Global warming and water eutrophication have led to the increased prevalence and extent of harmful algal blooms (cyanoHABs), including the potentially toxic cyanobacterial species Cylindrospermopsis raciborskii. Bacillus subtilis, one of the most commonly reported microorganisms that could inhibit the growth of cyanobacteria among microbial agents, provides an efficient, safe, and sustainable solution for controlling blooms. Most research has found that the primary or significant mechanism by which Bacillus subtilis inhibits the growth of cyanobacteria is through the release of biologically active substances with algicidal properties. In this study, the dose-effect relationship, inhibition mechanism and lasting time of Bacillus subtilis fermentation broth were systematically studied under different conditions. We also analyzed the concentrations of dissolved organic carbon, protein, and polysaccharides after treatment to assess the risk of intracellular organic matter release. In addition, we analyzed the mechanism by which microbial agents inhibit C. raciborskii by assessing its photosynthetic activity and superoxide dismutase activity (SOD) during treatment, as well as through scanning electron microscopy.

The results showed that the inhibition rate reached 90.48% with 10 μL/L Bacillus subtilis fermentation broth at the 4th day when the initial concentration is 2.26×10⁹ cells/L, and it grows closely to 100% at the following day. The pH and water temperature had the minimal effects on the inhibition effect. Under the treatment of 10—50 μL/L Bacillus subtilis fermentation broth, the maximum photochemical quantum yield (F_v/F_m) of C. raciborskii decreased to 0 after the first day, indicating that almost all the C. raciborskii cells lost their activity within one day. Low-dose treatment (5 μL/L) inhibited the photosynthetic system of C. raciborskii and caused a sharp increase in SOD activity. but C. raciborskii was able to resume growth after 2d. The high concentration of fermentation liquid resulted in the lysis of C. raciborskii and the release a large amount of organic matter, but the amount of intracellular organic matter decreased to 6.27 mg/L within five days. Large amounts of soluble proteins and polysaccharides were also detected after treated with 10—50 μL/L Bacillus subtilis fermentation broth. Scanning electron microscopy (SEM) showed increased shrinkage, significant folding and atrophy on the cell surface, as well as a large number of cell lysis residues.

Overall, the study demonstrates the potential of Bacillus subtilis (mainly its extracellular substances) as a sustainable method for controlling C. raciborskii blooms in lakes and reservoirs. Particular attention should be paid to the risk of the release of intracellular substances, especially toxins, from C. raciborskii during the treatment process. Subsequent studies will focus on the risk of toxin release during the microbial agent treatment of C. raciborskii blooms.