Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3 ) to obtain energy for growth on carbon dioxide (CO2 ) and can also produce nitrous oxide (N2 O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses ofN. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2 CO3 ) supplemented with atmospheric CO2 . IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2 CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3 -limited cultures. Rates of N2 O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3 -N that was transformed to N2 O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2 CO3 ). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2 O. Together, these data suggest thatN. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2 O production.IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2 ) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome ofNitrosomonas europaea , a model ammonia-oxidizing bacterium, and report on increased production of N2 O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2 O emissions from nitrification in soils and wastewater treatment.