Characteristic Analysis And Detoxification Evaluation Of Novel High-efficiency Thiocyanate Degradation Microbial Community

Thiocyanate（SCN^-）is a common pollutant in gold mining,textile,printing and dyeing and coking industries,with high toxicity and harm to biosafety.At present,it is urgent to treat thiocyanate wastewater pollution,and the common treatment methods mainly include chemical oxidation,physical adsorption and biological treatment.Biological treatment is regarded as one of the most promising remediation methods for thiocyanate wastewater as an efficient,pollution-free and low-cost way of pollutant disposal.However,low thiocyanate concentration and incomplete detoxification are commonly tolerated by biological treatment.Therefore,it is necessary to construct a microbial community of thiocyanate degradation with high efficiency,high salt tolerance and complete detoxification,which will promote the degradation of thiocyanate wastewater to a new height.In this study,thiocyanate,a common pollutant in industrial wastewater,was used as substrate to construct a group of microbial community with high degradation performance.In this study,16S r RNA high-throughput sequencing technology was used to analyze the microbial evolution of the complex flora,and functional microorganisms were excavated.In addition,in order to improve the degradation performance of the microbial community,the culture conditions were optimized and the salinity tolerance was orientated.In order to understand the degradation rule of thiocyanate by microbial community,the degradation kinetics of thiocyanate was studied,and the high concentration thiocyanate wastewater was also degraded to make it suitable for industrial application.In order to further explore the thoroughness of thiocyanate degradation by microbial community,toxicological evaluation of thiocyanate and its metabolites was carried out.The main research results are as follows:（1）A group of highly efficient thiocyanate degradation microbial community was constructed from activated sludge orientation,which could stably degrade 154.64 m M thiocyanate.Acid was generated during the degradation process,and the degradation products included SO₄^2-and NH₄⁺.Morphological observation showed that the microbial community was dispersed in the fermentation broth in the form of yellow radial spheres.Scanning electron microscopy showed that the microbial community was composed of rod-shaped,spherical and other strains of different shapes and sizes,which were closely attached to each other,and the surface of the bacteria was villous.The formation of spherical microbial community is beneficial to thiocyanate degradation.（2）The results of single factor experiment showed that the optimal inoculation amount was 6 g/L,p H value was 7.0,temperature was 30℃and shaking speed was 180 rpm.The concentration of ammonium sulfate affects the degradation of thiocyanate.The higher the concentration of ammonium sulfate is,the slower the degradation rate of thiocyanate is.The addition of exogenous carbon and nitrogen sources can effectively increase biomass,but the degradation rate of thiocyanate slows down.At the salt concentration of 20 g/L,the degradation rate of thiocyanate was more than 30%within 24 hours and can reach 100%in72 hours.（3）Response surface optimization results showed that the degradation rate of thiocyanate was the highest when p H value was 7.19,temperature was 32℃and shaking speed was 185 rpm,and the degradation rate of thiocyanate was more than 70%in 24 hours.Based on the results of RSA,the average degradation rate of thiocyanate was verified to be75.16%in 24 hours and reach 100%in 30 hours under the optimal conditions,which proved the feasibility and accuracy of the model.The results showed that the microbial community could degrade thiocyanate,phenol,quinoline,COD,indole and aniline,and the degradation rates within 36 hours were 100%,99.36%,98.49%,96.37%,94.72%and 98.58%,respectively.GC-MS results showed that the microbial community could degrade most organic substances in wastewater.It showed broad spectrum of degradation of complex flora.（4）The kinetics results showed that the degradation of thiocyanate by microbial community was in accordance with the substrate inhibition model-Haldane equation,and the maximum degradation rate of thiocyanate and the maximum growth rate of microbial community were 0.14911 g·L^-1h^-1 and 0.23646 g·L^-1h^-1,respectively.In addition,the microbial community could completely degrade 41.24 m M thiocyanate within 30 hours,while the efficiency of original activated sludge was only 13.41%.The maximum tolerance of the microbial community was 154.64 m M thiocyanate,and the degradation rate reached100%after 6 days of culture.The production of SO₄^2-and NH₄⁺was 121.52 m M and 80.74m M,respectively.（5）The results of 16S r RNA high-throughput sequencing showed that the diversity and structure of bacteria were changed after thiocyanate enrichment and acclimation,and the biodiversity decreased again.The dominant phyla in activated sludge were Proteobacteria,Acidobacteria,Bacteroidetes,Firmicutes and Planctomycetes,with the abundance of 25.88%,57.97%,12.5%,1.32%and 0.76%,respectively.The dominant phyla in microbial community were Proteobacteria,Bacteroidetes,Patescibacteria,Armatimonadetes and WPS-2,with the abundance of 81.07%,14.43%,1.8%,1.33%and0.76%,respectively.Uncultured＿bacterium＿f＿Blastocatellaceae,Alicycliphilus,uncultured＿bacterium＿f＿NS9＿marine＿group,uncultured＿bacterium＿f＿Mitochondria,Diaphorobacter and Thiobacillus are the main genera in the original sludge samples.Their abundance was 57.97%,12.37%,5.98%,2.92%,2.79%and 2.69%,respectively.Thiobacillus（77.78%）,uncultured＿bacterium＿f＿Chitinophagaceae（6.15%）,uncultured＿bacterium＿f＿NS9＿marine＿group（5.6%）,Dokdonella（2.07%）and uncultured＿bacterium＿f＿Saccharimonadaceae（1.8%）are the main genera in the microbial community samples.The abundance of Thiobacillus in the microbial community was77.78%,and the abundance of Thiobacillus in the activated sludge was 2.69%.Thiobacillus changed from the inferior bacteria in the activated sludge to the dominant bacteria in the complex bacteria,which is considered to be the key bacteria in the degradation of thiocyanate by the microbial community.（6）The results of plant toxicology showed that thiocyanate inhibited the germination of rice and mung bean seeds,the germination rate of mung bean and rice seeds germinated by 41.24 m M thiocyanate could only reach 82%and 76%,respectively,and the seeds would die after germination.The effect of thiocyanate on plants was not significant after the degradation of microbial community,the germination rate of seeds was 100%,and the growth was good at the later stage,which was obviously better than the detoxification effect of original sludge.The GI（%）of mung bean and rice seeds cultured after thiocyanate degraded by the microflora increased by 94%and 84.83%,respectively,compared with without thiocyanate degraded.（7）In vitro cytotoxicology tests showed that compared with distilled water group,41.24m M thiocyanate medium,the medium before and after activated sludge degradation,and the medium before and after microbial community degradation significantly inhibited the activity of Neuro-2a cells,induced ROS generation of Neuro-2a cells,increased calcium ion concentration of Neuro-2a cells,damaged the nucleus of Neuro-2a cells,and promoted the apoptosis of Neuro-2a cells,decreased mitochondrial membrane potential of Neuro-2a cells,increased cell number in G1 phase of Neuro-2a cells,and damaged DNA of Neuro-2a cells.There was no significant difference between the Neuro-2a cells cultured in distilled water and the cells cultured in culture medium at the later stage of thiocyanate degradation by the microbial community,and the microbial community had a good detoxification effect on thiocyanate.