The bloom-forming cyanobacteria were obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Supplementary Table S1). The test organisms were cultured in sterilized BG11 medium at 25°C under continuous fluorescent white light (50 μmol m⁻²s⁻¹) with a light/dark cycle of 12 h/12 h (Lee et al., 2015). The BG11 medium is commonly used for the large-scale cultivation and test of cyanobacteria, and the experimental results are generally consistent with those observed in real water (Pandey et al., 2023). The cultures were manually agitated every 8 h. The growth state of the organisms was monitored every 2 days by measuring the optical density of cells at 685 nm (OD₆₈₅) using a spectrophotometer (UV-2800, Unico, Shanghai, China). The concentration of cyanobacteria was consistently maintained at an OD₆₈₅ of 0.8 in all subsequent experiments. The cultures were harvested for experiments during the exponential growth phase (Randhawa et al., 2013). The pH value was set at 7.4 and changed with time (Supplementary Figure S1).

Microcystis Aeruginosa (MA), separated from the culture medium through a centrifugation process (4,000 rpm, 10 min), was diluted to OD₆₈₅ = 0.8 using newly prepared BG11 medium (without FeSO₄). A suitable volume of FeSO₄ and H₂O₂ was added to the algae solutions, with the H₂O₂ concentration set at 10 mg/L and Fe²⁺ concentrations of 0, 1, 2.5, 5, and 10 mg/L, respectively (Experiment started). Fv/Fm, a performance index of biological activity (Li et al., 2010), was measured at 0, 0.5, 1, 2, 4, 6, and 8 h after adding H₂O₂ and FeSO₄. All samples were incubated at 25°C under dark conditions and continuous fluorescent white light (50, 150, and 300 μmol m^-2s^-1). Each experiment was conducted in triplicate.

MA and Microcystis Panniformisin (MP) was collected from culture through centrifuge process respectively, and diluted to OD₆₈₅ = 0.8 by new-prepared BG11 (without FeSO₄). EDTANaFe and H₂O₂ were added to the algae solutions, so that the H₂O₂ concentration was 10 mg/L, and the Fe³⁺ concentration was 0, 1, 2.5, 5, 10, and 20 mg/L respectively. Fv/Fm was tested at 1.5 h, 4 h, 24 h or 4 days after adding the reagents. All the samples were incubated at 25°C, under dark and continuous fluorescent white light (50 μmol m^-2s^-1) conditions. Each experiment had three duplicates.

Eight kinds of common bloom cyanobacteria were separately collected from the culture through a centrifugation process and diluted to OD₆₈₅ = 0.8 using newly prepared BG11 medium (without FeSO₄). EDTANaFe and H₂O₂ were then added to every algae solution, resulting in an H₂O₂ concentration of 10 m/L and Fe³⁺ concentrations of 3 mg/L, respectively. Fv/Fm was measured at 24 h after adding the reagents, and photographs were taken for each type of algae after 4 days. All samples were incubated at 25°C under continuous fluorescent white light (50 μmol m^-2s^-1). Each experiment was conducted in triplicate.

Algae MA and MP were collected from culture through a centrifugation process and diluted to OD₆₈₅ = 0.8 using newly prepared BG11 medium (without FeSO₄). The samples were divided into two groups: group 1, which was treated with H₂O₂ and FeSO₄ (H₂O₂ = 10 mg/L, Fe²⁺ = 3 mg/L), and group 2, which was treated with H₂O₂ and EDTANaFe (H₂O₂ = 10 mg/L, Fe³⁺ = 3 mg/L). The samples were then incubated at 25°C under continuous fluorescent white light (50 μmol m^-2s^-1). Each group was manually agitated every 15 min, and after 2 hours, the samples were centrifuged (4,000 rpm) to obtain supernatant and algal body fractions.

The supernatant was filtered using a 0.45 µm microfiltration membrane, and then the concentrations of Fe²⁺, Fe³⁺ and total iron were measured (Fortune and Mellon, 1938). The algae body was washed with 0.01M PBS and dried for 24 h under vacuum freeze conditions and then analyzed by SEM-EDS. Meanwhile, FeSO₄ and EDTANaFe were separately added into a 0.01 M PBS solution (pH = 7.4) to achieve total iron concentrations of 5, 50, 500, and 2000 m/L. The solution was treated at 25°C under continuous fluorescent white light (50 μmol m^-2s^-1). The particle size of the solution was measured using a Malvern Particle Size Analyzer (ZS90).

The concentration of H₂O₂ was measured using the four-channel free radical analyzer TRB4100 (WPI). The TBR4100 free radical detector can convert the chemical signals produced by H₂O₂ on the detection probe into electrical signals and record and analyze them in real time (Young et al., 2011). Detailed procedures see Supporting Information (Supplementary Section S1). The yield of OH was determined using the rapid capture method with methylene blue (MB). Fenton regent (H₂O₂ was 10 mg/L, inorganic iron of FeSO₄ was 3 mg/L) and Fenton-like regent (H₂O₂ was 10 mg/L, organic iron of EDTANaFe was 3 mg/L) were added in methylene blue (MB, 60 μM) solution respectively under magnetic stirring condition. The reactions proceeded under dark and light (50 μmol m^-2s^-1) condition. After 5 min of reaction, the absorbance value (OD₆₆₀) was immediately measured to calculated the corresponding concentration of MB. Subsequently, the MB concentration was substituted into Equation 1 to calculate the amount of OH within 5 min, which was used to represent the yield of hydroxyl radical. · OH =   C k −   C 5 ⁡ min (1) C_k: Indicates the MB concentration without adding H₂O₂ group.

C_5min: Represents the MB concentration after treatment with the corresponding H₂O₂ dose for 5 min.

The algicidal effect of H₂O₂ catalyzed by FeSO₄ was evaluated using chlorophyll fluorescence (Fv/Fm), as shown in Figure 1. Significant differences were observed between dark and light conditions, with Fv/Fm values being lower under light (Figures 1A,B). Furthermore, as light intensity increased, the reduction in Fv/Fm became more pronounced (Figures 1A–D). When the light intensity is set at 50 μmol m^-2s^-1, the Fv/Fm values for the experimental groups with ferrous iron doses of 5 and 10 mg/L are above the death line (Figure 1B). The phenomenon shows that the addition of inorganic ferrous salts not only fails to enhance the algicidal effect of H₂O₂ but actually reduces its toxicity against algae. Fv/Fm is an important parameter for assessing photosynthetic performance and is highly sensitive. When photosynthetic organisms like cyanobacteria experience external stress, Fv/Fm responds immediately (Baker, 2008). If the Fv/Fm value drops below 0.1, it is highly likely that recovery to a normal state is difficult, and it can be considered a criterion for the death of cyanobacteria (Zhu et al., 2020). When the light intensity is increased to 150 μmol m^-2s^-1, all experimental groups have Fv/Fm values below the death line. At a light intensity of 300 μmol m^-2s^-1, all experimental groups show almost complete failure in Fv/Fm. This suggests that light significantly enhances the algicidal effect of Fenton reagents. These results are consistent with findings by Mikula, and Jia, who reported that light radiation stimulates the rapid production of OH by H₂O₂, thereby enhancing the algicidal effect (Mikula et al., 2012; Jia et al., 2018).

The phenomenon where the addition of a ferrous catalyst actually inhibited the oxidative performance of H₂O₂ contradicts traditional understanding (Huang et al., 2016). As a catalyst for H₂O₂, FeSO₄ played an adverse role from Figures 1A–D. Traditional Fenton reagents (H₂O₂+FeSO₄) require an acidic environment (pH < 4) for optimal reaction, as excessively high pH values (pH > 6) tend to suppress the formation of OH (Hermosilla et al., 2009). The progression of the Fenton reaction depended on the existence morphology of iron, as illustrated in Figure 2. Figure 2 illustrated the distribution of iron complexes with pH using the MINTEQ model (Le Calve et al., 1997). When the pH value was above 4, Fe³⁺ precipitated completely, primarily forming goethite (FeOOH), which adsorbed onto the extracellular polymeric substances (EPS) on the surface of Microcystis Aeruginosa. This hindered the further advancement of the Fenton reaction. Additionally, iron salts serve as nutrient substances required for the growth of algae (Chen et al., 2011). A small amount of Fe²⁺ could actually enhance biological activity. Consequently, traditional Fenton reagents cannot be effectively employed to control water blooms in natural lakes.

To prevent deactivation caused by inorganic iron precipitation under natural conditions, organically complexed iron was selected for subsequent experiments. Organic iron chelators, such as EDTA or DTPA, have been found to enhance the Fenton reaction and broaden the effective pH range (Zhang and Zhou, 2019; Clarizia et al., 2017), therefore, we used EDTANaFe as the representative organic iron compound for the experiments. Figures 3A,C illustrate that the Fv/Fm ratios measured at 1.5, 4, and 24 h, as well as 4 days, fluctuated with increasing concentrations of organic Fe³⁺. Regardless of the ferrous ion concentration, the Fv/Fm values are generally above 0.2 (MA: Fv/Fm > 0.2; MP: Fv/Fm > 0.3). This indicates that the algicidal effect of H₂O₂ catalyzed by EDTANaFe is ineffective under dark conditions.

In contrast, the results under light conditions (as depicted in Figures 2B,D) were markedly different from those in the dark. The Fv/Fm values of the experimental group were significantly lower than those of the control group (H₂O₂ =10 mg/L, Fe³⁺ = 0 mg/L), regardless of whether the algae were Microcystis Aeruginosa (MA) or Microcystis Panniformis (MP). This suggests that the addition of organic iron (EDTANaFe) was effective in killing cyanobacteria under light conditions.

Figures 2B,D also demonstrated that Fv/Fm reached its lowest point (below death line) when the organic iron (Fe³⁺) ranged from 2.5 to 10 mg/L. This indicates that the Fenton-like reagents caused lethal damage to the algae MA and MP under light conditions. Both insufficient (<2.5 mg/L) and excessive (>10 mg/L) concentrations of organic Fe³⁺ were not conducive to effectively eliminating the algae. In general, the parameters of H₂O₂ = 10 mg/L and organic Fe³⁺=2.5–10 mg/L were found to be suitable for controlling water bloom. The molar ratio of H₂O₂ to Fe^(II/III) in the photo-Fenton process affects the efficiency of ferric ion (Liu et al., 2018).

Low Fe³⁺ concentrations catalyze H₂O₂ decomposition to generate hydroxyl radicals, with OH concentration increasing as Fe³⁺ levels rise. However, an excess of Fe³⁺ can consume a significant amount of OH, leading to the ineffective decomposition of H₂O₂. Furthermore, Figures 2B,D showed that the Fv/Fm ratio gradually decreased over time (from 1.5 h to 4 days), indicating that the algicidal effect took several days to manifest. This suggests that the appearance of algicidal effects lagged by a few days.

Light radiation played a crucial role in the eradication of bloom-forming cyanobacteria using Fenton-like reagent, enhancing the toxicity of H₂O₂ (Sukenik and Kaplan, 2021). The use of Fenton-like reagents in the presence of light was markedly effective in killing algae because light radiation also served as a catalyst, promoting the production of OH. Additionally, light can increase the transformation efficiency between Fe²⁺ and Fe³⁺, thereby allowing the Fenton reaction to proceed to completion (Mao et al., 2024).

Based on the aforementioned findings, eight primary bloom-forming cyanobacteria were selected for treatment, and the results are depicted in Figure 4. After treatment with Fenton-like reagents (H₂O₂ = 10 mg/L; organic Fe³⁺=3 m/L), all eight selected cyanobacterial strains exhibited various reductions in Fv/Fm, indicating the varying efficacy of Fenton-like reagents in controlling different cyanobacterial species. With the exception of strain E, the other seven strains suffered fatal damage, leading to the subsequent death of the majority of cyanobacteria in the following days. These results confirmed that the combination of H₂O₂, organic iron, and light radiation exerted a significant algicidal effect. It is noteworthy that the elimination of cyanobacteria cells may lead to the rise of algae membrane permeability (Supplementary Figure S2) and the release of cyanotoxins (Schneider and Bláha, 2020), posing significant ecological and health risks to the aquatic environment.

To explain why organic iron catalyzes H₂O₂ to kill cyanobacteria more effectively than inorganic iron. Two hours after the addition of the Fenton reagents, iron concentration was measured, as shown in Figure 5A. Regardless of whether in the MA solution or the MP solution, Fe³⁺ remained the predominant species after 2 h of reaction in both groups (inorganic iron and organic iron). The total iron concentration in Group 1 (inorganic iron, FeSO₄) was significantly lower than that in Group 2 (organic iron, EDTANaFe). Figure 5A also indicated that only about 10% of Fe^(II) remained in solution for Group 1, whereas 35% of Fe^(II) was still present in the solution for Group 2. These findings suggest that the majority of the iron salt in Group 1 might have precipitated onto the surface of the algae cells, while the iron salt in Group 2 remained in a dissolved state. This aligns with several studies that have explored the interplay between Fe^(II) and Fe^(III) (Fujii et al., 2010; Wang et al., 2017).

To investigate the distribution of iron on the surface of algae cells, SEM images of the two sample groups were obtained. Figures 5B–E show complex compounds surrounding the cell surfaces, composed of EPS. Figures 5B,D reveal that the percentage of iron on the surface of MA and MP (inorganic iron, group 1) was 25.99% and 20.74%, respectively, exceeding the percentage of iron (9.72% and 6.58%) in group 2 (organic iron). This indicates a greater amount of iron deposition and adsorption on the surface of cyanobacterial cells in group 1. This difference can be attributed to the tendency of FeSO₄ to form larger particles (goethite) that are more readily adsorbed by the EPS on the cell surface, as demonstrated in Figure 2, while organic iron remained dissolved throughout the entire duration. These findings suggest that the physical characteristics of iron compounds, influenced by their chemical form, significantly affect their interaction with algal cells (Mishra et al., 2011).

Figure 6 further illustrates the particle sizes in the FeSO₄ and EDTANaFe solutions, revealing that the particle size in the FeSO₄ solution was significantly larger than that in EDTANaFe solution at the same iron concentration. In both solutions, particle size increased progressively with rising iron concentrations. Iron in chelated states, such as in the EDTANaFe solution, remained dissolved, whereas the inorganic iron in the FeSO₄ solution existed in particulate form. This difference explains why chelating agents like EDTANaFe are crucial for maintaining catalytic properties during Fenton-like processes (Wang et al., 2017).

Iron oxides, such as goethite, are more readily adsorbed by EPS surrounding the surface of algal cells (as shown in Figure 2). Daoyong Zhang’s research suggested that EPS from biofilms were important organic ligands for metal complexation and can significantly affect their chemical forms, mobility, bioavailability, and ecotoxicity (Zhang et al., 2010). As a result, there was a reduction in the catalytic components available in the solution. Conversely, the Fe³⁺ ions in the EDTANaFe solution were relatively difficult to adsorb onto the surface of the cells. This difference in adsorption behavior affected the availability of iron for catalytic reactions in the water, potentially impacting the efficacy of algae treatments involving these iron forms (Li et al., 2016). Therefore, we may confidently conclude that: Fe²⁺ in the FeSO₄ solution was oxidized to Fe³⁺ and subsequently precipitated as sediment (goethite), whereas the Fe³⁺ in the EDTANaFe solution remained in a chelated state, preserving its catalytic activity.

The specific interactions of chelates are relatively complex. However, for the Fe-EDTA chelate system, Mingqiong Wang (Wang and Xu, 2012) have detailed how EDTA interacts specifically with iron ions during the degradation of organic matter (as Equations 2, 3). The presence of EDTA acts like a crab, competing with other anions to capture iron ions. This competition alters its precipitation equilibrium state, as defined by the equilibrium constant Log K (Kontoghiorghes, 2020). Thus, EDTA ensures that iron remains in a hydrated ionic state, facilitating the continuous cycling of ferric (Fe³⁺) and ferrous (Fe²⁺) ions. This cycling process consistently generates reactive oxygen species (ROS) such as hydroxyl radicals (˙OH) and hydroperoxyl radicals (HO₂˙). Fe 2 + ⋯ EDTA + H 2 O 2 s → Fe 3 + ⋯ EDTA + ˙ OH s + OH − (2) Fe 3 + ⋯ EDTA + H 2 O 2 s → Fe 2 + ⋯ EDTA ⋯ H + + HO 2 ˙ s (3)

To elucidate the reasons why inorganic iron catalyzes the ineffectiveness of H₂O₂ in eliminating cyanobacteria in natural water bodies while organic iron catalyzes its effectiveness, we measured the variations of H₂O₂ concentration under six different conditions and monitored the corresponding yields of hydroxyl radicals, as depicted in Figure 7.

As shown in Figure 7A, in BG11 culture medium (with only H₂O₂ present), illumination (50 μmol m^-2s^-1) significantly enhanced the degradation rate of H₂O₂ and increased the production yield of hydroxyl radicals (Figure 7B). The addition of inorganic iron (FeSO₄) did not noticeably alter the trend of H₂O₂ degradation (Figure 7A). The addition of FeSO₄ also did not significantly increase the production of free radicals hydroxyl radicals (Figure 7B). Overall, it was difficult for H₂O₂ catalyzed by inorganic ferrous iron to produce hydroxyl radicals under neutral conditions (Wang et al., 2012).

The addition of organic iron (EDTANaFe) significantly accelerated the degradation rate of H₂O₂ under both dark and illuminated conditions, achieving complete degradation of 10 mg/L H₂O₂ within half an hour (Figure 7A). However, Figure 7B indicated that although H₂O₂ degraded rapidly, the production yield of hydroxyl radicals was low under dark conditions, suggesting that H₂O₂ was predominantly undergoing ineffective decomposition. Conversely, under illuminated conditions, the degradation of H₂O₂ produced a significant amount of OH (53.5 μM within 5 min), indicating effective decomposition. This is also why EDTANaFe exhibits significant algicidal effects only under light conditions. The main reason for the effective degradation of H₂O₂ might be that, under light radiation, the catalytic reaction activation energy (Ea) for the process generating free radicals is lower compared to that in the absence of light radiation (Fındık, 2022). This change in energy states influences the production of free radicals, affecting the overall algicidal effect. Consequently, it can be affirmed that in natural water bodies, achieving optimal cyanobacteria eradication with H₂O₂ (10 mg/L) necessitates the addition of a small amount of organic iron (EDTANaFe, 3 mg/L) under illuminated conditions.