Effects Of Sulfadiazine On Nitrogen Removal Performance,microbial Community And Functional Gene In Moving Bed Biofilm Reactor (MBBR)

Sulfadiazine（SDZ）is an antibiotic contaminant with high detection frequency and high concentration distribution in wastewater treatment and aquatic ecology systems in China.The research focus on influence of SDZ on the nitrogen removal efficiency and microbial community of moving bed biofilm reactor（MBBR）is still insufficient.For this purpose,the pure MBBR process was used to treat artificially formulated SDZ wastewater.The effects of influent SDZ concentrations（0,1,3,5,7 and 10 mg/L）on the operational performance of pure MBBR process were investigated under the conditions of maintaining influent COD of 400 mg/L,NH₄⁺-N of 30 mg/L,TP of 5mg/L,DO of 2.5-2.8 mg/L,HRT of 12 h and carrier filling rate of 25%.Fluorescence quantitative PCR and functional gene sequencing was used to analyze the influences of SDZ on absolute abundance of functional genes for nitrogen conversion and the structure of AOB population in biofilm,respectively.Scanning electron microscopy was used to monitor the effects of SDZ on the morphological characteristics of the carrier biofilm and the biomass of pure MBBR was measured.Metagenomic was used to investigate the effects of SDZ on biofilm population structure and nitrogen metabolism pathways,and the relation between relative abundance of nitrogen metabolism functional genes and simultaneous nitrification and denitrification（SND）was discussed.Metatranscriptomic was used to detect the impacts of SDZ on species and functional gene expression abundance in biofilm.According to the test results of water quality,the average removal efficiency of pure MBBR for 1～10 mg/L SDZ was 98.80±1.54%.However,SDZ significantly inhibited COD removal（P<0.05）,and the higher SDZ concentration,the stronger the inhibition effect.But SDZ of 1～3 mg/L significantly enhanced NH₄⁺-N removal performance（P<0.05）.Instead,SDZ strongly interfered with denitrification,and both SND performance and TN removal performance were significantly reduced（P<0.05）.pure MBBR was not well operated in phosphorus removal,but SDZ of 1～3 mg/L could promote phosphorus removal（P<0.05）.Based on fluorescence quantitative PCR and functional gene sequencing,the pure MBBR process had strong enrichment capacity for ammonia-oxidizing bacteria（AOB）,ammonia-oxidizing archaea（AOA）and nitrite-oxidizing bacteria（NOB）.Compared to AOA,AOB were detected as the dominator for ammonia oxidation in the pure MBBR.SDZ of 1-10 mg/L significantly suppressed the copies of AOB amo A gene（P<0.05）,but effectively promoted the copies of AOA amo A and NOB nxr B gene to up-regulation（P<0.05）.In contrast,SDZ of 10 mg/L significantly reduced the copies of nir S gene in denitrifying bacteria（P<0.05）.Nitrosomonadaceae was the main bearers of the ammonia oxidation function of biofilms and SDZ of 1～3 mg/L significantly promoted the diversification of AOB and Nitrosospira growth（P<0.05）.The supplementation of nitrogen sources,the alteration of the abundance of AOB and AOA,the promotion of microbial resistance and the interference with the structure of the AOB population are the main impact mechanisms by SDZ on ammonia oxidation process in pure MBBR.According to the changes of biofilm morphological and biomass,SDZ exposure caused the migration of attached biomass from the surface to the interior of sponge carrier and accelerated shedding and renewal of the aerobic biofilm layer.Based on the metagenomic sequencing results,biofilm communities at the species level were strongly shifted by SDZ（P<0.05）and the relative abundance of most species could not be restored to their initial levels.SND was the main nitrogen metabolism pathway in pure MBBR biofilm,and SDZ didn’t alter the primary nitrogen metabolism mechanism of biofilm.And the effect of SDZ on the ratio of denitrification functional genes to nitrification functional genes was the main mechanism by which it coerced the biofilm SND performance and thus inhibited the TN removal performance.According to the results of metatranscriptomic sequencing,SDZ strongly inhibited the expression abundance of aerobic heterotrophic microorganisms,which was the main reason for the inhibition of COD removal performance.But SDZ inhibited the activity of autotrophic bacterium Nitrosomonadaceae to a lesser extent than its against the heterotrophic bacterium.The effective expression of Alcaligenes faecalis strains which could degrade sulfonamides in the biofilm and the expression of potential SDZ degrading microorganisms,were the important reasons for the high removal efficiency of SDZ by the pure MBBR process.For nitrification functional genes,SDZ of 1 mg/L effectively promoted the expression of amo CAB and hao,which was the main reason for the promotion of ammonia removal.For denitrification functional genes,SDZ didn’t inhibit the expression of（nir K+nir S）and nos Z,but could inhibit the expression of nor BC.Meanwhile,increased expression of the sulfonamide’s antibiotic resistance genes Sul1 and Sul2 was the main mechanism by which biofilm microorganisms adapted to high SDZ concentrations.