The Release Of Biochar Colloids In The Environment And Its Effect On The Transformation Of Antibiotic Resistance Genes

Biochar is a kind of carbon-rich material produced after biomass pyrolysis.Because of its advantages such as fixing carbon and increasing soil fertility,it is being widely used in agriculture and environment.Biochar in the environment will be physically decomposed by the penetration of plant roots,dry-wet cycles,etc.,releasing biochar colloids.In recent years,antibiotic resistance genes（ARGs）in the environment have been recognized as a new type of pollutant that will spread between different species through horizontal gene transfer（such as transformation）,leading to the production of multi-drug-resistant pathogens,thereby reducing the effectiveness of antibiotics to treat bacterial infections.The biochar in the environment will inevitably come into contact with ARGs and affect the spread of ARGs.Previous studies mostly focused on the release behavior of biochar colloids in laboratory water distribution,and seldom paid attention to the release of biochar colloids in actual water and soil environments,and there are few studies on the effect of biochar colloids on the transformation behavior of ARGs.Therefore,this study mainly investigated the colloidal release/sedimentation behavior of biochar in real soil solutions and its effect on the transformation behavior of plasmid-mediated ARGs,and the following main conclusions were obtained:（1）The pyrolysis temperature and the source of biomass have an important effect on the release of biochar colloids in the soil aqueous solution.The release of biochar colloids prepared at lower temperature（400°C）was significantly higher than that of biochar prepared at high temperature（700°C）;the colloids release amount of biochar prepared from wheat straw was higher than that prepared from rice straw.In the soil aqueous solution,the average release of biochar colloids prepared at 400°C can reach 9.33–16.41 mg g^-1,and the average release of biochar colloids prepared at 700°C is less than 2 mg g^-1.The release of biochar colloids is affected by the comprehensive effects of various components in the soil aqueous solution,and has a close negative correlation with the soluble organic carbon（DOC）,Fe,and Al content in the soil solution.（2）The sedimentation curve of the biochar colloids in the soil solution conforms to the exponential model,showing high colloids suspension stability,and 40%of the biochar colloids remains stable in suspension when settlement reaches equilibrium.The sedimentation performance of biochar colloids has a closely positive correlation with the ionic strength of the soil solution and the content of K,Ca,Na,and Mg ions.（3）Wheat straw biochar prepared at 300℃,400℃,500℃,600℃,700℃pyrolysis temperature,whether it is bulk biochar（150μm）or biochar colloids（0.45-2μm）,has a significant inhibitory effect on the transformation behavior of plasmid-mediated ARGs in aqueous solution.Compared with the control group（without biochar）,the inhibition rate of bulk biochar on ARGs transformation was 34.9%–69.9%,and the inhibition rate of biochar colloids on ARGs transformation was 34.6%–77.7%.In general,the biochar prepared at higher temperature has a stronger inhibitory effect on the transformation behavior of ARGs than the biochar prepared at lower temperature,and the inhibitory effect of biochar colloids on the transformation behavior of ARGs is stronger than that of bulk biochar.（4）The inhibition of biochar on the transformation of ARGs is mainly caused by the adsorption of the plasmid carrying ARGs by the biochar during the transformation of ARGs.The ARGs adsorbed on the biochar are difficult to be taken up by the recipient bacteria（E.coli）because they are blocked outside the cell membrane,which in turn affects the plasmid-mediated transformation of ARGs.（5）Both bulk biochar and biochar colloids can inhibit the growth of competent bacteria（E.coli）to a certain extent.Compared with the control group,the growth inhibition rate of bulk biochar on competent bacteria was 22.6–29.4%,and the growth inhibition rate of biochar colloids on competent bacteria was 21.2–26.7%.The inhibitory effect of biochar on the growth of competent bacteria is one of the important reasons for the inhibition of plasmid-mediated ARGs transformation.（6）The biochar extract solution in all experiments had no significant effect on the growth of competent bacteria.Except for the bulk biochar extract solution at 300℃,which has a significant inhibitory effect on the transformation of plasmid-mediated ARGs,the other bulk biochar and biochar colloids extract solution have no significant inhibitory effect on the transformation behavior of ARGs.Gel electrophoresis and CD circular dichroism characterization showed that the 300℃bulk biochar extract solution had no significant effect on the integrity and configuration of the plasmid ARGs.Atomic force microscope characterization showed that the plasmid ARGs had a significant agglomeration in the 300℃bulk biochar extract solution,and the particle size of the plasmid DNA increased.Therefore,the inhibitory effect of the 300℃bulk biochar extract solution on the transformation behavior of ARGs is related to the aggregation of plasmid DNA.That is,the increase of the aggregate size will increase the difficulty of uptake of ARGs by competent bacteria,which will significantly inhibit the transformation behavior of ARGs.This study revealed the release and sedimentation laws of biochar colloids in the actual soil solution,and found that biochar has an inhibitory effect on the transformation behavior of plasmid-mediated ARGs.This will help people have a more comprehensive understanding of the stability of biochar in the environment,provide a new understanding of the interaction between biochar and plasmid ARGs,and provide a way for the use of biochar technology to prevent the spread of plasmid ARGs.