The Mechanism of Berberine Wastewater Treatment with Compound Microorganisms

Author:Liu Shi Yue

Supervisor:wang he li zeng ping


Degree Year:2019





Berberine is a broad-spectrum antibacterial drug.A large amount of wastewater containing berberine is discharged into the environment,which poses a great threat to the ecosystem,due to its large-scale production and use.To solve the problem that the berberine wastewater is difficult to be treated with conventional biological processes,10 bacterial strains with high degradability were obtained from the activated sludge of berberine integrated wastewater treatment system.The identification information of the strains was confirmed by microscopy,physical and chemical properties identification and biological 16S rDNA strain identification.Berberine was taken as the main target pollutant,the degradation ability of different strains to berberine was explored separately.The high-efficiency berberine degrading compound microorganism(BDCM)was constructed by orthogonal test,and the degradation parameters were optimized to achieve efficient degradation of berberine.In order to explore the berberine degradation mechanism and functional mechanism of microorganisms under BDCM,metabolomics and proteomics were synergistically used.The main conclusions of this study are as follows:1.10 strains were isolated and purified from activated sludge of berberine wastewater treatment system.They were identified as:A1:Citrobacter freundii,A2:Bacillus oleronius,A12:Bacillus endophyticus,B1:Stenotrophomonas maltophilia,B2:Bacillus pumilus,B4:Bacillus cereus,B7:Stenotrophomonas sp,B16:Sphingopyxis macrogoltabida,B17:Ochrobactrum sp.When the initial concentration of berberine is within the range of 0-20mg·L-1,B16 showed the highest removal efficiency of berberine.2.The high-efficiency BDCM was constructed for berberine degrading.BDCM was composed of strains A2,A3,B1,B4,B16 and B17,and the ratio between strains was 1:1:1:1:1:1.Under optimal conditions of 25°C,pH 7,150 rpm,10%microbial inoculum,BDCM degraded the berberine in solution at an initial concentration of 40mg·L-1.The removal rates of berberine and TOC reached 100%and 87%,respectively.The acute biological toxicity decreased from-98%to-2%within 48 hours.When the initial concentration of berberine was in the range of 0-80 mg·L-1,the degradation process of berberine by BDCM accords with the zero-order reaction kinetics model.Vmax=40.44 mg·(g MLSS·h)-1,Km=61.86 mg·L-1,and Ki=21.23 mg·L-1 were obtained,by the Haldane inhibition model fitting.The calculated results were well fitted with the experimental results.3.Real-time quantitative PCR was used to monitor the expression changes of various strains during berberine degradation by BDCM.The results showed that the abundance of A2,A3 and B4 varied significantly,followed by B1 and B17,and the bacterial abundance of B16 remained stable during the degradation process.The abundance of strains B17,A3 and B4 showed an upward trend during berberine degradation,it was concluded that some intermediate metabolites of berberine promoted their growth during the degradation process.The abundance of strains A2and B1 decreased slightly during berberine degradation,and it was inferred that its ability to utilize intermediate metabolites of berberine was not as good as the former.4.The metabolomics method of BDCM on berberine was successfully established.A total of 288 potentially differential metabolites were screened and detected as intracellular soluble substances,cellular metabolites and some berberine metabolites.It was confirmed that the four metabolites were intermediate metabolites of berberine,which were 13,13 a-dihydro-9,10-dimethoxy-2,3-(methylene dioxy)-methylpyridine;3-[benzo1,3 dioxane]-7,8 dimethoxyl-6,7,8,9 tetrahydro-2(3H)-naphthone;2,3-dimethoxybenzaldehyde;3,4-dimethoxybenzaldehyde.Therefore,the biodegradation pathway of berberine can be inferred.5.The label free proteomics method of BDCM on berberine was successfully established,and the expression pattern and regulation mechanism of protein degradation in berberine were analyzed.The results showed that 6160 peptides and4769 proteins were detected under the"1%FDR"filtration standard,and the proteins and their basic information during berberine degradation were identified.GO annotation results showed that 35.88%of the proteins have the molecular function of the regulatory genes,25%of the proteins are involved in the composition of the cellular components,and 39.12%of the proteins are involved in the biological process.The KEGG pathway information showed that the top three biological processes involved in protein are the biosynthesis,carbon metabolism and ribosome metabolism of antibiotics,which prove that BDCM can use berberine as a carbon source and energy for normal biological activities.A total of 1352 differentially expressed proteins were screened under the criteria of FC>3.00 and T-test P-value<0.05,of which 346 were attributed to BDCM.There are 135 differentially expressed proteins and 211 differential proteins in BDCM.Among the BDCM strains,B1(Stenotrophomonas maltophilia)plays a major role in the metabolism.