Exogenous Fe²⁺ alleviated the toxicity of CuO nanoparticles on Pseudomonas tolaasii Y-11 under different nitrogen sources

Cell cultivation and culture media

The aerobic denitrification and heterotrophic nitrification strain Pseudomonas tolaasii Y-11 (KP410741) used in this study was isolated from a winter paddy field by He & Li (2015).

The basal medium (BM) (Huang et al., 2020) was used to determine the nitrogen removal performance of strain Y-11 (1 L containing 0.31 g NaNO₃ (BM₁) or 0.25 g (NH₄)₂SO₄ (BM₂), 2.56 g CH₃COONa, 1.5 g KH₂PO₄, 0.42 g Na₂HPO₄, and 0.1 g MgSO₄ ⋅ 7H₂O). BM₁ and BM₂were the BM for two different nitrogen sources. We added 0.05 g/L FeSO₄ ⋅ 7H₂O to the BM to explore the mitigation effect of Fe²⁺ on CuO-NP toxicity. Before exposure to CuO-NPs, strain Y-11 was cultivated in Luria-Bertani (LB) medium (1 L containing NaCl 10 g, tryptone 10 g, and yeast extract 5 g) at 15 °C and 150 rpm/min for 36 h.

The initial pH of all the media was adjusted to 7.3 with 0.1 M NaOH or 0.1 M HCl. Each 250 ml conical flask contained 100 ml of medium. The medium was sterilized at 121 °C, 0.11 MPa for 20 min and cooled naturally to room temperature.

Preparation of CuO-NPs suspension

The CuO-NPs (40 nm, 99.5% purity) used in this study were purchased from Zewu Company (Chongqing, China) as a dry powder. The suspension of the CuO-NPs (2000 mg/L) was prepared by adding 100 mg of the CuO-NPs to 50 mL ultrapure deionized water. Subsequently, the CuO-NPs suspension was sonicated (600 W and 40 kHz) for 20 min to increase their dispersion (Huang et al., 2020). The morphology of CuO-NPs was observed by a scanning electron microscope (SEM, Phenom World, Holland). The hydrodynamic diameter of ZnO-NPs in the suspensions were measured by Dynamic Light Scattering (DLS, Brookhaven, USA). A SEM image and hydrodynamic diameter of the CuO-NPs (2,000 mg/L) used for this study are available in the Figs. S1 and S2. Different concentrations of CuO-NPs in simulated sewage were obtained by diluting the suspension with basal medium.

P. tolaasii Y-11 exposure to CuO-NPs with or without Fe

To investigate the difference in toxicity of CuO-NPs to strain Y-11 with different nitrogen sources, the strain was exposed to 1, 5, 10, and 20 mg/L CuO-NPs in the basal medium. Basal medium without CuO-NPs was used as a control. Strain Y-11 was inoculated into BM, and shaken for 48 h at 15 °C and 150 rpm. The inoculation volume of the above experiments was 1% (v/v), and the initial optical density (OD₆₀₀) was about 0.1. The above experiments were repeated three times. After 48 h, the OD₆₀₀ of the medium was measured. The medium was centrifuged (8000 rpm, 5 min) to determine the amount of NO₃⁻, NH₄⁺, and metal ions (Cu²⁺). Three parallel measurements were preformed per sample.

P. tolaasii Y-11 exposure to Cu²⁺ with or without Fe²⁺

To investigate whether the toxicity of CuO-NPs was caused by dissolved Cu²⁺, the concentrations of Cu²⁺ in BM₁ and BM₂ were set at 0.01, 0.1, 0.5, and 1 mg/L and 0.01, 0.05, 0.1, and 0.15 mg/L, respectively. The above experiments were repeated in triplicate. The culture conditions and measurement indexes were the same as above.

Analytical methods

The OD₆₀₀ was measured at an optical density of 600 nm. NO₃⁻ and NH₄⁺ were detected in the supernatant after centrifugation (8000 rpm, 5 min) according to the method of He et al. (2019). Specifically, the concentrations of NO₃⁻ and NH₄⁺ were determined by the hydrochloric acid photometric method and the indophenol blue method, respectively. The concentration of Cu²⁺ in the supernatant was analyzed via ICP-OES (ICP-OES 5110, Agilent, USA).

The removal efficiency of NO₃⁻ or NH₄⁺ was calculated as follows: (1)${\displaystyle R=\left(T_0-T_1\right)∕T_0\times 100.0\text{\%},}$ where R is the NO₃⁻ or NH₄⁺ removal efficiency (%), and T₀ and T₁ represent the initial and final NO₃⁻ or NH₄⁺ concentrations in the system, respectively.

Statistical analyses

The significance of the results was tested using the one-way analysis of variance (ANOVA) in SPSS Statistics 20. Three parallel measurements were performed per sample and the results were expressed as mean ± standard deviation.

Effects of CuO-NPs on proliferation and nitrogen removal of strain Y-11 with and without Fe²⁺ addition

The effect of CuO-NPs on the proliferation and nitrogen removal performance of strain Y-11 with or without added Fe²⁺ is shown in Fig. 1. Interestingly, 1 mg/L CuO-NPs promoted cell proliferation and NO₃⁻ removal (Fig. 1A). In the Fe ²⁺-free treatment, as the CuO-NP content increased, cell proliferation was inhibited. At 20 mg/L CuO-NPs, the proliferation of bacteria stopped completely. Meanwhile, the NO₃⁻ removal efficiency also decreased from 42.29% to 2.05%. Hou et al. (2015) found that 50 mg/L CuO-NPs inhibited the denitrification process and significantly reduced the nitrogen removal rate. The addition of Fe²⁺ significantly promoted the growth of bacteria and the removal of NO₃⁻. At 0, 1, 5, and 10 mg/L CuO-NPs, the OD₆₀₀ reached 1.33, 2.14, 1.87, and 0.83, which were significantly higher values than those of Fe²⁺-free treatment. At 20 mg/L CuO-NPs, the bacteria grew slowly. In the Fe²⁺-containing treatment, when CuO-NP content was 0, 1, 5, 10, and 20 mg/L, the removal efficiencies of NO₃⁻ were 69.77%, 88.93%, 80.51%, 36.17%, and 2.47%, respectively. The addition of Fe²⁺ increased the activity of the bacteria and enhanced the resistance to CuO-NPs. Song et al. (2016) found that exogenous Fe²⁺ could improve the microbial activity of water samples and promote the removal of NO₃⁻. Therefore, adding Fe²⁺ is a feasible method by which to increase bacterial tolerance to CuO-NPs.

In the Fe²⁺-free treatment, as the CuO-NPs content increased from 0 to 20 mg/L, OD₆₀₀ decreased from 1.17 to 0, and the NH₄⁺ removal efficiency decreased from 29.83% to 2.33% (Fig. 1B). Even 1 mg/L CuO-NPs were inhibitory to bacteria. This was consistent with the research results of Huang et al. (2020). Zhang et al. (2017a) showed through their research that 1 mg/L CuO-NPs slightly affected the removal of ammonia, and 3–10 mg/L promoted the removal of ammonia. This was different from the results of the present study. By comparing the cell proliferation under Fe²⁺-free treatment, it could be concluded that the cytotoxicity of CuO-NPs in NH₄⁺ wastewater was stronger than that of NO₃⁻ wastewater. The cytotoxicity levels of CuO-NPs in BM₁ and BM₂ were different, and nitrification-related enzymes were more sensitive to CuO-NPs than denitrification-related enzymes. Ye et al. (2020) found that the denitrification process was more sensitive to the toxicity of ZnO NPs than the nitrification process. This finding was consistent with the results of this study. In the Fe²⁺-containing treatment, when the CuO-NP contents were 0, 1, 5, 10, and 20 mg/L, OD₆₀₀ was 2.20, 2.39, 1.01, 0.41 and 0.08, respectively, and the NH₄⁺ removal efficiencies were 55.95%, 96.71%, 38.11%, 20.71%, and 7.43%, respectively. It is worth noting that in the Fe²⁺-containing treatment, 1 mg/L CuO-NPs significantly promoted cell proliferation and NH₄⁺ removal (compared to the control treatment). Fe plays an important role in the growth and metabolism of microorganisms and is a component of ferritin (Feng et al., 2020). Zhang et al. (2018b) found that Fe²⁺ (less than 5 mg/L) could significantly promote the removal of nitrogen in the ammonia oxidation system. Exogenous Fe²⁺ enhanced the resistance of strain Y-11 to CuO-NPs and promoted the removal of NH₄⁺.

Effect of Fe²⁺ addition on Cu²⁺ dissolution from CuO-NPs

Due to the large specific surface area of CuO-NPs, Cu²⁺ would be released in the aqueous environment (Luo et al., 2018). Figure 2 shows the effect of Fe²⁺ addition on the release of Cu²⁺ from CuO-NPs. In the Fe²⁺-free treatment, the amounts of Cu²⁺ released by 1, 5, 10, and 20 mg/L CuO-NPs in BM₁ were 0.008, 0.013, 0.432, and 1.739 mg/L, respectively (Fig. 2A). In the Fe²⁺- containing treatment, as the CuO-NPs content increased from 1 mg/L to 20 mg/L, the maximum release amount of Cu²⁺ of 1.575 mg/L occurred at 5 mg/L CuO-NPs. At 1 mg/L, 10 mg/L, and 20 mg/L CuO-NPs, the Cu²⁺ concentrations were 0.126 mg/L, 1.07 mg/L, and 1.09 mg/L, respectively. As the dose of NPs increased, the dissolved metal ions were adsorbed by the NPs, resulting in a decrease in the amount of metal ions released (Wang et al., 2016). Interestingly, although the addition of Fe²⁺ promoted the release of Cu²⁺ from CuO-NPs (except for 20 mg/L CuO-NPs), bacterial activity and the removal of NO₃⁻ were enhanced (Fig. 1A). Kang, Mauter & Elimelech (2009) found that higher concentrations of divalent cations (such as Mg and Ca) in wastewater may cause nano-metal oxide particles to accumulate more than in river water, thereby affecting their ecotoxicity. Adeleye et al. (2018) found that doping Fe in CuO-NPs significantly promoted the release of Cu²⁺, but this did not increase the toxicity of CuO-NPs to the marine phytoplankton. However, Naatz et al. (2017) found that the doping of Fe significantly reduced the dissolution of Cu²⁺ and reduced the toxicity of CuO-NPs to zebrafish embryos. Therefore, in BM₁, the toxicity of CuO-NPs to strain Y-11 was caused by the NPs themselves. Exogenous Fe²⁺ enhanced the tolerance of strain Y-11 to CuO-NPs.

CuO-NPs released very little Cu²⁺ in BM₂ (Fig. 2B). As the CuO-NPs content increased from 1 mg/L to 20 mg/L, the released Cu²⁺ content gradually increased. In the Fe²⁺-free treatment, the maximum release amount of Cu²⁺ reached 0.095 mg/L when the CuO-NPs content was 20 mg/L. This was much lower than the release amount of Cu²⁺ in BM₁ at the same concentration (1.739 mg/L). The dissolution of NPs is affected by their surface area (Liu et al., 2018). Under the action of electrostatic attraction, NH₄⁺ ions were adsorbed on the surface of CuO-NPs, which reduced the surface area of the CuO-NPs. In the Fe²⁺-containing treatment, less Cu²⁺ was released. The Cu²⁺ release amounts were 0.0003, 0.0075, 0.016, and 0.057 mg/L at 1, 5, 10, and 20 mg/L of CuO-NPs, respectively. Fe²⁺ played a role in inhibiting the dissolution of Cu²⁺ from CuO-NPs. Previous studies had shown that the composition of the solution (such as a high concentration of divalent cations) affected the dissolution of NPs (Chen et al., 2017; Kang, Mauter & Elimelech, 2009; Kunhikrishnan et al., 2015). Mao et al. (2020) found that the addition of metal cations promoted the aggregation of NPs. Exogenous Fe²⁺ increased the content of divalent cations in the medium and inhibited the dissolution of CuO-NPs. Although the solubility of CuO-NPs in BM₂ was lower, CuO-NPs had a higher inhibitory effect on bacterial proliferation and NH₄⁺ removal (Fig. 1B). Exogenous Fe²⁺ inhibited the dissolution of Cu²⁺, and the toxicity of CuO-NPs to strain Y-11 was reduced. Therefore, in BM₂, the toxicity of CuO-NPs on the strain Y-11 was mainly caused by the release of metal ions.

Effects of Cu²⁺ on growth and nitrogen removal of strain Y-11 with and without Fe²⁺ addition

Metal ions are released from NPs in aqueous environment. According to the concentration of Cu²⁺ released from CuO-NPs in different nitrogen sources, the effects of Cu ²⁺ on cell proliferation and nitrogen removal of strain Y-11 were discussed (Fig. 3). In BM₁, the release of Cu²⁺ exceeded 1 mg/L (Fig. 2A). Therefore, the concentrations of Cu²⁺ were set at 0, 0.01, 0.1, 0.5, and 1mg/L (Fig. 3A) for testing. Interestingly, when the concentration of Cu²⁺ exceeded 0.01 mg/L, severe toxicity was produced and cell proliferation stopped. At 0.01 mg/L Cu²⁺, the addition of Fe²⁺ improved the activity of bacteria, and the removal efficiency of NO₃⁻ reached 93.1% (66.8% for the control treatment). Low-dose CuO-NPs (1 mg/L) released less than 0.1 mg/L Cu²⁺ (Fig. 2A), which may be the main reason for the low dose of CuO-NPs (<1 mg/L) induced the synthesis of related enzymes (such as Cu-containing nitrite reductase) and material transfer to increase the activity of the bacteria (Huang et al., 2020; Wang & Chen, 2016; Zhang et al., 2017a). The addition of Fe²⁺ promoted the release of Cu²⁺. When Fe²⁺ was added, the toxicity of CuO-NPs did not increase but promoted cell proliferation and NO₃⁻ removal (Figs. 1A and 2A). However, the addition of Fe²⁺ in the treatment of Cu²⁺ did not achieve detoxification (Fig. 3A). Cu²⁺ had strong toxicity in cells. Therefore, we concluded that in BM₁ the toxicity of CuO-NPs on strain Y-11 was mainly caused by the NPs themselves. The addition of exogenous Fe²⁺ caused the Fe²⁺ to be adsorbed onto the CuO-NPs and inhibited the direct contact between the CuO-NPs and cells, thereby reducing the damage caused by CuO-NPs to strain Y-11. Although exogenous Fe²⁺ promoted the release of Cu²⁺, Cu²⁺ may undergo a hydrolysis process to generate hydroxides and reduce the poisonous effects of Cu²⁺ (Wang et al., 2016).

In BM₂, the maximum release amount of Cu²⁺ from CuO-NPs was less than 0.1 mg/L. We set the Cu²⁺ concentration gradient in BM₂ to 0, 0.01, 0.05, 0.1, and 0.15 mg/L (Fig. 3B). In Fe²⁺-free wastewater, 0.01 mg/L Cu²⁺ significantly increased cell proliferation to 2.12 (1.05 for the Cu²⁺-free treatment). As the concentration of Cu²⁺ increased, cell proliferation was inhibited. The NH₄⁺ removal efficiency decreased from 96.52% to 8.72% as Cu²⁺ concentration increased. The release of Cu²⁺ in BM₂ was less, but the CuO-NPs showed a higher inhibition effect (Figs. 1B and 2B). With the addition of Fe²⁺, cell proliferation was maintained at about 2.5, and the NH₄⁺ removal efficiency was higher than 96%. The addition of Fe²⁺ inhibited the release of Cu²⁺ from CuO-NPs and reduced its toxic effect on cells (Figs. 1B and 2B). In BM₂, the aggregation of CuO-NPs due to the electrostatic effect effectively inhibited the distribution of CuO-NPs in the wastewater, and the free Cu²⁺ made a major contribution to the cytotoxicity. The addition of Fe²⁺ effectively reduced the dissolution of Cu²⁺ from CuO-NPs and reduced the toxic effects of Cu²⁺ on cells.

Effect of exogenous Fe²⁺ on the aggregation behavior of CuO-NPs

The hydrodynamic diameter could reflect the aggregation state of CuO-NPs in water medium. The hydrodynamic diameter of CuO-NPs under different nitrogen sources was shown in Table 1. The hydrodynamic diameter increased gradually with the increase of the concentration of CuO-NPs, indicating that CuO-NPs had aggregated. This may be due to the increased collision frequency (Mwaanga, Carraway & Van den Hurk, 2014). The hydrodynamic diameter of CuO-NPs in BM₂ is larger than that of in BM₁, which further revealed that the amount of Cu²⁺ released in BM₂ is less than that of in BM₁. Furthermore, it has been reported that the aggregated NPs can reduce toxicity (Hou et al., 2016). Exogenous Fe²⁺ further promoted the aggregation of CuO-NPs, and the hydrodynamic diameter increases to 2–3 times of the original. The aggregation of CuO-NPs effectively reduced the possibility of contact with bacteria, and reduced the damage of CuO-NPs themselves to cells.