The Study Of Algicidal Bacteria And Flocculation Technique Against Microcystis Aeruginosa

Nowadays, harmful algal blooms (HABs) commonly occur in aquatic environments and Microcystis aeruginosa is the chief culprit of cyanobacteria pollution in China’s waters. Previous reports indicated that over 75% of lakes in China are seriously polluted by M. aeruginosa, which poses a grave threat to not only the regional economic development but also the safety of aquatic animals and humans. Therefore, it is increasingly urgent to develop useful strategies for terminating HABs and reducing their negative impacts. In the present study, investigations focused on algicidal bacteria and bioflocculants against M. aeruginosa bloom were carried out and these investigations showed:(1) The algicidal characteristics of Citrobacter sp. Rl against M. aeruginosa were studied when the sole carbon source was wheat bran or glucose, respectively. The results indicated that when glucose being the sole carbon source, the algicidal activity of strain R1 could achieve 81.63±2.18% within 72 h, while when wheat bran being the sole carbon source, the algicldal activity was only -0.08±3.34% within 72 h. In order to explore the underlying mechanism, a mutant of strain R1 was first constructed using the technique of transposon, and then Tail-PCR was employed to clone the functional gene. Finally, the functional gene was identified as a gene of glycogen synthase A, namely, glgA. We then explore the underlying mechanism under the level of transcription and translation. glgA used as the target, we first detected the expression quantity of glgA under two different carbon sources, and the results showed that relative expression quantity of glgA under the carbon source of glucose was slightly lower than that under wheat bran, indicating that it may not be a reason that different carbon sources caused the difference in expression quantity of functional gene. Then, we detected the expression quantity of the total proteins, and the results indicated that there was a significant difference in protein expression under two different carbon sources. When the carbon source was glucose, the protein expression quantity was relative small, while there was much more protein expressed when the carbon source was wheat bran, indicating that it may be the major reason that different carbon sources caused the difference in protein expression quantity.(2) We studied the algicidal activity of Alcaligenes aquatilis strain F8 against Microcystis aeruginosa. The maximal algicidal rate of the free bacterium reached 88.45±1.24% within 72 h. With a view to its better application to the control of M. aeruginosa in the natural environment, strain F8 was immobilized in sodium alginate beads, but immobilization of the strain decreased its algicidal rate compared to that of the free bacterium. However, addition of wheat bran to the sodium alginate matrix used to immobilize strain F8 not only eliminated the adverse effects of immobilization on the bacteria but also resulted in an 8.83% higher algicidal rate of the immobilized than free bacteria. Exclusion and recovery methods were used to identify key ingredients of wheat bran and gain insight into the mechanism underlying the observed enhancement of algicidal activity. This analysis indicated that certain factors in wheat bran, including vitamins B1, B2, B9 and E were responsible for promoting bacterial growth and thereby improving the algicidal rate of immobilized strain F8.(3) A newly isolated strain of Bacillus methylotrophicus, ZJU, was firstly used to control Microcystis aeruginosa. Analyses of relative reactive oxygen level, malondialdehyde content, superoxide dismutase activity, and fluorescence staining indicated that oxidative damage caused by the algicidal supernatant of strain ZJU mainly affected the cell membrane and consequently the membrane permeability, metabolic activity and membrane potential of M.aeruginosa cells. Furthermore, an embedded immobilization technique was employed to improve the practical application of strain ZJU as an algicidal agent. On this basis, we proposed a novel concept of enhancing the algicidal properties of immobilized ZJU by adding Fe3O4 nanoparticles and wheat bran in the process of immobilization. Our studies showed that Fe3O4 nanoparticles conferred the immobilized bacteria with a magnetization of 30.87 emu/g, and this magnetization enabled efficient re-collection of the immobilized bacteria by magnetic means. Moreover, wheat bran endowed the immobilized bacteria with 10.34% higher algicidal activity than immobilized bacteria without wheat bran.(4) A novel composite consisting of clay, bioflocculant and inorganic flocculant was designed and its flocculating effect on harmful algal blooms (HABs) was studied. The extracellular polymeric substances (EPS), produced by a newly isolated Pseudomonas aeruginosa ZJU1, was indicated to be a key component in the composite. The components and functional groups of the EPS were analyzed, and it showed that polysaccharides, proteins and nucleic acids are the main components; polar functional groups in the EPS are responsible for its flocculating activity. The novel composite was optimized by the response surface methodology and after optimization, the optical components and contents of the composite were Kaolin 2.38 g/L, CaCl20.28 g/L, KAl(SO4)20.09 g/L and EPS 1.75 mg/L. The flocculating rates of the composite were tested, and it could rapidly reach 100±0.13% within 2 min when OD680 of M aeruginosa was 0.1; it could reach 100±0.08% within 5 min for OD680 of M. aeruginosa in HABs up to 1.0.(5) A novel bioflocculant, EPS-1, was prepared and used to flocculate the kaolin suspension and Microcystis aeruginosa. An important attribute of EPS-1 was its protein content, with 18 protein types identified that occupied a total content of 31.70% in the EPS-1. Moreover, the flocculating activity of these protein components was estimated to be no less than 33.93%. Additionally, polysaccharides that occupied 57.12% of the total EPS-1 content consisted of four monosaccharides:maltose, D-xylose, mannose, and D-fructose. In addition, carbonyl, amino, and hydroxyl groups were identified as the main functional groups. Three main elements, namely C1s, N1s, and O1s, were present in EPS-1 with relative atomic percentages of 62.63%, 24.91%, and 10.5%, respectively. Zeta potential analysis indicated that charge neutralization contributed to kaolin flocculation, but was not involved in M. aeruginosa flocculation. The flocculation conditions of EPS-1 were optimized, and the maximum flocculating efficiencies were 93.34% within 2 min for kaolin suspension and 87.98% within 10 min for M. aeruginosa.