# Freshwater Salinization Impacts the Interspecific Competition between Microcystis and Scenedesmus

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Microalgae and Salinity Adaptation

^{−2}s

^{−1}. Then algae were inoculated in fresh culture media every 3 days to maintain an exponential growth phase prior to the experiment. Two algae were cultured in 1 L Erlenmeyer flasks containing 500 mL of BG-11 medium. The salinity of the algal culture was raised every two days at an interval of one until it reached six. The measured salinities of the three target salinities (0, 3, 6) were 0.99, 3.97 and 6.98, respectively. No difference was found between different mono- or cocultures.

#### 2.2. Experimental Protocol

^{−1}NaNO

_{3}and 0.039 mg L

^{−1}KH

_{2}PO

_{4}. Three salinities were set to simulate freshwater salinization: zero, three, and six, according to the predictive results of salinity levels and trends across and within the seven regional river basins [30]. Algal were cultured semi-continuously in different salt conditions (0, 3, 6) to remain in exponential growth prior to the experiments, and then exponential-phase cells were inoculated into Erlenmeyer flasks at different initial algal densities.

^{5}cells mL

^{−1}and S. obliquus was 1.0 × 10

^{5}cells mL

^{−1}. Three cocultures with a uniform initial total biovolume were set with the following biovolume ratios, and initial cell densities were set as below: (1) 75% Ma + 25% So: M. aeruginosa was 3.75 × 10

^{5}cells mL

^{−1}and S. obliquus was 0.25 × 10

^{5}cells mL

^{−1}; (2) 50% Ma + 50% So: M. aeruginosa was 2.5 × 10

^{5}cells mL

^{−1}and S. obliquus was 0.5 × 10

^{5}cells mL

^{−1}; (3) 25% Ma + 75% So: M. aeruginosa was 1 × 10

^{5}cells mL

^{−1}and S. obliquus was 0.75 × 10

^{5}cells mL

^{−1}. Both monoculture and coculture groups were incubated in climate-controlled chambers in 250 mL Erlenmeyer flasks at three different salinities (0, 3, and 6). The experiments were performed in triplicate. All the cultures were grown axenically in a climate-controlled chamber at a constant temperature of 20 °C, illuminated with a 14:10 h light:dark cycle at 60 μmol photons m

^{−2}s

^{−1}and shaken manually three times daily.

#### 2.3. Data Analyses

^{−1}) was assumed to be exponential and determined as the slope of ln cell density vs time. One-way ANOVA was used to compare the differences in growth rates at different salinities of the two algal monocultures. In the cocultures, the algal cell density versus time was fitted by the Gaussian distribution ${N}_{t}={N}_{max}\times {e}^{(-0.5\times {(\frac{t-{t}_{max}}{SD})}^{2})}$, where N

_{0}, N

_{t}and N

_{max}represent the algal abundance at time zero and t, and the maximal number of algal abundances, t and t

_{max}represent the cultural time and the time when reached N

_{max}, and SD was the width of the Gaussian distribution. In cocultures, the natural log of the ratio of the abundances of the two algae, $Y\left(t\right)=ln(\frac{{N}_{Microcystis}}{{N}_{Scnedesmus}})$, were calculated and regressed against time. The slope of the linear regression of Y(t) versus t is adopted as the competitive displacement rate. To reflect the changes of Y(t) with time, they were fitted by the Gaussian distribution: $Y\left(t\right)={Y}_{max}\times {e}^{(-0.5\times {(\frac{t-{{t}^{\prime}}_{max}}{{SD}^{\prime}})}^{2})}+{Y}_{0}$, where Y

_{0}, Y

_{max}represent the initial Y(t), Y(t) at time t, and the maximal value of Y(t), t and t′

_{max}represent the cultural time and the time when reached Y

_{max}, and SD′ was the width of the Gaussian distribution.

_{i}is the number of particles in different morphs.

#### 2.4. Chlorophyll Fluorescence Measurements

_{v}/F

_{m}$=\frac{{F}_{m}-{F}_{0}}{{F}_{m}}$, where F

_{m}and F

_{0}are the maximal and minimal chlorophyll fluorescence yields, respectively, of dark-adapted (15 min) algal suspensions. Three-way ANOVA tests were run to analyze the effects of salinity, initial algal composition, and incubation time on F

_{v}/F

_{m}of both algae, and Holm-Sidak tests were adopted for all multiple pairwise comparisons.

## 3. Results

#### 3.1. Response of Algae Growth and Competition to Salinity Stress

_{(2,6)}= 71.37, p < 0.0001) and S. obliquus (F

_{(2,6)}= 7.715, p = 0.0219). The algal population growth rate of M. aeruginosa significantly decreased with increasing salinity (Figure 1a), and the growth rate of S. obliquus was significantly lower under salinity stress but had no significant difference between the two salinity groups (3, 6) (Figure 1b).

_{max}) was slightly advanced under higher salinity conditions (Table 1). The results of two-way ANOVA tests showed a significant effect of salinity on N

_{max}and t

_{max}of S. obliquus, and t

_{max}of M. aeruginosa. The initial composition of two algal species had no effect on neither N

_{max}nor t

_{max}of the two algae. Furthermore, there was no significant interaction between salinity and initial composition (Table 2). In the treatments with an initial cell density ratio of 75% Ma + 25% So, M. aeruginosa occupied dominance in the early several days. The competitive advantage of M. aeruginosa was maintained for 6.546, 5.208, and 3.989 days, respectively, in zero, three, and six (Figure 2a,d,g). When the initial density proportion of M. aeruginosa decreased to 50%, the competitive advantage could only maintain for 2.950, 3.547 and 3.289 days, respectively, in zero, three, and six (Figure 2b,e,h). However, in treatments with an initial ratio of 25% Ma + 75% So, S. obliquus occupied dominance from second day (Figure 2c,f,i).

#### 3.2. Morphological Variations of S. obliquus

#### 3.3. Photosynthetic Performance of Both Algae in Cocultures

_{v}/F

_{m}) was significantly affected by the increase in salinity, the presence of competitors, and the incubation time for both M. aeruginosa and S. obliquus (Figure 5, Table 3). Additionally, there were statistically significant interactions on F

_{v}/F

_{m}of both algae between time, salinity, and initial proportion of algae. For M. aeruginosa, F

_{v}/F

_{m}differed significantly between the treatments of zero and three (p = 0.016), also zero and six (p = 0.006), but not significantly between the two high salinity treatments (p = 0.635). F

_{v}/F

_{m}increased with time when cultured at zero (Figure 5a), when at three and six, F

_{v}/F

_{m}increased more rapidly than at zero (Figure 5b,c). Interestingly, the treatment of 75% Ma + 25% So had the lowest F

_{v}/F

_{m}value in zero and six (Figure 5a,c). For S. obliquus (Figure 5d–f), similar to M. aeruginosa, F

_{v}/F

_{m}differed significantly between the treatments of zero and three (p < 0.001), also zero and six (p < 0.001), but not significantly between the two high salinity treatments (p = 0.230). The initial algal proportion affected F

_{v}/F

_{m}differently with M. aeruginosa: monoculture (100% So) differed significantly with all three cocultures, whereas F

_{v}/F

_{m}of the cocultures had no significant difference. Especially in the treatment of six, F

_{v}/F

_{m}was higher in monoculture than in cocultures (Figure 5f).

## 4. Discussion

^{+}extrusion, accumulation and synthesis of certain solutes, and metabolic modifications like synthesizing compatible solutes, such as sugars, amino acids, and fats, which act as osmoprotectants. The differences in interspecies sensitivity to salinity were likely due to the inherent morphological and physiological aspects of each species. S. obliquus can form colonies through the attachment of daughter cells during cell division and can produce a mucilage envelope, which serves as a protective mechanism against environmental stressors, such as salinity and heavy metals toxicity [32,33]. Zhu et al. [34] reported that the presence of M. aeruginosa affected the formation of morphological defence against grazers in S. obliquus. This suggested that competition can influence the ability of organisms to defend themselves against stressors. Nevertheless, the formation of cell colonies can relieve the stress caused by elevated salinity levels, although this mechanism of defence may have an impact on algal growth, such as reducing the surface-to-volume ratio and intensifying nutrient competition among cells. Small-sized unicells have a relatively high surface-to-volume ratio, which facilitates nutrient uptake and utilization of light [35]. M. aeruginosa has been found to have weak osmotic regulating abilities because of its limited ability to synthesize compatible solutes. Even decreases in microcystin production in M. aeruginosa under high osmotic conditions have been reported in former studies [36,37], but contrary change has been found in Planktothrix agardhii [38]. Further, the relatively thin cell wall of M. aeruginosa makes it susceptible to osmotic stress. In natural waters, M. aeruginosa can often form large colonies with a mucilage sheath, which could act as a hydrated matrix and maintain a more stable osmotic environment within the colonies [39]. In our study, M. aeruginosa maintained unicellular or bicellular morphs; no mucilage sheath was synthesized during the experiment, which might cause less tolerance to high salinity.

_{v}/F

_{m}, especially in the treatment of six. The influence of competition on photosynthetic efficiency has also been documented, Sun et al. [47] found that ultraviolet-B (UV-B) radiation stress changed the competitive outcome of algae and that this change was related to changes in photosynthetic efficiency. Light limitation from the shading effect of the coexisting algae might be responsible for the reduction of F

_{v}/F

_{m}[48]. Additionally, allelopathic substances secreted by competitors could also affect photosynthesis. Hernández-Zamora et al. [49] reported the diminishment of chlorophyll-a, b of M. aeruginosa and two green algae in combined cultures. The allelopathic activity of cyanobacteria on other microalgae can vary, Zak et al. [50] found that while Anabaena variabilis had a strong inhibitory effect on C. vulgaris, Nodularia spumigena mostly stimulated its growth. Further, Different species of Scenedesmus have been found to restrict the growth of cyanobacteria through various means of exposure, including the use of conditioned water and crude extracts [51,52].

## 5. Conclusions

## Author Contributions

## Funding

## Data Availability Statement

## Conflicts of Interest

## References

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**Figure 1.**Growth rates of M. aeruginosa (

**a**) and S. obliquus (

**b**) in monocultures at three salinities (0, 3, 6). The values were presented as mean ± standard error. Lowercase letters in italic style represented significant differences caused by different salinities).

**Figure 2.**The increase of cell density of M. aeruginosa (blue square) and S. obliquus (green circle) over the 17-day experiment in three different salinities (0, 3, 6).

**Figure 3.**Displacement rates for M. aeruginosa against S. obliquus with different initial algal compositions at different salinities ((

**a**,

**d**,

**g**): 75% Ma/25% So at 0, 3, 6; (

**b**,

**e**,

**h**): 50% Ma/50% So at 0, 3, 6; (

**c**,

**f**,

**i**): 25% Ma/75% So at 0, 3, 6).

**Figure 4.**Proportions of unicells and 2-, 4- and 8-celled colonies in S. obliquus populations of monocultures and cocultures with different initial algal compositions. The “rest” group represents 3-, 5-, 6-, and 7-celled colonies.

**Figure 5.**The maximal efficiency of PSII photochemistry (F

_{v}/F

_{m}) of M. aeruginosa and S. obliquus in monocultures and cocultures with different initial algal compositions at three salinities (0: (

**a**,

**d**), 3: (

**b**,

**e**), 6: (

**c**,

**f**)) on different days.

**Table 1.**Gaussian distribution formulae of the algal cell density versus time of both M. aeruginosa and S. obliquus in cocultures, and the formulae of competitive displacement rate against time of S. obliquus by M. aeruginosa.

Salinity | Algae | 25% So + 75% Ma | 50% So + 50% Ma | 75% So + 25% Ma |
---|---|---|---|---|

0 | Ma | ${N}_{t}=2.250\times {e}^{\left(-0.5\times {\left(\frac{t-12.59}{6.985}\right)}^{2}\right)}-0.248$ R ^{2} = 0.6799 | ${N}_{t}=1.351\times {e}^{\left(-0.5\times {\left(\frac{t-11.98}{6.004}\right)}^{2}\right)}-0.049$ R ^{2} = 0.6004 | ${N}_{t}=0.742\times {e}^{\left(-0.5\times {\left(\frac{t-14.14}{7.824}\right)}^{2}\right)}-0.113$ R ^{2} = 0.5069 |

So | ${N}_{t}=6.040\times {e}^{\left(-0.5\times {\left(\frac{t-14.27}{4.584}\right)}^{2}\right)}-0.143$ R ^{2} = 0.9180 | ${N}_{t}=6.413\times {e}^{\left(-0.5\times {\left(\frac{t-15.57}{6.286}\right)}^{2}\right)}-0.484$ R ^{2} = 0.9543 | ${N}_{t}=7.084\times {e}^{\left(-0.5\times {\left(\frac{t-15.28}{6.418}\right)}^{2}\right)}-0.640$ R ^{2} = 0.9140 | |

Y(t) | $Y\left(t\right)=10.46\times {e}^{\left(-0.5\times {\left(\frac{t+12.86}{9.568}\right)}^{2}\right)}-1.318$ R ^{2} = 0.9398 | $Y\left(t\right)=381.8\times {e}^{\left(-0.5\times {\left(\frac{t+54.23}{17.78}\right)}^{2}\right)}-1.793$ R ^{2} = 0.8693 | $Y\left(t\right)=17.73\times {e}^{\left(-0.5\times {\left(\frac{t+18.73}{9.850}\right)}^{2}\right)}-2.472$ R ^{2} = 0.8013 | |

3 | Ma | ${N}_{t}=1.380\times {e}^{\left(-0.5\times {\left(\frac{t-8.251}{5.333}\right)}^{2}\right)}-0.049$ R ^{2} = 0.6753 | ${N}_{t}=1.987\times {e}^{\left(-0.5\times {\left(\frac{t-7.999}{9.493}\right)}^{2}\right)}-1.132$ R ^{2} = 0.5851 | ${N}_{t}=0.399\times {e}^{\left(-0.5\times {\left(\frac{t-3.851}{0.148}\right)}^{2}\right)}+0.311$ R ^{2} = 0.1538 |

So | ${N}_{t}=6.213\times {e}^{\left(-0.5\times {\left(\frac{t-13.98}{5.072}\right)}^{2}\right)}-0.268$ R ^{2} = 0.9041 | ${N}_{t}=5.941\times {e}^{\left(-0.5\times {\left(\frac{t-13.62}{5.241}\right)}^{2}\right)}-0.274$ R ^{2} = 0.9003 | ${N}_{t}=8.253\times {e}^{\left(-0.5\times {\left(\frac{t-16.14}{9.598}\right)}^{2}\right)}-2.397$ R ^{2} = 0.9016 | |

Y(t) | $Y\left(t\right)=5.808\times {e}^{\left(-0.5\times {\left(\frac{t+3.185}{7.274}\right)}^{2}\right)}-2.547$ R ^{2} = 0.8627 | $Y\left(t\right)=4.695\times {e}^{\left(-0.5\times {\left(\frac{t+0.911}{5.128}\right)}^{2}\right)}-2.523$ R ^{2} = 0.8746 | $Y\left(t\right)=3.562\times {e}^{\left(-0.5\times {\left(\frac{t-1.157}{2.961}\right)}^{2}\right)}-2.832$ R ^{2} = 0.8574 | |

6 | Ma | ${N}_{t}=1.143\times {e}^{\left(-0.5\times {\left(\frac{t-9.356}{6.643}\right)}^{2}\right)}-0.061$ R ^{2} = 0.6174 | ${N}_{t}=0.812\times {e}^{\left(-0.5\times {\left(\frac{t-10.36}{7.320}\right)}^{2}\right)}-0.032$ R ^{2} = 0.5690 | ${N}_{t}=0.425\times {e}^{\left(-0.5\times {\left(\frac{t-10.48}{5.329}\right)}^{2}\right)}+0.049$ R ^{2} = 0.4568 |

So | ${N}_{t}=4.374\times {e}^{\left(-0.5\times {\left(\frac{t-11.82}{5.121}\right)}^{2}\right)}-0.482$ R ^{2} = 0.8361 | ${N}_{t}=4.351\times {e}^{\left(-0.5\times {\left(\frac{t-11.84}{4.567}\right)}^{2}\right)}-0.246$ R ^{2} = 0.9269 | ${N}_{t}=5.095\times {e}^{\left(-0.5\times {\left(\frac{t-12.42}{5.045}\right)}^{2}\right)}-0.320$ R ^{2} = 0.7749 | |

Y(t) | $Y\left(t\right)=6.225\times {e}^{\left(-0.5\times {\left(\frac{t+5.596}{5.858}\right)}^{2}\right)}-1.369$ R ^{2} = 0.9349 | $Y\left(t\right)=3.296\times {e}^{\left(-0.5\times {\left(\frac{t-0.417}{3.326}\right)}^{2}\right)}-1.656$ R ^{2} = 0.8829 | $Y\left(t\right)=3.199\times {e}^{\left(-0.5\times {\left(\frac{t-1.432}{1.915}\right)}^{2}\right)}-2.365$ R ^{2} = 0.7747 |

**Table 2.**Summary of two-way ANOVAs for the effects of salinity and initial algal composition on the maximal values of algal abundance (N

_{max}) and the time to N

_{max}(t

_{max}) of M. aeruginosa and S. obliquus.

Algae | Factors | N_{max} | t_{max} | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|

SS | DF | MS | F | p Value | SS | DF | MS | F | p Value | ||

Microcystis | Salinity | 2.014 | 2 | 1.007 | 0.1493 | 0.8624 | 173.7 | 2 | 86.85 | 19.14 | <0.0001 |

Composition | 5.766 | 2 | 2.883 | 0.4273 | 0.6587 | 2.169 | 2 | 1.085 | 0.2391 | 0.7898 | |

Interaction | 2.319 | 4 | 0.5799 | 0.08594 | 0.9857 | 44.16 | 4 | 11.04 | 2.434 | 0.085 | |

Scenedesmus | Salinity | 25.62 | 2 | 12.81 | 5.596 | 0.0129 | 47.52 | 2 | 23.76 | 10.43 | 0.001 |

Composition | 9.455 | 2 | 4.727 | 2.065 | 0.1558 | 7.698 | 2 | 3.849 | 1.689 | 0.2126 | |

Interaction | 2.877 | 4 | 0.7193 | 0.3142 | 0.8647 | 6.962 | 4 | 1.74 | 0.7640 | 0.5623 |

**Table 3.**Summary of three-way ANOVAs for the effects of salinity, proportion, and culture time on the maximal efficiency of PSII photochemistry (F

_{v}/F

_{m}) of M. aeruginosa and S. obliquus.

Algae | Source of Variation | SS | DF | MS | F | p Value |
---|---|---|---|---|---|---|

Microcystis | Time | 0.437 | 7 | 0.0625 | 47.108 | <0.001 |

Salinity | 0.0153 | 2 | 0.00765 | 5.773 | 0.004 | |

Proportion of Ma | 0.0531 | 3 | 0.0177 | 13.341 | <0.001 | |

Time × Salinity | 0.185 | 14 | 0.0132 | 9.973 | <0.001 | |

Time × Proportion of Ma | 0.0773 | 21 | 0.00368 | 2.778 | <0.001 | |

Salinity × Proportion of Ma | 0.0242 | 6 | 0.00403 | 3.043 | 0.007 | |

Time × Salinity × Proportion of Ma | 0.0847 | 42 | 0.00202 | 1.522 | 0.031 | |

Scenedesmus | Time | 0.152 | 7 | 0.0217 | 62.987 | <0.001 |

Salinity | 0.00898 | 2 | 0.00449 | 13.009 | <0.001 | |

Proportion of So | 0.00543 | 3 | 0.00181 | 5.243 | 0.002 | |

Time × Salinity | 0.0285 | 14 | 0.00203 | 5.889 | <0.001 | |

Time × Proportion of So | 0.0233 | 21 | 0.00111 | 3.216 | <0.001 | |

Salinity × Proportion of So | 0.0115 | 6 | 0.00191 | 5.539 | <0.001 | |

Time × Salinity × Proportion of So | 0.0421 | 42 | 0.001 | 2.906 | <0.001 |

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## Share and Cite

**MDPI and ACS Style**

Gao, T.; Li, Y.; Xue, W.; Pan, Y.; Zhu, X. Freshwater Salinization Impacts the Interspecific Competition between *Microcystis* and *Scenedesmus*. *Water* **2023**, *15*, 1331.
https://doi.org/10.3390/w15071331

**AMA Style**

Gao T, Li Y, Xue W, Pan Y, Zhu X. Freshwater Salinization Impacts the Interspecific Competition between *Microcystis* and *Scenedesmus*. *Water*. 2023; 15(7):1331.
https://doi.org/10.3390/w15071331

**Chicago/Turabian Style**

Gao, Tianheng, Yinkang Li, Wenlei Xue, Yueqiang Pan, and Xuexia Zhu. 2023. "Freshwater Salinization Impacts the Interspecific Competition between *Microcystis* and *Scenedesmus*" *Water* 15, no. 7: 1331.
https://doi.org/10.3390/w15071331