Introduction

Hydrogen peroxide (H2O2) is the simplest peroxide and is also a powerful oxidizing agent. This molecule can occur naturally in aquatic environments from dissolved chromophoric organic materials exposed to sunlight (Clark et al. 2014). However, the major natural source of H2O2 is biologically generated by phytoplankton (Diaz and Plummer 2018). The natural H2O2 concentrations in ponds or lakes are very low (below 5.3 μM) (Ndungu et al. 2019; Weenink et al. 2021). Reactive oxygen species, including H2O2, at a low concentration usually serve as important signaling molecules (Neill et al. 2002). Cyanobacteria are a diverse group of prokaryotic microorganisms that perform oxygenic photosynthesis and widely exist in freshwater, marine, and terrestrial ecosystems. The application of exogenous H2O2 in the treatment of toxic cyanobacteria is environmentally friendly because of its decomposition to O2 and H2O. The H2O2 dosage applied is crucial for the compositional changes in phytoplankton and zooplankton communities (Weenink et al. 2015, 2021). High doses of exogenous H2O2 (e.g., 0.06 ~ 0.29 mM) have been reported to selectively eliminate toxic cyanobacteria such as Microcystis aeruginosa and Planktothrix agardhii, with a minimal impact on eukaryotic phytoplankton and zooplankton (Matthijs et al. 2012; Spoof et al. 2020; Santos et al. 2021). A biocidal level of H2O2 is supposed to trigger intracellular reactive oxygen species and result in severe oxidative stress and subsequent cell death (Drábková et al. 2007; Foo et al. 2020).

Many cyanobacteria are known to be able to synthesize extracellular polysaccharides or exopolysaccharides (EPSs) (Philippis and Vincenzini 1998). Although the functions of microbial EPS vary with the species, it serves as one of the primary mechanisms for cell survival in extreme habitats and defense against toxins, heavy metals, and antagonists (Rossi and De Philippis 2016; Bhatnagar and Bhatnagar 2019). Cyanobacterial EPS also exhibits antioxidant activity (Huang et al. 2017; Zhong et al. 2019; Yuan et al. 2023), and antioxidant activity is associated with multiple factors, such as molecular weight, monosaccharide composition, sulfate position and sulfate degree (Mohan and Thirupathi 2021). It has been reported that H2O2 levels above 0.27 mM are lethal to M. aeruginosa (Foo et al. 2020). In contrast, another study showed that Microcystis populations with a colony size below 25 μm collapsed under 0.15 mM H2O2, while a H2O2 level of 0.59 mM was needed to control larger Microcystis colonies (Liu et al. 2017a). The formation of cyanobacterial colonies is mainly facilitated by EPS (Limoli et al. 2015). The difference in the biocidal level of H2O2 may hint that it is crucial for cyanobacterial cells to have a high abundance of EPS to combat external H2O2 stress.

A previous study reported that the extracellular polymeric substance of M. aeruginosa, mainly composed of polysaccharides and proteins (approximately 3:1 ratio in weight), showed the capability to buffer H2O2 (Gao et al. 2015). To clarify the crucial role of the EPS itself in combating exogenous high-dose H2O2, we evaluated the H2O2 buffering or scavenging capability as well as the protective role of the EPSs from four nonbloom-forming cyanobacteria with different thicknesses of extracellular polymers. This study deepens our understanding of the crucial role of EPS in combating external oxidative stress and provides reference information for guiding better application of H2O2 in environmental management.

Materials and methods

Cyanobacterial strains and culture conditions

The aquatic-living culture of Nostoc flagelliforme was previously prepared from a terrestrial natural sample in our laboratory (Cui et al. 2017). Its natural sample, presenting a filamentous colony form, consists of a large number of trichomes encased by dense EPS. Nostoc punctiforme PCC 73102 (hereafter referred to as N. punctiforme) was obtained from Pia Lindberg (Department of Chemistry-Ångström, Uppsala University), which can also produce a large amount of EPS (Soule et al. 2016). The N. flagelliforme and N. punctiforme strains were statically cultured in Blue Green-11 (BG-11) medium at 25 °C under constant white LED light of 40 µmol photons m−2 s−1. Two freshwater strains, Nostoc sp. PCC 7120 (hereafter referred to as Nostoc 7120) and Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis 6803), were statically cultured in BG-11 medium at 30℃ under the same light intensity. These cultures were gently shaken three times per day. Cell cultures at the exponential growth phase, with an optical density at 730 nm (OD730) of 0.2 ~ 0.3, were used for subsequent experiments.

Analysis of the H2O2 tolerance of four cyanobacteria

The H2O2 stock solution (30%, v/v; Damao®, Tianjin Damao Chemical Reagent Factory, China) was diluted to 100 mM with sterile water for use. Cell cultures of 20 mL were used to test the tolerance to exogenous H2O2 in 50 mL conical flasks. The cell cultures were first treated with 2 mM or 4 mM H2O2 (final concentration). For a rapid estimation of the physiological status of cells, the chlorophyll fluorescence parameter Fv'/Fm' was determined, which reflects the photochemical efficiency of open photosystem II centers under a given light acclimation status (Campbell et al. 1998). At different time points (0~24 h), 2 mL of cell culture was collected and subjected to detection using a portable Plant Efficiency Analyzer (AquaPen FP110, Czech Republic) according to the manufacturer’s instructions.

For the cell growth assay, cell cultures of 20 mL were treated with various concentrations of H2O2 (0 ~ 20 mM, final concentration). Then, the H2O2-treated cell cultures were cultivated for 4 days under the abovementioned conditions. Cell growth was monitored by measuring the concentration of chlorophyll a (Chl a). Chl a was extracted and quantified as previously described (Zhao et al. 2008). Briefly, 3 mL of cell suspension was concentrated by centrifugation at 6000 rpm for 5 min (TGL-16 M, Hunan Xiangyi Laboratory Instrument Inc., China) and then extracted with 3 mL 95% ethanol overnight at 4 °C. The absorbance was measured at 664.1 and 648.6 nm using an ultraviolet‒visible spectrophotometer (Shanghai Spectrum Instruments Inc., China). The concentration of Chl a was calculated using the following equation: Chl a (μg mL−1) = 13.36 × A664.1–5.19 × A648.6 (Lichtenthaler and Buschmann 2001).

Extraction and purification of EPS from cell cultures

The extraction of EPS was conducted with hot water as previously described (Huang et al. 1998) with slight modification. In brief, 10 mL of cell culture was extracted in 95 °C hot water for 2 h, and after centrifugation, the polysaccharide in the supernatant was precipitated overnight at 4 °C with ethanol (final concentration 80%, v/v). The crude polysaccharide was collected, dissolved in distilled water, and deproteinized with Sevag reagent (n-butanol:chloroform = 1:4, v/v) (Zheng et al. 2021). The purified EPS was freeze-dried, weighed, and dissolved in distilled water for use. The polysaccharide content was determined using the phenol‒sulfuric acid method (DuBois et al. 1956). To verify that there was no contamination of proteins, the polysaccharide solution (1 mg mL−1) was scanned by an ultraviolet‒visible spectrophotometer from 200 to 400 nm.

Analysis of the H2O2 scavenging ability of the EPSs

The purified polysaccharide was added to a 5 mL H2O2 solution (2 mM) with final concentrations of 1 and 5 mg mL−1. The H2O2 solution without the polysaccharide addition (0 mg mL−1) was used for comparison. Then, the H2O2 concentration in the solution was determined at different time points (0~3 h) using the Hydrogen Peroxide Assay Kit (Beyotime Biotechnology Inc, Shanghai, China) according to the manufacturer’s instructions (Zang et al. 2018). The kit uses H2O2 to oxidize Fe2+ to Fe3+, leading to the formation of a purple color in a specific solution. Absorbance was read at 560 nm using a microplate reader (Infinite M Nano, Tecan, USA). The relative changes (%) in H2O2 concentration were calculated.

Analysis of EPS protection of cells against exogenous H2O2

The above purified polysaccharides from the N. flagelliforme and N. punctiforme cultures were added into the cell cultures (20 mL) of Synechocystis 6803 and Nostoc 7120, respectively, with a final concentration of 0.05 or 0.10 mg mL−1. The control cultures were not supplemented with the polysaccharides (0 mg mL−1). Then, the cultures were treated with 1 mM H2O2 (final concentration). Subsequently, the physiological response of the treated cells, in terms of the Fv'/Fm' value, was analyzed at different time points (0~6 h). In addition, cell growth was monitored by measuring the Chl a content at 0, 2, and 4 days.

Analysis of the H2O2 scavenging ability of cell lysates

The cell lysates of four cyanobacteria were prepared as previously described (Weenink et al. 2021). In brief, 5 mL of cell culture was centrifuged at 6000 rpm for 5 min, and the cell pellets were resuspended in 5 mL phosphate buffered solution (0.01 M, pH 7.4). The cells were further disrupted at 4 °C in a 10 mL glass homogenizer (tissue grinding tube) and were ground manually with a ground glass pestle in the tube for 10 min. The cell debris was removed by centrifugation at 4 °C for 5 min. The supernatant (cell lysate) was collected and subjected to protein detection by the Bradford method (Bradford 1976) and polysaccharide detection by the phenol‒sulfuric acid method (DuBois et al. 1956). The ratio of the polysaccharide to protein (mg mg−1) in cell lysates was calculated. Furthermore, the scavenging abilities of cell lysates against H2O2 were assayed in 5 mL H2O2 solution (1 mM) by supplementing the cell lysate containing 0.5 mg soluble proteins. The H2O2 concentration in the solution was measured, and the relative change (%) was calculated.

Statistical analysis

For Fv'/Fm' detection, six replicates were performed. Other experiments except the growth assay in response to a wide concentration range of H2O2 were performed in three replicates. For column diagrams, the statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test (p < 0.05) (IBM SPSS Statistics 26).

Results

Different tolerances of four cyanobacteria to exogenous H2O2

In this study, four cyanobacteria available in our laboratory were used for experiments. Three of them, Synechocystis 6803, Nostoc 7120, and N. punctiforme, are widely used model strains; the last strain, N. flagelliforme, is becoming a new model strain, particularly along with its genome being sequenced (Shang et al. 2019). Synechocystis 6803 and Nostoc 7120 strains with thin EPS envelopes are referred to here as EPS-thin strains; N. punctiforme and N. flagelliforme strains are usually present as visible particles or colonies in culture due to their abundant EPS and are referred to here as EPS-rich strains. The responses of their cells to exogenous H2O2 were first assessed in a short-term test (Fig. 1). Fv'/Fm' serves as a quick indicator of the photosynthetic performance of algae and plants under abiotic stresses (Singh and Raja Reddy 2011; She et al. 2022). In terms of Fv'/Fm', the physiological activities of the two EPS-thin strains were rapidly reduced to zero after either 2 or 4 mM H2O2 treatment. However, the physiological activities of the two EPS-rich strains were rapidly inhibited in the initial several hours and then gradually recovered; unlike the N. punctiforme strain, the activity of the N. flagelliforme strain did not recover to its original level under 4 mM H2O2 conditions.

Fig. 1
figure 1

Photophysiological activities (in terms of Fv'/Fm') of four cyanobacteria in response to exogenous H2O2. A, the treatment of cultures with 2 mM H2O2. B, the treatment of cultures with 4 mM H2O2. Data shown are the mean ± SD (n = 6). S. 6803, Synechocystis sp. PCC 6803; N. 7120, Nostoc sp. PCC 7120; N. pun, Nostoc punctiforme PCC 73102; N. fla, Nostoc flagelliforme

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Furthermore, the cell growth of the four strains, in terms of Chl a content, was assessed in response to exogenous H2O2 (Fig. 2). In the relatively long-term treatment (4 days), an expanded concentration range of H2O2 was used. The Synechocystis 6803 and Nostoc 7120 strains showed similar tolerance to H2O2, and both strains were not tolerant to 2 mM H2O2. In contrast, N. punctiforme and N. flagelliforme strains were much more tolerant to H2O2. The N. punctiforme strain was not tolerant to H2O2 levels above 16 mM, while the N. flagelliforme strain was not tolerant to H2O2 levels above 4 mM. The appearances of these cultures in glass flasks after H2O2 treatment were consistent with the above observations (Supplementary Fig. S1).

Fig. 2
figure 2

The cell growth (in terms of Chl a content) of four cyanobacteria after treatment with exogenous H2O2. A and B, the growth of Synechocystis 6803 and Nostoc 7120, respectively. Cells were treated with 0 ~ 2.0 mM H2O2. C and D, the growth of N. punctiforme and N. flagelliforme, respectively. Cells were treated with 0 ~ 20 mM H2O2

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The scavenging ability of the EPSs against H2O2

The EPS encases the cyanobacterial cell and can also facilitate the formation of the colony; thus, its protective function by scavenging H2O2 should be important. The H2O2 scavenging abilities of the EPSs from four cyanobacteria were assayed (Fig. 3). The EPSs were extracted from the four cell cultures with proteins removed. A slow natural decomposition of H2O2 was observed, but all the EPSs could greatly accelerate the decomposition rate. At 3 h, 31.1% and 63.0% of the initial H2O2 was scavenged by 1 and 5 mg mL−1 Synechocystis 6803 EPS, respectively; the scavenging rates were 34.0% and 61.3% for 1 and 5 mg mL−1 Nostoc 7120 EPS, 43.8% and 72.1% for 1 and 5 mg mL−1 N. punctiforme EPS, and 35.0% and 70.7% for 1 and 5 mg mL−1 N. flagelliforme EPS, respectively. It was also noted that rapid H2O2 decomposition by these EPSs was observed within 1 h.

Fig. 3
figure 3

The scavenging ability of four EPSs against H2O2. A, the Synechocystis 6803 EPS. B, the Nostoc 7120 EPS. C, the N. punctiforme EPS. D, the N. flagelliforme EPS. The polysaccharide concentrations in 2 mM H2O2 solution were 0, 1.0, and 5.0 mg mL.−1. The relative changes (%) in H2O2 concentration in the solutions were calculated. Data shown are the mean ± SD (n = 3)

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Protection of the EPSs against exogenous H2O2 in cyanobacteria

As shown in Figs. 1 and 2, Synechocystis 6803 and Nostoc 7120 were more sensitive to H2O2. Thus, their cultures were chosen for physiological activity and cell growth assays upon H2O2 treatment after supplementation with additional EPSs. The physiological activity (in terms of Fv'/Fm') changes of Synechocystis 6803 and Nostoc 7120 cells against H2O2 were first assayed (Fig. 4). In the absence of additional EPSs (as the control), the physiological activities of the H2O2-treated Synechocystis 6803 and Nostoc 7120 cells rapidly decreased to zero or near zero within 3 h. In the presence of additional EPSs, the physiological activities of Synechocystis 6803 cells were stimulated within 1 h, slightly reduced at 3 h, and then recovered to a steady level at 6 h, while the physiological activities of Nostoc 7120 cells were rapidly reduced within 1 h and then recovered to some extent at 3 or 6 h. Furthermore, the cell growth (in terms of Chl a content) of Synechocystis 6803 and Nostoc 7120 after H2O2 treatment was assayed (Fig. 5). Cells were cultivated for 4 days following H2O2 treatment. With supplementation of additional EPSs, both Synechocystis 6803 and Nostoc 7120 cells showed better growth than their corresponding controls without EPS supplementation.

Fig. 4
figure 4

Photophysiological activities (in terms of Fv'/Fm') of two EPS-thin cyanobacteria after H2O2 treatment in the presence of additional EPSs. The concentrations of additional EPSs in the cultures were 0, 0.05, or 0.10 mg mL.−1, and then the cultures were treated with 1.0 mM H2O2. A and B, Synechocystis 6803 cells protected by N. punctiforme EPS and N. flagelliforme EPS, respectively. C and D, Nostoc 7120 cells protected by N. punctiforme EPS and N. flagelliforme EPS, respectively. Data shown are the mean ± SD (n = 6)

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Fig. 5
figure 5

The cell growth (in terms of Chl a content) of two EPS-thin cyanobacteria after H2O2 treatment in the presence of additional EPSs. The concentrations of additional EPSs in the cultures were 0, 0.05, or 0.10 mg mL.−1, and the cultures were treated with 1.0 mM H2O2. A and B, Synechocystis 6803 cells protected by N. punctiforme EPS and N. flagelliforme EPS, respectively. C and D, Nostoc 7120 cells protected by N. punctiforme EPS and N. flagelliforme EPS, respectively. The letters (a, b) in each group of data suggest significant differences (P < 0.05, Tukey’s test). Data shown are the mean ± SD (n = 3)

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The H2O2 scavenging ability of cell lysates from four cyanobacteria

When the H2O2 dosage exceeds the scavenging capacity of the total polysaccharide, protection from intracellular antioxidant substances should also be important. Here, we evaluated the H2O2 scavenging ability of cell lysates from four cyanobacteria (Fig. 6). The cell lysates were characterized by detecting the protein and polysaccharide contents. The content ratios of polysaccharides to proteins were evaluated (Fig. 6A). The EPS/protein ratios of N. punctiforme and N. flagelliforme cells were 2.6- and 3.0-fold higher than those (0.31) of Synechocystis 6803 and Nostoc 7120 cells, respectively. The H2O2 scavenging abilities of the cell lysates were further assayed (Fig. 6B). Overall, all the cell lysates could efficiently scavenge H2O2. Despite having a much higher EPS/protein ratio, the cell lysates of the N. punctiforme and N. flagelliforme strains only showed slightly higher scavenging activities (below 5%) than those of the Synechocystis 6803 and Nostoc 7120 strains.

Fig. 6
figure 6

The scavenging ability of cell lysates from four cyanobacteria against exogenous H2O2. A, the relative contents of EPS to protein in cell lysates. The letters (a, b, c) on the columns suggest significant differences (P < 0.05, Tukey’s test). B, the H2O2 scavenging ability of cell lysates. Each cell lysate with an equal protein level (0.5 mg) was added to the H2O2 solution. The relative change (%) in H2O2 content in the solution was calculated. Data shown are the mean ± SD (n = 3)

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Discussion

Although a biocidal level of H2O2 has been widely applied in controlling boom-forming cyanobacteria, the resilience of other cyanobacteria to cope with a high dose of exogenous H2O2 is not well known. Extracellular polymeric substances have been reported to be crucial for combating various environmental stresses (Gao et al. 2015, 2019; Tamaru et al. 2005; Liu et al. 2017b). In the extracellular polymeric substance of M. aeruginosa, the protein content varied in a large range, with the EPS/protein ratio being 0.24 ~ 2.88:1 (Gao et al. 2015; Ni et al. 2017). Both polysaccharides and proteins function in H2O2 decomposition (Gao et al. 2015). However, studies of terrestrial cyanobacteria have shown that extracellular polymeric substances contain only a minor proportion of exoproteins (Stuart et al. 2016; Shirkey et al. 2000). Aquatic toxic cyanobacteria seem to have a relatively larger amount of exoproteins in their extracellular polymers. Taking the four nonbloom-forming cyanobacteria with emphasis on the polysaccharide itself, we further clarified the importance of EPS in combating exogenous high-dose H2O2.

The EPS-rich cyanobacteria were much more tolerant to exogenous H2O2 than the EPS-thin cyanobacteria (Figs. 1 and 2). Further investigation showed that the EPSs of four cyanobacteria exhibited efficient H2O2 scavenging capability (Fig. 3) and seemed to provide rapid and nonselective protection for cells (Figs. 4 and 5). However, the physiological responses of Synechocystis 6803 and Nostoc 7120 were somewhat different (Fig. 4), implying a species/strain-dependent difference in combating H2O2 stress. The H2O2 decomposition rate was positively associated with the concentration of EPS (Fig. 3), implying that the polysaccharide amount plays a critical role. The H2O2 scavenging ability of the cell lysate was also observed (Fig. 6), which might help rescue the damaged cells to recover vitality. In previous studies, the activities of polysaccharides were mostly biochemically evaluated with the sensitive substrates 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, and hydroxyl radical (Zhong et al. 2019). With N. flagelliforme as an example, cultural conditions and environmental treatment could also affect the antioxidant activity of the produced EPS (Shen et al. 2018; Yuan et al. 2023). In contrast, this study demonstrated a similar capability of four EPSs in combating a high dose of H2O2. Due to its phycosphere distribution, the amount of EPS should play a crucial role in cells against exogenous strong oxidative stress.

Although the cyanobacteria used in this study were not bloom-forming strains, they should still shed light on the understanding of environmental management. As implied in Fig. 3, the scavenging ability of the EPSs for exogenous H2O2 was rapid but also limited. In other words, the EPS amount plays a pivotal role. H2O2 sensitivity has been reported to sometimes be very different in the same strains (Liu et al. 2017a; Foo et al. 2020). The H2O2 tolerance concentrations of the N. punctiforme and Nostoc 7120 strains shown in this study were also different from those reported by Samanta et al. (Samanta et al. 2022). The reasons might be largely attributed to the EPS content per cell, colony size (still relevant to the EPS content), or ambient polysaccharide amount. In addition, the H2O2 sensitivity of cyanobacterial cells also depends on other factors, such as nutrient availability, metal ions, light intensity, heterotrophic bacteria, and eukaryotic algae (Drábková et al. 2007; Shen et al. 2011; Xu et al. 2016; Sandrini et al. 2020; Weenink et al. 2021; Moreno-Andrés et al. 2022), which increases the difficulty in precisely assessing the H2O2 dosage for environmental use. Considering the first-line role of EPS in H2O2 decomposition, the total polysaccharide amount in freshwater may be a primary consideration in determining H2O2 dosage for application.

In summary, this study suggests that cyanobacterial EPSs play a general role against exogenous high-dose H2O2 and also the polysaccharide amount plays a crucial role in protecting cells against external strong oxidative stress. This study provides a useful reference for the application of H2O2 in environmental management.