Investigation on Lanthanum Modified Kaolinite for Control of Cyanobacterial Growth and Microcystin Production

Abstract

Eutrophication and its resultant cyanobacterial blooms are a severe environmental issue in global water bodies, and phosphate is regarded as one of the primary triggers. In this study, the in situ-synthesized heated kaolinite lanthanum hydroxide composite (HKL-LH) was used to treat cyanobacterial blooms through phosphate removal. A typical cyanobacteria species—Microcystis aeruginosa—was selected as the target organism. HKL-LH efficiently removed phosphate in the solution with the inoculation of M. aeruginosa over the course of one day. A good performance of HKL-LH on control cyanobacterial blooms with initial cell densities ranging from 10⁴ cells mL⁻¹ to 10⁵ cells mL⁻¹ was observed. Although the genetic expression relating to photosynthesis and cell division was upregulated under the stress of phosphorus deficiency, M. aeruginosa growth was significantly inhibited, i.e., the inhibition rate of up to 98% was achieved by 0.1g L⁻¹ of HKL-LH. In addition to cell growth, the photosynthetic activity and viability of M. aeruginosa cells were decreased by HKL-LH. Furthermore, the production of associated toxins (microcystins) and algal organic matters were effectively inhibited, which can reduce the ecological risk and challenges that follow water treatment. In this study, it is shown that HKL-LH has excellent application potential in the mitigation of cyanobacterial blooms in eutrophic water.

1. Introduction

Eutrophication in various water bodies is a critical environmental problem worldwide, leading to the frequent occurrence of cyanobacterial (blue–green algal) blooms [1]. These blooms can negatively impact aquatic environments [2], inducing the depletion of dissolved oxygen, death of fish and invertebrates, and reduction in biodiversity [3]. Microcystis aeruginosa (M. aeruginosa), one of the dominant bloom-forming species of cyanobacteria [4], can produce harmful metabolites including microcystins (MCs) which are potent liver toxins [5]. In addition, the algal organic matters (AOMs) produced via cyanobacteria may interfere with coagulation/flocculation, block filters/membranes, and act as the precursors of disinfection by-products (DBPs) during drinking water treatments [6,7,8,9]. Although various methods have been proposed for controlling M. aeruginosa blooms, they continue to occur in many countries such as Brazil, China, and the United States, causing huge economic loss [10,11,12]. Therefore, there is a significant need for approaches to develop efficient and low-cost techniques, which can inhibit cyanobacterial blooms and simultaneously alleviate the risk of secondary metabolites.

Reducing external nutrient loading is considered as a fundamental solution for suppressing cyanobacterial blooms [13,14]. However, it seems impractical to restrict nutrient inputs (i.e., phosphorus) in many countries or areas. Phosphorus usually exists in the form of phosphate in water [15,16]. Hence, using adsorption to remove phosphate is regarded as one of the most effective methods to mitigate eutrophication [17,18]. Nevertheless, some traditional phosphate adsorbents (e.g., aluminum-based salts) may lead to pH reduction and release of aluminum, negatively impacting aquatic ecosystems [19,20,21,22]. Although biodegradable materials (e.g., chitosan) are generally environmentally friendly, they have shown poor adsorption capability and have been ineffective in controlling M. aeruginosa blooms [23,24]. Thus, it is important to seek alternative methods to effectively restrict phosphorus for M. aeruginosa control.

Kaolinite is regarded as an abundant, economical, and eco-friendly natural material [25,26,27]. Its plentiful surface hydroxyl groups and negative charge make it suitable for the preparation of advanced composites [28,29]. Because of an inherently high affinity for phosphate, lanthanum (La) is known as a promising candidate for phosphate removal [30,31]. As documented in a recent study, an in situ-synthesized heated kaolinite–lanthanum hydroxide complex (HKL-LH) could remove ~99% of phosphate in a pure phosphate solution with concentrations of up to 20 mg L⁻¹, showing its potential as an effective adsorbent [32]. However, the presence of cyanobacteria and their metabolites may affect the efficiency of adsorbents for phosphate removal [33,34]. Moreover, phosphorus availability has been considered the principal nutrient that limits cyanobacterial biomass [35,36]; even a low phosphate concentration (0.02 mg L⁻¹) is adequate for stimulating cyanobacterial growth [37]. Thus, it is currently unclear whether HKL-LH can effectively remove phosphate in a solution inoculated with the cells of M. aeruginosa and inhibit their growth. Additionally, the possible effects of HKL-LH on MCs and AOMs produced by M. aeruginosa remain unknown and require further research.

Consequently, the aims of this study were as follows: (1) to determine the phosphate removal capability of HKL-LH in M. aeruginosa samples, (2) to access the effects of HKL-LH on M. aeruginosa growth and viability (photosynthetic activity and cellular integrity), and (3) evaluate the production of MCs and AOMs in M. aeruginosa treated by HKL-LH.

2. Materials and Methods

2.1. Materials and Reagents

M. aeruginosa (strain FACHB-905) was purchased from the Institute of Hydrobiology, Chinese Academy of Science. The strain was observed to produce MCs, primarily MC-LR [38]. It was cultivated in the sterilized BG-11 media with a modified phosphate concentration (0.6 mg L⁻¹), according to a eutrophication evaluation standard in natural waters [39]. NaOH and HCl were used to adjust the pH of BG-11 media to 7.5 ± 0.1. The strain was cultured under a cool fluorescent light flux (25 μmol photos m⁻² s⁻¹) with a 12 h: 12 h light/dark cycle at a constant temperature of 25 ± 1 °C. All solutions were prepared with ultra-pure water purified by a Milli-Q water purification system (Synergy, Merck Millipore, Darmstadt, Germany). All the chemicals utilized in this study were analytical grade. The preparation of heated kaolinite (HKL) and HKL-LH was detailed in a previous study [32].

2.2. Treatment of M. aeruginosa by HKL-LH

To investigate the effects of HKL-LH on M. aeruginosa, 0.023, 0.1, and 0.2 g L⁻¹ of HKL-LH were applied in M. aeruginosa samples, and HKL (0.2 g L⁻¹) was selected for comparison. Different eutrophication conditions were simulated as background water in this study, using initial phosphate concentrations of 0.6, 1.2, and 3.0 mg L⁻¹, respectively. The samples of M. aeruginosa were taken at specific intervals for cell counting (0, 6, 9, 12, 15, 18, 21, 24, 30, 36, 48, and 60 days) and determination of phosphate concentrations (0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, and 30 days). Fv/Fm and cell integrity of cyanobacterial samples were measured every 6 days. Samples were also collected to determine the amounts of MCs and dissolved organic carbon (DOC) on days 0, 6, 12, 24, and 36. In addition, the M. aeruginosa cells were obtained for real-time quantitative PCR (qPCR) testing after treatment for 12 days.

2.3. Analytical Methods

2.3.1. Kinetic Model

To describe the phosphate removal by HKL-LH in M. aeruginosa samples quantitatively, the second-order kinetic (Equation (1)) was applied for fitting the data [40], as follows:

$$C_t=C_c/\left(1+C_{c}kt\right)$$

(1)

where C_c (mg L⁻¹) means the phosphate concentration in the control at a time t; C_t (mg L⁻¹) means the phosphate concentration in M. aeruginosa treated by HKL-LH at a time t; and k (L mg⁻¹ d⁻¹) means the second-order kinetic constant of phosphate removal. The reduced phosphate value during the first three days in the control was taken into account in order to obtain a better fit.

2.3.2. Growth Assessment of M. aeruginosa

To determine the cell density of M. aeruginosa, 2 mL of each sample was treated with Lugol’s iodine and examined under a microscope (ECLIPSE E100, Nikon, Tokyo, Japan) with a total 100× magnification [41]. The growth inhibition rate of M. aeruginosa samples was calculated by Equation (2), as follows:

$$\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)=\left(N_0-N_t\right)/N_0\times 100$$

(2)

where N₀ presents the M. aeruginosa cell density in the control at a time t and N_t presents the M. aeruginosa cell density by HKL-LH treatment at a time t.

2.3.3. Fv/Fm and Cell Integrity Analysis

The chlorophyll fluorescence parameter Fv/Fm is a measure of the maximum quantum efficiency of photosystem II, which is used as a typical indicator of the photosynthetic activity in cyanobacteria [42,43]. The Fv/Fm values were quantified by a Phyto-PAM chlorophyll fluorometer (Walz, Effeltrich, Germany), after a modified dark incubation period of 15 min [44].

The cell integrity of M. aeruginosa cells was measured by a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA) equipped with a laser emitting at 488 nm wavelength. SYTOX Green nucleic acid stain (Invitrogen, Waltham, MA, USA) was used to identify the intact and damaged M. aeruginosa cells [41]. The detailed operation procedures were referred to a previous study [45].

2.3.4. Determination of Cyanobacterial Cell Morphology

M. aeruginosa suspensions were centrifuged at 5000 rpm for 10 min and the supernatant was subsequently removed. The cells were immobilized with 2.5% glutaraldehyde for 8 h and then washed with phosphate buffer solution (PBS, 0.1 M, pH = 7.0) for three times. The cells were dehydrated in a graded ethanol series (30% (v/v), 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min at each step. The processed samples were finally sprayed with gold and analyzed by a scanning electron microscope (SEM, Sigma 500, Zeiss, Oberkochen, Germany).

2.3.5. MC-LR Analysis

For the determination of MC-LR production, the sampled M. aeruginosa underwent three freeze–thaw cycles and then filtered through 0.45 μm and 0.22 μm glass fiber filters (Xingya, Shanghai, China) [45]. High-performance liquid chromatography (Symmetry C18, Waters, Milford, MA, USA) equipped with a C18 reverse phase column (4.6 × 150 mm) was used for the analysis of MC-LR concentrations [46]. The mobile phases were methanol and PBS (pH = 3.0) (56%/44%, v/v) at a flow rate of 1.0 mL min⁻¹.

2.3.6. Measurement of Phosphate and AOMs

The concentrations of phosphate were measured by the molybdenum blue method using a UV–Visible spectrophotometer (Evolution 300, Thermo Scientific, Waltham, MA, USA) [47]. AOMs (characterized by DOC content) include extracellular organic matter (EOM) and intracellular organic matter (IOM) [48]. M. aeruginosa samples were filtered with 0.45 μm glass fiber filter. The supernatant was used for EOM determination, and the cells on the membrane were suspended in ultrapure water and underwent three freeze-thaw cycles and was then filtered for IOM analysis. The concentrations of DOC in EOM and IOM were analyzed by a total organic carbon (TOC) analyzer (TOC–V CPH, Shimadzu, Kyoto, Japan).

2.3.7. Gene Expression Test

The M. aeruginosa cells were collected by centrifugation (5000 rpm, 5 min) at 4 °C, then washed with PBS (pH = 7.2) three times. After immediately being frozen in liquid nitrogen, the cells were stored at −80 °C until sequencing. To explore the effects of phosphate restriction on the physiological and biochemical functions of M. aeruginosa, mcyA, mcyD, ftsH, sphX, pstS, ntcA, atpB, psaB, and rbcL were chosen as the target genes, and quantified by qPCR. Primer sequences and functions for the housekeeping gene (16S rRNA) and target genes are listed in Table S1 (Supplementary Materials). The relative expression was calculated by the 2^−ΔΔCt method [49].

2.4. Statistical Analysis

A one-way ANOVA test was performed on Prism 8.0 (GraphPad Software, San Diego, CA, USA) to evaluate statistically significant differences between treatment and control groups. The figures and heatmap presented were created by Prism 8.0. All the experiments were conducted in triplicates, and the error bar represents mean ± standard deviation.

3. Results and Discussion

3.1. Phosphate Removal by HKL-LH in M. aeruginosa Samples

The changes in phosphate concentration in M. aeruginosa samples were investigated during HKL-LH treatment (Figure 1a,c,e). There were no significant differences found in the phosphate concentrations between the HKL-treated and control samples (p > 0.05). However, HKL-LH efficiently removed phosphate; i.e., reductions of 84–94% in phosphate concentration were achieved with 0.1 g L⁻¹ HKL-LH treatment for 3 days. The results were comparable to those in a study by Zheng et al. [32], documenting 91% of phosphate was removed by 1.0 g L⁻¹ HKL-LH in pure phosphate solution after 3 days. Previous studies have suggested that anions (e.g., CO₃²⁻, SO₄²⁻, Cl⁻, and NO³⁻), cations (e.g., Ca²⁺ and Mg²⁺), and humic substances in the background water may influence the performance of La-based adsorbents [50,51,52]. However, in this study, it was shown that these factors scarcely abated the effectiveness of HKL-LH in phosphate removal, indicating HKL-LH is suitable for application in much more complicated water environments.

The capacity of phosphate removal by HKL-LH in M. aeruginosa samples was promoted with its increasing dosage (

Table 1. Kinetic parameters of phosphate removal by HKL-LH with different initial phosphate concentrations in M. aeruginosa samples.

[HKL-LH] (g L−1)	Initial Phosphate Concentrations (mg L−1)
	0.6		1.2		3.0
	k (L mg−1 d−1)	R2	k (L mg−1 d−1)	R2	k (L mg−1 d−1)	R2
0.023	0.86	0.9908	0.21	0.9853	0.22	0.9721
0.1	2.68	0.9904	2.38	0.9704	1.67	0.9823
0.2	2.91	0.9943	6.93	0.9670	2.00	0.9943

). For instance, the constant values (k) for phosphate removal were 0.86, 2.68, and 2.91 for HKL-LH dosages of 0.023, 0.1, and 0.2 g L⁻¹, respectively, where an initial phosphate concentration of 0.6 mg⁻¹ was applied. Generally, the k value in HKL-LH treatment was increased with reduced initial phosphate concentration. For instance, the k values were 2.68, 2.38, and 1.67, respectively, in the samples treated by 0.1 g L⁻¹ HKL-LH, with 0.6, 1.2, and 3.0 mg L⁻¹ phosphate initially. It indicates that the phosphate removal capacity is positively correlated with HKL-LH dosages while it is negatively associated with initial phosphate concentrations. Similar findings were reported on other La-containing adsorbents, such as hydrated lanthanum oxide-modified diatomite composites [53].

3.2. The Impact of HKL-LH on the Growth of M. aeruginosa

The M. aeruginosa cell densities of the control samples increased to peak values of 1.5 × 10⁷ and 1.6 × 10⁷ cells mL⁻¹ on days 24 and 21, with initial phosphate concentrations of 0.6 and 1.2 mg L⁻¹, respectively (Figure 1b,d). For the control samples added with 3.0 mg L⁻¹ phosphate initially, higher cell density (3.0 × 10⁷ cells mL⁻¹) was achieved and maintained after 36 days (Figure 1f). The growth dynamic of M. aeruginosa by HKL treatment was similar to that of the control, with equivalent initial phosphate concentration. However, HKL-LH effectively inhibited the growth of M. aeruginosa (Figure 1b,d,f). The inhibition degree of cell growth increased as HKL-LH dosages increasing from 0.023 g L⁻¹ to 0.1 g L⁻¹, while no significant differences (p > 0.05) were observed at HKL-LH dosages > 0.1 g L⁻¹. For instance, with the initial phosphate concentration of 0.6 mg L⁻¹, the inhibition rates of M. aeruginosa were 48%, 98%, and 97% after 0.023, 0.1, and 0.2 g L⁻¹ HKL-LH treatments for 24 days, respectively (Figure 1b and Figure S1, Supplementary Materials). As a limiting factor, phosphorus plays a key role in the growth of cyanobacteria [54]. Thus, the growth of M. aeruginosa was inhibited with phosphate deficiency caused by HKL-LH (Figure 1a,c,e). It agrees with previous studies showing that phosphate adsorbents (e.g., lanthanum peroxide-loaded sepiolite nanocomposite and zero-valent iron-modified biochar) inhibited the metabolism of M. aeruginosa by preventing nutrient intake [55,56]. Notably, commercial phosphate fixatives such as Phoslock^® (a lanthanum-embedded bentonite) only showed less than 15% decrease in the growth rate of M. aeruginosa at the concentration of 0.1 g L⁻¹, while the cell growth was effectively inhibited by HKL-LH at the same concentration [57].

The effects of HKL-LH on the growth of M. aeruginosa with different initial cell densities were also investigated, initially with 0.6 mg L⁻¹ phosphate (Figure 2). Generally, the inhibition rate of M. aeruginosa decreased with increasing initial cell densities. For instance, the inhibition rate of M. aeruginosa (initial cell density = 2.8 × 10⁴ cells mL⁻¹) was 97% by 0.1 g L⁻¹ HKL-LH treatment (Figure 2a), while it was 75% at a higher initial cell density of 6.3 × 10⁵ cells mL⁻¹ (Figure 2c). As reported, cyanobacterial blooms with cell densities of 10⁴ cells mL⁻¹ are already visible at the water surface [58]. Therefore, HKL-LH is generally efficient to treat cyanobacterial blooms (with initial cell densities of 10⁴−10⁵ cells mL⁻¹). However, to achieve optimization, it would be recommended to apply HKL-LH at their early bloom stage with a relatively lower biomass (≤10⁴ cells mL⁻¹).

3.3. Impacts of HKL-LH on the Viability of M. aeruginosa

The Fv/Fm value of M. aeruginosa in the control gradually increased to 0.46 during the first 12 days and then began to decline (Figure 3a). The photosynthetic activity in the HKL-treated samples showed similar trends to those observed in control. The Fv/Fm value of samples treated by 0.023 g L⁻¹ HKL-LH was similar to that of control during the initial 18 days, but it became lower than the value in the control thereafter. The Fv/Fm value of M. aeruginosa by 0.1, and 0.2 g L⁻¹ HKL-LH treatment was 74.4% and 78.9% lower than the control samples on day 36, respectively, indicating the photosynthetic system was seriously damaged [59]. It may be attributed to the efficient removal of phosphate by HKL-LH (Figure 1a), which led to the reduction in electron transport rate and thus inhibition of photosynthetic performance in M. aeruginosa cells [60,61]. In addition, according to the SEM images, HKL-LH particles may decrease cell integrity, enhance the cell aggregation, and then block the light that can illuminate M. aeruginosa cells, thereby suppressing the photosynthetic activity (Figure S2, Supplementary Materials).

More than 98% of the M. aeruginosa cells maintained membrane integrity in the control, 0.2 g L⁻¹ HKL, and 0.023 g L⁻¹ HKL-LH treatments over the course of 36 days (Figure 3b). A slight decrease in the proportion of intact cells could be observed after 0.1 and 0.2 g L⁻¹ HKL-LH treatments for 36 days. Phosphorus is one of the important elements in the synthesis of phospholipids in cell membranes [62]. The membranes of M. aeruginosa cells are more likely to rupture when they are unable to take up sufficient phosphate over a long period [63]. SEM images also showed that the membranes of some cells in the groups treated by HKL-LH were wrinkled and weakened (Figure S2, Supplementary Materials). Similar results were reported that magnetite/lanthanum hydroxide composite caused the membrane rupture and morphological deformation of M. aeruginosa cells [64]. Therefore, HKL-LH can not only reduce the phosphate concentration and inhibit the growth of M. aeruginosa (Figure 1) but may also inactivate the cells through phosphate deficiency.

3.4. Changes in MCs and AOMs by HKL-LH Treatment

Accompanied by the increasing cyanobacterial biomass, the amount of MCs in the control gradually increased from 2.8 μg L⁻¹ (day 0) to 366.0 μg L⁻¹ (day 36) (Figure 4). The amount of MCs in the HKL-treated samples were similar to that of control, while they were significantly decreased after exposure to HKL-LH (p < 0.01). The concentrations of MCs were 186.1, 56.1, and 55.2 μg L⁻¹, with 0.023, 0.1, and 0.2 g L⁻¹ HKL-LH treatments for 36 days, respectively. Although the MC production was reduced by HKL-LH, the expression of mcyA (MC biosynthesis gene) was significantly upregulated (Figure 5). This may be attributed to a self-protective mechanism of M. aeruginosa cells in response to phosphate stress [65,66]. Thus, the reduced MCs may be owing to the decrease in M. aeruginosa biomass (Figure 1b). Previous studies have also shown that the production of MCs is positively correlated with the amounts of biomass [67,68]. Additionally, the amounts of MCs in cyanobacteria can be affected by the inhibition of photosynthetic activity, which decreases the availability of synthetic substrates and the energy required for MC synthesis (Figure 3a) [69,70].

The concentration of total DOC in the control reached 49.8 mg L⁻¹ on day 24 and then decreased to 35.5 mg L⁻¹ on day 36, which was consistent with the tendency in cell density of M. aeruginosa (Figure 6). The amounts of total DOC in the M. aeruginosa samples treated with HKL were similar to the control (p > 0.05). Conversely, HKL-LH significantly decreased the total DOC production in M. aeruginosa cells (p < 0.01), with decreases of 54.0%, 70.0%, and 74.2% by 0.023, 0.1, and 0.2 g L⁻¹ dosages on day 36, respectively, compared to the control. The extracellular DOC concentration in the control increased from 4.3 mg L⁻¹ (day 0) to 10.7 mg L⁻¹ (day 36), probably due to release of intracellular substances from decaying cells. The extracellular DOC of M. aeruginosa were only 3.9 and 4.2 mg L⁻¹ after 0.1 and 0.2 g L⁻¹ HKL-LH treatments for 36 days, respectively. These lower concentrations of extracellular DOC may be attributed to the significant reduction in total DOC in M. aeruginosa with decreased biomass (Figure 1b). The accumulation of extracellular DOC is considered a precursor of DBPs, which can pose a significant challenge to water treatment plants due to their risks to human health and negative impacts on treatment processes [71,72]. Therefore, a reduction in extracellular DOC by HKL-LH treatment can effectively mitigate the associated risks.

4. Conclusions

In this study, the effects of HKL-LH on M. aeruginosa treatment were investigated. HKL-LH showed an outstanding phosphate removal performance in M. aeruginosa samples, thereby significantly inhibiting the growth of M. aeruginosa cells with various initial cell densities. Although the related genes were upregulated in response to the stress caused by HKL-LH, the photosynthetic activity and vitality of M. aeruginosa was significantly suppressed. In addition, HKL-LH efficiently reduced the production of MCs and AOMs in M. aeruginosa samples, which can lighten the burden on subsequent water treatment processes. Therefore, the implementation of HKL-LH may be practically applicable for water authorities to control cyanobacterial blooms in various water bodies, including recreational waters, aquaculture ponds, and urban lakes. In this study, it was shown that HKL-LH could effectively control cyanobacteria in media with different phosphate concentrations, while its application in other environments with more complex water matrices remains to be further explored.