Characterizing the Metabolic Trade-Off in Nitrosomonas europaea in

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Characterizing the Metabolic Trade-Off in Nitrosomonas europaea in Response to Changes in Inorganic Carbon Supply D. Jiang,†,# W. O. Khunjar,*,†,‡,# B. Wett,∥ S. N. Murthy,⊥ and K. Chandran*,† †

Department of Earth and Environmental Engineering, Columbia University, 500 W. 120th Street, Mudd 918, New York, New York 10027-4711, United States ∥ ARA Consult, Innsbruck, Tyrol, 6020, Austria ⊥ DC Water, 5000 Overlook Avenue SW, Washington, D.C. 20032, United States S Supporting Information *

ABSTRACT: The link between the nitrogen and one-carbon cycles forms the metabolic basis for energy and biomass synthesis in autotrophic nitrifying organisms, which in turn are crucial players in engineered nitrogen removal processes. To understand how autotrophic nitrifying organisms respond to inorganic carbon (IC) conditions that could be encountered in engineered partially nitrifying systems, we investigated the response of one of the most extensively studied model ammonia oxidizing bacteria, Nitrosomonas europaea (ATCC19718), to three IC availability conditions: excess gaseous and excess ionic IC supply (40× stoichiometric requirement), excess gaseous IC supply (4× stoichiometric requirement in gaseous form only), and limiting IC supply (0.25× stoichiometric requirement). We found that, when switching from excess gaseous and excess ionic IC supply to excess gaseous IC supply, N. europaea chemostat cultures demonstrated an acclimation period that was characterized by transient decreases in the ammonia removal efficiency and transient peaks in the specific oxygen uptake rate. Limiting IC supply led to permanent reactor failures (characterized by biomass washout and failure of ammonia removal) that were preceded by similar decreases in the ammonia removal efficiency and peaks in the specific oxygen uptake rate. Notably, both excess gaseous IC supply and limiting IC supply elicited a previously undocumented increase in nitric and nitrous oxide emissions. Further, gene expression patterns suggested that excess gaseous IC supply and limiting IC supply led to consistent up-regulation of ammonia respiration genes and carbon assimilation genes. Under these conditions, interrogation of the N. europaea proteome revealed increased levels of carbon fixation and transport proteins and decreased levels of ammonia oxidation proteins (active in energy synthesis pathways). Together, the results indicated that N. europaea mobilized enhanced IC scavenging pathways for biosynthesis and turned down respiratory pathways for energy synthesis, when challenged with excess gaseous IC supply and limiting IC supply.

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INTRODUCTION The unchecked emission of reactive nitrogen (N) from wastewater streams into the environment is undesirable. Reactive N removal from wastewaters can be achieved using conventional nitrification and denitrification pathways or through predominantly lithoautotrophic pathways involving nitritation and anaerobic ammonia oxidation (anammox). Compared to conventional pathways, chemolithoautotrophic metabolism relies on inorganic energy sources (including ammonia and nitrite) to fix CO2 into cellular biomass, which allows wastewater treatment plants (WWTPs) to reduce energy and chemical consumption as well as biomass production.1 In chemolithoautotrophic ammonia oxidizing bacteria (AOB) such as Nitrosomonas europaea, the carbon and nitrogen cycles are intrinsically linked since a fraction of the energy produced by nitrogen oxidation must be diverted to carbon assimilation reactions to replenish key metabolic intermediates. Given that © 2014 American Chemical Society

inorganic carbon limitation could be encountered in high nitrogen-strength applications (e.g., reject water, leachate),2−6 there is merit in understanding how N removal is impacted by inorganic carbon (IC) availability, particularly as facilities shift toward embracing predominately lithoautotrophic nitrogen removal strategies. Thus far, studies have mostly focused on noncarbon stressors (including heavy metals, anoxia)7−14 and have not yet explored the impact of inorganic carbon on lithoautotrophic ammonia oxidation at levels beyond the reactor level response15−18 or expression of specific target genes.19 We hypothesized that ammonia oxidation activity is inversely related to the form and availability of IC supply. Therefore, as the Received: Revised: Accepted: Published: 2523

September 2, 2014 December 16, 2014 December 29, 2014 December 29, 2014 DOI: 10.1021/es5043222 Environ. Sci. Technol. 2015, 49, 2523−2531

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Environmental Science & Technology

Figure 1. Ammonia nitrite concentration (A), maximum specific oxygen uptake rate (B), and hydroxylamine concentration measurements (C) for N. europaea cultures subjected to condition 1 (40× stoichiometric requirement), condition 2 (4× stoichiometric requirement), and condition 3 (0.25× stoichiometric requirement). Error bars represent the standard deviation of technical replicates. (Replicate results are presented in the Supporting Information.)

was defined as three consecutive days with an average effluent ammonia concentration that was not statistically different at the 95% confidence level (α = 0.05) from the three previous time points. When steady state was attained, the culture was subjected to one of three conditions. Condition 1 was the control during which IC was supplied at 40× the stoichiometric requirement in both ionic and gaseous forms through 80 g/L NaHCO3 solution and air (where the stoichiometric requirement is 0.087 mol C/ mol N to fully oxidize ammonia and sustain biomass growth).22 Condition 2 was the excess gaseous IC supply at 4× the stoichiometric requirement using air, during which pH was controlled using a 1 N NaOH solution. Condition 3 represented limiting IC at 0.25× the stoichiometric requirement using CO2free air again with pH control using a 1 N NaOH solution and Tech Air (Please refer to the Supporting Information for calculations of IC supply). Conditions 1, 2, and 3 are also referred to as excess ionic and excess gaseous IC supply, excess gaseous IC supply, and insufficient IC supply, respectively, in this paper. Reactor performance was monitored by measuring cell concentrations, nitrogen concentrations (ammonia, nitrite, hydroxylamine (NH2OH), nitric oxide (NO), nitrous oxide (N2O)), total organic and inorganic carbon, and biokinetic parameters. Aqueous ammonia (NH3; gas sensing electrode), nitrite (NO2−; colorimetric), nitrate (NO3−; ion selective electrode), hydroxylamine (NH2OH; colorimetric), gaseous nitric oxide (NO; chemiluminescence, CLD-64, Ecophysics, Ann Arbor MI), and nitrous oxide (N2O; gas-filter correlation, Teledyne API320E, San Diego CA) as well as total inorganic carbon (TIC; heated-UV-Persulfate and NDIR, Shimadzu TOC5000, Japan) and cell counts (brightline hemocytometer, Hausser Scientific, Horsham PA) were directly measured using standard methods.23−26 Maximum specific oxygen uptake rates

form of IC supply is changed from ionic to gaseous and the availability of IC supply is reduced from excess to limiting, AOB activity, as characterized by ammonia oxidation, nitrogen reduction, and carbon fixation rates, would decrease. To test this hypothesis, we selected N. europaea (ATCC 19718)20,21 as the model AOB given its prevalence in engineered biological nitrogen removal (BNR) processes, particularly in systems with high ammonia concentrations. We utilized a multitiered approach to determining the impact of form and availability of IC by simultaneously examining results from bulk nutrient concentrations (ammonia, hydroxylamine, nitrite, inorganic carbon), whole cell activity, gene transcription, enzymatic activities, and global proteomics.

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MATERIALS AND METHODS Culture and Growth Conditions. Pure cultures of N. europaea ATCC 19718 were grown in duplicate chemostats (V = 7 L; dilution rate = 0.33 day−1; 23 °C) on sterilized feed medium devoid of organic carbon.14 Briefly, the influent feed medium contained the following: MgSO4·7H2O (200 mg/L), CaCl2· 2H2O (20 mg/L), K2HPO4 (87 mg/L), 3-[4-(2-hydroxyethyl)1-piperazine] propanesulfonic acid (EPPS, 1250 mg/L), Na2MoO4·2H2O (0.01 mg/L), MnSO4·H2O (0.017 mg/L), CoCl2·7H20 (0.0004 mg/L), CuCl2·2H2O (0.17 mg/L), ZnSO4· 7H2O (0.01 mg/L), chelated iron (1 mg/L), and (NH4)2SO4 (1330 mg/L).14 Sterile aeration was provided via air pumps fitted with 0.22 μm HEPA filters (gas flow rate = 3.3 L/min) while pH (7.5 ± 0.01) was controlled via automated addition of NaHCO3 (80 g/L). Culture purity was confirmed via direct microscopic examination (phase contrast, 400×, Nikon Eclipse, 80i, Melville, NY) and DNA sequencing.7 Changes in Inorganic Carbon Supply to Chemostat Cultures. Each chemostat was operated to steady state, which 2524

DOI: 10.1021/es5043222 Environ. Sci. Technol. 2015, 49, 2523−2531

Article

Environmental Science & Technology

Statistical analysis was performed using JMP 8.0. The leastsquares and Bonferroni correction were used to select for the statistically significant proteins. The proteins were normalized by the total ribosomal protein concentrations and characterized under metabolism, genetic information processing, environmental information processing, and cellular process proteins based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (please refer to details in SI-4, Supporting Information).30 Student’s t tests were done to compare the relative protein contents except when proteins were undetected or did not have enough replicates to allow for a t test. p-values for the t tests are listed in Table SI-3, Supporting Information.

(sOURmax) were calculated from oxygen uptake rate profiles as described elsewhere.27 Biomass samples for gene expression and proteomics analyses were obtained from replicate experiments representing conditions 1, 2, and 3. These samples corresponded with time points that represented the most significant changes in chemostat performance as measured by ammonia removal, which are henceforth denoted as P1, P2, and P3. Specifically, samples for condition 2-P1, P2, and P3 were taken on day 3, 23, and 32 (Figure 1A), which were days on which the ammonia concentration started to increase, peaked, and recovered, respectively. Samples for condition 3-P1, P2, and P3 were taken on day 2, 3, and 4 (Figure 1A), which represented prefailure, 1/3, and 2/3 into reactor failure, respectively. Nitrogen mass balances were performed from running averages of ammonia, nitrite, NO, N2O, and cell count profiles. Carbon mass balances were performed by estimating the mass of CO2 transferred from aeration using a two film model to estimate KLA,CO2 from the measured oxygen mass transfer coefficient (KLA,O2 = 0.0017 1/ s)28 and the mass of bicarbonate or sodium hydroxide added for pH control, as well as cellular carbon in the effluent stream (Table SI-2, Supporting Information). Enzyme Activity Measurements. Cell aliquots from chemostats were assayed for carbonic anhydrase (Cah) and ribulose 1,5-bisphosphate carboxylase (RubisCO) activities using previously documented methods (SI-1, Supporting Information).30,31 For this study, one unit of RubisCO activity was defined as the conversion of 1.0 μmole of D-ribulose 1,5 diphosphate and CO2 to 2.0 μmoles of D-3-phosphoglycerate per minute at pH 7.8 at 25 °C. One unit of carbonic anhydrase activity was defined as the drop in the pH of a 20 mM Veronal buffer from 8.0 to 6.8 per minute at 0 °C. Assays for RubisCO and carbonic anhydrase were done at room temperature and 0 °C, respectively. Functional Gene Expression. RNA from biomass samples (50−100 mL) was purified by centrifugation, bead beating, and an acid phenol procedure as described in the Supporting Information. The mRNA transcript concentrations of ammonia monooxygenase (amoA1, NE0944), hydroxylamine oxidoreductase (hao, NE2044), nitrite reductase (nirK, NE0924), nitric oxide reductase (norB, NE2004), ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (cbbL, NE1921), carbonic anhydrase (cah, NE0606), and ammonium transporter (NE0448) were quantified and normalized to expression of the 16S rRNA gene (copy number per copy number) at respective sampling points using optimized quantitative real-time reverse transcriptase polymerase chain reaction assays (refer to details in Table SI-1, Supporting Information). Student’s t tests were done to compare the relative transcript concentrations of the functional genes under different IC supply conditions to their transcription in the control condition. Label-Free Comparative Proteomics. Proteomics samples were obtained by first lysing cells with a French pressure cell followed by centrifugation (SI-1, Supporting Information). Analyses were performed using a Synapt G2 quadrupole-timeof-flight HDMS mass spectrometer (Waters Corp). Spectra from all samples were analyzed with a ProteinLynx Global Server (Vers. 2.5, RC9) (Waters Corp) and searched against a database of N. europaea sequences from the UniProtKB database29 with a false discovery rate set at 4%. The database also contained sequences for yeast alcohol dehydrogenase, porcine trypsin, and human keratins. This database was composed of 2460 protein sequences and 999 415 amino acid residues.

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RESULTS Excess Gaseous IC and Limiting IC Supply Negatively Impact Ammonia Conversion Efficiency. Complete ammonia removal was achieved under excess gaseous and ionic IC supply wherein the ammonia concentration, maximum specific oxygen uptake rate, and hydroxylamine concentration were 0.2 ± 0.3 mg NH3-N/L (Figure 1A), 4.9 ± 1.7 mg O2/mg biomass COD-day (Figure 1B), and 0.03 ± 0.02 mg NH2OH-N/L, respectively (two samples were measured under condition 1 and were not plotted). Upon removal of ionic IC and resorting only to excess gaseous IC supply (condition 2), ammonia and hydroxylamine accumulated to 15.3 ± 0.28 mg NH3-N/L and 0.34 ± 0.01 mg NH2OH-N/L, respectively, after 24 days but recovered to 2.27 ± 0.15 mg NH3-N/L and 0.16 ± 0.01 mg NH2OH-N/L after 30 days (Figure 1A,C). The maximum specific oxygen uptake rate started fluctuating between 6.0 ± 1.4 and 19.7 ± 2.0 mg O2/mg biomass COD-day immediately after imposition of excess gaseous IC but recovered to preperturbation levels after 10 days (Figure 1B). In contrast, permanent failure of reactor performance and cell washout were observed approximately 7 days after switching to insufficient IC supply (condition 3). Ammonia removal ceased completely (Figure 1A). The maximum specific oxygen uptake rate increased to 8.02 ± 0.01 mg O2/mg biomass COD-day on day 3 and dropped below the detection limit after 6 days (Figure 1B). NH2OH accumulated to 0.3 ± 0.1 mg NH2OH-N/L after 6 days (Figure 1C). Changes in IC Supply Can Elicit Increased Gaseous Nitrogen Oxide Production. Both excess gaseous IC and limiting IC led to a previously undocumented increase in NO and N2O emissions. From a mass balance perspective, the NO and N2O emissions increased from 0.41 ± 0.09% and 0.03 ± 0.06% of total influent ammonia-N load in condition 1 (excess ionic and gaseous IC) to 1.10 ± 0.11% and 1.41 ± 0.27%, respectively, 19 days into condition 2 (excess gaseous IC supply) (Figure 2). The NO and N2O emissions remained substantially higher than in condition 1 after 30 days in condition 2 (Figure 2), despite the recovery of the ammonia removal and maximum specific oxygen uptake rate at this time (Figure 1A,B). In condition 3 (limiting IC supply), the peak NO and N2O emissions reached 1.04 ± 0.65% (day 1) and 1.79 ± 0.59% (day 3) of the total influent ammoniaN load, respectively, but dropped to below the detection limit after 6 days due to the reactor failure and biomass washout (Figure 2). Excess Gaseous IC Supply and Limiting IC Supply Increase the Relative mRNA Concentrations of Genes Coding for Nitrogen Processing and Carbon Fixation. Excess gaseous IC supply (condition 2) led to significant increases in the relative concentration of ammonia oxidation and hydroxylamine oxidation genes, namely, amoA1 and hao, in 2525

DOI: 10.1021/es5043222 Environ. Sci. Technol. 2015, 49, 2523−2531

Article

Environmental Science & Technology

Activities of Carbon Fixation and Transportation Proteins Were Inversely Correlated. Interestingly, enzymatic assays indicated that carbonic anhydrase activity was not related to sOURmax (Figure 4A) while carbon fixation rates (as inferred from RubisCO activity) were the highest when sOURmax and carbonic anhydrase activity were the lowest (Figure 4B−C). Specifically, RubisCO activity increased from less than 0.001 U/ mg protein to 0.007 ± 0.002 U/mg protein when sOURmax increased from 5.5 ± 1.1 to 13.6 ± 0.8 mg O2/g biomass CODday (Figure 4B) and carbonic anhydrase activity decreased from 701 ± 39 to 95 ± 18 U/mg protein (Figure 4C). Excess Gaseous IC Supply and Limiting IC Supply Lead To Overall Decrease in the Relative Content of Ammonia Respiration and Electron Transport Proteins. The relative content of ammonia monooxygenase (AMO) subunits B1 (encoded by NE0943), C2 (encoded by NE1411), and AMO acetylene-binding proteins (encoded by NE0944) were significantly lower under condition 2-P2 (peak ammonia accumulation) and remained low in condition 2-P3 despite the recovery of ammonia removal efficiency at this time (Figure 5A). AMO subunits A1 (encoded by NE0944) and A2 (encoded by NE2063) became undetectable in conditions 2-P2 and P3 (Figure 5A). The changes in AMO levels were in agreement with established results that different AMO subunits could have significantly different levels and respond differently to the same perturbance (e.g., acetylene, light).31 The relative content of AMO subunit C, which was implicated in recovery from ammonia starvation,19 increased under conditions 2-P1 and P2, although a t test was not possible given that AMO subunit C was only detected in 1 out of 6 replicates in condition 1. The relative content of HAO significantly increased in condition 2-P2, which corresponded well to the peak hydroxylamine concentration (Figure 5A). NirK, which is involved in nitrite reduction to NO, became undetectable in conditions 2-P2 and P3 while NorQ, the NorB-associated ATPase involved in reduction of NO to N2O, did not show statistically significant trends in condition 2. No statistically significant trends in the relative protein content were detected for AMO subunits in condition 3 except the significant decrease in the relative content of AMO acetylenebinding protein in conditions 3-P1 and P2 (Figure 3B). The relative content of AMO subunit C also increased while AMO subunits A2 and C1 (encoded by NE0945) became undetectable in conditions 3-P2 and P3 (Figure 3B). No statistically significant difference was found for AMO subunit A1, HAO, and NirK throughout condition 3 (Figure 3B), although AMO subunit A1 remained detected in condition 3-P3.

Figure 2. Mass balance of NO and N2O emissions in conditions 1, 2, and 3. Error bars represent the standard deviation of measurements during the day at 1 min intervals.

conditions 2-P2 and P3 (Figure 3A) (p-value = 0.04, 0.03, 0.001, respectively), which coincided in time with the peak ammonia and hydroxylamine concentrations in condition 2-P2 (Figure 1A,C). Excess gaseous IC supply also led to significant increases in the relative concentration of nirK, which codes for nitrite reduction, during conditions 2-P1 and P2 and significant decreases (p-value = 0.04, 0.006, respectively) in the relative concentration of a putative ammonium transporter gene, NE0448, throughout condition 2 (Figure 3A, p-value = 0.02, 0.003, 0.002, respectively). norB, which codes for the catalytic subunit of nitric oxide reductase, Nor, did not show statistically significant trends in condition 2, despite the increased NO and N2O emissions (Figure 3A). Meanwhile, the relative concentration of cbbL, which codes for CO2 fixation, increased significantly in conditions 2-P2 and P3 after a slight initial decrease in condition 2-P1 (p-value