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Article

Antialgal Effects of Nonanoic and Palmitic Acids on Microcystis aeruginosa and the Underlying Mechanisms

by
Ning Hu
1,†,
Yaowen Tan
1,†,
Xian Xiao
1,*,
Yuexiang Gao
2,*,
Kaikai Zheng
3,
Wenhan Qian
1,2,
Yimin Zhang
2 and
Yuan Zhao
1
1
School of Environmental Science and Engineering, Changzhou University, Changzhou 213164, China
2
Nanjing Institute of Environmental Sciences, Nanjing 210042, China
3
Three Gorges Hydrology and Water Resources Survey Bureau, Hydrology Bureau of Changjiang Water Resources Commission, Yichang 443000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sustainability 2025, 17(3), 1207; https://doi.org/10.3390/su17031207
Submission received: 4 January 2025 / Revised: 23 January 2025 / Accepted: 31 January 2025 / Published: 2 February 2025
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Abstract

:
Algal blooms caused by Microcystis aeruginosa are a common occurrence and pose significant threats to freshwater ecosystems. This study investigates the antialgal effects and underlying mechanisms of two plant-derived fatty acids, nonanoic acid and palmitic acid, on Microcystis aeruginosa. The results show that the inhibitory effects of both fatty acids on M. aeruginosa increase with higher concentrations. Algal recovery occurs when nonanoic acid concentrations are below 0.5 mg/L and palmitic acid concentrations are below 50 mg/L. Acute toxicity tests indicate that the safe concentrations of nonanoic acid and palmitic acid are below 1.87 mg/L and 263.3 mg/L, respectively. The inhibitory effect of nonanoic acid is more pronounced under conditions of pH 5.5, 15 °C temperature, 0.75 mg/L nitrogen, and 2 mg/L phosphorus, with inhibition efficiency remaining unaffected by increased light intensity. Both fatty acids exert their strongest inhibitory effects in the early stages of addition (0–8 days), causing cell death and the release of extracellular organic matter primarily consisting of aromatic compounds and proteins. Oxidative stress analysis reveals that high concentrations of fatty acids can cause irreversible damage to the algae’s antioxidant defense system. These findings provide valuable insights for the prevention and control of cyanobacterial blooms, which can help promote the sustainable development of freshwater ecosystems.

1. Introduction

Global water bodies have faced increasing challenges related to eutrophication, resulting in the rapid proliferation of phytoplankton and the emergence of algal blooms. These blooms trigger a range of harmful environmental consequences, including water contamination and adverse effects on terrestrial and aquatic ecosystems, as well as human health [1]. They also cause significant economic losses, with a total cost of approximately USD 2.2 billion per year due to freshwater eutrophication in the United States [2]. The frequency and intensity of harmful algal blooms are exacerbated by the effects of global warming [3]. These blooms encompass a variety of algal species, including cyanobacteria, green algae, and diatoms, with cyanobacterial blooms being the most common and threatening. Cyanobacteria exhibit rapid growth and reproduction, quickly forming blooms under favorable conditions of light, temperature, hydrodynamics, and nutrient concentrations, which can severely disrupt aquatic ecosystems [4,5]. High concentrations of algal toxins pose serious risks to human health, with effects ranging from gastrointestinal distress to potential carcinogenicity [6]. Since the 1980s, 68% of large lakes and reservoirs worldwide have experienced an increase in the intensity of cyanobacterial blooms [7]. Cyanobacterial blooms dominated by Microcystis species rapidly deplete dissolved oxygen and release microcystin toxins, posing a serious threat to the ecological balance [8,9]. Microcystic aeruginosa is a dominant freshwater cyanobacterium; it is widely distributed and abundant [10]. The inhibition of M. aeruginosa to prevent algal blooms is vital for maintaining ecological balance and promoting sustainable management of water resources.
Research demonstrates that plants, particularly macrophytes, can effectively inhibit algal growth through allelopathic mechanisms by releasing compounds such as phenolic acids, fatty acids, terpenes, and alkaloids [11]. These allelochemicals target essential processes in algal cells, including photosynthesis, and membrane integrity, thereby reducing the occurrence of cyanobacterial blooms [12]. A wide range of aquatic plant species has been thoroughly studied, confirming their allelopathic effects on algae and successfully identifying the allelochemical profiles of many of these plants. For instance, Zhang et al. [13] observed that Acorus calamus L. exhibits strong inhibitory effects on M. aeruginosa, significantly hindering its growth dynamics. Nakai et al. [14] identified eight distinct allelochemicals produced by Myriophyllum spicatum that actively inhibit the growth and photosynthetic functions of M. aeruginosa. As interest in the allelopathic potential of fatty acids grows, research has identified over 40 fatty acid varieties, with 18 showing measurable inhibitory effects on cyanobacteria; notably; 77% demonstrate significant toxicity at low concentrations [15]. Studies have shown that low iron promotes oligomerization of Microcystins, leading to oxidative stress in cyanobacteria and worsening water pollution [16], whereas palmitic acid contains hydroxyl groups that can inhibit the growth of Microcystins by chelating heavy metal ions to form complexes and removing heavy metals from the water column [17]. However, our understanding of the inhibitory potentials and mechanisms of fatty acids remains limited [18,19], emphasizing the need for further research to inform effective management strategies for cyanobacterial blooms. Additionally, the intricate interactions within aquatic ecosystems affect the efficacy of these allelopathic agents in suppressing algal overgrowth. Therefore, a comprehensive understanding of the algal inhibitory potentials, safe dosage, and environmental factors affecting the efficacy of fatty acids is still required to facilitate their application in natural water bodies.
Fatty acids are widely present in natural environments and are primarily categorized as saturated or unsaturated [20]. Nonanoic acid and palmitic acid are notable unsaturated fatty acids released by the roots of aquatic plants used in water remediation efforts and recognized for their eco-friendliness and low toxicity. Nonanoic acid, an approved food additive [21], is found in roses, irises, and certain essential oils and there are trace amounts in milk and specific alcoholic beverages. It is safe for human consumption in small doses and degrades quickly in the environment, reducing the risk of secondary contamination. Palmitic acid is widely distributed in nature and is present in both animal and plant fats [22]. Animal sources like butter, mutton fat, and lard contain significant levels of palmitic acid, while plant oils such as palm oil and coconut oil also have considerable amounts.
In this study, we aimed to investigate: (1) the inhibitory effects of nonanoic acid and palmitic acid, both individually and in combination, on M. aeruginosa, as well as the safe thresholds for acute toxicity in zebrafish and large crayfish; (2) the impact of environmental factors (including water pH, temperature, light intensity, nitrogen, and phosphorus) on the algal inhibition effects of fatty acids; and (3) the mechanisms by which fatty acids suppress M. aeruginosa, focusing on the release of extracellular organic matter, changes in the algal photosynthetic system, and the effects on antioxidant enzyme activity in algal cells.

2. Materials and Methods

2.1. Cultivation of M. Aeruginosa

The M. aeruginosa (FACHB-905) used in this study was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. The protocol for cultivating M. aeruginosa is as follows: The algal strain is inoculated into a sterilized Erlenmeyer flask and placed in an illuminated incubator with a light–dark cycle of 12 h:12 h, at a temperature of 25 °C, and a light intensity of 4000 lx. The flask is gently shaken 3–5 times daily. On day 4, the algae are transferred to a sterilized 1000 mL Erlenmeyer flask, and approximately 100 mL of fresh culture medium is added daily. This cultivation process continues for one week, with all steps performed under a sterile laminar flow hood to maintain aseptic conditions. The composition of the culture medium is as follows: NaNO3 (1500 mg), K2HPO4 (40 mg), Na2CO3 (20 mg), CaCl2·2H2O (36 mg), MgSO4·7H2O (75 mg), citric acid (6 mg), ferric ammonium citrate (6 mg), EDTA-2Na (1 mg), H3BO3 (2.86 mg), MnCl2·4H2O (1.81 mg), ZnSO4·7H2O (0.22 mg), CuSO4·5H2O (0.079 mg), Na2MoO4·2H2O (0.039 mg), and distilled water to a final volume of 1000 mL.

2.2. The Effects of Nonanoic and Palmitic Acids on the Growth of M. Aeruginosa

The inhibitory effects of nonanoic acid and palmitic acid on M. aeruginosa were tested in flasks with an initial cell density of 9 × 104 cells/mL, using varying concentrations of nonanoic acid (0.5, 0.75, 1, 1.25, 1.5, 1.75, 2 mg/L) and palmitic acid (25, 50, 100, 150, 200, 250, 300 mg/L). A solvent control group without fatty acids was included, using dimethyl sulfoxide (DMSO) as the solvent. All experiments were conducted in triplicate. The algicidal effect on M. aeruginosa growth was assessed by comparing cell counts in treated and untreated groups. Algal cells were counted under a microscope, and the inhibition rate (%) was calculated using the formula: inhibition rate (%)  =  (1 − Nt/Nc)  ×  100, where Nc is the algal cell count in the control group and Nt is the count in the treatment group.
A combined algal inhibition assay was conducted at an iso–mass concentration ratio of 1:1 based on the half effective concentrations of nonanoic acid and palmitic acid at 16 days of the experimental cycle [23]. The experimental conditions were the same, and blood counting plates were examined at 2-day intervals over the 16-day period. The expected EC50 (median effect concentration) values for the two fatty acid mixtures were derived from the EC50 of the individual fatty acid algal inhibition experiments at 16 days. The ratio of the expected effective concentration of the fatty acid mixtures to that of the observed experiments was calculated to determine the type of combined effect of the two fatty acids. The formula for calculating the expected EC50 of a mixture of two fatty acids was (EC50mix, the expected EC50 value; EC50i, the ith EC50 value; Pi, the ith concentration percentage; n, the number of mixed components):
E C 50 mix = ( ∑ i = 1 n P i E C 50 i ) − 1

2.3. Acute Toxicity of Nonanoic and Palmitic Acids for Danio Rerio and Daphnia Magna

D. rerio were obtained from China Zebrafish Resource Center; D. magna were sourced from the College of Fisheries at Huazhong Agricultural University. The acute toxicity of fatty acids to D. rerio was assessed after 96 h of exposure in a static test, following OECD Guideline no. 203 [24]. The water temperature was maintained at 23 ± 1 °C, and the fish were kept under standard laboratory lighting with a 12 h light/dark cycle. Ten fish were placed in 5 L glass aquaria containing either the test solution or control solution, and the water was aerated to maintain dissolved oxygen at least 90% of air saturation. The D. magna test was conducted according to OECD Guideline No. 202 [25]. Testing was carried out at 20 °C in darkness for 24, 48, and 72 h, after which immobility was recorded. Ten organisms were used for each test concentration. For both the acute test of D. rerio and D. magna, a blank control group (without fatty acids and DMSO) and a solvent control group (with DMSO) were included. The concentrations of nonanoic acid and palmitic acid matched those outlined in Section 2.2.
The safe concentrations (SC) of nonanoic acid and palmitic acid for D. rerio and D. magna were calculated using the following formula (where LC50 refers to the lethal concentration for 50% of the test subjects):
S C = 24 h L C 50 × 0.3 ( 24 h L C 50 / 48 h L C 50 ) 3

2.4. Environmental Factors Influencing the Antialgal Activity of Fatty Acids

Previous findings in this study indicate that even at low concentrations nonanoic acid can significantly reduce algal density and is more cost-effective than palmitic acid. Thus, we investigated how five environmental factors—pH, temperature, light intensity, nitrogen, and phosphorus levels—affect the antialgal efficacy of nonanoic acid. The initial algal cell concentration in the culture was 1.3 × 105 cells/mL, with nonanoic acid added at 1.75 mg/L. The test conditions were set as follows: pH values of 5.5, 6.5, 7.5, 8.5, and 9.5; temperatures of 15, 20, 25, 30, and 35 °C; light intensities of 1700, 2500, 5000, 7500, and 10,000 lx; NaNO3 concentrations of 0.75, 1.25, 2.5, 5, and 10 mg/L; and KH2PO4 concentrations of 0.125, 0.25, 0.5, 1, and 2 mg/L. A solvent control group using 0.5% DMSO (v/v) was included. Each treatment had three replicates, and the experiment spanned 16 days, with algal cell counts taken every two days. The inhibition rate (%) was calculated as described in Section 2.2. The effective time (ET20) was defined as the time required to reach 20% inhibition, and the half-effective time (ET50) as the time required to reach 50% inhibition.

2.5. Determination of Extracellular Organic Matter and Photosynthesis of M. Aeruginosa

To investigate the effect of nonanoic and palmitic acid dosages on the release of extracellular organic matter from M. aeruginosa, we measured dissolved organic carbon (DOC) and conducted UV-visible spectra and three-dimensional fluorescence spectra of the algal extracellular filtrate. Briefly, the initial algal cell concentration in the culture was 1.3 × 105 cells/mL, using varying concentrations of nonanoic acid (0.75, 1, 1.25, 1.5, 1.75 mg/L) and palmitic acid (50, 100, 150, 200, 250 mg/L). A solvent control group without fatty acids was included, using DMSO as the solvent. All experiments were conducted in triplicate. Samples of the algal solution were collected on days 4, 8, 12, and 16 of cultivation, filtered through a 0.22 μm membrane, and the DOC of the filtrate was measured using a Shimadzu total organic carbon analyzer. UV-visible spectra were obtained by scanning wavelengths from 300 to 800 nm using a UV-2000 spectrophotometer. Three-dimensional fluorescence measurements were performed with a Duatta fluorometer under the following conditions: excitation wavelength (Ex) range of 250–550 nm with 5 nm intervals; emission wavelength (Em) range of 280–650 nm with 2 nm intervals; slit width set to 10 nm; and a scan speed of 1000 nm/min.
Photosynthetic changes in M. aeruginosa cells following fatty acid addition were evaluated by measuring chlorophyll a (Chl-a), maximum quantum yield of PSII (Fv/Fm), and electron transport rate (ETR). Chl-a was measured using the ethanol extraction method, while Fv/Fm and ETR were determined with a phytoplankton classification fluorometer (Phyto-PAM).

2.6. Oxidative Stress Analysis

The activities of superoxide dismutase (SOD) and catalase (CAT) and the level of the lipid peroxidation product malonaldehyde (MDA) in alga cells were determined to identify the oxidative stress of fatty acids to M. aeruginosa. The activities of SOD, CAT, and the level of MDA were determined by using commercial kits (NanJing Jiancheng Bioeng. Inst., NanJing, China).

2.7. Statistical Analysis

A one-way ANOVA test, conducted in R 4.2.0, was used to evaluate the statistical differences of biological parameters between control group and the treated groups. The results were expressed as mean ± standard deviation (SD). P-values less than 0.05 indicate statistically significant results (*, p < 0.05; **, 0.01 < p < 0.05; ***, p < 0.001). All figures were plotted using Origin 2018. The detailed research process is shown in Figure S1.

3. Results and Discussion

3.1. Inhibition of M. Aeruginosa by Nonanoic Acid and Palmitic Acid

Figure 1 illustrates the inhibition rates of M. aeruginosa at different concentrations of fatty acids. When nonanoic acid concentrations exceeded 0.75 mg/L, the inhibition rate of M. aeruginosa increased with concentration, reaching over 60% by day 10 (Figure 1a). However, at concentrations below 0.75 mg/L, the inhibition rate declined on day 10, likely because the inhibition was not sufficient to outpace the algal growth rate. This result aligns with previous findings that allelochemicals may promote growth at low concentrations but inhibit it at higher levels [26,27]. On day 16, when the nonanoic acid concentration was ≥1.75 mg/L, the inhibition rate of M. aeruginosa exceeded 90%. Overall, the growth inhibition of M. aeruginosa was positively correlated with the concentration of nonanoic acid, that is, higher concentrations led to more pronounced inhibition effects.
At palmitic acid concentrations of 25 and 50 mg/L, the inhibition of M. aeruginosa was moderate, with rates below 50% (Figure 1b). When the concentration reached ≥150 mg/L, inhibition became significant, achieving 60% by day 6. At 300 mg/L, inhibition reached 60% as early as day 4, indicating a marked inhibitory effect at higher concentrations. Concentrations ≥150 mg/L showed a positive correlation between palmitic acid levels and inhibition rates.
Inhibitory effects were judged using Keplinger reviews. Enhancing effect: ratio >1.75; Antagonistic effect: ratio <0.57; Additive effect: 0.57–1.75. Based on the actual experimental results, the EC50 value of the combined algal inhibition on the 16th day was calculated, and the expected EC50 of the combined algal inhibition of nonanoic acid and palmitic acid was calculated according to the formula and the combined inhibition effect was evaluated by using the Keplinger reviews. Table 1 shows that the combination of nonanoic acid and palmitic acid showed an antagonistic effect on M. aeruginosa. Hu et al. [23] showed that the effects of chemosensory substances with similar algal inhibitory effects are generally antagonistic, unless the algal inhibitory mechanism is not similar or a new algal inhibitory substance is produced to show synergism. Therefore, it can be shown that the inhibitory mechanisms of nonanoic acid and palmitic acid on M. aeruginosa are similar.

3.2. Acute Toxicity of Nonanoic Acid and Palmitic Acid for D. Rerio and D. Magna

For the blank control group (without fatty acids and DMSO), no D. rerio or D. magna cell death was observed during the test period. In the solvent control group (with DMSO), the cell death rates for D. rerio or D. magna were 3.67% and 6.67%, respectively, indicating that the solvent DMSO had minimal impact on the experiment. Overall, the cell death rates for D. rerio or D. magna increased with higher concentrations and longer exposure to nonanoic and palmitic acids (Table 2). Based on the 96 h LC50 for D. rerio and the 48 h LC50 for D. magna, nonanoic acid showed moderate toxicity to D. rerio, while palmitic acid exhibited low toxicity. For D. magna, nonanoic acid displayed low toxicity, whereas palmitic acid was classified as slightly toxic. Based on the calculated safe concentrations of fatty acids from experimental results, the levels of nonanoic acid applied for controlling blue-green algae in natural waters should not exceed 1.87 mg/L or 2.21 mg/L. For palmitic acid, concentrations should remain below 263.3 mg/L or 278.76 mg/L to ensure safe application.

3.3. Impact of Environmental Factors on the Antialgal Activity of Nonanoic Acid

To explore the impact of environmental factors on the antialgal activity of nonanoic acid, five environmental factors—pH, temperature, light intensity, nitrogen, and phosphorus—were included. As shown in Figure 2a, nonanoic acid demonstrates stronger antialgal effects in acidic conditions, with maximum inhibition at pH 5.5 and a decline in effectiveness as pH increases. This reduced inhibition in alkaline conditions may stem from the faster growth of M. aeruginosa in such environments [28]. Temperature also impacts the effectiveness of nonanoic acid, with significantly higher inhibition observed at 15 °C (Figure 2b), likely because higher temperatures promote algal photosynthesis and cell division [29]. Given that blue-green algal blooms often occur during warm summer months, these results suggest that applying nonanoic acid in cooler, pre-bloom conditions could enhance its efficacy. As shown in Figure 2c, an intensity of 10,000 lx did not exceed the light saturation point, so increasing light did not reduce inhibition efficiency. However, nonanoic acid’s effect lessened as light intensity increased, likely due to light’s influence on M. aeruginosa growth. In Figure 2d, inhibition remained relatively stable across varying nitrogen levels, with ET20 and ET50 shortest at 0.75 mg/L nitrogen; these increased with higher nitrogen concentrations, although they slightly declined at 10 mg/L, suggesting greater inhibition under low nitrogen conditions. Figure 2e shows minimal variation in inhibition across phosphorus levels, with the shortest ET20 and ET50 observed at 2 mg/L phosphorus, indicating that higher phosphorus concentrations enhance the inhibitory effect of nonanoic acid on M. aeruginosa.

3.4. Effect of Fatty Acids on the Release of Extracellular Organic Matter from M. Aeruginosa

To investigate the effect of nonanoic and palmitic acid dosages on the release of extracellular organic matter from M. aeruginosa, we measured DOC and recorded UV-visible spectra (Figure 3). As shown in Figure 3a,b, both nonanoic and palmitic acids at different concentrations significantly increased the DOC content in the algal extracellular filtrate compared to the control, indicating a profound inhibitory effect on M. aeruginosa growth. With higher fatty acid dosages, DOC content increased, suggesting that higher concentrations are more effective for algal inhibition. In the first four days, DOC levels rose most sharply, while from days 4 to 8, the increase slowed, implying that the DOC increase primarily resulted from inhibited cell growth and subsequent cell death, releasing organic matter into the medium. Beyond day 8, the DOC levels in the palmitic acid group decreased more noticeably than in the nonanoic acid group, suggesting a time-limited effect for palmitic acid. As the M. aeruginosa growth rate began to exceed the inhibitory effect of lower palmitic acid concentrations, the algae started utilizing the available nutrients for growth, leading to a decrease in DOC levels [30]. Zhang et al. [31] studied the algal inhibition effect of fatty acids with different carbon chain lengths, and the test results showed that various fatty acids have certain algal inhibition functions and that the strength of the algal inhibition effect is directly related to the structure of fatty acids.
The UV-visible spectra indicated that the inhibitory effects of nonanoic and palmitic acids on M. aeruginosa occur primarily during the initial phase of treatment (Figure 3c,d). The extracellular filtrate of M. aeruginosa treated with these acids shows two main absorption peaks: Peak A at 442 nm in the blue-violet region and Peak B at 682 nm in the red region. No peak appears in the green region (492–577 nm), consistent with the green color of M. aeruginosa. As the experiment progressed, both peaks A and B gradually decreased. From the start of the experiment to day 4, the absorbance at Peak A dropped by 13.4–15.6% and at Peak B by 11–13%; the largest declines occurred in the first four days. This trend aligns with the observed impact of nonanoic and palmitic acids on algal growth, confirming that their antialgal effects are most pronounced in the early stages of treatment.
A three-dimensional fluorescence spectrum scan of the algal extracellular filtrate showed that the extracellular organic substances were mainly aromatic proteins and humic-like substances (Figure 4). Three fluorescence peaks were detected: Peak T1, at Ex/Em = (270–290) nm/(320–370) nm, represents soluble microbial byproducts, primarily tryptophan-like substances. Peak T2, at Ex/Em = (325–370) nm/(400–450) nm, and peak T3, at Ex/Em = 275 nm/(400–450) nm, are both associated with humic substances. T1 reflects biochemical organics in M. aeruginosa, mainly proteins, peptides, amino acids, and DNA. T2 represents degradation products during algal cell death, with lower fluorescence indicating high cell viability. T3 reflects humic substances derived from organic materials after algal cell death [32].
The effects of nonanoic acid and palmitic acid on M. aeruginosa are shown in the 3D fluorescence spectra in Figure 4. On day 0, only the T1 peak, representing biochemical organics in M. aeruginosa and indicating high cell viability, was clearly observed in the filtrate. By day 4, the nonanoic acid group displayed low-intensity T2 and T3 peaks, while the palmitic acid group showed a low-intensity T3 peak but no T2 peak. On day 8, the intensity of T3 in the palmitic acid group increased compared to day 4, and by day 16, the T2 peak had appeared fully. The rise in T2 fluorescence suggests an increase in humic substances generated from cell death, with a higher humic acid content correlating with longer exposure times. The T3 peak, associated with byproducts of algal inhibition by fatty acids, gradually increased due to the chemical stability of fulvic acids, indicating that both humic and fulvic acids are products of algal inhibition by fatty acids [33,34,35].

3.5. Changes in Photosynthesis of M. Aeruginosa Cells with Fatty Acid Addition

Chl-a is a key indicator of algal cell photosynthetic activity, with changes in Chl-a levels directly reflecting algal cell vitality. Compared to the control group, the Chl-a content in the culture system significantly decreased with the addition of nonanoic acid (Figure 5a). Palmitic acid, by contrast, had a comparatively smaller effect on Chl-a levels (Figure 5b). When nonanoic acid reached 1.75 mg/L or palmitic acid exceeded 100 mg/L, the Chl-a content was notably suppressed.
Fv/Fm, an important measure of PS II system health in algal cells, indicates cell health status. Both fatty acids significantly reduced Fv/Fm values compared to the control group (Figure 5c,d). In general, as the fatty acid concentration and exposure time increased, the inhibition of Fv/Fm values became more pronounced.
The ETR (electron transport rate) reflects the photosynthetic rate of algal cells. Both fatty acids noticeably suppressed the ETR in M. aeruginosa compared to the control (Figure 5e,f). This suppression intensified with higher fatty acid concentrations and longer exposure times. However, at low palmitic acid concentrations, the effect on the ETR was minimal; between days 12 and 16 at 50 mg/L of palmitic acid, the ETR showed little reduction, consistent with the limited impact of low palmitic acid concentrations on M. aeruginosa growth. This may suggest a time-limited inhibitory effect in the low-concentration palmitic acid group.

3.6. Effect of Fatty Acids on the Antioxidant Enzyme System of M. Aeruginosa Cells

When M. aeruginosa is exposed to a new environment, its cells perceive environmental stress, which triggers antioxidant enzymes to initiate a stress response, elevating levels of SOD, CAT, and MDA. Once the environmental stress subsides, the enzyme activity gradually returns to normal. SOD, an essential component of the antioxidant enzyme system, helps balance the oxidative and antioxidative processes [36]. Significant variations in SOD activity during the experiment indicate a notable stimulatory effect of capric and palmitic acids on SOD in M. aeruginosa cells (Figure 6a,b), especially on days 4 and 8 following fatty acid addition. When capric acid was added at concentrations ≥ 1.25 mg/L and palmitic acid at concentrations ≥150 mg/L, SOD activity showed highly significant differences from the control group (p < 0.01). As the cultivation time increased, SOD activity declined. On day 16, the SOD activity in the group treated with 1.75 mg/L capric acid and the group treated with 250 mg/L palmitic acid remained significantly different from the control (p < 0.05), indicating irreversible damage to the antioxidant defense system at high fatty acid concentrations.
Similarly, CAT, another essential enzyme, catalyzes the breakdown of hydrogen peroxide, protecting cells from ROS-induced oxidative damage [37]. Its activity followed the same trend as SOD, with highly significant changes on days 4 and 8 in response to high levels of fatty acid treatment (Figure 6c,d). By day 16, CAT activity was lower than in the control group, further suggesting that high concentrations of capric and palmitic acids cause lasting damage to the antioxidant defense system in M. aeruginosa.
Malondialdehyde (MDA), a product of lipid peroxidation in algal cells, reflects the extent of lipid peroxidation in the cell membrane [38]. MDA levels rose on days 4 and 8 in all treatment groups, with a significant increase by day 8 in groups treated with capric acid at concentrations ≥1.25 mg/L and palmitic acid ≥200 mg/L (Figure 6e,f; p < 0.01). The MDA levels reached their peak in M. aeruginosa cells treated with 1.75 mg/L capric acid and 250 mg/L palmitic acid, measuring 16.21 mg/105 cells and 13.21 mg/105 cells, respectively. The increase in MDA levels following fatty acid addition indicates abnormal lipid peroxidation due to the inhibitory effects of fatty acids, compromising the antioxidant enzyme system and leading to cell death in M. aeruginosa.

4. Conclusions

In this study, we investigated the inhibitory effects and mechanisms of nonanoic acid and palmitic acid on M. aeruginosa. The results showed that both acids effectively inhibited M. aeruginosa, with inhibition intensifying as the concentration increased. When the concentration of nonanoic acid was below 0.5 mg/L and that of palmitic acid below 50 mg/L, M. aeruginosa could recover. Acute toxicity tests with D. rerio and D. magna indicated that the safe concentrations for nonanoic and palmitic acid are below 1.87 mg/L and 263.3 mg/L, respectively. Environmental factors (pH, temperature, light, nitrogen and phosphorus) influenced nonanoic acid’s inhibitory effect, with optimal inhibition at pH 5.5, 15 °C, a nitrogen concentration of 0.75 mg/L, and a phosphorus concentration of 2 mg/L; the inhibition efficiency did not decrease with increased light intensity. By examining the extracellular organic matter and photosynthetic activity during the inhibition process, we found that nonanoic and palmitic acids primarily inhibited the extracellular organic matter release in the early stages, leading to cell death and increased DOC in the culture medium. The main extracellular organic matter components were aromatic proteins and humic acid-like substances. The trends in Chl-a levels, Fv/Fm values, and the ETR in response to fatty acids aligned with changes in cell density. Analysis of oxidative enzyme activity revealed that fatty acids damage M. aeruginosa’s antioxidant enzyme activity, disrupt cell membrane permeability, and harm the unsaturated fatty acids in cell membranes. These findings provide technical support for using fatty acids to inhibit algal growth in natural water bodies, which helps to improve the health of aquatic ecosystems and promote the sustainable use of water resources. As a result, we can better protect freshwater ecosystems and realize the sustainable coexistence of the ecological environment and human activities. However, in real water bodies, algae species are not found in isolation, so the algal inhibition effects of nonanoic acid and palmitic acid on mixed algae should be considered. Secondly, this study is only a simulation experiment; however, fatty acids should be considered as algae control agents to be applied into actual water bodies and in situ experiments should be conducted in the field, so as to give the experiments more practical significance.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/su17031207/s1. Figure S1: Flowchart of the research process.

Author Contributions

N.H.: Visualization, Writing—original draft. Y.T.: Visualization, Writing—original draft. X.X.: Conceptualization, Visualization, Writing—original draft. Y.G.: Visualization, Writing—review and editing. K.Z.: Methodology, Writing—review and editing. W.Q.: Methodology. Y.Z. (Yimin Zhang): Writing—review and editing. Y.Z. (Yuan Zhao): Conceptualization. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Hydrology Bureau of Changjiang Water Resources Commission Innovation and Technology Fund (SWJ-24CJX05) and the Jiangsu Graduate Research and Innovation Program (SJCX23_1542).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data will be made available on request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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Figure 1. Inhibition rates of M. aeruginosa induced by varying concentrations of nonanoic acid (a) and palmitic acid (b).
Figure 1. Inhibition rates of M. aeruginosa induced by varying concentrations of nonanoic acid (a) and palmitic acid (b).
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Figure 2. The influence of environmental factors on the antialgal activity of nonanoic acid. (a) pH; (b) Temperature; (c) Light intensity; (d) Nitrogen concentrations; and (e) Phosphorus concentrations. *, p < 0.05; **, 0.01 < p < 0.05.
Figure 2. The influence of environmental factors on the antialgal activity of nonanoic acid. (a) pH; (b) Temperature; (c) Light intensity; (d) Nitrogen concentrations; and (e) Phosphorus concentrations. *, p < 0.05; **, 0.01 < p < 0.05.
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Figure 3. The effect of nonanoic and palmitic acids on the release of extracellular organic matter from M. aeruginosa. (a,b) Dissolved organic carbon concentration and (c,d) UV-visible spectra of the extracellular filtrate of M. aeruginosa treated with nonanoic and palmitic acids.
Figure 3. The effect of nonanoic and palmitic acids on the release of extracellular organic matter from M. aeruginosa. (a,b) Dissolved organic carbon concentration and (c,d) UV-visible spectra of the extracellular filtrate of M. aeruginosa treated with nonanoic and palmitic acids.
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Figure 4. Three-dimensional fluorescence spectrum scan of the algal extracellular filtrate. (a–e) The 3D fluorescence spectra of the M. aeruginosa filtrate treated with nonanoic acid on days 0, 4, 8, 12, and 16, respectively; (f–j) The spectra for the filtrate treated with palmitic acid on the same days.
Figure 4. Three-dimensional fluorescence spectrum scan of the algal extracellular filtrate. (a–e) The 3D fluorescence spectra of the M. aeruginosa filtrate treated with nonanoic acid on days 0, 4, 8, 12, and 16, respectively; (f–j) The spectra for the filtrate treated with palmitic acid on the same days.
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Figure 5. The influence of nonanoic acid and palmitic acid on the photosynthesis of M. aeruginosa cells. (a,b) Chlorophyll a (Chl-a); (c,d) Fv/Fm; (e,f) Electron transport rate (ETR).
Figure 5. The influence of nonanoic acid and palmitic acid on the photosynthesis of M. aeruginosa cells. (a,b) Chlorophyll a (Chl-a); (c,d) Fv/Fm; (e,f) Electron transport rate (ETR).
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Figure 6. The influence of nonanoic acid and palmitic acid on the photosynthesis of M. aeruginosa cells. (a,b) Chlorophyll a (Chl-a); (c,d) Fv/Fm; (e,f) Electron transport rate (ETR). *, p < 0.05; **, 0.01 < p < 0.05.
Figure 6. The influence of nonanoic acid and palmitic acid on the photosynthesis of M. aeruginosa cells. (a,b) Chlorophyll a (Chl-a); (c,d) Fv/Fm; (e,f) Electron transport rate (ETR). *, p < 0.05; **, 0.01 < p < 0.05.
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Table 1. Evaluation of the combined toxic effects of nonanoic acid and palmitic acid by half lethal dose index method.
Table 1. Evaluation of the combined toxic effects of nonanoic acid and palmitic acid by half lethal dose index method.
Co-Reagents16d Measured EC50
(mg/L)		Expected EC50
(mg/L)		RatioKeplinger Reviews
Nonanoic acid + Palmitic acid78.5442.580.54antagonistic
Table 2. Acute toxicity of nonanoic acid and palmitic acid for D. rerio and D. magna.
Table 2. Acute toxicity of nonanoic acid and palmitic acid for D. rerio and D. magna.
Tested OrganismsFatty AcidsLC50 (mg/L)Safe Concentration (mg/L)
24 h48 h96 h (D. rerio)/72h (D. magna)
D. rerioNonanoic acid57.5227.4317.431.87
Palmitic acid1176.081066.12975.51263.30
D. magnaNonanoic acid51.7626.5221.432.21
Palmitic acid1245.141124.211024.54278.76
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Hu, N.; Tan, Y.; Xiao, X.; Gao, Y.; Zheng, K.; Qian, W.; Zhang, Y.; Zhao, Y. Antialgal Effects of Nonanoic and Palmitic Acids on Microcystis aeruginosa and the Underlying Mechanisms. Sustainability 2025, 17, 1207. https://doi.org/10.3390/su17031207

AMA Style

Hu N, Tan Y, Xiao X, Gao Y, Zheng K, Qian W, Zhang Y, Zhao Y. Antialgal Effects of Nonanoic and Palmitic Acids on Microcystis aeruginosa and the Underlying Mechanisms. Sustainability. 2025; 17(3):1207. https://doi.org/10.3390/su17031207

Chicago/Turabian Style

Hu, Ning, Yaowen Tan, Xian Xiao, Yuexiang Gao, Kaikai Zheng, Wenhan Qian, Yimin Zhang, and Yuan Zhao. 2025. "Antialgal Effects of Nonanoic and Palmitic Acids on Microcystis aeruginosa and the Underlying Mechanisms" Sustainability 17, no. 3: 1207. https://doi.org/10.3390/su17031207

APA Style

Hu, N., Tan, Y., Xiao, X., Gao, Y., Zheng, K., Qian, W., Zhang, Y., & Zhao, Y. (2025). Antialgal Effects of Nonanoic and Palmitic Acids on Microcystis aeruginosa and the Underlying Mechanisms. Sustainability, 17(3), 1207. https://doi.org/10.3390/su17031207

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