Abstract
Cyanobacterial harmful algal blooms (cyanoHABs) have become an increasing problem for southwest Florida freshwater bodies. Microcystis is a notorious freshwater cyanobacterial genus that forms heavy, toxic blooms, particularly in eutrophic ecosystems. Their proliferation and ability to produce harmful cyanotoxins threaten wildlife, residents, and the economy. Although nutrient reduction is considered the best option for a long-term reduction in the occurrence of these blooms, fast-acting mitigation methods such as algaecides are often effective in removing the immediate risk these blooms pose. Hydrogen peroxide and L-lysine have been shown to selectively inhibit the growth of Microcystis and are considered ecologically friendly algaecide options because they don’t leave toxic residue in water and they can enhance conditions for non-target organisms. We explored the use of these two algaecides individually, as well as the combination of both chemicals, for Microcystis aeruginosa removal, hypothesizing the different growth inhibition mechanisms of each treatment in tandem would lead to quicker and more efficient removal of M. aeruginosa. To test this, we first assessed the susceptibility of seven Microcystis aeruginosa strains and six other phytoplankton commonly found in Florida to hydrogen peroxide (16.7 mg/L), L-lysine (8 mg/L), and mixed treatments (16.7 mg/L hydrogen peroxide: 8 mg/L L-lysine) in test tubes. All three treatments were effective at inhibiting the growth of M. aeruginosa. Mixed treatments were most effective with a median growth inhibition ratio of 94.2% on the last day of the experiment, while hydrogen peroxide (83.8%) and L-lysine (78.5%) were less so. Axenic M. aeruginosa were more sensitive to hydrogen peroxide when compared with nonaxenic strains (p < 0.01, n = 18). L-lysine was found to be significantly more toxic to M. aeruginosa than other examined cyanobacteria and chlorophyte strains at the end of the experiment (p < 0.001, n = 33), demonstrating its specificity to this cyanobacterium, while hydrogen peroxide and mixed treatments had varying effects on the other tested phytoplankton. We further assessed the three treatments in a 7-day mesocosm study using natural microbial communities from the Caloosahatchee River, FL, USA to better understand potential microbial succession patterns. The relative abundance of cyanobacteria rapidly declined in mixed (92%) and hydrogen peroxide mesocosms (82%) one day after application and the cyanobacteria were succeeded by the picoplankton Nannochloropsis, while many other eukaryotic phytoplankton were affected by treatment applications. L-lysine was more slow-acting on cyanobacteria, however, by day 7 their abundance had declined by 81% and succession by chlorophytes was observed. Transcriptomics revealed heterotrophic bacteria resilient to hydrogen peroxide and mixed treatments benefited from increased catalase expression, which allowed groups to uptake nutrients from ruptured cyanobacteria. After application, Exiguobacterium was highly abundant in both treatments, and displayed high catalase expressional activity. Microbial communities were diversely sensitive to L-lysine treatments, and transcriptomics showed this was likely a result of amino acid homeostasis disruption as well as abnormal lysine riboswitch and degradation activity. Overall, a combination cyanobacterial treatment approach using hydrogen peroxide and L-lysine successfully improved the removal efficiency of toxic cyanobacteria. Additionally, to further improve our understanding of molecular mechanisms inhibiting the growth of M. aeruginosa under hydrogen peroxide and L-lysine, we exposed two toxic and two nontoxic strains to these chemicals for RNA-seq analysis. We found both treatments led to increased sulfur and cysteine transcriptional activity, which supports antioxidant enzyme activity. These regulatory mechanisms along with methionine metabolism, were more enhanced in L-lysine treated strains, which may relate to abnormal L-lysine riboswitch activity affecting the biosynthesis of amino acids as well as sulfur recycling around the cell. Increased expression of oxidative stress regulatory genes was also observed under both treatments, which, along with the downregulation of microcystin genes by the two toxic strains, draws further support for the view microcystins do not offer protection against high levels of oxidative stress. Sulfur demands to produce antioxidant enzymes to alleviate oxidative stress likely outcompeted demands for microcystin, leading to its reduced expression. This has important implications for the mitigation of toxin-producing M. aeruginosa blooms, possibly suggesting high-dose hydrogen peroxide lowers microcystin synthetase gene expression, therefore likely lowering intracellular microcystin content which may be released upon cell lysis after treatments. To our knowledge, the reduced expression of microcystin synthetase genes after exposure to L-lysine has never been examined before. Seeing as this is a safe algaecide specifically targeting M. aeruginosa, this was another beneficial finding. In conclusion, we found through laboratory and field trials that M. aeruginosa is inhibited by the application of all three treatments, with the greatest growth inhibition observed from mixed treatments. Transcriptomics and metatranscriptomics showed both treatments led to oxidative stress for cyanobacteria, or M. aeruginosa specifically, and a lack of catalase genes may inhibit their ability to recover from such stressors. Findings of reduced microcystin production, along with fast degradation rates in water, provide further merit for consideration of these algaecides for use in M. aeruginosa cyanoHAB removal.
