Article pubs.acs.org/est
Temperature Decouples Ammonium and Nitrite Oxidation in Coastal Waters Sylvia C. Schaefer† and James T. Hollibaugh*,† †
Department of Marine Sciences, University of Georgia, Athens, Georgia 30602-3636, United States S Supporting Information *
ABSTRACT: Nitrification is a two-step process linking the reduced and oxidized sides of the nitrogen cycle. These steps are typically tightly coupled with the primary intermediate, nitrite, rarely accumulating in coastal environments. Nitrite concentrations can exceed 10 μM during summer in estuarine waters adjacent to Sapelo Island, Georgia, U.S.A. Similar peaks at other locations have been attributed to decoupling of the two steps of nitrification by hypoxia; however, the waters around Sapelo Island are aerobic and wellmixed. Experiments examining the response to temperature shifts of a nitrifying assemblage composed of the same organisms found in the field indicate that ammonia- and nitrite-oxidation become uncoupled between 20 and 30 °C, leading to nitrite accumulation. This suggests that nitrite peaks in coastal waters might be explained by differences in the responses of ammonia- and nitrite-oxidizers to increased summer temperatures. Analysis of field data from 270 stations in 29 temperate and subtropical estuaries and lagoons show transient accumulation of nitrite driven primarily by water temperatures, rather than by hypoxia. Increased climate variability and warming coastal waters may therefore increase the frequency of these nitrite peaks, with potential ecosystem consequences that include increased N2O production, NO2− toxicity, and shifts in phytoplankton community composition.
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INTRODUCTION
< 0.001), suggesting a functional link between net accumulation of nitrite and growth of the AOA population. The nitrite peaks reported in Hollibaugh et al.1 occurred in a well-mixed estuary and coincided with environmental conditions common during summer months, including higher temperatures, decreased DO, elevated pCO2, lower pH, and increased PO43− concentrations; hallmarks of net ecosystem heterotrophy.15 Water temperature strongly affects DO in estuaries, both through decreased oxygen solubility at higher temperatures and because respiration rates increase as a function of temperature. Respiration affects pCO2 and thus pH,16 and phosphorus regeneration.17 Statistical analyses1,18 reveal that these variables covaried in our data set, and a principle components analysis revealed that they all contributed to explaining variability in AOA abundance. We hypothesized that the seasonal increase in water temperature at the study site perturbs the coupling between ammonia and nitrite oxidation by enhancing growth of ammonia-oxidizers relative to nitriteoxidizers, resulting in nitrite accumulation. If indeed this coupling is driven primarily by the increase in water temperature; then climate change, which is expected to increase seasonal variability and further warm estuaries,8 could cause
Seasonally elevated nitrite concentrations have been observed in a number of estuaries.2−5 Such nitrite peaks are of concern as they may have consequences such as NO2− toxicity,6 enhanced N2O production,7 and may lead to shifts in phytoplankton community composition.8,9 These incidents have been attributed to hypoxia, which is thought to affect nitrite accumulation as a result of differences between the oxygen affinities of ammonia- and nitrite-oxidizers.10−12 Because both ammonia-oxidizing Archaea and ammonia-oxidizing Bacteria are relatively tolerant of low dissolved oxygen (DO) concentrations, particularly ammonia-oxidizing Archaea,13 they may be able to continue oxidizing ammonia when nitrite-oxidizers are inhibited, causing nitrite to accumulate.14 Studies performed in estuarine waters adjacent to Sapelo Island, Georgia, U.S.A. (31.45°, −81.30°) revealed regular, midsummer peaks in the abundance of nitrifying organisms,1 especially of ammonia-oxidizing Archaea (AOA or Thaumarchaeota, >104-fold seasonal variation in abundance; Supporting Information Table S1), but also of ammonia oxidizing Betaproteobacteria (AOB) and of Nitrospina, a NitriteOxidizing Bacteria (NOB) of the Nitrospinae phylum (∼103fold seasonal variation; Table S1). Accumulation of nitrite in the water column of this system correlates well with the abundance of AOA (Figure 1a, ρ = 0.78, p ≪ 0.001) but not of AOB (Figure 1b, p > 0.05) or Nitrospina (Figure 1c, ρ = 0.36, p © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
July 11, 2016 February 17, 2017 February 22, 2017 February 22, 2017 DOI: 10.1021/acs.est.6b03483 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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incubation temperatures using a nitrifying enrichment culture raised from an inoculum collected at Marsh Landing, Sapelo Island, Georgia (31°25.075′ N, 81°17.75′ W) in 2012. The enrichment was maintained at room temperature (22 ± 2 °C) in the dark with periodic transfers in medium composed of filter-sterilized coastal seawater amended with 50 μM NH4Cl. Ammonium concentrations were monitored between transfers20 and NH4Cl was added to 50 μM when ammonium levels were 99% similar to the coastal Georgia strain reported in Hollibaugh et al.1 (Figure S1; 61 of 62 cloned Archaea amplicons) and Nitrospina (33 of 74 cloned Bacteria amplicons, Table S2). The Bacteria library also contained 1 (of 74) Nitrosomonas sequence. The sequences obtained (Bacteria and Archaea) have been deposited in Genbank under accession numbers KX669088 to KX669223. The ammonium concentration in a 1 L subculture was monitored daily over ∼1 month. The culture was amended twice with NH4Cl (to 10 μM) when the [NH4+] in the culture dropped below ∼1 μM to ensure that it was growing rapidly before starting the experiment. After the second amendment had been depleted (measured NH4Cl < 1 μM), the culture was added to 8 L of the same filter-sterilized coastal seawater used to make culture medium. This culture was split into 2 aliquots. One aliquot was amended with NH4Cl to 50 μM while the second received NaNO2 to 50 μM. The culture in each of these aliquots was dispensed into 18 polycarbonate bottles (250 mL bottles each containing 200 mL, a total of 36 bottles) that were placed in small cardboard boxes (3 bottles per box) with a temperature logger (Onset HOBO TidbiT or Pendant). Pilot experiments revealed that ammonia oxidation by the enrichment was extremely slow at temperatures 40 °C or ≤30 °C, dissolved oxygen values >40 mg/L, salinities 2 mg L−1 (Figure 4c,d), as well as under hypoxic conditions. While χ2 tests on the entire data set revealed that elevated nitrite events occurred under hypoxia significantly more frequently than expected (Table S3), the significance of the test was much greater for water temperature (χ2 = 736) than for hypoxia (χ2 = 32). This was also the case for Chesapeake Bay stations (Figure S6a−d), including depths that exhibit regular, recurring hypoxia (Figure S6e,f; Table S3). Nor did we find a correlation between ammonium, which can accumulate under extreme hypoxia, and
nitrite concentration in these data. We conclude that water temperature affects nitrite accumulation directly, rather than indirectly via oxygen availability. Numerous factors contribute to nitrite accumulation in marine ecosystems10,30,32 and undoubtedly play a role in the interannual variability in the magnitude of elevated nitrite events at a site; however, the effects of temperature have generally been overlooked. Elevated temperatures (>30 °C) are known to induce decoupling between ammonia- and nitriteoxidation during wastewater processing, which can lead to substantial accumulation of nitrite,33 a characteristic exploited in the design of anammox-based nitrogen removal systems.34 Most such studies have focused on bacterial nitrification (e.g., ref 11), but recent work suggests that AOA communities may be more responsive to changes in temperature than AOB.35 Elevated nitrite concentrations thus indicate decoupling of the two steps of nitrification, but the underlying cause appears to be increased ammonia oxidation rates at higher temperatures rather than inhibition of nitrite oxidation by low DO. We hypothesized that the response of nitrifiers, and thus nitrite accumulation, is tied to temperature variability, so that estuaries with warm but stable temperatures might have high ammonia oxidation rates with no accumulation of nitrite, since NOB D
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Figure 4. (A) Nitrite concentration versus temperature; (B) heat map showing the number of observations per grid square of Z-scores of nitrite concentration versus temperature; (C) nitrite concentration versus dissolved oxygen concentration; (D) heat map showing the number of observations per grid square of Z-scores of nitrite concentration versus dissolved oxygen concentration. Color scale reflects log(10) of the number of observations per grid square (1 Z-score units × 1 °C or 1 mg L−1 dissolved oxygen). The horizontal black lines in panels B and D bound ±2 Z-score units to delineate extreme nitrite events. White indicates that no observations fell into that grid square.
at higher summer temperatures is responsible for decoupling ammonia- and nitrite-oxidation, leading to nitrite accumulation. Conversely, spikes in nitrite concentration at other sites may thus be an indication of rapid growth of ammonia oxidizers, especially AOA. A recent analysis of nitrification in the Gulf of Mexico hypoxic zone38 also identified temperature as the major factor leading to AOA growth and nitrite accumulation in that system. This contrasts with observations of midwinter peaks of AOA and nitrite in a time series from the North Sea.39 The timing of those events makes it unlikely that they are driven by temperature. Instead, release of AOA from photoinhibition or winter mixing, as discussed in Pitcher et al.;39 or advection of waters rich in AOA from elsewhere, as suggested by the coincidence of low salinity events with elevated AOA abundance in their data set, may explain the events. Our findings have several important implications. First, our results suggest that the growing trend by coastal monitoring programs to measure only total oxidized nitrogen (NOX) rather than differentiating between NO 2 − and NO 3 − misses documenting an important and interesting aspect of the nitrogen biogeochemical cycle. NO2− is an intermediate in other N cycle processes of interest, such as anammox and DNRA, as well as being central to nitrification. Unlike nitrate, nitrite contributes to biological oxygen demand and may be significant in hypoxic environments. Finally, measuring nitrite (which is simple) will also provide managers with valuable additional information on the health and functioning of estuarine ecosystems, particularly in areas where NO2 −
would adapt to consistently higher temperatures. However, we found only a weak correlation between the standard deviations of nitrite concentration and the standard deviations of either temperature (Spearman’s ρ = 0.20, p < 0.0001) or DO (Spearman’s ρ = 0.35, p < 0.0001) at the same station (Figure S7a,b), suggesting that the nitrifier community does not adapt to environmental variability at a site. We tested this further by examining the relationship between the rate of temperature change over ∼1 month prior to annual nitrite maxima and the maximum nitrite concentrations reported (Figure S8). We found no evidence of a strong relationship between this parameter and elevated nitrite concentrations, suggesting that the maximum temperature at a site is more important than its rate of change. Recent reports36,37 have documented that some members of the Nitrospira genus of NOB can carry out both steps of nitrification, so that nitrification does not necessarily require two different groups of organisms and thus might not undergo the same decoupling we report here. However, neither of these papers found any evidence for a significant contribution of this process to nitrification in the marine environment and Nitrospira (regardless of whether or not they possess the capability for complete nitrification) are not common at our study site and were not detected in the enrichment culture used above. Analysis of ammonia- and nitrite-oxidizer populations from Georgia coastal waters1 and the experiment shown in Figure 2 strongly suggest that accelerated growth of water column AOA E
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concentrations may become high enough to cause concern over toxicity. We thus encourage monitoring programs to report both nitrite and nitrate concentrations. Second, our analysis suggests that ammonia oxidation and the growth of ammonia oxidizer populations in estuarine and coastal waters are regulated primarily by temperature, with other factors of secondary importance. This conclusion may not be applicable to ammonia oxidation in sediments where the effects of DO40 and sulfide41 have been shown to be important, and it is clearly not the case for oceanic oxygen minimum zones where temperatures are stable and nitrogen geochemistry is tied to oxygen availability.42 This generality also appears to break down in shelf waters that do not contain the high concentrations of light attenuating substances (sediment or CDOM) that characterize estuarine15 or nearshore waters (e.g., the South Atlantic Bight43,44), and in which photoinhibition of ammonia oxidation25,45,46 or limitation by a substrate required for growth25,47,48 may prevent growth of ammonia oxidizers. We conclude that turbid estuarine and nearshore waters provide optimum conditions for growth of ammonia oxidizers that are otherwise limited by water temperature. Finally, estuarine temperatures can be expected to rise in the future as a consequence of climate change.49,50 The mean temperature of Narragansett Bay has increased by 1.4−1.6 °C over the past 5 decades, with an increased frequency of midsummer peak temperatures exceeding a 23 °C benchmark.8 Significant changes in the Narragansett Bay ecosystem, including shifts in phytoplankton community composition and seasonal dynamics, and a corresponding shift in benthic production, have accompanied this warming.19 Rates of bacterioplankton productivity and many other microbial processes in Chesapeake Bay have also been shown to respond strongly to temperature.29 Our analysis suggests that warmer water, and especially higher peak summer water temperatures, will result in higher estuarine ammonia oxidation rates and significant nitrite accumulation that may have wider impacts. Ammonia oxidation by Archaea may be a significant source of nitrous oxide in the ocean51 and elevated levels of nitrite in the water column have been shown to be more effective than nitrate in stimulating production of N2O, a potent greenhouse gas, in sediments.7 Coastal areas may contribute significantly to oceanic N2O emissions,52 and thus increased N2O emissions due to temperature-induced NO2− spikes in estuaries may contribute to a positive feedback loop for global warming. Another potential effect of increased nitrite concentrations is toxicity to aquatic organisms. Although marine organisms are considered more tolerant than freshwater organisms to NO2− exposure, some estimates of safe nitrite levels for aquatic organisms have been as low as 5−7 μM6, concentrations that are exceeded in approximately 2% of measurements in the data sets we analyzed. The work we report here focused on estuarine nitrification, but the effect of temperature on this process and on related biogeochemical transformations is likely to be as important in fresh water and terrestrial environments35,53as it is in estuaries, potentially enhancing loss to runoff of fixed nitrogen applied to crops. Humans have greatly altered the nitrogen biogeochemical cycle, resulting in increased fluxes of fixed nitrogen from land into coastal waters, with profound environmental consequences.54,55 Our analysis suggests that ongoing warming of coastal waters may further alter the nitrogen biogeochemical cycle, with consequences that are not easily predicted.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b03483. Figures S1−S7, Tables S1−S3, and list of data sources (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: 706/542-7671; fax: 706/542-5888; e-mail: aquadoc@ uga.edu (J.T.H.). ORCID
James T. Hollibaugh: 0000-0001-8037-160X Author Contributions
J.T.H. conceived the meta-analysis; S.C.S. performed the metaanalysis; J.T.H. performed microbial experiments and analyses; and S.C.S. and J.T.H. cowrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank all of the individuals and agencies who made their data freely available and without which this analysis would not have been possible. We would like to thank Mr. Jelani Cheek for his assistance with the temperature response experiment, and Ms Annie Bratcher and Erica Malagón for their assistance with the pilot experiments that led up to it. Qian Liu, Meredith Ross, Bradley Tolar and other members of the Hollibaugh laboratory were instrumental in collecting the data shown in Figure 1,. We would also like to thank G. M. King, J. E. Cloern, M. Alber, and 3 anonymous reviewers for helpful comments on earlier drafts of this manuscript. This work was supported by NSF OCE 1335838 and 12-37140.
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