Research On Characterisation Of Atrazine-degrading Strain Arthrobacter Ureafaciens ZXY-2 And Bioaugmentation Of Constructed Wetland

Increases in the production and application of pesticide and herbicide have caused rapid increases in contaminated soils and waters.The concentration of atrazine in contaminated surface water has beyond the standard of Chinese environmental quality of surface water.Among the application of herbicide,atrazine has been used extensively to control both broadleaf and grassy weeds in agriculture.Compared with the physical and chemical treatment,biological treatment was considered as a feasible and cost-effective approach to treat atrazine pollution.In this study,we aimed to isolate an atrazine-degrading strain with high degradation rate and the whole genome was sequenced to gain a better understanding of the strain.Furthermore,we investigated the bioaugmentation potential for treating atrazine wastewater in constructed wetland systems by dosing the constructed microbial inoculum.Firstly,a strain designated as ZXY-2,could utilize atrazine as the sole source of carbon and nitrogen,was selected from Jilin Pesticide Plant,China.The strain was identified as Arthrobacter by morphological characteristics,physiological and biochemical identification,Biolog analysis and 16 S rRNA sequences.Plackett-Burman?PB?multifactorial design and response surface methodology?RSM?were then employed to optimize the cultural conditions.Results showed that among the selected fifteen factors,six contributing factors were obtained.Subsequently,by employing the RSM to model and optimize atrazine degradation,a biodegradation efficiency of 12.73mg?L^-1?h^-1was reached under optimal conditions?34.04^oC,pH 9.0,inoculum size 10%?v/v?,2.212 g?L^-11 of sucrose,6 g?L^-11 of Na₂HPO₄·12H₂O,and 50 mg?L^-11 of atrazine?.Moreover,we used life cycle assessment and life cycle costing to examine whether the two techniques could be employed to evaluate the optimized conditions obtained from the PB-CCD design.Evaluation results from life-cycle approaches showed that the implementation of optimal conditions could reduce the total environmental impact by50.0%and the total economic impact by 24.5%.Secondly,we carried out studies investigating the degradation characterisation of strain ZXY-2.To be specific,we conducted the kinetics,elucidated the atrazine degradation pathway,and we utilised real-time quantitative PCR?RT-qPCR?analysis to measure the transcriptional expression of three atrazine-degrading genes throughout the entire degradation process.The kinetics results showed that the growth pattern of strain ZXY-2 followed the Haldane-Andrews model with an?_max,Ks and Ki of 0.6363h^-1,23.81mg/L and 52.76mg/L,respectively.Atrazine biodegradation fitted with the first-order kinetic equation when the initial atrazine concentration ranged from 0mg/L to40mg/L,and the zero-order kinetic equation was found to couple with atrazine biodegradation tendency as the initial atrazine concentration ranged from 50mg/L to120mg/L.The hydrolytic pathway metabolised atrazine to hydroxyatrazine,and the N-alkyl substituents were then eliminated from the s-triazine ring to yield cyanuric acid.The Real-time quantitative PCR?RT-qPCR?results showed a positive correlation between the atrazine degradation rate and the expression levels of three functional genes?trzN,atzB,and atzC?.Additionally,the atrazine degradation closely paralleled the increased gene expression levels of all three genes and decreased gene expression levels were observed after the atrazine degradation efficiency reached the highest level.Thirdly,the whole genome of strain Arthrobacter ureafaciens ZXY-2 was sequenced by PacBio RS ? platform.After annotated analysis,the genome size,GC content and CRISPR number was 5.73 M,63.50%and 7,respectively.The genome contained one chromosome and five plismids,in which the atrazine-degrading genes trzN,atzB and atzC were located on chromosome.21 sanlinity tolerate geness and 4cold shock genes were identified which were helpful for strain ZXY-2 to adapt to the high salinity and low temperature conditions.In addition,the genome contained 21 genonic islands.The restriction modification system,protein P450,and membrane surface related genes could not only ensure stability of the whole genome but also ensure stress survival.Finally,this study constructed the atrazine-degrading microbial inculum based on the cometabolism principle.Results showed that the dosed microbial inculum could enhance the removal effiencies of atrazine,cyanuric acid,total carbon and total nitrogen in bacth tests.When the unbioaugmented system and bioaugmented system reached to stabilization,the bioaugmented system was inoculated with the microbial inoculum.Results indicated that bioaugmentation could shorten the start-up time and the period of microbial inoculum was 11 days.Additionally,we explored the response of the original microbial community to such bioaugmentation via high-throughput sequencing.Notably,bioaugmentation could fine tune the bacterial population.Bioaugmented soil showed lower species richness and the introduced strains Arthrobacter and Pseudomonas played a significant role during the bioaugmentation treatment process.