Study On Impacts Of Silver Nanoparticles On Microbes Of Constructed Wetlands

The widespread use of silver nanoparticles（Ag NPs）leads to their inevitable entry into the natural environment during production,transportation,usage and disposal,which may pose a potential threat to safety of ecological environment and human society.Constructed wetlands（CWs）are widely used as a composite wastewater treatment technology consisting of substrate-plant-microorganisms,in which microorganisms play an important role in the process of pollutant transformation and removal.In order to assess the potential threat of Ag NPs to CWs and further explore the regulatory strategies,it is necessary to systematically and thoroughly investigate the ecological effects of Ag NPs in CWs and their stress mechanisms on key microorganisms.However,little research has been conducted on the stressful effects of Ag NPs on pollutant removal of CWs,especially on stress effects of microorganisms.To address above issues,this study combined the changes of decontamination performance and microscopic toxicity effects to investigate the migration and spatial distribution of Ag NPs in CWs and reveal the influence of long-term Ag NPs exposure to decontamination performance in order to explore the response mechanisms of key microorganisms in CWs to long-term Ag NPs stress.Firstly,this study investigated migration distribution of different concentrations of Ag NPs and their effects on key microorganisms in CWs.The results showed that CWs could achieve effective retention of Ag NPs（removal efficiency>96%）,while 93%of Ag NPs were retained in soil layer,and their migration abilities were weak.After short-term exposure,different concentrations of Ag NPs exhibited significant acute inhibitory effects on nitrogen and phosphorus removal in CWs,and the relative abundance of polyphosphate bacteria such as Rhodoplanes and functional genes ppx and pho A/pho D encoding exonuclease and polyphosphate kinase were inhibited,while decrease degrees in removal of ammonia nitrogen（NH₄⁺-N）and total nitrogen（TN）were significantly and positively correlated with exposure concentrations（p<0.05）,mainly due to the Ag NPs suppression on key microbial abundance,which altered the microbial community structure,and significantly downregulated the relative abundance of functional genes amo A,hao and nir K and nor B（p<0.05）,and decreased relative abundance of nitrifying bacteria such as Nitrosomonas and Nitrospira and denitrifying bacteria such as Flavobacterium,Comamonas and Azoarcus.The recovery of NH₄⁺-N and TN removal after long-term exposure to Ag NPs（450 days）was observed,especially significant recovery in soil layer,attributing to the toxic excitatory effect of Ag NPs stimulating urease（URE）,ammonia monooxygenase（AMO）and nitrite reductase（NIR）activities and up-regulating the nitrogen removal functional genes amo A and nos Z abundance（p<0.05）.Subsequently,this study investigated effects of the presence/absence of wetland plants on the migration and distribution of Ag NPs,and microbial community in CWs.The study showed that the distribution and removal of Ag NPs in two CWs were not significantly different,and the Ag NPs removal in CWs with plants（97%）was slightly higher than that in CWs without plants（95%）.Wetland plant exhibited a certain absorption and enrichment on Ag NPs,and Ag NPs mainly accumulated in the plant roots,while substrate retention played a major role in Ag NPs removal.Before adding Ag NPs,the decontamination performance of CWs with plants was significantly higher than that of CWs without plants（p<0.05）.After added Ag NPs（100μg/L）,removal of nitrogen and phosphorus significantly decreased（p<0.05）but decrease degree of NH₄⁺-N and TN removal in CWs with plants（14.67%,10.60%）was significantly lower than that in CWs without plants（30.66%,24.39%）（p<0.05）,indicating that wetland plants alleviated inhibitory effect of Ag NPs to some extent.Microbial community structure of CWs with and without plants significantly differed under Ag NPs exposure,and key wetland microorganisms such as Sulfuritalea,Dechloromonas,Pseudomonas and Comamonas had higher relative abundance in CWs with plants.Meanwhile,relative abundances of the functional genes amo A/nxr A,nir K/nir S and nos Z in CWs with plants were higher than that in CWs without plants,which indicated that plant played a synergistic anti-stress role on microorganisms and guaranted the stable operation of the CWs to some extent.This study also comparatively analyzed the migration of Ag NPs in CWs with different hydraulic flow directions（upstream and downstream）and their effects on key microorganisms.The results showed that both CWs could effectively remove Ag NPs（removal rate of 98%）,and the substrate type had a significant effect on Ag NPs retention,with the retention capacity of sand layer being greater than gravel layer.In upflow CWs,Ag NPs exposure led to inhibition of nitrification,decreased NH₄⁺-N removal,and significantly down-regulated of amo A,nxr A and annamox gene abundances（p<0.05）.And 0.5 mg/L Ag NPs significantly reduced NO₃^--N removal and down-regulated nir K,nir S and nos Z genes（p<0.05）,but 2 mg/L Ag NPs showed no adverse effect on denitrification and functional genes.In downstream CWs,Ag NPs exposure at 2 mg/L inhibited the nitrification process and decreased NH₄⁺-N removal,while the abundances of amo A and nxr A were significantly down-regulated（p<0.05）and the relative abundance of Nitrosomonas and Nitrospira decreased.Ag NPs exposure led to a significant decrease in NO₃^--N removal and abundances of nir K,nir S and nos Z were significantly down-regulated（p<0.05）.In addition,the microbial community structure differed significantly,with enriching denitrifying bacteria Azospira and Rhizobacte in downflow CWs,while enriching denitrifying bacteria Thauera and Pseudomonas in upflow CWs.Sulfur-reducing bacteria Desulfonema was enriched in upflow CWs,indicating that upflow CWs were in a hypoxic state and the difference of hydraulic flow direction changed the system microenvironment,which in turn led to the difference in the response of microorganisms to Ag NPs.Finally,this study comparatively revealed stress effects of long-term exposure of Ag NPs,Ag₂S NPs and Ag⁺on key microorganisms in CWs for advanced treatment,and initially explored toxicity mechanisms of Ag NPs.The results showed that CWs removed 98%of Ag NPs,Ag₂S NPs and Ag⁺,where TFs in order was Ag NPs>Ag₂S NPs>Ag⁺.The long-term exposure of Ag NPs,Ag₂S NPs and Ag⁺all led to downregulation of amo A and nxr A genes and reduced AMO activity,which in turn significantly inhibited nitrification process in sand layer,decreasing of NH₄⁺-N removal,with Ag⁺having the greatest inhibitory effect,followed by Ag₂S NPs and Ag NPs.Unlike Ag₂S NPs and Ag⁺,Ag NPs significantly inhibited the denitrification process,decreased NO₃^--N removal（p<0.05）,significantly down-regulated nir S and nos Z gene and inhibited the relative activities of the substrate binding protein responsible for translocating and uptaking NO₃^--N（Nrt ABCD）as well as the nitrate reductase（EC:1.7.99.-）（p<0.05）.Comparing with effects of Ag⁺and Ag₂S NPs on microbial denitrification,the stress effect of Ag NPs originated from the combined effect of released Ag⁺and its nanoparticles.In addition,the microbial communities differed significantly under different NPs exposures,with Ag NPs and Ag⁺being closer and farther from Ag₂S NPs.The relative abundances of biomarker microbials associated with denitrification such as Bacillus,Azospira and Geothrix in Ag NPs were lower than control,while Acidovorax,Sulfurimonas,Zoogloea,Desulfobacterium and Rhodanobacter in CWs with Ag₂S NPs were higher than control.In addition,Ag⁺-exposed CWs enriched denitrifying bacteria such as Dechloromonas and Comamonas.In summary,this study thoroughly investigated migration distribution of Ag NPs in CWs and their effects on key microorganisms,and preliminarily explored the stress mechanism of Ag NPs through comparison with Ag₂S NPs and Ag⁺,which would help to provide the necessary theoretical and technical support for the correct prediction and comprehensive evaluation of the potential threat of Ag NPs to CWs,the establishment of Ag NPs release limits and environmental risk control standards.