| p-toluenesulfonic acid(PTS)is widely applied as a raw material in the medicine and pesticide industries.Extensive studies have shown that PTS has strong biological toxicity and low biodegradability.Wastewater containing concentrated PTS will cause serious harm to the water ecological environment and human health after its inappropriate discharge into the environment.It is very meaningful to screen microorganisms which can degrade PTS from environmental media.Besides,it is theoretically significant to investigate on the degradation behavior and the metabolic mechanism.Unfortunately,very few studies report the biodegradation and metabolism of PTS up until now.Moreover,there is no report focusing on the development and application of the engineering-scale bioaugementation system for the remediation of PTS wastewater.In this study,a Hydrogenophaga strain QY7-2,which can efficiently degrade PTS,was isolated.The metabolic pathway,degradation genes and enzymes were investigated respectively.The PTS degrading microbial germplasm library was enriched.Strain QY7-2 was applied to the laboratory-scale sequencing batch biofilm reactor to analyze the colonization of QY7-2 and the optimal operating parameters of the reactor.Furthermore,the strain QY7-2 was applied to an engineering-scale treatment of PTS-containing wastewater from pesticide production,which achieved the effective removal of PTS in wastewater.The results of this study can provide technical support for the removal and standard discharge of typical pollutant in wastewater.The main contents and results are as summarized as follows:1.Isolation and Degradation Characteristics of PTS Degradation StrainsQY7-2 was isolated from activated sludge which can utilize PTS as the sole carbon and energy sources.Phylogenetic analysis based on 16S rDNA gene sequences revealed that strain QY7-2 formed a monophyletic lineage in the genus Hydrogenophaga sp.,and showed highest similarity to the model strain Hydrogenophaga atypical BSB 41.8T(98.34%).Strain QY7-2 could completely degrade 200 mg/L PTS within 21 h.The optimum degradation conditions were initial pH 6.0-7.0 and temperature 30?-35?.The metabolites of PTS degradation were further identified by liquid chromatography-mass spectrometry(LC-MS).The metabolic pathways were proposed as follows:first,the side chain methyl was oxidized and turned to carboxyl catalyzed by oxygenase;then,sulfonic groups were oxidized by dioxygense and generated protocatechuic acid.The degradation kinetic of PTS with strain QY7-2 was also studied and and the process conformed to the Monod equation.2?Cloning and Expression of PTS Degradation GeneThe draft genome of strain QY7-2 was sequenced using the Illumina Hiseq 2000 sequencing platform.The total length of the genome was 4911693 bp,the predicted ORF was 4590 and the(G+C)%content was 68.77%.In comparison with the reported PTS degradation-related genes,the suspected gene cluster ptsABCD associated with methyl oxidation was located in strain QY7-2.The alignment results showed that the protein sequences PtsA and PtsB encoded by the gene ptsA and ptsB had 99%and 97%homology to the oxidative and reducing components of the 4-methylbenzenesulfonic acid methyl monooxygenase in the previous reported strain T-2 respectively,whereas the protein sequences PtsC and PtsD encoded by gene ptsC and ptsD showed 99%homology with both 4-aldehyde benzenesulfonic acid dehydrogenase and 4-hydroxymethylbenzenesulfonic acid dehydrogenase in strain T-2 respectively.The four components of PtsA,PtsB,PtsC and PtsD were expressed in E.coli BL21(DE3)using pET28a expression system and purified by Ni-affinty chromatography.The results showed that PtsAB could oxidize p-toluenesulfonic acid to p-hydroxymethylbenzenesulfonic acid with specific activity of 161.5±3.3 nmol min-1 mg-1.PtsD could oxidize p-methylbenzenesulfonic acid to aldehyde benzene sulfonic acid with a specific activity of 429 ± 2.6 nmol min-1 mg-1.PtsC could oxidize aldehyde benzenesulfonic acid to p-carboxybenzenesulfonic acid with a specific activity of 481 ±2.9 nmol min-1 mg-1.3.Study on Treatment of PTS Wastewater by Sequencing Bio film Batch Reactor(SBBR)in Laboratory ScaleThe strain QY7-2 was applied as the microorganisms agent,to verify its effectiveness in the treatment of PTS containing wastewater by SBBR.The results showed that the removal efficiency of COD and PTS could be improved by adding the bioaugmentation agent.The colonization of strain QY7-2 in biofilm was monitored by fluorescence quantitative PCR.The effects of different innoculation methods on colonization of strain QY7-2 on biofilm were investigated.The three-factor and three-level experiments were designed by Design Expert 8.0.5 software.The removal rates of COD and PTS were used as the response values.The response surface methodology was adopted for the objective optimization of process operation parameters,and analyzed the influences of temperature,pH and hydraulic retention time(HRT)on the two response values.The optimal reaction conditions were determined by model prediction as follows:temperature 31.77?,pH 6.69 HRT 2.49d,COD 96.04%and PTS removal rates 91.43%,respectively.4?Engineering-Scale Bioaugmented Treatmen of PTS Containing Wastewater Based on the above investigation results,we applied the isolated high-efficiency PTS-degrading strain into a pesticide manufacturing company in Jiangsu wastewater treatment system(processing scale 700 m3/d),and improved the treatment efficiency of the original treatment facilities.The removal rates of COD and PTS before bioaugmentation were 78.32%and 7.07%,respectively,which increased to 97.12%and 89.24%after application of PTS degradation agent.The concentration of COD in effluent decreased from 950-1050mg/L to 100-200mg/L,and the concentration of PTS in effluent decreased from 450-550 mg/L to 50-80mg/L.The microbial community analysis of the activated sludge in different operating stages was carried out by high throughput sequencing.The results showed that the abundance and diversity of microbial populations in the stable-stage were significantly higher than those in the start-up stage.According to the sequence based on the phylum level classification,the microbial community structure of the sludge system showed some variations at the beginning of start-up stage,the end of the start-up stage and the stable operation stage.The relative abundance of Proteobacteria and Gammaproteobacteria were increased,and the relative abundance of Alphaproteobacteria was decreased significantly after a period time of stable operation.At the same time,the relative abundance of Bacteroidetes showed a downward trend all the time.According to the sequence in the classification at the genus level,the relative abundance of Hydrogenophaga was increased during the operation of the bioremediation system.At the end of the start-up phase,the relative abundance of Hydrogenophaga of inlet and outlet water reached 5.241%and 6.129%respectively,and at the stable operation stage,the relative abundance of Hydrogenophaga has reached 14.338%and 9.542%respectively.It can be seen that the high-efficient PTS-degrading strain can be well colonized in the bioaugmentation system and formed a stable microbial community system,which showed effective and stable treatment performance. |
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