ARTICLE https://doi.org/10.1038/s41467-020-17366-3 OPEN Substrate regulation leads to differential responses of microbial ammonia-oxidizing communities to ocean warming Zhen-Zhen Zheng1, Li-Wei Zheng2, Min Nina Xu 1,3, Ehui Tan 2, David A. Hutchins 3, Wenchao Deng1, ✉ Yao Zhang1, Dalin Shi 1, Minhan Dai 2 & Shuh-Ji Kao 1,2 1234567890():,; In the context of continuously increasing anthropogenic nitrogen inputs, knowledge of how ammonia oxidation (AO) in the ocean responds to warming is crucial to predicting future changes in marine nitrogen biogeochemistry. Here, we show divergent thermal response patterns for marine AO across a wide onshore/offshore trophic gradient. We find ammonia oxidizer community and ambient substrate co-regulate optimum temperatures (Topt), gen- erating distinct thermal response patterns with Topt varying from ≤14 °C to ≥34 °C. Substrate addition elevates Topt when ambient substrate is unsaturated. The thermal sensitivity of kinetic parameters allows us to predict responses of both AO rate and Topt at varying substrate and temperature below the critical temperature. A warming ocean promotes nearshore AO, while suppressing offshore AO. Our findings reconcile field inconsistencies of temperature effects on AO, suggesting that predictive biogeochemical models need to include such differential warming mechanisms on this key nitrogen cycle process. 1 State Key Laboratory of Marine Environmental Science, College of the Environment and Ecology, Xiamen University, Xiamen, Fujian, P. R. China. 2 State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, Fujian, P. R. China. 3 Marine and ✉ Environmental Biology, University of Southern California, Los Angeles, CA 90089, USA. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:3511 | https://doi.org/10.1038/s41467-020-17366-3 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17366-3 fi fi mmonia oxidation (AO), the rst step in nitri cation, response, which has never been reported before, with a Topt of ≤ – connects the most reduced and oxidized inorganic 14 °C (Fig. 1d f); and (III) a dome-shaped response with a Topt A – – nitrogen species in the ocean. It therefore replenishes the of 20 29 °C (Fig. 1c, g i). marine pools of nitrite and provides oxidized substrates for The Type I pattern was observed at two of the three estuarine denitrification and annamox, two primary nitrogen loss terms in stations (JLR1 and JLR2, Fig. 1a, b) where ammonium the ocean1,2. Thus, AO plays a crucial role in the marine nitrogen concentrations were high (≥24 μM), and the AOR increased cycle. In addition, AO interacts with the ocean carbon cycle from linearly as the temperature increased from 14 to 34 °C. In these various perspectives, and is therefore involved in multiple climate cases, the Topt was equal to or higher than the maximum feedback processes. For example, chemoautotrophic ammonia- experimental temperature of 34 °C (Fig. 1a, b). The Type II oxidizing organisms fix inorganic carbon3, while the end product pattern was observed at the shelf stations (N1, M1, and M2), + of nitrification provides approximately half of the nitrate con- where NH4 concentrations ranged from 45 to 550 nM 4 – sumed by growing phytoplankton on a global scale . On the other (Fig. 1d f). In contrast to the Type I pattern, the Topt of the 5 hand, AO generates nitrous oxide (N2O) as a by-product ,a Type II pattern was equal to or lower than the minimum potent greenhouse gas with a ~300-fold higher greenhouse gas experimental temperature of 14 °C, showing a continuously potential per molecule than carbon dioxide. Thus, AO helps to decreasing AOR as temperature increased. The Type III pattern make the ocean a net source of N2O to the atmosphere. In view of was observed at station JLR3 (outer estuary), N2 (shelf), N3 and global change and the rapid increases in the influx of anthro- J1 (basin), for which the Topt of the AOR varied from 20 to 29 °C, pogenic nitrogen into the marine environment6,7, factors like with rates decreasing toward both higher and lower temperatures fi 8,9 fi – + acidi cation , strati cation, deoxygenation, and especially (Fig. 1c, g i). The NH4 concentrations of the Type III stations warming that may affect the ammonia oxidation rate (AOR) and ranged from 14 to 5000 nM. Nevertheless, the highest Topt values AO microbial community structure, need to be addressed10,11. were observed at coastal sites with the highest ambient Only then can these environmental change factors be properly ammonium concentrations (Fig. 1). incorporated into nitrogen-driven biogeochemical models to make accurate climate predictions. Temperature is recognized as a primary global driver to tune Substrate regulates AOR and its thermal optimum tempera- biological metabolic rates12. However, knowledge of how marine ture. For those stations with low ammonium concentrations, the AO responds to warming remains underexplored, especially for AOR at in situ temperature increased when the substrate was 13,14 15 + areas having high AOR, such as estuaries, coastal zones , and enriched (AORenriched, additions of 2000 nM NH4 ) (Fig. 1f, i). 15–17 fi the base of the euphotic zone in the open ocean . These Meanwhile, the Topt of the AOR shifted signi cantly toward environments also appear to be the frontline of both anthro- higher values (t test, p < 0.05; Fig. 1d, f, g, i). Although the pogenic nitrogen disturbances and ocean warming. Pure cultures resolution of the temperature interval set in our incubation of three strains of ammonia-oxidizing archaea (AOA) isolated experiments was not high enough to identify a precise Topt for the from marine habitats have shown that AOA growth rates are AO community, the positive Topt shift induced by ammonium positively correlated with temperature until their optimum tem- enrichment was evident. 18 peratures (Topt) are reached . Paradoxically, the limited number To further explore how the substrate-regulated Topt of AOR in of field studies in marine environments19–22 produced incon- marine environments, we designed a Michaelis–Menten (M–M) sistent results, i.e. positive and insensitive responses to tempera- thermal kinetics experiment for J1 (substrate deprived sea basin) ture increase. Due to insufficient field information, how ammonia and JLR4 (substrate-replete upstream estuary) stations with oxidizers may respond to thermal stress in the vast ocean remains distinctive substrate concentrations (see details in Methods). enigmatic, as do possible synergistic effects with continuously The experimental results revealed that at a given temperature, the increasing anthropogenic nitrogen inputs into the ocean. responses of AOR along with substrate addition can be fitted by To better predict the future of marine nitrogen biogeochem- the classic M–M curve (Fig. 2a, b; Supplementary Table 2), i.e. the + istry, we performed manipulation experiments to characterize the rate increased as the NH4 concentration increased until the temperature responses of the marine AO microbial community substrate became saturated. These M–M curves are temperature- relative to substrate changes across a broad environmental gra- dependent, with the maximum rate (Vmax) and the half- dient. Using isotope labeling techniques and a series of tem- saturation constant (Km) increasing as the temperature increased perature/substrate manipulation incubations, we revealed distinct until the saturation optimum temperature (Topt-sat) was reached – fi temperature response patterns of marine AO communities along (Fig. 2c f). Note that the Topt-sat was de ned as the optimum a substrate gradient from coastal eutrophic waters to offshore temperature of Vmax at saturated substrate level (Topt-sat, ~26 °C oligotrophic regions (Supplementary Fig. 1; Fig. 1; Supplementary for station J1 and ~29 °C for JLR4 station; see Fig. 2c, d). Table 1). Our experimental results shed light on the substrate- From the above results, we suggest that the Topt-sat of a single- regulated thermal kinetics and parameterization of nitrification species derived from laboratory culture under a saturated 18 that are critically needed for marine biogeochemical models of the substrate concentration may not properly represent its Topt in rapidly changing marine nitrogen cycle. the field, where ammonium is not always saturated. Some published biogeochemical models with a nitrification component have assumed that nitrification follows the Arrhenius relationship 23,24 Results and discussion until temperature reaches the Topt . However, the Topt in these Distinctive temperature responses along a substrate gradient. models is derived from pure laboratory cultures typically grown Within the temperature range of ~14 to ~34 °C in our incuba- at saturating substrate concentrations (Topt-sat), which may cause tions, the observed AORs at the ambient substrate level an overestimation of rates in the warming ocean at ambient (AORambient, see Methods) varied over 3 orders of magnitude, unsaturated substrate concentrations. from 0.5 to ~4000 nM d−1, across a wide spectrum of ambient To examine the interactive effects of temperature and substrate μ ammonium levels ranging from 14 nM to 96 M (Fig. 1). Three on AOR and Topt, we adapted the Dual Arrhenius and – different types of temperature response of AORambient patterns at Michaelis Menten kinetics model (DAMM) developed by 25,26 estuarine, shelf, and sea basin stations were observed: (I) a Davidson et al. The temperature