Effect of Zinc on Microcystis aeruginosa UTEX LB 2385 and Its Toxin Production

Cyanobacteria harmful algal blooms (CHABs) are primarily caused by man-made eutrophication and increasing climate-change conditions. The presence of heavy metal runoff in affected water systems may result in CHABs alteration to their ecological interactions. Certain CHABs produce by-products, such as microcystin (MC) cyanotoxins, that have detrimentally affected humans through contact via recreation activities within implicated water bodies, directly drinking contaminated water, ingesting biomagnified cyanotoxins in seafood, and/or contact through miscellaneous water treatment. Metallothionein (MT) is a small, metal-sequestration cysteine rich protein often upregulated within the stress response mechanism. This study focused on zinc metal resistance and stress response in a toxigenic cyanobacterium, Microcystis aeruginosa UTEX LB 2385, by monitoring cells with (0, 0.1, 0.25, and 0.5 mg/L) ZnCl2 treatment. Flow cytometry and phase contrast microscopy were used to evaluate physiological responses in cultures. Molecular assays and an immunosorbent assay were used to characterize the expression of MT and MC under zinc stress. The results showed that the half maximal inhibitory concentration (IC50) was 0.25 mg/L ZnCl2. Flow cytometry and phase contrast microscopy showed morphological changes occurred in cultures exposed to 0.25 and 0.5 mg/L ZnCl2. Quantitative PCR (qPCR) analysis of selected cDNA samples showed significant upregulation of Mmt through all time points, significant upregulation of mcyC at a later time point. ELISA MC-LR analysis showed extracellular MC-LR (µg/L) and intracellular MC-LR (µg/cell) quota measurements persisted through 15 days, although 0.25 mg/L ZnCl2 treatment produced half the normal cell biomass and 0.5 mg/L treatment largely inhibited growth. The 0.25 and 0.5 mg/L ZnCl2 treated cells demonstrated a ~40% and 33% increase of extracellular MC-LR(µg/L) equivalents, respectively, as early as Day 5 compared to control cells. The 0.5 mg/L ZnCl2 treated cells showed higher total MC-LR (µg/cell) quota yield by Day 8 than both 0 mg/L ZnCl2 control cells and 0.1 mg/L ZnCl2 treated cells, indicating release of MCs upon cell lysis. This study showed this Microcystis aeruginosa strain is able to survive in 0.25 mg/L ZnCl2 concentration. Certain morphological zinc stress responses and the upregulation of mt and mcy genes, as well as periodical increased extracellular MC-LR concentration with ZnCl2 treatment were observed.


Introduction
Cyanobacteria harmful algal blooms (CHABs) are described as toxigenic or irritating biomasses of mostly oxygenic, photosynthetic bacteria on the rise worldwide due to anthropogenic eutrophication (via excessive P and N loading) and increasing climate-change conditions [1][2][3][4][5][6]. Oftentimes, metal pollutant runoff in water systems may also affect the ecological interaction of a given CHAB population [7]. Certain essential element processes, such as iron's regulation by (FUR) uptake regulators, percent ingestion [42]. Cyanobacteria sensitivity, resistance or adaptive sequestration of heavy metal concentrations has been documented within both colonial CHABs and unicellular species [7,[43][44][45]. Aside from the observed zinc metal-complexing potential of MCs (MC-LR-Zn = −617 ± 7 kcal mol −1 ; MC-RR-Zn =−777 ± 9 kcal mol −1 ) [46], metallothioneins (MTs) are well documented metal-cation chelating, cysteine-rich proteins (<10 kDa) ubiquitously found in prokaryotes and eukaryotes [47,48]. MTs have been shown to be upregulated in different species of cyanobacteria when exposed to Zn 2+ or Cd 2+ concentrations while remaining relatively constant at basal levels [49,50]. Because of the variability of the MC synthetase gene cluster (encoding mcyABC-mcyD-J) in different cyanobacteria clades and within strains [51] and a relative conservation of MT cysteine domain sequences and motifs across cyanobacteria and bacteria [48], these genes may be possible quantification method candidates involving heavy metal zinc response and resistance in identified MC producing Microcystis species and strains.
While the use of quantitative PCR (qPCR) has yielded both successes and noncorrelation in relating MC synthetase gene copy numbers or gene expressions with collection site MC concentrations [52], qPCR remains a very powerful and accessible technique for the study of gene regulation in known toxigenic or identified cyanobacteria species [53]. Along with other quantitative analysis (HPLC, LC/MS, ELISA) and sequencing profiles, it may lead to the development of known metal-response gene standards for important identified toxic cyanobacteria species and strains. These parameters may better assist in determining toxic vs. non-toxic cyanobacteria response and resistance to heavy metal pollution.
The aim of this study was (1) to study the growth and physiological effects of zinc concentrations on an established toxigenic M. aeruginosa strain; (2) to design mcyC, mcyE, and Mmt qPCR oligonucleotides to quantify mcyC, mcyE and Mmt relative gene expression profiles of this strain treated with varying Zn 2+ concentrations; and (3) to determine relative quantitation of MC-LR equivalents within ZnCl 2 -treated M. aeruginosa using intracellular and extracellular portions.

Growth Response to ZnCl 2 in M. Aeruginosa UTEX LB 2385
To better evaluate the zinc metal resistance and response mechanisms of a globally important toxigenic cyanobacteria species, M. aeruginosa UTEX LB 2385 cultures were used for the observation of physiological responses to long-term ZnCl 2 concentrations exposure via growth monitoring ( Figure 1). For M. aeruginosa-UTEX LB 2385 cells exposed to 0.1 mg/L ZnCl 2 , the cell concentration was similar to 0 mg/L ZnCl 2 (control) through 15 days ( Figure 1). However, the average turbidity for M. aeruginosa UTEX LB 2385 culture cells exposed to all ZnCl 2 concentrations decreased below the initial control measurement (optical density, OD 750 nm ≈ 0.47) for 0.1, 0.25, and 0.5 mg/L ZnCl 2 by Day 1 (Figure 1b). When compared to control cells, M. aeruginosa UTEX LB 2385 culture cell numbers exposed to 0.25 mg/L ZnCl 2 were reduced by~28% by Day 15 ((4.73 ± 1.243/8.41 ± 0.122) × 100) (Figure 1a). At the highest concentration treatment (0.5 mg/L ZnCl 2 ), M. aeruginosa UTEX LB 2385 culture cells were almost completely inhibited in comparison to 0.25 mg/L ZnCl 2 cells by the end of the growth monitoring period. This concentration (0.5 mg/L ZnCl 2 ) may therefore present as the ZnCl 2 minimum inhibitory concentration (MIC) for M. aeruginosa UTEX LB 2385 within this study ( Figure 1). Aside from these observations, M. aeruginosa UTEX LB 2385 culture cells treated with 0.25 and 0.5 mg/L ZnCl 2 were observed via hemocytometer to possess larger aggregate cell clusters and extracellular debris compared to control cells.

Phase Contrast Microscopy Imaging in ZnCl 2 Exposed Cells
M. aeruginosa UTEX LB 2385 culture cells exposed to 0.25 mg/L ZnCl 2 possessed morphological and size characteristics similar to the control cells through eight days as single cells (Figure 2), but also showed an increase in multi-paired cell aggregation and size by Days 5 and 8 when observed via phase contrast microscopy. Additionally, though 0.5 mg/L ZnCl 2 treatment greatly inhibited M. aeruginosa  Figure 2). It also appeared a greater observable amount of multi-cell aggregation within 0.5 mg/L ZnCl 2 treated cells versus control cells. Flow cytometry was used to further evaluate the population sizes of ZnCl 2 treated M. aeruginosa UTEX LB 2385.

Phase Contrast Microscopy Imaging in ZnCl2 Exposed Cells
M. aeruginosa UTEX LB 2385 culture cells exposed to 0.25 mg/L ZnCl2 possessed morphological and size characteristics similar to the control cells through eight days as single cells (Figure 2), but also showed an increase in multi-paired cell aggregation and size by Days 5 and 8 when observed via phase contrast microscopy. Additionally, though 0.5 mg/L ZnCl2 treatment greatly inhibited M. aeruginosa UTEX LB 2385 culture cell numbers by Day 8 compared to the control, there was an observable number of cells possessing similar M. aeruginosa UTEX LB 2385 morphology to control cells ( Figure 2). It also appeared a greater observable amount of multi-cell aggregation within 0.5 mg/L ZnCl2 treated cells versus control cells. Flow cytometry was used to further evaluate the population sizes of ZnCl2 treated M. aeruginosa UTEX LB 2385.

Phase Contrast Microscopy Imaging in ZnCl2 Exposed Cells
M. aeruginosa UTEX LB 2385 culture cells exposed to 0.25 mg/L ZnCl2 possessed morphological and size characteristics similar to the control cells through eight days as single cells (Figure 2), but also showed an increase in multi-paired cell aggregation and size by Days 5 and 8 when observed via phase contrast microscopy. Additionally, though 0.5 mg/L ZnCl2 treatment greatly inhibited M. aeruginosa UTEX LB 2385 culture cell numbers by Day 8 compared to the control, there was an observable number of cells possessing similar M. aeruginosa UTEX LB 2385 morphology to control cells ( Figure 2). It also appeared a greater observable amount of multi-cell aggregation within 0.5 mg/L ZnCl2 treated cells versus control cells. Flow cytometry was used to further evaluate the population sizes of ZnCl2 treated M. aeruginosa UTEX LB 2385.

Flow Cytometry
Flow cytometry was used over the eight day time-course to evaluate the relative morphology size of M. aeruginosa UTEX LB 2385 culture cell populations treated with the highest ZnCl 2 concentrations (0.25 and 0.5 mg/L). The flow cytometry measurement (FCM) histogram profiles were ungated for subpopulations to evaluate overall distribution of single cell + multi-cell aggregates.

Flow Cytometry
Flow cytometry was used over the eight day time-course to evaluate the relative morphology size of M. aeruginosa UTEX LB 2385 culture cell populations treated with the highest ZnCl2 concentrations (0.25 and 0.5 mg/L). The flow cytometry measurement (FCM) histogram profiles were ungated for subpopulations to evaluate overall distribution of single cell + multi-cell aggregates.

Quantitative Polymerase Chain Reaction (qPCR) Analysis of M. Aeruginosa UTEX LB 2385
The qPCR analysis results were calculated as fold change gene expressions of Mmt, mcyC, and mcyE relative to the M. aeruginosa UTEX LB 2385 16S ribosomal RNA internal control, within the specific time period and samples (Days 1, 5, 12, 15 for 0, 0.1, and 0.25 mg/L ZnCl2) and the initial 0 mg/L ZnCl2 Day 1 sample. Fold change gene expression values were expressed as [2 -ΔΔCt ] and evaluated as a combined [log2(RQ)] heatmap within RStudio statistical software. The genes expressed in log2 > 0 were represented within an orange-red color scale and indicate upregulation as a response to ZnCl2 treatment, and the genes in log2 < 0 were represented within a green color scale and indicated

Quantitative Polymerase Chain Reaction (qPCR) Analysis of M. Aeruginosa UTEX LB 2385
The qPCR analysis results were calculated as fold change gene expressions of Mmt, mcyC, and mcyE relative to the M. aeruginosa UTEX LB 2385 16S ribosomal RNA internal control, within the specific time period and samples (Days 1, 5, 12, 15 for 0, 0.1, and 0.25 mg/L ZnCl 2 ) and the initial 0 mg/L ZnCl 2 Day 1 sample. Fold change gene expression values were expressed as [2 −∆∆Ct ] and evaluated as a combined [log 2 (RQ)] heatmap within RStudio statistical software. The genes expressed in log2 > 0 were represented within an orange-red color scale and indicate upregulation as a response to ZnCl 2 treatment, and the genes in log2 < 0 were represented within a green color scale and indicated downregulation as a response to ZnCl 2 treatment. Welch two sample t-tests of 16S rRNA endogenous average C t values control were found to not be statistically different within each sample condition (all p values > 0.05). The specific sample treatment and days were abbreviated as D1_0 mg/L ZnCl 2 , D1_0.1 mg/L ZnCl 2 ,

ELISA Analysis
ELISA quantitative analysis of 0, 0.1, 0.25, and 0.5 mg/L ZnCl 2 treated M. aeruginosa UTEX LB 2385 cells was performed as MC-LR equivalents using an MC-LR standard curve generated with MC-LR standards (0-5 µg/L). A standard 2 nd -order polynomial equation (y = 7.5536x 2 − 54.927x + 96.06) was used to calculate MC-LR equivalents below the 2.5 µg/L standard range. The ELISA analysis was performed on extracted MC-LR intracellular and extracellular samples at time periods (Day: 1, 5, 8, 12, and 15). Total MC-LR (µg/cell) quota was calculated by adding intracellular and extracellular MC-LR equivalents and dividing by average cells/L. The Pearson's r correlation showed that total MC-LR (µg/cell) quota was positively correlated with ZnCl 2 concentration (p < 0.05, r = 0.6). The

Discussion
Zn is a classified essential, transitional metal ubiquitously involved in numerous cellular biochemical pathways, and is often found within enzyme cavities and protein infrastructures. Zn is often employed in galvanization processes, vulcanization procedures, automobile applications, coating alloy formulations, and various manufacturing components where it often enters the environment in a sequential fashion after ZnO is formed [54]. Environmental chloride ions and pH often form ZnCl 2 near man-made sources high in processed Zn [55].
The interactions of zinc and heavy metal pollution with organisms in naturally occurring and man-made aquatic environments presents its own immediate and long-term problems in our increasingly industrial societies. As an example, a surface river, estuary, and bay sediment study in highly industrial Jinzhou Bay, China found Cu, Zn, Pb, and Cd heavy metals concentrations to be correlated to man-made industrial activities, and has been identified as a polluted ecological risk [56]. Relatively, documented metal resistance and stress response mechanisms within cyanobacteria populations necessitates that focus shifts towards risk assessment, prevention, and treatment. Synechococcus sp. IU 625 cultures were shown to be highly tolerant to 25 mg/L ZnCl 2 treatment, and a similar strain showed comparable growth to control when treated with 0.1 mg/L and 0.5 mg/L HgCl 2 [43,50]. Relatively and conversely, a total-protein profile of an Anabaena flos-aquae isolate showed progressive decrease upon treatment with increasing concentrations of CdCl 2 and CuCl 2 -with the highest concentrations presenting the most considerable biophysiological and biomass damage [57].
CHABs-associated species and strains often found in metal polluted environments must be given special attention due to their potential survivability and resultant detrimental effects (i.e., cyanotoxin release). Although distinct environmental factors have been observed to contribute to favorable conditions conducive to cyanobacteria growth, the specific combinations of effects that causes heavy metal tolerance or resistance in cyanobacteria population are not well understood, or in fact, remain unknown.
Our results showed that M. aeruginosa UTEX LB 2385 is tolerant to ZnCl 2 concentrations of 0.1 and 0.25 mg/L, with 0.1 mg/L ZnCl 2 treated cell numbers and turbidity measurements being similar to control cells through 15 days (Figure 1a,b). All cell concentrations were found to be statistically similar through five days (student's t-test p > 0.05 for 0 mg/L and 0.5 mg/L ZnCl 2 treated cells/mL). Further, 0.5 mg/L ZnCl 2 concentration was found to largely inhibit cell concentration (cell/mL) by Day 15 {(0.01/8.41) × 100~99.9% inhibition} and decreased the turbidity of the culture. This result is similar to gradual cell density decrease with increasing Zn 2+ concentration [11]. Additionally, it was also similar to M. aeruginosa Kütz 854 cultures showing chlorophyll and phycobiliprotein decrease at CdCl 2 concentrations of 1 and 2 µM, and a bactericidal reaction at 4 µM CdCl 2 treatment [58]. Both a toxic M. aeruginosa (FACHB-905) and a non-toxic strain (FACHB-469) were shown to accumulate Zn 2+ and Cd 2+ intracellularly as an uptake metal concentration vs. four-hour time rate [59]. This previous study observed intracellular Zn concentration was a good predictor of Zn toxicity in M. aeruginosa. In a different study, a 10 nM treatment of Zn as a free ion concentration did not decrease microcystin/chlorophyll-a (µg/µg) in comparison to UVR and Cu 2+ metal treatments of M. aeruginosa strains (UTEX LB 2385 and LE3), but total added Zn to lake water did affect the final biomass and growth rates [60]. Different M. aeruginosa chlorophyll concentrations were also found to be more stable to CdNO 3 treatment than to Pb(NO 3 ) 2 , and only showed chlorophyll decrease at 20 mg/L Cd after 24 h [61]. Additionally, low levels of both Cd and Pb (1-5 mg/L) resulted in increases of chlorophyll fluorescence during 24-h incubation, and showed that specific M. aeruginosa strain was not inhibited at those concentrations. Conversely, Fe 2+ and Fe 3+ was shown to increase M. aeruginosa cell density with increasing concentrations up to Toxins 2020, 12, 92 9 of 17 12 mg/L [11]. Lastly, M. aeruginosa (FACHB-905) cultures were observed to show levels of resistance and recovery to treatment with arsenic (III) concentrations (0.01, 0.1, and 1 mg/L) after 48 h incubation, and only showed marked damaging effects to growth and carotenoids production at 10 mg/L [62]. These observations indicate that heavy metal inhibition, possible resistance, or resultant growth rate in M. aeruginosa is likely dependent on strain and metal species.
Cell counts, phase-contrast microscopy, and flow cytometry (FSC-A) histogram profile showed that M. aeruginosa UTEX LB 2385 treated with 0.25 and 0.5 mg/L ZnCl 2 possessed larger amounts of multi-cell aggregates compared to 0 and 0.1 mg/L ZnCl 2 treated cells at Day 8 (Figure 3c(D8)). Progressive positive shifting of histograms occurred from Day 5 to Day 8 referenced from FCM of (100) FSC-A for 0.25 and 0.1 mg/L ZnCl 2 treated cells (Figure 3). Future experiments should concentrate on the dynamics of aggregation formation as it relates to intracellular molecular profile, in response to increasing ZnCl 2 concentrations. Aggregation of many Microcystis species is thought to contribute to possible natural bloom formation, and subsequent protection from environmental factors such as heavy metals [63]. M. aeruginosa were shown able to survive in low concentrations of variable heavy metal compounds, and Microcystis blooms showed a capacity to bioaccumulate and sequester heavy metal compounds within historically eutrophic lakes [7,10,58].  [65], further analysis must be performed to evaluate mcyC gene relation to heavy metal stress response, MC-variant identification, and total MC release to the extracellular environment. The intracellular roles of MCs are still largely undetermined, but a possible siderophore-like function towards zinc and iron may exist under specific intracellular conditions [66]. Molecular modeling for MC-LR metal binding showed that the total potential energy (Kcal·mol −1 ) (relative stability) for MC-LR was: Zn > Cu ≥ Fe ≥ Mg > Ca [46].
ELISA quantitative analysis showed MC-LR (µg/L) were present in all ZnCl 2 treated intracellular and extracellular M. aeruginosa UTEX LB 2385 culture portions ( Figure 5). There was a higher correlation of MC-LR (µg/cell) yield to 0.5 mg/L ZnCl 2 treatment cells from Day One to Day 15 versus 0, 0.1, and 0.2 mg/L ZnCl 2 treated cells (p < 0.05, r = 0.6). This correlation was inversely associated with the decreased cell biomass through 15 days, indicating release of MC-LR into the extracellular environment. It was observed that intracellular microcystin production was decreased with increasing multiple metal concentrations, indicating a possible distribution of microcystins (intracellularly to extracellularly) due to cell lysis and/or cellular damage [10]. The 0.25 and 0.5 mg/L ZnCl 2 treated cells showed ã 40% and 33% increase of extracellular MC-LR equivalents, respectively, as early as Day 5 ( Figure 5 MC-LR (µg/L) concentration through 15 days was slightly higher for cultures treated with 0, 0.1, and 0.25 mg/L ZnCl 2 than for the 0.5 mg/L ZnCl 2 treated culture. These results are supported by observations that total MC presence is largely associated with overall cell biomass [67]. Extracellular MC-LR (µg/L) equivalent measurements for 0.25 mg/L ZnCl 2 treated cells fluctuated between Days 8, 12, and 15 (1.64, 0.15, 0.80 µg/L, respectively), but was decreased from Day 5 ( Figure 5). Interestingly, the presence of observed multi-cell aggregates in both 0.25 and 0.5 mg/L ZnCl 2 treated cells from Days 8 through 15 coincided with this observation. These structures may have possibly affected MC interactions with higher ZnCl 2 concentrations (0.25 and 0.5 mg/L ZnCl 2 ) in the extracellular environment for the measured time period. A further study of multi-cell aggregation, Exopolysaccharides (EPS) production, and the MC role in the extracellular environment must be evaluated to determine interaction dynamics and possible association. These observations are supported by the following studies. The application of 0.5 mg·L −1 Zn 2+ was shown to increase the dissolve organic carbon (DOC) concentration of M. aeruginosa (FACHB 469) into the surrounding media by a 18-day study [68]. Furthermore, it was shown that the addition of MC-RR (0.25-10 µg·L −1 ) significantly enlarged Microcystis colony size, specifically increasing EPS production [69]. There are a number of undetermined factors concerning the interaction of MCs-metal species in the extracellular environment, and these factors are complicated by the presence of EPS, amino acids, and miscellaneous molecules produced by stress-responsive cells.
Further associative evaluation of Microcystis aggregation and the associated multi-cell components to heavy metal stress is necessary to determine potential resistance mechanisms and survivability dynamics of this cyanobacteria. A future transcriptomic evaluation of Zn heavy metal response in M. aeruginosa is necessary to profile which intracellular and extracellular localizing genes are in regulation, and to distinguish this important CHAB-associated cyanobacteria species from other cyanobacteria species. Finally, the role of MCs in the extracellular environment must be further determined to understand cyanobacteria-cyanobacteria environment dynamics.

Conclusions
This study exhibits the ZnCl 2 metal stress response and resistance capabilities that a universal CHAB species, Microcystis aeruginosa (in this study with strain M. aeruginosa UTEX LB 2385) possesses, and the potential survivability this species demonstrates in increasing polluted aquatic environments. M. aeruginosa UTEX LB 2385 was observed to survive ZnCl 2 concentrations of up to 0.25 mg/L, with increasing biomass through 15 days. Though mostly inhibited, 0.5 mg/L ZnCl 2 treated cultures presented multi-cell aggregates and residual populations through 15 days. A persistent yield of the cyanotoxin MC-LR (µg/cell) was observed in all ZnCl 2 treated cells by 15 days, indicating that this cyanotoxin remains present in the environment even with low cell concentrations. This finding was supported by qPCR data gene expression profiles of Mmt and mcyC, suggesting that M. aeruginosa UTEX LB 2385 possesses several metal response mechanisms.

Growth Monitoring
Microcystis aeruginosa UTEX LB 2385 (NCBI Taxon ID: 1296356) was acquired from UTEX Culture Collection of Algae, TX, USA, and was grown to late exponential growth phase (OD 750 nm = 1.0; cells/mL = 6 × 10 7 ) in sterile 1X Cyanobacteria BG-11 Freshwater Medium (Sigma Life Sciences, St. Louis, MO, USA), within a sterile 250 mL Borosilicate Erlenmeyer flask at 21 ± 2 • C under constant 24 h cool-white fluorescent light (24 µmol·m −2 s −1 photons) at a constant agitation of 100 rpm (Innova 2000 Platform Shaker-New Brunswick Scientific, Edison, NJ, USA). The 1X Cyanobacteria BG-11 media was prepared with sterile deionized Milli-Q water (Milli-Q Plus ultra-pure water system-Millipore, Billerica, MA, USA) and the pH was adjusted to 8.0 ± 0.1 with 1 M NaOH. Duplicate sets of four 20 mL volumes of M. aeruginosa UTEX LB 2385 culture were centrifuged at 2900× g for 10 min, the supernatants were discarded, and the cell pellets were gently washed with sterile deionized Milli-Q water. These cells were repelleted and constituted in 20 mL fresh sterile 1X Cyanobacteria BG-11 media. The 20 mL volumes were aseptically transferred and diluted with 80 mL fresh sterile 1X BG-11 Medium into eight new sterile 250 mL Borosilicate Erlenmeyer Flasks.

Experimental Design
A sterile 1% zinc chloride (ZnCl 2 ) (Sigma-Aldrich, St. Louis, MO, USA) solution was added to each M. aeruginosa UTEX LB 2385 culture to yield relative ZnCl 2 concentrations: 0 mg/L, 0.1 mg/L, 0.25 mg/L, and 0.5 mg/L (0 µM, 0.734 µM, 1.835 µM, 3.669 µM, respectively), as described previously [50,70]. These ZnCl 2 concentrations were targeted through multiple growth curve analysis from past experiments, with concentrations as high as 10 mg/L ZnCl 2 (data not shown). The rationale was predicated on a previously studied cyanobacterium, Synechococcus sp. IU 625 with ZnCl 2 . All ZnCl 2 -treated cultures were maintained at the same culture growth parameters described above and were monitored by turbidity observation at optical density

Phase Contrast Microscopy
To determine the physiological effects of ZnCl 2 concentration on M. aeruginosa UTEX LB 2385 cultures, cell cultures were collected and imaged. All ZnCl 2 -treated M. aeruginosa UTEX LB 2385 cultures were collected during the set, predetermined time points (Days: 0, 1, 5, 8, 12, and 15) and immediately imaged with a Zeiss AxioLab A1 phase contrast microscope coupled with an AxioCam MrC camera (Carl Zeiss, Oberkochen, Germany) at 1000× total magnification.

Flow Cytometry
Sample cell size for ZnCl 2 -treated M. aeruginosa UTEX LB 2385 cultures was measured (FCM) in 10,000 events per sample within a calibrated MACSQuant Analyzer 10 (Miltenyi Biotec, Auburn, CA, USA) containing 405 nm , 488 nm , and 638 nm lasers, using the forward scatter setting (FSC-A) for a set short term and long term course period of 8 days (0, 0. 25, 0.5, 1, 5, 8). For each time point, 1 mL of each cell treatment was aseptically transferred into a 1.5 mL microcentrifuge tubes, and the parameters were set to gently resuspend before 100 µL was measured in FCM. Different cyanobacteria populations may exhibit distinct patterns of size, complexity, and autofluorescence in regards to colony formation and to their phycobilisome complex. Furthermore, the light harvesting components and phycobilisome complex has been shown to be affected by heavy metals in concentration dependent kinetics [71,72]. Allophycocyanin and phycoerythrin fluorescent intensities for M. aeruginosa UTEX LB 2385 cells were measured in a calibrated MACSQuant Analyzer 10 (Miltenyi Biotec, Auburn, CA, USA) for the set time course period of 8 days (data not shown). All flow cytometry histograms and statistical analysis of measurements were generated via FlowJo software analysis (FlowJo, Ashland, OR, USA) to compare the effects of varying concentrations of ZnCl 2 treatment within all cultures.

Total RNA Isolation and cDNA Synthesis
The ZnCl 2 treatment (0.5 mg/L) was found to inhibit M. aeruginosa UTEX LB 2385 cell number and decrease turbidity by Day 8. Therefore, total RNA (M. aeruginosa UTEX LB 2385-0, 0.1, and 0.25 mg/L ZnCl 2 for Days: 1, 5, 12, and 15) was isolated and purified using a modified Ambion ® RiboPure™ Kit (Ambion, Austin, TX, USA) approach. Homogenization and sample disruption preparation was prepared by adding a volume of 48 mL of 100% EtOH to 60 mL of Wash Solution Concentrate. One mL TRI Reagent was then aseptically mixed with 250 µL of cultures within sterile 2 mL microcentrifuge tubes. The mixtures were homogenized (by vortexing), sonicated 15 times with 3 s pulses (20% power) using a Branson Sonifier Cell Disruptor 200 (Emerson Industrial, St. Louis, MO, USA), and incubated for 5 min at room temperature. Homogenates were centrifuged at 12,000× g for 10 min at 4 • C, and supernatants transferred to new sterile 2 mL microcentrifuge tubes. RNA extraction was performed by aseptically adding a volume of 200 µL CHCl 3 solution to each sample, tightly capping and vortexing the sample tubes at 700× g for 15 s, and then incubating at room temperature for 5 min. Each sample was centrifuged at 12,000× g for 10 min at 4 • C before 400 µL of aqueous phase was aseptically transferred to sterile 2 mL microcentrifuge tubes. Then, 200 µL of 100% EtOH was aseptically added to the 400 µL of aqueous phase and immediately vortexed at 700× g for 5 s. The sample mixtures were aseptically transferred to a filter cartridge placed within a collection tube, capped, and centrifuged at 12,000× g for 30 s at room temperature. Each sample flow-through was discarded and the filter cartridge (with bound RNA) was replaced within the same collection tube. Next, 500 µL of wash solution was aseptically added to the filter cartridges, capped, and centrifuged at 12,000× g for 30 s at room temperature. Each sample flow-through was discarded and the filter cartridge was replaced within the same collection tube. The same wash solution step was repeated. Then, the filter cartridges were transferred to new sterile collection tubes and 100 µL elution buffer was added to each respective filter column. The samples were then incubated at RT for 2 min and centrifuged at 12,000× g for 30 s to elute RNA.
The concentration (µg/mL) and A260/280 ratios for total RNA samples were checked using a BioDrop UV/VIS Spectrophotometer (Denville Scientific, Metuchen, NJ, USA). The isolated RNA samples served as templates for cDNA synthesis with an ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems-Life Technologies, Camarillo, CA, USA), using random hexamers as per the manufacturer's specification. Briefly for each sample, 10 µL DNase-treated RNA was carefully mixed with 10 µL 2X RT MasterMix I (2 µL RT Buffer; 0.8 µL 25X dNTP Mix; 2 µL 10X RT Random Primers; 1 µL MultiScribe™ Reverse Transcriptase; 4.2 µL sterile nuclease-free H 2 O) within a 200 µL nuclease-free reaction tube. The sample tubes were placed and run in a Veriti 96 well Thermocycler (Applied Biosystems, Camarillo, CA, USA) via incubation at 25 • C for ten minutes, 37 • C for two hours, and RT inactivation at 85 • C for 5 m. cDNA sample concentrations and A260/280 ratios were checked using a BioDrop UV/VIS Spectrophotometer, and stored at −20 • C until prepared to use.
Microcystis aeruginosa Mmt, mcyC, and mcyE genes were chosen for primer design based upon phylogenetic analysis of 16S-23S rRNA ITS sequences, multiple alignment of Microcystis aeruginosa MC synthetase sequences, and multiple alignment of selected Microcystis MT amino acid sequences (T-Coffee Program) (data not shown).
The cDNA samples were diluted to a concentration of~100 µg/mL and measured with a BioDrop UV/VIS Spectrophotometer to determine A260/280 ratios. The comparative C T relative gene expression method was used with the final equation: Quantitative RT PCR reactions were performed within 96-well plate assays using an Applied Biosystems StepOnePlus™ Real Time PCR System (ThermoFisher Scientific, MA, USA) with a Luna ® Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, USA) (containing Hot Start Taq DNA Polymerase). SYBR green dye chemistry was used for reactions containing: 10 µL qPCR Master Mix, 7 µL nuclease-free H 2 O, 1 µL of forward and 1 µL reverse primers for a total final 1 µM concentration, and 1 µL < 100 ng final mass of diluted cDNA. The qPCR reactions were performed in triplicates through an initial incubation of 50 • C for two minutes, an initial one-cycle denaturation step at 95 • C for 60 s, and 40 cycles of 95 • C denaturation for 15 s with 60 • C extension for 30 s.

Enzyme-Linked Immunosorbent Assay (ELISA)
ZnCl 2 -treated M. aeruginosa UTEX LB 2385 cultures were collected for a predetermined course of 15 days (Days: 1, 5, 8, 12, and 15) by centrifuging at 2900× g for 10 min at RT. The supernatants were aseptically removed from the pellets and filtered into new sterile centrifuge tubes using sterile microfiltration apparatus (with sterile ≤ 0.22 µm membrane filters). All pellet and supernatant samples were frozen at −20 • C and stored for 15 days. Intracellular microcystin samples were extracted via modified extraction methods [22,73]. Briefly, frozen M. aeruginosa UTEX LB 2385 pellet samples were thawed, refrozen at −20 • C and thawed again, before 5 mL 75% methanol was added and samples were sonicated for 1 min at 20% power using a Branson Sonifier Cell Disruptor 200 (Emerson Industrial, St. Louis, MO, USA). The homogenized pellet samples were allowed to sit at RT for 5 min before they were transferred to a sterile microfiltration device/apparatus (with a sterile ≤ 0.22 µm membrane filter). For initial supernatant samples, 5 mL 75% methanol was added and mixed, before aseptically transferring to their respective sterile microfiltration device/apparatus. The total filtrates were diluted to final 1 mL 4% methanol working sample solutions.
ELISA quantitation for intracellular and extracellular microcystin-LR equivalents was performed using a Microcystin-LR ELISA kit (colorimetric) (Abnova, Taipei, Taiwan) as per manufacturer specifications. Final absorbances were read at 450 nm in duplicates using a Varioskan™ LUX multimode microplate reader (ThermoFisher Scientific, Waltham, MA, USA) with SkanIt Software.

Statistical Analysis
The growth analysis experiments were performed in triplicates and the standard deviation of means were used to generate growth curves. The statistical analysis of flow cytometry measurements (FCM) were generated via FlowJo software analysis (FlowJo, Ashland, OR, USA). The initial raw data was analyzed using the comparative C t method in the ABI StepOne Software (Life Technologies, Camarillo, CA, USA). To ascertain if the endogenous 16S rRNA reference gene varied due to experimental conditions, student's t-tests were performed for all accumulated averaged C t values organized as treatment groups [74]. The full form of the comparative C t method equation (1) was used to evaluate relative quantifications of mcyE, mcyC, and mt genes using the MA_UTEX LB 2385-specific 16S rRNA endogenous control for the calculations. A Welch two sample t-test was used to evaluate possible statistical significance between different, pooled ZnCl 2 treatment groupings of 16S rRNA endogenous control, using RStudio statistical software (RStudio Team, 2018). A standard 2 nd -order polynomial equation (y = 7.5536x 2 −54.927x + 96.06) was used to calculate MC-LR equivalents below the 2.5 µg/L standard range. Total MC-LR (µg/cell) quota was calculated by adding intracellular-extracellular MC-LR equivalents (µg/L) and dividing by average cells/L. A Pearson's product-moment r correlation was used to evaluate increasing ZnCl 2 concentration.