Effect of CO₂ on NADH production of denitrifying microbes via inhibiting carbon source transport and its metabolism

The potential effect of CO₂ on environmental microbes has drawn much attention recently. As an important section of the nitrogen cycle, biological denitrification requires electron donor to reduce nitrogen oxide. Nicotinamide adenine dinucleotide (NADH), which is formed during carbon source metabolism, is a widely reported electron donor for denitrification. Here we studied the effect of CO₂ on NADH production and carbon source utilization in the denitrifying microbe Paracoccus denitrificans. We observed that NADH level was decreased by 45.5% with the increase of CO₂ concentration from 0 to 30,000 ppm, which was attributed to the significantly decreased utilization of carbon source (i.e., acetate). Further study showed that CO₂ inhibited carbon source utilization because of multiple negative influences: (1) suppressing the growth and viability of denitrifier cells, (2) weakening the driving force for carbon source transport by decreasing bacterial membrane potential, and (3) downregulating the expression of genes encoding key enzymes involved in intracellular carbon metabolism, such as citrate synthase, aconitate hydratase, isocitrate dehydrogenase, succinate dehydrogenase, and fumarate reductase. This study suggests that the inhibitory effect of CO₂ on NADH production in denitrifiers might deteriorate the denitrification performance in an elevated CO₂ climate scenario.