Synergistic Effects Of Prodigiosin And Indigenous Microcystin-degrading Bacteria On Microcystis Aeruginosa

The eutrophication became increasingly serious in China nowadays, causing poisonous and harmful algal blooms and algal toxins, which pose a direct threat to humans and environment safety. Indigenous bacteria with abilities of algae-lysing and MC-degrading had many advantages on the pollution control, such as lowest cost, high efficiency, no secondary pollutants, and safety. The effects and mechanisms of prodigiosin on M. aeruginosa were investigated in this study. Then, an indigenous MC-degrading bacterium strain a7 was successfully isolated from Lake Taihu. Its MC-degrading genes, enzymes and pathways were detected. At last, the synergistic effects of prodigiosin, Serratia marcescens and MC-degrading bacteria on M. aeruginosa and MCs were explored.1. The effects and mechanisms of prodigiosin on Microcystis aeruginosaThe effects of prodigiosin on growth of M. aeruginosa were studied by cell counting and flow cytometry assay after exposed to six doses of prodigiosin between 0.156 and 5.000?g/mL. The effects on the content of ROS, SOD, DNA, intracellular and extracellular MCs of M. aeruginosa were investigated respectively after exposure to 1.25,2.50 and 5.00?g/mL of prodigiosin. The variation of density and MCs was detected after exposure 15 days. The results showed that the EC50 value of prodigiosin for growth inhibition of M. aeruginosa was 2.76?g/mL (95% confidence interval was 0.96 to 7.91?g/mL). The cell diameter was larger than normal cells after 24h exposure. Besides, the average content of DNA was more than normal cells. The content of ROS was significantly increased after 24h exposure to 2.50 and 5.00?g/mL of prodigiosin. But the activity of SOD was significantly decreased after exposure. The membrane integrity showed significant variations after exposure to different doses of prodigiosin. Furthermore, the MC-LR production of M. aeruginosa was inhibited after exposure to prodigiosin. It was reduced by 23.80% when exposed 72h to 2.50?g/mL of prodigiosin, which was in the most significant inhibitory effect. With the extension of exposure time, the growth inhibition was increased, causing the extracellular MC-LR increased with more cells death. Above all, Prodigiosin induced cell oxidative damage and proliferation inhibition so that it inhibited the growth of M. aeruginosa eventually. Prodigiosin reduced the production of MC-LR in short term. Besides that, more cells died and extracellular MC-LR concentration was increased for a long term exposure.2. Screening and MC-degrading characteristics of indigenous MC-degrading bacteriaThe V4 area of 16S rDNA of MC-degrading bacteria communities was detected by high throughput sequencing technology to analyze the composition of bacteria communities and the changes during domestication. The MC-degrading efficiency of MC-degrading bacterium strain a7 was detected by HPLC. Strain a7 was identified by 16S rDNA sequence technology. Different kinds of extractions were obtained from strain a7 to analyze MC-degrading material. The MC-degrading genes of strain a7 were detected by PCR, and the degradation intermediates were studied by HPLC and LC-TOF-MS. The results showed that the bacteria with MC-degrading abilities became dominant species after domesticated in MCs. The average degradation rates of MC-LR in extracts and standards were 0.60?g/(mL·h) and 3.33?g/(mL·h), respectively. The 16S rDNA sequence detection revealed that the strain a7 belonged to the genus Sphingopyxis sp. The active ingredients of MCs degradation was intracellular and was thermolabile. mlrA, mlrC, and mlrD were found in strain a7 by PCR. The Tetrapetide (m/z 615.3398) and others (m/z315.1955, m/z283.1700) were detected as intermediates when MC-LR was degraded by strain a7. In conclusion, Sphingopyxis sp. strain a7 had mlrA, mlrC, mlrD gene, and the Tetrapetide (m/z 615.3390) and Adda (m/z 332.2208) were intermediates when MC-LR was degraded by strain a7.3. Synergistic effects of prodigiosin and indigenous MC-Degrading Bacteria on M. aeruginosa.M. aeruginosa was cultivated to logarithmic phase, the density and extracellular MC-LR were detected to explore synergistic effects after co-exposure of 2?g/mL prodigiosin and 1/5(v/v) MC-degrading bacteria communities. Then, influence factors of synergistic effects were studied such as the content of MC-degrading bacteria communities, the concentration of prodigiosin, temperature and addition time interval. Synergistic effects and its influence factor were studied after adding 2.5?g/mL prodigiosin and different contents of MC-degrading bacterium strain a7. Mimetic lake water with LB medium, Serratia marcescens, Serratia marcescens+strain a7 and Serratia marcescens+bacteria communities were builded up as different treatment groups. Then, the changes of density, extracellular MC-LR, TN, TP and TOC were studied to verify the synergistic effects. The results showed that the density was lowest and the extracellular MC-LR was gradually reduced under synergistic effects of prodigiosin and MC-degrading bacteria communities. 1/5(v/v) MC-degrading bacteria communities, appropriate prodigiosin,28-37?, simultaneously or priority adding bacteria communities enhanced synergistic effects. The density was lowest and the extracellular MC-LR was missing in day 2 under synergistic effects of prodigiosin and MC-degrading bacterium strain a7. And the dosage of strain a7 was very less. The extracellular MC-LR of four treatment groups were all gradually reduced in mimetic lake water. And the density of LB medium group was lowest. The TN, TP and TOC all showed a trend of decline. In conclusion, the synergistic effects of prodigiosin, Serratia marcescens and indigenous MC-degrading bacteria inhibited the growth of M. aeruginosa but also degraded MCs released by M. aeruginosa.