Egeria densa organic extracts: an eco-friendly approach to suppress Microcystis aeruginosa growth through allelopathy

The growth of harmful cyanobacteria in freshwater poses numerous problems to the ecosystem, such as hypoxia, aquatic fauna mortality, and the release of toxins into the water, including microcystins, cylindrospermopsin, anatoxins, and saxitoxins (Dolman et al. 2012; Paerl & Otten 2013). There are three potential bloom-forming toxin-releasing cyanobacteria genera: Microcystis, Oscillatoria, and Anabaena. These species are more hazardous due to their ability to produce secondary metabolites and endotoxins. Microcystis spp., the most common species, known for its ability to form harmful blooms in freshwater bodies worldwide, has been extensively studied by researchers more than other cyanobacteria species (Dolman et al. 2012; Preece et al. 2017). The hepatotoxic microcystins produced by Microcystis spp. are the most common cyanotoxin and are known to cause serious health and environmental problems not only in aquatic organisms but also in human health as well (Preece et al. 2017).

Over the years, studies extensively aimed at mitigating cyanobacterial growth and harmful cyanobacteria blooms (HABs); among the various in situ control methods, leveraging the natural allelopathic abilities of plants, including macrophytes, to inhibit cyanobacterial growth has proven to be an effective method compared to many other conventional in situ control approaches. Conventional methods such as algaecide application, coagulant use, ultrasonic technology, aeration towers, and sediment dredging are practiced as cyanobacteria suppression methods (Paerl & Otten 2013; Sample & Fox 2013). However, all have costly initial application or maintenance and cause negative effects on ecosystem members (Paerl & Otten 2013; Chorus & Welker 2021). Particularly, the use of algaecides, such as copper sulfate, was prohibited due to the potential for these chemicals to accumulate in lake sediments and subsequently be released into the water, potentially harming aquatic organisms (Sukenik & Kaplan 2021). Moreover, ultrasound technology has the potential to affect fish skin due to ultrasound-induced cavitation (Renkel et al. 1999). Additionally, ultrasonic waves can lead to cell lysis in cyanobacteria, resulting in the release of intracellular toxins or an increase in toxin production as a defense mechanism against sonication (Zhang et al. 2006; Dehghani 2016). Furthermore, dredging activities typically involve significant expenses related to logistics, such as pumping, water drainage, water treatment, sediment transportation, and disposal (Oldenborg & Steinman 2019; Kibuye et al. 2021).

The presence of macrophyte beds has a significant impact on the abundance and composition of plankton communities in aquatic environments (Jasser 1995). Due to coexistence within the same environment, these communities are interdependent on shared resources. As a result, the presence of macrophyte vegetation leads to reduced light availability, decreased water turbulence, lower nutrient levels, and allelopathic interactions which suppresses the growth of plankton, including cyanobacteria (Vanderstukken et al. 2011; Senavirathna et al. 2021). An important factor to consider is the inhibitory effect exerted by allelochemicals produced by macrophytes, which additionally suppress the growth of cyanobacteria (Hu & Hong 2008; Amorim et al. 2019). Macrophytes possessing allelopathic potential play a crucial role in the remediation of water bodies affected by HABs, which is a significant environmental concern in both natural and artificial shallow water systems globally.

Allelopathy is a biological phenomenon characterized by the capability of plants to influence neighboring plants or other organisms through the release of chemical substances into their immediate surroundings, resulting in repressive or stimulative effects (Hu & Hong 2008). The production of allelochemicals represents a natural strategy employed by macrophytes to reduce competition for primary resources and ensure their well-being (Mulderij et al. 2007). Several macrophyte species, including Myriophyllum spp., Ceratophyllum demersum, Egeria densa, and Hydrilla verticillata, have been identified as strong producers of allelochemicals (Hu & Hong 2008; Vanderstukken et al. 2011). Studies by Amorim et al. (2019) have shown a significant reduction in Microcystis aeruginosa biomass in the presence of C. demersum. Similarly, Wang et al. (2017) and Nakai et al. (2000) have reported the successful inhibition of M. aeruginosa growth by the macrophyte genus Myriophyllum spp. Although many macrophytes inhibit cyanobacteria growth when present as live plants, the feasibility of utilizing such macrophytes is often limited owing to their invasive nature. Therefore, the utilization of biologically derived substances (BDS) extracted from cyanobacteria-inhibiting macrophytes represents a promising alternative for harnessing the potential of these plants.

Previous studies have shown that the extracts derived from macrophyte species have been found to suppress cyanobacteria growth (Wang et al. 2017). The inhibition ability of those allelochemicals present in macrophyte extracts on cyanobacteria is not only species-specific but depends on various other factors such as pretreatment method, utilized solvent, and extraction method on macrophytes. Pretreatments such as mechanical grinding and prolonged stirring are important to induce the release of allelochemicals in the plants (Chen et al. 2012). Various aqueous and organic solvents were employed to extract allelochemicals from different parts of macrophytes to inhibit harmful cyanobacteria growth. Distilled or pure water was utilized as a successful aqueous solvent by many researchers (Li et al. 2016; Hua et al. 2018; Wang et al. 2022). Some researchers used a combination of aqueous and organic solvents such as methanol (Gil et al. 2021), ethanol (Maredová et al. 2021), or dimethyl sulfoxide (DMSO) (Yi et al. 2012) to increase the solubility of the allelochemical compounds. On the other hand, some researchers exclusively employed organic solvents, such as hexane, ethyl acetate, and ethanol solutions (Zhang et al. 2015), acetone, and dichloromethane (Gao et al. 2020) to extract allelochemicals.

Studies investigated the allelopathic inhibitory activities of various macrophyte extracts against M. aeruginosa. Research conducted by Zhang et al. (2016) observed a sharp decrease in total algal biomass including the dominant M. aeruginosa by using the organic extracts derived from macrophyte Acorus calamus roots using 95% ethanol as the solvent. According to a study conducted by Li et al. (2016), an aqueous extract derived from Sagittaria trifolia tubers was able to inhibit M. aeruginosa growth by 90% after 6 days. In another study, M. aeruginosa density was reduced by 82%, and a significant increase in oxidative stress markers was evidenced in cyanobacterial cells by Ranunculus aquatilis-dried tissue extracts (Tazart et al. 2021). Submerged macrophytes Potamogeton pectinatus and C. demersum aqueous extracts with 50 and 100% acetone and ethanol solvents also inhibited M. aeruginosa (Ghobrial et al. 2015). Furthermore, the extracts from Chara globularis, Ceratophyllum submersum, Elodea nuttallii, H. verticillata, Myriophyllum heterophyllum, M. spicatum, and Vallisneria americana demonstrated the ability to inhibit the growth of cyanobacteria. Most importantly, all these species are submerged macrophytes (Maredová et al. 2021).

E. densa is another submerged macrophyte, widely recognized for its ability to produce allelochemicals with anti-cyanobacterial properties as a live plant when they coexist in the same environment (Hu & Hong 2008; Vanderstukken et al. 2011; Senavirathna et al. 2021). In a mesocosm study conducted by Vanderstukken et al. (2011), it was demonstrated that E. densa is recognized as an effective inhibitor of cyanobacteria. The results of our previous experiment (Wijesinghe et al. 2023) also exhibited that the fresh tissue aqueous extracts obtained from E. densa co-existed with M. aeruginosa performed a considerable suppression than the extracts obtained from E. densa monoculture. However, there was limited research on the application of E. densa extracts on cyanobacteria control. E. densa is an invasive, perennial submerged macrophyte species found in water bodies around the world (Zehnsdorf et al. 2015). This submerged macrophyte has become a threat to the natural vegetation in temperate, subtropical and tropical ecosystems because of its invasive nature, the rapid growth which eventually crowds out other natural flora (Dutartre et al. 1999). The extraction of natural allelochemicals from invasive macrophyte species is a sustainable method, given their abundance in water bodies. Thus, in this study, we selected E. densa as the subject to investigate a potential approach for utilizing extracts from dried plants to suppress M. aeruginosa. The cyanobacterium M. aeruginosa was selected for this study due to its ubiquitous and its tendency to form toxic blooms nature in water bodies worldwide (Chen et al. 2012). The selection of aqueous or organic solvents to obtain the allelopathic compounds from the plant materials is crucial, as some compounds are readily dissolved in water, while others need organic solvents to increase their water solubility. Therefore, the effects of E. densa extracts derived from both aqueous (pure water) or organic (DMSO) solvents on M. aeruginosa growth were assessed. Further, the study examined the effect of E. densa organic extracts on oxidative damage and antioxidant responses in M. aeruginosa.

M. aeruginosa strains (NIES-111) were obtained from the National Institute of Environmental Studies (NIES), Japan. The cultures were pre-cultured in a sterile BG-11 medium (Rippka et al. 1979) using autoclaved 300-mL Erlenmeyer flasks. The cultures were maintained in an incubator (LH-55RDS, NK Systems Limited, Tokyo, Japan) at 20 °C under a light intensity of 25–30 μmol m⁻² s⁻¹ photosynthetically active radiation (PAR) on a photoperiod of 12:12 h light: dark cycle which was maintained using an automatic setup device (REVEX PT7, Saitama, Japan). The cultures were covered with porous stoppers (SILICOSEN, Shin-Etsu Polymer Co., Ltd, Japan) for better aeration and gently shaken every day. The stock cultures in the exponential growth stage were used for the experiments.

E. densa certified for agrochemicals and chemicals-free were commercially purchased and cultured in 20-L (25 cm × 24 cm × 40 cm, height × width × length) glass tanks in a temperature-controlled room at 25 ± 2 °C. Macrophytes were cultured in nutrient-removed river sand as the substrate. A commercial aquaponic solution (Hyponex concentrated nutrient solution, Hyponex, Osaka, Japan) was provided as the growth medium. The cultures were exposed to a 12:12 h light: dark cycle with a light intensity of 80–85 μmol m⁻² s⁻¹ PAR. The water levels of the culture tanks were kept constant by replenishing them with deionized water.

The aqueous and organic extracts of E. densa were prepared according to Shi et al. (2020) and (Yi et al. 2012), respectively with modifications. Healthy E. densa plants were selected and washed gently once with tap water and three times with distilled water to remove any kind of detritus and oven dried at 60 °C for 48 h. The dried plant materials were cut into 2 cm small pieces and homogenized in a blender (IFM-C20G, Iwatani Cooperation, Osaka, Japan) and the obtained powder was utilized to prepare the extracts.

For the aqueous solvent, 10 g of dry E. densa powder was mixed with 400 mL of ultra-pure water (1:40 w/v) to get 25 g/L stock and stirred at 150 rpm for 24 h in dark conditions. The solution was filtered through 0.45 μm and the obtained filtrate was utilized as the E. densa aqueous extract and stored at 4 °C. The dry plant powder (10 g) was mixed with 99% DMSO maintaining a 1:2 ratio (sample/solvent) and then topped up with ultra-pure water to obtain 25 g/L stock solution with 5% (v/v) final DMSO. The mixture was stirred at 150 rpm continuously for 24 h in dark conditions before passing through 0.45 μm filter units. The obtained filtrates were utilized as the E. densa organic extract and stored at 4 °C.

A preliminary experiment was conducted to assess the effect of DMSO on M. aeruginosa before commencing the main experiments. The concentration gradient of prepared E. densa extracts ranged from 0.5 to 6 g/L, with DMSO concentration ranging from 0.1 to 2%. Consequently, we exposed M. aeruginosa cultures at a cell density of 1 × 10⁵ cells/mL to corresponding DMSO percentages without E. densa extracts under similar experimental conditions as described in 2.3.1. This experiment was conducted for a duration of 12 days, during which we monitored cell densities at 72-h intervals.

To investigate the impact of low concentrations of E. densa organic extracts on the growth of M. aeruginosa, an experiment was conducted using a gradient of extract concentrations at 0, 0.1, 0.2, 0.3, and 0.4 g/L. We exposed M. aeruginosa cultures, initially at a cell density of 1 × 10⁵ cells/mL, to the varying concentrations of E. densa organic extract under identical experimental conditions as described in 2.3.1. This experiment was conducted for a duration of 10 days, during which we monitored cell densities at 48-h intervals.

To assess the sustained inhibitory effect of E. densa organic extracts, the most M. aeruginosa inhibitive concentrations were chosen. The M. aeruginosa cultures were subjected to continuous exposure to selected extract concentrations at regular intervals of 24 h for 4 days. The experimental setups were carried out under similar conditions as described in Section 2.3.1.

To assess the impact of the available phosphate (⁠⁠) in the organic extract on the growth of M. aeruginosa, we exposed M. aeruginosa cultures to concentrations equivalent to the available levels in E. densa extracts at 0.5, 3, and 6 g/L (Figure 6(b)), without adding E. densa extracts. Additionally, another group of M. aeruginosa cultures was exposed to equivalent concentrations in groups of 0.5, 3, and 6 g/L, along with 0.5 g/L of E. densa extract. A control group of cells was maintained without either or E. densa extracts. We monitored cell density every 48 h, and the experiment was conducted for 10 days. The experimental setups were carried out under similar conditions as described in Section 2.3.1. This experiment was conducted for a duration of 10 days, during which we monitored cell densities at 48-h intervals.

Before starting each set of experiments M. aeruginosa cultures were acclimatized for 3 days. The experiments were conducted under consistent conditions, with triplicates maintained to ensure reliable and consistent results.

Cell counts were determined by microscopically (BZ-X810, KEYENCE Corporation, Osaka, Japan) counting with a hemocytometer (DHC-N01, C-Chip, NanoEnTec Inc, Guro-gu, Seoul, Korea). The cell count was expressed as cells/mL.

and stands for the cell density of the treatment sample and cell density of the control sample, respectively.

To determine the total protein content of M. aeruginosa, the Bradford method (Bradford 1976) was employed. The M. aeruginosa cell pellets were mixed with 0.5 M NaOH and heated for 10 min at 70 °C in a water bath. The cooled mixture was then centrifuged at 10,000× g for 10 min using a centrifuge (TOMY MX-105), and the resulting supernatant was mixed with Bradford reagent (Wako Chemical, Tokyo, Japan). The mixture was incubated in the dark for 10 min at 25 ± 2 °C, and the absorbance was measured at 595 nm using a spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan). The protein content was estimated using a standard curve and expressed as μg/mL.

and stands as the absorbance values at 665 and 649 nm, respectively, and the Chl-a concentration was expressed as μg/μg of protein.

To quantify the cellular H₂O₂ content, a method proposed by (Jana & Choudhuri 1984) was followed with modifications. Specifically, 1 mL of 0.1 M phosphate buffer (pH 6.5) was added to the M. aeruginosa cell pellets, which were then vortexed (Vortex-Genie 2, Scientific Industries, Inc., Bohemia, NY, USA) and centrifuged at 10,000× g for 10 min at 4 °C to extract the cellular content of H₂O₂. The supernatants were collected and mixed with a reaction mixture containing 0.1% titanium chloride in 20% H₂SO₄ (v/v), and the mixture was incubated for 1 min before centrifugation at 25 ± 2 °C. The supernatant's absorbance was measured at 410 nm using a UV-1280 spectrophotometer. The H₂O₂ concentration per protein was expressed as μmol/μg.

The rate constant (⁠⁠) is considered a direct measure of the catalase concentration, and is the higher first value of the linear extinction, and is the lower final value of absorption. Also, is the time interval.

The total organic carbon analyzer (TOC-2300, Hiranuma Sangyo Co., Ltd, Ibaraki, Japan) was utilized to measure the concentration of DOC of the aqueous and organic E. densa extracts. The catalytic oxidation method was used: TOC = TC (total carbon) – IC (inorganic carbon) to measure the DOC concentrations in the samples filtered through 0.45 μm filters.

The UV₂₅₄ absorbance was measured using a UV-1280 spectrophotometer. Extracts were filtered through 0.45 μm filters and distilled water was utilized as the control before taking the absorbance values.

Three-dimensional excitation-emission matrix (3D EEM) is another method that can be used to identify the compounds present in the organic extract. The mechanism of fluorescence spectrometry is that organic molecules in the sample instantly (∼ 10⁻⁸ s) absorb light energy at certain wavelengths and produce emission light with greater wavelengths than the excitation wavelengths (Zhang et al. 2019). The excitation and emission wavelengths of each organic compound are unique and fixed (Hu & Yin 2017). Therefore, this method is widely used to identify the groups of various organic compounds in a sample (Hu & Yin 2017; Zhang et al. 2019; Yin et al. 2022). A 3D EEM of the E. densa organic extract was obtained using a fluorescence spectrophotometer (LS 45, Perkin Elmer, Waltham, MA, USA) to identify the presence of organic compounds in E. densa extracts. The excitation wavelengths were changed from 200 to 600 nm and the emission wavelength was from 300 to 625 nm at a scanning speed of 1,500 nm/min. Their possible functional classes were determined by comparing the 3D EMMs with those published in the literature (Hu & Yin 2017; Zhang et al. 2019). The humification index was calculated according to the regions where the peaks were produced to understand the characteristics of compounds available in the extract.

The concentrations of E. densa aqueous and organic extracts filtered through 0.45 μm filters were evaluated using PAKTEST Phosphate (Range 2–100 mg/L) and Digital pack test meter (DPM-MTSP, KYORITSU CHEMICAL Corp, Japan).

The concentrations of E. densa aqueous and organic extracts filtered through 0.45 μm filters were evaluated using PACKTEST Nitrate (Range 1–45 mg/L) and Digital pack test meter (DPM-MTSP, KYORITSU CHEMICAL Corp, Japan).

Data were analyzed using SPSS statistical software (Version 20.0, IBM SPSS) with a significance level of 5% (p ≤ 0.05). Growth and stress parameters under different extract conditions were compared using one-way analysis of variance (ANOVA) with the post-hoc Tukey HSD (honestly significant difference) test. Each experiment was performed in triplicates. All of the graphs were created using OriginLab software 2022 (OriginLab Corporation, Northampton, MA, USA). All graphical images were created with BioRender.com.

After a 48-h exposure period, it was observed that the cultures treated with high concentrations of aqueous extracts (3 and 6 g/L) exhibited an approximately similar cell count compared to the control culture (Figure 3(a)). However, as the treatment progressed, the cell count in all cultures treated with aqueous extracts significantly increased compared to both the initial cell density (Day 0) and the cell density of the control culture (p < 0.05). In other words, M. aeruginosa cultures treated with aqueous extracts did not show any inhibition throughout the entire exposure period.

Table 1 compares the organic carbon, ⁠, and contents of the stocks of both aqueous and organic extracts of E. densa.

Table 1

Organic carbon, ⁠, and contents of E. densa aqueous and organic stock extract solutions

Parameter .	Aqueous extract (mg/L) .	Organic extract .
Nitrate (⁠⁠)	9.6	4.6
Phosphate (⁠⁠)	315.5	420
Dissolved organic carbon (DOC)	2,360	4,780

Parameter .	Aqueous extract (mg/L) .	Organic extract .
Nitrate (⁠⁠)	9.6	4.6
Phosphate (⁠⁠)	315.5	420
Dissolved organic carbon (DOC)	2,360	4,780

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The aqueous extract exhibited higher concentrations than the organic extract, with the 6 g/L aqueous extract recording the highest value of 2.7 ± 0.2 mg/L (Figure 6(a)). Particularly, concentrations were below the detection limit of 1 mg/L in both aqueous and organic extracts for lower concentrations (0.5 and 1 g/L). In contrast, E. densa organic extracts showed higher concentrations compared to the aqueous extract. Both extracts exhibited a consistent increase in concentration as the extract dosage increased, with the highest concentration of 124 ± 1.2 mg/L observed in the 6 g/L group of organic extract (Figure 6(b)). The results indicated that the higher concentrations of extracts contained high amounts of favorable nutrients, especially and which may have suppressed the inhibitive effect of allelochemicals by assisting the fast growth of M. aeruginosa. Dissolved phosphorus is known to be the key nutrient for algal growth (House 2003). The initial suppression of growth in the cultures treated with high extract concentrations (3 and 6 g/L) can be attributed to the inhibitive effect of high concentrations of allelochemicals. Nevertheless, prolonged exposure to high concentrations of extracts may have triggered the survival of M. aeruginosa cells. This response could potentially result from the availability of abundant nutrients in the medium, leading to rapid growth after day 4 (Figure 3(b)).

Furthermore, DOC concentration in the extracts is an indicator of the availability of organic compounds within them. In the organic extract (excluding the DOC values of DMSO), the DOC concentration was approximately three times higher than that recorded in the corresponding aqueous extracts at the same extract concentration (Figure 6(c)). As a result, the aqueous extract did not suppress the growth of M. aeruginosa compared to the control culture at any extract concentration level during the exposure period (Figure 3(a)).

The inability of the aqueous extract to suppress the M. aeruginosa growth indicates a lower availability of anti-cyanobacterial organic chemicals dissolved in the solvent. Consequently, aqueous solvents may not be the most suitable option for extracting allelochemicals from the E. densa. On the other hand, following 48 h of exposure to organic extract, high concentrations (3 and 6 g/L) resulted in growth suppression of M. aeruginosa, with the 6 g/L concentration exhibiting the lowest cell density (3.48 ± 0.16 × 10⁵ cells/ mL). Furthermore, after 6 days, cultures treated with 0.5 and 1 g/L concentrations exhibit a sustained suppression of M. aeruginosa growth, indicating that extracts at low concentrations are more effective. Yi et al. (2012) stated that numerous compounds produced by plants have limited water solubility. The results obtained in this study proved that allelochemicals in E. densa exhibit better solubility in DMSO. Multiple previous studies have similarly reported successful inhibition of cyanobacteria growth by utilizing various organic plant extracts (Hu & Hong 2008; Yi et al. 2012; Zhang et al. 2015). The extracts of aquatic macrophytes consist of numerous compounds, including phenolics, tannin, lignin, and aromatic compounds. These substances collectively contribute to the overall composition of available humic substances (Kang et al. 2020; Yin et al. 2022). According to Zhou et al. (2019), phenolic compounds are the most common and vital plant allelochemicals. SUVA is a widely used method for evaluating the aromaticity and composition of DOC in a sample. The corresponding DOC values of both aqueous and organic stocks are presented in Table 1. The elevated SUVA values occur owing to an increase in the proportion of recalcitrant organic compounds such as phenolic compounds and humic substances (Kang & Mitchell 2013). However, SUVA values of aqueous and organic extract stocks observed in the study were 0.46 and 0.22, respectively. A lower SUVA value was reported for organic extract may be due to the high concentration of non-aromatic compounds present in the organic extracts.

The results of the 3D EEM analysis on the aqueous extract of E. densa produced four peaks, while the organic extract produced five peaks, as shown in Figure 6(a) and 6(b), respectively. These peaks elucidate the availability of different organic compounds present within extracts. The corresponding excitation and emission wavelengths of each peak are shown in Table 2.

The Chl-a concentration mirrored the growth variations showed by cell counts as a whole by exhibiting low Chl-a concentrations in cultures treated with low organic extract concentrations. The 0.5 and 1 g/L treatments showed significantly lower Chl-a concentrations than the control (p < 0.05) by the end of the exposure time (Figure 9). This indicates that the low concentrations of E. densa organic extracts affected the photosynthesis systems of M. aeruginosa cells disturbing the Chl-a production of the cells. However, the high organic extract concentrations (3 and 6 g/L) exhibited higher Chl-a concentrations than the control by the end of exposure time. This exhibits the opposite of what Wang et al. (2022) witnessed in the inhibitory effects of Vallisneria aqueous extract, where high extract concentrations inhibit M. aeruginosa growth while low concentrations stimulated it. The high nutrient-absorbing nature of E. densa (Vanderstukken et al. 2011) may have been attributed to higher nutrient availability in the extract with the extracted allelochemicals. Which created an additional nutrient effect, that surpassed the allelochemical effect of high concentrations on the M. aeruginosa cells.

The reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂) are naturally generated and continuously involved in many reactions in plant metabolism and plant growth as a normal phenomenon (Sharma & Dubey 2005). However, when cells are under various environmental stresses ROS production was stimulated resulting in the accumulation of H₂O₂ and other radicals (Sharma & Dubey 2005; Wang et al. 2022). Which leads to oxidative stress and when not regulated by antioxidative activities, eventual cell death (Sharma & Dubey 2005). Antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) act as defense mechanisms to protect the cells by eliminating excessive H₂O₂ (Hua et al. 2018; Wang et al. 2022). Numerous studies have investigated the oxidative damage and the antioxidant defense activities in cyanobacteria caused by various allelochemicals derived from plants (Li et al. 2016; Hua et al. 2018; Tazart et al. 2021; Wang et al. 2022). Polyphenols and alkaloids are commonly known compounds found in the various plant tissue extracts that have been studied for their potential inhibitory mechanisms on cyanobacteria. Previous research suggests that the suppression of alkaline phosphatase and disruption of the electron transfer chain within photosystem II (PS II) could potentially serve as inhibitory mechanisms of polyphenols against Microcystis species (Dziga et al. 2006; Shao et al. 2013). The deexcitation of excited energy through a non-photochemical pathway increases due to the interruption of the electron transfer via PSII, consequently leading to an elevation in ROS within the cells (Shao et al. 2013).

The H₂O₂ concentration was investigated as a measure of oxidative stress in M. aeruginosa cultures treated with E. densa organic extracts. On day 4, the cultures treated with a concentration of 0.5 g/L E. densa organic extract showed a significantly high H₂O₂ concentration of 1.11 ± 0.04 μmol/μg protein, compared to the control (F(4, 10) = 32.357, p < 0.001) (Supplementary material, Figure S1). Indicating lowest extract concentration caused the highest oxidative damage in the cultures. However, the cultures treated with both lower concentrations (0.5 and 1 g/L) exhibited a decrease in H₂O₂ concentration over time during the subsequent exposure period. The H₂O₂ concentration of the culture treated with 6 g/L concentration exhibited the highest value (0.77 ± 0.11 μmol/mg) on day 2 which tally with the lowest growth on the same day by 6 g/L culture. The high concentration of allelochemicals in the extract may have induced the H₂O₂ production in the cells, thus oxidative damage, and destroying cells. The high H₂O₂ concentration in cultures treated with low extract concentration could be an effect of allelochemicals catching up when the received low amount of nutrients were consumed by the M. aeruginosa. Therefore, it was clear that the exposure time gets longer the cultures treated with low concentrations exhibited a decreasing trend of H₂O₂ concentration while the high extract-treated cultures showed higher H₂O₂ concentrations. Even though the high extract concentration did not reflect an impact on growth in M. aeruginosa, the increased H₂O₂ concentration exhibited that M. aeruginosa cells were subjected to oxidative stress. This elucidates that the allelochemical effect provided by high extract concentrations has been overpowered by the nutrient effect of the extracts in terms of oxidative damage as well.

The production of antioxidant enzymes is reported to increase in response to the rapid accumulation of H₂O₂, targeting to reduce oxidative damage to cells by breaking down H₂O₂ into H₂O and O₂ (Sharma et al. 2012). In this study, we also observed that a significantly high (F(4, 10) = 55.5, p < 0.001) CAT activity rate of 8.7 ± 0.8 × 10⁻⁴ sec⁻¹ at day 4 in 0.5 g/L concentration corresponds to the high H₂O₂ concentration in the culture on the same exposure day. The CAT activity also mirrors the pattern of H₂O₂ concentration in cultures treated with high extract concentrations (3 and 6 g/L) by day 10 (Supplementary material, Figure S2), indicating the induction of antioxidant enzyme production to mitigate oxidative stress. However, the CAT activity rate decreased with the exposure time after reaching its peak on day 4. According to Wang et al. (2022), this reduction could have occurred as a result of the effect of allelochemicals, which may induce severe stress in the cells and lead to a rapid production of H₂O₂ that suppress the limits of cells' antioxidant defense mechanisms, resulting in a reduction of CAT activity.

During continuous exposure to both selected concentrations, the cultures of M. aeruginosa exhibited lower cell densities compared to the control group for the first 72 h (Figure 10(a)). The cultures exposed to the initial dose of 1 g/L showed the lowest cell density after 24 h, measuring 0.8 ± 0.02 × 10⁵ cells/ mL. The cultures treated with a concentration of 1 g/L displayed the highest IR of 29% after 24 h of continuous exposure, which subsequently decreased over time (Figure 10(b)). On the other hand, the cultures treated continuously with a concentration of 0.5 g/L exhibited an IR of 24% at 72 h. After 72 h, the cell count of both concentration groups started to exceed that of the control culture. A similar trend was observed in Chl-a concentration. The lowest Chl-a concentration was shown by the culture, continuously exposed to 0.5 g/L at 24 h as 5.37 ± 0.3 × 10⁻³ μg/μg protein (Figure 10(c)). This continuous exposure approach was conducted to understand the dominance of nutritional effects over allelochemical inhibitory effects. The results also confirmed that following three doses, M. aeruginosa showed a higher growth rate than control cell culture. This observation confirms that a high concentration of nutrients overpowers the influence of allelochemicals. Interestingly, H₂O₂ concentration (Figure 10(d)) and CAT activity rates (Figure 10(e)) remained consistently high throughout the 96 h of exposure to both concentrations (0.5 and 1 g/L). Particularly, the culture exposed to 1 g/L concentration showed the highest oxidative damage, accompanied by the greatest antioxidant concentration. The M. aeruginosa cells may have been stressed because of the continuous addition of extracts that consisted of both nutrients and allelochemicals. The elevated CAT activity rate indicates that the increased H₂O₂ concentration is effectively regulated by the antioxidative defense mechanisms of the cells. The increased CAT activity serves to neutralize H₂O₂, effectively scavenging free radicals and preventing further oxidative damage within the cells (Wang et al. 2022).

The results revealed that low concentrations (0.5 and 1 g/L) of the organic extracts showed inhibitory effects on M. aeruginosa, while aqueous extracts stimulate the growth of M. aeruginosa. This difference was further supported by the DOC concentrations and distinct fluorescence fingerprints of organic compounds observed in EEM. The coexistence of higher nutrient levels such as in both extracts contributes to enhancing the growth of M. aeruginosa. The absence of organic compounds that are responsible for anti-cyanobacterial activities in the aqueous extracts limited its ability to suppress M. aeruginosa growth. The DMSO organic extract of E. densa demonstrated the potential for bioremediation application. Specifically, the application of low concentrations (0.5 and 1 g/L) of organic extracts effectively suppressed the growth of M. aeruginosa and induced significant oxidative damage by the fourth day of exposure. However, higher concentrations (3 and 6 g/L) of organic extracts promoted the growth of M. aeruginosa due to high nutrient availability, while simultaneously inducing oxidative damage over time, which was continuously regulated by an antioxidant mechanism. The results confirm that the high availability of nutrients in the E. densa organic extracts at high concentrations disrupts the inhibitory effect of allelochemicals. Therefore, compared with an aqueous extract, the organic extract at low concentrations appears to be successful in suppressing M. aeruginosa growth. These results provide an approach to the sustainable use of strong anti-cyanobacterial properties in invasive macrophyte species that cannot be introduced as live plants for bioremediation in aquatic ecosystems.

This method is exceptionally well-suited for water bodies where the use of live macrophytes is impractical, such as boat yards, man-made shallow ponds in parks, or ornamental fountains, though not so for the application for large water bodies such as lakes and large reservoirs. However, the ecotoxicity of the extracts with strong inhibitory abilities needs to be investigated prior to the consideration of field applications as a crucial matter since there can be negative influence on non-target beneficial species in the ecosystem, whereas extracts obtained from Corallina pilulifera exhibited a nonselective toxicity of several microalgae species (Jeong et al. 2000). An extract obtained from eucalyptus leaves showed a toxicity on zebra fish at higher concentrations (Zhao et al. 2019).

A.W. was involved in conceptualization; methodology; investigation, writing – original draft, M.D.H.J.S. was involved in conceptualization; methodology; investigation; resources; writing – review and editing; supervision, T.F. was involved in resource collection and supervision.

This research was supported by the annual budget allocation of Saitama University.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.