Introduction

The presence of toxic cyanobacteria in natural waters poses a threat to animal and human health because they can produce many types of toxic compounds1,2, which can cause serious and even fatal human liver, digestive, neurological, and skin diseases2,3. Besides this, the water quality can also be greatly diminished, for example, the aesthetics of drinking water can be compromised by taste and odor compounds produced by cyanobacteria such as geosmin and 2-methylisoborneol. Recently, due to nutrient over-enrichment of surface waters by urban, agricultural, and industrial development, cyanobacterial blooms have quickly become a global epidemic, including Lake Victoria in Africa, Lake Winnipeg in Canada, Lake Taihu in China, and the Baltic Sea in Europe4,5,6,7. Although most of the cyanotoxins are present within cells8,9 - with the exception of Cylindrospermopsis sp., which can have a large amount of the toxin extracellularly10,11 - the intracellular toxins can be released into surrounding waters under certain external stresses responsible for cell lysis. The release of intracellular toxins poses a more serious hazard to water safety because of their more difficult removal compared to the whole cells12,13. Therefore, it has become very important to find an effective method of removing cyanobacteria without cell damage.

Coagulation, the key step in conventional drinking water treatment for pollutant removal, has been verified as an effective approach to remove intracellular toxins with intact cells, without causing additional release of intracellular toxins12,14. The successful removal of intact algal cells can avoid the release of intracellular material, especially the toxins, into the supernatant, decreasing the burden on subsequent processes and decreasing production of toxic disinfection byproducts15,16. After coagulation/flocculation and sedimentation processes, the intact cyanobacteria cells are transferred into a solid phase, namely the drinking water sludge.

Large amounts of drinking water sludge will be produced during the production of drinking water, equivalent to 4–7% of the total drinking water produced17,18. Furthermore, due to the global water scarcity in recent years, more and more drinking water sludge is dewatered, with the extracted water being recycled into the production stream. However, in the treatment of cyanobacteria-containing drinking water sludge, damage to cyanobacterial cells will result in the release of intracellular material, especially toxins, into the recycled water, thus increasing the burden of toxin removal17. Therefore, knowledge of cell viability, integrity and intracellular metabolite release during the sludge treatment process is required. How do cyanobacteria cells behave when transferred to the sludge? Up to now, little attention has been drawn to it.

Based on our previous study, cyanobacteria cells coagulated by the conventional coagulants (including AlCl3, FeCl3, and PAFC) lysed on different days during floc storage19,20,21, which means that there may be different responses or behavior of cyanobacteria present in the various sludges. Hence, in this study we will systematically explore the trends of cell viability and integrity of cyanobacteria in the sludge, and then reveal the possible mechanisms of these processes.

Results and Discussion

Effects of coagulant species on M. aeruginosa cell integrity during sludge storage

To investigate the cell integrity of M. aeruginosa during the sludge storage process, we analyzed the changes of extracellular microcystins (MCs), polysaccharide, TP, and TN after storage for 0, 2, 4, 6, 8, and 10 d, and the results are given in Fig. 1(a–d). The extracellular MCs concentration in the four systems (without coagulation, AlCl3, FeCl3, and PAFC coagulation, respectively) initially increased and then declined with prolonged storage time, however, the variation of the extracellular MCs in the system without coagulation was more obvious than that in the systems with coagulation (Fig. 1(a)). The initial increase in the extracellular MCs may be due to the secretion of MCs by natural processes22, or the release of intracellular MCs from the broken M. aeruginosa cells. Furthermore, the extracellular MCs concentration reached 108.5 ± 2.9 μg/L (the maximum level) on the sixth day in the system without coagulation, which means that the lysis of M. aeruginosa cells might occur and most of the intracellular MCs were released out after 6 d storage. For the systems with coagulation, the extracellular MCs were lower than that in the system without coagulation (P < 0.05). The main reason may be that the algal cells can be coated with a layer of coagulant owing to the charge neutralization between the negatively charged cells and the positively charged coagulant, which may have inhibited the release of intracellular substances and prevented the lysis of algae cells to some extent19,20,23,24. Another potential reason may be that the extracellular polymeric substances produced by M. aeruginosa cells increased due to the exposure of cells to external stimulus, and thus the protective shield formed by extracellular polymeric substances was strengthened for cells responding to the external stress20. In addition, the extracellular MCs concentration of cyanobacteria-containing AlCl3 sludge increased gradually during days 4–6, then decreased slightly during days 8–10. The extracellular MCs concentrations of cyanobacteria-containing FeCl3 and PAFC sludges had no significant increase during sludge storage, indicating that the damage of the cells in AlCl3 sludge is higher than that in FeCl3 and PAFC sludge. Besides this, the extracellular MCs levels decreased from 108.5 ± 2.9 μg/L (the sixth day) to 4.7 ± 0.1 μg/L within 2 d in the system without coagulation, which might be due to the biodegradation by bacteria. Rapid degradation of MCs by coexisting bacteria of M. aeruginosa, such as Bacillus cereus, Burkholderia sp. and Pseudomonas sp., in natural lakes, reservoirs, and laboratory culture has been reported25,26,27. In addition, despite decline in the level of extracellular MCs for AlCl3, FeCl3, and PAFC sludges upon prolonged storage, the remaining MC concentrations were still 54.6 ± 1.5, 24.7 ± 1.5, and 27.8 ± 0.3 μg/L after 10 d storage. Even though extracellular MCs could be degraded by the aforementioned bacteria, the slightly damaged algal cells could release intracellular MCs outside gradually during the whole storage process. The combination of these two opposing effects may account for the observation that extracellular MCs remained at a relatively high level for up to 10 d of storage.

Figure 1
figure 1

Extracellular concentrations of (a) MCs, (b) polysaccharide, (c) TP, and (d) TN after storage for 0, 2, 4, 6, 8, and 10 d. Data are shown as the mean ± SD (n = 3). Asterisks above the bars indicate significant differences with respect to the system without coagulation (P < 0.05).

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Figure 1(b) shows the levels of extracellular polysaccharide during the storage time. The extracellular polysaccharide concentration in the four systems also initially increased and then declined with increasing storage time, and the degree of change in the extracellular polysaccharide in the system without coagulation was higher than that in the systems with coagulation (P < 0.05), which is a similar trend to that seen with the extracellular MCs (Fig. 1(a)). As the storage time increased, the extracellular polysaccharide reached the maximum value of 28.7 ± 0.3 mg/L in the uncoagulated system on the sixth day, while the extracellular polysaccharide only reached 12.9 ± 0.5 mg/L at the same storage time in the cyanobacteria-containing AlCl3 sludge. Furthermore, there was no obvious increase of extracellular polysaccharide in the cyanobacteria-containing FeCl3 and PAFC sludge during the sludge storage period. These results were consistent with the results of extracellular MCs described above.

As storage time increased, the concentrations of TP in the four systems increased (see Fig. 1(c)). Moreover, the TP level in the system without coagulation rose significantly from 0.13 ± 0.01 mg/L on the fourth day to 0.55 ± 0.02 mg/L on the sixth day, and then increased to 0.79 ± 0.03 mg/L after 10 d storage. This result also illustrated that the damage of M. aeruginosa cells began to occur on the sixth day and the lysis of M. aeruginosa cells may be more serious with prolonged storage. Compared to the system without coagulation, the concentrations of TP were 0.33 ± 0.01, 0.17 ± 0.01, and 0.2 ± 0.01 mg/L in the cyanobacteria-containing AlCl3, FeCl3 and PAFC sludge after 10 d storage, respectively, which showed that (i) the flocs had the protection of M. aeruginosa cells, (ii) the lysis of algal cells in AlCl3 sludge was higher than the cells in FeCl3 and PAFC sludges, and (iii) there was basically no breakage of M. aeruginosa cells in FeCl3 and PAFC sludge. In addition, the variation of TN in Fig. 1(d) also showed a similar trend, and further verified the results above.

As previously reported, SEM has been an intuitive and indispensable tool for the estimation of cell damage in recent years28. It is a visual method that images a sample producing signals that contain information about the sample’s surface topography, composition, and other properties29. Thus, to further verify the above inference, four systems were established to directly detect the M. aeruginosa cell changes in different conditions: (A) without coagulation, (B) AlCl3 coagulation, (C) FeCl3 coagulation, and (D) PAFC coagulation, and after the cells were stored for (a) 0 d, (b) 6 d, and (c) 8 d. The samples in these systems were taken for SEM analysis, as shown in Fig. 2. For the system without coagulation, the M. aeruginosa cells were intact and the surfaces were smooth with 0 d storage (see Fig. 2(A-a)), however, the M. aeruginosa cells began to lyse after 6 d (Fig. 2(A-b)) and were almost completely damaged after 8 d (Fig. 2(A-c)). Although the M. aeruginosa cells remained intact after 6 d storage following AlCl3 coagulation, some cells were no longer spherical and seemed to have collapsed (Fig. 2(B-b)), and some M. aeruginosa cells were damaged when stored for 8 d (Fig. 2(B-c)). In addition, although some collapse was found on the surface of the FeCl3-coagulated M. aeruginosa cells after 8 d storage (Fig. 2(C-c), the M. aeruginosa cells remained intact and no lysis occurred until 8 d in the systems with FeCl3 and PAFC coagulation (Fig. 2(C-c,D-c)). Therefore, the lysis of M. aeruginosa cells varied in the following sequence for a given storage time: M. aeruginosa without coagulation > AlCl3 sludge > FeCl3 sludge ≈ PAFC sludge, which was consistent with the variations of extracellular MCs, polysaccharide, TP, and TN described above.

Figure 2
figure 2

SEM micrographs of M. aeruginosa cells before and after coagulation.

(A) Cells with no coagulant, (B) cells with AlCl3 coagulation, (C) cells with FeCl3 coagulation, (D) cells with PAFC coagulation. Flocs were stored for (a) 0 d, (b) 6 d, (c) 8 d.

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Effects of coagulant species on M. aeruginosa cell viability during sludge storage

The cell viability of M. aeruginosa cells in sludge represents the risk of M. aeruginosa cells regrowth. Therefore, the effect of coagulant species on M. aeruginosa cell viability during sludge storage was investigated so as to inform the safe treatment of coagulation sludge in drinking water treatment plants. According to our previous study, there was a strong linear relationship between the chlorophyll a concentration and cell viability for M. aeruginosa cells20. Thus, chlorophyll a concentration served as an index of M. aeruginosa cell viability in our study. The changes of chlorophyll a in the systems without coagulation and with AlCl3, FeCl3 and PAFC coagulation were measured at 2 d intervals for the whole storage period up to 10 d, and the results are shown in Fig. 3(a). The content of chlorophyll a in the four systems decreased with increasing storage time, indicating that the cell viability decreased. Because of the obvious protection that flocs provide to M. aeruginosa cells19,20,21, the decrease of chlorophyll a in the systems with coagulation was lower than that in the system without coagulation (P < 0.05). Moreover, as noted in Fig. 3(a), the content of chlorophyll a in AlCl3 and FeCl3 sludges was 2.16 ± 0.06 mg/L and 2.31 ± 0.13 mg/L when stored for 10 d, respectively, however, the chlorophyll a concentration still remained at 2.97 ± 0.04 mg/L in PAFC sludge after the same storage time, indicating that the cell viability varied in the following sequence when stored for 10 d: PAFC sludge > AlCl3 sludge ≈ FeCl3 sludge. As we know, chlorophyll a auto-fluorescence analysis can show the cell viability directly, hence, auto-fluorescence of M. aeruginosa was detected in the four systems (Figure S1). Furthermore, to quantify the fluorescence intensity, the mean fluorescence intensity of cells, which was calculated using total fluorescence intensity divided into the number of cells in each image in Figure S1, in different samples was calculated using Image J program (NIH, USA, Version: ij150-win-jre6-32-bit) and the results are shown in Fig. 3(b). It can be observed that the fluorescence intensity decreased as the storage time prolonged in the four systems, and the decrease of fluorescence intensity in the system without coagulation was higher than that in the systems with coagulation, especially after 6-d storage (P < 0.05). Furthermore, it can also be observed that the cell viability varied in the following sequence when stored for 10 d: PAFC sludge > AlCl3 sludge ≈ FeCl3 sludge, which is in line with the results of chlorophyll a described above.

Figure 3
figure 3

Chlorophyll a concentrations (a), mean chlorophyll a autofluorescence intensity (b), RuBisCO (c), and PEPCase activity (d) in the four systems at different floc storage times (0, 2, 4, 6, 8, and 10 d). Data are shown as the mean ± SD (n = 3). Asterisks above the bars indicate significant differences with respect to the system without coagulation (P < 0.05).

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It is well known that chlorophyll a content is an important factor in determining the photosynthetic rates. To verify the variation of the chlorophyll a, the activity of the other two important photosynthetic enzymes, RuBisCO and PEPCase, was investigated during the storage period. RuBisCO is a rate-limiting enzyme in the photosynthetic carbon reduction cycle and catalyzes the first step of the carbon assimilation process30, while PEPCase plays a key role during C4 photosynthesis31. As shown in Fig. 3(c), the activity of RuBisCO in the four systems also decreased with increasing storage time and the RuBisCO activity in coagulated M. aeruginosa cells was higher than that in the system without coagulation (P < 0.05), in agreement with the observed variation of chlorophyll a. Furthermore, the RuBisCO activity in PAFC coagulation sludge was higher than in AlCl3 and FeCl3 coagulation sludges after storage for 10 d (P < 0.05), consistent with the results for chlorophyll a above. In addition, the change of PEPCase activity in the four systems during the storage time was also measured (Fig. 3(d)), and the results were similar to those for the RuBisCO.

To further test the effects of coagulant species on M. aeruginosa cell viability during the sludge storage process, a re-suspended culture experiment was conducted. After the cyanobacteria-containing sludge was stored for a period of time (0, 4, 6, and 8 d), the cyanobacteria-containing sludge was re-suspended and then cultured for 24 d, the results are given in Fig. 4(a–d). As shown in Fig. 4(a), the chlorophyll a of the system without coagulation gradually increased from 2.2 ± 0.02 mg/L to 12.1 ± 0.33 mg/L within 20 days’ incubation and then decreased with increasing culture time. Because the growth of M. aeruginosa may be inhibited, in part, by the flocs, the chlorophyll a level in the PAFC-coagulated system increased from 2.2 ± 0.03 mg/L to 10.9 ± 0.45 mg/L within the tested 20 days, however, the chlorophyll a of the systems with AlCl3 and FeCl3 coagulation only rose from 2.2 ± 0.03 mg/L to 5 ± 0.25 mg/L and 3.6 ± 0.15 mg/L, respectively, when the systems were cultured for 20 days. Figure 4(b) shows the growth of M. aeruginosa cells which were resuspended after the sludge was stored for 4 d. The cell viability in the uncoagulated system has obviously declined, and the chlorophyll a level only reached 4.37 ± 0.16 mg/L (the maximum level) after 8 d of culturing, and then declined. However, chlorophyll a content with PAFC coagulation increased to 7.52 ± 0.06 mg/L within 22 days’ culturing. In contrast, the concentration of chlorophyll a with AlCl3 and FeCl3 coagulation rapidly declined to 0 within 10 days’ incubation. Figure 4(c) further verified these results. Despite the sludge having been stored for 6 d, the M. aeruginosa cells in PAFC flocs were still viable and chlorophyll a concentration rose to 4.83 ± 0.07 mg/L after 18 days’ incubation. When the sludge had been stored for 8 d, the cell viability was further decreased as shown before (see Fig. 3(a)). Due to the low viability of M. aeruginosa cells after storage for 8 d, the M. aeruginosa cells cannot regrow and the chlorophyll a content rapidly decreased to 0 after culturing the resuspended material for a few days (Fig. 4(d)). Therefore, these results confirmed the conclusion that the cell viability in PAFC sludge was higher than FeCl3 and AlCl3 sludges, and the cell viability in AlCl3 sludge was similar to FeCl3 sludge for the same storage time.

Figure 4
figure 4

Chlorophyll a concentrations in the four systems when the flocs were re-suspended and cultured after storage times of (a) 0 d, (b) 4 d, (c) 6 d, (d) 8 d. Data are shown as the mean ± SD (n = 3).

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The relationship between cell integrity and cell viability during sludge storage

Interestingly, the cell viability of M. aeruginosa cells in sludge varied as follows for the same storage time: AlCl3 sludge ≈ FeCl3 sludge < PAFC sludge, whereas the lysis of M. aeruginosa cells varied in the following sequence at the same storage time: AlCl3 sludge > FeCl3 sludge ≈ PAFC sludge. Though the cell viability of M. aeruginosa in FeCl3 sludge gradually declined with increased storage time, there was basically no breakage of algae cells for up to 10 d of storage. These results may be related to the different properties of the flocs, such as floc size and density, produced by different types of coagulants.

To test this hypothesis, the characteristics of the flocs during the coagulation process were evaluated and the results are summarized in Fig. 5 and Table 1. The initial median diameter (d50) in the bloom water was about 5 μm, and grew rapidly once coagulants were dosed (Fig. 5). AlCl3 and PAFC flocs grew rapidly in the first 5 minutes and then remained stable, and the final d50 values of the flocs were about 618 μm and 719 μm, respectively. In contract, the flocs formed by FeCl3 gradually grew to become much larger (805 μm) than those of AlCl3 and PAFC. Therefore, the better protective effect of FeCl3 flocs might be partly due to the larger floc size.

Table 1 The fractal dimension, pH, and Al and Fe concentrations before coagulation and during the AlCl3, FeCl3, and PAFC coagulation processes.
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Figure 5
figure 5

The formation and growth of M. aeruginosa flocs formed by AlCl3, FeCl3, and PAFC during the coagulation process.

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In addition, the fractal structure of flocs was analyzed through the measurement of fractal dimension (Df), which could be obtained by the exponential relationship of mass and particle size32 and could indicate the development of aggregate structure during the formation of flocs33. As presented in Table 1, the fractal dimension of AlCl3, FeCl3 and PAFC flocs were 1.88, 2.24, and 1.79, respectively. Bridgeman et al.34 reported that higher Df values indicated more compact structures, while flocs with lower Df values were looser and more open. Therefore, these results demonstrated that FeCl3 flocs were more compact than AlCl3 and PAFC flocs, which also indicated FeCl3 flocs have a better protective effect on the M. aeruginosa cells than do AlCl3 and PAFC flocs. Hence, although the cell viability of M. aeruginosa in FeCl3 sludge was lower than that in PAFC sludge, there was basically no damage of algae cells for up to 10 d of storage (similar to PAFC sludge).

Proposed mechanisms involved in the actions of coagulant species on M. aeruginosa cells during sludge storage

As discussed above, the M. aeruginosa cell viability varied in the following sequence for equal storage times: AlCl3 sludge ≈ FeCl3 sludge < PAFC sludge. The initial pH value has been identified as an important factor affecting the growth and cell viability of algae35,36, and Qian et al.36 reported that low-pH stress (pH < 5) could cause the lysis of algae cells and metabolite release. To explore whether our result is related to the initial pH value, we examined the initial pH value in the four systems after coagulation, and the results are shown in Table 1. As noted in Table 1, the pH value of the bloom water was 8.49 ± 0.05, and due to the hydrolysis of Fe3+ and Al3+ during the coagulation process, the pH value decreased to 8.02 ± 0.04, 7.42 ± 0.1, and 8.35 ± 0.06 after AlCl3, FeCl3, and PAFC coagulation, respectively. McLachlan and Gorham37 reported that M. aeruginosa could grow well in the pH range of 6.5–10. Therefore, the pH value should not be the main factor affecting the cell viability in this study.

In addition, it is well known that Al is toxic to freshwater algae38,39,40, and Gensemer and Playle40 reported that aluminum likely reduces P uptake rates in algae, perhaps by inhibition of the acid phosphatase enzyme. Because iron is a key component of chromophore synthesis, and biosynthesis of chlorophyll and phycobilin pigments includes iron-dependent steps even though neither of them contain iron41, iron is usually essential for cell growth, for example, Imai et al.42 and Wang et al.43 reported that the growth rate of M. aeruginosa increased with increasing Fe concentration when the concentration of Fe was low. However, the growth would obviously be inhibited when the Fe concentration was higher than 1377 μg/L43. Furthermore, Kumawat et al.44 reported that iron affects chlorophyll synthesis indirectly by affecting its precursor, δ-aminolevulinic acid (ALA), thereby affecting the growth of algae cells. To explore whether the concentrations of Al and Fe influence the activity of M. aeruginosa cells during storage, we analyzed Al and Fe concentrations after coagulation (Table 1). The content of Al and Fe in the bloom water before coagulation was about 28.6 ± 2.3 μg/L and 11.7 ± 3.9 μg/L, respectively (Table 1). Correspondingly, the concentration of Al increased to 705 ± 26.4 μg/L after 15 mg/L AlCl3 coagulation. According to the U.S. Environmental Protection Agency (US-EPA), total Al concentrations ranging from 460 μg/L to 6480 μg/L are toxic to freshwater algae40,45. Therefore, the high level of Al observed (705 ± 26.4 μg/L) is hazardous to the growth of M. aeruginosa in our study. Because 50 mg/L FeCl3 was added, the Fe concentration drastically increased to 2999 ± 35.4 μg/L after coagulation, higher than 1377 μg/L, which could obviously inhibit M. aeruginosa growth. Moreover, the Al concentration only increased to 240.2 ± 15.1 μg/L after 15 mg/L PAFC was added, which has little or no toxic effect on M. aeruginosa cells, whilst the concentration of Fe increased to 80.7 ± 7.4 μg/L, which may be of benefit to M. aeruginosa growth.

To further test the effect of Al and Fe concentration on M. aeruginosa cells during storage, the physiological characteristics of M. aeruginosa cells were investigated. Wang et al.46 and Xia et al.47 reported that the intracellular reactive oxygen species (ROS) would be increased in M. aeruginosa cells after treatment by either CuO or TiO2 nanoparticles. Furthermore, superoxide radicals and hydrogen can be consumed by SOD to produce hydrogen peroxide and oxygen, which has been described as the first line of resistance against ROS48. Thus, to explore whether high concentrations of Fe or Al could induce the increase of ROS in M. aeruginosa cells, ROS level and the responses of SOD were investigated at 2 d intervals during the whole sludge storage time up to 10 d.

As shown in Fig. 6(a), the level of ROS increased dramatically as the storage time prolonged in the four systems, and the increase of ROS in the systems with coagulation was significantly lower than that in the system without coagulation (P < 0.05), owing to the protection of flocs. Moreover, it is notable that the increase of ROS in the system with PAFC coagulation was obviously lower than that in the systems with AlCl3 and FeCl3 coagulation (P < 0.05). The reason may be due to the toxicity of Fe and Al ions aforementioned.

Figure 6
figure 6

ROS level (a), SOD activity (b) and MDA content (c) of the M. aeruginosa cells in the four systems at different floc storage times (0, 2, 4, 6, 8, and 10 d). Data are shown as the mean ± SD (n = 3). Asterisks above the bars indicate significant differences with respect to the system without coagulation (P < 0.05).

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SOD activity of M. aeruginosa increased initially and then decreased as storage time was extended (Fig. 6(b)). The increase of SOD in the initial stage might be ascribed to the initial stresses, such as the toxicity of Fe and Al ions, and the subsequent SOD decrease implies that the enhanced ROS level had exceeded the scavenging ability of the antioxidant enzymes49. Due to the protection of flocs, the increase of SOD in coagulated systems was lower than in the uncoagulated system (P < 0.05). In addition, the SOD activity reached 309.7 ± 2.36 and 291.6 U/mgprot (the maximum level) on the fourth day with AlCl3 and FeCl3 coagulation, respectively, whereas the SOD activity only reached 149.5 ± 14.9 U/mgprot (the maximum level) on the sixth day with PAFC coagulation, which means that the ROS level in M. aeruginosa cells with AlCl3 and FeCl3 coagulation was more severe than in the cells with PAFC coagulation. Therefore, from the result above, we verified that high concentrations of Al and Fe were harmful to the growth of M. aeruginosa cells. Furthermore, a moderate Fe level was beneficial to M. aeruginosa growth, and low level of Al has no effect on M. aeruginosa cells.

MDA, one of several low-molecular-weight end products formed by the decomposition of polyunsaturated fatty acid hydroperoxides, is usually used as a biomarker of physiological stresses and cellular oxidative damage50. To verify the results above (Fig. 6(a,b)) and further investigate whether the oxidative stress, such as toxicity of Al and Fe at high concentrations, could cause oxidative damage on M. aeruginosa cells, the variation of MDA was examined (Fig. 6(c)). As shown in Fig. 6(c), the MDA content increased with increasing storage time, and the MDA content in M. aeruginosa cells with coagulation was lower than that in the system without coagulation (P < 0.05), which illustrated that the cellular oxidative damage in M. aeruginosa cells with coagulation was lower than that in the system without coagulation, and the reason might be attributed to the protection of the flocs. As storage time increased, it was found that the MDA content with PAFC coagulation was lower than that with AlCl3 or FeCl3 coagulation (P < 0.05), indicating that the cellular oxidative damage in M. aeruginosa cells with AlCl3 or FeCl3 coagulation was more severe than in the cells with PAFC coagulation. In addition, due to the FeCl3 flocs being larger and more compact than AlCl3 flocs, there was a stronger protective effect for FeCl3 flocs than AlCl3 flocs. Hence, the MDA content in M. aeruginosa cells with FeCl3 coagulation was slightly lower than that with AlCl3 coagulation for equal storage times, indicating that the cellular oxidative damage in the M. aeruginosa cells of FeCl3 sludge was lower than that in AlCl3 sludge.

Based on the discussion above, due to the protection of flocs, the integrity and viability of M. aeruginosa cells with coagulation was better than for uncoagulated cells. Furthermore, coagulant species have an obvious effect on cell integrity and viability during sludge storage. As shown in Fig. 7, for cyanobacteria-containing AlCl3 and FeCl3 sludges, the high levels of Al and Fe may inhibit the activity of acid phosphatase enzyme and hinder the synthesis of chlorophyll in M. aeruginosa cells, respectively. The higher floc size and density for FeCl3 flocs may restrict the absorption of light for M. aeruginosa cells, which also prevents the synthesis of chlorophyll for cells in FeCl3 flocs. Thus the normal metabolism of M. aeruginosa cells would be destroyed. Furthermore, due to the adverse environmental stressors, such as high concentration of Al and Fe, excess ROS may be produced in M. aeruginosa cells with increased storage time. Because SOD was the first line of resistance against ROS, SOD activity was then induced in response to the oxidative stress. However, when ample ROS was produced in M. aeruginosa cells, the activity of SOD would be limited and the cell membrane would be damaged by lipid peroxidation. Then the viability of M. aeruginosa cells would be decreased and the integrity of the cell membrane would be damaged. More suitable Fe concentrations could promote the growth of M. aeruginosa cells in cyanobacteria-containing PAFC sludge, whilst low concentration of Al has little or no effect on M. aeruginosa cells. There was no excess ROS accumulated in M. aeruginosa cells, and basically no lipid peroxidation on the cell membrane, hence, the M. aeruginosa cells maintained relatively high activity and integrity. Correspondingly, the M. aeruginosa cells in AlCl3 sludge began to lyse after 6 d of storage and the M. aeruginosa cells in PAFC sludge remained intact after up to 10 d storage. However, the M. aeruginosa cells in FeCl3 sludge also remained intact after up to 10 d storage (similar to PAFC sludge). The reason may be ascribed to the higher floc size and density for FeCl3 flocs, which can more effectively prevent the damage of M. aeruginosa cells.

Figure 7
figure 7

Schematic diagram depicting the effect of AlCl3, FeCl3, and PAFC on M. aeruginosa cell viability and integrity during storage of cyanobacteria-containing sludges.

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Conclusions

In summary, coagulant species have a significant effect on the cell viability and integrity of M. aeruginosa cells during sludge storage processes. Because high concentrations of Al and Fe are toxic to M. aeruginosa, while appropriate amount of Fe is beneficial to the growth of M. aeruginosa, the M. aeruginosa cells in PAFC sludge have higher cell viability than those in AlCl3 and FeCl3 sludges after the same storage times. Furthermore, we also found that the lysis of M. aeruginosa cells in AlCl3 sludge occurred earlier and was more severe than the M. aeruginosa cells in the systems with FeCl3 and PAFC coagulation. Consistent with the protective function of the large floc size and high density of the FeCl3 sludge, the M. aeruginosa cells remained basically intact in that sludge after 10 d storage, even though the cell viability was low. Hence, FeCl3 may be an ideal coagulant that can not only reduce the viability of M. aeruginosa cells but also prevent the lysis of the cells. Overall, this study provided not only a systematic analysis of M. aeruginosa cells’ death and lysis in AlCl3, FeCl3 and PAFC sludges upon increasing storage time, but also guidance for the safe treatment of coagulation sludges in drinking water treatment plants.

Materials and Methods

Algal culturing

Microcystis aeruginosa (M. aeruginosa, FACHB-905, obtained from the Institute of Hydrobiology, Chinese Academy of Sciences) was selected as the experimental cyanobacteria, and was grown in BG11 medium (pH 7.5). The cultures were maintained in a constant temperature incubator (GXZ-280C, Ningbo Jiangnan Instrument Factory, China) at 25 °C under 2000 lux with a light–dark cycle of 12 h/12 h and harvested at the late exponential phase of growth with a final cell yield up to about 107 cells/mL.

Raw water and bloom water

The raw source water investigated in this research was sampled from the Queshan Reservoir, one of the important drinking water sources in Jinan, China, and filtrated through a 0.45 μm cellulose acetate membrane (Shanghai Mili Membrane Separation Technology Co., China) to remove natural algae. The main characteristics of the raw water were: temperature 16.7 °C, pH 8.4, turbidity 1.31 NTU, UV254 0.047 cm−1, DOC 4.67 mg/L, NH3-N 0.19 mg/L, TN 2.2 mg/L, TP 0.03 mg/L, alkalinity 133.9 mg/L, residual Al 91.5 μg/L, and residual Fe 58.3 μg/L. To simulate the algal bloom in a high algae-laden period according to the guidance value of WHO12, the raw water was spiked with M. aeruginosa cultures to achieve a final cell density of about 106 cells/mL (bloom water).

Chemicals

Ferric chloride hexahydrate (FeCl3·6H2O, AR grade) and aluminum chloride hexahydrate (AlCl3·6H2O, AR grade) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Polymeric aluminum ferric chloride (PAFC) was purchased from Gongyi Yongxing Biochemical Materials Co., China. The main parameters of the PAFC were: relative density 1.25 (20 °C), alumina content 8–10%, iron oxide content 1–2%, basicity 65–90%. The AlCl3, FeCl3 and PAFC stock solutions were prepared to be 3 g/L (pH 3.22), 10 g/L (pH 1.65) and 5 g/L (pH 4.44), respectively.

Coagulation experiments

Coagulation experiments were performed at room temperature (25 ± 2 °C) in a six-paddle stirrer (ZR4-6, Zhongrun Water Industry Technology Development Co., China). For the coagulation experiment, each bloom water sample (1 L) was dosed with the stock solution when the rapid mixing began. The optimum coagulation conditions for the effective removal of M. aeruginosa cells by AlCl3, FeCl3, and PAFC, respectively, are listed in Table S1. After coagulation, samples were quiescently settled for 30 min to obtain the flocs (formed into sludge) and supernatants.

Floc storage experiments

After removing 930 mL of supernatant, 70 mL of cyanobacteria-containing sludge remained. For a control (without coagulation), the bloom water sample (1 L) was centrifuged at 6000 rpm for 3 min, and the cells were then re-suspended into the raw water to make up 70 mL, forming a coagulant-free sludge. All cyanobacteria-containing sludge samples were then housed in an incubator at 25 °C under 2000 lux illumination with the 12 h/12 h (light/dark) cycle for up to 10 d. The sludge samples were drawn every two days for analysis during the whole storage period.

Cell integrity analysis during the storage process

The intracellular materials would be released when the cell membrane is damaged. Therefore, the change of extracellular MCs, polysaccharide, total nitrogen (TN), and total phosphorus (TP) was measured, to investigate the integrity of M. aeruginosa cells during storage. The sludge samples were drawn every two days during storage and filtered through 0.45 μm cellulose acetate membranes. Extracellular MC content was measured using a Beacon Microcystin ELISA kit (Beacon Analytical Systems, Maine, USA). The method used for the detection of MCs by ELISA was performed as previously reported20. Extracellular polysaccharide concentration was determined by the phenol–sulfuric acid method51. Extracellular TN and TP were conducted according to Chinese state standard testing methods52.

To directly assess the surface information and morphology of M. aeruginosa cells during the storage time, scanning electron microscopy (SEM) was used. The sludge samples were centrifuged at 4000 rpm for 5 min, the pellets were pre-fixed with 2.5% glutaraldehyde overnight, washed by phosphate buffer solution three times and post-fixed with 1% osmium tetraoxide for 1 h, then again washed with phosphate buffer solution. After that, samples were consecutively treated with 50%, 75%, 90% and 100% ethanol solutions (15 min each) and dried with a vacuum drier. The completely dry samples were then mounted on a copper stub, coated with gold and examined with an SEM (S-4100, Hitachi, Japan) at 3 kV.

Cell viability analysis during the storage process

To explore the change of cell viability during the sludge storage period, chlorophyll a content, chlorophyll a auto-fluorescence, activity of Ribulose-1,5-bisphosphate carboxylase (RuBPCase) and Phosphoenol-pyruvate carboxylase (PEPCase) in M. aeruginosa were measured. The chlorophyll a content was measured according to the method of Pancha et al.53. A fluorescence microscope (NIKON TE2000, Japan) fitted with filters including exciter filter EX510-560, dichroic mirror DM575, barrier filter BA590, was used for chlorophyll a auto-fluorescence observation. The red emission spectra were captured using a CCD camera. Activity of RuBPCase and PEPCase in M. aeruginosa cells was measured by utilizing the plant RuBPCase ELISA Kit (R&D, USA) and the plant PEPCK ELISA kit (R&D, USA), respectively. The activity of RuBPCase and PEPCase in M. aeruginosa cells were expressed as the ratios of RuBPCase and PEPCase to intracellular protein (pg/mgprot), respectively.

In addition, to further study the viability of the M. aeruginosa cells in the sludge formed by AlCl3, FeCl3, and PAFC after different storage time, a re-suspended culture experiment was conducted according to the method of Li and Pan54. After storage of the AlCl3, FeCl3, and PAFC cyanobacteria-containing sludge for 0, 4, 6, and 8 d, 70 mL of BG11 medium (double concentrated) was added to each sludge sample. The re-suspended cyanobacteria-containing sludge samples were maintained in the illuminated incubator at 25 °C under 2000 lux illumination on the 12 h/12 h (light/dark) cycle. The recovery and regrowth of the M. aeruginosa cells were monitored by detecting the chlorophyll a concentration in the culture over the following 24 days.

Floc properties, pH, Al and Fe during the coagulation process

A laser diffraction instrument, Mastersizer 2000 (Malvern, UK), was used to monitor dynamic floc size. Size measurements of the flocs were taken every 30 s in the process of coagulation and the results were recorded automatically by the computer. The size data were expressed as equivalent volumetric diameters, and the floc size was characterized by the median volumetric diameter (d50).

The fractal dimension (Df) of aggregates was measured by a small-angle laser light scattering (SALLS) method by the Mastersizer 2000 with a 632.8 nm laser light beam55. The total scattered light intensity I, the scattering vector Q, and Df followed a power law as shown in Eq. (1)55:

The scattering vector Q is the difference between the incident and scattered wave vectors of the radiation beam in the medium as shown in Eq. (2)56:

where n, θ and λ are the refractive index of the medium, the laser light wavelength in vacuum, and the scattering angle, respectively.

Furthermore, after coagulation, the pH of the bloom water was measured using a digital pH-meter (pHS-3C, Leici, China). The concentration of residual aluminum and iron in the bloom water and the cyanobacteria-containing sludge were determined using an inductively-coupled plasma optical emission spectrometer (180-80, Hitachi, Japan). Samples were filtered through 0.45 μm cellulose acetate membranes and acidified to pH < 2 with HNO3, and digested for 2 h by a Lianhua 5B-1B digestion device (Lianhua Tech. Co., Beijing, China) before analysis.

ROS, SOD and MDA analysis in M. aeruginosa cells during the storage process

The reactive oxygen species (ROS) level in M. aeruginosa cells during storage was determined as previously reported57. The ROS level in the presence of algae was expressed as the ratio of fluorescence emission intensity to intracellular protein.

To extract antioxidation enzymes, 2 mL of each sludge sample were centrifuged at 8000 rpm for 10 min. The cell pellets were then re-suspended with phosphate buffered saline solution (50 mM, pH 7.0) and homogenized by an ultrasonic cell pulverizer (Scientz-IID, China) at 600 W for a total time of 10 min (cycle: 2 s ultrasonication, 8 s rest) in an ice bath. Finally, the homogenate was centrifuged at 12000 rpm for 10 min at 4 °C to obtain the supernatant for assays of the enzyme activity and the level of lipid peroxidation. Superoxide dismutase (SOD) activity in M. aeruginosa cells was determined by the inhibition of nitro blue tetrazolium reduction according to the method of Beauchamp and Fridovich58. Malondialdehyde (MDA) content in algal cells was measured from material reacting to thiobarbituric acid, according to the thiobarbituric acid reaction as described by Dogru et al.59. MDA and SOD results were expressed as μmol/mgprot and U/mgprot, respectively. Furthermore, the intracellular protein was determined by the method of Bradford60 using bovine serum albumin as a standard and the results are shown in Figure S2.

Statistical analysis

All experiments performed were performed in triplicate and the data were expressed as the means ± standard deviation (SD). All of the parameters were compared across treatments with one-way ANOVA using the SPSS software (version 16.0), and the significance was set to P < 0.05. All statistical analyses were carried out using Origin 9.1.

Additional Information

How to cite this article: Xu, H. et al. Behaviors of Microcystis aeruginosa cells during floc storage in drinking water treatment process. Sci. Rep. 6, 34943; doi: 10.1038/srep34943 (2016).