Toxicity And Influence On Nitrogen Fixation Activity Of Metal-organic Framework Material MOF-199 On Azotobacter Vinelandii

Metal organic framework material(MOF)is an emerging material with unique structure and performance.It has been widely used in many fields due to its simple preparation,low production cost and large pore volume.Before large-scale production and application of MOF,it is necessary to thoroughly study its toxicity and environmental safety,and avoid the old path of"pollution first,treatment later".However,compared with the application research in full swing,the environmental biosafety of MOF materials is rarely reported.Biological nitrogen fixation accounts for 80%of the total nitrogen fixation,which is a key part of the nitrogen cycle;in biological nitrogen fixation,nitrogen-fixing bacteria play an irreplaceable role.Therefore,systematic research on the toxicity and environmental impact of MOF materials on nitrogen-fixing bacteria is of great significance,and will provide important basic data for the environmental safety evaluation of MOF materials.This thesis uses the Azotobacter vinelandii as a model to study the effects of a typical MOF material MOF-199 on its growth,structure and nitrogen fixation activity,explore ways to reduce the toxicity of MOF-199 through carbonization and remove copper,and expand the bacterial toxicity of MOF-199 mechanism research.Using Cu(NO₃)₂ and H₃BTC as raw materials,MOF-199 was synthesized by hydrothermal method.The product was characterized by scanning electron microscope(SEM),infrared spectroscopy(IR),Raman spectroscopy(Raman),X-ray diffraction(XRD),X-ray photoelectron spectroscopy(XPS),etc.,to obtain information on its structure and chemical composition.After MOF-199 was co-incubated with Escherichia coli,Staphylococcus aureus,and Azotobacter vinelandii,the growth curve(OD₆₀₀),colony forming unit(CFU)count,cell mortality,and ultrastructure were used to study MOF-199's Toxicity,and explore the mechanism of MOF-199 bacterial toxicity by measuring membrane damage,DNA damage,oxidative stress parameters and Cu release.The effect of MOF-199 on the nitrogen-fixing activity of Azotobacter vinelandii was determined by the isotope dilution method and the acetylene reduction method,and the gene of Azotobacter vinelandii was sequenced to analyze the mechanism that affects the nitrogen-fixing activity.Based on the above experiments,the research results obtained in this paper are:(1)MOF-199 has certain toxicity to Escherichia coli and Staphylococcus aureus,and the release of Cu²⁺by MOF-199 is the main source of its toxicity.MOF-199inhibits the growth of bacteria only at high concentrations,and cell death,ultrastructural damage and DNA damage are observed.The toxicological mechanism of MOF-199 is attributed to oxidative stress,mainly from Cu²⁺.The toxicity of MOF-199 to bacteria is between the raw materials Cu(NO₃)₂ and H₃BTC.In addition,a large amount of Cu²⁺eluted from MOF-199 was detected in the medium.(2)MOF-199 inhibits the growth of Azotobacter vinelandii and inhibits its nitrogen-fixing activity.At 40?g/mL and higher concentrations,MOF-199 caused a slowdown in OD₆₀₀ growth,a decrease in CFU,and an increase in cell death rate.The ultrastructure shows obvious cell shrinkage,a decrease in the number of vesicles in the cytoplasm,and damage to the edge of the cell membrane.Similar to the toxicity mechanism of Escherichia coli and Staphylococcus aureus,MOF-199 releases Cu²⁺in the medium of Azotobacter vinelandii,causing oxidative stress.MOF-199 causes the reduction of nitrogen-fixing activity of nitrogen-fixing bacteria,and its molecular mechanism is related to the inhibition of phosphorylase-related energy metabolism pathways by MOF-199.(3)DMOF-199 is obtained after carbonization treatment and copper removal.The toxicity of this material is significantly lower than that of MOF-199.Within the range of 0-60?g/mL,DMOF-199 does not inhibit the growth of Azotobacter vinelandii,nor does it induce cell death.Scanning electron microscopy and transmission electron microscopy observations showed that DMOF-199 did not damage the cell wall and cell membrane of Azotobacter vinelandii,nor did it cause cytoplasmic loss.There is no significant change in oxidative stress parameters,indicating that DMOF-199 does not cause oxidative damage.DMOF-199 does not inhibit the nitrogen-fixing activity of Azotobacter vinelandii.