]. As a solid waste, PG has been recycled and used as an alternative for natural gypsum [34]. Beyond its conventional uses, PG contains activated P that is able to immobilize HMs [35]. Meanwhile, A. niger can produce low molecular weight organic acids (LMWOAs) to improve the solubility of phosphate. It has been proposed that the combination of A. niger and PG improved the immobilization efficiency of Pb [36]. Furthermore, our previous studies demonstrated that Cd²⁺ removal by A. niger improved with the co-existence of Pb²⁺ [30]. Therefore, it is meaningful to explore the effect of PG addition to the remediation of Pb and Cd co-contamination by A. niger.

The aim of this study was to investigate the bioactivity of the phosphate-solubilizing fungus A. niger under the coexistence of Pb and Cd. Further, the immobilization of HMs was also studied.

2. Materials and Methods

2.1. PG Preparation and A. niger Activation

The phosphogypsum (PG) was collected from Yichang, Hubei Province. The accession number of A. niger is M 2023240 in China Center for Type Culture Collection (CCTCC). The information about A. niger (Nanjing, China) in this experiment can be found in a previous study [30]. The fungi stored in the −80 °C refrigerator were activated before the experiment. The spore suspension was defrosted at 28 °C, and then inoculated on Potato Dextrose Agar (PDA) medium in a clean bench. Intensive spores formed after 5 days of extended incubation at 28 °C. The surface of the medium was washed with sterile water in a clean bench and the spores were carefully scraped off with a sterile inoculation ring. Finally, three layers of sterile gauze were used to filter the spore suspensions to remove the broken mycelium fragments.

2.2. A. niger Incubation

The modified Pikovskaya (PVK) medium used in this experiment contained 10.0 g Dextrose, 0.03 g FeSO₄·7H₂O, 0.03 g MgSO₄·7H₂O, 0.3 g NaCl, 0.3 g KCl, 0.5 g (NH₄)₂SO₄, 0.03 g MnSO₄·H₂O, and 1000 mL ultrapure water. No P source was added to the PVK.

Before incubation, 50 mL PVK medium was added to 150 mL serum bottle and sterilized at 121 °C for 20 min. Then, 1.0 mL suspension of A. niger spores and 1.0 g PG were added to each serum bottle [36]. Cd²⁺ and Pb²⁺ cations were added by Cd (NO₃)₂ and Pb (NO₃)₂ powder (Xilong Scientific Ltd., Shantou, China). Nitrates were used in the incubation to avoid the influence of sulfate and chlorides [37]. Five treatments were performed in this study, i.e., Control (no HMs), Pbs (sole 1 mmol/L Pb²⁺), Pb-CdL (1 mmol/L Pb²⁺ + 0.01 mmol/L Cd²⁺), Pb-CdM (1 mmol/L Pb²⁺ + 0.1 mmol/L Cd²⁺), and Pb-CdH (1 mmol/L Pb²⁺ + 1 mmol/L Cd²⁺). Each treatment was performed with three replicates.

The serum bottles were sealed with a parafilm (BS-QM-003, Biosharp, Hefei, China) containing a 0.22 μm air breathable membrane. Then, A. niger was incubated for six days (28 °C, 180 rpm).

2.3. Chemical Properties and Spectral Analysis

After incubation, the serum bottles were filled with air for five minutes. The CO₂ emissions were measured. The medium was filtered using a 0.22 μm polyethersulfone (PES) to separate the solid phase from the liquid phase for subsequent testing. After filtration, the filtrate was obtained to measure the pH. Finally, Pb, Cd, and water-soluble P concentrations in the filtrate were measured using ICP-MS. In addition, the immobilization rates of Pb²⁺ and Cd²⁺ were calculated based on the following formula:

$${\displaystyle \frac{C_T-C_S}{C_T}}\times 100\%$$

(1)

where the C_T (mg/L) refers to the initial added Pb²⁺ and Cd²⁺ concentrations, while the C_S (mg/L) represents Pb²⁺ and Cd²⁺concentrations after incubation [36].

One-way ANOVAs were used to test significant differences in CO₂ emission rates; filtrate pH values; water-soluble Pb, Cd, and P concentrations; and heavy metal immobilization rates. The significance level (α) was set at 0.05. All these analyses were performed using IBM SPSS Statistics 27.

A portion of the solid phase obtained by filtration for each treatment was collected. The solid phase above was ground into a powder after drying for attenuated total reflection infrared spectroscopy (ATR-IR) and X-ray diffraction (XRD) tests.

In addition, the remaining solid phase above was used for the SEM-EDS test. Firstly, it was placed in 2.5% glutaraldehyde solution for 12 h (25 °C) to fix the microbial skeleton. Then, it was dehydrated using ethanol at concentrations of 25%, 50%, 75%, 90%, and 100%. Finally, it was dried in a freeze-dryer for 48 h, and it was glued on conductive tape for SEM-EDS analysis.

2.4. Instrumentation

The CO₂ emissions of the system were measured by gas chromatography (Agilent 7890B with Agilent OpenLab CDS ChemStation Edition) [38]. Then, the pH of the filtrate was measured using Mettler pH meter (Pro-ISM-IP67). Finally, ICP-MS (NexION 5000 with Syngistix 2.5) was used for the measurement of Pb²⁺, Cd²⁺, and water-soluble P concentrations in the filtrate. The filtrates were dried in a vacuum freeze-dryer (Genscience Instrument Pro-4055, Nanjing, China).

The XRD test of the solid phase was conducted using a Bruker D8 diffractometer (Cu-Kα; 40 kV; 40 mA; scanned from 10 to 80° at a speed of 0.02°/s). The results of the XRD test were analyzed using MDI Jade 6 and Origin module. The ATR-IR test was applied on a Nicolet iS5 Fourier-transform infrared spectrometer (ThermoFisher Scientific Inc., USA) with Thermo Scientific OMNIC 9.3 software. The results of the ATR-IR test were analyzed using an Origin module to determine the functional groups in the solid phase.

The morphology of the microorganisms was observed through SEM and the elements contained in the solid phase were analyzed using EDS. The SEM image acquisition was tested using the Carl Zeiss SUPRATM 55 system. Semi-quantitative analysis was performed using Oxford Aztec X-Max 150 energy dispersive X-ray spectrometer (EDS). For SEM imaging, the operating voltage was set at 5 kV to obtain images with higher clarity, whereas for the EDS analysis, a voltage of 15 kV was applied to enhance elemental signal detection.

The solid phase of the Pb–CdH treatment was not determined by ATR-IR, XRD, and SEM-EDS, as the growth of all strains was almost fully inhibited.

3. Results

3.1. CO₂ Emission Rate and pH Value

In the control treatment, CO₂ emission rates were 3319 μg·C·kg·h⁻¹ after incubation (Figure 1A). After the addition of Pb²⁺, the respiration intensity significantly increased to 8515 μg·C·kg·h⁻¹ for the Pbs treatment. The respiration intensity of the Pb–CdL treatment was significantly higher than that of the Pbs treatment, i.e., 10,701 μg·C·kg·h⁻¹ (Figure 1A). This also suggests a stimulating effect. Then, the respiration intensity was reduced to 4475 μg·C·kg·h⁻¹ for the Pb-CdM treatment, and finally dramatically decreased to 449 μg·C·kg·h⁻¹ for the Pb-CdH treatment.

For the treatments without Cd²⁺, the pH value increased from 2.58 (control treatment) to 3.75 (Pbs treatment). With the addition of Cd²⁺, the pH value increased from 3.78 (Pb-CdL treatment) to 4.48 (Pb–CdM treatment), and finally reached 6.32 in the Pb–CdH treatment after incubation (Figure 1B).

3.2. Pb, Cd, and P Concentrations

In the Pbs treatment, the Pb concentration was 9.55 mg/L (Figure 1C). After the addition of Cd²⁺, the Pb concentration decreased to 7.20 mg/L for the Pb–CdL treatment. The Pb concentration of the Pb–CdM treatment (4.44 mg/L) was lower than that of Pb–CdL treatment (Figure 1C). Then, the Pb concentration was significantly reduced to 0.45 mg/L for the Pb–CdH treatment (Figure 1C). The Cd concentration of the Pb–CdL treatment was 1.05 mg/L (Figure 1D). The value increased to 10.05 mg/L for the Pb–CdM treatment (Figure 1D). Finally, the Cd concentration increased significantly to 89.14 mg/L for the Pb–CdH treatment (Figure 1D).

The water-soluble P concentration in the control treatment was 41.86 mg/L (Figure 1E). After the addition of both Pb²⁺ and Cd²⁺, the concentration of water-soluble P in the solution decreased dramatically from 3.09 mg/L (Pbs treatment) to 1.25 mg/L (Pb–CdL treatment). Moreover, there was no significant difference in treatments with the addition of Cd, i.e., from 1.74 mg/L (Pb–CdM treatment) to 1.44 mg/L (Pb–CdH treatment) (see Figure 1E).

In the Pbs treatment, the removal rate of Pb²⁺ was 95.39% after incubation (Figure 1F). With the increasing concentration of Cd²⁺, the removal rates of Pb²⁺ were relatively stable, i.e., 96.52% (Pb–CdL treatment), 97.96% (Pb–CdM treatment), and 99.78% (Pb–CdH treatment). Compared with the removal rates of Pb²⁺, the removal of Cd²⁺ showed significant changes. The values increased significantly from 6.22% (Pb–CdL treatment) to 10.63% (Pb–CdM treatment). In the Pb–CdH treatment, the removal rate of Cd²⁺ was 20.70%, nearly twice that of Pb–CdM treatment (Figure 1F).

3.3. ATR-IR Analysis

The peak at 598 cm⁻¹ in the ATR-IR spectra was assigned to the ν4 P-O vibration (Figure 2) [39]. The peak at 1027 cm⁻¹ was assigned to the ν1 and ν3 P-O vibration of phosphates (Figure 2) [40,41]. Meanwhile, the peak at 780 cm⁻¹ was ascribed to the P-O vibration from HPO₄⁻. This peak disappeared in the Pbs, Pb-CdL and Pb-CdM treatment (Figure 2). The representative peaks at 1315 and 1616 cm⁻¹ could be assigned to symmetric stretching vibrations of C-O from C₂O₄²⁻ and asymmetric stretching vibrations from HC₂O₄⁻, which were usually associated with oxalic acid (Figure 2) [42]. The 1683 cm⁻¹ peak represented the C=O stretching vibration (Figure 2) [43,44]. In addition, the 1104 cm⁻¹ peak was a combination of P-O (for PO₄³⁻) and S-O (for SO₄²⁻) vibrations, which reflected both the ν3 P-O stretching vibration of phosphate and the sulfate content in PG (Figure 2) [39,45].

3.4. XRD Analysis

The peaks located at 23.31° and 29.05° were the two representative peaks of gypsum [45]. These two peaks indicated that gypsum was abundant in the solid phase regardless of the addition of HM (Figure 3). The peaks at 20.68° and 26.53° were associated with anglesite [36]. The representative peak of pyromorphite was differentiated at 29.56 (Figure 3). It appeared after the addition of 0.1 mmol/L Cd²⁺, whose peak intensity was relatively weak. In the Pb–CdM treatment, A. niger was stressed by Pb and Cd, which resulted in decreased oxalic acid secretion. Then, Pb²⁺ began to be mineralized into pyromorphite (Figure 4C,F). Moreover, the peak located at 31.17° ascribed to lead oxalate could be observed (Figure 3) [36]. The intensity of this peak in Pbs treatment was relatively strong (Figure 3).

3.5. SEM-EDS Analysis

The solid phases obtained from the Pbs treatment and Pb-CdM treatment were observed using SEM (Figure 4). The SEM image and EDS result of the Pbs treatment indicated that Pb²⁺ replaced Ca²⁺ in PG and combined with SO₄²⁻ to induce the mineralization of anglesite (Figure 4A,D) [36]. SEM imaging demonstrated that the hypha of A. niger adsorbed a large amount of gypsum (Figure 4A). Anglesite formed by the reaction was tightly wrapped in mycelium (Figure 4A). In addition, lead oxalate and pyromorphite were observed from the solid phase of Pb–CdM treatment (Figure 4B,C,E,F). Both of them were attached to the ends of A. niger mycelium (Figure 4A). The minerals observed above indicated that the A. niger complex PG system was capable of providing sufficient anions to ensure the immobilization of Pb²⁺.

4. Discussion

It was proposed that 1 mmol/L Pb²⁺ stimulated the bioactivity of A. niger and enhanced its metabolism during incubation (Figure 1A), which was consistent with our previous study [15]. This phenomenon was further confirmed by the significant increase in the biomass of A. niger after Pb addition (Figure 1A,E). Concurrently, water-soluble P decreased dramatically, which was primarily due to the consumption for the intense growth of fungus. Additionally, a small portion of P in PG participated in the mineralization of Pb²⁺ to form pyromorphite (Figure 4C,F). The toxicity of Pb²⁺ was relatively weak, attributed to its low charge density and mobility [46]. The low toxicity provides more chances for microorganisms to survive.

The presence of low Cd²⁺ (0.01 mmol/L) also had a stimulating effect on A. niger (Figure 1A). A. niger could activate antioxidative systems to reduce oxidative damage generated by Cd²⁺ [47]. Thus, the respiration of the fungus was significantly elevated (see Figure 1A). Therefore, the co-exposure of Pb and low-level Cd could benefit the long-term remediation of both HMs. Moreover, the immobilization rate of Pb²⁺ maintained >95% (Figure 1F). This can be attributed to the fact that Pb²⁺ was more readily adsorbed and fixed by A. niger with respect to Cd²⁺. From a chemical perspective, Pb²⁺ exhibited a higher adsorption affinity due to its higher standard electrode potential and smaller covalent radius [48,49,50]. As a result, A. niger preferentially adsorbed more Pb²⁺ to minimize direct contact with Cd²⁺. Although Cd²⁺ itself showed only a modest remediation effect, its presence made the immobilization process of Pb²⁺ in solution relatively stable (Figure 1F).

A. niger secreted a large amount of oxalic acid when growing rapidly, triggering the anions (e.g., SO₄²⁻) in PG to be released (Figure 3). Most of them combined with Pb²⁺ to form anglesite, resulting in more pronounced peaks for anglesite and oxalic acid with respect to Pbs treatment (Figure 3). This process further reduced the Pb concentration in solution. Compared with anglesite (K_sp = 1.82 × 10⁻⁸), cadmium sulfate is soluble, lowering its mineralization. Instead, it is highly probable that the oxalic acid secreted by A. niger combined with Cd²⁺ to form cadmium oxalate (K_sp = 1.51 × 10⁻⁸) (Figure 1F). In actual scenarios of soil contamination, Cd²⁺ typically co-exists with Pb²⁺, with the concentration of Pb²⁺ invariably exceeding that of Cd²⁺ [51]. The concentration of 0.01 mmol/L Cd²⁺ was suitable for many pollution scenarios (the background levels of Cd in rice soils are 0.37 μg/g). This indicates that the application of the A. niger complex PG system could effectively address the issue of HM contamination by Pb and Cd.

In the Pb–CdM treatment (0.1 mmol/L Cd²⁺), the high toxicity of Cd²⁺ inhibited the respiration of A. niger. This suggests the strong toxicity of Cd²⁺ on A. niger, which counterbalanced its stimulating effect (Figure 1B). In the absence of Cd²⁺, the biomass of A. niger grew significantly, accompanied by a large amount of secreted oxalic acid (Figure 1B). That led to the formation of more lead oxalate in the solution (Figure 4B,E). Under 0.1 mmol/L Cd²⁺, the secretion of oxalic acid declined, resulting in less free C₂O₄²⁻. However, Pb²⁺ on the surface of A. niger occupied the potential sites for Cd²⁺ [30], thereby maintaining a relatively stable immobilization rate for Pb²⁺ (Figure 1B). The XRD results showed that only Pb–CdM treatment had the representative peak of pyromorphite (Figure 3). The A. niger complex PG system primarily induced Pb²⁺ in the environment mineralized into anglesite (Ksp = 1.6 × 10⁻⁸) [36]. Pb²⁺ was more inclined to be combined with SO₄²⁻, and then be precipitated as anglesite [39]. However, anglesite was not as stable as pyromorphite [52], which was inclined to redissolve in acidic environments [53]. In this study, the pH of the incubation system increased with increasing Cd²⁺ concentrations (Figure 1C,D). Therefore, the secondary release of Pb²⁺ was not evident (Figure 1E,F).

When the Cd concentration reached 1 mmol/L (i.e., 112.41 mg/L), Cd²⁺ exhibited strong toxicity to the fungal cells [31]. Our previous studies demonstrated that A. niger tolerance to Cd²⁺ was around 100 mg/L [28,54]. However, due to the presence of Pb²⁺ in the system, the antagonism between the two HMs weakened their toxicity [18,30,55]. Then, some A. niger was able to survive, which was confirmed by the consumption of P by Pb–CdH treatment (Figure 1E). Nevertheless, the metabolic process of A. niger was seriously stressed [56]. Notably, the immobilization rate of Cd²⁺ increased significantly from 10% to 20% (Figure 1F). Although the biological activity of the fungus was significantly reduced after six days of incubation under Cd stress, the organic matter from the debris (e.g., glucans, lipids, chitins, and chitin derivatives in the cell wall) could still bind Pb²⁺ and Cd²⁺ in a short time [57]. This suggested that even if the microorganism was not fully biologically functional, Cd²⁺ could still be immobilized by biological debris from A. niger. Additionally, PG itself could dissociate phosphate [58], generating cadmium phosphate (K_sp = 2.53 × 10⁻³³).

In previous studies, phosphorus-soluble bacteria (PSB) Ochrobactrum sp. J023 were combined with biochar for the remediation of Pb pollution [59]. The experiment was conducted in a Ca₃(PO₄)₂ medium with a Pb concentration of 100 mg/L. It was reported that 71.30% Pb was immobilized by the combination of biochar and PSB. In contrast, the combination of PSF A. niger and PG demonstrated evident promotion of Pb remediation (Figure 1F). This is because fungi usually have a higher biomass than bacteria. Meanwhile, the spores of the A. niger promise its sustainable growth under stress.

5. Conclusions

The experimental results show that A. niger and PG had a better remediation on Pb pollution compared with Cd. When Cd was present in the environment, the remediation effect on Pb remained stable. The immobilization of Cd slightly increased with the elevation of Cd²⁺ concentration. Meanwhile, the presence of Pb²⁺ would stimulate the bioactivity of A. niger during incubation. In addition, a trace amount of Cd²⁺ had a similar effect. The combination of PSF and PG has a vitally broad prospect for remediating the environment polluted by Pb and Cd.