**Jose L. Perez and Tinchun Chu \***

Department of Biological Sciences, Seton Hall University, South Orange, NJ 07079, USA **\*** Correspondence: tin-chun.chu@shu.edu

Received: 1 January 2020; Accepted: 28 January 2020; Published: 30 January 2020

**Abstract:** 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.

**Keywords:** cyanobacteria; cyanotoxins; microcystin; metal; zinc; *Microcystis aeruginosa*

**Key Contribution:** Zinc stress causes cyanotoxin production increase in *M. aeruginosa* UTEX LB 2385.

### **1. 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 [1–6]. 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, may act as growth-limiting factors in established cyanobacteria populations [1,8]. Additionally, the effects of different heavy metal compounds and concentrations on varying cyanobacteria populations and cyanotoxin production have been demonstrated in a few studies [9–11].

The rising occurrence of global CHABs may lead to a greater probability of human exposure and animal detrimental effects through environmental interactions [12,13]. Generally, humans may be exposed to CHABs cyanotoxins or irritants via recreation activities in contaminated water sources, imbibement or infusion (via medical dialysis) of contaminated water, ingestion of biomagnified cyanotoxins through food sources, and possible long-term chronic exposure through ingestion [14–16]. Cyanotoxins may structurally present as alkaloids, polyketides, cyclic peptides, and amino acid complexes, and are potently classified as neurotoxins, hepatotoxins, or cytotoxins [17,18]. The toxic health effects of cyanotoxins in humans are both varied and dependent on the class, but effects may display from mild symptoms to severe and fatal [16,19].

Aside from escalating incidences of detrimental effects to organisms, CHABs will most likely play an increasing role in global economic dynamics. The greater occurrence of global CHABs have reported some monitoring and contingency plan estimate costs of 10,000 s–1,000,000 s USD per country per year [20]. While these numbers do not often reflect loss of recreation revenue, cost of treatment actions, or socio-economic strategies (for anthropogenic P-N load reduction), an estimate report that took these components into account (within the United States) calculated potential losses in the billions USD per year [21].

*Microcystis* spp. and the hepatotoxins microcystins (MCs), have been identified as several of the most commonly encountered freshwater CHABs species and cyanotoxins, respectively, on a global scale and within the U.S. [22,23]. As such, studies evaluating both *Microcystis* and MCs have increased in importance in the recent years. Adding to the gravity of these considerations, examples of toxigenic *Microcystis* outcompeting non-toxigenic strains within elevated surface water temperature parameters have been observed [24,25]. This indicates a potentially competitive selection of toxigenic strains over non-toxigenic strains under climate change conditions. *Microcystis aeruginosa* is a commonly identified species of *Microcystis* within many CHABs, and have been found on all continents except for the Antarctic [26]. *M. aeruginosa* are typically 2–8 μm wide, unicellular planktonic cyanobacteria that possess variable, intracellular gas vesicles and often form colonies in natural and eutrophic conditions [27–29]. MCs (~995 Da for MC-LR) are water soluble, monocyclic heptapeptides that can be produced by freshwater, terrestrial, and benthic cyanobacteria. The most common genus and species producing a given variant(s) of the ~ 200 identified MCs are: *Microcystis, Chroococcus, Planktothrix, Anabaena, Nostoc, Oscillatoria, Hapalosiphon,* and *Phormidium* [18,30,31]. MCs are biosynthesized by a non-ribosomal peptide synthetase/polyketide synthase complex (known as microcystin synthetase) [18], and vary in toxicity depending on the L-amino acid components and binding capacity to receptor sites [32]. The role of MCs in the extracellular environment remains largely undetermined, but several studies have proposed an intercell signal-like characteristic where environmental conditions and introduced MCs enhanced *mcy* gene and toxin production in established cell populations [33,34]. Therefore, further evaluation of stimulatory MC release to the extracellular environment and their possible extracellular functions with environmental factors is of great importance. Zinc (Zn) is an important mineral integral to the physiological functions of all organisms. Zn naturally occurs as a trace element in world average river waters (0.27–27 μg/L), as ionic complexes in continental crust and world soil averages (70 mg/kg, respectively), and as precipitating minerals, organic, and inorganic compounds in water (~3.25 μg/L in worldwide, clean drinking water), but organic and inorganic zinc compounds are oftentimes identified as metal runoff contaminants within aqueous environments [35–37]. A comparison of the average Zn levels originating from a continental crustal average (52 ppm) was found to be significantly different to an anthropogenic impacted region, New York Harbor, United States (188–244 ppm), showing a further trend in heavy metal accumulation within water environments proximal to urbanized and industrial sources [38,39]. The toxicity of ZnCl2 has been known in many organisms [37,40,41] and can cause external irritation, severe inflammation, and gastrointestinal toxicity dependent on

percent ingestion [42]. Cyanobacteria sensitivity, resistance or adaptive sequestration of heavy metal concentrations has been documented within both colonial CHABs and unicellular species [7,43–45]. Aside from the observed zinc metal-complexing potential of MCs (MC-LR-Zn = <sup>−</sup>617 <sup>±</sup> 7 kcal mol<sup>−</sup>1; MC-RR-Zn =−777 <sup>±</sup> 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 Zn2<sup>+</sup> or Cd2<sup>+</sup> 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 Zn2<sup>+</sup> concentrations; and (3) to determine relative quantitation of MC-LR equivalents within ZnCl2-treated *M. aeruginosa* using intracellular and extracellular portions.

### **2. Results**
