Simultaneous Removal of the Freshwater Bloom-Forming Cyanobacterium Microcystis and Cyanotoxin Microcystins via Combined Use of Algicidal Bacterial Filtrate and the Microcystin-Degrading Enzymatic Agent, MlrA

Abstract

Freshwater cyanobacterial blooms (e.g., Microcystis blooms) constitute a major global environmental problem because of their risks to public health and aquatic ecological systems. Current physicochemical treatments of toxic cyanobacteria cause the significant release of cyanotoxin microcystins from damaged cells. Biological control is a promising eco-friendly technology to manage harmful cyanobacteria and cyanotoxins. Here, we demonstrated an efficient biological control strategy at the laboratory scale to simultaneously remove Microcystis and microcystins via the combined use of the algicidal bacterial filtrate and the microcystin-degrading enzymatic agent. The algicidal indigenous bacterium Paenibacillus sp. SJ-73 was isolated from the sediment of northern Lake Taihu, China, and the microcystin-degrading enzymatic agent (MlrA) was prepared via the heterologous expression of the mlrA gene in the indigenous microcystin-degrading bacterium Sphingopyxis sp. HW isolated from Lake Taihu. The single use of a fermentation filtrate (5%, v/v) of Paenibacillus sp. SJ-73 for seven days removed the unicellular Microcystis aeruginosa PCC 7806 and the native colonial Microcystis strain TH1701 in Lake Taihu by 84% and 92%, respectively, whereas the single use of MlrA removed 85% of microcystins. Used in combination, the fermentation filtrate and MlrA removed Microcystis TH1701 and microcystins by 92% and 79%, respectively. The present biological control thus provides an important technical basis for the further development of safe, efficient, and effective measures to manage Microcystis blooms and microcystins in natural waterbodies.

Keywords: Microcystis blooms; MlrA enzyme; algicidal bacteria; biological removal; microcystin release.

Figure 1

Scanning electron microscopy (SEM) image of morphological characteristics of algicidal bacterium SJ-73.

Figure 2

The algicidal efficiency of the culture, filtrate, and washed cell and of Paenibacillus sp. SJ-73 against M. aeruginosa PCC 7806 after 7 days’ treatment. Bars represent the standard errors of the means for triplicate. Significant differences are shown by asterisks: **, p < 0.01.

Figure 3

Effects of bacterium SJ-73 on the concentration of Chl-a (A,D), photosynthetic activities (B,E) and extracellular MCs (C,F) of unicellular M. aeruginosa PCC 7806 and colonial M. aeruginosa TH1701 cultures. Bars represent the standard errors of the means for triplicate. Significant differences are shown by asterisks: *, p < 0.05; **, p < 0.01.

Figure 4

Cell morphology and ultrastructures of M. aeruginosa PCC 7806 visualized by scanning electron microscopy (SEM) and transmission electric microscopy (TEM) after treatment with SJ-73 filtrates for 0 days (A,B), 3 days (C,D), and 7 days (E,F).

Figure 5

(A) HPLC chromatographic presentation of the MlrA activity of the cell-free extracts of recombinant pET28h-mlrA BL21(DE3). Peaks with retention time of 6.5 min and 4.2 min correspond to [D-Asp³]MC-LR and linearized [D-Asp³]MC-LR, respectively. (B) MS spectra of [D-Asp³]MC-LR (m/z: 981.5393) and linearized [D-Asp³]MC-LR (m/z: 999.5492); (C) degradation curve of [D-Asp³] MC-LR by 0.8 mg/L MlrA (30 °C, pH = 7), and (D) changes in different concentrations of [D-Asp³]MC-LR concentration treated with 0.8 mg/L MlrA. Bars represent the standard errors of the means for triplicate.

Figure 6

Changes on the concentration of extracellular MCs in M. aeruginosa PCC 7806 culture treated with 5% bacteria SJ-73 filtrates and 0.8 mg/L MlrA. Bars represent the standard errors of the means for triplicate. Significant differences are shown by asterisks: **, p < 0.01.

Figure 7

Changes on extracellular MCs concentration in colonial M. aeruginosa TH1701 culture treated with bacteria SJ-73. Black arrows represent the the day for adding MlrA in algal culture. The (A) MlrA was added into algal culture at the beginning of the experiment, and (B) the MlrA was added into algal culture at the beginning of the experiment and on the third day. Bars represent the standard errors of the means for triplicate. Significant differences are shown by asterisks: **, p < 0.01.