3.5.2. Diurnal Changes of Algal *Fv/F<sup>m</sup>*

Compared with normal growth conditions, diurnal changes of algal *Fv/F<sup>m</sup>* were similar under nutrient enrichment conditions (Figure 8). However, the decline degrees of algal *Fv/F<sup>m</sup>* after UV-B radiation were lower (17.5–50.8%), and the recovery efficiency of *Fv/F<sup>m</sup>* was better with nutrient enrichment. For example, *Fv/F<sup>m</sup>* of two *M. aeruginosa* species could both totally recover to the initial values after UV-B radiation on Day 2, and *Fv/F<sup>m</sup>* of *C. pyrenoidosa* on Day 6 and Day 8 totally recovered to the initial values within 16 h and 8 h after UV-B radiation, respectively. For three species in the UV-B treatment, the decline degree of *Fv/F<sup>m</sup>* was also lower for toxic *M. aeruginosa* and it exhibited a faster recovery rate. This result was consistent with that under normal growth conditions.

**Figure 7.** Cell density (line and scatter) and *Fv/Fm* (vertical bar) of three species in the PAR and UV‐ B treatments under nutrient enrichment conditions (the arrow indicates the initial value of *Fv/Fm*). **Figure 7.** Cell density (line and scatter) and *Fv/Fm* (vertical bar) of three species in the PAR and UV-B treatments under nutrient enrichment conditions (the arrow indicates the initial value of *Fv/Fm*).

#### 3.5.2. Diurnal Changes of Algal *Fv/Fm* Compared with normal growth conditions, diurnal changes of algal *Fv/Fm* were sim‐ *3.6. Antioxidant Responses of Algal Species under Nutrient Enrichment Conditions* 3.6.1. ROS in Algal Cells and SOD Activity

ilar under nutrient enrichment conditions (Figure 8). However, the decline degrees of al‐ gal *Fv/Fm* after UV‐B radiation were lower(17.5–50.8%), and the recovery efficiency of *Fv/Fm* was better with nutrient enrichment. For example, *Fv/Fm* of two *M. aeruginosa* species could both totally recover to the initial values after UV‐B radiation on Day 2, and *Fv/Fm* of *C. pyrenoidosa* on Day 6 and Day 8 totally recovered to the initial values within 16 h and 8 h after UV‐B radiation, respectively. For three species in the UV‐B treatment, the decline degree of *Fv/Fm* was also lower for toxic *M. aeruginosa* and it exhibited a faster recovery rate. This result was consistent with that under normal growth conditions. Under nutrient enrichment conditions, the variation patters of ROS in algal cells and algal SOD activity were also similar for three species (Figure 9). More specifically, PARtreatment did not cause great oxidative stresses on algae, but ROS and algal SOD activity gradually increased at the later stage of incubation. In the UV-B treatment, ROS in algal cells also increased gradually, and they only showed higher values (*p* < 0.05) than those in the PAR treatment after Day 10. For algal SOD activity in the UV-B treatment, they all exhibited a sharp increase and decreased gradually to maintain a stable value. For both PAR and UV-B treatments, ROS in algal cells were lower (*p* < 0.05) than those under normal growth conditions on a specific day.

**Figure 8.** Diurnal changes of *Fv/Fm* of three species in the PAR and UV‐B treatments under nutrient enrichment conditions on Day 2, Day 6, and Day 8. **Figure 8.** Diurnal changes of *Fv/F<sup>m</sup>* of three species in the PAR and UV-B treatments under nutrient enrichment conditions on Day 2, Day 6, and Day 8.

#### *3.6. Antioxidant Responses of Algal Species under Nutrient Enrichment Conditions* 3.6.2. Contents of Photosynthetic Pigments

3.6.1. ROS in Algal Cells and SOD Activity Under nutrient enrichment conditions, the variation patters of ROS in algal cells and algal SOD activity were also similar for three species (Figure 9). More specifically, PAR treatment did not cause great oxidative stresses on algae, but ROS and algal SOD activity gradually increased at the later stage of incubation. In the UV‐B treatment, ROS in algal cells also increased gradually, and they only showed higher values (*p* < 0.05) than those in As shown in Table 2, similar patterns were overserved for the algal synthesis of photosynthetic pigments on Day 1. Compared to the initial values, Chl-a contents of algal single cells were comparable in the PAR and UV-B treatment (*p* > 0.05), but CAR and PC in single cells increased greatly (*p* < 0.05) in the UV-B treatment, resulting in the higher CAR/Chl-a and PC/Chl-a ratios of three species on Day 1. Moreover, CAR and PC contents, CAR/Chl-a and PC/Chl-a ratios were all higher (*p* < 0.05) with nutrient enrichment compared to those under normal growth conditions.

the PAR treatment after Day 10. For algal SOD activity in the UV‐B treatment, they all exhibited a sharp increase and decreased gradually to maintain a stable value. For both PAR and UV‐B treatments, ROS in algal cells were lower (*p* < 0.05) than those under nor‐ mal growth conditions on a specific day. On Day 8, despite the fact that the Chl-a contents of algal single cells were lower (*p* < 0.05) in the UV-B treatments, they showed an increasing trend compared with those on Day 1. This was consistent with the patterns of cell density. In addition, CAR and PC in single cells were also higher (*p* < 0.05) in the UV-B treatment at this moment, and CAR/Chl-a and PC/Chl-a ratios were promoted with UV-B radiation. This pattern was remarkably different from that under normal growth conditions.

**Figure 9.** ROS in the cells of three species (vertical bar) and algal SOD activity (line and scatter) during the incubation in the PAR and UV‐B treatments under nutrient enrichment conditions (the arrow indicates the initial value of ROS contents). **Figure 9.** ROS in the cells of three species (vertical bar) and algal SOD activity (line and scatter) during the incubation in the PAR and UV-B treatments under nutrient enrichment conditions (the arrow indicates the initial value of ROS contents).

#### 3.6.2. Contents of Photosynthetic Pigments *3.7. Interspecific Competition in the Co-Cultures*

As shown in Table 2, similar patterns were overserved for the algal synthesis of pho‐ tosynthetic pigments on Day 1. Compared to the initial values, Chl‐a contents of algal single cells were comparable in the PAR and UV‐B treatment (*p* > 0.05), but CAR and PC in single cells increased greatly (*p* < 0.05) in the UV‐B treatment, resulting in the higher CAR/Chl‐a and PC/Chl‐a ratios of three species on Day 1. Moreover, CAR and PC con‐ tents, CAR/Chl‐a and PC/Chl‐a ratios were all higher (*p* < 0.05) with nutrient enrichment compared to those under normal growth conditions. On Day 8, despite the fact that the Chl‐a contents of algal single cells were lower (*p* < 0.05) in the UV‐B treatments, they showed an increasing trend compared with those on Day 1. This was consistent with the patterns of cell density. In addition, CAR and PC in single cells were also higher(*p* < 0.05) in the UV‐B treatment at this moment, and CAR/Chl‐ a and PC/Chl‐a ratios were promoted with UV‐B radiation. This pattern was remarkably Algal growth patterns were comparable in the PAR treatment (Figure 10), i.e., *C. pyrenoidosa* grew rapidly after the lag period and soon outcompeted non-toxic or toxic *M. aeruginosa*, while the cell density of *C. pyrenoidosa* decreased when that of *M. aeruginosa* started to increase. Compared with mono-cultures, the maximum cell densities of three species were all lower (*p* < 0.05) in the co-cultures (Table S2). However, the maximum cell density of *C. pyrenoidosa* seemed to decrease more in the PAR treatment, and the decline was great in the co-cultures of *C. pyrenoidosa* with toxic *M. aeruginosa*. In comparison, two *M. aeruginosa* species gained the obvious dominance and maintained competitive advantages from the beginning in the UV-B treatment, and the growth of *C. pyrenoidosa* was also markedly inhibited, which achieved 42.1% and 31.4% of the maximum cell density in the mono-cultures. Meanwhile, despite the faster growth of *C. pyrenoidosa* in the mono-cultures, µmax of *C. pyrenoidosa* decreased greatly when it was co-cultured with *M. aeruginosa* in

different from that under normal growth conditions.

the PAR and UV-B treatments. However, µmax of non-toxic *M. aeruginosa* only decreased slightly and µmax of toxic *M. aeruginosa* even increased slightly.


**Table 2.** Contents of photosynthetic pigments of three species in the PAR and UV-B treatments on Day 1 and Day 8 under nutrient enrichment conditions.

\* Bold values with \* indicated significant higher contents in the UV-B treatment compared with PAR treatment at *p* < 0.05, while those bold and underlined values with \* indicated significant lower contents in the UV-B treatment compared with PAR treatment at *p* < 0.05. *Int. J. Environ. Res. Public Health* **2022**, *19*, x FOR PEER REVIEW 16 of 23

**Figure 10.** Cell densities of algal species in the co‐cultures of (**a**) *C. pyrenoidosa* & non‐toxic *M. aeru‐ ginosa*, and of (**b**) *C. pyrenoidosa* & toxic *M. aeruginosa* in the PAR and UV‐B treatments under nutrient enrichment conditions. **Figure 10.** Cell densities of algal species in the co-cultures of (**a**) *C. pyrenoidosa* & non-toxic *M. aeruginosa*, and of (**b**) *C. pyrenoidosa* & toxic *M. aeruginosa* in the PAR and UV-B treatments under nutrient enrichment conditions.

#### **4. Discussion 4. Discussion**

#### *4.1. Effects of UV‐B Radiation and Algal Responses 4.1. Effects of UV-B Radiation and Algal Responses*

Although UV irradiance usually constitutes a few percent of solar radiation (5.85– 8.51% in China), many studies have analyzed the effects of UV radiation on algal growth Although UV irradiance usually constitutes a few percent of solar radiation (5.85–8.51% in China), many studies have analyzed the effects of UV radiation on algal growth and

and negative effects were often reported [46,47]. Based on field and laboratory experi‐ ments, the main influencing mechanisms of UV‐B radiation on algae include cell vitality

radiation could exert negative effects on typical algal species in freshwater ecosystems,

Since the growth and vitality of photosynthetic organisms are mainly governed by photosynthetic activity and the photosynthetic apparatus is an important damage target of UV‐B radiation [50,51], daily *Fv/Fm* of all three species in the UV‐B treatment were often lower compared to those in the PAR treatment. This result indicated that ambient UV‐B might cause damages to D1 or D2 protein in algal photosystems [52] and 50 μmol m−<sup>2</sup> s−<sup>1</sup> of PAR did not have similar effects. However, different from many other studies using high‐dose UV‐B radiation whereby algal photosynthetic systems were greatly damaged [53,54], algal *Fv/Fm* gradually increased during Day 2–10 under two different growth con‐ ditions. Based on algal Chl‐a contents on Day 1 and the release rates of K+, the adopted UV‐B treatment in our study did not have direct lethal effects on algae, and changes of algal *Fv/Fm* and growth could be the balance between the light‐induced effects and adap‐ tive physiological processes of cells. This was confirmed by the diurnal changes of algal *Fv/Fm*, as *Fv/Fm* of three species recovered with different rates after UV‐B exposure, which could have resulted from processes, such as oxidation resistance, nucleotide resynthesis,

which also showed adaptative responses to UV‐B radiation.

ATP supply, or the repair of damaged proteins [55,56].

negative effects were often reported [46,47]. Based on field and laboratory experiments, the main influencing mechanisms of UV-B radiation on algae include cell vitality impairment, ROS production, DNA damages, changes of nutrient utilization, etc. [26,27,48,49]. Our results are consistent with these findings, namely in that ambient UV-B radiation could exert negative effects on typical algal species in freshwater ecosystems, which also showed adaptative responses to UV-B radiation.

Since the growth and vitality of photosynthetic organisms are mainly governed by photosynthetic activity and the photosynthetic apparatus is an important damage target of UV-B radiation [50,51], daily *Fv/F<sup>m</sup>* of all three species in the UV-B treatment were often lower compared to those in the PAR treatment. This result indicated that ambient UV-B might cause damages to D1 or D2 protein in algal photosystems [52] and 50 µmol m−<sup>2</sup> s −1 of PAR did not have similar effects. However, different from many other studies using highdose UV-B radiation whereby algal photosynthetic systems were greatly damaged [53,54], algal *Fv/F<sup>m</sup>* gradually increased during Day 2–10 under two different growth conditions. Based on algal Chl-a contents on Day 1 and the release rates of K<sup>+</sup> , the adopted UV-B treatment in our study did not have direct lethal effects on algae, and changes of algal *Fv/F<sup>m</sup>* and growth could be the balance between the light-induced effects and adaptive physiological processes of cells. This was confirmed by the diurnal changes of algal *Fv/Fm*, as *Fv/F<sup>m</sup>* of three species recovered with different rates after UV-B exposure, which could have resulted from processes, such as oxidation resistance, nucleotide resynthesis, ATP supply, or the repair of damaged proteins [55,56].

UV radiation could cause the overexcitation of substances and produce excess ROS in algal cells or in the cultures [57,58], leading to the impairment of algal photosynthetic systems and normal growth. Consistently, ROS contents in UV-B radiated algal cells were higher during the incubation under normal growth conditions. The increase of ROS in the later period in the PAR treatment was consistent with the work of Latifi et al. [59] in that some environmental factors, such as nutrient deficiency and light limitation, could indirectly generate ROS at multiple sites of the photosynthetic electron transport chain in algal cells. However, as mentioned above, the oxidative stresses and resulting damages could be mitigated with algal adaptive strategies. In our study, EPS production, the up-regulation of SOD activity, CAR and PC synthesis, and recovery of *Fv/F<sup>m</sup>* by three species, could act as their effective adaptation mechanisms, resulting in decreased sensitivity to UV-B exposure and increased self-repair efficiency. For example, algal EPS consisted of polysaccharides, proteins, lipids, and humic substances and often appeared as a structureless slimy layer around cells, which was helpful to algal aggregation and its resistance to environmental stresses [38,42,60]. Meanwhile, higher CAR and PC in cells could adsorb UV-B light and quench ROS to alleviate damage to algal photosynthetic systems and DNA [50,61]. Moreover, higher CAR in the cells could increase the algal utilization efficiency of light and promote its generation of ATP and other substances [50,62], such as antioxidant enzymes, nucleotides, and proteins, to repair damaged apparatus in algal cells [18,30]. In the UV-B treatment, the high SOD activity and enhanced production of CAR and PC by algae in the early stage could partly explain the gradual increase of algal *Fv/F<sup>m</sup>* and cell density. However, these adaptive responses of three species might be not enough to remove UV-induced oxidative stresses at the later stage under normal growth conditions, and inhibition on algal growth could occur. Our previous study indicated that algal adaptation to UV-B radiation required energy and essential nutrient substances [26], and this could be the possible reason for algal decay at the later stage in the UV-B treatment. Especially, cumulative ROS might damage the antioxidant systems of UV-radiated algae after Day 8 under normal growth conditions and result in low SOD activity and algal death.

#### *4.2. Comparison of Algal Adaptation to UV-B Radiation*

In previous studies, scholars have often investigated the strategies of cyanobacteria to alleviate the harmful effects of UV-B radiation, such as the production of UV-absorbing compounds (UVCs) to mitigate photo-induced damages, vertical migration of cells to decrease the irradiation stress, enhanced self-repair, etc. [46,51,63]. In this study, three species exhibited strain-specific responses to UV-B radiation, when toxic *M. aeruginosa* was more tolerant and showed a higher adaptation capability, including lower sensitivity to UV-B radiation and better self-repair efficiency.

Firstly, *C. pyrenoidosa* grew faster, whereas toxic *M. aeruginosa* had similar µmax in the PAR and UV-B treatments, which might indicate the stronger plasticity of toxic *M. aeruginosa* to maintain a stable growth potential. The lower growth rate of toxic *M. aeruginosa* was probably caused by the excess energy cost for microcystin production [64]. Secondly, EPS production by toxic *M. aeruginosa* could provide a better adaptation to UV-B radiation. In this study, toxic *M. aeruginosa* produced more BEPS in the early stage in the UV-B treatment, when tryptophan-like substances in BEPS could absorb UV-B radiation and play the role of precursor to UV-absorbing metabolites [65]. The decrease of BEPS and increase of SEPS at the later stage could be explained as some UV-absorbing compounds were degraded, and this contributed to the decreased adaptation of algae to UV radiation under normal growth conditions [49,57]. Meanwhile, toxic *M. aeruginosa* excreted more SEPS, and organic matter in SEPS had a positive effect on algal aggregation [41,66]. The aggregated morphology of algal cells could be beneficial to reduce photo-induced damage by shading [67,68] and this was regarded as one kind of defense against UV-B in the natural waters. Furthermore, the high iron availability for algal cells could decrease UV-induced damages [27] and higher EPS could serve as an important iron reservoir that helps toxic *M. aeruginosa* to better cope with UV-B radiation [11,60].

Moreover, toxic *M. aeruginosa* exhibited a better antioxidant response in the UV-B treatment. In our study, two *M. aeruginosa* species could promote the synthesis of CAR and PC in the UV-B treatment, which was further enhanced with nutrient enrichment. As mentioned above, the beneficial effects of CAR and PC included the alleviation of photo-induced damage and the promotion of self-repair [15,69]. Moreover, microcystin synthesis by toxic *M. aeruginosa* could contribute to a higher fitness of cells under UV-B irradiation through a covalent interaction with the cysteine residue of proteins [70]. Consequently, *Fv/F<sup>m</sup>* decline was lower and the recovery rate was faster for toxic *M. aeruginosa* under two different conditions. Xu et al. [18] also indicated that toxic *M. aeruginosa* had a competitive advantage relative to non-toxic strain in a changing light environment via stronger antioxidant capacity (higher SOD activity and the synthesis of microcystin) and quicker PSII recovery capacity The decrease of CAR and PC on Day 8 under normal growth conditions was related to the photooxidation and photodegradation of pigments, when the biological resources in the cultures might be not enough for the algal resynthesis of pigments and other efficient metabolic processes [71]. Compared to PAR treatment, the higher CAR/Chl-a and PC/Chl-a ratios of two *M. aeruginosa* species under normal growth conditions and higher CAR/Chl-a and PC/Chl-a ratios of all three species under nutrient enrichment conditions probably indicated their increased acclimation to prolonged UV-B exposure [13,72]. This was consistent with results obtained by Jiang et al. [50]. Although increased cell density partially reduced UV-B radiation at the later stage of incubation, our results could be mainly ascribed to the adaptation capability of algae to UV radiation in the 2-cm depth dishes.

#### *4.3. Effects of Nutrient Enrichment and Algal Competition Characteristics*

Whereas scholars have often studied the influences and mechanisms of UV radiation on algae, fewer studies have focused on the effects of nutrient enrichment. Meanwhile, the role of UV-B radiation in determining interspecific competition has not been clearly elucidated.

Combining the diurnal changes of algal *Fv/F<sup>m</sup>* and algal growth patterns under different growth conditions, nutrient enrichment alleviated the negative effects of UV-B radiation on three species in our study. This was in accordance with our previous findings that higher P availability could enhance algal adaptation to UV radiation [26]. Zheng et al. [29] also reported that UV-induced inhibition of algal growth and photosynthetic production

changed in accordance with the changes of the chemical environment in the water. In our study, the beneficial effects of nutrient enrichment also included decreasing algal sensitivity to UV-B radiation and increasing its self-repair efficiency. For example, higher contents of CAR and PC in cells with nutrient enrichment could help algae to counteract UV-induced damages [50,73], which resulted in lower ROS in cells and lower decline degrees of algal *Fv/F<sup>m</sup>* on each day. Meanwhile, three species did not require a great deal of energy and biological resources to deal with UV-B radiation, and they could better promote their growth after self-repair with more nutrients in the medium, such as the photo-reactivation of DNA or resynthesis of D1 proteins [18,22]. Therefore, algal µmax values were higher and three species persistently grew in the UV-B treatment under nutrient enrichment conditions. Since toxic *M. aeruginosa* exhibited a higher adaptation capability to UV-B radiation, as previously discussed, the beneficial effects of nutrient enrichment were best for toxic *M. aeruginosa*, and its growth was comparable between PAR and UV-B treatment during the whole incubation.

Wind-induced mixing of water and sediment resuspension could cause pulse fluctuations of irradiation conditions and high nutrient availability in the water, where different algal species coexist. Thus, the co-cultures under nutrient enrichment conditions might partly explain the competitive advantages of typical species in the field. Different from the mono-cultures, *C. pyrenoidosa* was not always the fastest-growing species in the co-cultures, and exposure to UV-B radiation could enhance the growth advantages of *M. aeruginosa*. Our previous study indicated that the augmentation of algal P quota could alleviate or eliminate the negative effects of UV radiation on algae [26]. Considering that *M. aeruginosa* had a faster and better P accumulation ability compared to other typical species in freshwater ecosystems [74], *M. aeruginosa* might have a stronger adaptation capability to UV-B radiation and a stronger competitive advantage in the co-cultures. However, since nutrients were not limited under nutrient enrichment conditions, allelopathy effects between species might have a more important role in the co-cultures [19,75]. In our study, two *M. aeruginosa* species demonstrated a greater inhibition effect on *C. pyrenoidosa* growth compared with the negative effects of *C. pyrenoidosa* on *M. aeruginosa*. For example, when the secondary metabolites of green algae showed declining inhibitory effects as incubation progressed, the extracts of cyanobacteria and microcystins were often more effective to inhibit the growth of other species [24,76]. Therefore, µmax of *C. pyrenoidosa* decreased greatly and two *M. aeruginosa* species outcompeted *C. pyrenoidosa* at the later stage in the PAR treatment. Meanwhile, toxic *M. aeruginosa* showed a greater competitiveness to maintain high µmax and inhibit *C. pyrenoidosa* growth in the co-cultures. As mentioned above, the higher EPS contents and microcystin of *M. aeruginosa* cells were conducive to the adaptation of *Microcystis* to UV-B radiation [11,70]. Furthermore, the aggregation of *Microcystis* might prevent *C. pyrenoidosa* to utilize PAR for self-repair or recovery after UV-B radiation [77]. Consequently, non-toxic and toxic *M. aeruginosa* were dominant from the beginning in the UV-B treatment, and toxic *M. aeruginosa* also had a greater impact in depressing the growth of *C. pyrenoidosa*. In this sense, the dominance of cyanobacteria and advantages of toxic *M. aeruginosa* could be enhanced in UV-radiated waters with severer eutrophication. However, the complexities and likely influence of coexisting yet unexamined factors deserve a further in situ study in the future.

#### **5. Conclusions**


In addition to stable µmax in two treatments, higher production of EPS, and enhanced production of CAR and PC under UV-B radiation, toxic *M. aeruginosa* showed a better recovery of its photosynthetic efficiency.

(3) Nutrient enrichment could alleviate the negative effects of UV-B radiation on algae, and the growth of toxic *M. aeruginosa* was comparable between PAR and UV-B treatment. In the co-cultures with nutrient enrichment, *M. aeruginosa* gradually outcompeted *C. pyrenoidosa* in the PAR treatment, and UV-B treatment enhanced the growth advantages of *M. aeruginosa*, when toxic *M. aeruginosa* showed a greater competitiveness to maintain high µmax and inhibit the growth of *C. pyrenoidosa*.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19095485/s1, Figure S1. The spectral power distribution and weighted UV radiation of UV- B lamps (TL20W/01RS, Philips) used in the irradiation experiment. Figure S2. A schematic diagram of the irradiation experiments. Figure S3. Fluorescence EEM spectra for EPS produced by three species. Figure S4. Whole-cell absorption spectra of algal cultures at the beginning of mono-cultures. Cell cultures with OD<sup>680</sup> of 0.10 were used for measurement and adsorption values were normalized to the optical density at OD680. Table S1. Composition of the modified BG<sup>11</sup> medium under different growth conditions in our experiment. Table S2. The maximum growth rate (µmax, d−<sup>1</sup> ) and maximum cell density (10<sup>6</sup> cells/mL) of three species in the mono-cultures and co-cultures under nutrient enrichment conditions and the percentage change of maximum cell density showing in parentheses. References [57,78–80] are cited in the supplementary materials.

**Author Contributions:** Conceptualization, J.H. and L.R.; methodology, K.D.; data curation, Y.W.; writing—original draft preparation, L.R.; writing—review and editing, Y.Y. and L.Z.; investigation, H.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Natural Science Foundation of Jiangsu Province (BK20210933) and National Natural Science Foundation of China (22006066).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author, upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

