Investigation On The Effect And Mechanism Of Phytohormones On Nutrient Removal In Wastewater By Microalgae And Harmful Algae Control By Allelochemicals

Aiming at the control of eutrophication in water,the source and in-situ control technology and their mechanisms were researched and developed.To solve the problems of low efficiency of nitrogen and phosphorus removal,high ammonia-nitrogen stress of algae in wastewater treatment and unclear mechanism of algal inhibition by allelochemical.Taking microalgae as the research object,from the perspective of plant hormones,on the one hand,the effects of exogenous plant hormones on the efficiency of deep dephosphorization and denitrification by microalgae,the activity of algal photosynthesis and nitrogen metabolism system were investigated.At the same time,the anti-stress effect and mechanism of exogenous plant hormones on microalgae under high ammonia nitrogen stress were investigated.The effects of exogenous plant hormones on photosynthetic activity,antioxidant capacity,nitrogen metabolic activity and gene transcription level of microalgae under high ammonia nitrogen stress were investigated by using molecular biological methods.On the other hand,the effects of terpenoids on endogenous plant hormone synthesis in cyanobacteria cells were investigated.Biochemical and molecular biological methods were used to analyze the response mechanism of plant hormones in the process of algal inhibition by natural terpenoids from the perspectives of photosynthetic activity,oxidative stress and antioxidant properties.The main results are as follows:（1）Exogenous plant hormones Indole-3-acetic acid（IAA）,Zeatin（ZT）and Brassinosteroids（Br）significantly promoted the growth and biomass production rate of microalgae.The results of nutrient removal showed that plant hormones could significantly increase the removal efficiency of NO₃^--N,NH₄⁺-N,TN,and PO₄^3--P by microalgae for advanced sewage treatment.After stimulated by IAA,the removal efficiency of NH₄⁺-N,NO₃^--N,TN,and PO₄^3--P by algae were 75.9%,41.1%,43.6%,and82.1%,which were 0.9,1.0,1.5,and 0.5 times higher than that of the control group,respectively.The removal efficiency of NH₄⁺-N,NO₃^--N,TN,and PO₄^3--P by microalgae exposed to ZT was 87.5%,39.4%,43.9%,and 85.7%,which were 1.2,0.9,1.5 and 0.5 times higher than that of the control group,respectively;Under the stimulation by Br,the removal efficiency of NH₄⁺-N,NO₃^--N,TN,and PO₄^3--P by microalgae were 87.5%,46.6%,50.4%and 84.5%,respectively,which were increased by 1.2,1.3,1.8 and 0.5times compared with the control group.（2）Exogenous plant hormones significantly promote the activity of photosynthetic and nitrogen metabolism of microalgae.The results of chlorophyll fluorescence found that the chlorophyll fluorescence Q_Y and Fv/Fm were 1.09,1.13,and 1.16 times and 1.06,1.08 and 1.1 times higher than that of the control after 4 d of exposure to 10^-5 M IAA,10^-7 M ZT,and 10^-9 M Br.Enzyme activity results indicated that,in comparison with the control group,activities of nitrogen metabolism related enzymes nitrate reductase（NR）and glutamine synthase（GS）of microalgae was increased by 1.0,0.4 and 0.9 times and1.4,1.1 and 0.8 times,respectively,after 4 d of exposure to 10^-5 M IAA,10^-7 M ZT,and10^-9 M Br.Real-time quantitative PCR results showed that the transcriptional abundance of photosynthesis-related gene rbc L and nitrogen metabolism-related gene GS of microalgae were increased by 46.1%and 30.9%,respectively,after 4 d of exposure to 10^-5 M IAA,10^-7 M ZT,and 10^-9 M Br.This results proved the positive role of plant hormones affect on gene transcription of microalgae.（3）IAA,ZT and Br significantly increased the resistance of microalgae cells to high ammonia nitrogen stress.IAA,ZT and Br can significantly improve the biomass concentration,chlorophyll content,PSII photochemical efficiency of microalgae cells under the environment of high ammonia nitrogen stress(500 mg L^-1 NH₄⁺-N)（increase Fv/Fm and Fv/Fo）,and alleviate photoinhibition（significantly reduce DIo/RC and ABS/RC）.After treated by 10^-5 M IAA,10^-9 M ZT and 10^-9 M Br for 1 day,the concentration of microalgae biomass increased by 32.44%,33.32%and 34.79%respectively compared with the control group.After treated by 10^-5 M IAA,10^-9 M ZT and 10^-9 M Br for 7 days,the content of Chl a in microalgae significantly increased by 20%,25.83% and 30.83% respectively.Chlorophyll fluorescence Fv/Fm increased by16%,19.68%and 19.75%,respectively.Fv/Fo increased by 19%,26.64%and 24.41%,respectively.Br significantly promoted the expression of rbc L in microalgal cells under high ammonia nitrogen stress(500 mg L^-1 NH₄⁺-N),which increased by 10.95 times compared with the control group.（4）After the treatment by IAA,ZT,and Br,compared with algae exposed to 500 mg L^-1 NH₄⁺-N,MDA content separately decreased by 20.4%,22.37%,and 20.8%and CAT activity increased by 53.69%,56.92%,and 117.3%,respectively.The results showed that exogenous plant hormones could eliminate the excessive production of ROS,reduce the lipid oxidation of algal cell membrane,and activate the antioxidant system in vivo.（5）Plant hormones can alleviate the inhibition of high ammonia nitrogen stress on gene transcription of nitrogen metabolism system in microalgae.After treated by IAA,ZT and Br for 4 d,GS activity increased by 2.6,2.62,and 5.72 times,respectively.And Br increased the transcriptional abundance of GS-related genes by 64%.These results showed that plant hormones alleviate the toxicity of high ammonia nitrogen stress on GS.（6）Plant hormones regulate the inhibitory effect of terpenoids on M.aeruginosa.Terpenoid monomers terpinolene（TER）and eugenol（EUG）inhibited the growth of M.aeruginosa,and significantly changed its photosynthetic activity,cell structure,extracellular organism（EOM）and membrane protein gene expression.Results of ELISA test showed that after 4 d of the exposure to 1.47 m M TER,the content of endogenous IAA,Br,ZT,SA and JA in M.aeruginosa increased by 0.98 times,0.21 times,0.8 times,2.08 times and 1.47 times,respectively;After 4 d of the exposure to 0.16 m M EUG,the contents of endogenous IAA,Br,ZT,SA,and JA in M.aeruginosa increased by 0.33times,0.07 times,0.43 times,2.32 times,and 1.0 times,respectively.Inhibitors of IAA,ZT,Br,SA and JA further enhanced the inhibitory effect of TER and EUG on cells.Results of chlorophyll fluorescence showed that after treated with TIBA,UNI,PCZ,PAC,and IBU for 4 days,Fv/Fm and Fv/Fo were further inhibited.Plant hormone inhibitors could also significantly enhance MDA content while inhibiting SOD activity.Exogenous IAA,Br,ZT,SA,and JA can significantly reduce the inhibitory effect of TER and EUG on M.aeruginosa.Which further explained that the algae-inhibiting process of TER and EUG triggers the signal transduction cascade response in M.aeruginosa initiated by plant hormones,and slso inducing abiotic stress tolerance.（7）TER and EUG could significantly promote the synthesis of NO in M.aeruginosa.After 4 d of the exposure to TER and EUG,the NO concentration in the cells was significantly increased by 9.12 and 3.04 times compared with the control group.Further research found that after 4 d of the exposure to exogenous NO,Fv/Fm and Fv/Fo decreased by 57.72%and 56.1%,in comparison with the TER treatment group,and MDA content increased by 46.88%and 26.86%compared with the TER and EUG treatment groups.Compared with the TER and EUG treatment groups,the reduction of SOD activity was 18.86%and 25.85%.The effect of NO scavengers on the cells partially relieved the inhibitory effects of TER and EUG,indicating that the algae inhibitory effects of terpenoids are regulated by NO.