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Article

Changes in Extracellular Microcystins (MCs) Accompanying Algae/Cyanobacteria Removal during Three Representative Algae/Cyanobacteria Inactivation Processes and an MC Diffusion Model in Still Water

School of Resources & Environment, Anhui Agricultural University, Hefei 230036, China
*
Author to whom correspondence should be addressed.
Water 2023, 15(20), 3591; https://doi.org/10.3390/w15203591
Submission received: 11 August 2023 / Revised: 5 October 2023 / Accepted: 5 October 2023 / Published: 13 October 2023

Abstract

:
This study addresses the lack of comparative research on algae/cyanobacteria elimination technologies in the existing literature. Our investigation focused on evaluating the performance of three commonly used algae/cyanobacteria removal processes: ultrasound (20.8 kHz), copper sulfate and biotic algicide (Bacillus subtilis). The evaluation considered both algae/cyanobacteria removal efficacy and the consequent changes in extracellular microcystins (MCs). To achieve this, we employed real eutrophic water as the test water. The ultrasound treatment demonstrated effective algae/cyanobacteria removal, with an average rate of algae/cyanobacteria decreasing (RAD) ranging from 0.50 to 0.99 µg chlorophyll a per liter per minute (µg chlorophyll a/L·min). On the other hand, the copper sulfate and biotic algicide treatments exhibited relatively lower rates of algae/cyanobacteria removal, with average RAD values of 0.21 to 0.38 µg chlorophyll a per liter per day (µg chlorophyll a/L·d) and 0.10 to 0.13 µg chlorophyll a per liter per day (µg chlorophyll a/L·d), respectively. Moreover, we observed significant increases in extracellular MCs in the ultrasound and copper sulfate treatments. The corresponding values of the increment of extracellular MCs accompanying removal per microgramme (µg) chlorophyll a (IEMARMC) were 0.34 to 2.43 µg MCs per µg chlorophyll a (µg MCs/µg chlorophyll a) and 18.13 to 185.08 µg MCs per µg chlorophyll a (µg MCs/µg chlorophyll a), respectively. However, in certain conditions where sufficient dosages (0.5 to 2 mg/L) and reaction time (≥8 days) are provided, the biotic algicide treatment could result in a decrease in MCs compared to an untreated control group (IEMARMC: −43.94 to −32.18 µg MCs per µg chlorophyll a). This suggests that the biotic algicide effectively degraded the MCs. In addition, we developed a one-dimensional MC diffusion model in still water based on Newton’s second law, which exhibits excellent simulation capabilities.

1. Introduction

The widespread eutrophication of water bodies has significantly increased the frequency of algae/cyanobacteria blooms in China. Decomposing algae/cyanobacteria blooms consume a massive amount of dissolved oxygen in water [1,2], resulting in deteriorated water quality [3]. In addition, Microcystis aeruginosa is one of the cyanobacteria most prone to blooms [4]. Microcystis can produce microcystins [5], which are potent hepatotoxins, neurotoxins and possible carcinogens [6,7,8]. Microcystins are recalcitrant because of their cyclic structures and a spacer double bond therein [9]; thus, they could pose a major threat to drinking water and water ecosystem safety. Once a bloom occurs, the algae/cyanobacteria need to be removed promptly. To date, many studies have focused on a variety of algae/cyanobacteria elimination technologies based on chemical methods. For example, Ma et al. (2023) evaluated the UV/Fenton system for the removal efficiency of Microcystis aeruginosa; a great removal (97.3%) of Microcystis aeruginosa cells was achieved [10]. Lin et al. (2021) investigated the effect of sodium hypochlorite (NaOCl) oxidation-assisted polyaluminum chloride (PACl) coagulation on algae removal; the results demonstrated that the highest algae removal was around 99% [11]. Chen et al. (2021) investigated two solid peroxides and H2O2 for the removal of Microcystis aeruginosa and found that H2O2 and peroxymonosulfate (PMS) effectively removed algae in 2 d at pH 5.0, 7.0 and 9.0, while peroxydisulfate (PDS) was only effective at pH 5.0 [12]. A study conducted by Fan et al. (2021) showed that self-floating photocatalytic hydrogel had excellent photocatalytic activity, with a 99.4% removal efficiency of chlorophyll a within 4 h; it can still remove above 95% chlorophyll a after six consecutive recycles [13]. Even though effective algae/cyanobacteria removal has been achieved using these chemical technologies, Microcystis elimination processes likely cause algae/cyanobacteria cell lysis and damage, resulting in the release of microcystins; the release of algal/cyanobacteria toxins is of great concern [14]. Some studies explored algae/cyanobacteria elimination methods with no/little algae/cyanobacteria cell lysis. For example, Song et al. (2020) applied Fe2+/persulfate to generate sulfate radicals and simultaneously form in situ Fe (III) for integrated oxidation–coagulation, leading to a notable enhancement in Microcystis aeruginosa removal without cell breakage and better control of microcystins [15]. These advanced technologies provide novel ways for algae/cyanobacteria elimination; however, more effort is still needed for their practical application. Copper sulfate is a frequently used, low-cost chemical reagent for bloom control and has been used to treat many lakes and reservoirs [16,17]. However, the release of intracellular microcystins caused by copper sulfate has also been observed in some studies [18]. Zhang et al. (2019) observed a drastic release of microcystins at a relatively low dose of 3 μg/mL of copper sulfate [18]. Zhou et al. (2013) found that increasing the copper sulfate dose resulted in a significant release of MCs [19]. Tsai (2013) concluded that the minimum amount of copper required to inhibit Microcystis aeruginosa populations could effectively reduce the total microcystin concentration; with the minimum amount of copper (160 µg Cu/L as copper sulfate; 80 µg Cu/L as copper–ethanolamine), despite the release of intracellular microcystins during applications of copper sulfate, microcystin degradation via copper compounds was not observed to cause an increase in extracellular microcystin concentrations [20]. However, there is still no consistent conclusion regarding the risk of extracellular microcystin changes caused by copper sulfate algicide.
Physical technologies for algae/cyanobacteria removal reported by the existing studies include coagulation, air flotation, ultrasonic irradiation, etc. Coagulation was commonly used for drinking water treatment. Ma et al. (2023) evaluated the enhanced coagulation performance for algae removal by using novel Al/Fe-based covalently bonded composite coagulants coupled with potassium permanganate oxidation; the results show that 99.6% OD686 (used for the measure of algal cell density) was removed from extracellular organic matter algae-laden water at a CAFM dosage of 5.4 mg/L and a KMnO4 dosage of 1 mg/L [21]. One study investigated the algae removal process using a low-pressure air flotation (LAF) system installed on a ferryboat in an experiment conducted in the Han River and Daecheong Dam; the LAF system ferryboat obtained chlorophyll a removal efficiency of 99% [22]. Ultrasonic irradiation is considered an environmentally friendly algae/cyanobacteria removal approach that requires no additional chemicals [23]. However, the release of intracellular toxic substances has become more and more of a concern since this technology was developed; there have been many studies indicating that microcystins are released during algae/cyanobacteria removal using ultrasonic irradiation. For example, Zhang et al. (2006) found that 5 min of sonication at 80 W (80 kHz) increased extracellular microcystins from 0.87 µg/L to 3.1 µg/L. In addition to the release of intracellular toxins via cells breaking down, two possible pathways for changes in extracellular microcystin concentrations are the secretion of more toxins by algae/cyanobacteria cells as a defensive countermeasure to sonication and potential aqueous toxin degradation via free radical reactions caused by cavitation [14]. Limited variations in extracellular microcystin concentrations were observed when sonication levels were below 48 W in the study; accordingly, sonication below 48 W is considered safe in terms of microcystin release [14]. Chen et al. (2020) found that high-power ultrasound (1200 W) can effectively degrade microcystins; consequently, no increase in microcystins was observed after using 1200 W ultrasound to treat Microcystis [24].
The removal of algae/cyanobacteria via ultrasound and the associated release of microcystins are believed to be dependent on ultrasonic frequencies, ultrasonic powers, ultrasonic irradiation time, the physiological state, the structure of the algae/cyanobacteria cells, etc. [25,26]. Ideally, algae/cyanobacteria cells are removed with low energy consumption, and only a small number of intracellular toxic substances are released. To obtain optimal ultrasound parameters for this purpose, more efforts are needed. Moreover, most of the existing studies used cultured Microcystis aeruginosa in their experiments; thus, there is a lack of understanding of the performance of ultrasound technology in treating real eutrophic water.
In addition to physical and chemical algicide, biotic algicide has been reported in recent years. For example, Liu et al. (2023) screened two algicidal strains, identified as Bacillus sp. and Brevibacillus sp., and performed a contrastive study on algicidal properties against Microcystis aeruginosa of these two strains with that of two preferred algicidal strains, Paenibacillus polymyxa and Paenibacillus alvei; it was concluded that the algicidal mode of these four strains was indirect. Brevibacillus sp. showed potential to treat eutrophic water containing harmful algae based on its significant algicidal activity and strong stability [27]. Jia et al. (2023) evaluated the algicidal potential of Paebubacillus sp. A9 (A9) cells, supernatants and cultures with flocculation properties against Microcystis aeruginosa. The results showed that different fractions of A9 exhibited algicidal activity in a dose- and time-dependent manner. Initially, polysaccharides with a series of carboxyl groups released by A9 caused flocculation of Microcystis aeruginosa cells, which was followed by the lysis of algal cells by the algicidal active compounds present in the A9 culture. The algicidal process activated the antioxidant system of Microcystis aeruginosa, as evidenced by a dramatic increase in the activities of catalase (CAT) and superoxide dismutase (SOD) enzyme [28]. Liu et al. (2023) verified the algicidal efficacy of Brevibacillus sp. in the practical application scenario. Results indicated that the algicidal threshold of Brevibacillus sp. culture was 3‰ inoculation concentration, and the removal rate of Microcystis aeruginosa reached 100% [29]. To the best of our knowledge, studies about microcystin release and degradation caused by biotic algicide are rather rare.
The release and degradation of microcystins resulting from algicide application are influenced by various factors, with the composition of algae/cyanobacteria species being particularly important. However, accurately replicating the algae/cyanobacteria species composition in real eutrophic water is challenging, highlighting the need for investigations into the changes in microcystin concentrations when algicides are applied to real eutrophic water. Unfortunately, the existing studies on this subject are insufficient in their scope and findings.
To identify the most effective processes for removing algae/cyanobacteria, it is essential to compare representative algae/cyanobacteria removal technologies. However, only a limited number of studies have addressed this topic to date. Therefore, the primary objective of this study is to examine the changes in microcystin concentrations during algae/cyanobacteria removal from real eutrophic water using three commonly employed removal processes: sonication, copper sulfate, and biotic algicide (Bacillus subtilis). By comparing these processes based on their efficiency in algae/cyanobacteria removal and the alterations in extracellular microcystins, we aim to determine the optimal algae/cyanobacteria removal technique.
Additionally, the diffusion processes of microcystins in water, including their migration, have not been adequately investigated. Consequently, the secondary objective of this study is to develop a mathematical model that can describe these diffusion processes in water. This model will serve as a valuable tool for controlling microcystins after implementing an algae/cyanobacteria removal process.

2. Materials and Methods

2.1. Test Water

Test eutrophic water was collected from western Chaohu Lake on 5 and 28 September 2018 (when a water bloom was breaking out) for algae/cyanobacteria removal experiments and to simulate MC diffusion in still water. Some of the physicochemical parameters of eutrophic water are shown in Table 1. The concentrations of some critical ions that were not detected by us can be found in the literature [30].
Via microscopic examination, we determined that Microcystis was the dominant cyanobacteria genus. The eutrophic water was used in the experiments after a short period of storage (2~4 h) at the temperature of 28 ± 1 °C indoors. Aeration with a micro-porous aerator was conducted to maintain the DO at 2.5 mg/L during storage. According to our preliminary tests, copper sulfate and biotic algicide exhibited poor algae/cyanobacteria removal, even at high dosages (copper sulfate > 10 mg/L; biotic algicide > 10 mg/L) when treating the raw eutrophic water, possibly owing to the dense algae/cyanobacteria hindering the algicide mass transfer; hence, the raw eutrophic test water was diluted 4 times for the copper sulfate and biotic algicide treatments. Of note, the concentrations of MCs in the diluted eutrophic test water did not decrease proportionally to that of chlorophyll a. The possible reason is that the raw eutrophic test water for ultrasound treatment and the other two treatments (copper sulfate and biotic algicide treatments) were collected from different points (the raw eutrophic test water for ultrasound treatment: point 1; the raw eutrophic water of the other two treatments: point 2) of the lake at which the concentrations of MCs might be different (but the concentrations of chlorophyll a are close) for complex reasons. The two sampling points were about 150 m apart.

2.2. Algae/Cyanobacteria Removal Experiments

To investigate changes in the concentrations of extracellular MC and chlorophyll a in the three algae/cyanobacteria removal processes, a series of algae/cyanobacteria removal experiments were conducted. Several plastic pails were used in the experiments; each pail was filled with 5 L of test water. In the ultrasound algae/cyanobacteria removal experiment, an ultrasonic apparatus with a low-frequency probe (20.8 kHz) was employed (Shenzhen Xinhe Ultrasonic Equipment Co., Ltd., Shenzhen, China). The acoustic power, P (W), was calculated via the following equation:
P = m C p d T d t
where T is temperature (°C); t is time (seconds); Cp is the heat capacity of water at 25 °C (J ·kg−1 ·K−1); and m is the molar mass of H2O (kg).
The algae/cyanobacteria removal and changes in extracellular MCs were investigated using three ultrasonic power densities (UPD) (2.6 W/L, 10.62 W/L and 16 W/L) as well as an untreated control. Algae/cyanobacteria removal experiments using copper sulfate/biotic algicide (Bacillus subtilis) (Beijing Green Kingline Technology Development Co., Ltd., Beijing, China) were conducted using five different dosages, including an untreated control (0 mg/L, 0.5 mg/L, 2 mg/L, 4 mg/L, 8 mg/L for copper sulfate; 0 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L for biotic algicide). A constant temperature shock incubator (QHZ-98A) (Taicang Huamei Biochemical Instrument Co., Ltd., Suzhou, China) was used in the treatments with a chemical/biotic algicide. The shock incubator shook at an oscillation frequency of 100 rpm. The temperature inside the chambers was 27 °C; the light intensity during the day was 80,000 LX for the treatments with the chemical/biotic algicide.
Samples from the pails were collected at 0, 1, 3, 5, 10 and 15 min during the ultrasound treatments and at 0, 1, 2, 4, 6 and 8 days during the treatments with the chemical/biotic algicide. Concentrations of chlorophyll a and extracellular MCs in each sample were analyzed.
All tests were conducted in triplicate. Data were presented as the mean of three parallel tests, shown as the mean ± standard deviation (SD).

2.3. Simulation of MC Diffusion in Still Water

A device (Figure 1) to simulate MC diffusion in still water (lakes or reservoirs) was designed for the current study. The device was made of plexiglass and had a length, width, and height of 2 m, 2 m and 0.3 m, respectively. A movable plexiglass cylinder with a diameter of 0.3 m and a height of 0.28 m was placed in the center of the device, forming an operation area for centralized algae/cyanobacteria removal. Sampling points were set at the circles with distances of 0.45 m, 0.75 m and 1 m from the center point; each circle contained four sampling points. Diluted eutrophic water and dechlorinated tap water were added to the inside and outside of the cylinder before the experiment, respectively, leveling the water surface on both sides. To produce microcystins, the eutrophic water inside the cylinder was irradiated with ultrasound (20.8 kHz, 16 W/L) for 15 min, followed by the quick removal of the cylinder with a slight disturbance to the water. Water samples from all sampling points were collected 1 min, 10 min, 30 min, 4 h and 12 h after the cylinder was removed. MC concentrations in these water samples were measured; the parameters of the developed MC diffusion model were estimated, and model verification was performed based on the MC concentration data.

2.4. Analytic Methods

Since all the living algae/cyanobacteria contained chlorophyll a, algae/cyanobacteria biomass was indicated by chlorophyll a [31]. Chlorophyll a was measured as follows: A 5 mL sample was filtrated using a 0.45 μm cellulose acetate membrane in a dark room to retain the algae/cyanobacteria. After the filtration, we cut the membrane into pieces and put them into a centrifuge tube with a stopper, followed by the addition of 90% acetone and a small amount of MgCO3. The sample was frozen for 60 min at −20 °C and then thawed at room temperature; this process was repeated three times. We vibrated the centrifuge tube in a thermostatic oscillator for 2 min after diluting the sample to 10 mL with 90% acetone. The sample was then refrigerated at 4 °C in a refrigerator and taken out after 30 min to be shaken; the operation was repeated three times in darkness. Before being measured, the sample was centrifuged at 3500 r/min for 15 min and diluted to 10 mL with 90% acetone. The OD750, OD663, OD645 and OD630 values of the sample were measured using a UV-visible spectrophotometer. The chlorophyll a concentration was calculated using Equation (2):
Chlorophyll   a   ( μ g / L ) = [ 11.64 ( O D 663 O D 750 ) 2.16 ( O D 645 O D 750 ) + 0.10 ( O D 650 O D 750 ) ] V 1 V δ
where V is the volume of the water sample, V1 is the volume of the extract, and δ is the cuvette optical path.
Extracellular MC concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Suwei Microorganism Co., Ltd., Suzhou, China) following the manufacturer’s instructions. The limit of detection for this kit is 0.1–2 µg/L. To prevent the MC concentrations in the samples from exceeding the detection limit, the samples were diluted 20–150 times before measurement.

2.5. Assessment of Changes in Extracellular MCs Accompanying Algae/Cyanobacteria Removal

The increment of extracellular MCs (µg) accompanying removal of per microgramme (µg) chlorophyll a (IEMARMC), which should be determined based on the comprehensive effects of algae/cyanobacteria cell ruptures and MC degradation caused by ultrasound/algicide during algae/cyanobacteria inactivation, was employed to present changes in extracellular MCs accompanying algae/cyanobacteria removal. The IEMARMC was calculated using Equation (3):
IEMARMC   ( μ g   MCs /   μ g   chlorophyll   a ) = C M C s - t r C M C s - c o C c h l - c o C c h l - t r
where CMCs-tr and CMCs-co are the concentrations of extracellular MCs in an ultrasound/copper sulfate/biotic algicide treatment and that of untreated control, respectively; Cchl-tr and Cchl-co are the total concentration of chlorophyll a in the water in an ultrasound/copper sulfate/biotic algicide treatment and that of an untreated control, respectively.

3. Results and Discussion

3.1. Dynamic Change in Extracellular MCs and Chlorophyll a Concentrations during the Three Algae/Cyanobacteria Removal Processes

Figure 2 shows the dynamic concentrations of chlorophyll a and extracellular MCs during the three algae/cyanobacteria removal processes. No nutrients for algae/cyanobacteria were added during the experiments; as a result, natural attenuation in the algae/cyanobacteria was inferred via an observable decline in chlorophyll a in the untreated control groups during a relatively long test time (Figure 2e,f). Chlorophyll a concentrations decreased more sharply in the ultrasound/copper sulfate/biotic algicide treatments compared with the untreated controls (Figure 2), indicating the effectiveness of the three methods in inactivating algae/cyanobacteria.
The ultrasound treatments resulted in a greater increase in extracellular MC concentrations compared with the untreated control (Figure 2a), likely because of algae/cyanobacteria cell ruptures caused by the ultrasound [14,23]. Ultrasound can physically damage the cell wall/membrane through high shear forces and solution jets rising from cavitation, leading to the release of intracellular toxins [23]. Cavitation bubbles are considered the main cause of algae/cyanobacteria cell ruptures [32]. Considering the disadvantages of high-frequency ultrasound (energy declines faster with distance and always requires high power to produce better effects) [26,33], a low-frequency (20.8 kHz) ultrasound was employed in this study. Some studies have indicated that lower ultrasonic frequencies are favorable for generating cavitation [26], which tends to aggravate ruptures in the algae/cyanobacteria cells. High-frequency ultrasound generally generates less cavitation yet likely produces a larger proportion of free radicals, which could chemically attack and weaken the algae/cyanobacterial cell walls [23]. Therefore, a higher frequency does not necessarily alleviate algae/cyanobacteria cell lysis. Yamamoto et al. (2015) found that high-frequency sonication could disrupt algae/cyanobacteria cells more violently compared with low-frequency sonication [34]. Further studies are still needed to explore the effect of ultrasonic frequency on algae/cyanobacteria cell lysis, especially when ultrasound is employed to treat real eutrophic water.
In our study, improving ultrasonic power density was favorable to algae/cyanobacteria removal (Figure 2d), which was in agreement with the conclusions of other studies [26,35]. However, a higher ultrasonic power density resulted in a greater extracellular MC increase at the same time (Figure 2a). Table 2 shows that a higher ultrasonic power density resulted in a greater IEMARMC value, suggesting that improving ultrasonic power density might aggravate algae/cyanobacteria cell damage. This conclusion was also implied by a study that observed a more intense immediate release of microcystin at a higher ultrasonic power density [36].
Hydroxyl radicals are capable of degrading MCs [37]. Some studies have reported that a higher ultrasonic power density could produce a greater number of hydroxyl radicals [26,37]. However, these produced hydroxyl radicals are likely insufficient to degrade all the released MCs, according to the results of this study. Thus, an increase in extracellular MCs is likely inevitable in ultrasound algae/cyanobacteria removal processes under operational conditions similar to those of the current study. Chen et al. (2020) found that when Microcystis was treated with ultrasound at 28 kHz (1200 W), the levels of microcystins in the water did not increase [24]. The discrepancy between the results of our study and that research might be attributed to the difference in ultrasonic power density adopted in the two studies. The complex quality of the test water (real eutrophic water) used in our study was different compared with that of the test water containing only cultured Microcystis aeruginosa used in Chen et al. [24], which significantly influenced our results. Adjusting the ultrasonic frequency, the ultrasonic irradiation time and other ultrasonic parameters may alleviate MC releases caused by ultrasound, but a high energy cost might be necessary [26]. In the future, it may be worth investigating advanced methods to help mitigate algae/cyanobacteria cell ruptures caused by ultrasound algae/cyanobacteria removal processes and developing effective technologies for in situ algae/cyanobacteria toxin elimination should be encouraged.
As shown in Figure 2e,f, the copper sulfate/biotic algicide algae/cyanobacteria removal processes were observably impacted by the dosages of the algicide. Increasing the dosages was favorable to algae/cyanobacteria removal, but the effects were not noticeable over a certain value (4 mg/L for copper sulfate; 2 mg/L for biotic algicide), implying that an optimal dosage of copper sulfate/biotic algicide exists for a specific quality of raw water.
Some studies have found that Cu2+ could damage algae/cyanobacteria cells through copper chelates/lipid peroxidation, leading to algae/cyanobacteria ruptures [18,38]; cell lysis could facilitate intracellular microcystin release [39]. In the current study, obvious MC releases seemed to occur during the copper sulfate treatments, given the larger increase in extracellular MCs compared with the untreated controls. A larger dose of copper sulfate resulted in a higher IEMARMC overall (Table 3), indicating that improving the copper sulfate dosage likely accelerated algae/cyanobacteria ruptures. Shen et al. (2019) also concluded that a higher copper concentration could result in faster cell lysis [40].
Cu2+ is capable of degrading microcystins by inducing oxidation in microcystins [18,20]. A previous study conducted with a synthetic culture medium concluded that extracellular MCs in copper sulfate treatments could be degraded to less than those of an untreated control at a proper range of copper sulfate dosage (80~1000 µg/L) 8 days after the treatments [20]. Conversely, the extracellular MC concentrations in this study’s copper sulfate treatments maintained their higher levels compared with those of the untreated controls and rose throughout the testing period (lasting 8 days) at all dosages (Figure 2b). Wu et al. (2017) found that some variations related to Microcystis aeruginosa removal and microcystin release were species-dependent [16]. Thus, the specific result of the current study can likely be correlated with the complex composition of the algae/cyanobacteria species in the test water, as well as the relatively large algae/cyanobacteria concentration.
In theory, improving the dosage of Cu2+ could promote the MC degradation induced by Cu2+. Nevertheless, a larger Cu2+ dosage resulted in a greater increase in extracellular MCs in this study (Figure 2b), implying that the number of increased extracellular MCs caused by increasing the Cu2+ dosage was far greater than the number of extracellular MCs degraded by the increased Cu2+. Thus, it can be concluded that increased extracellular microcystins are inevitable if the Cu2+ dosage is increased to achieve greater algae/cyanobacteria removal under conditions similar to those of the current study.
Biotic algicide may inhibit the growth of algae/cyanobacteria through the following factors: competition for nutrients/survival space with microalgae/cyanobacteria; the secretion of algicidal substances; and the flocculation of microalgae/cyanobacteria [41]. Some bacterial strains can produce polysaccharide lyase capable of disrupting algae/cyanobacteria cell walls [42]. Figure 2c shows that the extracellular MC concentrations in the biotic algicide treatments were greater than those of the untreated control starting on the second day of treatments, indicating that algae/cyanobacteria cell damage occurred during the biotic algicide algae/cyanobacteria removal. A larger dosage of biotic algicide likely resulted in more serious cell damage, which is implied by the result showing that a greater dosage corresponded to a greater IEMARMC overall (Table 4). Figure 2c shows that extracellular MC concentrations in the untreated control monotonically increased during the experiments. Conversely, the extracellular MC concentrations in the biotic algicide treatments increased first and then declined, and they were smaller than those of the untreated control 6 days after biotic algicide treatments at dosages of less than 4 mg/L. Based on these results, the biotic algicide seemed to have a good ability to degrade MCs.

3.2. Comparison of the Three Algae/Cyanobacteria Removal Processes

The three processes for algae/cyanobacteria removal were compared based on the above experimental data in terms of the rate of algae/cyanobacteria decreasing (RAD) and IEMARMC values. In this study, the average RAD values at different ultrasonic power densities/dosages in ultrasound, copper sulfate, and biotic algicide treatments were 0.5–0.99 µg chlorophyll a/L·min, 0.21–0.38 µg chlorophyll a/L·d, and 0.1–0.13 µg chlorophyll a/L·d, respectively. The ultrasound treatment achieved a much greater RAD compared with the other two treatments. Ultrasound (20.8 kHz) at a power density of 16 W/L could remove chlorophyll a at a removal rate of up to 78.8% in 15 min. The copper sulfate treatments were also capable of achieving a great removal of up to 96% at a dosage of 4 mg/L, but this required a relatively long reaction time (≥8 days in the current study). The biotic algicide exhibited the lowest algae/cyanobacteria removal effectiveness in this experiment, with 34.6% in 8 days. The ultrasound IEMARMC values were relatively low (Table 2); however, of note, the ultrasound treatments only lasted for 15 min. The continuous release of microcystins from damaged algae/cyanobacteria cells after ultrasonic irradiation was not investigated in the current study but should be considered in further studies. Of the three algae/cyanobacteria removal processes, the copper sulfate treatment seemed to pose the greatest risks related to MCs, considering it has the largest IEMARMC value overall (Table 3). Given a sufficient reaction time (≥8 days in the current study), the IEMARMC of the biotic algicide treatment could be decreased to a negative value with a biotic algicide dosage of 0.5~2 mg/L (Table 4), which was expected given the biotic algicide’s good ability to degrade MCs, as mentioned above.
In conclusion, ultrasound and copper sulfate can achieve effective algae/cyanobacteria removal under appropriate operational conditions; ultrasonic irradiation is likely suitable for rapidly removing algae/cyanobacteria, given its good RAD value. However, the secondary pollution of MCs caused by these two algae/cyanobacteria removal processes seems to be non-negligible; thus, methods/techniques for MC elimination might be necessary in cases where these two processes are employed to treat real eutrophic water. The biotic algicide employed in this study seemed to have the potential for MC elimination and algae/cyanobacteria removal, given a sufficient reaction time. Therefore, the biotic algicide could be used to further inactivate algae and eliminate released MCs after using another algae/cyanobacteria removal process.
The current study just focused on changes in extracellular microcystins caused by algae/cyanobacteria inactivation processes; however, intracellular MCs and total MCs are also important for a better understanding of those algae/cyanobacteria removal processes. Further studies are needed to compare the three typical algae/cyanobacteria removal processes in terms of intracellular MCs and total MCs.

3.3. Development and Validation of an MC Diffusion Model in Still Water

MC diffusion in still water was assumed to be a one-dimensional diffusion process in this study. A diffusion model was developed based on Newton’s second law, as described in Equation (4):
C t = D 2 C x 2
where C is the concentration of MCs (µg/L); t is the diffusion time (h); x is the distance from the MC source to an arbitrary point in the water; and D (m2/h) is the diffusion coefficient. Based on the simulation experiment for MC diffusion in still water (see Section 2.3), after the quick removal of the cylinder, MCs produced by the algae/cyanobacteria removal process could be considered a pollutant source that is injected into the central area in an instant. Thus, the general solution for Equation (4) is as follows:
C = M 4 π D t exp ( x 2 4 D t )
where M (µg) is the total mass of MCs per unit volume in the central area. In a bounded water area, considering the complete reflection of the boundaries, MC concentrations at an arbitrary point should be in a superposition over the true source and image sources; they can be described with Equation (6):
C = n = M 4 π D t exp [ ( x + 2 n L ) 2 4 D t ]
where L is the distance from the MC source to the water boundary. Based on the results of the microcystin diffusion simulation device (Figure 1), L = 1 m. Reflections were considered twice. The CFTOOL of MATLAB was employed to fit the MC data at the source point and the temporal MC concentrations at x = 1 with Equation (6) to obtain Equation (7):
C = n = 2.25 4.76 t exp [ ( x + 2 n ) 2 1.516 t ] . ( R 2   =   0.982 )
Diffusion coefficient D was estimated to be 0.379 m2/h using the experimental data obtained from the MC diffusion simulation in still water. The surface corresponding to Equation (7) is shown in Figure 3. Figure 3 shows that the MC concentrations at all x (>0) values rose first and then declined. A smaller x seemed to result in greater MC concentration fluctuations with time, starting at the beginning of the MC diffusion process. This might be due to a greater MC concentration gradient at the point closest to the MC source. MC concentrations were nearly equal in the diffusion area after two hours of diffusion, indicating fast MC diffusion in still water. Accordingly, quickly eliminating MCs after an algae/cyanobacteria removal operation is necessary.
Data at x = 0.45 m and x = 0.75 m were used to verify the model. Figure 4 shows that the relative errors between the simulated and measured MC concentrations were within 3.7~19.9%, indicating that the model simulated MC diffusion in a still water well, and this could help predict the distribution of MC concentrations in a given diffusion area. However, it should be noted that the developed model only attempted to simulate the MC diffusion process in still water, and the model did not take natural physicochemical conditions and the effect of water movement into account, so more efforts are still needed to complete the model for accurate simulation of change in MC concentration in water bodies under natural conditions. The current study provides useful information for further investigation about the simulation of the change in MC concentration in water, which should be helpful for MC pollution control during algae/cyanobacteria removal processes.

4. Conclusions

Using real eutrophic water, the performance of three typical algae/cyanobacteria removal processes (ultrasound (20.8 kHz), copper sulfate and biotic algicide (Bacillus subtilis)) was investigated in terms of algae/cyanobacteria removal and changes in extracellular MCs caused by said processes. The results show that all three algae/cyanobacteria removal processes can effectively reduce algae/cyanobacteria mass to some extent (indicated by chlorophyll a concentrations). The ultrasound treatment reduced algae/cyanobacteria at a high rate (0.50–0.99 ·g chlorophyll a/Lžmin); a chlorophyll a removal rate of 78.8% was achieved by ultrasound treatment for 15 min at a power density of 16 W/L. Accordingly, ultrasonic irradiation is likely a suitable method for rapidly removing algae/cyanobacteria. The RAD values (0.21–0.38 µg chlorophyll a/L·d) in the copper sulfate treatments were much lower compared with the ultrasound treatments. As a result, although the copper sulfate treatments achieved a high removal rate (up to 96% at a dosage of 4 mg/L), a long reaction time—greater than 8 days—was required. Obvious increases in MCs were caused by the ultrasound and copper sulfate treatments in this study; the IEMARMC values of these two processes were 0.34~2.43 µg MCs/µg chlorophyll a and 18.13~185.08 µg MCs/µg chlorophyll a, respectively. The copper sulfate treatments seemed to pose the greatest risk related to MCs, given that they had the highest IEMARMC values. The biotic algicide treatment had the lowest algae/cyanobacteria removal effectiveness of the three processes, with RAD values of 0.10–0.13 µg chlorophyll a/L·d and algae/cyanobacteria removal rates of less than 34.6% over 8 days of reaction. However, the biotic algicide treatments seemed to degrade MCs efficiently; given a sufficient reaction time, biotic algicide was able to achieve a decrease in MCs compared to that for an untreated control (IEMARMC values: −43.94~−32.18 µg MCs/µg chlorophyll a at a dosage of 0.5~2 mg/L in 8 days). Accordingly, we suggest using biotic algicide to further inactivate algae/cyanobacteria and eliminate released MCs after other algae/cyanobacteria removal processes. One limitation of this study is that we only focused on changes in extracellular microcystins. Further studies are suggested to compare the three typical algae/cyanobacteria removal processes in terms of intracellular MCs and total MCs. A one-dimensional MC diffusion model in still water was developed based on Newton’s second law. The simulation results of the model showed fast MC diffusion in still water. The model was verified to have a good simulation ability regarding MC diffusion in still water, and it could help predict MC distributions in a given diffusion area. The relative errors between the simulated and measured MC concentrations were between 3.7% and 19.9%. The developed model did not take natural physicochemical conditions and the effect of water movement into account, and more efforts are still needed to complete the model for accurate simulation of change in MC concentration in water bodies under natural conditions.

Author Contributions

Conceptualization, C.S. and W.X.; methodology, W.F. and J.Y.; writing—original draft preparation, C.S. and W.X.; software, validation, investigation, W.F. and C.S.; formal analysis, data curation, M.M. and J.Y.; writing—review and editing, C.S., W.F. and W.X.; visualization, supervision, project administration, funding acquisition, W.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Major Natural Science Research Projects of Colleges and Universities in Anhui Province (KJ2017ZD16).

Data Availability Statement

Data will be made available on request.

Acknowledgments

We thank Jiangsu Suwei Microorganism Co., Ltd. for assistance with material supplies and assay analysis.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic diagram of the device for microcystin diffusion simulation.
Figure 1. Schematic diagram of the device for microcystin diffusion simulation.
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Figure 2. Dynamic changes in extracellular MCs during the three algae/cyanobacteria removal processes: (a) ultrasound; (b) copper sulfate; (c) biotic algicide. Dynamic changes and dynamic change in total chlorophyll a during the three algae/cyanobacteria removal processes: (d) ultrasound; (e) copper sulfate; (f) biotic algicide; data are presented as the mean ± SD, n = 3.
Figure 2. Dynamic changes in extracellular MCs during the three algae/cyanobacteria removal processes: (a) ultrasound; (b) copper sulfate; (c) biotic algicide. Dynamic changes and dynamic change in total chlorophyll a during the three algae/cyanobacteria removal processes: (d) ultrasound; (e) copper sulfate; (f) biotic algicide; data are presented as the mean ± SD, n = 3.
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Figure 3. Surface of the MC diffusion model in still water.
Figure 3. Surface of the MC diffusion model in still water.
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Figure 4. Simulated and measured values for temporal MC concentrations at x = 0.45 m (a) and x = 0.75 m (b); the measured data are presented as the mean ± SD, n = 3.
Figure 4. Simulated and measured values for temporal MC concentrations at x = 0.45 m (a) and x = 0.75 m (b); the measured data are presented as the mean ± SD, n = 3.
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Table 1. Physicochemical parameters of the sampling points.
Table 1. Physicochemical parameters of the sampling points.
Physicochemical Parameters5 September 201828 September 2018
Point 1Point 2Point 3
TN(mg/L)2.24 ± 0.182.51 ± 0.282.36 ± 0.25
TP(mg/L)0.070 ± 0.0080.073 ± 0.0060.08 ± 0.004
PO43−-P(mg/L)0.013 ± 0.0020.017 ± 0.0050.012 ± 0.004
NH4+-N(mg/L)0.855 ± 0.0121.042 ± 0.0230.276 ± 0.013
NO2-N(mg/L)0.016 ± 0.00130.042 ± 0.00180.057 ± 0.0013
NO3-N(mg/L)1.462 ± 0.0921.12 ± 0.0831.374 ± 0.087
DO(mg/L)7.82 ± 0.407.17 ± 0.338.32 ± 0.35
Secchi depth/(cm)39.8 ± 6.424.7 ± 3.847.6 ± 8.3
T/°C28.0 ± 1.028.0 ± 1.226.0 ± 1.3
pH8.01 ± 0.037.98 ± 0.058.21 ± 0.05
Chlorophyll a/(µg/L)15.46 ± 0.3318.82 ± 0.4816.42 ± 0.52
Notes: Data are presented as the mean ± SD, n = 3.
Table 2. IEMARMCs (µg MCs/µg chlorophyll a) caused by ultrasound.
Table 2. IEMARMCs (µg MCs/µg chlorophyll a) caused by ultrasound.
Ultrasonic Power Density (W/L)1 min3 min5 min10 min15 min
2.650.51 ± 0.070.43 ± 0.090.55 ± 0.060.48 ± 0.020.55 ± 0.05
10.620.34 ± 0.040.54 ± 0.030.82 ± 0.211.18 ± 0.191.36 ± 0.11
161.51 ± 0.421.21 ± 0.271.63 ± 0.182.12 ± 0.532.43 ± 0.17
Notes: Data are presented as the mean ± SD, n = 3.
Table 3. IEMARMCs (µg MCs/µg chlorophyll a) caused by CuSO4.
Table 3. IEMARMCs (µg MCs/µg chlorophyll a) caused by CuSO4.
Dosage of CuSO4 (mg/L)1 d2 d4 d6 d8 d
CuSO40.558.0 ± 8.725.81 ± 1.676.48 ± 4.518.13 ± 1.919.2 ± 3.9
256.28 ± 6.5167.85 ± 22.159.02 ± 8.737.07 ± 4.624.14 ± 2.5
4102.11 ± 13.1178.65 ± 16.787.67 ± 9.948.31 ± 2.840.92 ± 5.5
895.98 ± 10.5185.08 ± 13.874.59 ± 6.660.99 ± 7.743.02 ± 3.2
Notes: Data are presented as the mean ± SD, n = 3.
Table 4. IEMARMCs (µg MCs/µg chlorophyll a) caused by biotic algicide (BA).
Table 4. IEMARMCs (µg MCs/µg chlorophyll a) caused by biotic algicide (BA).
Dosage of BA
(mg/L)
1 d2 d4 d6 d8 d
BA0.5−61.38 ± 3.213.2 ± 1.6−16.4 ± 3.2−32.12 ± 4.5−43.94 ± 8.1
1−94.61 ± 8.280.58 ± 6.2−0.77 ± 0.1−14.33 ± 2.6−32.18 ± 5.03
299.44 ± 7.3121.74 ± 10.16.24 ± 2.1−9.72 ± 2.9−20.14 ± 3.7
4133.62 ± 18.9230.19 ± 28.817.7 ± 4.74.09 ± 0.566.99 ± 1.5
Notes: Data are presented as the mean ± SD, n = 3.
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Shi, C.; Fang, W.; Ma, M.; Xu, W.; Ye, J. Changes in Extracellular Microcystins (MCs) Accompanying Algae/Cyanobacteria Removal during Three Representative Algae/Cyanobacteria Inactivation Processes and an MC Diffusion Model in Still Water. Water 2023, 15, 3591. https://doi.org/10.3390/w15203591

AMA Style

Shi C, Fang W, Ma M, Xu W, Ye J. Changes in Extracellular Microcystins (MCs) Accompanying Algae/Cyanobacteria Removal during Three Representative Algae/Cyanobacteria Inactivation Processes and an MC Diffusion Model in Still Water. Water. 2023; 15(20):3591. https://doi.org/10.3390/w15203591

Chicago/Turabian Style

Shi, Chengcheng, Weijian Fang, Mengru Ma, Wei Xu, and Jingjing Ye. 2023. "Changes in Extracellular Microcystins (MCs) Accompanying Algae/Cyanobacteria Removal during Three Representative Algae/Cyanobacteria Inactivation Processes and an MC Diffusion Model in Still Water" Water 15, no. 20: 3591. https://doi.org/10.3390/w15203591

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