Lead immobilization assisted by fungal decomposition of organophosphate under various pH values

Organic phosphates (OP) account for approximately 30–90% of total soil P. However, it is too stable to be utilized by plants as available P source. Aspergillus niger (A. niger) has considerable ability to secret phytase to decompose OP. Meanwhile, mineralization of lead (Pb) is efficient to achieve its remediation. This study hence investigated Pb immobilization by A. niger assisted decomposition of OP under variable acidic environments. A. niger can survive in the acidic environment as low as pH = 1.5. However, alternation of environmental pH within 3.5–6.5 significantly changed fungal phytase secretion. In particular, weakly acidic stimulation (pH of ~5.5) increased phytase activity secreted by A. niger to 0.075 µmol/min/mL, hence elevating P release to a maximal concentration of ~20 mg/L. After Pb addition, ATR-IR and TEM results demonstrated the formation of abundant chloropyromorphite [Pb5(PO4)3Cl] mineral on the surface of mycelium at pH = 5.5. Anglesite, with a higher solubility than pyromorphite, was precipitated massively in other treatments with pH lower or higher than 5.5. This study elucidated the great potential of applying OP for Pb immobilization in contaminated water.


Acidity of the medium and biomass of A. niger. Final pH values of EI treatments were shown in
. The final pH value slightly decreased to 1.36 for the treatment with initial pH = 1.5, whereas the final pH values in the medium maintained around 2.0 when initial pH of 2.5-6.5. In addition, the final pH value (2.22) for the treatment with initial pH = 2.5 were significantly higher than those (2.04-2.08) of the treatments with initial pH values from 3.5 to 6.5. Therefore, A. niger has a strong ability to induce environmental acidity.
Biomass can be applied to evaluate the growth of fungi in the medium. In the EC treatments, A. niger had the lowest biomass (28 ± 2 mg) at pH = 1.5 after five-days incubation (Fig. 1A). The biomass of A. niger reached the maximal value of 148 ± 5 mg as pH = 2.5. Then, the biomass (from 78 ± 4 to 123 ± 4 mg) of A. niger increased slowly as the pH values increased from 3.5 to 6.5.
In the EI treatments, A. niger had the biomass of 29 ± 2 mg at pH = 1.5, which was significantly lower than those of other treatments (Fig. 1B). This is consistent with the above EC treatments. The biomass of A. niger varies from 116-192 mg as pH of 2.5-6.5. In addition, the treatments with initial pH = 2.5-4.5 have the relatively high Treatments 1.5 2.5 3.5 4.5 5.5 6.5 Final value 1.36 ± 0.01c 2.22 ± 0.01a 2.05 ± 0.05b 2.04 ± 0.02b 2.04 ± 0.03b 2.08 ± 0.05b The oxalic acid secretion. Oxalic acid is the dominant organic acid secreted by A. niger 42 . In the EC treatments, the concentration of oxalic acid was the lowest (below the detection line) when the initial pH = 1.5 ( Fig. 2A). Additionally, when the pH values of the medium increased from 2.5 to 6.5, the concentrations of oxalic acid secreted by A. niger elevated from 68 ± 2 to 882 ± 34 mg/L. These results confirmed that the neutral environment promoted the secretion of oxalic acid from A. niger.
In the EI treatments, the concentrations of oxalic acid were below the limit of detection at pH = 1.5, and there was a slight increase in oxalic acid concentration when the pH value increased to 2.5 ( Fig. 2B). At pH = 3.5, the value increased six times compared with the treatment with initial pH = 2.5. However, as the pH increased from 3.5 to 6.5, oxalic acid concentrations secreted by A. niger had no significant difference. Phytase activity. In the EC treatments, phytase activities at the initial pH values of 1.5 and 2.5 are extremely low (<0.005 µmol/min/mL) (Fig. 2C), consistent with the above results of biomass and oxalic acid concentration (Figs 1A, 2A). However, the activity of phytase increased substantially as the pH value increased from 3.5 to 5.5. The phytase activity was 0.075 µmol/min/mL when pH = 5.5, which was the maximum value in all the EC treatments. Then, it decreased to 0.060 µmol/min/mL with pH value elevated to 6.5. Therefore, phytase activity increased to a high level when the medium pH near neutral (Fig. 2C), which suggests that delicate pH changes may cause considerable variation of phytase activity.
The variation trend of phytase activities in the EI treatments was different from that in the EC treatments (Fig. 2D). With the initial pH values of 1.5-3.5, the activities of phytase were all at low levels, i.e., <0.020 µmol/ min/mL. It then gradually increased during pH values elevated from 4.5 to 6.5, and finally reached a maximum value (0.056 µmol/min/mL) at pH of 6.5. The value was significantly lower than those of the EC treatments as initial pH of 3.5-5.5. pb 2+ removal in the medium. The removal of Pb 2+ by P source was efficient and widely accepted. In the EC and EI treatments with Pb addition, A. niger was able to remove >80% Pb 2+ from media with addition of Pb(NO 3 ) 2 (initial Pb 2+ concentration = 500 mg/L) and calcium phytate when pH = 5.5 and 6.5 (Fig. 3A). www.nature.com/scientificreports www.nature.com/scientificreports/ EI treatments showed a higher Pb 2+ removal percentage (>92%) than EC treatments (~84%), probably due to the formation of anglesite via the reaction between SO 4 2+ in PVK and Pb 2+ 11 . Moreover, it indicated that Pb 2+ removal percentages for EI treatments were significantly enhanced from ~92% to ~97% with the increase of initial pH.
In the EC treatments after Pb addition, the P concentration of the medium was 0.15 mg/L at pH = 5.5 (Fig. 3B). Then, it has a slight rising trend (P from 0.15 to 0.18 mg/L) as the pH increased from 5.5 to 6.5, respectively. There was no significant difference between pH = 5.5 and pH = 6.5 (P > 0.05). However, in the EI treatments, the highest P concentration was present at pH = 5.5 (0.18 mg/L), which was almost doubled compared with the value at pH = 6.5 (Fig. 3B).
ATR-IR analysis. The peaks of ATR-IR spectra located at ~593 and 630 cm −1 can be both assigned to S-O vibration of sulfate, which were present in all treatments 43,44 . For pH EC = 5.5, the ~539 cm −1 peak was attributed to the ν 4 mode of the PO 4 group 45 . Compared with other three treatments, the ~593 and 630 cm −1 peaks at pH EC = 5.5 were stronger (Fig. 4). The peak at ~965 cm −1 and ~1031 cm −1 respectively represented ν 1 (PO 4 3− ) and 46,47 , which were consistent with our previous research 48 . Moreover, the intensity of the ~965 cm −1 peak was substantially elevated at pH EC = 5.5 (Fig. 4). Besides, the representative peaks at ~539, 965, and 1031 cm −1 indicated that pyromorphite was formed in the EC treatments at pH = 5.5. Compared to other treatments, the new peaks at 680 and ~1396 cm −1 were two typical peaks for pH EC = 5.5, which are represented C-O vibration in carbonates 38,49 . The presence of these peaks may indicate the formation of Pb carbonate after Pb 2+ adsorption 43 .
The peaks at 539, 680, 965, 1031 and 1396 cm −1 disappeared in the EC treatments when the pH value increased from 5.5 to 6.5, confirming that no carbonate and pyromorphite were formed (Fig. 4). The most evident peak appeared at 1024 cm −1 represented ν 3 (PO 4 3− ) band 50,51 . Specifically, asymmetric stretching vibrations ν as (COO − ) appeared between 1634 and 1651 cm −1 52 . For EI treatments at pH = 5.5 and 6.5, their spectra had the similar peaks as those of pH EC = 6.5. Figure 5A,B showed mineralization on surface of the mycelium under TEM. TEM-EDS demonstrated identifiable Pb, P, and Cl signals in the area with dense plate-like minerals (Fig. 5C). It was then selected for detailed SAD analyses. The strongest diffraction can be identified in Fig. 5D (the calculated d value is  www.nature.com/scientificreports www.nature.com/scientificreports/ 2.99 Å), which can be assigned to the (2 1 1) diffraction of pyromorphite 53 , suggesting that pyromorphite could be formed at pH EC = 5.5.

TEM analysis.
Some needle-like minerals formed were observed under TEM (Fig. 5E). EDS analyses also confirmed abundant Pb, P, and Cl in these particles. Based on the SAD pattern, the characteristic (1 1 2) diffraction (d value = 2.96 Å) of pyromorphite can be identified in Fig. 5F. Therefore, their various shapes under TEM can be ascribed to the different orientation of crystallites.

Discussion
A. niger is one of the most representative PSF, which is efficient in increasing available P. The released P enhances formation of Pb-contained minerals. Subsequently, the bioavailability (uptake of Pb 2+ by plants or animals) of Pb 2+ will be decreased. Meanwhile, environmental pH value is critical for microbial activities. The subsequent biomineralization is hence also significantly affected.
PSF can secrete organic acids ten times more than bacteria, lowering environmental pH as low as to 1 ~ 2 35,40 . In this study, the lowest biomass of A. niger was observed at pH = 1.5. This suggests that A. niger can survive but not prefer strongly acidic environment. A. niger had the highest biomass in the medium with pH of 2.5 in both EC and EI treatments (Fig. 1), whereas P concentration and phytase activity were the lowest among all the treatments (Figs 1, 2). This could be due to that A. niger consumes available P for their own growth, resulting in less P released into the environment 54,55 . Moreover, we also confirmed that A. niger is sensitive to pH alternation between 1-6. Therefore, it might be critical to control environmental pH delicately for microbial decomposition of phosphates and subsequent Pb remediation. www.nature.com/scientificreports www.nature.com/scientificreports/ The low pH of the medium after incubation confirmed the secretion of abundant oxalic acid 56 . The prominent oxalate production in fungi occurs by a process called glyoxylate oxidation 57,58 . However, the trend of oxalic acid secretion is inconsistent with that of phytase. Especially, for pH EC = 5.5, the high phytase activity and relatively low oxalic acid concentration were observed in solution, which suggested preference of solubilizing organic phosphates for the fungus at this pH.
Geochemical modeling based on our results in water also confirms that pyromorphite, anglesite, and cerussite appear as three major species after the incubation (Fig. 6). Pyromorphite could be easily formed as long as the H 2 PO 4 − concentration > 10 −11 mM at pH = 5.5 compared to pH = 6.5. Therefore, precipitation of Pb 2+ to pyromorphite is an effective way to immobilize Pb 2+ and reduce Pb contamination at pH EC = 5.5.
The typical limiting factor for successful remediation of heavy metals is the lack of available P. Weak acid environment promotes the decomposition of OP by fungi. It is hence an effective and efficient way for most Pb-contaminated environmental media (either for water or soil). Moreover, this study demonstrated that OP can be mineralized by microorganisms and transformed into pyromorphite mineral with addition of Pb 2+ . Therefore, this study promotes a further understanding of microorganism interactions with toxic metals and OP, as well as providing a new insight into the Pb remediation.

Materials and Methods
Preparation of strains and medium. A. niger (CGMCC No. 11544, Nanjing Agricultural University) was isolated from the soybean rhizosphere soil in Nanjing, China, and the preparation of spores has been discussed in detail in our previous research 37 .
All subsequent experiments were carried out in the modified liquid Pikovskaya (PVK) medium (the phosphate-solubilizing medium). The major ingredients of the modified PVK medium were the same as our previous study 11 , where 0.5 g TCP substituted by 0.1 g calcium phytate (Sigma Aldrich, St. Louis, MO) as P source for all the experiments.
pH adjustment experiments. The experiments with continuous pH adjustment treatments (EC treatments, to simulate the environment with stable pH) were conducted to quantify P concentration, organic acids, and phytase activities influenced by A. niger in the medium under variable acidic environments. The mediums were adjusted (per 24 h) to pH values of 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 with hydrochloric acid and sodium hydroxide. Then, the solution was sterilized at 121 °C for 20 min. Finally, 1 mL spore suspension of A. niger (1.25 × 10 7 spores/mL) was transferred to 100 mL modified PVK media. The media were shaken for five days at 28 °C at 180 r/min. All the treatments have three replicates. www.nature.com/scientificreports www.nature.com/scientificreports/ The experiments with initial pH adjustment treatments (EI treatments, to simulate the environment with pH disturbance) were also conducted to study the ability of A. niger to change the various acidic environment. The experiment was a parallel experiment and was performed by setting the initial pH of media between 1.5 and 6.5 at an interval of 1.0. Most of the preparation is the same as EC treatments, except that no further pH adjustment was performed during the incubation. This parallel experiment was also continued for five days with three replicates.
After five-days incubation, the medium was filtered, and biomass of A. niger was weighed after drying 10 h at 80 °C. The pH values of medium were analyzed by SG98 InLab pH meter with an Expert Pro-ISM-IP67 probe (Mettler Toledo Inc.). P concentrations were analyzed after centrifugation at 4000 rpm for 30 min and by filtered through 0.22 μm membrane for Agilent 710 ICP-OES analysis. The oxalic acid concentration was analyzed by Agilent 1200 HPLC. Phytase activities were extracted by phytase extraction kit (Cominbio, Suzhou, China) and were quantified spectrophotometrically using the Spectramax i3x multi-mode microplate reader (Molecular Devices, Austria). enhanced pb immobilization by A. niger. EC and EI treatments with higher oxalic acid concentration and phytase activity (at pH of 5.5 and 6.5) were selected to demonstrate the influences of OP on Pb immobilization via A. niger. The experiment was carried out with a mixture of Pb(NO 3 ) 2 and PVK medium with initial Pb 2+ concentration adjusted to 500 mg/L. The experiments in the fungal solution were performed with four pH adjustment treatments, i.e., pH EC = 5.5, pH EC = 6.5, pH EI = 5.5, and pH EI = 6.5. All the treatments were conducted with three replicates.
The chemical and morphological properties of precipitates after the centrifugation were determined by ATR-IR and TEM. ATR-IR technique was performed by a Nicolet iS5 Fourier transform infrared spectrometer (ThermoFisher Scientific Inc., Madison, USA) with an attenuated total reflection accessory. IR spectra were collected in the 500-1900 cm −1 range, with a 4 cm −1 resolution and 64 scans at room temperature. High resolution TEM images were collected on a FEI Tecnai G2 F20 with an accelerating voltage of 200 kV. The selected area diffraction (SAD) was also carried out under TEM.

Modeling of Pb speciation by GWB.
Module Phase2 of Geochemist's Workbench (GWB, Version 12.0, Aqueous Solutions LLC.) was used to verify the stability of Pb minerals and the predominance of Pb chemical species after reaction. The parameters were set as shown in Table 2, based on the values of our analysis 11 .

Data Availability
The data sets generated during this study are available from the corresponding author upon reasonable request.  Table 2. Parameters setting in Phase2 module of GWB modeling. The units of ion contents are mol/L.