This is the accepted version of the article: Hu, Minjie; Peñuelas, Josep; Sardans i Galobart, Jordi; [et al.]. «Denitrification rates in tidal marsh soils : the roles of soil texture, salinity and nitrogen enrichment». European journal of soil science, 2020. DOI 10.1111/ejss.12956 This version is avaible at https://ddd.uab.cat/record/224214 under the terms of the license 1 Denitrification rates in tidal marsh soils: The roles of soil texture, salinity, and 2 nitrogen enrichment 3 M.J. HUa, b, J. PEÑUELASc, d, J. SARDANSc, d, J.F. HUANGa, K. XUa & C. TONGa a 4 Key Laboratory of Humid Sub-tropical Eco-geographical Process of Ministry of Education, Fujian 5 Normal University, Fuzhou 350007, China; bCollege of the Environment and Ecology, Xiamen 6 University, Xiamen 361102, China; cCSIC, Global Ecology CREAF-CSIC-UAB, Bellaterra, 08193 7 Barcelona, Catalonia, Spain; and dCREAF, Cerdanyola del Vallès, 08193 Barcelona, Catalonia, 8 Spain 9 Correspondence: C. TONG. E-mail: tongch@fjnu.edu.cn 10 Running title: Denitrification rates in tidal marsh soils 1 11 Summary 12 The denitrification rates of freshwater and oligohaline tidal marsh soils with different textures (loam and sandy 13 soils) in a subtropical estuary, and their responses to nitrogen (N) loading, were investigated. In both marshes, 14 the denitrification rates varied significantly with the season only in loam soil. The denitrification rates were 15 highest in oligohaline marsh loam soil and lowest in freshwater marsh sand soil. NH4NO3 addition significantly 16 increased the denitrification rates of all the marsh soils. Our findings suggest that soil texture, soil organic 17 matter (SOM) content and low-level increases in salinity all had large effects on denitrification, indicating that 18 the dynamics of denitrification rates in estuarine marshes with low-level salinity were controlled by the 19 interaction of salinity and soil texture but mainly depended on SOM content. We propose that denitrification 20 in tidal marshes plays an important role in regulating current and future N loading into estuary and inshore 21 coastal waters, especially for tidal freshwater marshes, which introduces great uncertainty to the N dynamics 22 of estuaries under global changes. 23 Keywords: soil texture, salinity, nitrogen enrichment, SOM, denitrification, estuarine tidal marsh 2 24 Highlights: 25  Denitrification rates were higher in loam than sandy soil, independent of salinity. 26  Denitrification rates were higher in oligohaline marsh loam than freshwater marsh loam. 27  Exogenous N input enhanced denitrification in estuarine marsh soil. 28  Loam with high-N enhanced N removal, especially in freshwater environments. 3 29 Introduction 30 Denitrification is the dominant natural pathway for N-cycling transformation in wetlands. However, 31 the factors that control this pathway are multifaceted and interactive (Day et al., 2018; Neubauer et 32 al., 2019; Zhang et al., 2019). Estuarine marshes play a vital role in denitrification because they 33 consist of a combination of anaerobic environments and include carbonaceous substrates in the soil 34 (Marks et al., 2016). Previous studies confirmed that denitrification in estuaries is variable and 35 potentially regulated by various factors, such as the soil texture, salinity and N loading (Lee et al., 36 2017; Wang et al., 2017; Hinshaw et al., 2019). Owing to the influence of upstream runoff and tidal 37 saltwater intrusion, the salinity and soil texture change significantly in estuary systems, and, at the 38 same time, large amounts of exogenous N derived from fertilization are introduced into estuaries from 39 runoff, tides and deposition (Hu et al., 2019; Neubauer et al., 2019). However, few studies have 40 explicitly linked potential denitrification with the above factors simultaneously, and this knowledge 41 gap currently limits the understanding of the geochemical processes that drive N cycling and the 42 associated environmental responses. 43 The Min River estuary tidal marsh undergoes a clear shift from a freshwater to an oligohaline 44 environment (Tong et al., 2017) and has a high N input from agricultural and industrial activities (Hu 45 et al., 2019). Such an estuarine system provides an ideal environment for studying the responses of 46 soil denitrification to the roles of soil texture, salinity and N enrichment. Herein, we conducted a 47 seasonal incubation experiment to assess the interactive effects of soil texture, salinity and N loading 48 on the denitrification of marsh soils. We hypothesized (1) that the denitrification rate of freshwater 49 marshes is higher than that of oligohaline marshes, and (2) in both freshwater and oligohaline marshes, 50 the denitrification rate is higher in loamy than in sandy soils, especially in the soils with additional N. 4 51 Materials and methods 52 Soils were collected from a subtropical estuarine marsh in the Min River estuary in southeastern 53 China (Figure S1). We selected two tidal marshes: the freshwater Longxiangdao Marsh (26°1.8′52.8″ 54 N, 119°18′17.8″ E) and the oligohaline Shanyutan Marsh (26°1.8′13″ N, 119°37′46″ E), with average 55 salinities of 0.08±0.02 and 2.70±0.12 ppt, respectively. In the Longxiangdao marsh, we collected soil 56 samples with different textures from stands of the same plant species (Cyperus malaccensis). In the 57 Shanyutan Marsh, the loam soil samples were also collected in C. malaccensis stands, but the sandy 58 soil samples were only collected in Spartina alterniflora stands near the sea. The biomass of C. 59 malaccensis was not significantly different between the two marshes (Wang et al., 2017; Luo et al., 60 2019). In the Shanyutan marsh, the total above- and belowground biomasses were 4230 g m-2 for the 61 C. malaccensis stand and 4620 g m -2 for the S. alterniflora stand, which were nearly identical. Two 62 soils (0–15 cm; in triplicate) of contrasting textures were selected at each marsh, namely sandy 63 (coarse-textured) and loam (fine-textured) soils. The soils were sieved to 2 mm, homogenized, and 64 then divided into two subsamples for measuring the denitrification rate and physicochemical 65 properties. Tidal water was also collected simultaneously for incubation and analysis. The main 66 properties of the soils and tidal water, along with their values, are listed in Table 1. 67 The denitrification potential was measured using the modified chloramphenicol-amended 68 acetylene (C2H2) inhibition technique (Magalhães et al., 2005; Marton & Craft, 2012; Ballantine et 69 al., 2014). Although the acetylene inhibition technique may result in an underestimated ambient 70 denitrification rate due to an inhibitory effect on nitrification (Rudolph et al., 2006; McCrackin & 71 Elser, 2010; Palta et al., 2016), this technique has nevertheless been employed by many previous 72 studies (Ullah & Zinati, 2006; McCrackin & Elser, 2010; Marton & Craft, 2012; Lishawa et al., 2014; 5 73 Tomasek et al., 2017) to compare denitrification rates among different sites and treatments. Briefly, 74 30 g of fresh soil was transferred to 140 mL flasks; chloramphenicol (0.3 mg) and glucose (1.2 mg) 75 were added as substrate and enzyme inhibitor, respectively. Then, 30 mL of the treatment solutions 76 were injected into each flask, in which N was added as an NH4NO3 solution (0.5 mg·g -1 dry soil); 77 tidal water was added instead of the NH4NO3 solution in the control samples (six replicates for each 78 treatment). Each treatment sample was sealed and made anoxic by filling with pure N 2 for 5 min. 79 Then, the samples were divided into two subgroups (triplicate): with and without acetylene (10% vol: 80 vol). All samples were then incubated at four temperatures (21, 11, 17 and 28 °C representing the 81 seasonal conditions of the autumn, winter, spring and summer samples, respectively) in a dark 82 shaking incubator for 6 h. Gas samples were taken after 0.5, 1.5, 3.5 and 6 h of incubation, and the 83 N2O concentrations were analyzed using a gas chromatograph (Shimadzu Corporation, Kyoto, Japan) 84 equipped with an electron-capture detector. The denitrification rates were calculated as the difference 85 between the N2O produced with and without acetylene (Magalhães et al., 2005) and were expressed 86 in μg N·kg-1·h-1 based on their dry weight. The physicochemical properties of soils and tidal waters 87 were measured as described in our previous study (Tong et al., 2017; Hu et al., 2019). 88 When necessary, data (i.e., denitrification rates and environment variables) were log- 89 transformed to meet the analysis of variance (ANOVA) assumption of normality and 90 homoscedasticity. The differences in denitrification rates and environment variables between the 91 different seasons and soils were tested using a one-way ANOVA. In cases where significant fixed 92 effects were detected, pairwise comparisons among groups were conducted via Tukey’s post hoc test. 93 A two-way ANOVA was used to identify the effects of soil texture and season on the denitrification 94 rates using R version 3.5.1 (R Development Core Team, 2008). Overall distributions and variations 95 in soil denitrification rates and environmental parameters among the study sites were summarized 6 96 using a principal components analysis (PCA) in R platform. 97 Results and discussion 98 The pH of the soils varied significantly but irregularly with the season (Table S1). Soil ammonium- 99 N (NH4+-N) concentrations varied substantially with the season but were less variable in response to 100 salinity and texture. Soil nitrate-N (NO3--N) concentrations had similar seasonal patterns among the 101 sites and were larger in the loam than the sandy soil. First, these larger nitrate concentrations should 102 drive increased nitrification in the fine-texture soils because they continue to have larger nitrate 103 concentrations despite having greater nitrate losses due to increased denitrification rates. Second, the 104 larger total C and N contents in the loam soils together with the higher soil C/N ratios (Table 1) clearly 105 indicates less mineralization of organic N, allowing more substrates for nitrification (Janssen, 1996). 106 The concentrations of dissolved organic carbon (DOC) and organic matter (OM) in the oligohaline 107 marsh with loam soil were the largest and had clear seasonal patterns (Table S1). The high DOC and 108 OM concentrations in the loam soil were assumed to be caused by the physical protection and large 109 surface area of the bigger fine fraction in the loam compared to that of the sand, which protected OM 110 against decomposition and improved adhesion (Perryman et al., 2011). 111 The denitrification rates of both freshwater and oligohaline marsh soils varied significantly with 112 the season (F=12.59, P<0.01, and F=17.23, P<0.01, respectively; Figure 1; Table S2), which is 113 consistent with the results of a previous study in which optimum temperatures triggered high 114 denitrification rates by stimulating substrate availability and denitrification potential (Wang et al., 115 2017). The denitrification rates were higher during the winter than during the spring and autumn, 116 probably owing to the increased NO3--N availability in the winter (Table S1), which can supply more 117 N substrates to denitrifiers. The denitrification rates were significantly higher in the loam soil than in 7 118 the sandy soil (Figure 1). These findings may be mainly attributed to a variety of circumstances. First, 119 the lower aeration in the fine-textured loam soil, due to the high level of soil moisture and the low 120 bulk density (Table 1), favours denitrification processes by reducing redox potentials during 121 anaerobic incubation. Second, the larger concentrations of NO3--N, DOC and OM in the loam soil 122 than in the sand (Table S1) accelerate denitrifier growth and enzyme synthesis by supplying organic 123 substrates and inorganic electron acceptors (Gu et al., 2013; Palta et al., 2016), thereby enhancing 124 soil denitrification capacity. Soil OM provides not only electrons for NO3--N denitrification through 125 mineralization, but also decreases soil redox potential, thus resulting in high substrate availability and 126 suitable environment for the growth and activity of denitrifiers (McLain & Martens, 2006; Xu & Cai, 127 2007). Moreover, NO3--N leaching, which is necessary for microbial growth, is less in loam soil, 128 which contributes to denitrification (Lee et al., 2014). 129 The denitrification rate of the loam soil was significantly higher in the oligohaline marsh than 130 the freshwater marsh (F=52.01, P<0.001; Figure 1). This finding is inconsistent with earlier studies 131 where denitrification decreased strongly as salinity increased (Osborne et al., 2015; Marks et al., 132 2016). This is most likely because the small variation of salinity in this estuary (0-3‰) cannot 133 substantially affect the activity of denitrifiers, which in turn also implies that the denitrifiers were 134 acclimated to the soils with low-level increases in salinity. Moreover, the concentrations of NO3--N, 135 DOC and OM in the loam soil were greater in the oligohaline than the freshwater marsh (Table S1), 136 which provides sufficient substrate for denitrifiers. A review of the literature also indicated that 137 denitrification rate patterns change with fluctuations in salinity (salinity range 0‰–30‰; Table S3), 138 suggesting that the effect of salinity is site-dependent. The overall PCA indicated that the groups of 139 samples of each soil type are clearly separated in the 2-dimension layout generated by the two main 140 axes (Figure 2). Soil denitrification rates consistently correlate positively with soil N, DOC, OM and 8 141 pH and negatively with bulk density. Overall, our data indicate that the variable salinity and texture, 142 and the interaction between them, have a significant effect on denitrification (F=21.92, 271.54, and 143 5.27; P<0.001, <0.001, and <0.05, respectively). 144 The addition of NH4NO3 substantially changed the denitrification rates, but the impact varied 145 with the soil texture and salinity (Figure 3). Specifically, NH4NO3 addition significantly increased the 146 denitrification rate relative to the control in the sandy soil (F=9.58, P<0.05) and loam soil (F=7.33, 147 P<0.05) of the freshwater marsh, and in the loam soil (F=1.99, P<0.01) of the oligohaline marsh by 148 212, 102, and 125%, respectively. Nitrogen is often the limiting nutrient in this estuary system (Wang 149 et al., 2014); therefore, NH4NO3 addition provided abundant NO3--N as the substrate for direct 150 denitrification (Figure S2b) and also contributed to nitrification by increasing NH 4+-N availability 151 (Figure S2a), which can lead to high rates of coupled nitrification-denitrification (Gu et al., 2013). 152 Our findings initially indicated that soil texture might be a critical factor controlling N cycling 153 in wetland systems, regardless of salinity. They suggested that fine-textured soils with low porosity 154 and large contents of NO3--N and OM would fuel the denitrification process and would be 155 accompanied by the removal of N. Further, different soil textures in the estuary system are formed at 156 different sedimentation times due to sea-land interactions (Wallace et al., 2005). The longer the 157 sedimentation time, the finer the soil texture, and the associated increases in the content of substrates 158 such as OM eventually leads to increases in denitrification capacity. Our data also clearly 159 demonstrated that an increased N loading could potentially promote the denitrification rates of soils 160 with contrasting textures and salinities. These results indicate that denitrification could be an 161 important pathway to regulate current and future N loading into estuarine and inshore coastal waters, 162 which reduces the negative effects of exogenous N on water eutrophication and acidification but, on 9 163 the other hand, may increase the risk of global warming. Further, we conclude that tidal marshes with 164 fine-textured loam soil had relatively higher N removal potential, especially for high N-enrichment 165 environments. This partly supports our hypothesis that much uncertainty is introduced in the N 166 dynamics of estuaries under longer-term, climate change-mediated sea level rise and N deposition. 167 Thus, the detailed mechanisms and processes that control estuarine denitrification and, subsequently, 168 N cycling need further consideration. 169 Acknowledgments 170 This research was jointly funded by the National Science Foundation of China (41877335, 41801062), 171 the Key Science Foundation of Fujian Province (2019J02008), China Postdoctoral Science 172 Foundation (2018M630731, 2019T120556), European Research Council Synergy grant (ERC-SyG- 173 2013-610028 IMBALANCE-P), Spanish Government grant (CGL2016-79835-P), and Catalan 174 Government grant (SGR 2017-1005). 175 Data Availability Statement 176 The data that support the findings of this study are available from the corresponding author upon 177 reasonable request. 178 Conflict of interest 179 The authors declare that they have no conflicts of interest in this work. 10 180 References 181 Ballantine, K.A., Groffman, P.M., Lehmann, J. & Schneider, R.L. 2014. 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Different lowercase letters on the bars indicate significant differences between seasons (P<0.05); the absence of letters indicates an absence of significant differences; the asterisks (*) indicate significant differences between the soil types (P<0.05). FS and FL represent sand and loam soil in the freshwater marsh, respectively; OS and OL represent sandy and loam soil in the oligohaline marsh, respectively. Figure 2 Principal component analysis of soil denitrification rates and main environmental parameters in different soil texture and salinity conditions. The means of PC scores of each one of the four soil types are depicted by small circles and the corresponding area around these circles depicts confidence intervals at 95%. BD: bulk density; EC: electrical conductivity; NH4+-N: ammonium nitrogen; NO3--N: nitrate nitrogen; DOC: dissolved organic carbon; SM: soil moisture; OM: organic matter. TC: total carbon; TN: total nitrogen. FS and FL represent sandy and loam soil in the freshwater marsh, respectively; OS and OL represent sandy and loam soil in the oligohaline marsh, respectively. Figure 3 Responses of the potential denitrification rate to nitrogen addition in soils with different textures and salinities. Different letters on the bars indicate significant differences between soils (P<0.05). Asterisks indicate significant differences between the control and N treatments (*P<0.05 and **P<0.01). C: control; N: NH4NO3 addition. FS and FL represent sand and loam soil in the freshwater marsh, respectively; OS and OL represent sandy and loam soil in the oligohaline marsh, respectively. Figure 1 Denitrification rate (g N kg h ) FS FL OS OL -1 25      -1 20 a a 15 10 b b 5 a a 1.6 ab 0.8 b 0.0 autumn winter spring summer overall Time Figure 2 Loading -2 -1 0 1 22 1.0 FS 1.4 1.4 FL OS [C:N] OL 0.5 0.7 0.7 SM TC PC2 (13.6%) Loading OM Scores 0.0 NH4+-N 00.0 pH BD TN DOC DR -0.5 - NO3 -N -0.5 -0.7 EC -1.0 -1.0 -1.4 -1.0 -1.0 -0.5 -0.5 0.0 0.5 1.0 1.0 Scores PC1 (54.5%) Figure 3 Denitrification rate (g N kg h ) C  -1 N 30 -1 a 20  a 10   b 3 2 b b 1 b b b 0 FS FL OS OL overall Type