Single And Combined Pollution Of Three Antibiotics And Copper Affect On Soil Microbial Community Structure And Function

With the development of livestock and poultry industry,China has gradually become the world's largest producer and user of antibiotics.Because of the excessive use of antibiotics in feed and their inability to absorb completely by animals and poultry,most antibiotics are excreted in the form of raw drugs and metabolites.Heavy metals play an important role in the growth and development of livestock and poultry.A certain amount of heavy metals will be added to livestock and poultry feed.However,the amount of heavy metals in feed is often larger than the demand.Heavy metals that cannot be absorbed will be discharged with livestock and poultry manure.With the agricultural use of livestock and poultry manure,antibiotics and heavy metals enter the soil with the agricultural use of livestock and poultry manure and accumulate to produce compound pollution.In order to analyze the effects of antibiotics and heavy metals on soil activity,the study selected three kinds of antibiotic SM2?sulfadimidine?,ENR?enrofloxacin?,OTC?oxytetracycline?and one kind of heavy metal?copper,Cu?as pollutant.According to the molar ratio of 1:2,1:8 and 1:32,the study designed 24 treatments which including four levels for antibiotics(0,0.05,0.20,0.80 mmol?kg^-1)and two levels for Cu(0,1.60 mmol?kg^-1).The study regarded soil microorganisms as the research object,combining with traditional research methods and modern molecular biology methods,and studyed the changes of microbial community structure,functional genes?three carbon cycle genes:cbbM,cbbLR,cbblG,four nitrogen cycle genes:apr,narG,nirS,nifH?and antibiotic resistance genes?ARGs?in different experimental cycles?7,14,21 and 28 days?.The study evaluated the ecological toxicological effects of these three antibiotics and copper,and provided scientific basis for environmental hazard assessment.The results were obtained as follows:?1?The response of different genes to antibiotics was different when the antibiotics were single contaminated.When SM2 was single contaminated,the content of cbbM gene decreased significantly compared with CK group at 7 d and 28 d.When the cbbLR gene was treated with0.8SM2,the gene content decreased in the early stage of the experiment and restored to the level of CK group at the later stage of the experiment.There are also different effects on gene content,such as cbbLG gene,when ENR was single contaminated,its content increased significantly at 14 d and 21 d,and returned to CK group level at 28 d?except 0.2ENR?.But when OTC single pollution,its content is higher than CK group;RubisCO activity was activated in ENR and OTC low concentration groups(0.05 and 0.2 mmol?kg^-1),while it was inhibited in high concentration group at the later stage of the experiment.It was found that the concentration of ARGs increased significantly in single contamination of three antibiotics.In the T-RFLP test,single antibiotic contamination had a greater effect on different length fragments,and different antibiotics had different effects on the same fragment.On 14th day,the proportion of 66 bp fragments in SM2 single contaminated group decreased gradually with the increase of antibiotic concentration,from 65%?0.05SM2?to 47%?0.8SM2?.With the increase of OTC concentration,the proportion of 66 bp fragments decreased and then increased,from 73%?0.8SM2?.05 OTC decreased to 45%?0.2OTC?and increased to 64%?0.8 OTC?.?2?With the single pollutant of Cu,except that the activity of enzyme decreased on 14th day?inhibition rate was 33.33%?,the activity of RubisCO increased in the other three periods,and the content of soil ARGs was also promoted by Cu.The content of four nitrogen cycle genes?apr,narG,nirS,nifH?was increased.Heavy metal pollution may be a potential cause of ARG transmission.?3?When Combined contamination of antibiotics and copper was used,the effects of Combined contamination on gene content were different at different time.For example,when SM2 and Cu were co-contaminated,the narG gene content decreased with the increase of the concentration on 7th day,and increased with the increase of the concentration on 14th day.The response of different genes to Combined contamination was also different.For example,the content of narG gene in 0.2ENR+Cu treatment group was significantly lower than that in CK group on 28th day,while the content of nirS gene was significantly increased.In the experiment of RubisCO activity,at the later stage of the experiment,the combined pollution of three antibiotics and Cu showed a synergistic effect,which weakened the activation of enzyme activity.In the T-RFLP experiment,the compound contamination had a great influence on some microorganisms.For example,when the fragment 51 bp was contaminated by SM2+Cu,OTC+Cu on 14 d,the content decreased to zero with the increase of antibiotic concentration.Heavy metals and antibiotics in soil have compound selective pressure on ARGs.Combined pollution of OTC and Cu,0.8OTC+Cu group has greater influence on tetM than 0.05OTC+Cu and 0.2OTC+Cu.