| Poultry and livestock feeds are frequently supplemented with heavy metals,however,heavy metals are adsorbed poorly in the animal gut and much of it is excreted unchanged in the urine and feces.Aerobic composting is an important approach to treat livestock manure,and microorganisms play major roles in the degradation of organic matter during the composting process,which is an important factor affecting the efficiency of composting.Microbes involving in nitrogen cycle are the main role in the turnover of nitrogen,which effects the quality of the compost product.However,compost product contains resistance genes can enter the soil following directly land application is an important pathway for transmitting resistance genes to the natural environment and pose a great health risk to humans.Therefore,using copper（Cu）as a typical pollutant in manure,and to investigate the effects of Cu on the bacterial communities,microbes of nitrogen cycle（including nitrification,denitrification,and nitrogen fixation）,and resistance genes（including copper resistance genes（CRGs）,antibiotic resistance genes（ARGs）,and the class 1 integron-integrase gene（int I1））,and the relationships among Cu,bacterial communities,microbes of nitrogen cycle,and resistance genes were also analyzed.The aim of this study was to explore the effects of different concentrations of Cu on the function of microbial communities and resistance genes and to elucidate the underlying microbiological mechanisms.The main results and conclusions are as follows.（1）Actinobacteria,Firmicutes,and Proteobacteria were the dominant bacterial taxa,and a high copper concentration decreased the abundances of bacteria that degrade cellulose and lignin（e.g.,class Bacilli and genus Truepera）,especially in the early phases.The abundance of bacteria was inhibited by high concentration（2000 mg/kg）of copper during middle and late phases,and principal component analysis clearly separated high concentration of copper treatment from control and low concentration（200 mg/kg）.Furthermore,results obtained using PICRUSt showed that copper had a significant negative correlation with the predicted genes related to lignocellulose degradation.Moreover,network analysis showed that copper might alter the co-occurrence patterns of bacterial communities by changing the properties of the networks,the keystone taxa,and increase the competition during composting.Temperature,water-soluble carbohydrates,and copper significantly affected the variations in the bacterial community according to redundancy analysis.The copper content mainly contributed to the bacterial community in the thermophilic and cooling phases,where it had positive relationships with potentially pathogenic bacteria（e.g.,Corynebacterium1 and Acinetobacter）.（2）The pH,NH4+-N,NO3--N,and potential ammonia oxidation（PAO）values were inhibited significantly by the addition of Cu.Furthermore,the abundances of the ammonia-oxidizing archaea（AOA）amoA gene and ammonia-oxidizing bacteria（AOB）amoA gene were determined by quantitative PCR,and their compositions were evaluated by denaturing gradient gel electrophoresis（DGGE）.AOA was the dominant ammonia oxidizing microorganism,of which the abundance was much higher than AOB during composting.Cu50 and Cu500 had significant inhibitory effects on the abundance of the amoA gene.The DGGE profile and statistical analysis showed that Cu changed the AOA and AOB community structure and diversity,where Nitrosomonas and Crenarchaeota dominated throughout the composting process.（3）High concentration（2000 mg/kg）of copper enhanced the abundance and diversity of nirS,nirK,and nosZ in second compost phase.Proteobacteria dominated in nirS,nirK,and nosZ types of denitrifier community,and Cu could alter the denitrifier community composition.Steroidobacter,Nitrosomonas,and Paracoccus dominated the changes of nirS,nirK,and nosZ abundance via network analysis and these genus correlated with increasing Cu based on redundancy analysis（RDA）.Interestingly,the result of co-occurrence patterns among denitrifying bacteria of each treatment shown Cu might increase competition of the denitrifying bacteria communities and the stability.（4）The nifH gene copy number was relatively high in the early stage of composting and Cu had a significant inhibitory effect on the nifH copy number.Furthermore,Cu decreased the diversity of nifH and changed the microbial community structure in the early stage.The nifH genes from members of Firmicutes and Clostridium were most abundant,in addition,low concentration（200 mg/kg）and high concentration（2000 mg/kg）of copper decreased the abundance of Desulfotomaculum,Desulfovibrio,and Paenibacillus,while increased Cupriavidus abundance.Co-occurrence ecological network analysis showed that the Cu treatments affected the co-occurrence patterns of diazotroph communities and reduced the associations between different diazotrophs.Interestingly,Cu may weaken symbiotic diazotrophic interactions and enhance the roles of free-living diazotrophs.（5）The absolute abundances of pcoA,tcrB,erm（A）,erm（B）and intl1 were reduced,whereas those of cop A and cusA increased after swine manure composting.High concentration（2000 mg/kg）of copper slowed down the loss of CRGs,ARGs,and intl1during composting.Redundancy analysis showed that NH4+and NO3-play an important role in the distribution of CRGs,ARGs,and intl1,and temperature significantly affected the reduction in CRGs,ARGs,and intl1.The pcoA,tcrB,erm（A）,erm（B）,and intl1 were positively correlated with the bio-available Cu levels.At the genus level,Clostridiumsensustricto1 dominated mesophilic and thermophilic phases,while Pseudomonas and Corynebacterium1 dominated cooling and maturity phases.Network analysis determined that the co-occurrence of CRGs,ARGs,and int I1,and the bacterial community were the main contributors to the changes in CRGs,ARG,and intl1.Thus,temperature,copper,and changes in the bacterial community composition had important effects on the variations in CRGs,ARGs,and intl1 during manure composting in the presence of added copper. |
