Transformation And Toxic Effects Of Graphene Oxide Nanomaterials In Culture System Of Nitrogen-fixing Bacteria

Graphene is the most important carbon nanomaterials in recent years.Graphene industry is highly encouraged by the government and several producing lines with annual production of hundreds of tons have been built.Graphene battery,graphene facial mask,graphene tire,graphene touch screen and other products are produced for commercial purposes.With the study,production,usage and discharge of graphene products,graphene is entering the environment,which might induce unknown hazards to the environment.Therefore,the environmental bio-effects of graphene should be fully investigated.Biological nitrogen fixation is the most important step of nitrogen cycle,where nitrogen fixation bacteria play crucial role.When graphene enters the environment,graphene would interact with nitrogen fixation bacteria,which would influence the growth of nitrogen fixation bacteria and also the properties of graphene itself.Such influences might consequently lead to different graphene environmental effects.Thus,the study of transformation and bio-effects of graphene in nitrogen fixation bacteria culture systems becomes an important direction in the environmental biosafety evaluations of graphene materials.In this thesis,we selected classical azotobacter(Azotobacter chroococcum)as the model for nitrogen fixation bacteria to measure the toxicity of graphene oxide and the transformation of graphene in A.chroococcum culture system for the environmental bio-effect evaluations of graphene materials.Graphene oxide was prepared by modified Hummers method and systematically characterized by transmission electron microscopy,atomic force microscopy,infrared spectroscopy,X-ray photoelectron spectroscopy,Raman spectroscopy for the structural and chemical component information.Different concentrations of graphene oxide(0-1.0 mg/mL)was added to A.chroococcum culture system and cultured for 2 h-14 d.The growth curves were recorded.The morphological and ultrastructural information was obtained by checking the A.chroococcum samples under transmission electron microscopy and scanning electron scopy.The growth of A.chroococcum in soil culture system was also measured.Then,we separated graphene oxide from the A.chroococcum culture system,measured the samples by infrared spectroscopy,X-ray photoelectron spectroscopy,X-ray diffraction,Raman spectroscopy,transmission electron microscopy and scanning electron microscopy,and analyzed the functional groups,chemical components and morphology of graphene samples.According to the aforementioned experiments,we obtained the following results:(1)Graphene oxide influenced the growth and structure of A.chroococcum with obvious dose effects.The toxicity of graphene oxide to A.chroococcum largely depended on the exposure concentration.Low concentration of graphene oxide stimulated the growth of A.chroococcum,while inhibition was observed at high concentrations.At low graphene oxide concentration,A.chroococcum kept the morphology just as that of the control group.At high concentration,A.chroococcum showed cell wall break and loss of cytoplasm.In addition,graphene oxide easily bound to the secretion of A.chroococcum cells to form large aggregates that hindered the observation of A.chroococcum under scanning electron microscopy.Even at low graphene oxide concentration,the size of A.chroococcum cells became smaller under transmission electron microscopy.At high concentration,the ultrastructure of A.chroococcum cells was destroyed seriously.Some graphene sheets could be observed surrounding cell wall of A.chroococcum.The dose effect was also observed in the soil culture systems,where graphene oxide stimulated the growth of A.chroococcum at low concentration and inhibited at high concentration.(2)Graphene oxide was fast reduced after entering A.chroococcum culture system and was slightly oxidized in the followed culture period.Graphene oxide was reduced by the culture media and secretion of A.chroococcum immediately after entering the A.chroococcum culture system.The oxygen content of graphene oxide decreased,and accordingly the carbon content increased.Due to the remaining oxygen containing groups,the infrared spectra showed similar characteristics before and after reduction.The color became dark after the reduction,and the stacking of graphene sheets was observed,which was confirmed by the shift of peaks in X-ray diffraction toward larger angle,implying the shortening of layer distances.The reduction increased the defects on graphene sheets,indicated by the larger I_D/I_G values in Raman spectra.With the extension of culture period,the changes of graphene samples were small,including the increase of oxygen contents and decrease of defects.The stacking of graphene sheets was still serious,which was observed under transmission electron microscopy and scanning electron microscopy.The graphene samples lost the sharp single layers under both microscopies.