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

Abstract

ABSTRACT Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH 3 ) to obtain energy for growth on carbon dioxide (CO 2 ) and can also produce nitrous oxide (N 2 O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. 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 (Na 2 CO 3 ) supplemented with atmospheric CO 2 . IC-limited cultures oxidized 25 to 58% of available NH 3 to nitrite, depending on the dilution rate and Na 2 CO 3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH 3 -limited cultures. Rates of N 2 O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH 3 -N that was transformed to N 2 O-N from 0.5% (replete) up to 4.4% (0.2 mM Na 2 CO 3 ). 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 C 1 metabolism, including the genes for RuBisCO ( cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C 1 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 N 2 O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH 3 oxidation from growth and increased N 2 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 (CO 2 ) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea , a model ammonia-oxidizing bacterium, and report on increased production of N 2 O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N 2 O emissions from nitrification in soils and wastewater treatment.