Microbial Mechanisms Of Sulfamethazine On Greenhouse Gases（CO₂,CH₄ And N₂O）Emissions From Farmland Soil

The manure application is the main source of soil respiration（CO₂）,CH₄ and N₂O which mainly produced by soil microbial process.In recent years,the abuse of veterinary antibiotics in breeding industry has resulted in high levels of maternal and metabolite in livestock manure,which has become the main source of antibiotic pollution in farmland.The veterinary antibiotics entering soil can change the structure and function of soil microbial community through their antibacterial or bacteriostatic action and biodegradation process,so that the emissions of CO₂,CH₄ and N₂O are influenced.Sulfonamides are one of the most commonly used antibiotics,and they have a long half-life in soil.It is important to study the direct and indirect effects of sulfonamides on microorganisms related to CO₂,CH₄ and N₂O under different moisture and fertilizer conditions to understand their environmental ecological effect and risk of manure application.Sulfamethazine（SMZ）is one of the typical examples of sulfonamides.In order to analyze the degradation of sulfamethazine and its effect on greenhouse gases and soil microorganisms after 47 and 61d of SMZ（30mg/kg）being synchronous applied to paddy soil with different basal fertilizers（pig manure and compound fertilizer）,a field experiment was conducted.Based on the main degradation product of SMZ was 2-amino-4,6-dimethylpyrimidine（ADPD）in rice field experiment,Indoor pure incubation experiment was carried to further study the microbial mechanisms of soil greenhouse gases emissions of SMZ and its degradation product ADPD with different moisture and fertilizers.Four treatments were set in field experiment,including:pig manure（CM）used as basal fertilizer,with and without 30mg/kg SMZ（RCM and RCM+SMZ30）;compound fertilizer used as basal fertilizer,with and without 30mg/kg SMZ（RCF and RCF+SMZ30）,urea was applied as topdressing in all fertilizing treatments.Fifteen treatments were set in the Indoor simulated dry land（D）and flooded（F）incubation experiments,including:no fertilizers and no antibiotics applied（CK）;pig manure used as the first fertilizer source,with the additions of 0,0.01,0.1,1,5mg/kg SMZ and 0.1,1mg/kg ADPD respectively（CM,CM+SMZ0.01,CM+SMZ0.1,CM+SMZ1,CM+SMZ5,CM+ADPD0.1,CM+ADPD1）;urea used as the first fertilizer source,with the additions of 0,0.01,0.1,1,5mg/kg SMZ and 0.1,1mg/kg ADPD respectively（CF+SMZ0.01,CF+SMZ0.1,CF+SMZ1,CF+SMZ5,CF+ADPD0.1,CF+ADPD1）.urea was applied as the second fertilizer source in the all fertilizing treatments.In the paddy field,At the two sampling time points,2-amino-4,6-dimethylpyrimidine（ADPD）and 4-（2-imino-4,6-dimethylpyrimidine-1（2H）-ly）aniline（AN）were produced by the degradation of SMZ whether basic fertilizer was pig manure or compound fertilizer,and the former was the dominant intermediate product.The soil treated with SMZ and pig manure significantly decreased the relative abundances of Gemmatimonadetes/Gemmatimonadaceae by 0.70%～0.81%（P<0.05）after 47d,While it significantly promoted the relative abundances of Actinobacteria,Firmicutes（P<0.05）,and Burkholderiaceae（P<0.01）by 2.66%,0.71%,and 0.25%,respectively,after 61d of SMZ application.The soil treated with SMZ and compound fertilizer had a significant inhibitory effect on Nitrospirae and Thermodesulfovibrionia（P<0.05）after 61d.The changes of relative abundances of these bacteria indicated that the function of soil microorganism,especially the function related to carbon and nitrogen transformation,had changed.Under simulated dry land condition,the mid-low concentration（0.1mg/kg）SMZ promoted the soil respiration rate in the first fertilization stage,while other concentrations had no effects on soil respiration rates,and all SMZ treatments had no significant effects on cumulative CO₂ emissions（P>0.05）.Under simulated flooded condition,except for the low concentration（0.01mg/kg）SMZ,other concentrations（0.1～5mg/kg）SMZ significantly inhibited the soil respiration rates（P<0.05）,and the higher concentration of SMZ,the stronger the inhibition.The results of ADPD treatments of pig manure group under dry land condition were consistent with those in SMZ treatments under flooded condition.Under simulated dry land condition,The early（on the 3rd day after SMZ input）relative abundances of Proteobacteria,Bacteroidetes and Nitrospira were significantly reduced by 0.20%to 11.60%（P<0.05）in low concentration SMZ treatment with pig manure as fertilizer source,and the relative abundances of Acidobacteria and Cyanobacteria conspicuously increased by 13.66%and 0.16%（P<0.05）.The early relative abundance of Verrucomicrobia significantly increased by 1.20%（P<0.05）in soil treated with high concentration（5mg/kg）SMZ and urea.High concentration（1mg/kg）of ADPD was identical to the SMZ,with inhibiting the activity of Proteobacteria,and promoting the activities of Cyanobacteria and Verrucomicrobia.Under simulated flooded condition,the early（On the 7th day after SMZ input）relative abundances of Proteobacteria,Bacteroidetes,Actinobacteria and Gemmatimonadetes were significantly increased by 2.11%～18.97%（P<0.05）in mid-low concentration SMZ treatment with pig manure as fertilizer source,while the prophase relative abundances of Acidobacteria,Chloroflexi and Verrucomicrobia were significantly reduced by 1.81%～13.39%（P<0.05）.The later（On the 27th day after SMZ input）relative abundance of Firmicutes significantly increased by 15.16%（P<0.05）and Planctomycetacia decreased markedly by 0.53%（P<0.05）in high concentration SMZ treatment with urea as fertilizer source.The effect of high concentration ADPD treatment of urea group was consistent with that in high concentration SMZ treatment.According to the correlation,soil respiration was significantly related to Proteobacteria,Bacteroidetes and Nitrospira at the dryland condition（P<0.05）,while the decrease of CO₂ emissions in SMZ treatments may be the increase of relative abundance of Firmicutes and the decreases of relative abundances of Acidobacteria and Planctomycetacia under the flooded condition and the changes of CO₂ emissions in ADPD treatments were also related to the relative abundance of Firmicutes.Under simulated flooded condition,the soils treated with SMZ and pig manure significantly inhibited CH₄ emissions at the first fertilization stage（P<0.05）,while there were no significant effects in the second fertilization stage（P>0.05）.When the urea was used as the first fertilizer source,except for the significant promoting effect of CH₄ emission in mid-low concentration SMZ treatment（P<0.05）and conspicuously inhibition of CH₄ emission in middle（1mg/kg）concentration SMZ treatment（P<0.05）,other SMZ treatments had no significant effects on CH₄ emission（P>0.05）.During the whole cultivation time,the cumulative CH₄ emission in FCF+SMZ0.01 treatment increased significantly by 1.12 times（P<0.05）,while other concentrations only showed a downward trend at the average level（P>0.05）.ADPD treatments promoted CH₄ emissions in the second fertilization stage,but they had no significant effects on the total amount（P>0.05）.The mid-low and high concentration SMZ with pig manure as fertilizer source had a early promoting and late inhibited effect on CH₄-16s rRNA,pmoA and mcrA,while they mainly inhibited the three genes when urea was used as fertilizer source.The correlation showed that CH₄ emission was significantly positively correlated with the relative abundances and ratios of CH₄-16s rRNA and pmoA（P<0.01）,and negatively correlated with Firmicutes（P<0.01）,it indicated the direct and indirect causes of CH₄ emission changed by SMZ were the changes of the CH₄-16s rRNA/pmoA gene ratio and Firmicutes activity.At the same time,the ADPD results also showed that SMZ degradation products had a medium-and long-term effect on CH₄ emission.Under simulated dry land condition,different concentrations of SMZ promoted N₂O emissions in two stages of fertilization.Under simulated flooded condition,high concentration SMZ significantly inhibited N₂O emission at the first fertilization stage（P<0.05）and promoted it at the second fertilization stage（P<0.05）,the mid-low concentration（0.1～1mg/kg）of SMZ also significantly promoted N₂O emission at the second fertilization stage（P<0.05）.During the whole cultivation time,the cumulative N₂O emissions of SMZ treatments increased to a certain extent.The results of N₂O emissions in ADPD treatments were consistent with SMZ treatments.Under the dry land condition,except for the early significant inhibition of nirS gene in DCF+SMZ0.01 treatment（P<0.05）and the late promotion of nirK gene in DCM+SMZ5 treatment（P<0.05）,other SMZ treatments had a early promoting and late inhibited or no effect on AOA amoA,AOB amoA,nirK,nirS and nosZ genes.Under the flooded condition,soils added with SMZ and pig manure promoted the AOA amoA,AOB amoA,nirK,nirS and nosZ genes all the time,while soils added with SMZ and urea had a early promoting and late inhibited effect on nitrification and denitrification genes.The results of nitrification and denitrification genes of high concentration ADPD treatment were basically consistent with those in SMZ treatment under same water and fertilizer source conditions.According to the analysis of correlation and gene abundances of N₂O production and consumption,the increase of N₂O emission in SMZ and ADPD treatments under simulated dry land condition was mainly due to the increases of AOB amoA gene abundance and nirK/nosZ ratio,while the prophase decrease of N₂O emission in SMZ and ADPD treatments under simulated flooded condition was mainly due to the decrease of（nirK+nirS）/nosZ ratio.The ADPD results also showed that the SMZ degradation products had a long-term effect on NO₂ emission and related genes.