Temperature-Dependent Growth Characteristics and Competition of Pseudanabaena and Microcystis

Abstract

Global warming has been considered to accelerate the expansion of cyanobacterial blooms, which are frequently composed of the bloom-forming genera, Microcystis and Pseudanabaena, in freshwater ecosystems worldwide. Nonetheless, the impact of changes due to toxin production or lack thereof on the growth of co-existing strains, both arising from increasing temperature, has remained unknown to date. We conducted competition experiments involving toxic Microcystis PCC7806, a non-toxic mcyB⁻ mutant, and two strains of Pseudanabaena (TH-1 and DC-1) identified as P. mucicola. In mono-culture, the specific growth ratio of Pseudanabaena increased; nevertheless, the maximum cell density declined with increasing temperature. The maximum growth ratios of Pseudanabaena TH-1 and Pseudanabaena DC-1 were 0.512 day⁻¹ in the 30 °C group and 0.440 day⁻¹ in the 35 °C group, respectively. The optimum temperature for the growth of Pseudanabaena was 25 °C. Remarkably, there was no significant disparity in the maximum cell density of Microcystis PCC7806 and the mcyB⁻ mutant across varied temperature groups, even though their maximum growth rates differed marginally, reaching 0.280 day⁻¹ and 0.306 day⁻¹ in the 30 °C group, respectively. In co-cultures, the growth of Pseudanabaena TH-1 was uniformly inhibited, whereas that of DC-1 was somewhat influenced by co-culturing with toxic and non-toxic Microcystis, except for the 35 °C group, where inhibition was absent amongst DC-1 and Microcystis. Moreover, the growth of Microcystis was promoted with a co-culture of TH-1 in the 20 °C groups. Conversely, the co-culture of Microcystis with Pseudanabaena DC-1 led to the inhibition of the former in the 30 °C and 35 °C groups. With a Lotka–Volterra competition model, the results showed that Microcystis dominated or co-existed with Pseudanabaena, conforming to expectations from the wild.

1. Introduction

In recent years, eutrophication has caused continuous outbreaks of cyanobacteria blooms around the world, seriously endangering the health of water ecosystems. Among all the species of cyanobactiera, Microcystis stands out as the most extensively prevalent and commonplace form of cyanobacterial bloom in China. Furthermore, Pseudanabaena, a diminutive, filamentous, non-heterocystous cyanobacteria, has captivated the attention of researchers by virtue of its ability to generate the typical taste and odor compound, 2-methyl isoalcohol (2-MIB), which can subversively undermine the quality of drinking water [1,2]. Recent evidence suggests that Pseudanabaena distribution is widespread and has been observed in disparate aquatic ecosystems [3,4,5].

In freshwater, Microcystis typically manifests as a colonial organism in cyanobacterial blooms, while Pseudanabaena was often ignored in early studies because of difficulties in observation due to its small size [6]. However, in recent years, Pseudanabaena has been increasingly observed during bloom outbreaks [7,8], occupying a dominant position in certain freshwater environments in the United States, Japan, and Malaysia [9,10,11]. In China, Pseudanabaena limnetica has emerged as a prevailing species in lakes that are located alongside the South-to-North Water Diversion Project [12,13], the Maixi River [14], and some reservoirs in southwest China [15]. Furthermore, with the development of high-throughput sequencing methods in cyanobacterial bloom research, Pseudanabaena has often been determined to make up a significant proportion of biomass within the overall process of cyanobacteria bloom [16], even sometimes surpassing the common bloom-forming species [17]. It is noteworthy that, in some subtropical and temperate lakes, Pseudanabaena has been discovered to co-exist with Microcystis, occasionally even being found in joint succession or dominance patterns [18,19,20]. Studies have shown that the Pseudanabaena biomass was significantly correlated with the Microcystis biomass [19,21], which may be related to an epiphytic relationship between Pseudanabaena and colonial Microcystis [22,23]. During bloom periods, Pseudanabaena mucicola has been identified as epiphytic to colonial Microcystis, leading to the formation of a Microcystis–Pseudanabaena phycosphere [18,24,25]. Nonetheless, the unclear mechanisms underlying co-existence or competition between Pseudanabaena and Microcystis, which share similar ecological niches with cyanobacterial blooms, persist as a substantial and pressing enigma.

Various environmental factors, such as a temperature, light, nutrients, pH, and trace elements, influence phytoplankton growth. Temperature affects the metabolism and nutrient utilization rate of algae, which in turn affects their growth [26]. It also influences cell size and the length of the algae filament segment, which determines the advantage of cyanobacteria [27]. Climate warming is expected to have an impact on the proliferation of harmful algal blooms (HABs) in lakes and reservoirs [28,29,30], since increased temperature might promote more harmful species by promoting the growth rates of harmful cyanobacterial species relative to other species [29,31,32], extending the summer growing season [33,34] and causing greater vertical stratification, all of which confer greater fitness to cyanobacteria that can regulate their buoyancy. Cyanobacteria are more resistant to other eukaryotic algae and grow faster in warm water environments above 25 °C, especially in the optimal temperature range of 27 °C to 37 °C [29], which may differ between species and strains with the same phylum [35]. Rising temperatures heavily favor Microcystis over the filamentous species Dolichospermum and Aphanizomenon in Taihu Lake, Chaohu Lake, and Dianchi Lake [36,37]. In Erhai Lake, Pseudanabaena dominates from June to September during the rainy season, while an increased biomass of Microcystis is found in autumn [20]. The optimal temperature range of Microcystis is 25 to 35 °C [26,38,39], while that for Pseudanabaena in freshwater is between 20 and 30 °C [40]. Both genera consist of multiple strains with different morphologies [6,41]. Interactions between Pseudanabaena and Microcystis range from neutral or antagonistic depending on the strains [25]. Moreover, their relationship can be genotype-specific [18]. P. mucicola can inhabit the surface of mucilage of planktonic M.aeruginosa [42]. Hence, although Microcystis and Pseudanabaena can co-exist, dominance by one or the other depends on differences in their growth rates and physical and morphological characteristics as temperatures vary. To better understand cyanobacterial community succession, this study aims to explore interspecific competition between Pseudanabaena and Microcystis under increasing temperatures and examine the growth characteristics of Pseudanabaena and toxic and non-toxic Microcystis under various temperatures to determine whether elevated temperatures in the future might affect the succession of these two genera.

2. Materials and Methods

2.1. Stain Selection and Culture Condition

Toxic Microcystis PCC7806 and the non-toxic Microcystis mcyB⁻ mutant were obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology, while Pseudanabaena TH-1 and DC-1 were isolated from Taihu Lake (Jiangsu, China) in June 2017 and Dianchi Lake (Yunnan, China) in October 2017, respectively. Pseudanabaena were picked using a Pasteur pipette and cultured with BG-11 in a 24-well plate. The culture was then diluted from 10⁻¹ to 10⁻⁷, and different multiples were inoculated on to sterile BG-11 agar medium and cultured for 15–20 days. Single colonies of Pseudanabaena were picked using a Pasteur pipette under a microscope and subsequently transferred into a 24-well plate with 2–3 mL BG-11 medium.

All the strains were cultured using BG-11 medium under standardized culture conditions of 25 °C, 25 μmol m⁻² s⁻¹, and a 12:12 h light/dark cycle.

2.2. DNA Extraction, Sequencing, and Phylogenetic Analysis

Based on rbcLX gene sequence analysis, two strains of Pseudanabaena were identified as TH-1 and DC-1. Additionally, we conducted an analysis of the strains FACHB1277, 1998, and 2209 acquired from the Freshwater Algae Culture Collection located in Wuhan, China. To extract DNA, cultured cells were collected via centrifugation at 4 °C with a force of 4000× g for 10 min, and we utilized an EZNA^TM water DNA kit D5525 (Omega, Bio-Tek, Winooski, VT, USA) following the manufacturer’s protocol. The PCR reaction involved the amplification of the rbcLX fragment with the CX

(5′-GGCGCAGGTAAGAAAGGGTTTCGTA-3′) and CW (5′-CGTAGCTTCCGGTGGTATCCACGT-3′) [43] with a Bio-rad PCR system. The program consisted of a preliminary denaturation step at 95 °C 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing 55 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 5 min. The amplified PCR product (800 bp) was cloned into a PMD-18T vector (Takara Bio, Kusatsu, Japan), and the fragments were sequenced by Tsingke Biotechnology Co., Ltd., Beijing, China. Afterward, sequence similarity searches were conducted through the BLAST procedure, and comparisons were made against GenBank. Subsequently, using the MEGA11 program package, phylogenetic trees were constructed employing neighbor-joining (NJ), genetic distances were calculated using p-distance, and bootstrapping (1000 iterations) was used to compare the sequences and produce the corresponding phylogenetic trees.

2.3. Experimental Design

To investigate the growth characteristics of Pseudanabaena TH-1, DC-1, and Microcystis PCC7806 along with the mcyB⁻ mutant in mono-culture, we conducted experiments at different temperatures ranging from 10 to 35 °C. Prior to each group experiment, each strain was pre-cultured to adapt to the corresponding temperature. Each strain that reached the exponential stage was then placed in 100 mL Erlenmeyer flasks in quintuplicate and pre-cultured under different temperature conditions for 7 days while keeping the irradiance and photoperiod constant.

In the mono-culture experiment, we inoculated pre-cultured Pseudanabaena and Microcystis strains into 50 mL of sterilized BG-11 medium in 100 mL Erlenmeyer flasks, with the initial cell density of each strain being adjusted to 1.0 × 10⁶ cells. mL⁻¹ for each strain.

To examine the growth trend of both genera when co-cultured together under various temperatures, we conducted cross co-culture experiments using two strains of Pseudanabaena (TH-1 and DC-1) and Microcystis (PCC7806 and mcyB⁻ mutant), again setting the experimental temperature as identical to the mono-culture. The starting ratio was fixed at 1: 1, with an approximate cell density of 2.0 × 10⁶ cells mL⁻¹. The experiments were conducted in triplicate.

We measured the cell densities periodically in a preliminary stage every two or three days, and in the last stage, every five to seven days until saturation or constancy for most of the groups. For the 10 °C group, measurements were only taken after twenty days due to the minimal increase in cell density, prompting no further measurements. Cells counts were performed using an optical microscope (Nexcope NE910), and cell concentrations were calculated by counting four 10 μL samples from each flask in a 0.1 mm deep hemocytometer with a 40× objective and 10× ocular lenses. If the four counts varied by <10%, we designated the average value as the final cell concentration.

2.4. Growth Characteristics in Mono-Culture

The growth curves derived from our study of cells in the 20, 25, 30, and 35 °C groups were fitted with the logistic function. To obtain the maximum growth rate, we utilized a modified logistic model represented by Equation (1):

$$\lg \left({\mathrm{N}}_{\mathrm{t}}/{\mathrm{N}}_0\right)=\frac{\mathrm{a}}{1+\exp \left[-\mathrm{k}\times \left(\mathrm{t}-{\mathrm{x}}_{\mathrm{c}}\right)\right]}$$

(1)

$${\mathsf{\mu }}_{\max }=\mathrm{a}\times \mathrm{k}/4$$

(2)

where Nt and ${\mathrm{N}}_0$ are the cell density (cells. mL⁻¹) at day t of the exponential phase and the initial time, respectively; k is the relative growth rate (the slope) in time x_c; ${\mathsf{\mu }}_{\max }$ is the maximum growth rate; a is the deviation value of cells between stationary phases and initial time; x_c is the time to relative maximum growth rate (d).

Based on the maximum growth rate, we established a temperature-dependent growth rate using the Ratkowsky equation [44]:

$$\sqrt{{\mathsf{\mu }}_{\max }}=\mathrm{b}\left(\mathrm{T}-{\mathrm{T}}_{\min }\right)\{1-\exp \left[\mathrm{c}\left(\mathrm{T}-{\mathrm{T}}_{\max }\right)\right]\}$$

(3)

where b and c are the regression coefficients in the low-temperature range and the high-temperature range, respectively. The temperature is expressed as T(°C) in Equation (3), and T_min (°C) and T_max (°C) are the temperatures when the square root of the growth rate trend to zero.

2.5. Interspecies Competition Analyses

The interspecific competition of the two genera of cyanobacteria in co-cultures was analyzed using the Lotka-Volterra competition model [45]:

$$\frac{N_{Mn}-N_{Mn-1}}{t_n-t_{n-1}}=\frac{r_M\times N_{Mn-1}\times \left(K_M-N_{Mn-1}-\alpha N_{Pn-1}\right)}{K_M}$$

$$\frac{N_{Pn}-N_{Pn-1}}{t_n-t_{n-1}}=\frac{r_P\times N_{Pn-1}\times \left(K_P-N_{Pn-1}-\beta N_{Mn-1}\right)}{K_P}$$

where $N_{Mn}$, $N_{Mn-1}$, $N_{Pn,}$ and $N_{Pn-1}$ are the cell densities of Microcystis and Pseudanabaena, respectively, when co-cultured at day $t_n$ or $t_{n-1}$; $r_M$ and $r_P$ represent the intrinsic growth rates of Microcystis and Pseudanabaena in mono-cultures, respectively; $K_M$ and $K_P$ are the carrying capacities of Microcystis and Pseudanabaena in the mono-culture, respectively, which was estimated via logistic function; $\alpha$ and $\beta$ are the competitive coefficients in the co-cultures, where $\alpha$ indicates the inhibition of Pseudanabaena on Microcystis and $\beta$ indicates the inhibition of Microcystis on Pseudanabaena.

2.6. Statistical Analysis

In this study, all data were presented as mean ± SD (standard deviation). We used Data SPSS 20 software to analyze data differences via one-way ANOVA. Furthermore, regression analyses were conducted using Origin 8.0 software developed by Originlab Corporation in America.

3. Results

3.1. Identification of Pseudanabaena

According to the description given, Pseudanabaena TH-1 appeared as pale brown, and Pseudanabaena DC-1 appeared as pale blue-gray. Both had 3–6 cells in every short trichome, and the cells showed clear constriction at cross-walls. Phylogenetic analysis was conducted using NJ (neighbor-joining) trees based on rbcLX, which suggested that Pseudanabaena TH-1 and DC-1 were grouped with Pseudanabaena mucicola (Figure 1). FACHB1277, FACHB1998, and FACHB-2209 were sequenced simultaneously and used as references in the study. The relevant information is presented in Figure 1.

3.2. The Growth of Pseudanabaena in Mono-Culture Experiments at Different Temperature

The graph in Figure 2 displays the changes in cell density for Pseudanabaena TH-1 and DC-1 at different temperatures. The experimental results indicated that both strains could grow at set temperatures ranging from 20 to 35 °C. The cell density of both strains decreased at 10 °C within 20 days. The maximum cell density of Pseudanabaena TH-1 and DC-1 was observed at 25 °C on day 28, with cell densities of 4.27 × 10⁸ ± 4.99 × 10⁷ cells mL⁻¹ and 4.05 × 10⁸ ± 2.94 × 10⁷ cells mL⁻¹, respectively. On the other hand, TH-1 and DC-1 rapidly reached a lag phase on day 20 and day 16, respectively, at 35 °C. The cell density of both strains reached a maximum concentration on day 24 and day 28 in the 30 °C group, while reaching saturation on day 33 and day 40 in the 20 °C group.

The logarithmic phase in the 35 °C experimental group was shorter than that in the 20 °C, 25 °C, and 30 °C groups, and the cell density was significantly lower compared to the other groups. The maximum cell density of TH-1 and DC-1 at 35 °C was 2.07 × 10⁸ ± 3.29 × 10⁷ and 9.13 × 10⁷ ± 1.84 × 10⁷ cells. mL⁻¹, respectively. The cell density of both strains was higher at 20 °C than in the 30 °C group, but there was no significant discrepancy between the two groups for Psudanabaena TH-1.

The logistic model was used to fit the growth curve of Pseudanabaena TH-1 and DC-1, and the specific growth rates (μ) were calculated using Equation (2) for both strains from the 20 and 35 groups. The minimum specific growth rate values of TH-1 and DC-1 were 0.024 ± 0.006 and 0.016 ± 0.003 in the 10 °C group, respectively. The maximum specific growth rate values of TH-1 and DC-1 were 0.512 in the 30 °C group and 0.440 in the 35 °C groups, respectively.

The μmax values for Pseudanabaena at each temperature were plugged into Equations (4) and (5) for linear regression analysis to obtain the coefficients. The correlation curve of growth rate and temperature is plotted in Figure 3, and the fitting formula is expressed as follows:

$$\sqrt{\mathsf{\mu }}=0.0291\left(T-0.6\right)\left\{1-\mathrm{e}\mathrm{x}\mathrm{p}\left[2.52\left(T-35\right)\right]\right\}$$

(4)

$$\sqrt{\mathsf{\mu }}=0.0178\left(T+4.6\right)\left\{1-\mathrm{e}\mathrm{x}\mathrm{p}\left[0.44\left(T-41\right)\right]\right\}$$

(5)

Based on the correlation curve, the highest temperature-dependent growth rate for TH-1 and DC-1 was estimated to be 0.818 day⁻¹ and 0.656 day⁻¹, with a corresponding temperature of 34 °C. The coefficient of determination (R²) of TH-1 and DC-1 was 0.99 and 0.55, respectively, indicating that the correlation curve explains 99% and 55% of the variability in growth rate for TH-1 and DC-1.

3.3. The Growth of Microcystis in Mono-Culture Experiments at Different Temperatures

The data in Figure 4 show the changes in the cell density of Microcystis PCC7806 and the mcyB⁻ mutant at different temperatures. It was observed that both strains could not grow at 10 °C but grew well at temperatures ranging from 20 to 35 °C. The maximum cell density for these strains occurred at 35 °C with 1.54 × 10⁸ ± 8.13 × 10⁶ cells mL⁻¹ for Microcystis PCC7806 and 1.59 × 10⁸ ± 1.27 × 10⁷ cells mL⁻¹ for the mcyB⁻ mutant.

Both strains had similar growth curves from 20 to 35 °C, and there was no significant difference in the cell density during the lag phase at 25, 30, and 35 °C. However, during the logarithmic phase, the cell density of Microcystis PCC7806 was significantly lower at 20 °C than at 30 °C and 35 °C (p < 0.05).

The logistic model was used to fit the growth curve of both strains, and the specific growth rates (μ) were calculated using Equation (2) in the 20, 25, 30, and 35 °C groups. The minimum specific growth rate values of both strains were recorded at 10 °C, with 0.022 ± 0.003 and 0.020 ± 0.001 day⁻¹ for Microcystis PCC7806 and the mcyB⁻ mutant, respectively. On the other hand, the maximum specific growth rate values of Microcystis PCC7806 and the mcyB⁻ mutant were reported in the 30 °C group, with values of 0.280 day⁻¹ and 0.306 day⁻¹, respectively.

The μmax values at different temperatures for Microcystis were used to obtain coefficients by fitting Equations (6) and (7) using linear regression analysis. The correlation curve of growth rate and temperature was plotted in Figure 5, with the corresponding fitting formulas expressed as

$$\sqrt{\mathsf{\mu }}=0.0257\left(T-5.4\right)\left\{1-\mathrm{e}\mathrm{x}\mathrm{p}\left[0.22\left(T-39\right)\right]\right\}$$

(6)

$$\sqrt{\mathsf{\mu }}=0.0217\left(T-4.3\right)\left\{1-\mathrm{e}\mathrm{x}\mathrm{p}\left[0.80\left(T-36\right)\right]\right\}$$

(7)

Based on the correlation curve, the highest temperature-dependent growth rates for Microcystis PCC7806 and the mcyB⁻ mutant were estimated to be 0.287 day⁻¹ and 0.338 day⁻¹, respectively, with a corresponding temperature of 30 °C and 32 °C. The coefficient of determination (R²) for Microcystis PCC7806 and mcyB⁻ mutant was 0.97 and 0.99, respectively, indicating that the correlation curve explains 97% and 99% of the variability in growth rate for both strains.

3.4. Dominant Characteristics of Pseudanabaena and Microcystis in Co-Culture at Different Temperatures

In the co-culture, the growth of Microcystis was not affected by Pseudanabena in the 20 °C and 25 °C groups, but it was significantly affected by Pseudanabaena DC-1 in the 30 and 35 °C groups (Figure 6). Conversely, the growth of Pseudanabaena TH-1 was significantly inhibited by Microcystis (Figure 7). In the co-culture with Microcystis PCC7806, the cell density of Pseudanabaena TH-1 was greater than that in the co-culture with the mcyB⁻ mutant. Although Pseudanabaena DC-1 was mostly inhibited by Microcystis (toxic or non-toxic), there was no difference between the mono- and co-culture at 35 °C.

Using the Lotka–Volterra model parameters, the competition-inhibition parameters $\alpha$ (Pseudanabaena against Microcystis) and $\beta$ (Microcystis against Pseudnabaena) were calculated based on the data in

Table 1. Competitive parameters of Microcystis and Pseudanabaena according to the cell density in co-cultures under different temperature conditions.

Temperature (°C)	Competitive Parameters
	$\alpha$1	$\alpha$2	$\alpha$3	$\alpha$4	$\beta$1	$\beta$2	$\beta$3	$\beta$4
20	−0.31	−1.20	0.14	0.05	2.54	3.37	3.81	3.77
25	0.15	1.77	0.21	0.46	5.53	5.66	4.75	6.66
30	0.09	−1.16	0.27	0.18	2.93	2.83	2.84	2.79
35	0.02	0.41	0.70	1.18	1.35	0.96	0.69	2.34

Notes: $\alpha$₁ and $\alpha$₂ represent Pseudanabaena TH-1 on PCC7806 and mcyB⁻ mutant; $\alpha$₃ and $\alpha$₄ represent Pseudanabaena DC-1 on PCC7806 and mcyB⁻ mutant; $\beta$₁ and $\beta$₂ represent PCC7806 and mcyB⁻ mutant on Pseudanabaena TH-1; $\beta$₃ and $\beta$₄ represent on PCC7806 and mcyB⁻ mutant on Pseudanabaena DC-1.

. The results indicated that both species exhibited inhibitory competition as the values of $\alpha$ and $\beta$ values were positive. The competition-inhibition parameters of Pseudanabena DC-1 against Microcystis increased with increasing temperature. Moreover, the higher value of $\beta$ compared with $\alpha$ indicated that Microcystis exhibited greater competitive inhibition against Pseudanabaena than Pseudanabaena did against Microcystis in most conditions, indicating that Microcystis dominated in most conditions.

4. Discussion

The curves reveal that the span of the conceptual growth temperature range for Pseudanabaena, determined by Tmin and Tmax (TH-1: 0.6–35 °C; DC-1: −4.7–41 °C), exceeds that of Microcystis (PCC7806: 5.4–39 °C; mcyB⁻ mutant: 4.3–36 °C), which implies the wide temperature of Pseudanabaena. Indeed, Pseudanabaena manifests wide distribution, from tropic zones to frigid zones [46,47,48,49]. This investigation further illuminates that Pseudanabaena expands in summer and autumn when water temperatures are generally around 20~30 °C, conducive to its prosperity in lakes or reservoirs. Notably, under laboratory conditions, the maximum values of specific growth rate for TH-1 and DC-1 occur at 30 °C and 35°, respectively. One study showed that Pseudanabaena grew fastest in 25 °C, second fastest in 30 °C, and slowest in 10 °C [50]. Under 40 °C, the growth of Pseudanabaena was inhibited [40]. Our study showed that the growth rate of Pseudanabaena was higher in high-temperature water (30 and 35 °C), but the carrying capacity of cells was lower than that in low-temperature groups (20 and 25 °C). Most remarkably, the peak of cells coincided with the optimal laboratory temperature setting of 25 °C. An elevated temperature shortened the duration of the exponential growth phase and correspondingly lowered the biomass, as observed in our findings. Correspondingly, the mono-culture of P. geleata produced a growing rate directly proportional to the temperature increase [51]. Taken together, Pseudanabaena displays an extraordinary range of temperature tolerance, with its biomass peaking during summer and autumn, albeit displaying diminished proportional growth [52]. In our study, Pseudanabaena kept a higher growth rate in high-temperature water and a shorter period of growth, which was in accordance with observations in the wild.

Laboratory experiments have extensively investigated the growth responses of Microcystis strains to a wide range of temperature conditions. The optimum temperature for the growth of Microcystis was found to be over 20 °C [53,54], indicating that the optimal temperature for the growth of M. aeruginosa cells should be close to 30 °C [55]. A high temperature at 30 °C promoted the growth of five tropical Microcystis species, but an extremely high temperature at 36 °C inhibited the growth of M. ichthyoblabe, M. flosa-quae, and M. viridis [26]. An analysis of 62 cyanobacteria species revealed that the optimum mean temperature for their growth is 27.2 °C [56]. Similarly, high temperatures at 30 and 35 °C significantly inhibited the growth of M. aeruginosa cells [57]. Microcystis begins recruitment at 11–14 °C, and the optimum growth temperature is between 20 and 30 °C [58]. Temperature is also shown to influence the colony density of Microcysits, which regulates its buoyancy, and Microcystis colonies have higher buoyant velocities in the optimal growth temperature of 28 °C than in the low temperature of 17.5 °C [55]. Moreover, some studies have indicated that with increasing temperature, the relative abundance of less toxic or non-toxic Microcystis increases compared to toxic Microcystis [59]. In our study, the Ratkowsky equation showed that the T_min of PCC7806 and the mcyB⁻ mutant was about 5.4 °C and 4.3 °C when the square root of the growth rate reached zero. Additionally, the corresponding temperature of the highest growth rate for Microcystis PCC7806 and the mcyB⁻ mutant was found to be 30 °C and 32 °C, respectively. There was a similar growth trend observed for Microcystis PCC7806 and the mcyB⁻ mutant.

In Microcysits blooms, diverse bacterial communities such as Actinobacteria and Proteobacteria dominate [19,60]. In addition, filamentous cyanobacteria like Dolichospermum, Aphanizomenon and Planktothrix could co-occur or successively dominate with Microcystis in eutrophic freshwater [61,62,63]. Temperature has a significant impact on the growth of Pseudanabaena sp. and Microcystis sp., as well as their competitive interactions with species-specific effects. For example, under a rising water temperature, Microcystis proves to be a stronger competitor than the filamentous species Dolichospermum and Aphanizomeno [36,37]. The abundance proportion of Microcystis also increases compared to Pseudanabaena in high temperatures [54]. In the present study, the co-culture of Microcystis and Pseudanabaena TH-1 resulted in the promotion of Microcystis growth at 20 °C, while its growth was seriously inhibited when co-cultured with Pseudanabaena DC-1 at 35 °C. Conversely, both strains of Pseudanabaena were inhibited when co-cultured with toxic or non-toxic Microcystis strains across all groups (Figure 7 and Table 1). However, toxic Microcystis PCC7806 induced a higher cell density of Pseudanabaena most of the time compared to the non-toxic Microcystis mcyB⁻ mutant (Figure 7). Microcystis was proven to inhibit Pseudanabaena more strongly than vice versa at most given temperatures, especially at 25 °C (Table 1). Additionally, Pseudanabaena DC-1 was not only inhibited by Microcystis but was also affected by high temperatures. Furthermore, Pseudanabaena DC-1 influenced Microcystis at high temperatures (Figure 6). Other authors have also observed that the growth of M.a is enhanced when co-cultured with Pseudanabaena, and conversely, Pseudanabaena was inhibited in a co-culture with a certain proportion of M.a [64]. These findings indicate that the growth responses of Microcystis with a co-culture of Pseudanabaena were not only species-dependent but also temperature-dependent. Other the other hand, Pseudanabaena was inhibited by Microcystis regardless of toxicity.

In the present study, the Lotka–Volterra model was utilized to predict community succession, considering environmental capacity and inter-specific and intra-specific inhibition. The environmental carrying capacity of Microcystis is K_M, while that of Pseudanababaena is K_P; Microcystis inhibits the growth of its own population by 1/K_M, and Pseudanabaena inhibits the growth of its own population by 1/K_P. The influence of Pseudanabaena on the Microcystis population is indicated as $\alpha$/K_M, and influence of Microcystis on the Pseudanabaena population is indicated as $\beta$/K_P. In the Lotka–Volterra model, there were four kinds of competitive results: when the effect of Microcystis on itself is less than that of Microcystis on Pseudanabaena (1/K_M < $\beta$/K_P) and the effect of Pseudanabaena on itself is greater than that of Microcystis (1/K_P > $\alpha$/K_M), Microcystis dominates. Otherwise, when 1/K_M > $\beta$/K_P and 1/K_P < $\alpha$/K_M, Pseudanabaena dominates. When 1/K_M > $\beta$/K_P and 1/K_P > $\alpha$/K_M, the two cyanobacteria genera can co-exist steadily. Furthermore, when 1/K_M < $\beta$/K_P and 1/K_P < $\alpha$/K_M, the two cyanobacteria co-exist unstably. In this study, the toxic strain PCC7806 predominated most of the time, although Pseudanabaena could either co-exist stably or unstably with non-toxic Microcystis in most cases (Table S1). As we know, cyanobacterial blooms in the field are often composed of toxic and non-toxic Microcystis in the field, which have a combined influence on other genera, such as Pseudanabaena.

5. Conclusions

In conclusion, the present study showed that the carrying capacity of Pseudanabaena was significantly inhibited in high-temperature (>30 °C) groups. Pseudanabaena growth was not only affected by a co-culture with Microcystis, but also by temperature. In a word, Microcystis out-competed Pseudanabaena or co-existed in the experimental groups.