Effects Of Silver Nanoparticles On Soil Nitrogen Transformation By Bacteria And The Mechanisms

Currently,the use of silver nanoparticles?AgNPs?in consumer products is increasing with the development of engineered nanoparticles technology.During the production,manufacturing,used and disposal of AgNPs containing products,AgNPs would be released into the terrestrial system.AgNPs as an antimicrobial agent could also pose a threat to the microbial communities in the environment,and the majority of nitrogen entering ecosystems mediated by bacteria in soil.AgNPs as the new environmental pollution entered into farmland ecosystem,inhibited the activity of nitrification and nitrifying bacteria in soil had high sensitivity to them.The aim of the study was investigated the transfort,chemical properties,and toxicological effects of AgNPs on soil microorganism in the environment.The mechanisms of AgNPs inhibited the activity of bacteria,and effects of AgNPs on the expression of function genes copies needed to study,which were helpful for AgNPs pollution control in farmland ecosystems.Under laboratory condition,adopting batch processing method studied the effects of AgNPs on soil bacteria communities'structure diversity,the key process of soil nitrogen transformation by microbial,the mechanisms for killed bacteria and nitrogen gross transformation rates.At first,determined the soil microbial communities'structure diversity under exposure on AgNPs environment though Illumina platform;studied the effects of AgNPs on the intensity of ammonification and nitrification in soil,and used fluorescent quantitation PCR the expression of the key genes copies in soil nitrogen transformation;the model ammonia oxidation bacteria Nitrosomonas europeae was used to study the toxicity of AgNPs for different dose,size and the release of Ag⁺from AgNPs;based on ¹⁵N dilute technology determined the soil gross N nitrification rates,gross N mineralization rates and nitrogen transformation,the activities of enzymes were also studied under the condition exposure on AgNPs.The objective of this study was to investigate the change in soil microbial communities,microorganism-oriented toxicity of AgNPs in soil,and the effect of AgNPs on nitrogen fertilizer.The changes of function genes copies,the potential mechanism of bacterial toxicity and the nitrogen fertilizer transformation under the condition exposure to AgNPs in soil,the main results as following:1.Under laboratory condition,soil samples were incubated at 28?for 7 d under exposure on different concentrates of AgNPs?10,50,100 mg/kg?.After incubation,used chloroform fumigation exaction method and substrate induced respiration to determine soil microbial biomass nitrogen and the activity of soil microorganisms,respectively.AgNPs added in soil inhibited the activity of soil bacteria.The soil microbial biomass nitrogen were determined as exposed to the concentration of AgNPs 10,50,100 mg/kg in soil,respectively,compared with the control the inhibition were 33.1%,58.7%,71.1%,respectively;while substrate-induced respiration were determined at the same following exposure concentrations for AgNPs,and compared with the control the inhibition were 1.17%,17%,29.3%,the results shown that at low dose of AgNPs no signification inhibition for bacteria.Analysis the soil microbial communities'abundance under the condition soil exposure to different dose of AgNPs.For genus level determined by soil microbial,the number of genus groups were negative correlation with the dose of AgNPs,but the number for dominate groups were the same.The concentrations of AgNPs applied to soil were not affect the dominate phylum?Proteobacteria,Acidobacteria,Actinobacteria?,the abundance of Proteobacteria was negative correlation with the dose of AgNPs,while the abundance of Acidobacteria and Actinobacteria were positive correlation with the dose of AgNPs.The abundance for?-,?-,?-,?-Proteobacteria which exposed to AgNPs were higher than control.Acidobacteria were dominate abundance for each level?order,family and genus?and negative correlation with the content of AgNPs.2.The nitrification intensity were determined at the condition exposure on AgNPs under laboratory condition,used fluorescent quantitative analysed the effects of AgNPs on soil nitrification function genes copies?amoA and Arch-amoA?expression.AOB?ammonia oxidizing bacteria?model bacteria Nitrosomonas europeae was used to study the mechanisms for AgNPs inhibited the activity of nitrifying bacteria in soil,and incubated the bacteria to the medium contained different dose and size AgNPs to investigated bacteriostatic effect.Soil samples exposure to AgNPs inhibited the activity of nitrification,and higher concentration of AgNPs?100 mg/kg?in soil had stronger inhibition effect.As the dose of AgNPs were 50 mg/kg and 100 mg/kg in soil,the inhibition ratio for nitrification were almost the same.While the amoA gene copies were inhibited compared to control 34.4%,68.8%and 91.8%,follow the content of AgNPs were 10,50,100 mg/kg,respectively.The signification nitrification inhibition was caused by 100 mg/kg AgNPs content in soil.Nitrosomonas europeae is the model ammonia oxidizing bacteria,the sub-MIC?minimum inhibitory concentration?for N.europeae exposed to AgNPs were 5-10 mg/L.N.europeae incubated to the medium containing AgNPs?5-10 mg/L?,after 5-8 h incubation time the inhibition ratio were declined.Incubated N.europeae to the medium containing 20 mg/L AgNPs,the inhibited ratio of bacteria were more than 77%,and the ratio not declined with the incubation time.Ag⁺released from AgNPs caused 45%cell death rates at the condition exposure to AgNPs;at the same dose for AgNPs the smaller size caused higher cell death rates,and at lower concentration the small size AgNPs caused significant cell death.3.Soil dilution-plate method was used to calculate the number of cultivable ammonifying bacteria and denitrifying bacteria in soil under exposure on AgNPs,and study the effects of AgNPs on the activity of protease and the denitrification function genes copies.The model denitrification bacteria Achromobacter denitrificans and Pseudomonas stutzeri were incubated to the medium containing AgNPs,respectively,to investigate the effects of AgNPs on denitrifying bacteria.AgNPs caused inhibition effect on the activity for ammonifying bacteria and protease,and the dose of AgNPs in soil had a positive correlation with inhibition rate.But denitrifying bacteria in soil had strong resistance for AgNPs.The number of cultivable denitrifying bacteria in soil and the copies of function gene of denitrification nosZ were not reduced by added AgNPs in soil.Achromobacter denitrificans and Pseudomonas stutzeri were incubated to the medium containing AgNPs?5 mg/L?had no effect on growth and capsule were generated to protect the denitrifying bacteria.When denitrifying bacteria exposed to high content of AgNPs?20 mg/L?,the cell wall were completed and capsule were generated.4.Determined the activity of nitrate reductase and nitrite reductase for soil samples which the AgNPs content in soil were 10,50 and 100 mg/kg,respectively,added mineral fertilizer CO?NH₂?₂,KNO₃ and?NH₄?₂SO₄ or organic fertilizer straw and pig manure in soil investigated the effects of AgNPs on soil nitrogen transformation.Based on ¹⁵N isotope dilution method,primary N nitrification and mineralization rates were reckoned in-lab aerobic incubation under the condition exposure to different dose of AgNPs in soil.As the results shown,the activity of nitrate reductase and nitrite reductase were decreased as exposure to AgNPs.As the content of AgNPs in soil was 10 mg/kg had no significance effect on nitrate reductase.As the content of AgNPs in soil was 100 mg/kg,the activity of nitrate reductase was significant reduced,and inhibition caused by AgNPs were not changed with the incubation time.The inhibition rates for soil almost the same under the condition as AgNPs content in soil were 50 mg/kg and 100 mg/kg,and had significant difference compared to control.Mineral fertilizer CO?NH₂?₂ and?NH₄?₂SO₄ added in soil increased the urease activity under the condition exposed to AgNPs,but the urease activity also affect by the dose of AgNPs.Nitrate,and amide nitrogen promoted the activity for nitrate reductase,but CO?NH₂?₂ and?NH₄?₂SO₄ had no significant effect on nitrite reductase compared to control and exposure to low concentration of AgNPs?10 mg/kg?;nitrate added in soil exposure to AgNPs could increase the activity of hydroxylamine reductase.The activity of decomposition bacteria was inhibited by AgNPs,added organic fertilizer?straw,pig manure?in soil increased the content of NH₄⁺and NO₃^-than added AgNPs as compared.The rates of primary N nitrification and mineralization had negative correlation with the content of AgNPs in soil.At the later stage of incubation,the primary transformation rates were higher in the soil samples which added AgNPs.At incubation periods 2 h-1 d,the content of AgNPs in soil were 10 mg/kg,50 mg/kg and 100 mg/kg,suppressed gross nitrification rates 9.3%,22.63%and 34%,also suppressed gross mineralization rates 24.1%,62.3%and 82.2%,respectively.NH₄NO₃ were added to soil exposure to AgNPs,the content of NO₃^--N in soil had negative correlation with the dose of AgNPs.The function gene amoA and Arch-amoA for nitrification were increased expression as added NH₄NO₃ for control and under the exposure concentration for AgNPs were 10 mg/kg,while the gene expression were decreased for the concentration for AgNPs were 50 mg/kg and 100 mg/kg.