Removal mechanism of Microcystis aeruginosa in Fe2+/sodium percarbonate and Fe2+/sodium persulfate advanced oxidation-flocculation system

Advanced oxidation process (AOPs) can be used for the treatment of harmful algal blooms (HABs). In this study, two systems of Fe2+/sodium percarbonate (Fe2+/SPC system) and Fe2+/sodium persulfate (Fe2+/PS system) were established to explore the removal mechanism of Microcystis aeruginosa (M. aeruginosa). The results indicated that the Fe2+/SPC system catalyzed H2O2 to generate a large amount of ∙OH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bullet \mathrm{OH}$$\end{document} for oxidation by Fe2+ and formed Fe3+ to promote the flocculation of M. aeruginosa. The persulfate was activated by Fe2+ to generate SO4∙-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{SO}}_{4}{\bullet }^{-}$$\end{document} with super-oxidizing properties, and Fe3+ was generated to realize the oxidation and flocculation of M. aeruginosa in the Fe2+/PS system. Compared with the traditional method in which the pre-oxidation and flocculation processes are carried out separately, the method in this study effectively improves the utilization rate of the flocculant and the removal effect of M. aeruginosa. The absolute value of zeta potential of Fe2+/PS system (|ζ|= 0.808 mV) was significantly lower than that of Fe2+/SPC system (|ζ|= 21.4 mV) (P < 0.05), which indicated that Fe2+/PS system was more favorable for the flocculation of M. aeruginosa cells than the Fe2+/SPC system.


Introduction
Over last decades, there has been a growing concern about the impact of Microcystis aeruginosa (M. aeruginosa) and was harmful algal blooms (HABs) due to increasing pollution and eutrophication (Lei et al. 2021). Cyanobacteria will produce microcystins (MCs) into the aquatic environment, while the decayed harmful algae not only produce foul odors but also cause damage to biological systems (Fang et al. 2020), which posing a threat to water quality and human's health (Zhou et al. 2018). M. aeruginosa are negatively charged; the concentration effect (Liu 2020), hydrophilic effect, and electrostatic repulsion effect made the algal cells form stable particles in water (Ribau and João 2007;Henderson et al. 2008). The natural organic substances such as humic acid can be absorbed on the algal surface, which making the properties of M. aeruginosa more stable than the individual algal cells (Tian 2020). M. aeruginosa can move freely in the vertical direction, and it has the characteristics of wide spectral absorption range, fast growing rate, and the ability of releasing MCs (Yan et al. 2020). Some traditional oxidation methods (such as O 3 , ClO 2 , and KMnO 4 ) may lead algal cells to rupture and release a large amount of MCs (Lei et al. 2021). In recent years, AOPs (such as hydroxyl radical ( •OH ) and sulfate radical ( SO 4 • − )) have been recognized as a promising approach for wastewater treatment by destroying organic pollutants with strong oxidizing agents . Among AOPs, the catalytic oxidation of Fe 2+ / sodium percarbonate (SPC) is a promising water treatment method . The molecular formula of SPC is Na 2 CO 3 • 1.5H 2 O 2 , which can be decomposed into hydrogen peroxide and sodium carbonate when dissolved in water ( 2Na 2 CO 3 • 3H 2 O 2 → 2Na 2 CO 3 + 3H 2 O 2 ). As a solid carrier of H 2 O 2 , SPC is with the advantages of stability and it has good resistance to impact (Tian et al. 2021); substituting liquid H 2 O 2 with SPC can form a new Fenton-like system . With the wide range of pH value and reactive oxygen radical generation potential, the SPC system was given outstanding oxidative properties (Tian et al. 2021). In addition, ferrous sulfate and SPC are low cost; the decomposition products of the reaction system are CO 2 and water which are harmless, so SPC is suitable for water treatment process.
With the properties of good redox performance and long halfperiod, the sulfate radicals ( SO 4 • − ) pre-oxidation and coagulation system were widely used to treat the water where HABs are rampant . The iron (Fe), which is a transition metal, was incorporated into the SO 4 • − system for activation (Liu et al. 2017). With the advantages of non-toxic, environmentally friendly, and low cost, Fe is and widely used in SO 4 • − oxidation system instead of other transition metals (Liu et al. 2017). Both SO 4 • − and in situ Fe 3+ can be formed via persulfate activation that Fe 2+ acts as an activator and a flocculant in the reaction and will promote integration of pre-oxidation and flocculation (Dong et al. 2020). Therefore, there is no need to separate the pre-oxidation and flocculation, nor need add the external flocculant, which is feasible for the treatment of HABs in natural aquatic environment (Chuang et al. 2017;Zhao et al. 2020).
The method in the present study has greatly improved the accuracy of algal pollutant removal. The design of reactors and the important parameters throughout the reaction process were obtained. The conclusions help in making harmful algal bloom treatment efficient and fit for the purpose of environmental protection. Unlike the current researches which ignored the intracellular MC and IOM release, this study focused and examined the situation of algal cell rupture during AOPs in the Fe 2+ /PS system and Fe 2+ /PSC system. The Fe 2+ /PS system was selected from the two AOP systems of Fe 2+ /PS and Fe 2+ /PSC. It has the double advantage of both high-efficient algal removal and avoided rupturing releasing the microcystins (MCs) and harmful intracellular organic matters (IOMs) into extracellular environment.

Algae cultivation
The algal species used in this experiment was M. aeruginosa (FACHB-315) purchased from Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. The algae were cultivated in sterile BG11 medium. The algal inoculum was placed in a thermostatic and illumination incubator; the culture conditions were set as follows: 28 ± 2 °C and 120 μmol/ (m 2 ·s), and light-dark ratio was 12 h/12 h (Kong et al. 2019). The algal suspension solution during the logarithmic phase (the 6th d) was collected and prepared for experimental process with the density of 1 × 10 6 cell/mL.

Detection pre-oxidation-flocculation effect
According to the previous studies, the recommended feeding ratio of Fe 2+ /SPC system construction is 1:1 (the molar ratio of Fe 2+ to SPC). The concentration of SPC in the system is 0.2 mmol/L (Tian 2020). The feeding ratio of Fe 2+ /PS system construction was 1:1 (molar ratio of Fe 2+ to SPC), and the concentration of PS in the system is 0.1 mmol/L (Song 2020). 250 mL 0.1 mmol/L Na 2 S 2 O 8 solution, 250 mL 0.2 mmol/L NaH 2 CO 3 solution, 250 mL 0.1 mmol/L Fe 2 SO 4 solution, and 250 mL 0.2 mmol/L Fe 2 SO 4 solution were prepared with sterile water. All solutions mentioned above were prepared when it will be used (Liu et al. 2017;Cheng et al. 2021). Na 2 S 2 O 8 and Fe 2 SO 4 solutions (PS system) and NaH 2 CO 3 and Fe 2 SO 4 solutions (SPC system) were added to the prepared algae solution (1 × 10 6 cell/mL), respectively, and two different kinds of treatment systems were obtained. Each kind system was divided into control group (only M. aeruginosa at a concentration of 1 × 10 6 cell/mL), SPC experimental group (250 mL 0.2 mmol/L NaH 2 CO 3 solution and 250 mL 0.2 mmol/L Fe 2 SO 4 solution), and PS experimental group (250 mL 0.1 mmol/L Na 2 S 2 O 8 solution and 250 mL 0.1 mmol/L Fe 2 SO 4 solution) (Fig. 1). The stirring speed of the reaction system was set at 300 rpm, and the duration was 30 min. Samples were collected from 2 cm below the surface of the liquid at the beginning of standing, after 15 min, 30 min, 1 h, 2 h, 3 h, and 10 h. The quenching reaction was carried out using Na 2 S 2 O 3 before detections.

Algae removal rate detection
The concentration of M. aeruginosa was linearly correlated with the optical density (OD) at wavelength of 680 nm (Sun et al. 2018) which was determined by the spectrophotometer (T6 Xinyue, China). The removal rate of M. aeruginosa was measured and calculated as follows: where OD 680i and OD 680t refer to the initial and final densities of M. aeruginosa, respectively.
The concentration of M. aeruginosa cell could be positively reflected by the Chl-a. Algal cells' Chl-a was extracted using a 95% ethanol solution, and then, absorbance values at 663 nm and 645 nm were measured using a spectrophotometer (T6 Xinyue, China). Equations were used to determine the content of Chl-a : where A 663 and A 645 represent the absorbance at wavelengths of 663 nm and 645 nm, respectively.

SEM observation and zeta potential detection
The algae cells were enriched by centrifugation, centrifuged at 4000 g for 10 min, and the collected algae cells were treated as follows: The algal cells were washed 3 times with phosphate buffer saline (PBS) with a concentration of 0.01 mol/L at pH 7.0. The glutaraldehyde solution with mass fraction of 2.5% was added to the samples, then followed with vortex shaking. Subsequently, the samples were fixed at 4 °C for an overnight incubation. The 500 μL of 0.01 mol/L PBS solution were added to the samples to wash the algal cells for 3 times by centrifugation (4000 g, 5 min). Finally, the ethanol solutions with concentration 30%, 50%, 75%, 90%, 100%, and 100% were used to dehydrate. After the dehydration treatment was completed, 200 μL of tert-butanol was added for replacement. The samples were placed in a critical point dryer (Quorum K850, UK) for thorough drying. The dried samples were transferred to conductive glue, and the morphology of M. aeruginosa in the two pre-oxidation systems was observed using a scanning electron microscope (Zeiss Sigma 300, Germany) after gold spraying (Lu et al. 2021). The zeta potentials of the samples before and after the two pre-oxidation systems were measured using a Malvern Zeta potential (Malvern Zetasizer Nano ZS90, UK) meter.

3D-EEM detection method
The changes of extracellular substances of samples in Fe 2+ / SPC system and Fe 2+ /PS system were measured using a threedimensional fluorescence spectrometer (Hitachi F7000, Japan). Set the emission wavelength (Em) in the range of 250-550 nm with a step interval of 5 nm, the excitation wavelength (Ex) in the range of 220-450 nm with a step interval of 10 nm, and the scan speed at 2400 nm/min ). Using sterile water as a blank control, the resulting spectrum is the result after deducting Raman scattering.

Statistical analysis
All trials were done three times, and the results were provided as means with standard deviations (SD). The SPSS 19.0 was used to conduct the statistical analysis (IBM, USA).The variance was calculated using a one-way analysis of variance (ANOVA) with a significance threshold of 5% (P ≤ 0.05).

Analysis of flocculation effect of M. aeruginosa in Fe 2+ /PS and Fe 2+ /SPC systems
Under different treatment conditions, after Fe 2+ /PS and Fe 2+ / SPC integrated pre-oxidation-flocculation treatment and standing for 10 h, the removal rate of M. aeruginosa was 100% (Fig. 2a). Significant flocculation and sedimentation of M.aeruginosa cells occurred in both systems (Fig. 2b).
As can be seen from Fig. 2a, the concentration of algae cells in the control group did not change significantly. In the SPC group, the removal rate of OD 680 reached 88% after 1 h and 99% after 2 h, and the residual rate of M. aeruginosa was close to 0 after 3 h. In contrast, the residual rate of algae cells in the PS group was still 15% after 3 h, and the removal rate of M. aeruginosa in the PS group reached 100% after 10 h of flocculation, but the flocculation time was 3.3 times longer than that in the SPC group. As shown in Fig. 2(b1, b2, and b3), the control group (Fig. 2(b1)) was homogeneous and untreated algal fluid, and both the Fe 2+ /PS and Fe 2+ /SPC systems showed precipitation of supernatant and flocculated algal cells at the bottom. This indicated that both Fe 2+ /PS and Fe 2+ /SPC systems effectively broke the stable state of M. aeruginosa suspension, and the flocculation of M. aeruginosa in Fe 2+ /PS system was relatively stable, and it was not easy to break algae cells. It can be concluded that Fe 2+ /PS system is more conducive to maintaining the integrity of M. aeruginosa cells (Gu et al. 2017).

Effects of Fe 2+ /PS and Fe 2+ /SPC systems on morphology of M. aeruginosa cell
The SEM images was a clear and intuitive way to identify the cell damage upon flocs storage (Ma et al. 2014). To explore the Fe 2+ / PS and Fe 2+ /SPC effects of the system on the cell integrity of M. aeruginosa was led by the observation and analysis of the external morphology of anterior and posterior algal cells using SEM. The change in M. aeruginosa cell morphology for both control and pre-oxidation and coagulation treatment is presented in Fig. 3.
In the control group (Fig. 3a), the cells of M. aeruginosa were ellipsoidal, and the outside of the cells was complete and smooth. Without any treatment, this group was the original form of normal M. aeruginosa cells (Lei et al. 2021). M. aeruginosa cells treated with Fe 2+ /PS system were slightly damaged (Fig. 3b), without large area cell rupture, and were wrapped and adsorbed by floc. Therefore, it can be inferred that there are fewer phytotoxins and harmful extracellular substances released by algal cell rupture in this system (Lei et al. 2021). In contrast, M. aeruginosa cells treated with the Fe 2+ /SPC system showed a large area of cell rupture (Fig. 3c), with the white arrow in Fig. 3c indicating completely damaged cells. Therefore, it can be inferred that a large number of M. aeruginosa cells in the Fe 2+ /SPC system will rupture and release MCs (Liu et al. 2017;Tian 2020).

Zeta potential change of M. aeruginosa suspension in Fe 2+ /PS and Fe 2+ /SPC systems
The efficiency and treatment mechanism of wastewater treatment depend on the characteristics of pollutants and water treatment agents, such as flocculants, and the adsorption of flocs can also enhance the removal effect of pollutants. Zeta potential is usually used to describe charge neutralization in coagulation mechanisms (Wang 2021). In this study, zeta potential is used to reflect the charge on the surface of algae cells. The higher the net value is, the greater the electrostatic repulsion between algae cells is and the more stable the algae cells are (Lei et al. 2021). Zeta potential changes of M. aeruginosa system after treatment with Fe 2+ /SPC or Fe 2+ /PS are shown in Fig. 4.
The zeta potential of the control group was the highest in this three groups, with an absolute value of 34.4 mV, and shows that the system has a strong stability (Lei et al. 2021). This phenomenon occurs because there are a large amount of negatively charged algal organic matters (AOMs) in untreated M. aeruginosa cells. Compared with the control group, the zeta potential of M. aeruginosa suspension treated with Fe 2+ /SPC system decreased significantly, and the absolute value was 21.4 mV. This result shows that the stability of the system is significantly reduced (Yi et al. 2021). Compared with the control group, the absolute value of zeta potential of M. aeruginosa suspension decreased significantly to 0.808 mV after Fe 2+ /PS treatment. And the algae cell suspension system became extremely unstable and was very prone to flocculation and sedimentation (Yi et al. 2021). The above data indicate that SO 4 • − and •OH produced by Fe 2+ /PS system can oxidize negatively charged AOMs, and in situ Fe(III) formed in the system can cause flocculation and sedimentation of algae cells (Min et al. 2012;Liu et al. 2017). Compared with the control group, the absolute value of zeta potential of M. aeruginosa suspension in Fe 2+ /PS system changed more than that in Fe 2+ /SPC system, indicating that the charge neutralization ability of Fe 2+ /PS system was stronger than that of the latter, indicating that Fe 2+ /PS system could provide an environment and conditions easier for flocculation and sedimentation of M. aeruginosa (Song 2020).

Analysis on changes of algal organic matter in Fe 2+/ PS and Fe 2+ /SPC systems
Low concentration of algal organic matters (AOMs) can promote adsorption bridging and enhance the flocculation process. But high AOM content will increase the negative charge of M. aeruginosa cells in water, improve the stability of algal cells, inhibit flocculation, and thus reduce the sedimentation effect of M. aeruginosa in the system (Pivokonsky et al. 2006;Xie et al. 2016). Therefore, AOM produced by oxidized algae cells is a key step in enhancing flocculation and improving the sedimentation effect of M. aeruginosa. A three-dimensional fluorescent excitation emission matrix (EEM) was used to analyze changes in EOMs during flocs storage in this study, as illustrated in Fig. 5. And in this study, three-dimensional fluorescence spectroscopy (3DEEM) was used to further study the chemical composition and changes of AOMs in the treatment of M. aeruginosa with Fe 2+ /SPC and Fe 2+ /PS systems.
There are three peaks were identified by 3DEEM analysis (Fig. 5). The peak T1 at Ex/Em of 280/310 nm was associated with and peak T2 (230/325 nm) was associated with represent tyrosine-like and fulminate-like substances, respectively (Fig. 5a). As shown in Fig. 5c, peak T3 at Ex/ Em of 230/330 nm was associated with and was related to intermediate dissolved microbial metabolites, such as humus (protein-like substances) (Fu et al. 2005;Hao et al. 2007). The formation of the fluorescence peak of fulminate-like substances is mainly due to the presence of some organic substances (such as naphthalene) with low molecular weight and high fluorescence efficiency (Baker and Curry 2004). The results of this investigation demonstrate that there was a difference in peak intensities in the pre-oxidation and coagulation systems when compared to the control. All tyrosine-like and fulvic acid-like substances were oxidized and decomposed in Fe 2+ /PS and Fe 2+ /SPC systems, indicating that tyrosine-like and fulvic acid-like substances were more easily oxidized. It might due to the fact that the free radicals were prone to decompose macromolecular organic matter with unsaturated bonds (Tian et al. 2018). In comparison between the Fe 2+ /SPC system and the control group, T1 and T2 peaks disappeared, while T3 peaks appeared (Fig. 5b and c). This was because M. aeruginosa cells were under chemical environment and physiological  (Song 2020), resulting in a significant increase in the concentration of humus (proteins) (Hao et al. 2007). This also proved from the side that Fe 2+ /SPC system caused large numbers of M. aeruginosa rupture. Similarly, the SEM morphology characterization also confirmed this point. The cell rupture of M. aeruginosa would result in the release of a large amount of humus and algal toxins in the system, while there was no release of a large amount of intracellular substances in the Fe 2+ /PS system.

Comparison between Fe 2+ /PS and Fe 2+ /SPC systems
Fe 2+ /PS and Fe 2+ /SPC were utilized for pre-oxidation and coagulation in this work to further investigate the practical environmental impact of applying Fe 2+ /PS and Fe 2+ /SPC on suppressing M. aeruginosa cells. After a certain concentration of drugs was prepared, they were added into M. aeruginosa suspension and stirred for 30 min before standing. Flocculation sedimentation occurred in both systems. The difference is that Fe 2+ /PS system produces green flocs, while Fe 2+ /SPC system produces yellow-green flocs. The removal rate of algae cells in Fe 2+ /PS system was 100% after standing for 10 h, while the removal rate of algae cells in Fe 2+ /SPC system was 100% after standing for 3 h. However, compared with the Fe 2+ /PS system, the Fe 2+ /SPC system had more broken M. aeruginosa cells, which was more likely to cause the rupture of algae cells, thus leading to the release of various MCs into the water. The results of 3DEEM showed that although tyrosine-like and fulminate-like peaks disappeared in M. aeruginosa suspension in the control group in Fe 2+ /SPC system, new pollutants appeared. Therefore, it can be inferred that the new pollutants are caused by the rupture of M. aeruginosa cells.
As shown in Fig. 6, in Fe 2+ /PS and Fe 2+ /SPC systems, sodium percarbonate and sodium persulfate degrade algal organic substances by forming •OH and •SO − 4 (Sun 2020). As for the Fe 2+ /SPC system, sodium percarbonate dissolved in water, H 2 O 2 was oxidized to O 2 and H + through electron loss, Fig. 5 Changes of algae-derived organic matter in Fe 2+ /PS system and Fe 2+ /SPC system. a is the control group; b is Fe 2+ /PS system; c is Fe 2+ / SPC system Fig. 6 Removal mechanism of M. aeruginosa by Fe 2+ /SPC or Fe 2+ /PS system and Fe 2+ in aqueous solution was oxidized to Fe 3+ , resulting in precipitation, which destroyed the stability of M. aeruginosa suspension and improved the flocculation treatment effect of the system. Simultaneously, hydrogen peroxide will generate hydroxyl free radicals with strong oxidability through electron acquisition, which will destroy other pollutants by oxidation, so that the harmless part of pollutants can be understood as harmless fragments. At the same time, Fe 2+ in the aqueous solution was oxidized to Fe 3+ , resulting in precipitation, and the M. aeruginosa cells were flocculated and settled along with the precipitation. In conclusion, Fe 2+ /PS and Fe 2+ /SPC systems constructed an "integrated oxidation and flocculation" system by generating in situ Fe(III). This kind of system reduces the dosage of flocculant, can effectively reduce the economic cost of advanced oxidation system construction, and has better flocculation effect (Song 2020). Fe 2+ /SPC system has the advantages of the cost lower, easy storage, good oxidation effect, short standing time, and so on. However, the oxidation of the system is too strong, and it is easy to cause the fragmentation of M. aeruginosa cells and then release the intracellular toxins and other harmful intracellular substances, which have potential harm to the water environment and human health (Sun 2020). In contrast, Fe 2+ /PS system was not easy to cause M. aeruginosa cell rupture and could effectively promote the flocculation and precipitation of algae cells (Song 2020). Therefore, Fe 2+ /PS system is more suitable for flocculation, precipitation, and removal of M. aeruginosa in water environment than Fe 2+ /SPC system.

Conclusion
In this study, concentration of M. aeruginosa, content of chlorophyll a (Chl-a), appearance and morphology of algal cell, zeta potential, and extracellular dissolved organic matter of M. aeruginosa of Fe 2+ /SPC system and Fe 2+ /PS system were detected and analyzed. The results indicated that both two systems can remove M. aeruginosa thoroughly. However, the rupture rate of algal cells in the Fe 2+ /SPC system is significantly higher than that in the Fe 2+ /PS system. The absolute value of zeta potential of Fe 2+ /PS system (|ζ|= 0.808 mV) is significantly lower than Fe 2+ /SPC system (|ζ|= 21.4 mV) (P < 0.05). It indicates that the Fe 2+ /PS system is more feasible to remove M. aeruginosa. This is the first study comparing two methods of pre-oxidation and coagulation system, Fe 2+ /PS and Fe 2+ /SPC. The removal rate of Fe 2+ /PS and Fe 2+ /SPC pre-oxidation and coagulation system to M. aeruginosa was 100%, and the absolute value change of zeta potential of M. aeruginosa suspension in Fe 2+/ PS system was larger than that of Fe 2+ /SPC system. These results indicated that the charge neutralization ability of the former was stronger than that of the latter, and the Fe 2+ /PS system could provide an environment and conditions easier to flocculate and settle for M. aeruginosa. Fe 2+ /PS system can more effectively promote the sedimentation of M. aeruginosa and the removal of extracellular organic matter and at the same time can effectively avoid the release of harmful intracellular substances of M. aeruginosa. Therefore, Fe 2+ /PS system causes less secondary toxic substances and less secondary pollution to water body and is more suitable for flocculation, precipitation, and removal of M. aeruginosa in water environment. Compared to Fe 2+ / SPC, Fe 2+ /PS has been shown to be a promising and environmentally safe technique for the eradication of M. aeruginosa with real-world applications.