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

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. IMPORTANCENitrification, 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. During nitrification in aerobic environments, ammonia (NH 3 ) oxidation to nitrite (NO 2 Ϫ ) is initiated by ammonia-oxidizing bacteria (AOB) and archaea (1). These microorganisms are predominantly chemolithoautotrophs that derive energy for growth from NH 3 and meet their carbon (C) needs by fixing carbon dioxide (CO 2 ) (1). All known AOB fix CO 2 via the CalvinBenson-Bassham (CBB) cycle for growth, utilizing ribulose bisphosphate carboxylase/oxygenase (RuB...

Genome-Scale, Constraint-Based Modeling of Nitrogen Oxide Fluxes during Coculture of Nitrosomonas europaea and Nitrobacter winogradskyi

“…NH 3 oxidation and growth rate data generated in previous studies suggests that an increased NH 3 oxidation rate leads to uncoupling of NH 3 oxidation from growth, forcing N. europaea to direct electrons to NIR and NOR to regenerate reductant (21, 46–48). In addition, the integrative model reported here suggests different energy outcomes for the cell based on abiotic oxidation of NO to NO 2 − .…”

“…Extracellular NH 2 OH concentration was measured by chemical assay as previously described (58, 59). NO and NO 2 (NO x ) concentrations in the headspace were measured using a portable NO 2 analyzer/NO x converter (LMA-3D and LNC-3D; Unisearch Associates Ltd., Concord, Ontario, Canada), and N 2 O concentration was measured by gas chromatography as previously described (16, 21). …”

“…The genome-scale models required non-growth-associated maintenance (NGAM) energy requirements to be calculated based on energy model assumptions and chemostat experiments. The NGAM for N. europaea was 52.82 mmol ATP gDCW −1 h −1 (where gDCW stands for gram [dry cell weight]) based on previous chemostat experiments (21) and using the energy model shown in the supplemental material (Data Set S4). The NGAM for N. winogradskyi was 8 mmol ATP gDCW −1 h −1 for 3 h, followed by 18.52 mmol ATP gDCW −1 h −1 for the rest of the experiment.…”

“…The magnitude of nitrifier-derived emissions of N oxides generated by nitrification from soils and engineered environments are extremely variable and depend on a variety of environmental conditions such as the rate of nitrification, pH, temperature, and oxygen (O 2 ), among other factors (4, 13, 18–21). Recent modeling efforts by Perez-Garcia et al and others have sought to understand conditions that generate N oxides through single and multispecies metabolic network models of wastewater treatment systems (17, 22–24).…”

A consilience model to describe N2O production during biological N removal

“…29 However, at the same NH 3 oxidation rates, low IC levels increase the fraction of N 2 O produced. 30 Depending on the nitrogen removal system, wastewaters can have varying IC levels. Due to the heterotrophic oxidization of the organic content of conventional urban wastewater, IC is typically in excess for autotrophic growth, but high N-strength wastewaters with a lower C/N ratio may result in IC limited AOB growth.…”