Transformation And Environmental Bio-effects Of Carbon Nanomaterials In Culture System Of Nitrogen-fixing Bacteria

Biological nitrogen fixation dominates 80% of the nitrogen fixation from atmosphere,which an essential step of nitrogen cycle.Azotobacter chroococcum is a type of bacteria that dominates the nitrogen cycle and plays an irreplaceable role in biological nitrogen fixation.In recent decades,nanoscience has made great progress and been applied in many fields,such as information,environment,biomedicine,food health,and so on.Among these nanomaterials,carbon nanomaterials are widely produced and used due to their unique physical and chemical properties,and they are inevitably released into the environment.Carbon nanomaterials are chemically stable and difficult to be biodegraded,which may cause the continuously accumulatione in the environment and potential environmental risks.After entering the environment,carbon nanomaterials may affect the growth of A.chroococcum,the expression level and activity of nitrogen-fixing enzymes,and cause changes in their biological nitrogen-fixing capacity,which in turn interferes with the nitrogen cycle and leads to serious ecological consequences.Therefore,it is necessary to evaluate the environmental biological effects of carbon nanomaterials on A.chroococcum,so as to more comprehensively understand the environmental biological safety of carbon nanomaterials.In this study,we focused on sp~2 carbon nanomaterials,including pristine multi-walled carbon nanotubes(MWCNTs)and graphene oxide(GO),The influences of GO and MWCNTs on the growth,structure,and activity of autogenous A.chroococcum were investigated.The regulating effect of carbon nanomaterials on the biological nitrogen fixation was also revealed.Firstly,the modified Hummers method was used to prepare GO and MWCNTs were purchased.The two carbon nanomaterials were systematically characterized,by usingatomic force microscopy,infrared spectroscopy,X-ray photoelectron spectroscopy and Raman spectroscopy for the structural and chemical component information.After the carbon nanomaterials were incubated with A.chroococcum,the toxicity was monitored by colony-forming unit(CFU)counting,growth curve,live/dead staining and ultrastructural observations.The nitrogen fixation activity of A.chroococcum was measured by acetylene reduction assay and the contents of different nitrogen forms in the soil were determined.In addition,after incubation with A.chroococcum,GO was separated,washed,and characterized by infrared spectroscopy,X-ray photoelectron spectroscopy,X-ray diffraction,Raman spectroscopy,transmission electron microscopy,and scanning electron microscope,to reveal the reduction of GO in the A.chroococcum culture system.Based on the aforementioned experiments,the major achievements were listed as follows:(1)GO could be quickly reduced by the extracellular secretions of autogenous A.chroococcum.GO was rapidly reduced by the A.chroococcum secretion,and interacted with the A.chroococcum in a partially reduced form in the culture system.After the GO was reduced,the oxygen content decreased,the electrostatic repulsion between the graphene layers decreased,and the stacking was more compact.Raman characterization showed that the defects of graphene increased after reduction,and the carbon skeleton remained as sp~2 hybrid.(2)GO had significant toxic effects on autogenous A.chroococcum.The growth of autogenous A.chroococcum was stimulated at low concentrations and showed an inhibitory effect at high concentration.High concentration of GO induced A.chroococcum cell death and cell wall rupture.Its toxicological mechanism was related to membrane damage and oxidative stress,which was manifested by increased levels of lactate dehydrogenase and changes in oxidative stress indicators.GO also showed a concentration-dependent toxic effect in the soil with reduced toxicity.GO slightly inhibited the nitrogen-fixing activity of autogenous A.chroococcum,but high concentrations of GO caused an increase in soil nitrogen content.(3)MWCNTs were of low toxicity to autogenous A.chroococcum.MWCNTs stimulated the growth of A.chroococcum at a low concentrations and showed a slight inhibitory effect at high concentration.Under transmission electron microscopy,MWCNTs even at high concentrations did not significantly induce the death of A.chroococcum cells and cell wall rupture,and no cytoplasmic loss was found.The level of lactate dehydrogenase in the medium did not change significantly compared with the control group,indicating that MWCNTs did not cause membrane damage.There was no significant change in oxidative stress-related indicators,indicating that MWCNTs did not trigger a severe oxidative stress response.Fluorescent staining method was used to detect the survival rate of A.chroococcum.After 6 and 14 days of exposure,MWCNTs did not cause serious cell deaths.MWCNTs slightly inhibited the nitrogen fixation activity of autogenous A.chroococcum,but high concentration of MWCNTs caused the increase of soil nitrogen content.