Transient Accumulation of NO2- and N2O during Denitrification Explained by Assuming Cell Diversification by Stochastic Transcription of Denitrification Genes

Denitrifying bacteria accumulate NO2-, NO, and N2O, the amounts depending on transcriptional regulation of core denitrification genes in response to O-2-limiting conditions. The genes include nar, nir, nor and nosZ, encoding NO3-, NO2-, NO- and N2O reductase, respectively. We previously constructed a dynamic model to simulate growth and respiration in batch cultures of Paracoccus denitrificans. The observed denitrification kinetics were adequately simulated by assuming a stochastic initiation of nir-transcription in each cell with an extremely low probability (0.5% h(-1)), leading to product- and substrate-induced transcription of nir and nor, respectively, via NO. Thus, the model predicted cell diversification: after O-2 depletion, only a small fraction was able to grow by reducing NO2-. Here we have extended the model to simulate batch cultivation with NO3-, i.e., NO2-, NO, N2O, and N-2 kinetics, measured in a novel experiment including frequent measurements of NO2-. Pa. denitrificans reduced practically all NO3- to NO2- before initiating gas production. The NO2- production is adequately simulated by assuming stochastic nar-transcription, as that for nirS, but with a higher probability (0.035 h(-1)) and initiating at a higher O-2 concentration. Our model assumes that all cells express nosZ, thus predicting that a majority of cells have only N2O-reductase (A), while a minority (B) has NO2--, NO- and N2O-reductase. Population B has a higher cell-specific respiration rate than A because the latter can only use N2O produced by B. Thus, the ratio B/A is low immediately after O-2 depletion, but increases throughout the anoxic phase because B grows faster than A. As a result, the model predicts initially low but gradually increasing N2O concentration throughout the anoxic phase, as observed. The modelled cell diversification neatly explains the observed denitrification kinetics and transient intermediate accumulations. The result has major implications for understanding the relationship between genotype and phenotype in denitrification research.