Response of the Invasive Cyanobacterium Raphidiopsis raciborskii to Iron and Phosphorus Concentrations in the Habitat: Effects on Growth and Cellular Phosphorus Distribution

Abstract

Harmful Raphidiopsis raciborskii blooms threaten aquatic ecosystems via toxin production, hypoxia induction, and biodiversity loss. To elucidate the synergistic regulatory mechanisms of Fe³⁺ and phosphorus (P) in cyanobacterial growth, we used a sterile pure culture system under laboratory conditions. We set different phosphorus sources (organic phosphorus and inorganic phosphorus) and low phosphorus concentration of R. raciborskii culture medium for culture, and set different Fe³⁺ addition amount to determine the basic growth index of cyanobacteria cells and the phosphorus content of different components. The results revealed that under conditions of sufficient inorganic phosphorus, there was a logarithmic relationship between ferric ammonium citrate (Fe³⁺) and the specific growth rate of R. raciborskii. Fe³⁺ > 2 mg/L enhanced IPS enrichment and biomass accumulation. However, in oligotrophic or mesotrophic environments with low inorganic phosphorus concentrations, the effect of Fe³⁺ on the growth of R. raciborskii contrasted with that observed in high-IP (eutrophic) environments, exhibiting a pattern of ‘low promotion and high inhibition’. Under organic phosphorus conditions, R. raciborskii converted phosphorus by increasing alkaline phosphatase activity (APA), but this metabolic compensation failed to restore physiological functions, resulting in growth suppression and enhanced cellular phosphorus reserves. Our results establish quantitative linkages between Fe³⁺-P co-limitation thresholds and algal adaptive responses, providing mechanistic insights for controlling bloom dynamics through targeted manipulation of Fe-P bioavailability.

1. Introduction

With the intensification of global warming and eutrophication, blooms of the invasive cyanobacterium Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii) have become increasingly frequent and have expanded rapidly to subtropical and temperate regions, seriously threatening water ecology and human health [1,2,3]. R. raciborskii has a high ability to absorb and utilize nutrients, as evidenced by its rapid absorption and storage of phosphate (P) [4,5], more likely occupying a dominant position in the phytoplankton community [3]. Bonilla et al. [6] found that R. raciborskii exhibited broad adaptability to varying P environments across 28 lakes in diverse climatic regions, maintaining dominance even under low-phosphorus conditions with concentrations below 50 μg L⁻¹. Phosphorus is widely regarded as the primary limiting factor for R. raciborskii blooms, given its ability to directly uptake dissolved inorganic phosphorus (DIP) and enzymatically hydrolyze organic phosphorus (OP) via alkaline phosphatase secretion [7,8]. Under phosphorus-deficient conditions, R. raciborskii adopts a phosphorus accumulation-dominant strategy over growth-oriented utilization in natural competition, enabling sustained dominance within cyanobacterial communities and prolonged growth phase maintenance [9]. Therefore, regulating phosphorus bioavailability in aquatic systems still represents an effective strategy for mitigating R. raciborskii bloom formation.

Iron (Fe³⁺), an essential micronutrient for photosynthetic cyanobacteria, regulates algal growth through core biochemical functions spanning nitrogen fixation, photosynthetic electron transport, and enzymatic catalysis, and further influences ecosystem structure and biogeochemical cycles [10,11,12]. Cyanobacteria demonstrate uniquely elevated Fe³⁺ acquisition demands, particularly for sustaining their photosynthetic apparatus [13,14]. For example, Fe³⁺ is widely present in the thylakoid membranes of all oxygen-evolving photosynthetic organisms and participates in the entire process from water to NAD(P)⁻, the photosynthetic linear electron transport chain, which is essential for cell function and the balance of ATP/NAD(P) H supply and demand [15]. Fe³⁺ modulates nutrient assimilation dynamics in algal cells; for instance, it enhances nitrogen fixation in the filamentous cyanobacterium Lyngbya majuscula [16] and simultaneously regulates the bioavailability and uptake of other essential nutrients [17,18]. Spijkerman et al. [19] found that iron concentration affects the uptake rate of phosphorus by algae cells, affecting the growth of algae. Based on these fundamental mechanisms, the multifaceted roles of Fe³⁺ are particularly pronounced in tropical eutrophic reservoirs, where elevated dissolved iron concentrations (DFe > 0.025 mg/L) not only directly stimulate the growth of nitrogen-fixing cyanobacteria (e.g., R. raciborskii, Dolichospermum spp.) but also enhance their competitive advantage through regulating nitrogen and phosphorus availability, ultimately driving significant bloom formation [20].

In natural waters, Fe³⁺ predominantly exists in its stable form, where various iron species significantly mediate P uptake by algae, particularly under Fe³⁺-limited conditions [18,21,22]. Previous studies have shown that low concentrations and bioavailability of specific iron forms strongly inhibit cyanobacterial growth and reproduction [23], whereas dissolved iron enrichment significantly increases phytoplankton biomass [24]. Although the individual roles of Fe³⁺ and P in cyanobacterial growth regulation have been well established, the interdependent regulatory mechanisms governing their synergistic effects on R. raciborskii—specifically how Fe³⁺ concentration thresholds and phosphorus source availability co-modulate physiological responses and resource allocation—remain poorly characterized.

Our study investigated how Fe³⁺ influences the growth and phosphorus uptake/distribution dynamics in R. raciborskii under varying phosphorus conditions. We hypothesized the following: 1. Fe³⁺ exhibits a dose-dependent effect on R. raciborskii growth; 2. R. raciborskii employs distinct phosphorus acquisition strategies depending on phosphorus speciation (inorganic vs. organic); and 3. Fe³⁺ enhances algal growth and fitness under phosphorus limitation via adaptive intracellular phosphorus reallocation. By examining Fe³⁺–P interactions in R. raciborskii’s phosphorus uptake process, this study addresses a key knowledge gap in how metal ions and nutrient forms jointly shape cyanobacterial ecological strategies.

2. Materials and Methods

2.1. R. raciborskii Cultures and Pretreatment

Raphidiopsis raciborskii FACHB-1503 was purchased from the Freshwater Algae Species Bank of the Institute of Aquatic Biology, Chinese Academy of Sciences, and cultured in our laboratory at 25 °C, 3000 Lx, 12 h:12 h (D: L), until logarithmic growth. Algal cells were harvested via centrifugation at 5000 rpm for 15 min, washed three times using Fe-free BG11 medium (Table S1) to remove residual Fe³⁺, and subjected to 10-day Fe starvation to deplete intracellular iron, thereby obtaining Fe-depleted cultures. All treatment and control groups had a uniform biomass of 20 mg/L of cells, and the flasks were shaken regularly 2–3 times a day during the experiment, in addition to changing the position of the culture flasks to minimize the effect of uneven light exposure.

2.2. R. raciborskii Growth Response to Fe³⁺ Concentration

Ammonium ferric citrate (C₆H₈FeNO₇) was added to the iron-free BG11 medium as an additional iron source, and the initial Fe³⁺ concentration range was set at 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 4, 8 mg/L for the same batch of culture. R. raciborskii was inoculated into BG11 media containing varying Fe³⁺ concentrations (0–8 mg L⁻¹) and standard BG11 medium (Table S1) for 17 days without medium replenishment. Algal cell biomass and chlorophyll a (Chl a) were determined for 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, and 17 d, and specific growth rates were calculated.

2.3. Effect of Fe³⁺ on Algal Growth and P Distribution with Different P Sources

Dipotassium hydrogen phosphate (K₂HPO₄) was the inorganic phosphorus (IP) source, Beta-glycerol phosphate disodium salt pentahydrate (C₃H₁₂NaO₇P) was the organic phosphorus (OP) source, and the initial phosphorus concentration was 7.13 mg/L with standard BG11 as reference. Meanwhile, Fe³⁺ concentration was added, affecting the growth of algal cells, and a blank control without Fe³⁺ addition was set up. R. raciborskii was inoculated in the set medium with triplicates per group, and the experimental period was 7 d. Different P contents and alkaline phosphatase activities in soluble microbial products (SMPs), extracellular polymeric substances (EPSs), and intracellular polymeric substances (IPSs) of algal cells were measured daily, and algal growth and photosynthetic activity were measured every 2 d.

2.4. Effect of Fe³⁺ on Algal Growth and P Distribution with Low P Concentration

The phosphorus source more favorable to the growth of R. raciborskii was selected, and two low P concentrations of 0.02 mg/L (P1) and 0.07 mg/L (P2) were set based on the eutrophication evaluation method and grading of lakes (reservoirs) recommended by the China Environmental Monitoring General Station (Table S2) [25], with 7.13 mg/L phosphorus concentration as the high-phosphorus control group. Fe³⁺ concentrations were set to match the baseline levels reported in Section 2.2, and incorporated an additional concentration of 0.3 mg/L specifically included. The additional concentration accounts the range of Fe³⁺ concentrations (<0.3 mg/L) observed in the upper portion of the Tropical Reservoir Lake. R. raciborskii was inoculated in the set medium with triplicates per group, and the experimental period was 7 d. Detection indicators were consistent with those in Section 2.3.

2.5. Determination of R. raciborskii Growth Parameters

A series of range volumes of experimental R. raciborskii were diluted to 15 mL with Fe-free BG11, and the absorbance of the algal solution at 680 nm was measured using a UV-2800 spectrophotometer (N5000S Plus, Shanghai, China). An amount of 10 mL of the diluted algal solution was taken and filtered onto a dried and weighed 0.7 μm pore size Whatman GF/F glass fiber filter membrane. It was then dried again at 105 °C until constant weight and weighed. The difference between the two weights was recorded as the dry weight of R. raciborskii in 10 mL solution. A standard curve of cell dry weight versus absorbance was fitted. The absorbance of the culture solution of each treatment and control group was determined, and the dry weight of the cells was calculated based on the standard curve. The formula for calculating the specific growth rate μ is as follows:

μ = (lnC_t2 − lnC_t1)/(t₂ − t₁)

where C_t2 and C_t1 are the biomass of R. raciborskii at t₂ and t₁, respectively.

Chlorophyll a was determined using a phytoplankton fluorescence analyser (Phyto-PAM, Walz, Germany, Bavaria) after taking 3 mL of algal samples and darkening them in a measuring cup for 3 min.

2.6. Extraction of P Distribution Fractions and Determination of P Parameters

The SMP, EPS, and IPS extraction methods referred to Zhou et al. (2017) with slight modifications [26]. The culture solution was centrifuged at 4000 rpm, 4 °C for 15 min, and the supernatant was taken and centrifuged at 12,000 rpm, 4 °C for 10 min to collect the supernatant containing SMPs. The precipitate from the two centrifugations was prepared as a suspension using borate buffer, heated at 60 °C for 20 min, and then centrifuged at 4000 rpm for 15 min at 4 °C. The supernatant was taken at 4 °C and centrifuged at 12,000 rpm for 10 min to obtain the supernatant containing EPSs. The two precipitates were resuspended using borate buffer, heated at 100 °C for 60 min, and then centrifuged at 4000 rpm for 15 min at 4 °C. The supernatant was taken at 4 °C and centrifuged at 12,000 rpm for 10 min to obtain the supernatant containing IPSs. TP and IP were determined by taking treated samples. TP and IP were determined according to [27,28]. TP determination was first digested with potassium persulfate (K2S208), and IP was measured as molybdate-reactive phosphorus. OP = TP − IP. The formula for phosphorus distribution per unit biomass phosphorus is as follows:

P = P_t/C_t

where C_t is the biomass of R. raciborskii at t, P_t is the total phosphorus content.

2.7. Determination of Alkaline Phosphatase Activity (APA)

Alkaline phosphatase activity (APA) was determined spectrophotometrically. An amount of 500 µL of culture medium was taken and 400 µL of p-nitrophenyl phosphate disodium salt (3.6 mM), 100 µL of MgCl₂, and 2 mL of tris-HCl buffer (0.2 M) were added to give a final volume of 3 mL. The reaction was terminated by the addition of 300 µL NaOH (4 M) after 2 h at 37 °C, followed by centrifugation at 7000× g for 4 min, and the absorbance value of the supernatant at 405 nm was measured by a UV spectrophotometer. Enzyme activity was defined as the number of micromoles of p-nitrophenol released per hour per μg chlorophyll a at 37 °C (unit: μmol pNP released μg Chl a h⁻¹).

2.8. Data Analysis

Significance analyses were performed using one-way analysis of variance (ANOVA), which was preceded by a test for homogeneity of variance and a normal distribution test, and differences were considered significant when p < 0.05. Statistical analyses were performed using SPSS 27.0 analysis software and plotted using Origin 2021.

3. Results and Discussion

3.1. Correlation Between Growth of R. raciborskii and Fe³⁺ Concentration

The growth response of R. raciborskii to Fe³⁺ exhibited a concentration-dependent biphasic pattern (Figure 1). At low Fe³⁺ concentrations (<0.2 mg/L), R. raciborskii growth was significantly inhibited, as evidenced by visible yellowing of the culture and reduced biomass accumulation compared to the control (Figure 1a). Low concentrations of Fe³⁺ also inhibited the growth of other cyanobacteria [29]. In contrast, Fe³⁺ concentrations ≥ 8 mg/L markedly enhanced both the biomass production and chlorophyll a content of R. raciborskii (Figure 1a,b). Notably, the chlorophyll a content per unit biomass increased by 15–40% (p < 0.05) within the 0.5–8 mg/L Fe³⁺ range after 8 days of exposure (Figure 1c), indicating improved photosynthetic efficiency under Fe-replete conditions. Previous studies have demonstrated a positive correlation between Fe³⁺ availability (0–2 mg L⁻¹) and R. raciborskii growth rates [30,31]. Moreover, our study found that a logarithmic model effectively described the relationship between Fe³⁺ concentration and the algal growth rate of R. raciborskii (R² = 0.96, p < 0.001; Figure 1d). R. raciborskii growth rates escalated sharply within the 0–2 mg/L Fe³⁺ range, followed by saturation kinetics at higher concentrations (>2 mg/L) (Figure 1d), suggesting a threshold for Fe³⁺ uptake or metabolic utilization. A large number of studies have confirmed that the content of Fe³⁺ in the environment has a key limiting effect on the growth of cyanobacteria. The lack of Fe³⁺ concentration led to a sharp decrease in the chlorophyll a content of various Microcystis, accompanied by a significant decrease in the activity of photosynthetic system II [32,33,34].

3.2. Fe³⁺ Promotes IP Uptake Efficiency in R. raciborskii

The growth response of R. raciborskii to Fe³⁺ exhibited contrasting patterns depending on P sources (Figure 2). Under the inorganic phosphorus source, the supplementation of Fe³⁺ significantly increased algal biomass and chlorophyll a content (p < 0.05) (Figure 2a,b). Especially in the high-Fe³⁺ treatment group (8 mg/L), the biomass at 144 h increased by 90% compared with the control group. Fe³⁺ enhanced R. raciborskii growth dose-dependently without compromising photosynthetic integrity, likely through its dual roles in electron transport and phosphorus assimilation. This is because Fe³⁺ is not only involved in both growth and metabolic pathways such as photosynthesis, respiration, and protein and nucleic acid synthesis in cells [35], but also affects the phosphorus uptake rate of algal cells [19]. Zou [36] found that high Fe³⁺ concentration (1.80 mg/L) enhanced the inorganic phosphorus uptake rate of Dolichospermum flos-aquae (formerly Anabeana flos-aquae). In addition, although the organic phosphorus source initially promoted an increase in the actual photosynthetic rate, biomass, and chlorophyll a in the short term, these indicators decreased over time. Notably, the chlorophyll a level decreased by 87% relative to controls, and Fe³⁺ acted as a ‘catalyst’ in this process (Figure 2a–c). The morphological differences in organic phosphorus sources had significant differences in the growth effects of R. raciborskii. Meanwhile, Fe³⁺ may form complexes with organic phosphorus, which will increase the stability of organic phosphorus and make it more difficult to be affected by APA, thus affecting the hydrolysis and bioavailability of organic phosphorus [37].

3.3. Fe³⁺ Enhances IP Translocation and OP Storage in R. raciborskii

Under IP conditions, Fe³⁺ facilitated phosphate translocation from extracellular polymeric substances (EPSs) to intracellular pools (IPSs) in R. raciborskii; under organic phosphorus conditions, Fe³⁺ significantly promoted an increase in different forms of phosphorus unit biomass in R. raciborskii cells. Under the inorganic phosphorus source, the contents of SMP_OP and SMP_IP were almost unchanged (Figure 3a,d,g). Fe³⁺ supplementation elevated the EPS_IP depletion rate from 35.9% to 66.8% of the initial levels within 96 h, concurrent with a 63.9% increase in IPS_IP accumulation (p < 0.05) (Figure 3e,f). Zhou et al. [26] proposed that the unicellular cyanobacterium Synechocystis transports exogenous P via extracellular polymer adsorption/enrichment followed by intracellular energy storage system transport along the ‘SMP-to-EPS-to-IPS’ pathway. Similarly, R. raciborskii appears to follow this phosphorus transport trajectory, supporting the universality of this mechanism in cyanobacterial phosphorus utilization. When inorganic phosphorus was sufficient, R. raciborskii could use resources more effectively for population expansion (Figure 2a), rather than preferentially allocate resources to phosphorus absorption or storage [38], which indicates that although a sufficient phosphorus source is a necessary condition for its growth, it is not a sufficient condition.

Under the organic phosphorus conditions, the SMP_IP content gradually increased and the SMP_OP content gradually decreased (Figure 3a,d,g). The ‘OP-to-IP’ conversion process exhibited particular prominence at Fe³⁺ concentrations of 2 mg/L. Compared with the inorganic phosphorus environment, Fe³⁺ in the organic phosphorus environment promoted the secretion of APA, and the secretion amount reached 1.04–1.16 μmol pNP released μg Chl a h⁻¹ (Figure 3j). This may be due to the fact that organic phosphorus is usually not directly utilized by R. raciborskii, and cells are required to secrete more APA to achieve conversion. In this process, Fe³⁺, as a Lewis acid, can activate APA activity through electronic docking [39,40]. Fe³⁺ promoted organic phosphorus hydrolysis by polarizing C-O-P bonds, enhancing nucleophilic attack on ester bonds [41]. In addition, the organic phosphorus absorption of R. raciborskii showed a special adaptation mechanism, where the addition of Fe³⁺ significantly promoted the storage of phosphorus (Figure 3). At Fe³⁺ concentrations of 2–8 mg/L, the content of EPS_IP and IPS_IP per unit biomass increased significantly (p < 0.05). EPS_IP rose from 0.015–0.021 mg/mg to 0.066–0.075 mg/mg, and IPS_IP increased from 0.007–0.011 mg/mg to 0.031–0.038 mg/mg. Meanwhile, higher-Fe³⁺ treatments accelerated the conversion of OP to IP in EPSs (Figure 3e,h). Rabouille et al. [42] demonstrated that the unicellular N₂-fixing cyanobacterium Crocosphaera watsonii accumulates higher cellular phosphorus when utilizing adenosine monophosphate (AMP) compared to DL-α-glycerophosphate (α-GP), highlighting the influence of organic phosphorus chemical structure and degradability on phosphorus assimilation. Building on this, we posited that R. raciborskii employed a distinct survival strategy under organic phosphorus conditions, prioritizing intracellular phosphorus storage and cellular maintenance over population expansion.

3.4. IP Levels Modulate Fe³⁺ Inhibition Threshold in R. raciborskii

In the low-inorganic phosphorus (P1 = 0.02 mg/L, P2 = 0.07 mg/L) conditions, the cell biomass and chlorophyll a content of R. raciborskii were significantly lower than those in the inorganic phosphorus source-sufficient environment (p < 0.05) (Figure 2a,b). The growth of R. raciborskii exhibited an interaction between inorganic phosphorus concentration and Fe³⁺ levels. At extremely low inorganic phosphorus levels (P1), high Fe³⁺ concentrations (2–8 mg/L) significantly inhibited growth (p < 0.05). Under medium nutrient inorganic phosphorus levels (0.07 mg/L), 2 mg/L Fe³⁺ promoted growth, while 8 mg/L Fe³⁺ remained inhibitory (p < 0.05). Meanwhile, low inorganic phosphorus levels did not impair R. raciborskii photosynthesis, with Fe³⁺ treatments maintaining stable actual photosynthetic rates (Yield) throughout the experiment (Figure 4c). Shi et al. [43] reported a similar conclusion in their study on Microcystis aeruginosa in Taihu Lake, where high Fe³⁺ concentrations inhibited phosphorus uptake by this cyanobacterium. This inhibition likely arose from Fe³⁺-PO₄³⁻ precipitation under low-inorganic phosphorus conditions, which further depleted bioavailable phosphorus [44], thereby inhibiting the growth of algal cells and chlorophyll a synthesis, forming a vicious cycle. However, when inorganic phosphorus is sufficient, the complexation of Fe³⁺ with PO₄³⁻ would not be sufficient to cause phosphorus shortage, and could play an active role in the growth of R. raciborskii [15]. In summary, Fe³⁺ is an inhibitor of R. raciborskii growth under low inorganic phosphorus concentrations, but the inhibition threshold of Fe³⁺ increases with the increase in environmental inorganic phosphorus content.

3.5. Fe³⁺ Regulates OP-IP Metabolic Conversion in R. raciborskii

Compared to the high-inorganic phosphorus conditions (Figure 3), the SMP_TP content under low-phosphorus conditions increased from 0.03 ± 0.02 mg/L to 0.14 ± 0.02 mg/L, followed by a decline to 0.06 ± 0.04 mg/L, with SMP_OP being the dominant contributor to these fluctuations (Figure 5a–c). In the low-phosphorus group (p = 0.02 mg/L), SMP_OP and SMP_IP levels were higher than those in the mesotrophic group at 48 h and 96 h, respectively. Meanwhile, the unit biomass content of EPS_TP and IPS_TP exhibited an overall decreasing trend during the experiment, primarily driven by organic phosphorus dynamics. The addition of Fe³⁺ (8 mg/L) rapidly reduced EPS_OP to below 0.0045 mg/mg within 6 h (Figure 5h). The addition of high Fe³⁺ concentrations significantly enhanced the biosynthesis of APA in R. raciborskii, resulting in a marked increase in APA content (p < 0.05) (Figure 5j). Previous studies have demonstrated an inverse correlation between DIP availability and algal APA [45]. In their study of Phaeodactylum tricornutum, Wang et al. (2021) proved that PAP can maintain its physiological and biochemical functions in a substrate- and growth-dependent manner under phosphorus-deficient conditions [46]. Extracellular inorganic phosphorus is internalized via phosphate-specific transporters (Pst system) and subsequently partitioned into metabolic pools or polyphosphate storage bodies. One part of the inorganic phosphorus will be adsorbed by the -NH₃⁺ group of the organic component in the IPS, and the other part is involved in the conversion of ADP to ATP. At the same time, it is converted into intracellular organic phosphorus during biomass synthesis [47,48,49]. Fe³⁺ is widely present in all oxygen-evolving photosynthetic thylakoids. Fe³⁺ is essential for the balance of ATP/NAD(P)H supply and demand. The conversion process of IP to OP in algal cells is closely related to ATP [50], which may be the reason for the increase in IPS_OP per unit biomass of R. raciborskii in the Fe³⁺ treatment group.

4. Conclusions

This study revealed trends in the dynamic response of R. raciborskii growth and phosphorus transformation under different Fe³⁺ concentrations and phosphorus conditions. We found a dual dose-dependent effect of Fe³⁺ on R. raciborskii growth, characterized by high-concentration promotion and low-concentration inhibition. Under inorganic phosphorus-sufficient conditions, Fe³⁺ supplementation not only enhanced biomass accumulation but also drove efficient phosphorus translocation from EPSs to IPSs. Under inorganic phosphorus-limited conditions, R. raciborskii regulated intracellular phosphorus distribution by up-regulating APA activity, preferentially storing phosphorus as polyphosphate, especially at high Fe³⁺ concentrations. Our findings further elucidate the underlying mechanisms of R. raciborskii thriving under phosphorus-limited conditions and provide novel insights into iron–phosphorus co-regulation for controlling harmful cyanobacterial blooms.