The lysis and regrowth of toxic cyanobacteria during storage of achitosan–aluminium chloride composite coagulated sludge: implications for drinking water sludge treatment

Abstract

Coagulation is a key unit operation for cyanobacterial cell removal; however, the potential danger of cyanobacterial cells transferred into sludge is not well understood. In this study, the fate of Microcystis aeruginosa (M. aeruginosa) and secondary metabolites in chitosan–aluminium chloride (CTSAC) coagulated cyanobacteria-laden sludge were investigated during the sludge storage period. The extracellular microcystins (MCs) can be adsorbed onto the CTSAC flocs for six days with a reduced biodegradation rate. Less M. aeruginosa cell lysis was observed in the coagulated system than in the natural cell system, due to the protection of M. aeruginosa by the CTSAC. Furthermore, the residual Al content decreased in the cyanobacteria-laden sludge supernatant. The amount of extracellular organic matter (EOM) stayed low in the coagulated system for four days. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that coexisting bacteria reduced in the sludge during the initial four days storage time. Interestingly, the CTSAC degradation favored the growth of the M. aeruginosa cells. This study will be helpful for better understanding and managing secondary metabolite pollution problems related to coagulation-generated cyanobacteria-laden sludge during the sludge supernatant recycling process. The use of CTSAC composite coagulant is of practical value in reducing secondary pollution during cyanobacteria-laden sludge storage.

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1. Introduction

Outbreaks of cyanobacterial blooms have become a big challenge in providing safe drinking water. Microcystis aeruginosa, one of the most commonly found cyanobacteria in fresh water, releases toxic microcystins (MCs) that can cause nausea, liver cancer and death in humans and animals.^1,2 The disinfection by-products (DBPs) derived from chlorination/chloramination of algal organic matters (AOM) also impose a health risk.^3,4 Most AOM, including MCs contained in cyanobacterial cells, are intracellular organic matter (IOM), and the metabolites excreted from cyanobacterial cells into the environment are extracellular organic matter (EOM).¹ The IOM can also be released into the environment if the integrity of the cell is compromised.⁵ It is harder to remove the EOM, including released MCs, than to remove those MCs contained in M. aeruginosa cells.⁵ Therefore, to eliminate toxin release and algal related DBPs, it is the best to remove intact M. aeruginosa cells.

Conventional coagulation treatment, the key unit operation for removing cyanobacterial cells in drinking water treat plants (DWTPs), is efficient at removing cells; however, its effects on cell integrity are questionable. Many coagulants, such as ferric salt and clay coagulants are effective in removing cyanobacterial cells, but may cause cell lysis.^6,7 Our previous study focused on investigate the effect of chitosan (CTS) which is a non-toxic and biodegradable natural polymer on M. aeruginosa cells removal, and found the chitosan coagulant was able to effectively remove M. aeruginosa cells without cell damage meanwhile adsorb an amount of MCs.⁸ However, the high cost of chitosan has limited its comprehensive application as a single coagulant in drinking water treatment. Further investigation found that the combination of CTS and aluminium chloride (AC), at low dosages, could not only remove M. aeruginosa without cell lysis, but also adsorb a significant amount of EOM, especially extracellular MCs, while the individual coagulant was not as effective as the combined one.⁹ Therefore, there are advantages for the water treatment facilities to adapt the CTSAC composite coagulant as an effective way of removing cyanobacteria and adsorbing their metabolites.

Each DWTP has to manage its own sludge, and attention has increasingly been paid to the sludge management process.¹⁰ The large amount of water consumed has promoted an increase in the production of drinking water sludge.¹¹ However, very little is known about the treatment of cyanobacteria-laden sludge, so there is a great need for knowledge to transform coagulation into a practical solution in treating algae-contaminated drinking water sources.

Although the M. aeruginosa cells can be removed intact by a composite coagulation process, cell lysis can still occur in the sludge generation and accumulation processes, and release large amount of EOM, especially microcystin (MC) metabolites, into the sludge supernatant. It will pose a threat to drinking water quality if supernatant containing EOM and MC metabolites is recycled to the head of the plant, where it is blended with the raw water about to undergo treatment. To our knowledge, few studies have addressed the fate of M. aeruginosa cells, and EOM release in cyanobacteria-laden sludge, despite the fact that the cyanobacteria cells rapidly lost their viability in sludge.¹² Certain authors have identified some of the bacteria coexisting with M. aeruginosa that have the capability to degrade MCs and EOM.^13,14 However, several of the coexisting bacteria, such as Bacillus cereus and Burkholderia sp., are also pathogens.^13–17

CTS has been widely used as an antimicrobial agent against pathogens.^18,19 But the antibacterial ability of CTS when combined with AC is unclear. Furthermore, our previous study showed that CTS degradation would accelerate M. aeruginosa cell lysis.⁸ CTS degradation was known to be influenced by many factors including chemical modification and molecular weight.²⁰ The residual Al concentration in the water has become a subject of concern, as it could cause human neurological disorders such as Alzheimer's disease.²¹ But the variation of residual Al in the coagulation-generated cyanobacteria-laden sludge remains unknown so far. Therefore, upon the addition of CTSAC, the fate of M. aeruginosa cells, variation of EOM, change of residual Al and the coexisting bacteria in CTSAC-coagulated cyanobacteria-laden sludge could be a very intricate topic. To date, no research has focused on the fate of CTSAC-coagulated cyanobacteria-laden sludge.

The objectives of this work were: (1) to determine the levels of toxic MC release, M. aeruginosa cell lysis, and variations of EOM and residual Al for different sludge storage times; (2) to evaluate the change of microbial community in cyanobacteria-laden sludge generated from coagulation by CTSAC; (3) to study the fate of CTSAC composite and to elucidate the mechanisms of the interaction between microbial variation and changes in the composite coagulant.

2. Experimental procedures

2.1. Materials

2.1.1. Algal culturing. M. aeruginosa FACHB-905 (Institute of Hydrobiology, Chinese Academy of Sciences) was used as the model algae in this study. It was cultured in BG11 medium at 25 °C, underwent a 12 h diurnal cycle with 2800 lux illumination in the laboratory, and was harvested at the late exponential growth phase.

2.1.2. Preparation of CTSAC coagulant. CTS, with 95% degree of deacetylation and 50 000 g mol⁻¹ viscosity-average molecular mass, was purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). CTS was dissolved in 1.0% acetic acid solution and stirred overnight at 100 rpm to obtain 1.3 mg mL⁻¹ stock solution. The composite coagulant, denoted ‘CTSAC’, was prepared by adding 50 mL of 3.75 mg mL⁻¹ AC (AR grade) into 50 mL CTS stock solution with continuous stirring for 24 h to obtain a completely mixed solution.

2.1.3. Water sample. Natural water (residual Al = 91.5 μg L⁻¹) collected from Queshan Reservoir (Jinan, Shandong province) was filtered through a 0.45 μm glass fibre membrane for experiment within 120 min. Before coagulation, the concentration of M. aeruginosa cells was adjusted to 2 × 10⁶ cells per mL by diluting the cells using the filtered water. The main characteristics of natural water quality were as follows: temperature 18.5 °C, pH 8.4, turbidity 4.7 NTU, and DO 9.13 mg L⁻¹.

2.2. Coagulation experiment

Systems were set up and examined for physiochemical and biological changes of the system as well as the M. aeruginosa cells under two conditions: (A) without coagulation (natural cell system), (B) with CTSAC coagulation. Samples were taken from 12 × 1 L of water containing resuspended M. aeruginosa (2 × 10⁶ cells per mL) treated with and without coagulation. The pH of resuspended M. aeruginosa cell samples was adjusted to 8.4 by adding HCl (1 mol L⁻¹) or NaOH (1 mol L⁻¹). Six coagulation tests were simultaneously conducted in 1.0 L beakers using a program-controlled jar test apparatus (ZR4-6, Zhongrun Water Industry Technology Development Co., China). 4 mL of CTSAC coagulant (2.6 mg L⁻¹ CTS plus 7.5 mg L⁻¹ AC) was pipetted into the 1.0 L volume of water containing resuspended M. aeruginosa (2 × 10⁶ cells per mL) for coagulation. This dosage was determined by our previous research which studied the effect of CTS and AC dual/composite coagulants on the removal of M. aeruginosa cells, and found the optimal coagulation performance was obtained when CTSAC composite coagulant was applied as 2.6 mg L⁻¹ CTS plus 7.5 mg L⁻¹ AC.⁹ The coagulation process comprised firstly rapid mixing at 250 rpm for 2 min after dosing composite coagulant, followed by slow mixing at 20 rpm for 20 min. After settling for 60 min, the supernatant was collected from 1 cm below the surface for measurement of cell removal. The coagulation parameters utilized were all determined by our previous research.⁹ Meanwhile another 6 L of M. aeruginosa resuspended water was distributed equally between the six 1 L beakers of the jar test rig without coagulation treatment. After coagulation, samples were respectively settled for 0, 2, 4, 6, 8 and 10 days, the supernatants of the sample were filtered through 0.45 μm glass fiber membranes for analysis of extracellular MCs, the release of K⁺, residual Al, and fluorescence excitation–emission matrices (EEMs). The same operations were conducted in natural cell systems (without coagulation system). All tests were performed in triplicate. The flocs were also drawn every two days for SEM evaluation and PCR-DGGE measurement.

2.3. Analysis methods

2.3.1. M. aeruginosa cell density. The M. aeruginosa density was measured by a UV/vis spectrophotometer (U-3010, Hitachi Co., Japan) at a wavelength of 680 nm.

2.3.2. Dissolved (free extracellular) MCs. The extracellular MCs were measured by a Microcystin ELISA kit (Beacon Analytical Systems, Maine, USA). The detection of MCs was based on the method of Lei et al.²²

2.3.3. Scanning electron microscope (SEM). The floc precipitates were washed in a phosphate buffer solution and preserved for SEM analysis. Coated specimens were observed and photographed using a scanning electron microscope (S-3000 N, HITACHI, Japan) as a previous study described.⁸

2.3.4. The release of K⁺ and residual Al analysis. Measurements on the release of K⁺ and residual Al were conducted by an inductively coupled plasma optical emission spectrometer (180-80, HITACHI, Japan). Samples were filtered through 0.45 μm glass fiber membranes (MFS, Japan) and acidified to pH < 2 with HNO₃ (analytical grade, Beijing Chemical Reagents Company, China) before analysis.

K⁺ release was calculated as described in the literature.⁸ In brief, the release of K⁺ in each sample was calculated as follows:

where C_N is the K⁺ concentration in the supernatant sample obtained from floc samples stored for N = 0, 2, 4, 6, 8 and 10 d, for each system, and C₀ is the K⁺ concentration of supernatant obtained from resuspended M. aeruginosa cells at 0 d.

2.3.5. Three-dimensional excitation–emission matrix (EEM). Fluorescence spectra of the supernatant of settled sludge were measured using a fluorescence spectrophotometer (F-4600, Hitachi, Japan). Excitation wavelengths were incrementally increased from 220 to 450 nm in 5 nm steps, and the emission spectra ranged from 250 to 550 nm in 1 nm steps. The slits for both Ex and Em were 5 nm, with scan rate fixed at 2400 nm min⁻¹. All data were analyzed using MATLAB 2013b (MathWorks, Natick, USA).

2.3.6. DNA extraction and PCR-DGGE. Total genomic DNA was extracted from 2 g of M. aeruginosa-laden sludge using Soil DNA Fast Extraction Kit (Spin-column) with a final extract of 50 μL, according to the manufacturer's instructions (Bio Teke, China). Community analysis was conducted using PCR-DGGE as described in our previous study.²³ The V3 region of the 16S rDNA gene was amplified using touchdown PCR methods as previously described.²³ Three parallel PCR products were mixed for each sample.

Denatured Gradient Gel Electrophoresis (DGGE) was performed on a DCode Universal Mutation Detection System (Bio-Rad, USA) under the same conditions as described in the literature,²⁴ with 30 μL of composite PCR products loaded. Gels were stained by ethidium bromide for 120 min (FMC Bio Products, Rockland, USA) and visualized by UV trans-illumination. Photographs were captured by Quantity One 4.3.0 gel analysis software (Bio-Rad, USA) to detect the diversity indices of the microbial community calculated from the DGGE band profiles. After re-amplification and purification, the representative DGGE bands were sequenced by Sangon Biotech (Shanghai). The identified sequences were compared with the reference using ‘BLAST’ from the GenBank database. The phylogenic tree was constructed using the MEGA 5 and CLUSTAL-X programs by the neighbor-joining method with 1000 bootstrap replicates.

3. Results and discussion

3.1. Release and biodegradation of MCs

The initial concentration of extracellular MCs in the coagulated system was 9.01 μg L⁻¹, which was 53.08% lower than in the system without coagulation. This indicates that CTSAC coagulation flocs can reduce the level of MCs through adsorption. Even though the extracellular MCs increased to 18.78 μg L⁻¹ after 2 d, the concentration was still less than the uncoagulated control. The same trend remained for six days before it was reversed (Fig. 1). Thus the adsorption of the extracellular MCs through coagulation could last about six days. In comparison, the adsorption of extracellular MCs lasted for four days when CTS was used as sole coagulant,⁸ indicating CTSAC coagulation was a better solution for MC adsorption. Compared to the single coagulant, CTSAC demonstrated a stronger entrapment and bridging ability, as reported in our previous study.⁹ Therefore, the combination seemed to enhance the MC adsorption ability during coagulation and the extended sludge-holding period.

Fig. 1 The extracellular MC concentrations for (A) cells without coagulation and (B) cells with CTSAC coagulation stored for 0, 2, 4, 6, 8 and 10 d.

On day ten, the level of extracellular MCs in system that contained coagulant decreased about 59.05% compared to that on day six, while the level of the extracellular MCs in system containing no coagulant decreased about 89.86%. Thus the adsorption of MCs onto the coagulant is related to the duration of the sludge holding. Rapid degradation of MCs by coexisting bacteria in natural lakes, reservoirs, and laboratory culture of M. aeruginosa has been reported.^14,25,26 The above results indicated that MC-degraders may also exist in our systems and the CTSAC inhibited known MC-degraders, such as Pseudomonas sp., Bacillus cereus and Burkholderia sp., as a result, more MCs present in the coagulated system after six days.^8,13,27 On the other hand, it is also possible that, after six days, more intracellular MCs started to be released due to the onset of serious cell damage in the coagulated system than in the natural cell system. To confirm the hypotheses, the fate of M. aeruginosa cells was explored.

3.2. Fate of M. aeruginosa cells in the sludge

SEM micrographs were taken to analyze the cell changes of the two systems after floc storage of (a) 0 d, (b) 2 d, (c) 4 d, (d) 6 d and (e) 8 d (Fig. 2). Compared to the non-coagulated control, the membrane of M. aeruginosa cells retained intact and morphology did not change after coagulation at day 0, thus the CTSAC coagulation did not cause damage to M. aeruginosa cells. Even though cells started to become ruptured after two days in the control system, no obvious cell ruptures were observed in the coagulated system until after four days. After eight days, more M. aeruginosa cells were packed in flocs in the coagulated system than after six days. A possible mechanism is that the CTSAC coagulation can first form protective shields outside of the cells and then accelerate their proliferation. The SEM images after ten days were omitted as they show a similar phenomenon to that observed after eight days.

Fig. 2 SEM micrographs of M. aeruginosa cells before and after coagulation. (A) Cells with no coagulation, (B) cells with CTSAC coagulation. Flocs were stored for (a) 0 d, (b) 2 d, (c) 4 d, (d) 6 d and (e) 8 d.

The release of K⁺, a known indicator of M. aeruginosa cell integrity, was measured to further investigate the level of cell damage.²⁸ Our previous study further confirmed that the coagulation flocs would not adsorb K⁺, and the release of K⁺ could indicate the integrity of M. aeruginosa cells during floc storage.⁸ As shown in Fig. 3, the release of K⁺ in the control samples rapidly increased from 23.5% (day two) to 54.1% (day ten), indicating a rapid process of cell lysis. For the coagulated system, 8.5% release of K⁺ was observed after two days, significantly less than that of the control. Even though the release of K⁺ increased to 17.7% after four days, it was still much less than that of the control, and the same trend lasted until the end of the experiments. Of note, the release of K⁺ increased only 10.1% from days six to ten, which indicated cell damage was not severe during the storage between days six and ten. These results demonstrated that M. aeruginosa cells were not significantly damaged through a ten-day CTSAC-coagulated sludge storage process. This was consistent with a similar study that used AC as coagulant, which formed a protective shield for M. aeruginosa cells during a ten day sludge storage process.⁵ In comparison, CTS as sole coagulant accelerated the cell lysis of M. aeruginosa as a result of the degradation of CTS polymers after two days.⁸ Thus, the combination of CTS and AC may reduce the degradation rate of CTS.²⁰

Fig. 3 The release of K⁺ from (A) cells without coagulation and (B) cells with CTSAC coagulation stored for 0, 2, 4, 6, 8, and 10 d.

3.3. Characteristics of the EOM variation

In this study, the changes of EOM were evaluated using the fluorescence EEM. As illustrated in Fig. 4, the brightest peak (peak A) observed at an Ex/Em of 280/335 nm is for protein-like substances.²⁹ The second brightest peak (peak B) occurred at an Ex/Em of 230/350 nm, representing dissolved microbial metabolites.²⁹ Moreover, two other peaks located at an Ex/Em of 280/340 nm (peak C) and 340/430 nm (peak D) were ascribed to humic-like substances and fulvic-like substances, respectively.²⁹

Fig. 4 Fluorescence excitation–emission matrix (EEM) for extracellular organic matter (EOM) from uncoagulated cells stored for 0, 2, 4, 6, 8, and 10 d.

Of note, it is obvious that the protein organics disappeared in the CTSAC-coagulated system on day zero (Fig. 5). This might be due to the adsorption of EOM onto the CTSAC flocs. In addition, even though the protein and dissolved microbial metabolites increased as the storage time increased, they did not exceed that in the control during four days' storage time. Protein-like substance is primarily responsible for formation of haloacetamides which are highly toxic nitrogenous disinfection byproducts (N-DBPs).³ Therefore, CTSAC coagulation could largely reduce the amount of DBP precursors for at least four days during the process of composite coagulation. After four days, the intensities of the peaks of protein-like substances and dissolved microbial metabolites increased for the coagulated system (Fig. 5). As little cell damage was observed in the coagulated system, a possible explanation of the increased level of protein-like substances is that the addition of CTSAC could stimulate the proliferation of M. aeruginosa cells in sludge, and thence more metabolites excreted from cyanobacterial cells.²⁹ The adsorbed EOM may be released as the degradation of CTSAC occurred during sludge storage. However, to avoid DBP precursors being released again, the sludge should be disposed of within four days.

Fig. 5 Fluorescence excitation–emission matrix (EEM) for extracellular organic matter (EOM) from CTSAC-coagulated cells stored for 0, 2, 4, 6, 8, and 10 d.

The removal of humic- and fulvic-like materials was ineffective, since they are not able to crosslink with CTSAC, as previously illustrated.²⁸ As expected, the peak intensity of humic- and fulvic-like materials, derived from the decomposition of macromolecular organics, gradually increased with time.²⁹ However, by comparing Fig. 4 with 5, it can be found that there are less humic- and fulvic-like substances in the coagulated system than in the control samples for the same time period. Humic- and fulvic-like substances are the potential precursors of trihalomethanes (THMs) and haloacetic acids (HAAs).¹ This study implies that the application of CTSAC could reduce the risk of forming THMs and HAAs during the treatment of sludge laden with M. aeruginosa cells.

3.4. The variation of residual Al

As shown in Fig. 6, the concentration of residual aluminium in the control material gradually increased from 28.3 μg L⁻¹ to 80.1 μg L⁻¹ during the experiment. The residual Al after coagulation at day 0 was high in this study, suggesting there is still some dissolved Al species at pH 8.4 solution. This echoes the findings of Yang et al. (2013) who documented that an amount of residual Al concentration at pH > 8 after coagulation was reasonable owing to the existence of soluble Al species (Fig. 6).^21,30 The level of residual Al in the CTSAC-coagulated system decreased as sludge storage time was prolonged, as depicted in Fig. 6, with a sharp decrease down to 100.9 μg L⁻¹ on day four, a lower level than the standard drinking water limit in China (200 μg L⁻¹), and it remained lower than the standard thereafter.

Fig. 6 Residual Al concentration of (A) cells with no coagulation and (B) cells with CTSAC coagulation stored for 0, 2, 4, 6, 8, and 10 d.

Studies noted that EOM (e.g., proteins and humic-like substances), which possesses abundant functional groups, plays a crucial role in this adsorption process.³¹ It has been identified that the cell walls of cyanobacteria, which are composed of carbohydrates and polysaccharides, are also able to adsorb metals.³² Furthermore, metals can simultaneously be incorporated into aragonite or vacuole structures of cyanobacterial cells, and precipitated internally.³² With abundance of both cyanobacteria cell walls and EOM, the coagulated cyanobacteria-laden sludge has the ability to decrease residual Al content in aqueous solution. Indeed, a previous study has identified cyanobacteria as the key agent in a long-term strategy to remove metal pollutants through algal growth.³² On the other hand, the chemical condition may change upon prolonged storage, causing a change in Al speciation from Al³⁺ to Al(OH)₃ (gel), and finally to Al(OH)₄⁻, which are amorphous particles or sediments formed in flocs.²¹ Hence, with the negatively-charged EOM in the supernatant increased with time as discussed in Section 3.3, residual Al in aqueous solution was absorbed or transferred into amorphous particles species. Therefore, some of the residual Al could be removed in the coagulated cyanobacteria-laden sludge as storage time was prolonged. Unfortunately, significant cell lysis occurred in the natural system after two days, which could lead to desorption of Al from cell walls. Furthermore, the EOM decreased, which could cause desorption of Al from EOM in the natural cell system (Fig. 6). Correspondingly a gradual increase of residual Al was observed in the control samples.

3.5. Microbial community composition during sludge storage

The microbial communities from the control samples after 0 day and the sludge in the coagulated system after 0, 2, 4, 6, 8 and 10 days were examined using DGGE analysis of the 16S rDNA gene (Fig. 7). There were multiple bands on the DGGE gel, indicating numerous bacteria coexisting with M. aeruginosa, as expected.¹⁴ The sequence of excised DGGE bands were compared with the 16S rDNA database in GenBank using BLAST and the closest matches were used for phylogenetic analysis (Fig. 8). Analysis of acquired 16S rDNA genes revealed that major numbers of the phyla Cyanobacteria, Firmicutes, Bacteroidetes, and Proteobacteria were present in the overall microbial community (Fig. 8).

Fig. 7 (a) DGGE band profiles for Microcystis aeruginosa culture and CTSAC coagulation flocs for different storage times and (b) band peak intensities of mcyD fragments from Microcystis aeruginosa FACHB-905 with CTSAC coagulation for different floc aging times. Lane “M”: Microcystis aeruginosa culture without any treatment; Lane “0 d”–“10 d”: cells with coagulation stored for 0, 2, 4, 6, 8 and 10 days.

Fig. 8 Phylogenic tree of DGGE band sequences and reference sequences. Clones corresponding to the bands marked in Fig. 7.

The sequences of bands 1 and 13 were grouped into Cyanobacteria and associated with M. aeruginosa and uncultured Microcystis sp., respectively (Fig. 8). The peak intensities of band 1, which was closely related to M. aeruginosa (99%), were calculated by Quantity One v4.6.8 (Fig. 7b) in the coagulation system as previously reported.⁵ The peak intensities slightly decreased within the first two days and then increased significantly and continually for the rest of the experiment. Thus the number of M. aeruginosa cells started to increase after four days in the coagulated samples.

In our previous study, when AC was used, even after eight days the DGGE analysis showed that the majority of the M. aeruginosa cells were still present in the AC flocs and in the system without coagulant (control).⁵ However, no increase in cell numbers was observed in either the natural cell system or the AC-coagulated system in the previous study.⁵ Therefore, the growth of M. aeruginosa observed in the CTSAC coagulation flocs could be due to the addition of CTS. It has been reported that CTS-modified poly(L-lactic acid) substrate could promote cell proliferation.³³ An alginate-CTS hybrid was also reported to have increased cell adhesion and proliferation in vitro compared with alginate alone.³⁴ To date however, no studies have investigated whether a CTS-based composite could promote the growth of M. aeruginosa cells.

In the Proteobacteria phylum, band 12 represents bacteria related to Pseudomonas sp. and bands 11, 14 and 16 are affiliated with Burkholderia sp. (Fig. 8). Researchers reported Pseudomonas sp. existed in uni-cyanobacteria laboratory culture and Burkholderia sp. which was obtained from a coastal lagoon in Brazil both were microcystin-degrading bacteria.^13,35 Thus, the presence of Pseudomonas sp. and Burkholderia bacteria may be responsible for the rapid degradation of MCs in the control specimens. However, in the CTSAC-dosed system, band 12 was faint for the first 4 days and then became bright after 6 days (Fig. 7). Bands 11, 14 and 16 followed the same trend (Fig. 7). This might explain why the level of MCs in the CTSAC system increased at first, and then decreased as the degraders of MCs were not present at the beginning. Furthermore, the other sequences grouped into the Proteobacteria phylum in the control samples were identified as bacteria related to phosphate and organic pollutant removal.³⁶

The Firmicutes (band 7 and band 8) and Bacteroidetes (band 6 and band 9) phyla have also been reported to be able to degrade organic materials (Fig. 8).³⁷ Therefore, these organic degraders could be the reason for the reduction of protein-like substances and the increase of humic substances in the control samples. In comparison, the same bands in the coagulated system were actually diminishing during the storage process (Fig. 7). Therefore, less humic- and fulvic-like substances were produced in the coagulated system. The MC degraders such as Bacillus cereus, Pseudomonas sp. and Burkholderia sp. were also important pathogens.^13,15–17 Based on the above results, it can be concluded that the addition of CTSAC would decrease the pathogenic bacteria to a large extent. Yang et al.³⁸ prepared a quaternary ammonium salt grafted carboxymethyl CTS (CMC-g-PDMC) flocculant and found it also has pathogenic bacteria removal ability. However, they also noted that the high cost for preparation of CMC-g-PDMC limited its practical application. Comparatively, the easier preparation of CTSAC makes its application more economic and practicable. Future work will investigate the stability of the CTSAC composite coagulant (microbial decomposition) to further illustrate the practicability of this composite.

3.6. Implication of this work

After a simple preparation process, the novel CTSAC composite coagulant could effectively control the risk associated with secondary metabolites of cyanobacteria-laden sludge during 4 days storage, combining the advantages of CTS and AC coagulants. The dose of CTS in CTSAC composite coagulant reduces two-thirds compared to the CTS applied alone, and the amount of composite applied is lower than that of AC applied individually, making CTSAC composite a competitive and feasible coagulant. Therefore, the application of CTSAC is a promising way to treat cyanobacteria-laden source water during drinking water treatment, and the supernatant of CTSAC-coagulated cyanobacteria-laden sludge should be recycled during 4 days' sludge storage time.

This study analyzed the variations of microbial community in the CTSAC-coagulated cyanobacteria-laden sludge. The results showed that the degradation of CTSAC will support the persistence and proliferation of cyanobacteria as well as its coexisted bacteria. Thus, the CTSAC-coagulated cyanobacteria-laden sludge was suggested to dispose within 4 days to reduce the risk associated with the proliferation of cyanobacteria and its coexisted bacteria. Furthermore, it could also be concluded that the level of cell lysis and secondary metabolites release during cyanobacteria-laden sludge storage is largely related to the reagents (AC, CTS, or CTSAC) used in the coagulation/flocculation process. In the light of this finding, it is recommended that the risk associated with recycling sludge supernatant of different coagulation-generated cyanobacteria-laden sludge should be measured before bringing the supernatant return to the head of the plant.

4. Conclusion

The present results demonstrated that the MCs can be adsorbed onto the CTSAC coagulants for up to six days, along with a reduction in the biodegradation rate of MCs. The CTSAC flocs first showed evidence of providing a protective shield to M. aeruginosa cells, and then the degradation of the flocs would favor growth of the cells. This is the first study to report the occurrence of growth of M. aeruginosa cells in a CTS-based composite treated system. This work also showed that the EOM could be adsorbed by the CTSAC coagulants and there was no significant release of EOM in four days, thus a negligible level of DBP precursors was expected. Interestingly, the concentration of the residual Al in the coagulated system decreased sharply to 100.9 μg L⁻¹ after four days. The PCR-DGGE analysis based on the 16S rDNA gene indicated that the relative concentration of pathogens, such as Bacillus cereus, Pseudomonas sp. and Burkholderia sp., all being MC degraders, was reduced during the first four days but increased afterwards. Taken together, the CTSAC coagulation was initially (0–4 days) able to reduce the level of MCs and AOM through adsorption, but the reduction could not last due to the degradation of CTSAC after six days. In summary, it would be better to dispose of the sludge within four days for a clearer supernatant with less secondary metabolites. This study not only improves the understanding of the cyanobacterial variation, metabolites and biological risks during storage of CTSAC-coagulated cyanobacteria-laden sludge, but also is likely to facilitate improvements at DWTPs in terms of cyanobacteria-laden sludge management.

Acknowledgements

This work was supported by the Program for New Century Excellent Talents in University of the Ministry of Education of China (Grant No. NCET-12-0341), Natural Science Foundation of China (51478251), the International Cooperation Research of Shandong Province (2011176), Science and Technology Development Project of Shandong Province (2012GHZ30020), the International Science & Technology Cooperation Program of China (2010DFA91150) and National Science Fund for Excellent Young Scholars (51322811). The authors thank Dr David I. Verrelli of Medicine and Health Sciences, Macquarie University for revising the English in the manuscript.

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