Effects Of Silver Nanoparticles On The Toxicity Of Pseudomonas Stutzeri And Soil Denitrification

Silver nanoparticles have become one of the most commonly used nanomaterials in consumer products for their broad antibacterial properties.The extensive production,transportation and use of silver nanoparticles lead to their inevitable entry into the soil environment,posing potential risks to microorganisms.Biological denitrification in soil,driven by denitrifying microorganisms,is an important part of the nitrogen cycle in natural ecosystems.However,denitrifying microorganisms associated with this process respond to the stimulation of silver nanoparticles is unknown.Therefore,understand the effects of silver nanoparticles exposure on denitrification process and the toxicity mechanism of denitrifying microorganism is imminent,and to provide theoretical basis for ecological toxicity assessment of silver nanoparticles.The ion conversion in denitrification process under exposure to silver nanoparticles was studied to explore the influence of silver nanoparticles and released silver ions on the content changes of NO₃^--N,NO₂^--N and N₂O in denitrification process.The results showed that the denitrification rate was as high as 94.91%in the blank group without silver,while the denitrification rate was reduced to 87.66%,60.51%and 36.10%in the exposure group with different doses of silver nanoparticles?3.125,6.25 and 12.5 mg/L?.The removal process of NO₃^--N slowed down under the exposure of silver nanoparticles,the peak time of NO₂^--N is delayed,and finally the accumulation phenomenon occurs,the N₂O production is reduced,and the denitrification process is inhibited and delayed by silver nanoparticles.The results of silver ion release experiment showed that the release amount of silver nanoparticles exposed to denitrification medium was small,and the maximum release amount was about 2%.The highest released silver ions were added to the medium and cultured for 24 h,and no accumulation of NO₃^--N and NO₂^--N were found.The released silver ions had little effect on the denitrification process.The toxic effect of silver nanoparticles on soil denitrification model strain Pseudomonas stutzeri was targeted to explore the effects of silver nanoparticles on the activity,apoptosis,cell morphology,interaction with cell membrane,production of reactive oxygen species and cell Ag internalization,key denitrification enzyme activity and gene expression of p.stutzeri.Flow cytometry analysis indicated that the early apoptotic rates of P.stutzeri were 8.72%,30.60%,and 48.60%with treatments of 3.125,6.25,and 12.5 mg/L Ag NPs,respectively.Results for scanning electron microscope?SEM?imaging,?-potential analysis,lactate dehydrogenase?LDH?release and malonyldialdehyde?MDA?production assays demonstrated adsorption of Ag NPs on the cell surface,which altered membrane potential and mediated lipid peroxidation;these events eventually resulted in the aberration of cell morphology.Transmission electron microscopy?TEM?images and measurements of Ag content distribution by ICP-MS indicated that Ag NPs were easily internalized by P.stutzeri,which increased the accumulation of reactive oxygen species?ROS?.Furthermore,the presence of Ag NPs also greatly inhibited expression of genes nap A,nir S,cnor B,and nos Z,thereby reducing the activities of nitrate reductase?NAR?and nitrite reductase?NIR?.These findings will help further our understanding of the mechanism underlying Ag NPs cytotoxicity,and provide the means to evaluate the negative effect of nanoparticles in the environment.The toxicity of silver nanoparticles to p.stutzeri in the presence of two organic matters?FA and HA?was studied to explore the effect of silver nanoparticles on soil denitrification microorganisms in the presence of organic matters.The results showed that FA and HA could attenuate the toxicity of silver nanoparticles to p.stutzeri,and the moderating effect of HA was stronger than that of FA.After the addition of FA/HA,the Fourier infrared spectra showed that the silver nanoparticles could combine with carboxyl groups.Adding FA/HA reduced the inhibition of silver nanoparticles on p.stutzeri activity,but after 24 h of culture,the bacterial inhibition rate increased after adding 50 mg/L FA/HA and silver nanoparticles.In the presence of two kind of organic matters,the damage of silver nanoparticles to the membrane of p.stutzeri could be attenuated.The results of silver internalization after exposure of 50 mg/L FA/HA and silver nanoparticles for 24 h showed that the content of silver in cells was higher than that in cells when silver nanoparticles was added alone,and the high concentration of organic matter promoted the internalization of cells to silver.