Oxidative Stress and Antioxidant Responses of Phormidium ambiguum and Microcystis aeruginosa Under Diurnally Varying Light Conditions

Two harmful cyanobacteria species (Phormidium ambiguum and Microcystis aeruginosa) were exposed to diurnal light-intensity variation to investigate their favorable and stressed phases during a single day. The photosynthetically active radiation (PAR) started at 0 µmol·m−2·s−1 (06:00 h), increased by ~25 µmol·m−2·s−1 or ~50 µmol·m−2·s−1 every 30 min, peaking at 300 µmol·m−2·s−1 or 600 µmol·m−2·s−1 (12:00 h), and then decreased to 0 µmol·m−2·s−1 (by 18:00 h). The H2O2 and antioxidant activities were paralleled to light intensity. Higher H2O2 and antioxidant levels (guaiacol peroxidase, catalase (CAT), and superoxidase dismutase) were observed at 600 µmol·m−2·s−1 rather than at 300 µmol·m−2·s−1. Changes in antioxidant levels under each light condition differed between the species. Significant correlations were observed between antioxidant activities and H2O2 contents for both species, except for the CAT activity of P. ambiguum at 300 µmol·m−2·s−1. Under each of the conditions, both species responded proportionately to oxidative stress. Even under maximum light intensities (300 µmol·m−2·s−1 or 600 µmol·m−2·s−1 PAR intensity), neither species was stressed. Studies using extended exposure durations are warranted to better understand the growth performance and long-term physiological responses of both species.


Introduction
The growth and spread of cyanobacteria have increased, thus threatening today s water bodies and supplies worldwide [1,2]. Global warming and abundant nutrition supply have promoted the spread of cyanobacteria, which, among others, generate bad odors by producing substances such as 2-methylisoborneol, releasing cyanotoxins and forming blooms, thus making many water bodies unusable [3][4][5]. In addition, some cyanobacterial species can produce allelochemicals that are harmful to other aquatic species [6][7][8]. Therefore, numerous studies have focused on suppressing or preventing their growth, globally [9][10][11].
During cyanobacteria control efforts, chemical control measures are discouraged due to their potentially harmful secondary effects on ecosystems [12][13][14], while non-chemical methods require knowledge of the interactions of cyanobacteria with the natural environment, their responses to changing environmental factors or stresses, and their interaction with other species (allopathy). Currently, this approach is being extensively studied by various research groups [15][16][17][18][19][20][21]. In addition, many studies have focused on the physiology and morphology of cyanobacteria under natural and laboratory-derived conditions [22,23]. However, despite those findings, knowledge gaps remain to be filled.

Experimental Setup and Procedure
Following the 14-day incubation, 3 replicate conical flasks (500 mL Pyrex clear glass conical flasks) from each of the P. ambiguum and M. aeruginosa cyanobacteria cultures were made, maintaining the 0.6 ± 0.02 optical density measured at 730 nm (OD 730 ) using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). The dilution of the cyanobacteria culture was accomplished with BG11 nutrient medium. In all experiments, the temperature was maintained at 20 • C in an incubator, whereas the lighting conditions changed from 0 µmol·m −2 ·s −1 (at 06:00 h) to 300 µmol·m −2 ·s −1 or 600 µmol·m −2 ·s −1 (at 12:00 h) by changing the lighting intensity by~25 µmol·m −2 ·s −1 or 50 µmol·m −2 ·s −1 every 30 min with a VBP-L24-C2 light (Valore, Kyoto, Japan). The light intensity was then decreased at the same rate (until 18:00 h). The lighting condition was controlled with warm light-emitting diode panel lights, and the light intensity was measured using a quantum flux meter (Apogee, MQ-200, Logan, UT, USA). Cyanobacteria samples from each flask were collected for analysis every 3 h, at 06:00 h, 09:00 h, 12:00 h, 15:00 h, 18:00 h, and 21:00 h. To facilitate mixing, each flask was manually shaken at the time of sampling.

H 2 O 2 Concentration
Cellular H 2 O 2 contents were estimated according to standard methods [43]. Briefly, 1 mL was collected from each flask and the supernatants were removed by centrifugation at 10,000× g for 10 min at 4 • C. The cell pellets were washed once with ultrapure water (Milli-Q direct 5, Merck KGaA, Darmstadt, Germany). To extract cellular H 2 O 2 , cell pellets were homogenized in 1 mL of 0.1 M pH 6.5 phosphate buffer and centrifuged at 10,000× g for 10 min at 4 • C. A total of 750 µL of 1% titanium chloride in 20% H 2 SO 4 (v/v) was then added to initiate the reaction. The optical absorption was measured at 410 nm using a spectrophotometer (UVmini-1240), following centrifugation (10,000× g for 5 min) at room temperature (25 ± 2 • C). The H 2 O 2 concentration was determined using a standard curve, prepared using a series of samples with known H 2 O 2 concentration.

GPX-Activity Assay
The GPX activity was assayed as described by Hoda et al. [44] and MacAdam et al. [45], with modifications. Cyanobacteria cells were harvested by centrifuging 1 mL samples at 10,000× g at 4 • C for 10 min and removing the supernatant and cell pellets, which were homogenized in 1 mL potassium phosphate buffer (100 mM, pH 7.0). A total of 65 µl of enzyme extract was then mixed with 920 µL of potassium phosphate buffer (100 mM, pH 7) containing 20 mM guaiacol. With the addition of 15 µL of 0.6% H 2 O 2 , the reaction was then started, and the absorbance change was recorded at 470 nm every 10 s for 3 min using UV mini-1240. GPX activity was calculated using an extinction coefficient of 26.6 mM/cm.

CAT-Activity Assay
CAT activity was measured using the method described by Aebi [46]. A total of 1 mL of each culture was centrifuged at 10,000× g at 4 • C for 10 min. The supernatant was removed, and the cell pellets were homogenized in 1 mL potassium phosphate buffer (50 mM, pH 7.0), containing 0.1 mM EDTA. After centrifuging again (10,000× g at 4 • C for 10 min), the supernatant was collected as the enzyme extract. The CAT activity was measured by reacting 15 µL of 750 mM H 2 O 2 , 920 µL of potassium phosphate buffer, and 65 µL of extract supernatant. Optical absorption was measured at 240 nm using UV mini-1240. The measurements were recorded every 10 s for 3 min, and the CAT activity was calculated using an extinction coefficient of 39.4 mM/cm.

APX-Activity Assay
APX activity was assayed, as described by Nakano and Asada [47]. The decrease in absorbance at 290 nm was recorded every 10 s for 3 min using UV mini-1240. Each reaction mixture was performed in a 1-mL volume. Initially, 920 µL of 50 mM phosphate buffer (pH 7.0), containing 5 mM EDTA, was mixed with 15 µL of 0.5 mM ascorbic acid. Each reaction was then started routinely by adding 15 µL of 1 mM H 2 O 2 . Calculations were performed using a molar extinction coefficient for ascorbate of 2.8 mM/cm.

SOD-Activity Assay
SOD activities were determined by performing nitro blue tetrazolium (NBT) assays, as described by Ewing and Janero [48]. Each sample was mixed with 10 µL of 750 µM NBT, 10 µL of 130 mM methionine, 70 µL of 50 mM phosphate buffer with 100 µM EDTA (pH 7.8), and 10 µL of 20 µM riboflavin solution. The reactions were carried out for 5 min, and the absorbances were recorded at 560 nm using UV mini-1240. Blank reactions were prepared by substituting the sample with an equal volume of 50 mM phosphate buffer (pH 7.8). One unit of SOD activity was defined as the amount of SOD that inhibited the rate of formazan production by 50% at 25 • C.

Data Analysis
One-way analysis of variance (ANOVA), followed by Tukey's post-hoc test, was performed to test the statistical significance of variations among the means of sample groups. Data were normalized relative to the starting group (06:00 h), by dividing the results of each group by the corresponding 06:00 h group for each replicate. Significant differences between experimental groups of P. ambiguum and M. aeruginosa were evaluated using a Student's t-test, assuming equality of variance. Pearson's correlation analysis was used to evaluate correlations between parameters. Statistical analyses were performed by using IBM SPSS Statistics for Windows, Version 25.0. (IBM Corp, Armonk, NY, USA).

Data Analysis
One-way analysis of variance (ANOVA), followed by Tukey's post-hoc test, was performed to test the statistical significance of variations among the means of sample groups. Data were normalized relative to the starting group (06:00 h), by dividing the results of each group by the corresponding 06:00 h group for each replicate. Significant differences between experimental groups of P. ambiguum and M. aeruginosa were evaluated using a Student's t-test, assuming equality of variance. Pearson's correlation analysis was used to evaluate correlations between parameters. Statistical analyses were performed by using IBM SPSS Statistics for Windows, Version 25.0. (IBM Corp, Armonk, NY, USA).

Discussion
The H 2 O 2 contents and the antioxidant activities of P. ambiguum and M. aeruginosa were highly responsive to the diurnal variations in light intensity. In this study, the only variable factor was the light intensity, where H 2 O 2 levels were high during times of higher light intensities and decreased at lower light intensities. When cellular H 2 O 2 level increases, the antioxidant activities correspondingly increase to prevent damage induced by oxidative stress [49,50]. As observed with the H 2 O 2 levels, the antioxidant activities also varied during the same time frame and followed the H 2 O 2 levels, which increased at higher light intensities and decreased at lower light intensities. The antioxidant activities of both species were correlated linearly with the H 2 O 2 contents. Although the H 2 O 2 -antioxidant relationships were varied from strong to weak (depending on the antioxidant species), overall, our findings suggest that the antioxidant levels of both species responded to the cellular H 2 O 2 level accordingly.
The H 2 O 2 and antioxidant responses followed the same trends for both maximum light intensity conditions (PAR intensities of 300 µmol·m −2 ·s −1 or 600 µmol·m −2 ·s −1 ). Under the maximum PAR intensity of 600 µmol·m −2 ·s −1 , the cyanobacteria received approximately twice the photon energy of the group with a maximum PAR of 300 µmol·m −2 ·s −1 . Therefore, it can be anticipated that the experiment groups, which receive higher photon energy, undergo an enhanced rate of photosynthesis. This is evidenced by the increased H 2 O 2 formed after exposure to a higher light intensity [51,52]. However, at higher light intensities, where the photon energy exceeds tolerable levels for the photosystem, photoinhibition occurs to prevent photodamage [53,54], during which H 2 O 2 production is reduced with higher light exposure [55][56][57]. As the H 2 O 2 contents correlated directly with light intensity, even at higher intensities, PAR intensities under 600 µmol·m −2 ·s −1 did not subject either cyanobacterial species to photo stress. However, this study only involved a single day diurnal variation, and the H 2 O 2 levels of the cells did not reach the starting H 2 O 2 conditions at 06:00 h (even at 21:00 h) for either species. The antioxidant activities almost decreased to the initial conditions by 21:00 h. Therefore, cells may undergo oxidative stress during dark conditions due to the lack of antioxidant activities. If the H 2 O 2 was continued to be presence in cells, the protein synthesis of photosystems will be inhibited [36] and, in long duration, cell function will be reduced and even cell deaths may occur [58]. Therefore, an extended exposure period is required to better understand the fate of the remaining H 2 O 2 and adaptation responses.
The antioxidant levels differed between the two species, where the response level was lower for M. aeruginosa than P. ambiguum, except for GPX. Under high H 2 O 2 contents, the AOX activity was highly elevated in P. ambiguum, but in the dark, both species reached the starting AOX activity level at 21:00 h. This finding suggests that M. aeruginosa is less tolerant to oxidative stress than P. ambiguum [36,59]. Concerning the correlation between antioxidant responses and H 2 O 2 contents, both species demonstrated significant linear relationships (with the only exception being for GPX of P. ambiguum under a maximum PAR of 300 µmol·m −2 ·s −1 ). Therefore, despite the high AOX content of the P. ambiguum, both species were able to maintain balanced antioxidant activity under every light condition of the single day exposure.
The difference in antioxidant levels of the two species can be related to their behavioral characteristics. The M. aeruginosa is a buoyant species that floats in a range of depths and might have higher tolerance to oxidative stress [60,61] than benthic P. ambiguum. However, in the present study, both species experienced same light intensity variance as the cultures were mixed periodically. The non-different H 2 O 2 contents between the two species suggested that both species experienced similar levels of oxidative stress. Therefore, less oxidative stress tolerance of P. ambiguum triggered the antioxidant activity at a relatively higher rate. Conversely, nonenzymatic antioxidants, primarily carotenoids, protect against ROS in phototrophs, including cyanobacteria [36,62]. The nonenzymatic antioxidants can neutralize ROS prior to triggering the antioxidant enzymes. However, the carotenoid content is reported to be higher in P. ambiguum than M. aeruginosa [63][64][65]; therefore, it is challenging to determine whether the low antioxidant activity reported in M. aeruginosa is due to involvement of nonenzymic antioxidant over the P. ambiguum.
Our previous study on the effects of 8 days of exposure to non-varying, high-light intensities (300 µmol·m −2 ·s −1 and 600 µmol·m −2 ·s −1 ) confirmed that the OD 730 and chlorophyll-a contents of cyanobacteria (Pseudanabaena galeata and M. aeruginosa) were significantly reduced, which was associated with oxidative stress [60]. Although the present research confirmed the relationships between varying oxidative stress and antioxidant responses with light intensity, further investigation into the longer-term effects on the growth and pigmentation of cyanobacteria is warranted. Longer exposure duration will help to better understand the growth performance and physiological responses of cyanobacteria to diurnally varying light conditions. Further, there can be a circadian rhythm in the physiology of cyanobacteria, for which the cellular conditions can be changed diurnally, regardless of the prevailing conditions [66]. In future studies, the circadian rhythm of the cyanobacterial species should also be considered.