Increasing anthropogenic CO2 emissions have been reported to influence global biogeochemical processes; however, in the literature the effects of CO2 on denitrification have mainly been attributed to the changes it causes in environmental factors, while the direct effects of CO2 on denitrification remain unknown. In this study, increasing CO2 from 0 to 30 000 ppm under constant environmental conditions decreased total nitrogen removal efficiency from 97% to 54%, but increased N2O generation by 240 fold. A subsequent mechanistic study revealed that CO2 damaged the bacterial membrane and directly inhibited the transport and consumption of intracellular electrons by causing intracellular reactive nitrogen species (RNS) accumulation, suppressing the expression of key electron transfer proteins (flavoprotein, succinate dehydrogenase, and cytochrome c) and the synthesis and activity of key denitrifying enzymes. Further study indicated that the inhibitory effects of CO2 on the transport and consumption of electrons were caused by the decrease of intracellular iron due to key iron transporters (AfuA, FhuC, and FhuD) being down-regulated. Overall, this study suggests that the direct effect of CO2 on denitrifying microbes via inhibition of intracellular electron transport and consumption is an important reason for its negative influence on denitrification.

Over the past few decades, human activities have accelerated the rates and extents of water eutrophication and global warming through increasing delivery of biologically available nitrogen such as nitrate and large emissions of anthropogenic greenhouse gases. In particular, nitrous oxide (N2O) is one of the most important greenhouse gases, because it has a 300-fold higher global warming potential than carbon dioxide. Microbial denitrification is a major pathway responsible for nitrate removal, and also a dominant source of N2O emissions from terrestrial or aquatic environments. However, whether the release of zinc oxide nanoparticles (ZnO NPs) into the environment affects microbial denitrification is largely unknown. Here we show that the presence of ZnO NPs lead to great increases in nitrate delivery (9.8-fold higher) and N2O emissions (350- and 174-fold higher in the gas and liquid phases, respectively). Our data further reveal that ZnO NPs significantly change the transcriptional regulations of glycolysis and polyhydroxybutyrate synthesis, which causes the decrease in reducing powers available for the reduction of nitrate and N2O. Moreover, ZnO NPs substantially inhibit the gene expressions and catalytic activities of key denitrifying enzymes. These negative effects of ZnO NPs on microbial denitrification finally cause lower nitrate removal and higher N2O emissions, which is likely to exacerbate water eutrophication and global warming.

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