Investigation of cellular structure and metabolic activity of Microcystis cells treated by chlorination

Water eutrophication has become common in recent years as a result of global warming and excessive nutrient loads, and frequent outbreaks of cyanobacterial blooms were observed in many lakes and reservoirs (Smith 2003; Griffith & Gobler 2020). The advent of cyanobacterial blooms can induce a series of environmental issues, including the deterioration of water quality and the collapse of ecological systems (Xia et al. 2008; Jia et al. 2011). Cyanobacteria-laden source waters can challenge the water treatment process in drinking water treatment plants (DWTPs), such as the blocking of filtering materials, increased coagulant usage, membrane hole plugging, membrane contamination, and so on. Besides, cyanotoxins produced by cyanobacterial cells can pose a hazard to the safety of drinking water worldwide (Codd et al. 2005; Merel et al. 2013).

Chlorine has been widely employed as a pre-, inter- and post-oxidant in DWTPs for the overall elimination of cyanobacterial cells and cyanotoxins due to its low cost and user-friendly. Pre-chlorination is employed to enhance the coagulation process to remove cyanobacterial cells (Plummer & Edzwald 2002). Post- or inter-chlorination is used to degrade cyanotoxins to some degrees (Acero et al. 2008; Fan et al. 2016). However, chlorination can induce the destruction of cellular membrane of cyanobacteria and the release of intracellular cyanotoxins is observed when cyanobacterial cells are inactivated (Daly et al. 2007; Zamyadi et al. 2012; Fan et al. 2013, 2016; Li et al. 2020a, 2020b).Fan et al. (2014) found that the rate constant of extracellular cyanotoxin degradation was higher than the rate constant of intracellular cyanotoxin release throughout the chlorination process, resulting in a continuous decrease of extracellular cyanotoxins. In contrast, other investigations found that chlorination resulted in an increase of extracellular cyanotoxins (Ding et al. 2010; Li et al. 2020a). These inconsistent results are ascribed that previous studies did not well determine how different chlorination treatments affect the cellular structures of cyanobacterial cells, especially for the cellular surface responsible for membrane integrity. Thus, in this study, the effects of chlorination on the cellular structure of cyanobacterial cells should be carefully studied.

Besides, chlorine can be used as an algicide to inactivate cyanobacterial cells, with the aim to control cyanobacterial blooms in lakes or reservoirs. Generally, the chlorination destroys cellular contents to inactivate cyanobacterial cells leading to the cytolysis, corrosion and wrinkling of the cellular wall and cytoplasmic membrane (Ou et al. 2011). Many field studies found that the application of chlorine inhibited cyanobacterial growth for a period of time, but cyanobacterial blooms re-emerged within 2–4 weeks. These field cases indicated that these cyanobacterial cells have a strong resistance to chlorination, and remain alive after chlorination treatments. In other words, the metabolic activity of cyanobacterial cells is not completely inactivated after chlorination. Cyanobacterial cells are autotrophic prokaryotic bacteria and they utilize the photosynthetic apparatus to mediate light-energy conversion to synthesis organic matters. Cyanobacterial cells have the respiration process to produce ATP via organic matter utilization. The two metabolic processes are quite important to support cyanobacterial growth. Previous studies certified that chlorination destroyed the photosynthetic process of cyanobacteria (e.g., Microcystis aeruginosa, Oscillatoria sp., Lyngbya sp.) (Zamyadi et al. 2012; Wert et al. 2013; He & Wert 2016; Greenstein & Wert 2019), but whether the chlorination affects the respiration process remains unknown. To well address the influence of chlorination on the metabolic process of cyanobacteria, this study should analyze the photosynthetic and respiratory activity of cyanobacterial cells after chlorination.

To date, no studies have systematically investigated the influences of chlorination on the cellular structure and metabolic activity of cyanobacterial cells. In this study and after chlorination with various initial dosages (0.5–8.0 mg L⁻¹), the cellular size, cellular surface, and cellular structure of Microcystis cells were investigated using a laser particle size analyzer, scanning electron microscopy (SEM), and transmission electron microscope (TEM), respectively. Meanwhile, both photosynthetic and respiratory activity of Microcystis cells were measured via the analyses of chlorophyll fluorescence kinetics and using an enzyme linked immunosorbent assay, respectively. This study would provide a guideline of chlorine as an oxidant or algicide to inactivate cyanobacterial cells and control cyanobacterial blooms in reservoirs or lakes.

Microcystis aeruginosa is the most dominating species for cyanobacterial blooms (He et al. 2016), and thus, M. aeruginosa FACHB-915 was used for the chlorination experiments. This strain was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. The strain was cultivated in the BG11 media under light operating at 12 h day, 12 h night cycles at a temperature of 25 °C in an incubation chamber fitted with a cold light source Light Emitting Diode (LED) (GXZ-280C; China) (Li et al. 2020a, 2020b).

The BG11 medium's reagents were bought from Sinopharm (Shanghai, China). The pH was adjusted using sodium hydroxide (NaOH, Sinopharm, Shanghai, China) and hydrochloric acid (HCl, Sinopharm, Shanghai, China). The cell density of Microcystis samples was measured by microscopic counting after staining with the SYTOX green nucleic acid dye (Thermo Fisher, USA). Sodium hypochlorite commercial solutions (active chlorine content > 5.2%) were used to prepare a chlorine stock solution. In order to neutralize the residual chlorine, sodium thiosulfate (AR, Sinopharm, Tianjin, China) was used to terminate this reaction. To examine the morphology of Microcystis cells, the following chemicals were used, including phosphate buffer saline (PBS = 10 mM, pH = 7, Solarbio, China), glutaraldehyde (25% (v/v), Sinopharm, China), ethanol (Sinopharm, China), acetone (Sinopharm, China), embedding solution (Solarbio, China), uranium acetate, and lead citrate. The respiratory activity of Microcystis cells was measured using a Micro Na⁺/K ⁺-ATPase Assay Kit (Solarbio, Beijing, China). All solutions were made with deionized water that had been filtered with a Milli-Q water purification system to a resistivity of 18 MΩ cm (Millipore Pty Ltd, USA). All reagents were analytical grade.

To conduct the chlorination experiments, Microcystis cells were collected in the exponential phase. International Joint Commission and Ohio Environmental Protection Agency (EPA) have set chlorophyll at a threshold of 50 mg L⁻¹ for severe blooms, and the corresponding cell density was greater than 1.0 × 0⁵ cells mL⁻¹ (Watson & Boyer 2014; Kasich et al. 2014). Besides, previous researchers have performed extensive chlorination experiments using Microcystis cells of 10⁵–10⁶ cells mL⁻¹ (Table S1) (Daly et al. 2007; Zamyadi et al. 2012, 2013; Fan et al. 2013, 2014; Qi et al. 2016). Thus, Microcystis cells were diluted with double-distilled water (ddH₂O) to achieve a cell suspension concentration of 1.0 × 10⁶ cells mL⁻¹, as the same studies of Li et al. (2020a, 2020b). In addition, chlorination efficiency was highly dependent on the pH value of source waters and it could be significantly reduced above 8.0 (Nicholson et al. 1994), and thus, the pH value of the Microcystis samples was adjusted to 7.5 ± 0.1 using 0.1 M hydrochloric acid or sodium hydroxide (Daly et al. 2007; Fan et al. 2013).

For chlorination experiments, previous studies used the applied dosages of <8 mg L⁻¹ for Microcystis cells at exponential phase (Table S1) (Daly et al. 2007; Zamyadi et al. 2012, 2013; Fan et al. 2013, 2014, 2016; He & Wert 2016; Qi et al. 2016). Herein, Microcystis samples were treated with various chlorine doses (0.5, 1, 2, 4, and 8 mg L⁻¹). Microcystis cultures were incubated without chlorination as a control group. All chlorination studies were done in amber glass vials in the dark at 25 °C. During chlorination, Microcystis sample of 300 mL was taken at specified contact times of 0, 10, and 60 min. At each time interval, Microcystis samples with specific volumes were quenched with sodium thiosulfate at a stoichiometric ratio specified in Standard Methods (APHA et al. 1998), and prepared for the following analysis of cellular structures and metabolic activity of Microcystis cells.

A flow cytometry (Merck Millipore, USA) was used to quantify the cell-density of Microcystis samples, and more details are described in Li et al. (2020b).

The N, N, diethyl-p-phenylene-diamine (DPD) colorimetric method was used to measure the free chlorine concentration (APHA et al. 1998).

The dispersion of cellular grain size reflects the changes of morphological size. A beaker was filled with 200 mL of Microcystis solutions, and these samples were fully dispersed using ultrasonic methods. Then, the particle size distribution of these suspensions was measured using a laser particle size analyzer (Malvern Matersiser 2000, UK). The analytical range was set as 0.02–2000 μm to determine the particle size distribution of Dx(10), Dx(50), and Dx(90), as well as the volume average particle size D[4,3].

The effects of chlorination on the cellular surface of Microcystis cells were investigated using a scanning electron microscopy (SEM) (Mitra et al. 2018). Microcystis cells of 10 mL were harvested using 6000 g for 5 min at 4 °C, subsequently washed twice using the phosphate buffer saline (PBS = 10 mM, pH = 7). These cells were fixed overnight at 4 °C with 2.5% glutaraldehyde and then dehydrated with a graded series of ethanol solutions for 10 min each in 30, 50, 70, 80, 90 and 100% (twice) ethanol. Cells were dried with a critical point drier for 24 h, coated with a thin layer of gold and examined by the SEM (Hitachi S-4800, Japan) (Hitachi S-4800, Japan).

The transmission electron microscope (TEM) (Hitachi H-7650, Japan) was used to examine the microstructures of Microcystis cells (Li et al. 2020a). Cells were collected (10 mL), fixed, and dehydrated, followed by the same steps of SEM. The TEM samples were cured in an oven at 45 °C for 12 h after being buried overnight in pure acetone and embedding solution (1:2). With an ultrathin slicer, these samples were sliced into 50–60 nm slices and dyed with 3% uranium acetate and lead citrate. Finally, TEM observation was carried out for the cellular structures of Microcystis cells.

These data were processed using Excel 2017. Prism8.2, Origin2017. SPSS25 were used for data analysis and statistical tests for graphs and tables. All experiments were conducted in triplicate, and error bars in the graphs represent the standard deviation (SD) values from triplicate analyses.

Meanwhile, when the dosage of chlorine increased from 0.5 to 8.0 mg L⁻¹, the value of D[4,3] of Microcystis cells increased from 4.21 to 26.9 μm, especially for the chlorination of 8.0 mg L⁻¹ (Table 1). These was no significant difference of Dx(10) of Microcystis cells after chlorination of 0.5–8.0 mg L⁻¹ (P> 0.05) (Table 1). However, the value of Dx(50) of Microcystis cells increased from 2 to 29% and the value of Dx(90) strikingly increased from 37 to 850% (p< 0.05) (Table 1).

Meanwhile, the changes in Y(II) values of Microcystis cells after chlorination at 10 and 60 min, were further measured (Figure 4(c) and 4(d)). The trend of Y(II) values was almost the same for the chlorination at 10 and 60 min (Figure 4(c) and 4(d)). The value of Y(II) went down to zero when chlorine of 4.0 and 8.0 mg L⁻¹ were used to treat Microcystis cells (Figure 4(c) and 4(d)). The Y(II) of Microcystis cells with chlorination of 0.5 mg L⁻¹ was higher than the control (p< 0.05), but the Y(II) of Microcystis cells treated by the chlorination of 1.0 mg L⁻¹ did not significantly change compared with the control treatment (p> 0.05) (Figure 4(c) and 4(d)). Moreover, the Y(II) value of Microcystis cells treated by the chlorination of 2.0 mg L⁻¹ was significantly lower than the control treatment (p<0.05) (Figure 4(c) and 4(d)).

The respiration activity of Microcystis cells was characterized using the value of the Na⁺/K⁺ ATPase activity. The Na⁺/K⁺ ATPase activity of Microcystis cells was greatly affected by the chlorination, and their values showed a tendency of increase-reduction over the elevated dosages of chlorine (Table 2). In comparison with the control, the chlorination of 0.5 mg L⁻¹ decreased the activity of Na⁺/K⁺ ATPase at 10 and 60 min (p< 0.05) (Table 2). The activity of Na⁺/K⁺ ATPase began to rise after the chlorination of 1.0 and 2.0 mg L⁻¹ and its value was greater than the control treatment (p< 0.05) (Table 2). The Na⁺/K⁺ ATPase activity peaked at chlorination of 2.0 mg L⁻¹, which was higher by 3.78 times (10 min) and 1.81 times (60 min) than the control treatment (p< 0.05) (Table 2). When the chlorination of 4.0 mg L⁻¹ was used to treat Microcystis cells, the Na + /K + ATPase activity strikingly reduced (p< 0.05) (Table 2).

Chlorination of 0.5 mg L⁻¹ did not change the particle size distribution of Microcystis cells, and it presented a symmetrical normal curve of 2–10 μm (Figure 1). However, a small convex approximate normal distribution curve of 10–100 μm was observed, forming an asymmetric bimodal curve after chlorination of 1.0–8.0 mg L⁻¹, (Figure 1). Especially for the chlorination of 8.0 mg L⁻¹, the value of Dx(90) was more than 10 times the control treatments (Figure 1). This finding suggested that chlorination improved the particle size distribution of 10–100 μm that was not found by previous studies. Generally, chlorination destroyed the cellular surfaces (e.g., gelatinous layer) of Microcystis cells, as evidenced in Figure 2. Theoretically, the cellular size of these Microcystis cells became lower after chlorination, compared with the control treatment, suggesting that chlorination made Microcystis cells weak. The phenomenon of Microcystis cells aggregates were also observed, and found to be spherical or ellipsoidal shape treated by the chlorination (0.5–8.0 mg L⁻¹) (Figure 2).

Although the cellular surface of Microcystis cells was destroyed via the oxidation of polysaccharides/peptides (Figure 2), the majority of Microcystis cells maintained a roughly spherical or ellipsoidal shape treated by the dosages of chlorination (0.5–8.0 mg L⁻¹). It indicated that chlorination did not split the intact Microcystis cells into various fragments, and thus, Microcystis cells had a good resistance to cell lysis. This finding was different with treatments of electrochemistry (Zhou et al. 2019), ultrasound (Kumon et al. 2009), and other approaches for cell lysis of Microcystis. Notably, chlorination did not induce cell lysis, but it caused the destruction of intracellular structures to some degrees, in agreement with Kumon et al. (2009) and Ou et al. (2011), since chlorination has a wide oxidative capacity for various organics, including peptides, fatty acids etc. (Oyekunle et al. 2021; Zhang et al. 2021). The photosynthetic lamellar was destroyed mainly due to the oxidative reaction with their organic composition observed by the TEM. Moreover, when Microcystis cells were treated by the chlorination of 4.0 and 8.0 mg L⁻¹, these cells showed an apparent plasmolysis mainly ascribed to the decrease of osmotic pressure (Figure 3(d)–3(f)). As is known, the stability of osmotic pressure of Microcystis cells was determined by the intracellular organic matters, inorganic salts, and liquids. After chlorination, the membrane destruction resulted in the release of these intracellular organic matters and inorganic salts (e.g., K⁺) (Ma et al. 2012a, 2012b; Wert et al. 2013; He & Wert 2016; Li et al. 2020a), leading to a significant decrease of the cellular osmotic pressure.

The rise of PAR promoted the value of rETR, suggesting that Microcystis cells were sensitive to the light intensity radiation and elevated light intensity radiation improved the photosynthetic rate of Microcystis cells. Interestingly, the chlorination of 0.5 mg L⁻¹ significantly improved the value of rETR, demonstrating that low-level chlorination had a stimulating effect on the electron transport rate of photosynthesis process in Microcystis cells, which was not reported in previous studies. In contrast, elevated dosages of chlorination (2 mg L⁻¹) exhibited an inhibitory effect on the electron transport rate of the photosynthesis process, since it has destroyed the photosynthetic center. Moreover, Microcystis cells produced amounts of reactive oxygen species (ROS) to impede the photochemical process (Wei et al. 2020). With chlorination of 4.0 and 8.0 mg L⁻¹, the value of rETR was close to zero, suggesting that the photosynthetic activity of Microcystis cells were completely inactivated via blocking its electron transport. The similar results were also observed in previous studies of Wert et al. (2013); He & Wert (2016); Greenstein & Wert (2019) . The Y(II) of Microcystis cells also showed the same pattern. Overall, low dosages of chlorination (<0.5 mg L⁻¹) promoted photosynthetic activity of Microcystis cells, but high dosages of chlorination (2, 4, and 8 mg L⁻¹) effectively suppressed the photosynthetic activity of Microcystis cells.

The Na⁺/K ⁺-ATPase is the most important ion pump to transport Na⁺/K⁺ on the cellular membrane, which is quite important to maintain the cellular metabolism of microbes (Gorini et al. 2002; Magda et al. 2021). The respiratory activity of Microcystis cells can be well characterized by the Na + /K + ATPase activity. Similar to the photosynthetic activity of Microcystis cells, the influence of chlorination on Na⁺/K⁺ ATPase activity was not affected by the contact time of 10 and 60 min (Figure 4; Table 2). When Microcystis cells were treated by an initial chlorine dosage of 0.5 mg L⁻¹, the ATPase activity was reduced, since Microcystis cells had to consume more energy for various physiological responses under the oxidative pressure caused by chlorination. When the initial dosages of chlorine reached to 1.0 and 2.0 mg L⁻¹, the cellular membrane of Microcystis cells was destroyed, or the membrane permeability was improved (Figures 2 and 3). Then, the ATPase was released to the extracellular solutions, resulting in a considerable increase in the ATPase activity (Hammes et al. 2008; Ramseier et al. 2011; Xu et al. 2017). This finding was opposite to the photosynthetic activity treated by chlorination. It must be noted that the Na⁺/K ⁺-ATPase activity is a cell membrane protein, and HClO and ClO⁻ are effective chlorinated components of chlorine that can inactivate these proteins via creating stable N-Cl bonds (Zhang et al. 2011; Duan et al. 2018). When Microcystis cells were treated by a higher dosage of chlorination (8.0 mg L⁻¹), the Na⁺/K ⁺-ATPase as a function protein was oxidized and decomposed, and thus, the respiratory activity of Microcystis cells was strikingly reduced.

This study found that low-level chlorination (0.5 pg per cell, 0.5 mg L⁻¹ for Microcystis cells of 1.0 × 10⁶ cells mL⁻¹) improved the photosynthetic activity of Microcystis cells, but their respiratory activity was reduced. The photosynthetic process can synthesize organic matters that are utilized by the respiratory process, and thus their difference value shows the survival potential at various environmental stress. At this treatment, this value was the highest among all chlorination treatments, suggesting that low-level chlorination was not sufficient to inactivate Microcystis cells and these cells might regrow at proper environmental conditions. Hence, the application of low-level chlorination is not a proper algicide to control cyanobacterial blooms in lakes or reservoirs. However, this study observed the slight destruction of the cellular surface of Microcystis cells in this treatment (Fig. S1) that may be beneficial to the coagulation process to remove these cells. Consequently, low-level chlorination may be proper to use as pre-chlorination to treat cyanobacteria-laden source water in DWTPs.

In contrast, chlorination (1–2 pg per cell, 1–2 mg L⁻¹ for Microcystis cells of 1.0 × 10⁶ cells mL⁻¹) improved the respiratory activity, but its difference value of photosynthetic activity and respiratory activity significantly decreased. It indicated that these treatments effectively inhibited the metabolic activity of Microcystis cells to some degree. Moreover, cell aggregates were observed after these chlorination treatments, which could assist the post-coagulation to remove Microcystis cells (Song et al. 2021). These findings suggested that it can be used as an effective algicide for cyanobacterial control in lakes or reservoirs, and it is also proper to employ as a pre-, inter-, and post-oxidant to inactivate cyanobacterial cells in DWTPs. Nevertheless, at the decay stage of Microcystis bloom, the potential formation of ammonium could react with chlorine and chloramine is formed. Chloramine is less effective to inactivate Microcystis than chlorine (Wert et al. 2013). Thus, prior to the application of chlorination to treat cyanobacteria-laden source waters, the formation of chloramine should be carefully assessed.

This study demonstrated that sufficient chlorination (4–8 pg per cell, 4–8 mg L⁻¹ for Microcystis cells of 1.0 × 10⁶ cells mL⁻¹) completely inactivated the metabolic activity of Microcystis cells, and cellular structures were also severely damaged. This finding implied that these treatments were sufficient to eliminate the risk of the survival or regrowth of Microcystis cells. However, the release of cellular contents (e.g., cyanotoxins, algal organic matters) would occur after the severe destruction of cellular membrane (Fig. S1), and the formation of disinfection by-products was not to be ignored via chlorination with amounts of algal organic matters. Overall, advantages and drawbacks of chlorination as an oxidant or algicide to treat cyanobacteria-ladens source waters or control cyanobacterial blooms should be carefully assessed, and its effectiveness was mainly determined by the cellular structure and metabolic activity of Microcystis cells after chlorination.

This study demonstrated that the cellular structure and metabolic activity of Microcystis cells were strongly affected to some degree by chlorination. Sufficient chlorination of 1–8 mg L⁻¹ effectively decreased the metabolic activity of Microcystis cells. Cellular structures were also severely destroyed, and Microcystis aggregates induced an increase of the particle size distribution of 10–100 μm. In contrast, low-level chlorination of 0.5 mg L⁻¹ did not change the particle size distribution with a slight destruction of cellular structures. Interestingly, this treatment induced an increase of a photosynthetic activity of Microcystis cells, implying that insufficient chlorination might not be a proper algicide to inactivate cyanobacterial cells. Overall, this study would contribute to the application of chlorine as an effective oxidant or algicide, to inactivate cyanobacterial cells in DWTPs and control cyanobacterial blooms in reservoirs or lakes.

This work was supported by the Natural Science Foundation of China (42207071), and the Natural Science Foundation of Fujian Province of China (2020J01417). Special thanks for a project supported by the Special Research Assistant of Chinese Academy of Sciences.

Data cannot be made publicly available; readers should contact the corresponding author for details.

The authors declare there is no conflict.