**1. Introduction**

With the rapid economic development and pollutant discharge, eutrophication has seriously affected aquatic ecosystems over the last several decades [1,2]. Frequent outbreaks of harmful algal blooms (HABs) represent one of the most serious outcomes of eutrophication [3,4] and many studies have investigated the effects of environmental factors, such as temperature, light, and nutrients, on the growth of typical species and the development of HABs [5,6]. These factors could partly explain the underlying mechanism of HAB formation and the seasonal succession of species. For a long time, cyanobacteria gained

**Citation:** Ren, L.; Huang, J.; Ding, K.; Wang, Y.; Yang, Y.; Zhang, L.; Wu, H. Comparative Study of Algal Responses and Adaptation Capability to Ultraviolet Radiation with Different Nutrient Regimes. *Int. J. Environ. Res. Public Health* **2022**, *19*, 5485. https:// doi.org/10.3390/ijerph19095485

Academic Editor: Nansheng Chen

Received: 24 February 2022 Accepted: 28 April 2022 Published: 30 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

much attention from environmental degradation and human health perspectives [7–9]. Especially, many scholars have focused on *Microcystis* in recent years, which is a dominant cyanobacterial genus in many eutrophic waters and often exhibits a greater threat to the microcystins produced by toxic species [10–12].

Light could directly affect the photosynthesis and growth of cyanobacteria [13,14], which were closely associated with light intensity, exposure time, and light wavelength. Especially, other than necessary photosynthetically active radiation (PAR; 400–700 nm), enhanced ultraviolet (UV) radiation is reported for many aquatic ecosystems throughout the world due to serious stratospheric ozone depletion [15,16]. Therefore, the effects of UV radiation on typical cyanobacterial species have received considerable attention in recent years, and most studies have used *Microcystis* as a model species [17–19]. Freshwater ecosystems in the middle and lower reaches of Yangtze River are susceptible to enhanced UV radiation due to the lack of depth refuge [20], and *Microcystis* often occur as the surface blooms that encounter higher irradiance [21,22]. It is assumed that *Microcystis* should be more threatened and suffered greater UV-induced damage. However, the frequency and intensity of the dominance of *Microcystis* continue to increase in typical eutrophic lakes in China, such as Lake Taihu. Hence, it is crucial to investigate and compare the responses of *Microcystis* and other algal species to UV radiation.

The composition of HABs in freshwater ecosystems is varied and often includes cyanobacteria and green microalgae as the major components [21,23]. For example, *Microcystis* and *Chlorella* were the most dominant species in eutrophic lakes in China, although their cell densities fluctuated wildly during different seasons [12]. Meanwhile, *Microcystis* blooms were often formed by mixed species when the seasonal succession and competition between the non-toxic and toxic species have been widely studied [24,25]. For example, the toxic *Microcystis aeruginosa* was determined to be more harmful to *Chlorella vulgaris* than the non-toxic species at higher temperatures [19]. Although numerous studies have focused on the effects of UV on algae in recent years, relatively few studies have deeply examined and compared the adaptive strategies to the ambient UV radiation of non-toxic and toxic *M. aeruginosa* and other species [16,26]. Some scholars investigated the effects of nutrient enrichment on algal responses to UV radiation and the results are varied. For example, Li et al. [27] reported that the effects of UV-B on phytoplankton productivity might be underestimated in iron-deficient ecosystems, and Yang et al. [28] reported that the negative impact was most pronounced when UV-B exposure and P limitation were combined. Meanwhile, Zheng et al. [29] reported that impacts of solar UV radiation on algal growth differed significantly at different N concentrations. However, the influence mechanisms of nutrient enrichment on algal adaptation and biotic interactions to UV radiation also remain unclear. In addition, many studies have investigated the effects of UV radiation on algal growth in the pure mono-culture systems [13,30], and it remains unclear how the coexistence of algae was affected by UV radiation, despite the fact that algal species coexist in the natural ecosystems. In this regard, the co-cultures with different nutrient conditions may provide useful information to address cyanobacterial blooms and algal competition in the natural waters and to better explain the synergistic effects of eutrophication and irradiation in mixed communities.

In this study, we selected *C. pyrenoidosa* and non-toxic and toxic *M. aeruginosa* to investigate their various physiological responses with ambient irradiation treatment under different nutrient regimes. The main goals were to: (i) analyze the effects and mechanisms of ambient UV-B radiation on three species, (ii) compare and explore the responses of the adaptation capability of three species to UV radiation, and (iii) study the effects of nutrient enrichment on algal growth and competition.

#### **2. Materials and Methods**

#### *2.1. Algal Culture*

*C. pyrenoidosa* (FACHB 5), non-toxic *M. aeruginosa* (FACHB 469), and toxic *M. aeruginosa* (FACHB 905) were obtained from the Freshwater Algae Culture Collection of the Institute

of Hydrobiology, Chinese Academy of Sciences (FACHB). For the three algal species, *M. aeruginosa* is a dominant genus during the outbreaks of HABs, and *C. pyrenoidosa* was selected because of its common distribution and frequent co-existence with cyanobacteria in many Chinese eutrophic ecosystems [14]. All strains were pre-cultured separately and exponential growth was maintained by transferring 5 mL of growing cultures to fresh standard BG<sup>11</sup> medium in Erlenmeyer flasks every 8–10 days for enlargement [31]. Preculture was performed under sterile conditions and the flasks were placed at 25 ◦C under 40 µmol photons m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PAR with cool white fluorescent lamps (light/dark regime of 12 h:12 h) in the illuminated incubator (GZX-250BS-II). All flasks were shaken three times per day to prevent the cells from adhering to inner walls, and the position of flasks was exchanged randomly to ensure uniform light exposure.

## *2.2. Experimental Setup*

After pre-culture and enlargement–cultivation, the exponentially growing algal cells were collected and suspended in phosphate buffer solution (PBS, pH = 7.4) for washing and reservation prior to running our formal experiments. After 3–4 days, algal cells were collected again and inoculated into 500-mL flasks containing 300–400 mL of modified BG<sup>11</sup> medium for experiments in the mono-cultures and co-cultures. In the first scheme of modified BG<sup>11</sup> medium, the composition was as shown in Table S1, and concentrations of nitrogen (N), phosphorus (P), and iron were comparable to those in the natural waters, representing normal growth conditions. In the second scheme of modified BG<sup>11</sup> medium, the initial N, P, and iron concentrations were appropriately increased (Table S1), representing nutrient enrichment conditions. The initial cell density of three species was 1.0 <sup>×</sup> <sup>10</sup><sup>6</sup> cells mL−<sup>1</sup> , which approximated the cell number at the beginning of HABs in most eutrophic lakes in China [32]. Meanwhile, co-cultures were conducted to simulate natural conditions and to explore the characteristics of algal competition. To this end, *C. pyrenoidosa* was co-cultured with non-toxic and toxic *M. aeruginosa*, when the inoculation ratio was 1:1 and the cell density of each strain was 1.0 <sup>×</sup> <sup>10</sup><sup>6</sup> cells mL−<sup>1</sup> .

On each day, algal cultures in the flasks were transferred into sterilized petri dishes with quartz glass covers (20 cm in diameter) for PAR or UV-B exposure after slightly shaking the flasks, representing PAR treatment and UV-B treatment, and the two treatments both lasted for 4 h (9:00–13:00). In the PAR treatment, petri dishes were maintained in another illumination incubator and continuously irradiated with 50 µmol photons m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PAR. In the UV-B treatment, petri dishes were stored in clean chambers and subjected to highpressure mercury UV-B lamps with the dominant wavelength of 313 nm (TL20W/01RS, Philips, Eindhoven, Netherlands, Figure S1). In the UV-B treatment, light exposure was restricted to UV-B and no photosynthetically active wavelengths were given to algal cells. The effective irradiation intensity of PAR and UV-B in our study was 70 and 0.8 W m−<sup>2</sup> , respectively. After the irradiation treatment for 4 h, algal cultures were returned to flasks and incubated under the conditions as described in the pre-cultures for the rest time on each day (dark during 0:00–6:00 and 18:00–24:00, 40 µmol photons m−<sup>2</sup> s <sup>−</sup><sup>1</sup> PAR during 6:00–9:00 and 13:00–18:00). The incubation lasted for 14 days in our study and a schematic diagram of the experiment is shown in Figure S2.

Based on field monitoring, the adopted PAR and UV-B in different treatments was in accordance with the natural conditions at noon in the middle and lower reaches of the Yangtze River [26]. The vertical sides of petri dishes were all covered with aluminum foil to ensure vertical radiation and the irradiance was measured using a miniature fiber optic spectrometer (FLA4000A+, Flight, Hangzhou, China).

#### *2.3. Analytical Methods of Parameters*

2.3.1. Cell Density and Photosynthetic Efficiency

Subsamples were regularly taken for determining cell density in the mono-cultures and co-cultures. Cells were both enumerated by using a flow cytometer (CytoFLEX S, Beckman Coulter, Fullerton, CA, USA), when *M. aeruginosa* and *C. pyrenoidosa* could be

clearly differentiated by autofluorescence in the co-cultures [33]. Then, the algal growth rate was determined as follows: µ = (ln*N*<sup>2</sup> − ln*N*1)/(t<sup>2</sup> − t1), where *N*<sup>1</sup> and *N*<sup>2</sup> was the cell density on days t<sup>1</sup> and t2, respectively. The maximum µ during the whole incubation period was defined as µmax, which is an important index to indicate algal growth potential.

A Phyto-PAM fluorometer (Hein Walz, Effeltrich, Germany) was adopted to determine the effective quantum yield (*Fv*/*Fm*) of algal species. The Phyto-PAM fluorometer has been increasingly used in laboratory and in situ experiments, and *Fv*/*F<sup>m</sup>* can effectively indicate the efficiency of algal photosynthesis apparatus [34–36].
