(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure [1]

['Emma T. Brentjens', 'Oak Ridge Institute For Science', 'Education Research Participation Program Hosted U.S. Environmental Protection Agency', 'Oak Ridge', 'Tn', 'United States Of America', 'Elizabeth A. K. Beall', 'Robert M. Zucker', 'Public Health', 'Integrated Toxicology Division']

Date: 2023-12

M. aeruginosa fluorescent changes were observed using a Cytek Aurora spectral flow cytometer that contains 5 lasers and 64 narrow band detectors located between 365 and 829 nm. Cyanobacteria were treated with different concentrations of H 2 O 2 and then monitored after exposure between 1 and 8 days. The red fluorescence emission derived from the excitation of cyanobacteria with a yellow green laser (550 nm) was measured in the 652–669 nm detector while green fluorescence from excitation with a violet laser (405 nm) was measured in the 532–550 nm detector. The changes in these parameters were measured after the addition of H 2 O 2 . There was an initial increase in red fluorescence intensity at 24 hours. This was followed by a daily decrease in red fluorescence intensity. In contrast, green fluorescence increased at 24 hours and remained higher than the control for the duration of the 8-day study. A similar fluorescence intensity effect as H 2 O 2 on M. aeruginosa fluorescence emissions was observed after exposure to acetylacetone, diuron (DCMU), peracetic acid, and tryptoline. Minimal growth was also observed in H 2 O 2 treated cyanobacteria during exposure of H 2 O 2 for 24 days. In another experiment, H 2 O 2 -treated cyanobacteria were exposed to high-intensity blue (14 mW) and UV (1 mW) lights to assess the effects of light stress on fluorescence emissions. The combination of blue and UV light with H 2 O 2 had a synergistic effect on M. aeruginosa that induced greater fluorescent differences between control and treated samples than exposure to either stimulus individually. These experiments suggest that the early increase in red and green fluorescence may be due to an inhibition in the ability of photosynthesis to process photons. Further research into the mechanisms driving these increases in fluorescence is necessary.

Funding: The funding for this research was provided through the intermural research funds of the Office of Research and Development, U.S. Environmental Protection Agency, Center for Public Health and Environmental Assessment, Public Health and Integrated Toxicology Division. All the funding for this project was derived from internal ORD sources. Robert Martin Zucker (RMZ) conducted this research as a component of his regular job assignments at the USEPA. ETB and EAKB were supported by an appointment to the ORISE Research Participation Program for the U.S. Environmental Protection Agency, Office of Research and Development, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. EPA. The funding for this research was entirely supported with internal funds from the USEPA. RMZ is a principal investigator at the EPA and supported by the EPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The data that was obtained is the sole property of the EPA and is not owned by any commercial company. We use FCS express software (third-party) as it has added features that can be used to analyze Cytek spectral data that are not available in the Cytek Spectra Flo operating system. The data belongs to the EPA and not the Cytek company or a third-party company. The data is collected on a Cytek Aurora flow cytometer which is owned by the EPA. The standard procedure that we use with the generated data is to convert the Spectra Flo data into FCS 3.0 files which can be used by an EPA scientist or by other investigators that have third-party software. Converting data from a flow cytometer to FCS files is a standard procedure used by the flow cytometry community to analyze data for further analysis. The authors did not have any special access privileges from the Cytek company or a third-party software company. The Spectra Flo data can be used by individuals that have a Cytek flow cytometer or have access to any third-party software that can read FCS 3.0 files.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

The goal of this study is to examine the impact of H 2 O 2 and light stress on M. aeruginosa fluorescence using flow cytometry (FCM). We ask the following questions: 1) how do different doses of H 2 O 2 affect M. aeruginosa fluorescence and morphology over time? 2) how does H 2 O 2 compare to the effects of other photosynthesis inhibitory chemicals, and 3) how does M. aeruginosa fluorescence and morphology change with the addition of light exposure to H 2 O 2 treatments? We hypothesize that the effect of H 2 O 2 on M. aeruginosa cells results in an increased green fluorescence and decreased red fluorescence intensity in a dose- and time-dependent manner. Exposing H 2 O 2 -treated cells to bright UV-A or blue light will increase damage to cells based on wavelength, duration of exposure, and light intensity. We expect that autofluorescence data from this flow cytometry experiment may provide an indicator of stress and perturbation in M. aeruginosa.

High intensity light can also disrupt photosynthesis. Multiple studies have shown that excessive light exposure results in increased fluorescence emissions from cyanobacteria [ 6 , 26 , 27 ]. The combination of light and chemical exposure results in increased toxicity to cyanobacteria, especially at low wavelengths [ 17 , 18 , 20 , 22 , 28 ]. For example, UV radiation is known to be harmful to a variety of organisms and ecosystems [ 29 ]. Due to the increased efficacy of pairing light with H 2 O 2 treatments, multiple researchers have suggested that implementing H 2 O 2 in natural water systems on sunny days may enhance the treatment [ 30 , 31 ].

H 2 O 2 also reduces photosynthetic yield and electron transport rates of PSI and PSII, inhibiting cyanobacteria growth [ 17 , 25 ]. Previous research suggests that H 2 O 2 does not damage PSII proteins directly but targets the D1 protein located in the PSII reaction center, which regulates PSII repair [ 17 ]. A damaged PSII reaction center cannot absorb light as efficiently, which affects fluorescence [ 17 ]. Thus, analyzing fluorescence can help researchers better understand how H 2 O 2 impacts cyanobacteria physiology.

H 2 O 2 damages cyanobacteria cell structure, causing increased permeability of the cell membrane and destruction of the thylakoid membrane [ 17 , 21 ]. This occurs when H 2 O 2 forms hydroxyl radicals, which can damage membranes, proteins, and DNA [ 22 , 23 ]. H 2 O 2 is also an oxidizing agent that disrupts electron transport, resulting in the inhibition of photosynthesis [ 16 , 24 ].

Hydrogen peroxide (H 2 O 2 ) is one chemical commonly added to water systems to reduce cyanobacteria growth. H 2 O 2 is a reactive oxygen species (ROS), making it an effective chemical for targeting cyanobacteria, which are sensitive to oxidative stress [ 20 ].

Chemicals and intense light have been proposed to affect the functioning of cyanobacteria. These treatments seem to interfere with D1 protein synthesis, the transfer of light from phycobilisomes to PSII and/or the transfer of electrons from PSII to PSI [ 14 – 19 ]. Inhibitions to photosynthesis in cyanobacteria are likely to increase fluorescence emissions from pigments located prior to the spectra where they are utilized for energy production and glucose generation.

Cyanobacteria PBSs contain three main pigments: phycocyanin (PC) and allophycocyanin (APC), which are found in all cyanobacteria, and occasionally phycoerythrin (PE), which is mainly present in marine cyanobacteria [ 5 ]. PE in cyanobacteria absorbs blue and green light (absorption max. of ~565 nm), emits green light (~580 nm), and transfers energy to PC [ 10 ]. PC emits red fluorescence and transfers energy to APC [~640 nm; 11 ]. APC predominantly excites with red light and emits red light [~660 nm; 11 , 12 ]. Energy is then transferred from APC to the reaction center in photosystem II [PSII; 11 ], the first stage of the light dependent reactions in photosynthesis [ 13 ]. Energy transfer from APC to chlorophyll a in PSII results in fluorescence emissions at 695 nm [ 8 ]. These photons are used to produce energy, generate oxygen, and drive the Calvin cycle. There is a large Stokes shift of around 100–200 nm during this process depending on the excitation wavelength. This transfer of photons from the visible range to the far-red range by phycobilisomes is essential for photosynthesis to work in cyanobacteria.

Light absorbed by cyanobacteria may be used for photosynthesis, dissipated as heat, or emitted as fluorescence from pigments related to photosynthetic processes [ 4 ]. Phycobilisomes (PBSs), the light harvesting antennae of cyanobacteria, contain pigments that mainly excite with green and orange light and emit peak red (660- nm) fluorescence [ 5 , 6 ]. Chlorophyll also emits red (685–695 nm) fluorescence predominantly when excited with blue light [460 nm; 7 – 9 ]. Changes in fluorescence emissions from these pigments may provide information regarding photosynthetic and physiological changes in cyanobacteria.

Cyanobacteria, which are photosynthetic prokaryotes, proliferate rapidly to form harmful algal blooms (HABs) with detrimental effects on environmental conditions and drinking water quality. Cyanobacterial blooms constitute a public health threat due to the cyanotoxins they produce, which can cause acute and chronic illness in humans and even death in domestic animals [ 1 – 3 ]. Therefore, detecting and reducing the presence of cyanobacteria in water systems is critical to human and ecosystem safety.

Data was acquired in the Cytek Aurora flow cytometer using the Spectro Flo operating system software. The data was exported into FCS 3.0 files to enable the utilization of FCS Express (version 7.16, De Novo Software, Pasadena, CA) software that can be used to analyze the data. The samples were initially gated using a region defined by forward scatter area (blue laser) and side scatter area (violet laser). These cells from the scatter gate were then observed in a cytogram defined by the fluorescence channels YG4 (652–669 nm) on the y-axis and V7 (533–550 nm) on the x-axis to study the population of cyanobacteria. Mean light scatter and mean fluorescent data were exported into Microsoft Excel for analysis.

To assess how H 2 O 2 and light exposure interact to affect M. aeruginosa fluorescence, control and H 2 O 2 -treated M. aeruginosa cells were placed under blue and green NIGHTSEA lights (Electron Microscopy Sciences, Hatfield, PA) and 1-foot-long UV LED lamps. The cells were exposed to blue (440–460 nm) light at intensities of 14 and 7 mW, green (510–540 nm) light at intensities of 15 and 6 mW, and UV-A (395 nm) light at 1 and 0.5 mW. Different light intensities were achieved by placing the sample tubes at different distances from the light source. Light intensity was measured using a Laser Mate-Q detector (Coherent, Santa Clara, California) with either a visible or UV probe dependent on the wavelength measured. Each light treatment included control M. aeruginosa cells and H 2 O 2 -treated samples at concentrations of 750, 75, and 7.5 μg/mL. Cell fluorescence was measured on the flow cytometer after an initial 2.5 hours of continuous light exposure, an overnight recovery period under a white grow light at 350 LUX, and a second 2.5-hour light exposure, and then at 24 hours, 48 hours, 72 hours, and 8 days after initial light and H 2 O 2 exposure. When the cells were not exposed to green, blue, or UV light, they were incubated under a standard grow light with a 12-hour light-dark cycle.

Acetylacetone (product number: P7754), diuron (DCMU, product number: D2425), peracetic acid (product number: 269339), and tryptoline (product number: 300764) were obtained from Sigma-Aldrich (St. Louis, MO) added to M. aeruginosa samples to compare their effect on fluorescence to that of H 2 O 2 . The chemicals were added to 1mL M. aeruginosa samples at the following concentrations: 740, 74, 7.4 μg/mL acetylacetone (diluted to 2.97% in DI water); 0.5, 0.2, 0.1 μg/mL DCMU (diluted to 1% in ethanol); 800, 80, 8, 0.8 peracetic acid (dilute to 3.2% in DI water); and 75, 7.5, 0.75 μg/mL tryptoline (diluted to 1% in DMSO). Samples were measured on the flow cytometer at 48 and 72 hours. The different chemical treatments added to M. aeruginosa are outlined in Table 1 .

To determine the growth of samples treated with different doses (75–750 μg/mL) of H 2 O 2 , cells were counted with the flow cytometer at 0, 5, 8, 17 and 24 days after the addition of H 2 O 2 . The growth curves of control and treated cells in addition to the spectral charts and cytograms are displayed in the results.

Samples were measured on the flow cytometer at 24 hours, 48 hours, 72 hours, and 8 days to observe how M. aeruginosa cell fluorescence and morphology changed over time in response to different H 2 O 2 concentrations. 200 uL of each sample was measured between day 1 and day 3. At day 8, 200 uL of DI water was added to reduce the cell count to less than 1,500 per second, which also decreased the abort rate.

Five mL of a growing M. aeruginosa culture was diluted around 3x (to 16 mL) with BG-11 growth media (Sigma, St. Louis, MO). Ten doses of 3% commercial H 2 O 2 were added to 1 mL M. aeruginosa samples at the following concentrations: 3000, 1500, 750, 300, 150, 75, 30, 15, 7.5, and 3 μg/mL. Two 1 mL control samples with no chemicals added were also included.

Twenty-thousand cells for each sample were counted at a slow flow rate (10–20 μL/min) in a defined region of interest (ROI) in a FSC and SSC-violet cytogram. Counts were recorded within the ROI. A dual FSC and SSC threshold was used to remove the noise in the system. The flow cytometer underwent a quality assurance procedure with Spectro Flo 2000 series (lot 2003) 3 μm QC beads to check for acceptable CV values daily prior to use. The CVs of the beads measured in the majority of the 64 detectors were usually around 2–3%.

A Cytek Aurora flow cytometer was used to obtain fluorescent data from M. aeruginosa. The system contains five lasers (355 nm UV, 405 nm violet, 488 nm blue, 561 nm yellow-green, and 640 nm red) and 64 detectors. The order, wavelength, and power of the five lasers are as follows: yellow-green (561 nm, 50 mW), violet (405 nm, 100 mW), blue (488 nm, 50 mW), red (638 nm, 80 mW), and UV (355 nm, 20 mW). The laser excitation of the cells is separated by 20 μs. The emission spectrum from each laser was collected with 8–16 detectors, each consisting of 17–25 nm in size (Cytek Biosciences Inc., Fremont, CA). The machine also measures forward scatter (FSC) with the blue laser and measures two side scatter (SSC) channels with the blue and violet lasers. FSC refers to the scatter of light around the cell and is often used as a proxy measurement for cell size. SSC is a measure of cell granularity, determined by the reflection of light off structures inside the cell [ 32 ].

Lab-grown cultures of Microcystis aeruginosa, a species commonly found in cyanobacterial blooms, were used to test the effects of H 2 O 2 on cyanobacteria fluorescence. M. aeruginosa are round cells ranging 4–5 μm in size. This cyanobacteria species produces multiple toxins including microcystin [ 2 ]. Cells were grown in BG-11 media in 15 mL polystyrene conical tubes under a 300–350 LUX grow light (T5 Grow Light Bulbs 2 ft, 24W 6500K).

Results

The addition of H 2 O 2 to cyanobacteria results in dose dependent changes of fluorescence and scatter between 24 and 72 hours. For these studies photosynthesis emission ranges of 600 to 829 nm were designated as “red fluorescence” and the spectral ranges between 450 and 580 nm were designated as “green fluorescence.” There was an initial increase in both red and green fluorescence at 24 hours. At higher doses the red fluorescence decreases after 48 and 72 hours while the green fluorescence does not decrease (Fig 1). The forward light scatter generally decreases at most concentrations compared to control cells.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Dose dependent changes in fluorescence and forward scatter from cyanobacteria treated with H 2 O 2 . Fig 1 shows the dose dependent change in red fluorescence in one detector (652–669 nm)from a yellow green laser and green fluorescence in one detector (533–550 nm) from a violet laser for doses between 3 and 3000 μg/mL during a 3-day period (24–72 hours). There is an increase in green and red fluorescence at 24 hours. At higher doses there is an increase in green fluorescence and decrease in red fluorescence and forward light scatter relative to control cells. https://doi.org/10.1371/journal.pwat.0000177.g001

The decrease in forward light scatter may influence the detection of green and red fluorescence from the cyanobacteria. To normalize for the effect that size may have on fluorescence intensity emission after incubation of cyanobacteria with H 2 O 2 , the fluorescence measurements were divided by the relative change in forward scatter. Even after this normalization procedure, an increase in fluorescence after treatment with H 2 O 2 was apparent (Fig 2).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Dose dependent changes in fluorescence from cyanobacteria treated with H 2 O 2 after normalization with forward scatter. By dividing the green fluorescence (lower panels) and red fluorescence (upper panels) by the relative forward light scatter parameter shown in Fig 1,the graphs show that the resultant fluorescence actually increased with almost all doses with the highest values being at around 3000 μg/mL for red fluorescence. Green points represent values derived from the violet laser using the green detector (533–550 nm) and red points represent intensity values derived from the yellow-green laser in the red detector range (652–669 nm). https://doi.org/10.1371/journal.pwat.0000177.g002

The flow cytometer detected an increase in both red (615–829 nm) and green fluorescence (458–588 nm) in M. aeruginosa after 48 hours of exposure to 750 μg/mL H 2 O 2 relative to the control as shown in the cytogram (Fig 3). The data for 48-hour control and H 2 O 2 treated cells are displayed as a cytograms consisting of green fluorescence emitted from violet laser excitation (533–550 nm) vs red fluorescence emitted from yellow-green laser excitation (652–669 nm) (Fig 3).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. The detection of red (652–669 nm) and green (533–550 nm) fluorescence from yellow-green and violet laser excitation in control and H 2 O 2 -treated M. aeruginosa after 48 hours. Density cytograms show red fluorescence from 561 nm laser excitation and green fluorescence from the 405 nm laser excitation. There is an increase in both red and green fluorescence in the 750 μg/mL H 2 O 2 sample (B) relative to the control (A) after 48 hours of exposure. The red color in the figure represents higher density of cells while dark blue represents lower density of cells. The purple outline indicates the control region (based on the population in A). B shows the movement of cells out of the control region (A) after incubation with H 2 O 2 for 48 hours. https://doi.org/10.1371/journal.pwat.0000177.g003

The increase in red and green fluorescence was displayed as Cytek fluorescence intensity spectra (Fig 4). Intensity curves were generated by calculating the mean relative fluorescence intensity in each channel using FCS Express version 7.16 The x-axis shows the individual detectors designated by Cytek. Intensity curves show relative fluorescence intensity (y-axis) with UV (355 nm), violet (405 nm), blue (488 nm), yellow-green (561 nm), and red (640 nm) laser excitation of control (black) and 750 μg/mL H 2 O 2 (red) cells at 48 hours of exposure. The emissions occur from the following ranges: UV: 372 to 829 nm in 16 detectors; violet: 420 to 829 nm in 16 detectors; blue: 498 to 829 nm in 14 detectors; yellow-green: 567 to 829 nm in 10 detectors; and red (660 to 829 nm in 8 detectors.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Fluorescence intensity curves of control and 750 μg/mL H 2 O 2 M. aeruginosa samples from UV, violet, blue, yellow-green, and red laser excitation. Intensity curves show relative fluorescence intensity (y-axis) with UV (355 nm), violet (405 nm), blue (488 nm), yellow-green (561 nm), and red (640 nm) laser excitation of control (black) and 750 μg/mL H 2 O 2 (red) cells at 48 hours of exposure. Intensity curves were generated by calculating the mean relative fluorescence intensity in each channel using FCS Express version 7.16 The x-axis shows the individual detectors designated by Cytek. The emissions occur from the following ranges: UV: 372 to 829 nm in 16 detectors; violet: 420 to 829 nm in 16 detectors; blue: 498 to 829 nm in 14 detectors; yellow-green: 567 to 829 nm in 10 detectors; and red (660 to 829 nm in 8 detectors. There are ~660 nm fluorescence peaks from the H 2 O 2 -treated sample, while the control sample does not exhibit this increase in fluorescence emissions at 660 nm. H 2 O 2 -treated cells showed an increase in the green fluorescence ranges with violet and blue laser excitation. https://doi.org/10.1371/journal.pwat.0000177.g004

Fig 5 demonstrates a summary of the time- and dose-dependent effect of H 2 O 2 on M. aeruginosa fluorescence. The increase in red fluorescence was followed by a decrease after 72 hours, while the increased green fluorescence relative to the control was maintained after 8 days of H 2 O 2 exposure (Fig 5). The addition of H 2 O 2 caused a dose- and time-dependent change in red and green fluorescence, with red fluorescence emissions initially increasing by up to 240% relative to the control before eventually decreasing by about 1000 times as shown in Fig 5. In contrast to red fluorescence, green fluorescence increased at 24 hours by up to about 570% and remained higher than the control for the duration of the 8-day study.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. Pattern of M. aeruginosa fluorescence change with exposure to 750 μg/mL H 2 O 2 . A is a density cytogram showing red fluorescence (emission: 652–669 nm, excitation: 561 nm) on the y-axis and green fluorescence (emission: 533–550 nm, excitation: 405 nm) on the x-axis of control and 750 μg/mL H 2 O 2 M. aeruginosa samples after 24 hours, 72 hours, and 8 days. Red represents high density of cells and dark blue represents low density. B is a diagram showing the direction of M. aeruginosa fluorescence changes. Control M. aeruginosa populations began with high red fluorescence and low green fluorescence emissions; red and green fluorescence increased 24 hours later, then red fluorescence decreased after 72 hours and 8 days. https://doi.org/10.1371/journal.pwat.0000177.g005

The red fluorescence of most treated samples was greater than the control after 24 and 48 hours of H 2 O 2 exposure (Table 2). On day 8, almost all treated samples had a lower red fluorescence (ratio < 1) than the control (Table 2). Green fluorescence emissions in most of the treated samples increased steadily in the first 72 hours of exposure. Green fluorescence began to decline in samples with H 2 O 2 concentrations of 300 μg/mL and greater by day 8. However, these samples still exhibited a higher green fluorescence (ratio > 1) than the control on day 8 (Table 2).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Table 2. Red and green fluorescence of M. aeruginosa cells exposed to H 2 O 2 over time relative to control. H 2 O 2 -treated M. aeruginosa cells exhibited changes in red (emission: 652–669 nm, excitation: 561 nm) and green (emission: 533–550 nm, excitation: 405 nm) fluorescence in a dose- and time-dependent manner as shown in Fig 1. Values indicate ratio of fluorescence to the control sample (0 μg/mL H 2 O 2 ). Treated cells initially emitted increased red fluorescence (24–48 hours) followed by a decline starting at 72 hours for high dose samples (300–3000 μg/mL) and 8 days for the remaining treated samples. Green fluorescence increased in most treated samples for the first three days before declining in high dose samples after 8 days. The green fluorescence of all treated samples remained higher than the control. Numbers highlighted red in the chart indicate at least 30% lower fluorescence than control. Ratios of 0.00 indicate numbers too small to show with two decimal places. https://doi.org/10.1371/journal.pwat.0000177.t002

Fig 6 shows a spectral map of the five lasers across all the detectors that correlates to the cytograms in Fig 5. At 24 hours, green and red fluorescence emissions from the H 2 O 2 -treated cells was greater than the control in all 64 diode detectors than the control. At 72 hours, the fluorescence in the red range was less than the control while the fluorescence in the green range was greater than the control cells. At 8 days, the green fluorescence from the UV, violet, and blue lasers was greater while the red fluorescence was much lower than the control. The increased fluorescence after H 2 O 2 exposure appears more pronounced in the blue and green emission spectral ranges at all time points with the UV and violet, lasers excitation (Fig 6). The intensity curves also reveal a fluorescence peak at around 660 nm from the violet, blue, and yellow-green lasers (Fig 6). This peak is not present in control cells with violet and blue laser excitation (Fig 6). Treated and control cells exhibit peaks at 660 nm and 697 nm with yellow-green laser excitation, which are higher in treated samples after 24 and 48 hours (Fig 6).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Fluorescence intensity curves of control and 750 μg/mL H 2 O 2 M. aeruginosa samples from UV, violet, blue, yellow-green, and red laser excitation. Intensity curves show fluorescence emission intensity (y-axis) derived with five lasers: UV (355 nm), violet (405 nm), blue (488 nm), yellow-green (561 nm) and red (640 nm). The excitation of control (black, 24 hour) and 750 μg/mL H 2 O 2 cells at 24 hours (red), 72 hours (blue), and 8 days (green). Intensity curves were generated by calculating the mean fluorescence intensity in each channel using FCS Express version 7.12. The x-axis shows the individual detectors designated by Cytek. The emissions occur from the following ranges: UV: 372 to 829 nm in 16 detectors; violet: 420 to 829 nm in 16 detectors; blue: 498 to 829 nm in 14 detectors; yellow-green: 567 to 829 nm in 10 detectors; and red: 660 to 829 nm in 8 detectors. There are ~660 nm fluorescence peaks from the H 2 O 2 -treated sample, while the control sample does not exhibit this increase in fluorescence emissions at 660 nm. H 2 O 2 -treated cells showed an increase in the green fluorescence ranges with UV, violet, and blue laser excitation at all time points. https://doi.org/10.1371/journal.pwat.0000177.g006

Overall, red fluorescence eventually decreased with greater H 2 O 2 exposure time while green fluorescence generally increased over the same period. Fig 7 further illustrates dose- and time-dependent effects of H 2 O 2 on M. aeruginosa fluorescence. Over time and with higher doses of H 2 O 2 , treated populations show increased fluorescence intensity and shift farther to the right (greater green fluorescence) from the control region. In the higher concentration (750 μg/mL), 0% of the population remains in the control region after 72 hours while about 32% of the lower concentration (7.5 μg/mL) sample remains in the control region after 8 days.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. Dose- and time-dependent response of M. aeruginosa fluorescence to H 2 O 2 exposure. The density cytograms show red fluorescence (emission: 652–669 nm, excitation: 561 nm) on the y-axis and green fluorescence (emission: 533–550 nm, excitation: 405 nm) on the x-axis of control, 750 μg/mL, 75 μg/mL, and 7.5 μg/mL H 2 O 2 M. aeruginosa samples after 24 hours, 72 hours, and 8 days. Red represents a high density of cells and dark blue represents low density of the cells. The blue gate shown in all the cytograms represents the region containing control cells and serves as a reference for changes in fluorescence of H 2 O 2 treated cells. The population located within the control region decreased to 0% after 24 hours for both the 750 and 75 μg/mL H 2 O 2 samples. In contrast, the percentage of cells treated with low dose (7.5 μg/mL) H 2 O 2 found in the control region decreased each day and remained around 30% on day 8. https://doi.org/10.1371/journal.pwat.0000177.g007

M. aeruginosa cells also exhibited changes in FSC and SSC in response to H 2 O 2 exposure. Both FSC and SSC generally decreased in relative size with increasing H 2 O 2 concentration (S1 Table). At 48 hours, each dose used with the exception of the 30 μg/mL dose had lower FSC and SSC values compared to that of the control. FSC was also negatively associated with exposure time, decreasing over the 8-day study period in most samples (S1 Table).

The observed pattern of increased red and green fluorescence shown in the cytograms (Figs 5 and 7) was consistent (Table 3). The cyanobacteria were treated with H 2 O 2 in three separate experiments. The green and red fluorescence of the control and two doses of H2O2 was averaged to derive the mean fluorescence and standard deviation. These experiments show an increase in green fluorescence which was maintained at 48 and 72 hours. An initial increase in red fluorescence intensity was followed by a decrease in intensity.

Fig 8 shows that M. aeruginosa fluorescence was affected by acetylacetone (AA), diuron (DCMU), peracetic acid (PAA) and tryptoline (tryp) in a similar manner as H 2 O 2 at various doses. Each chemical caused increased fluorescence emissions in the green spectral range and all samples except tryptoline caused increased red fluorescence at 48 hours of exposure compared to control. The largest green fluorescence effects were observed with the violet laser, although the trend of increased fluorescence could be observed with the UV, blue and yellow-green lasers, as well. The H 2 O 2 , acetylacetone, DCMU, and peracetic acid all caused a similar peak at 660 nm relative to the control with all the lasers (Fig 8). It should be emphasized that only one dose of these chemicals is displayed; the point of Fig 8 was primarily to show that the effect observed with H 2 O 2 also occurred with other chemicals that affected cyanobacteria.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 8. Fluorescence intensity curves of M. aeruginosa treated with PSII inhibitory chemicals from violet, blue, and yellow-green laser excitation. Intensity curves show fluorescence detected from UV (355 nm), violet (405 nm), blue (488 nm), yellow-green (561 nm), and red (640 nm) laser excitation for control (black) and 750 μg/mL H 2 O 2 -treated cells (red) after 48 hours compared to 740 μg/mL acetylacetone (blue), 200 μg/mL DCMU (green), 8 μg/mL peracetic acid (purple), and 75 μg/mL tryptoline (pink). Intensity curves were generated by calculating the mean relative fluorescence intensity in each channel. The x-axis shows the individual detectors designated by Cytek. The emissions occur from the following ranges: UV: 372 to 829 nm in 16 detectors; violet: 420 to 829 nm in 16 detectors; blue: 498 to 829 nm in 14 detectors; yellow-green: 567 to 829 nm in 10 detectors; and red: 660 to 829 nm in 8 detectors. Similar to H 2 O 2 , the acetylacetone, DCMU, and peracetic acid samples exhibited fluorescent emission peaks at around 660 nm with excitation from all five lasers, but to a lesser extent. These three compounds and H 2 O 2 showed an increase in the green fluorescence ranges from UV, violet, and blue laser excitation. This peak at 660 nm was also observed with the violet laser in the tryptoline sample, but not with the blue or yellow-green lasers. https://doi.org/10.1371/journal.pwat.0000177.g008

Wavelengths below 460 nm had the greatest effect on H 2 O 2 -treated M. aeruginosa fluorescence. The effect of the H 2 O 2 + light exposure treatments varied by light intensity, wavelength, exposure time, and H 2 O 2 dose. Green light treatments did not noticeably impact M. aeruginosa fluorescence emissions while blue (440–460 nm) and UV-A (395 nm) light had a synergistic effect on changes in M. aeruginosa fluorescence when paired with H 2 O 2 treatments (Fig 9). The effect of blue and UV-A light alone without H 2 O 2 was not as great as the effect of H 2 O 2 alone. However, when paired together, H 2 O 2 and light caused greater changes in fluorescence, with green fluorescence emissions increasing and red fluorescence emissions decreasing at a faster rate. The greatest effect in these studies was observed in the 1 mW UV-A + 750 μg/mL H 2 O 2 sample. Red fluorescence had already begun to decrease to levels below the control after the second 2.5-hour light exposure (Fig 9). At 72 hours, red fluorescence decreased to levels below the control (Fig 9).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 9. Red and green fluorescence of M. aeruginosa after exposure to different wavelength light and H 2 O 2 at 72 hours. Density cytograms show red fluorescence (emission: 652–669 nm, excitation: 561 nm) on the y-axis and green fluorescence (emission: 533–550 nm, excitation: 405 nm) on the x-axis. In the cytograms, red represents higher density of cells and dark blue represents a lower density of cells. The purple gate represents the control region. Panel A consists of control cells (no treatment) and panel C consists of cells treated for 72 hours with 750 μg/mL H 2 O 2 . Panel B consists of cells treated with 1mw UV-A light and panel D consist of cells treated with 1 mW UV-A light and 750 μg/mL H 2 O 2 . Samples were exposed to UV-A light for 2.5 hours immediately after H 2 O 2 exposure and for another 2.5 hours at 24 hours. After the cells were exposed to UV-A lights for 2.5 hours, they were put under grow lights with a 12-hour light-dark cycle. UV light had a synergistic effect with H 2 O 2 on M. aeruginosa fluorescence. https://doi.org/10.1371/journal.pwat.0000177.g009

The high light intensity treatments (14 mW blue light and 1 mW UV-A light) had a greater effect on fluorescence than the low intensity treatments (7 mW blue light and 0.5 mW UV-A light; Table 4). Additionally, like control + H 2 O 2 cells, H 2 O 2 in light-exposed cells had a dose- and time-dependent effect on M. aeruginosa fluorescence, with higher doses of H 2 O 2 causing a greater increase in green fluorescence and decrease in red fluorescence over time (Table 4).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Table 4. Red and green fluorescence of M. aeruginosa cells exposed to H 2 O 2 and UV, blue, and green light over time relative to control. Cells were exposed to 3 different concentrations of H 2 O 2 and different illumination conditions with UV-A (0.5-mW and 1-mW), blue light (7-mW and 14-mW), and green light (6-mW and 15-mW). These data indicate a synergistic effect of H 2 O 2 + UV-A light (395 nm) and blue light (440–460 nm) dependent on H 2 O 2 dose and exposure time, and light intensity, which correlate with the data shown in Fig 7 for the 24- and 72-hour time points. Red fluorescence refers to 652–669 nm fluorescence with 561 nm excitation, and green fluorescence refers to 533–550 nm fluorescence with 405 nm excitation. Numbers highlighted red indicate at least 30% lower fluorescence than control. Ratios of 0.00 indicate numbers too small to show with two significant figures. https://doi.org/10.1371/journal.pwat.0000177.t004

The values in Table 4 indicate the ratio of red (emission: 652–669 nm, excitation: 561 nm) and green (emission: 533–550 nm, excitation: 405 nm) fluorescence relative to the control sample (0 μg/mL H 2 O 2 , no light treatment). The effect of H 2 O 2 and light exposure on red fluorescence was most pronounced in the 1 mW UV-A treatment, followed by the 14-mW blue treatment. The same treatments also elicited the greatest increase in green fluorescence. Green fluorescence intensity had begun to decrease after 72 hours in the 750 μg/mL H 2 O 2 + 1 mW UV treatment and 8 days in the 750 μg/mL H 2 O 2 + 14 mW blue light treatment, but still remained higher than green fluorescence in the control sample. Similar changes were observed at 24 and 48 hours but the magnitude of the change was not as great as that observed with the 72-hour point that showed minimal effects with only UV-A light but a synergistic effect when UV-A light was combined with H 2 O 2.

Cells were treated with 1) UV-A + H 2 O 2 2) blue light + H 2 O 2 and 3) H 2 O 2 and their fluorescence were compared. Fig 10 further demonstrates the synergistic effect of 750 μg/mL H 2 O 2 paired with 14 mW blue or 1 mW UV-A light. The treatments with light and H 2 O 2 showed lower red fluorescence, indicating the synergistic effect of light interacting with H 2 O 2 (Figs 9 and 10). Each curve shows singular peaks at 660 nm with UV, violet, and blue laser excitation and peaks at 660 nm and 697 nm with yellow-green excitation.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 10. Fluorescence intensity curves of M. aeruginosa after H 2 O 2 and light treatments from UV, violet, blue, yellow-green, red laser excitation. Intensity curves show relative emission fluorescence intensity (y-axis) derived with five lasers: UV (355 nm), violet (405 nm), blue (488 nm), yellow-green (561 nm) and red (640 nm). Cells were treated with 1) UV-A + H 2 O 2 2) blue light + H 2 O 2 and 3) H 2 O 2 and their fluorescence were compared. Cells exposed to both 14 mW blue (blue) and 1 mW UV-A light (green) showed increased fluorescence in the blue and green spectral ranges from UV, violet, and blue laser excitation compared to cells treated only with 750 μg/mL H 2 O 2 (red). The UV-A + H 2 O 2 -treated cells had the greatest decline in red fluorescence from all five lasers. Intensity curves were generated by calculating the mean relative fluorescence intensity in each channel using FCS Express version 7.16. The x-axis shows the individual detectors designated by Cytek. The emissions occur from the following ranges: UV: 372 to 829 nm in 16 detectors; violet: 420 to 829 nm in 16 detectors; blue: 498 to 829 nm in 14 detectors; yellow-green: 567 to 829 nm in 10 detectors; and red: 660 to 829 nm in 8 detectors. https://doi.org/10.1371/journal.pwat.0000177.g010

Among the light and H 2 O 2 -treated samples, FSC and SSC also varied in a dose- and time-dependent manner. The ratio of both FSC and SSC to the control decreased over time in most samples. Additionally, the highest H 2 O 2 concentrations (750 μg/mL) generally exhibited the lowest FSC and SSC values relative to the control at a given time point (S2 Table). One exception to this trend was the 7-mW blue light + 750 μg/mL H 2 O 2 sample, which had considerably higher FSC and lower SSC measurements than the rest of the samples at every stage of data collection.

Cyanobacteria cell counts were measured on a flow cytometer from samples treated with different concentrations of H 2 O 2 (75–750 μg/mL) to compare proliferation, cell death, and changes in fluorescence. It was found that H 2 O 2 treated samples did not increase in cell count for the 24- hour incubation period, in contrast to the control samples, which continued to proliferate (Fig 11).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 11. Cyanobacteria growth of treated and control cells. Cyanobacteria growth over 24 days is displayed for control and for 3 doses (75, 187.5, and 750 μg/mL) of H 2 O 2 treated cells in a line graph and table. The H 2 O 2 -treated cells did not grow, while the control cells proliferated. https://doi.org/10.1371/journal.pwat.0000177.g011

Control cells didn’t demonstrate a change in fluorescence over the 24 days. The H 2 O 2 treated cells experienced a sequential decrease in red fluorescence and an increase in green fluorescence, which remained higher than the control for the duration of the experiment. The magnitude of fluorescence intensity changes was dependent on H 2 O 2 dose and time after the chemical was added. The fluorescence data was derived from the lowest dose (75 μg/mL) and is displayed graphically using the five laser Cytek spectra and cytograms that compare the green fluorescence emissions (533–550 nm) from violet laser excitation and red fluorescence emissions (652–669 nm) from excitation with the yellow-green laser (Fig 12). Changes in red and green fluorescence can be observed even after cyanobacteria growth is inhibited.

[END]
---
[1] Url: https://journals.plos.org/water/article?id=10.1371/journal.pwat.0000177

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.