Isotopic constraints confirm the significant role of microbial nitrogen oxides emissions from the land and ocean environment

Abstract Nitrogen oxides (NOx, the sum of nitric oxide (NO) and N dioxide (NO2)) emissions and deposition have increased markedly over the past several decades, resulting in many adverse outcomes in both terrestrial and oceanic environments. However, because the microbial NOx emissions have been substantially underestimated on the land and unconstrained in the ocean, the global microbial NOx emissions and their importance relative to the known fossil-fuel NOx emissions remain unclear. Here we complied data on stable N isotopes of nitrate in atmospheric particulates over the land and ocean to ground-truth estimates of NOx emissions worldwide. By considering the N isotope effect of NOx transformations to particulate nitrate combined with dominant NOx emissions in the land (coal combustion, oil combustion, biomass burning and microbial N cycle) and ocean (oil combustion, microbial N cycle), we demonstrated that microbial NOx emissions account for 24 ± 4%, 58 ± 3% and 31 ± 12% in the land, ocean and global environment, respectively. Corresponding amounts of microbial NOx emissions in the land (13.6 ± 4.7 Tg N yr−1), ocean (8.8 ± 1.5 Tg N yr−1) and globe (22.5 ± 4.7 Tg N yr−1) are about 0.5, 1.4 and 0.6 times on average those of fossil-fuel NOx emissions in these sectors. Our findings provide empirical constraints on model predictions, revealing significant contributions of the microbial N cycle to regional NOx emissions into the atmospheric system, which is critical information for mitigating strategies, budgeting N deposition and evaluating the effects of atmospheric NOx loading on the world.


INTRODUCTION
Atmospheric nitrogen oxides (NO x ) loading influence human health (e.g. respiratory and cardiovascular diseases, acute bronchitis) [1], tropospheric chemistry (e.g. precipitation acidity, aerosol and ozone formation) [2][3][4], climate [4] and economic development [5]. In past decades, anthropogenic NO x emissions have significantly increased the fluxes of atmospheric NO 3 − deposition [6][7][8], altered N cycles in both terrestrial and marine ecosystems [9][10][11][12] and thus affected microbial NO x emissions to the atmosphere [13]. Hence, it is pivotal to accurately constrain land and ocean NO x emissions to the atmosphere to mitigate humaninduced NO x emissions, budget NO 3 − deposition fluxes and evaluate the eco-environmental and climatic effects of atmospheric NO x loading. However, it has long been challenging to accurately constrain land-and ocean-to-atmosphere NO x emissions due to uncertainties over microbial N cycles in both land and ocean.
In marine environments, the oil combustion of marine traffic transportation is a known source of NO x emissions [14][15][16][17][18][19][20]. According to the European Monitoring and Evaluation Programme Meteorological Synthesizing Centre West model, NO x emissions from oil combustion in the ocean averaged 6.4 ± 0.8 Tg N yr −1 (5.0-7.8 Tg N yr −1 ) [14][15][16][17][18][19][20]. However, the microbial N cycle occurring in the ocean is the other significant source of NO x emissions [21][22][23][24]. First, earlier studies based on molecular analysis and lab culture experiments have confirmed that multiple kinds of bacteria associated with several processes of microbial N cycles C The Author(s) 2022. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. can produce NO, e.g. ammonium-oxidizing bacteria, nitrite-oxidizing bacteria, methanotrophic bacteria and denitrifying bacteria [25][26][27][28][29]. Second, nitrification in the oxic layer of the ocean is a significant source of NO [22] and NO can be produced in biofilms and marine sediments [30]. Third, Ulva prolifera (forming a belt on a vertical concrete wall in the upper intertidal zone at low tide) was the primary contributor to the high NO concentrations during the late-bloom period [31]. Meanwhile, the photolysis of NO 2 − and NO 3 − (in the surface water and on particles) or alkyl nitrates or dissolved organic matter may also be the sources of atmospheric NO in the ocean [32][33][34][35]. However, due to its high reactivity [36], NO would be involved quickly into the NO x cycle in the atmosphere [34]. Accordingly, it has long been difficult to accurately observe microbial NO emissions in the ocean [24]. Until now, microbial NO x emissions from the ocean and their fractional contribution to total NO x emissions from the ocean have not been quantified [21][22][23][24]. Hitherto, owing to the lack of microbial NO x emissions, the NO x from oil combustion has long been assumed as the total ocean NO x emissions in reports of the Intergovernmental Panel on Climate Change (IPCC) [20].
In the land environment, NO x emissions are mainly derived from coal combustion, oil combustion, biomass burning and microbial N cycles in substrates such as waters, soils and wastes [3,[37][38][39][40]. Currently, emission amounts of NO x from coal combustion [10,41], oil combustion [42] and biomass burning [43,44] have been reported explicitly in national statistic yearbooks and emission inventories [45][46][47]. However, land NO x emissions from microbial N cycles have been observed chiefly for soils under natural vegetation and agriculture [40,43,48]. Therefore, estimates of NO x emissions from the land are based on limited empirical observations combined with process and statistical models and satellites used to scale up emissions [40,49,50]. Based on IPCC reports, microbial NO x emissions were budgeted at 5.6 Tg N yr −1 before 2001, increasing to 11.0 Tg N yr −1 when incorporating more observational data in the report of 2013 [40,49,50]. This doubling of emissions highlights a substantial underestimation of microbial NO x emissions in the land, which has shifted with additional measurements and better models. New methods are strongly needed to comprehensively constrain microbial NO x emissions from soils and many other unconsidered substrates (such as the surface water of rivers, lakes, swamps, etc.) and emission sources (such as wastewater, water treatment systems, solid wastes).
Here we provided a unique evaluation of the relative importance of the microbial NO x emissions in the land and ocean to the known fossil-fuel NO x emissions and then made a new budget for global microbial NO x emissions. First, we compiled stable N isotopes (δ 15 N values) of NO 3 − in atmospheric particulates (denoted as δ 15 N p-NO3-hereafter) in the land and ocean, respectively (detailed in 'Materials and methods' section) ( Fig. 1 and Supplementary Table S1). Second, based on concentrations and δ 15 N of NO x , HNO 3 and p-NO 3 − over the land, we estimated the δ 15 N of the initial NO x mixture from different emission sources in the atmosphere (denoted as δ 15 N i-NOx , Supplementary Fig. S1) and the  Table S2), we estimated the relative contributions of dominant NO x sources from the land and ocean, respectively, by developing a model of Stable Isotope Analysis in R code (detailed in 'Materials and methods' section). Finally, combining fractional contributions with corresponding amounts of fossil-fuel NO x emissions from the land and the ocean, we calculated the amount of microbial NO x emissions in the land and ocean, respectively (detailed in 'Materials and methods' section).

Different δ 15 N signatures of atmospheric p-NO 3 − between the land and ocean
Mean δ 15 N p-NO3-observed over terrestrial sites (4.7 ± 3.6 ; n = 91) was significantly higher (p < 0.05) than that observed for ocean sites (-3.5 ± 3.9 ; n = 134) (Fig. 2). This finding implied that human activities contributed relatively more 15 N-enriched NO x to atmospheric NO x loading on the land than in the ocean.
First, the δ 15 N p-NO3-signal observed at land sites can represent land NO x emissions without a significant overprinting of marine sources. The net water vapor flux transported from the ocean to the land accounted for only 10% of the total water evaporation over the ocean [51,52]. According to the existing oceanic NO x emissions (6.4 ± 0.8 Tg N yr −1 based on the known oil combustion) [14][15][16][17][18][19][20] and the land NO x emissions (53.3 ± 4.6 Tg N yr −1 ) [43,[53][54][55][56][57][58], the ocean-to-land atmospheric transport of NO x accounts for only 1.2% of land NO x emissions and thus is often assumed negligible [35]. Accordingly, the δ 15 N p-NO3-values observed at land sites can be directly used to differentiate dominant sources of NO x emissions (Equation 5 in the online Supplementary Data).
However, the δ 15 N p-NO3-signal observed at ocean sites cannot represent the NO 3 − purely derived from ocean NO x emissions. Because the land has much higher NO x emissions and a smaller area, and thus a higher concentration than the ocean [57,59,60], the net transportation of atmospheric NO x occurs from the land to the ocean. The modeled NO y (the sum of NO x , inorganic and organic nitrates in the atmosphere) transportation (11. are the dominant form of the land-to-ocean NO y transportation and between them, the p-NO 3 − is the main type to be transported because the lifetime of NO x is much shorter [35,61,62]. So far, no substantial isotope effect was assumed for the physical processes of atmospheric transportation [63,64]. Thus, we thought that the ocean p-NO 3 − produced by the land-derived NO x did not differ isotopically from the land p-NO 3 − and used isotope mass-balance calculations to obtain the δ 15 N values of p-NO 3 − derived only from the ocean NO x emissions (Equation 2 in the online Supplementary Data).
The calculated results revealed that the δ 15 N of p-NO 3 − purely derived from ocean NO x emissions averaged -12.5 ± 8.2 (Fig. 2), which was much lower than the δ 15 N p-NO3-observed for the land sites (4.7 ± 3.6 ; Fig. 2). The increase in 15 15 i-NOx→p-NO3-values ( Supplementary Fig. S2) and thereby constructed isotope mass-balance models to further evaluate the contribution of dominant NO x sources to p-NO 3 − in the land and ocean, respectively (detailed in 'Materials and methods' section).
For source δ 15 N end-members, we considered coal combustion, oil combustion, biomass burning and the microbial N cycle as dominant NO x sources of p-NO 3 − over the land [65], while oil combustion and the microbial N cycle are dominant NO x sources to p-NO 3 − over the ocean [2,20]. The δ 15 N of such sources differ significantly from each other (p < 0.05, Supplementary Table S2), which is a prerequisite to differentiating their relative contributions isotopically. We assumed the same δ 15 N value of each NO x source for both land and ocean sites due to no δ 15 N observations on NO x from oil combustion and microbial N cycle in the ocean (detailed in 'Materials and methods' section). We did not consider lightning a dominant NO x source because the NO x produced by lightning in the land and ocean atmosphere is negligible. First, the global NO x production from lighting is 5.2 ± 1.0 Tg N yr −1 (Supplementary Text S1), which accounted for ∼9.7% and ∼7.2% of global NO x emissions by modeling methods (51.9-58.0 Tg N yr −1 ) and by isotopic methods in this study (Fig. 3). Moreover, the meridional distribution of global lightning in the atmosphere shows three main lightning centers of the Americas, Africa and the maritime continent in Southeast Asia. The minima represent the oceanic regions where little lightning is observed [66]. This baseline assumption of the dominant NO x sources is supported by emission inventory and deposition modeling [10,41,42,[45][46][47].
Regarding isotope effects, we estimated 15 i-NOx→p-NO3-values under two independent scenarios (detailed in 'Materials and methods' section) and found no significant differences between them (11.3 ± 2.1 and 13.1 ± 3.8 , respectively) (Supplementary Fig. S2). Accordingly, we used the mean 15 i-NOx→p-NO3-estimate (12.2 ± 2.2 ) in our subsequent isotope mass-balance calculations (Supplementary Fig. S2). The mean 15 i-NOx→p-NO3value in this study (12.2 ± 2.2 ) did not differ from the ε NO→p-NO3-value estimated by Li et al. [67] (∼15 ) and was also comparable with the global mean 15 i-NOx→p-NO3-value (16.7 ± 2.3 ) [65]. The calculation of the global mean 15 i-NOx→p-NO3value by Song et al. [65] was based on the theoretical framework of computation established by Walters and Michalski [68,69], which combined natural 15 N and 17 O isotopes with environmental parameters relating to the NO x oxidization to p-NO 3 − . Relative contributions of dominant NO x sources were calculated using the Stable Isotope Analysis model in R programming language (detailed in 'Materials and methods' section). Results showed that the NO x from coal combustion, oil combustion, biomass burning and microbial N cycle accounted for 23 ± 7%, 27 ± 11%, 26 ± 10% and 24 ± 4% on the land, respectively (Supplementary Fig. S3a). In contrast, the NO x from oil combustion and microbial N cycle accounted for 42 ± 3% and 58 ± 3% in the ocean, respectively ( Supplementary  Fig. S3a). Generally, high fractions of microbial NO x emissions revealed the vital contribution of this pathway to both land and ocean NO x emissions into the global atmosphere.

Total and microbial NO x emissions on the land
Based on statistical data on quantities and NO x emission factors of coal and oil combustions in the land system, previous studies have estimated global fossil-fuel NO x emissions with a relatively high degree of certainty [7,43,50,70,71]. Global fossil-fuel NO x emissions averaged 28.4 ± 1.8 Tg N yr −1 , showing a relatively low variation over past decades (25.6-30.0 Tg N yr −1 ) [7,43,50,70,71]. By using the fraction and amount of fossil-fuel NO x emissions in the land (50 ± 14% and 28.4 ± 1.8 Tg N yr −1 , respectively, Supplementary Fig. S3a), we estimated that total land NO x emissions were 56.8 ± 18.6 Tg N yr −1 (Fig. 3 and Supplementary  Fig. S3b). Our estimate falls in the range of the total land NO x emissions (50.0-61.4 Tg N yr −1 ; averaging 55.6 ± 2.9 Tg N yr −1 ) estimated by optimized modeling methods by considering more microbial sources of NO x emissions [54,57,58]. However, our estimate is higher than the total land NO x emissions (39.7-51.0 Tg N yr −1 ; averaging 43.8 ± 5.0 Tg N yr −1 ) estimated using the global NO 2 satellite column concentrations [43,55,56]. Due to no consideration of the influences of atmospheric NO 2 transformations, the estimates based on the satellite data were thought to underestimate global NO x emissions [72][73][74].
Based on the fraction and amount of total land NO x emissions (24 ± 4% and 56.8 ± 18.6 Tg N yr −1 , respectively, Fig. 3 and Supplementary Fig. S3), microbial NO x emissions on the land were calculated as 13.6 ± 4.7 Tg N yr −1 (Fig. 3 and Supplementary Fig. S3b). So far, observations on microbial NO x emissions on the land showed a relatively lower flux of 7.9 ± 1.5 Tg N yr −1 (5.0-11.0 Tg N yr −1 ; data compiled from Refs [43,55,[75][76][77][78][79][80][81][82][83][84]) than our estimate, because these observations have been conducted mainly on fertilized soils and merely on unfertilized soils and other land substrates. Besides, few modeling studies showed distinctly higher fluxes of land microbial NO x emissions ≤20.4 Tg N yr −1 [80] and 23.6 Tg N yr −1 [85] than the observation results and our estimate, due to overestimated N inputs in cropland and natural ecosystems and largely overlooked the influence of NO x sink uncertainties on the satellite-derived NO x fluxes. However, by considering more substrates of microbial N cycles on the land to optimize the modeling methods, some studies showed the land microbial NO x emissions as 11.5-13.6 Tg N yr −1 (12.4 ± 0.7 Tg N yr −1 ) [53,71,86,87], which is very comparable with our estimate. The isotopic method in our study offers a comprehensive and accurate constraining on microbial NO x emissions.

Total and microbial NO x emissions in the ocean
Based on statistical data of quantities and NO x emission factors of oil combustions in the ocean system, ocean fossil-fuel NO x emissions have been estimated as 6.4 ± 0.8 Tg N yr −1 on average (5.0-7.8 Tg N yr −1 ; compiled from [14][15][16][17][18][19][20]). Using the fraction of the ocean fossil-fuel NO x emissions in our study (42 ± 3%, Supplementary Fig. S3a), we estimated the total ocean NO x emissions as 15.2 ± 2.3 Tg N yr −1 (Fig. 3 and Supplementary  Fig. S3b). The ocean NO y deposition averaged 21.3 ± 1.8 Tg N yr −1 (18.0-23.0 Tg N yr −1 ; compiled from Refs [35,61,62,[88][89][90]), which includes the land-to-ocean NO y transportation of 11.0 Tg N yr −1 [61]. Accordingly, the oceanic NO y deposition derived from oceanic NO x emissions was 10.3 ± 1.8 Tg N yr −1 , which is lower than our study's total ocean NO x emissions. The generally higher NO x emissions than NO y deposition in the ocean might be attributed to other fates such as biological NO x uptake and atmosphere retention. Further, we calculated ocean microbial NO x emissions as 8.8 ± 1.5 Tg N yr −1 on average ( Fig. 3 and Supplementary Fig. S3b). Our results updated the total and microbial NO x emissions in the marine environment.

Total and microbial NO x emissions in the globe
By integrating the land and ocean values together (detailed in 'Materials and methods' section), we calculated global total NO x emissions as 72.0 ± 18.1 Tg N yr −1 (Fig. 3 and Supplementary  Fig. S3b). Before this work, the modeled total land NO x emissions (39.7-61.4 Tg N yr −1 ; compiled from Refs [43,[53][54][55][56][57][58]) have been assumed as the global NO x emissions because the ocean NO x emissions have been unconstrained. Our results showed that oceanic NO x emissions accounted for ∼21% of the global NO x emissions. The global NO x emissions have been underestimated by 15-45% because oceanic NO x emissions have been unconsidered.
Moreover, we found that microbial NO x emissions accounted for 31 ± 12% of the total NO x emissions globally and reached up to 22.5 ± 4.7 Tg N yr −1 (Fig. 3 and Supplementary Fig. S3b). By comparison, microbial NO x emissions in the land (13.6 ± 4.7 Tg N yr −1 ), ocean (8.8 ± 1.5 Tg N yr −1 ) and globe (22.5 ± 4.7 Tg N yr −1 ) are ∼0.5, 1.4 and 0.6 times fossil-fuel NO x emissions in the land, ocean and globe, respectively ( Fig. 3 and Supplementary  Fig. S3b). Our results highlight a vital role of the microbial N cycle in global NO x emissions. In addition to the direct impacts of fossil-fuel combustion on global NO x emissions, other human activities such as inefficient fertilizer use in cropping systems, wastes and sewage discharge and treatments, N deposition and water N enrichment all can accelerate microbial NO x emissions in the land, inland water bodies, estuaries and ocean [13,91].
Our results offer an updated and isotopically grounded estimate of land-and ocean-toatmosphere NO x emissions. Notably, our results revealed that previous reports have largely underestimated land-based microbial NO x emissions, constrained long-missing uncertainties over ocean microbial NO x emissions and therefore elevated the recognition of the substantial contribution of the microbial N cycle to global NO x emissions. Moreover, our findings highlight the unique significance of natural records of atmospheric N isotopes for understanding global N biogeochemical cycles. Currently, reducing NO x emissions to alleviate N pollution while sustaining economic development is a major challenge in the twenty-first century. Owing partly to unclear contributions of microbial processes to NO x emissions, many countries have been engaging in developing technologies and measures for reducing fossil-fuel NO x emissions to reduce airborne and water N pollution, with a focus on adjusting energy systems and increasing the chemical conversion of NO x to reduce emissions during fossil-fuel combustion. Our findings point to the need to consider the substantial contribution of the microbial N cycle to atmospheric NO x loadings while reducing fossil-fuel NO x emissions. Accordingly, the potential costs and impacts of reducing fossil-fuel NO x emissions need to be re-assessed when making more effective emission mitigation strategies-including the indirect effects of anthropogenic N on terrestrial and marine microbial processes. Moreover, the isotopically constrained microbial NO x emissions and updated total NO x emissions we provide are helpful for benchmarking atmospheric and earth system models that project the feedback between the biosphere, climate and global N cycle.
In summary, based on large-scale isotope observations of p-NO 3 − in the atmosphere, we established a simple but effective approach for estimating NO x sources in the atmosphere. Before, isotope mass-balance models have been constructed to successfully partition continental hydrologic fluxes and quantify the contributions of local evaporation and ocean-to-land water transportation to the land moisture [92,93]. Accordingly, the framework established in our study enriches the application of isotopic mass-balance approaches in quantifying processes and fluxes of global biogeochemical cycles. However, our method can only consider dominant sources of NO x emissions. Additional work on detailed measurements of δ 15 N values for all NO x emission sources could further refine our estimates. Isotope observations of p-NO 3 − in the atmosphere across more sampling areas will be critical to reducing uncertainties in our estimation and offering spatial tools to pinpoint source regions of great concern.

MATERIALS AND METHODS
Detailed materials and methods are given in the online supplementary materials.

SUPPLEMENTARY DATA
Supplementary data are available at NSR online.