Effects Of Graphene-based Nanomaterials On Marine Microorganisms

Graphene-based nanomaterials have been widely used due to their excellent properties.Graphene-based nanomaterials have strong adsorption capacity and can be combined with other nanomaterials(heavy metal ions or metal oxides).These nanomaterials entering the environment will not be degraded and will eventually be collected into the ocean through various ways,which will have an impact on the future marine ecological environment.In order to evaluate the toxic effects of graphene-based nanomaterials on the marine environment,effects of functionalized graphene on the growth of marine microorganisms,related bacteriostatic mechanism and the effect of mixed contamination of graphene oxide and nano-titanium dioxide on marine bacteria were analyzed.In this study,the growth and BOD of a variety of common marine microorganisms were first investigated,including Marinobacter sp.Halomonas sp.,Alteromonas sp.,Fibrio parahaemolyticus,Synechococcus sp.and Rhodotorula sp.,meanwhile,Bacillus subtilis and Escherichia coli were selected as Gram-positive and Gram-negative bacteria respectively for reference strains.Cell biomass and respiration intensity of marine microorganisms were used as important indicators to evaluate the bacteriostatic performance of functionalized graphene.The results showed that functionalized graphene with different concentrations(1-50 mg/L)had significant effects on Marinobacter sp.,Halomonas sp.,Alteromonas sp.,V parahaemolyticus,E.coli,B.subtilis and Rhodotorula sp.,and the inhibition degree was positively related with concentration of functionalized graphene,namely the high concentration of functionalized graphene(50 mg/L)to the strongest inhibitory effect of marine microorganisms;low concentration(1 mg/L)functionalized graphene promoted the growth of Synechococcus sp.to some extent,while high concentration(10 mg/L and 50 mg/L)functionalized graphene inhibited the growth of Synechococcus sp.to some extent.To further understand the effects of functionalized graphene on marine microorganism,the microbial populations in seawater and marine sediments were exposed to functionalized graphene for 2 weeks by simulating the biochemical conditions of the marine ecosystem.The high-throughput sequencing of bacterial 16S rRNA gene was used to investigate the response of marine bacterial community structure to functionalized graphene.The results showed that functionalized graphene had different effects on the bacterial community composition in seawater(50 mg/L)and marine sediments(50 mg/kg).After the exposure of functionalized graphene,the species diversity and species abundance of bacteria in seawater decreased,while the species diversity and species abundance of bacteria in sediments increased.In order to understand the bacteriostatic mechanism of functionalized graphene on marine bacteria,we analyzed their effects on cell wall and cell membrane integrity.The results showed that the integrity of cell walls and cell membranes of Marinobacter sp.,Halomonas sp.,Alteromonas sp.,V.parahaemolyticus,E.coli and B.subtilis decreased by 20%-50%after 24 h exposure to functionalized graphene.Cellular morphological analysis showed that functionalized graphene could fully contact bacterial cells and destroy cell walls and cell membranes through penetration and wrapping.Further,oxidative damage assay results showed that functionalized graphene can stimulate marine bacteria to produce excessive reactive oxygen species(ROS),leading to oxidative stress.Genetic damage detection results showed that the content of genetic damage markers(8-hydroxydeoxyguanosine)in bacterial cells in the functionalized graphene treatment groups increased by 3.72 times compared with the control groups.Thus,functionalized graphene can cause oxidative DNA damage in bacterial cells.Finally,we investigated the effects of graphene oxide in combination with nano titanium dioxide on B.subtilis that derived from marine environment The results showed that the two nanomaterials have strong bacteriostatic performance when mixed together.The bacteriostatic performance of the two nanomaterials mixed were stronger than those used alone at the same concentration.The interaction model of the two factors was established by t test,which proved that the two nanomaterials had synergistic effect in bacteriostatic performance.Cell wall integrity analysis,cell membrane integrity analysis and oxidative damage detection further proved that these two nanomaterials mainly caused damage to bacterial cells through mechanical damage and oxidative damage.In conclusion,our results preliminarily reflected the effects of graphene-based nanomaterials on marine microorganisms and their bacteriostatic mechanism,which are of value in marine ecological safety assessment of graphene-based nanomaterials.