Response of Nitrate Processing to Bio-labile Dissolved Organic Matter Supply Under Variable Oxygen Conditions in a Sandy Beach Seepage Face

fmars-08-642143 June 18, 2021 Time: 11:11 # 1 ORIGINAL RESEARCH published: 23 June 2021 doi: 10.3389/fmars.2021.642143 Edited by: Christian Lønborg, Aarhus University, Denmark Reviewed by: Sara Benelli, University of Parma, Italy Soeren Ahmerkamp, Max Planck Institute for Marine Microbiology (MPG), Germany Kaijun Lu, University of Texas at Austin, United States *Correspondence: Shan Jiang [email protected] Specialty section: This article was submitted to Marine Biogeochemistry, a section of the journal Frontiers in Marine Science Received: 15 December 2020 Accepted: 01 June 2021 Published: 23 June 2021 Citation: Jiang S, Jin J, Wu Y, Zhang Y, Wei Y, Rocha C, Ibánhez JSP and Zhang J (2021) Response of Nitrate Processing to Bio-labile Dissolved Organic Matter Supply Under Variable Oxygen Conditions in a Sandy Beach Seepage Face. Front. Mar. Sci. 8:642143. doi: 10.3389/fmars.2021.642143 Response of Nitrate Processing to Bio-labile Dissolved Organic Matter Supply Under Variable Oxygen Conditions in a Sandy Beach Seepage Face Shan Jiang1*, Jie Jin1, Ying Wu1, Yixue Zhang1, Yongjun Wei2, Carlos Rocha3, Juan Severino Pino Ibánhez3,4 and Jing Zhang1,5 1State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China, 2Laboratory of Synthetic Biology, Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China, 3Biogeochemistry Research Group, School of Natural Sciences, Trinity College Dublin, Dublin, Ireland, 4Instituto de Investigacións Mariñas, Consejo Superior de Investigaciones Científicas (IIM-CSIC), Vigo, Spain, 5School of Oceanography, Shanghai Jiao Tong University, Shanghai, China Supply of bio-labile dissolved organic matter (DOM) has been assumed to be a key factor for the intensity of nitrate (NO3−) removal in permeable coastal sediments. In the present study, a series of flow through reactor experiments were conducted using glucose as a N-free bio-labile DOM source to permeable sediments from a sandy beach seepage face to identify its effect on benthic NO3−removal. The results revealed a shift from the dominance of NO3−production to removal processes when NO3−input concentration increased from 10 to 80 µM under oxic conditions. Sediment microbiota information suggests that nitrification (e.g., Nitrosomonas and Nitrososphaera) and denitrification (e.g., Marinobacter and Bacillus) were dominant pathways for benthic NO3−production and removal in the studied sediment. Compared with the active response of sediment microbiota to NO3−additions, the supply of glucose (approximately 300 µM final concentration added) did not significantly change the NO3−removal efficiency under aerobic conditions (dissolved oxygen saturation approximately 100%). Similarly, an insignificant increase of NO3−removal rate after glucose amendment of the circulating water was obtained when dissolved oxygen (DO) saturation decreased to approximately 70% in the input solution. When DO at the input solution was decreased to 30% saturation (sub-oxic conditions), the removal rate of NO3−in the group amended with glucose increased, suggesting that glucose stimulated denitrifiers. These results revealed that NO3−removal relied mainly on the anaerobic environment at particle surfaces, with a dependence on the sedimentary organic matter as an electron supplier under bulk aerobic conditions, while the bio-labile DOM was consumed mainly by aerobic respiration instead of stimulating NO3−reduction. However, the respiration triggered by the over-supply of bio-labile DOM reduced the Frontiers in Marine Science | www.frontiersin.org 1June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 2 Jiang et al. Nitrate Processing in Seepage Face DO in the porewater, likely depressing the activity of aerobic reactions in the permeable sediment. At this point, the benthic microbiota, especially potential denitrifiers, shifted to anaerobic reactions as the key to support nitrogen metabolism. The glucose amendment benefited NO3−reduction at this point, under sub-oxic conditions. Keywords: nitrate, dissolved organic matter, denitrification, subterranean estuary, flow through reactors INTRODUCTION Coastal permeable sediments, mainly composed by coarse silicate or carbonate particles with a permeability >10−12 m2, cover more than 70% of global coastal areas (Huettel et al., 2014). In this permeable environment, advection is the dominant pathway for the transport and dispersion of solutes in porewater (Jiang et al., 2021a), and feeds a wide range of biogeochemical reactions (Huettel et al., 2014;Mouret et al., 2020). Bioavailable nitrogen (N) has been intensively introduced into terrestrial ecosystems via different anthropogenic activities. The indiscriminate use of fertilizers, manure and waste sludge in land together with industrial spillages has led to the widespread accumulation of nitrate (NO3−) in coastal groundwater on a global scale (Rivett et al., 2008). Coupled with the transport of groundwater from aquifers to coastal oceans driven by hydraulic gradients, a significant portion of land-derived NO3−reaches permeable coastal sediments in subterranean estuaries (STEs; defined as the underground mixing zone between continental groundwater and infiltrating seawater; Moore, 1999) prior to discharge to the coast (Jung et al., 2020;Ibánhez et al., 2021a). High groundwater-borne NO3−loadings frequently trigger a series of negative feedbacks on coastal ecosystems such as blooms of toxic phytoplankton, accumulation of organic matter, and oxygen depletion (TroccoliGhinaglia et al., 2010;Amato et al., 2016;Rocha et al., 2016). As active reactors in coastal belts, permeable sediments have a strong capability to modulate NO3−mass transport via a wide range of biogeochemical reactions, such as autotrophic nitrification, microbial assimilation, and heterotrophic denitrification (Marchant et al., 2014;Kuypers et al., 2018; Ahmerkamp et al., 2020;Jiang et al., 2021c). The main pathway for NO3−removal in permeable coastal sediments is frequently identified as denitrification (Ibánhez et al., 2013;Marchant et al., 2014;Ibánhez and Rocha, 2017). Denitrification rates in permeable sediments are deemed to be deeply influenced by environmental factors, including the supply of organic matter, dissolved oxygen (DO) concentration, and advection rate, etc. (Santos et al., 2012;Ibánhez and Rocha, 2017;Ahmerkamp et al., 2020). As a heterotrophic microbial pathway, organic matter availability has widely been acknowledged to be a key factor for benthic denitrification (Crawshaw et al., 2019). In intertidal permeable sediments, the organic matter inventory includes both dissolved organic matter (DOM) and sedimentary organic matter (SOM). There, DOM shows high mobility due to seawater intrusion caused by tidal pumping, wave setup and temperature-driven porewater movement (Suryaputra et al., 2015;Meredith et al., 2020), while the SOM concentration is frequently related to adsorption and retention of particulate organic matter near the infiltration zones (Jiang et al., 2020). Both DOM and SOM can feed carbon-dependent reactions (Weston and Joye, 2005). In terms of denitrification, previous research outcomes frequently assumed that bio-labile DOM would control sediment/soil denitrification capability according to the requirement of electron transfer from carbon compounds (Cornwell et al., 1999;Bernhardt and Likens, 2002;Surey et al., 2020). However, permeable sediments are frequently enriched in DO by porewater exchange with DO saturated seawater and DO diffusion from the atmosphere (Berg et al., 2013;Ibánhez and Rocha, 2016). The bio-labile DOM might be consumed in aerobic mineralization prior to be available for denitrification (Jiang et al., 2018), suggesting a possible spatial isolation between bio-labile DOM enrichment and denitrifiers (e.g., spatial mismatch in coastal aquifers; Siemens et al., 2003). In addition, DO availability might directly hamper the denitrifying bacterial activity, leading to depression of denitrification process rates under oxic conditions (Evard et al., 2012). Alternatively, the consumption of bio-labile DOM in aerobic reactions triggers the decline of DO in porewater (Heiss et al., 2020), creating sub-oxic or anaerobic environments that are suitable for benthic denitrifiers along the porewater transport path (Kessler et al., 2012;Heiss et al., 2020). Nevertheless, our understanding of the interrelations between bio-labile DOM and DO supply and denitrification, i.e., the dominant pathway of NO3−removal, in coastal sands is still limited and hinders our understanding of NO3−cycling in coastal permeable environments and the resulting NO3−fluxes across the land-ocean boundary. Targeting this knowledge gap, in the present study, a series of laboratory simulations were conducted using flow through reactor (FTR) cells to mimic the dominant advective transport pathways of coastal sands. Glucose was added to the pumped solution as a source of bio-labile DOM to sediment microbes. In addition, the DO concentration in the input solution was adjusted by injecting nitrogen gas. Rates of NO3−removal and production were evaluated under different levels of glucose and DO availability. The main hypothesis of the present study was that glucose amendment could significantly stimulate NO3− removal rates. The research aim was to test the hypothesis and evaluate the possible mechanisms underlying observed reaction patterns. MATERIALS AND METHODS Sample Collection Permeable coastal sediments were collected from a sandy beach located at the inner part of Sanggou Bay (China). The porosity of the beach sediment was approximately 0.31 and the mean hydraulic conductivity of the sampled sandy sediments was Frontiers in Marine Science | www.frontiersin.org 2June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 3 Jiang et al. Nitrate Processing in Seepage Face 7.5 ×10−5m s−1. There, the outflow of low salinity porewater into the bay was verified during ebb tide (Jiang et al., 2020). This brackish water discharge is associated to continental groundwater inputs that also trigger the porewater enrichment in radium isotopes and nutrients (Wang et al., 2014). Around Sanggou Bay, agriculture activities, mainly focused on apples, corn and wheat are intensively developed. These activities lead to a significant accumulation of NO3−in the terrestrial groundwaters surrounding the bay, with concentrations >350 µM in coastal wells (Wang et al., 2014). This continental groundwater enriched with NO3−reaches the sampled seepage face, leading to a variation of NO3−concentration from 20 to 60 µM in porewater. DOC concentrations locally vary between 180 and 390 µM, while the P concentration in the porewater was much lower (below 2µM) due to adsorption on sandy particles (Wang et al., 2014). Apart from agriculture, the local economy is also supported by intensive marine culture activities conducted in the bay (Fang et al., 2016). High-biomass primary producers, such as diatoms and kelp, introduce a significant amount of pelagic DOM into the bay water via basic metabolism (e.g., secretion), diffusion across the cell membrane and decomposition (Thornton, 2014; Mahmood et al., 2017). This pelagic DOM is injected into the seepage face with seawater infiltration, creating a local mixing zone between bio-labile DOM and terrestrial NO3−. Sediment cores (20 cm depth, 10 cm diameter) were collected in the seepage face during ebb tide in April (spring) and October (autumn) 2018. Sediment at the sampling site presented a large accumulation of bivalve debris at the surface originated from the intense bivalve aquaculture in the area. This surface layer of bivalve debris (typically 0–1 cm depth) was removed before the collection of sediment cores. Laboratory FTR Simulation Flow through reactor cells were used to mimic in situ advective conditions and thus explore benthic reactions in the seepage face (Figure 1). FTR cells of 10 cm length and approximately 8 cm of diameter were used (total volume for sediments: 500 cm3). These include two collimators and fine meshes (pore size: 30 µm) to ensure an even transversal distribution of the pumped solutions through the incubated sediments. After collection, the sediments were gently repacked into the reactor cells after removing large pieces of debris and the inner walls of the cells were scrubbed to increase the contact between sediment particles and the containers and hence to eliminate the build-up of preferential flow paths along the walls. Aluminum foil was wrapped around each cell to eliminate the influence of light exposure on benthic microbial activity or primary producers. After setting up, all FTR cells were placed into a thermo-constant incubator at 20◦C, which was comparable to the porewater temperature during both sampled seasons (Jiang et al., 2020). FTR cells were then flushed with seawater overnight (approximately 10 h) at a constant rate of 3 mL min−1(discharge rate as 11.5 cm h−1), equivalent to the advection rate quantified in the seepage face during active seepage (Jiang et al., 2020). With these conditions, porewater retention time was calculated as the quotient of system flow rate and total porewater volume (product of total sediment volume and porosity) in a cell (approximately 52 min). Afterward, the flushed sediment from three reactors was kept for further physico-chemical analyses. After conditioning of the FTR cells, the working solutions were pumped through the test reactor cells using a highaccuracy peristaltic pump (BT-100, LongerR , China) at the same pumping rate as that used during the flushing period. The series of sediment incubations included seven groups (one control and six treatments) with different levels of NO3−, dissolved organic carbon (DOC) and DO in the input solution, as outlined in Table 1. The carrier solution used was aged seawater (storage >100 days) with a salinity of 33 to maintain strict control on the labile DOM content of the circulating porewater. In the six treatments, NO3−was kept constant (∼80 µM), while DOC concentrations ranged from ca. 140 (i.e., without amendment) to 480 µM via the addition of glucose. Additionally, three different DO levels in the input solution were employed (Table 1). In the DO saturation group (approximately 240 µM), the working solution was bubbled with air before the incubation. For the DO unsaturation groups (70% saturation: nearly 170 µM), nitrogen gas (99.999% purity) was bubbled into the working solution from the bottom of the container at a constant rate for the partial removal of DO. For groups with 30% DO saturation (nearly 75 µM), the input solution was treated in an ultrasonic bath for 3 min prior to the start of the bubbling process. For each treatment, triplicate reactor cells were used. During the experiments, the complete replacement of the porewater inside the FTR cells was determined by dye tracer release-balance (input concentration was identical to output concentration), and it was complete after approximately 3 h at the settled pumping rate (3 mL min−1) based on the inputoutput balancing experiments using Rhodamine tracer (the input and output balance displayed in Supplementary Figure 1). Accordingly, sampling of the input and outflow solutions from each reactor took place after pumping the working solution for 6 h to guarantee solute dispersion in sediments (Figure 1). In each reactor, four samplings were conducted at 1 h intervals. The collected samples were filtered with Millipore syringe filters (0.22 µm pore size) and stored in acid prewashed plastic bottles for dissolved inorganic nitrogen (DIN) species and precombusted glass vials for DOC and chromophoric dissolved organic matter (CDOM) determinations and kept at −20◦C prior to laboratory analyses to avoid potential microbial degradation of the samples (Jiang et al., 2017). Besides the collection of water samples, DO concentrations at both the input solution and the outflow stream were directly measured with a portable probe (WTW Multi 3630). Reaction rates for DIN species, DOC and DO were calculated from the difference in concentration between the input and the output solutions, porewater residence time (52 min) and the sediment volume contained in the FTR cell (500 cm−3) according to Ibánhez and Rocha (2014). Negative reaction rates indicate consumption of the target solute within the sediment, while positive values represent benthic production. Sediment Physico-Chemical Analyses Total organic carbon (TOC) content in the sediment samples was determined via a CHNOS Elementar cube (Vario EL III) Frontiers in Marine Science | www.frontiersin.org 3June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 4 Jiang et al. Nitrate Processing in Seepage Face FIGURE 1 | Sketch of flow through reactor (FTR) experiments established in the laboratory. This set highlights the injection of nitrogen gas into the reaction liquid to regulate the oxygen content level. This sketch is modified from Jiang et al. (2018). TABLE 1 | Initial concentrations of DOC, NO3−, and DO in each batch of the flow through reactor (FTR) incubation experiment. Group DOC Spring DOC Autumn NO3−Spring NO3−Autumn DO Spring DO Autumn Low control 142 ±2.5 133 ±2.4 10.5±0.2 10.2±0.3 101 ±1.5 100 ±2.6 NO3−-high DO 144 ±1.9 131 ±3.5 79.5±0.8 76.8±1.0 99.8±1.2 99.2±2.2 NO3−-medium DO 141 ±2.2 135 ±2.8 77.4±1.1 80.2±1.3 70.4±4.1 71.9±3.9 NO3−-low DO 145 ±1.9 132 ±3.7 79.4±1.9 76.7±1.2 32.7±5.2 35.1±3.4 Glucose-high DO 479 ±6.1 451 ±5.2 80.5±1.2 78.2±0.8 100 ±1.6 99.4±1.9 Glucose-medium DO 486 ±4.7 455 ±4.9 79.5±1.2 80.4±1.3 72.4±4.8 73.5±4.4 Glucose-low DO 481 ±5.8 460 ±4.8 78.4±1.4 79.6±1.5 30.6±5.7 32.5±4.9 The unit for dissolved organic carbon (DOC) and NO3−in the table is µM and unit for dissolved oxygen (DO) is%. It shows the mean ±stand deviation on the basis of triplicate measurements. after removing inorganic fractions using 2 M hydrochloric acid (Wu et al., 2019). Sediment total nitrogen (TN) concentration was measured with the same equipment without the acid pretreatment. The equipment accuracy for TOC and TN determinations in sandy sediments was approximately 5%. Exchangeable DIN, DOC, and CDOM from sediment particles (approximately 4 g dry weight) was extracted with a 0.5 M potassium chloride (KCl, approximately 40 mL) solution for 12 h under constant agitation, with a rotation rate of 200 rpm (20◦C). Subsequently, the supernatant was filtered with syringe filters and kept at −20◦C until analysis. Sediment grain size was measured using a Coulter LS 100Q particle analyzer (Coulter Company, United States). The particle size <2µm was defined as clay, 2–63 µm as silt, and >63 µm as sandy grains (Wu et al., 2019). Sediment samples from both sampled seasons were also used in a series of adsorption experiments to quantitively determine the influence of adsorption on glucose and NO3−concentrations under aerobic conditions. In particular, the sediment used in the adsorption was autoclaved for 30 min at 121◦C to eliminate the influence of sediment prokaryotes (Jiang et al., 2018). After gently washing with filtered seawater, the autoclaved sediments (10 g) were mixed with aged seawater (40 mL) spiked with NO3−(final concentration: 80 µM) and glucose (final DOC concentration: 480 µM), respectively. The adsorption experiments covered incubation periods ranging from 0.5 to 12 h. The spiked solutions before and after adsorption experiments were collected and stored for laboratory analyses. The adsorbed quantity of glucose and NO3−was calculated from the solute concentration difference in these two solutions divided by the sediment dry weight (Ibánhez and Rocha, 2017). Sediment Microbial Analyses Total DNA from approximately 0.30 g of sediment was extracted using the DNeasy PowerSoil Kit (Qiagen, Germany). Primers for the V3–V4 regions for 16S rDNA were designated to identify microbial diversity (Wei et al., 2021). The 16S rDNA gene amplicon library from PCR cycling was constructed with the KAPA Hyper Prep Kit (Roche). The built library was pairedend sequenced on an Illumina Miseq system (Illumina). The raw reads have been deposited in the NCBI database. The diversity indices from the spring and autumn samples, including the Shannon index and Equitability, were analyzed with USEARCH alpha_div (Edgar, 2010). Water Analyses Dissolved inorganic nitrogen species (NH4+, NO2−, and NO3−) in the samples collected from the FTR experiments, the solutions for extraction of the sediment exchangeable compounds and Frontiers in Marine Science | www.frontiersin.org 4June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 5 Jiang et al. Nitrate Processing in Seepage Face adsorption experiments (only NO3−) were quantified using a flow injection system (SAN++ plus, SKALAR Analytical B.V., Netherlands). DOC concentrations in these water samples were analyzed by high-temperature catalytic combustion using a Shimadzu TOC-LCPH TOC analyzer (TOC-LCPH, Shimadzu, Japan) with an ASI-L autosampler (Jiang et al., 2020). CDOM of the collected water samples was analyzed on a dual channel spectrophotometer (PERSEER , China, TU-1901) with ultrapure water as blank (water resistivity: 18.2 Mcm). After equipment stabilization, CDOM samples were continuously scanned from 400 to 250 nm on the spectrophotometer with 1 nm intervals. CDOM content in the tested water samples was estimated by the absorption coefficient at 355 nm (a355,Zhang et al., 2007). In addition, the spectral slope from 275 to 295 nm was determined as a proxy of the molecular weight of CDOM (S275−295; terrestrial materials frequently show small slopes, while pelagic materials tend to show larger slopes; Zhang et al., 2007). The isotopic composition of NO3−(δ15N/18O–NO3−) was determined in the collected samples to understand reaction pathways and rates in place (Jiang et al., 2019, 2021b). The isotopic ratios were measured by the bacterial reduction method (Sigman et al., 2001) using a Thermo-Fisher Delta V advantage analyzer coupled with a PreCon system (more details in Jin et al., 2020). The method reproductivity was approximately 0.2hfor δ15N–NO3−and 0.5hfor δ18O–NO3−. Statistical Analyses To explore the effect of glucose supply on NO3−removal rate under different DO saturations, a series of one-way ANOVAs and Tukey Honestly Significant Difference (HSD) test were performed with the Minitab software (version 17.0) with the statistical significance as p<0.05. Linear correlation between variables was also explored with Minitab. RESULTS Sediment Physico-Chemical Factors and Variability During the two sampled periods, the mean grain size of the collected sediment, mostly composed by sand, was highly similar (204 µm mean grain size in spring to 199 µm mean grain size during autumn, Table 2). The grain skewness was 0.23 in spring and 0.31 in autumn and the kurtosis was ∼2.4 during both seasons. Sediment TOC concentration, determined before the use of the working solutions in the FTR experiments, was 92.5 µmol g−1during spring, and decreased to 72.4 µmol g−1in autumn (Supplementary Figure 2). TN content was 7.2 and 5.9 µmol g−1during spring and autumn, respectively. No significant variations of TOC and TN were observed among treatments after the FTR incubations (Figures 2A,B). Compared with TN content, concentrations of exchangeable DIN species on sediment particles were much lower (Table 2). In addition, the FTR incubations slightly decreased the exchangeable NH4+ concentration in the control group as well as DO saturation and 70% saturation treatments during autumn, while the exchangeable NH4+content increased in all the FTR groups containing NO3−amended circulating water during spring (groups B–D, Figure 2C). Compared with extracted NH4+, the exchangeable NO3−(E–NO3−) content was 3–4 times lower (Supplementary Figure 2). During spring, the offset in E–NO3− became smaller as DO increased, while a reverse trend was found in autumn (Figure 2D). As a minor contributor to the DIN pool, extracted NO2−concentrations were <1 nmol g−1 (Supplementary Figure 2). Solute Adsorption and Heterogeneous Equilibrium in the Studied Sediments During spring, the adsorbed glucose in the tested sediment was ∼0.03 µmol glucose g−1after 0.5 h incubation regardless of the adsorption of other DOC solutes (Supplementary Figure 2). The adsorbed amount logarithmically increased when the incubation time prolonged, reaching 0.14 µmol glucose g−1after 12 h. During autumn, the sediment still retained glucose from the reaction solution via adsorption, similarly to the pattern observed during spring. The NO3−content remained relatively constant after 4 h of incubation in spite of the weak NO3−adsorption capacity shown by the sediments (Supplementary Figure 2), indicating that adsorption was fast. Dissolved Oxygen and DOM Variation The DO content in the working solutions was set at three different levels. In the DO saturated group, the DO concentration at the input solution was approximately 230 µM (Supplementary Figure 3). After flowing through the sediments, DO concentrations decreased (Supplementary Figure 3). In the control group, the outflow stream DO content decreased approximately 30 µM DO, implying benthic respiration rates of 12.9 and 11.6 nmol g−1h−1during spring and autumn, respectively. Introducing NO3−did not significantly change the DO consumption rate (group B), while the DO consumption was negatively influenced by the decreasing DO concentration in the input solution (groups C and D). The amendment of the circulating solution with glucose significantly increased DO consumption rates (group E; p<0.01) while a decay in benthic respiration rates at suboxic conditions (groups F and G) was observed for both seasons. Different from DO, the sediment acted as a net source of DOC in the control group, especially during spring (Figure 3B). The addition of NO3− enhanced the DOC production rate (group B), while decreasing DO in the input solution slightly reduced DOC production rates. In contrast, glucose addition to the circulating solution completely changed the sediment mediation of DOC fluxes into a strong DOC sink due to the rapid consumption of glucose. For CDOM (a355), sediments in all groups showed a production trend (Figure 3C), especially at the group B during both seasons (DO saturation, spiked with NO3−). After flowing through the sediment, S275−295, the index of DOM structure, increased in all groups, revealing increases of small-molecular DOM in the porewater via benthic reactions. The most active addition still occurred at group B during both seasons, while the slowest addition was found at the groups run under suboxic conditions (Figure 3D). Frontiers in Marine Science | www.frontiersin.org 5June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 6 Jiang et al. Nitrate Processing in Seepage Face TABLE 2 | Sediment physico-chemical characteristics and microbial indices determined during spring and autumn. Spring Autumn Physico-chemical Factors Gran size (µm) 204 ±4.5 199 ±6.4 Sandy grains (%) 91.2±1.2 90.7±0.7 TOC (µmol g−1) 92.5±1.6 72.4±1.4 TN (µmol g−1) 7.2±0.3 5.9±0.2 E-NH4+(nmol g−1) 88 ±2.8 72 ±1.9 E-NO2−(nmol g−1) 0.6±0.02 0.7±0.05 E-NO3−(nmol g−1) 19.2±0.6 18.0±0.4 Microbial factors OTUs 1288 2454 Shannon 7.33 8.29 Equitability 0.709 0.736 Dominant phylum Proteobacteria Proteobacteria Opportunistic phylum Bacteroidetes,Firmicutes Actinobacteria,Acidobacteria Key genus Sphingobium Nitrosomonas Marinobacter Gp10 Thioprofundum Thioprofundum Potential denitrifiers Marinobacter Marinobacter Bacillus Bacillus Lactobacillus Hyphomicrobium Physico-chemical characteristics in the table include the mean ±standard deviation of triplicate measurements. E-NH4+/NO2−/NO3−is the exchangeable NH4+/NO2−/NO3−species extracted from the sediment particles with KCl. FIGURE 2 | The offset of sediment total organic carbon (TOC) (A),TN(B), exchangeable NH4+(E-NH4+,C) and exchangeable NO3−(E-NO3−,D) before and after a series of incubations during spring and autumn. Positive values indicate solute concentration increases after incubation and negative is concentration decreases. Frontiers in Marine Science | www.frontiersin.org 6June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 7 Jiang et al. Nitrate Processing in Seepage Face FIGURE 3 | Reaction rates for dissolved oxygen (DO) (A) and dissolved organic carbon (DOC) (B) during spring and autumn, as well as offsets for chromophoric dissolved organic matter (CDOM) characters (Cfor a355;Dfor S275−295) before and after FTR incubations during both seasons. Positive values indicate production/offset enhancement after incubation and negative is removal/offset decrease. Dissolved Inorganic Nitrogen Species and NO3 −Isotopes Similarly, the sediment acted as a net source of NH4+in the control group during both seasons (Figure 4A). The amendment of NO3−decreased the NH4+production rate during spring, although the sediment still acted as a net NH4+source (group B). The NH4+production was stimulated by NO3−addition during autumn. In contrast, the decrease of DO concentration in the input solution significantly decreased the NH4+production rate during both seasons (p= 0.01), especially after the concomitant amendment with glucose (group G). Compared with NH4+, NO2−concentrations in both input and output solutions were much lower, ranging from 0.31 to 0.47 µM (Supplementary Figure 4). Such low concentrations led to minor variations among groups (Figure 4B). Different to the mediation of NO2−fluxes, the sediment acted as a net source of NO3−in the control group during both seasons (Figure 5A), leading to a decrease in both δ15N–NO3−(Figure 5B) and δ18O–NO3−(Supplementary Figure 5). The sediment became a net NO3−sink in response to amendment with NO3−, while the NH4+production and DO consumption rates remained similar under oxic conditions (Figures 4C,D). Amongst the different treatments, the glucose addition did not significantly decrease the NO3−concentration in the output solutions under aerobic conditions (group B vs. E). Accordingly, the isotope offset of both δ15N–NO3−and δ18O–NO3−(difference between the input and output fractions) was statistically similar between these two groups. Even when the DO concentration in the input solution dropped to 70% saturation, the glucose stimulation of NO3−removal rates was still insignificant (group C vs. F) and the isotopic response was similar. In the sub-oxic groups (D and G), NO3−removal rates significantly increased compared to the oxic group (p= 0.01), leading to a linear relationship between NO3−reduction rates and δ15N–NO3−increase (Figure 5C). In terms of the offset between δ15N–NO3−and δ18O–NO3−(Figure 5D), a linear correlation was observed, suggesting that the dominant NO3− consumption pathway remained the same from B to G groups. Benthic Prokaryotes During spring, the operational taxonomic units (OTUs) in the sediment were 1,288, increasing to 2,454 during autumn (Table 2). Concurrently, the Shannon index and Equitability also increased during autumn. In both periods, the dominant phylum was Proteobacteria while important opportunistic groups varied between seasons. The key genera were Sphingobium, Marinobacter, and Thioprofundum during spring, while the relative abundance of Nitrosopumilus and Gp10 increased during autumn. For the potential metabolic functionality (Supplementary Figure 6), a wide range of functions were Frontiers in Marine Science | www.frontiersin.org 7June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 8 Jiang et al. Nitrate Processing in Seepage Face FIGURE 4 | Reaction rates of NH4+(A) and NO2−(B) during spring and autumn. Positive rates indicate production after incubation and negative is removal. It also shows the correlation between NH4+and NO3−reaction rates (C) and DO and NO3−reaction rates (D). present with benthic carbon, nitrogen and sulfur metabolism, suggesting a significant requirement of electron donors. The nitrogen metabolism was also taken as a key function with a minor seasonal variation. For the microbiota involved in nitrogen transformations, nitrifiers, e.g., Nitrosopumilus, were observed in the seepage face. Denitrifying bacteria, characterized at the genus level, such as Marinobacter,Bacillus,Hyphomicrobium, or Lactobabillus, were also observed in the sediment (Table 2). The microorganisms involved in Anammox and DNRA were not identified at the genus level in the studied seepage face, indicating that NO3−removal was mainly conducted by denitrification. DISCUSSION Methodological Considerations of the Use of FTR Flow through reactor simulations under pre-defined environmental conditions, have been identified as an effective tool to quantitively determine nitrogen transformations in permeable coastal sediments, and have been widely applied in many research sites, e.g., Ria Formosa Lagoon, Portugal (Ibánhez and Rocha, 2014, 2016); South Atlantic Bight (Rao et al., 2007) and the North Sea (Marchant et al., 2016). FTR can effectively mimic 1-D advective flow in permeable sediments, adequately simulating the essentially vertical nature of porewater transport in the seepage face during ebbing tide (Ibánhez et al., 2011). The use of FTR with permeable sediments requires a “checklist” prior to laboratory incubations. Apart from flow rate, light exposure and reasonable temperature, the preferential flow paths that might form inside the cells are one of the pivotal factors in FTR simulations, and may lead to uneven transport and dispersion of the target solutes within the reactor cells, questioning the reliability of the reaction rates obtained. In the present study, these environmental variables were constrained at laboratory conditions with limited fluctuations during incubation experiments to reduce the potential errors. To further identify the reliability of our simulation results, a comparison of the obtained reaction rates with in situ rates was undertaken. During in situ field surveys at the sampling site, the DOC production ranged from 12.5 to 68.9 nmol cm−3h−1during spring and 2.8–62.7 nmol cm−3h−1during autumn, calculated from solute distribution at the seepage face together with measured seepage rates and applying mass balance approaches (data from Jiang et al., 2020). The reaction rates obtained in the FTR experiments were similar to the lower boundary of these in situ data. Moreover, on a global scale, the DOC production rates obtained in the present study are in line with the results found in a subterranean estuary seepage face in the Ria Formosa Lagoon, Portugal (FTR experiments; Ibánhez and Rocha, 2014). The DO consumption rate in the control group was 12.9 nmol cm−3h−1during spring, which was in line with Frontiers in Marine Science | www.frontiersin.org 8June 2021 | Volume 8 | Article 642143 fmars-08-642143 June 18, 2021 Time: 11:11 # 9 Jiang et al. Nitrate Processing in Seepage Face FIGURE 5 | Reaction rates NO3−during both seasons (A) and offsets for δ15N–NO3−from a series of incubations (B). Positive values indicate production/offset enhancement after incubation and negative is removal/offset decrease. It also shows the correlation between NO3−reaction rates and δ15N–NO3−(C) as well as δ15N–NO3−and δ18O–NO3−offsets (D). those determined by Rao et al. (2007) in the South Atlantic Bight using FTRs (3.8–16.6 nmol cm−3h−1). The rapid DO consumption in permeable sediments after glucose amendment was also comparable to the results obtained by Rusch et al. (2006) with sediments from the Atlantic Bight (35–57% enhancement). NO3−removal rates obtained in the present study are similar to those calculated from field survey data in the same STE (13.6 nmol cm−3h−1, unpublished data) and those measured with FTR in the Ria Formosa Lagoon (0.7–6.8 nmol cm−3h−1; Jiang et al., 2018). Bio-labile DOM Supply and Benthic NO3 − Transformations The effects of bio-labile DOM availability on benthic aerobic respiration and mineralization have been widely documented as a key factor for a wide range of heterotrophic transformations in many coastal permeable environments, such as in Skagerrak, North Sea (Hulthe et al., 1998), St. George Island, Gulf of Mexico (Chipman et al., 2010) or the Ria Formosa Lagoon, Portugal (Ibánhez and Rocha, 2016). In our FTR experiments, the addition of glucose enhanced aerobic respiration rates as expected. Nevertheless, despite DO being more energetically favorable as an electron acceptor than NO3−for sediment microbes, net NO3−reduction is observed after the addition of NO3−to the circulating porewater even at DO saturated conditions, revealing the temporal synchronism of both reactions. In the present study, nitrogen transformations were identified as a crucial function of sediment microbial metabolism (Supplementary Figure 6). Substantial microbial groups were identified as potential nitrifiers and denitrifiers in our sampled site based on the 16S rDNA information. For potential nitrifiers, the dominant genus was Nitrosomonas (Table 2). During laboratory incubations, Nitrosomonas genus showed a significant reliance on the supply of organic carbon during their growth stages in culture solutions despite the autotrophic character of the nitrification process (Clark and Schmidt, 1967). Similarly, the potential key denitrifiers found in the studied seepage face, included Bacillus,Hyphomicrobium,Lactobacillus and several of these were identified as facultative denitrifiers with different metabolism functions at variable DO levels (Ji et al., 2015). They have previously shown strong dependence on organic carbon supply (Harder and Attwood, 1978;Condón et al., 1996; Surono et al., 2008), likely due to their aerobic reaction pathway catalyzed by cytochrome o oxidase (Ji et al., 2015). Often, nitrification and facultative denitrification are closely coupled in coastal sediments (Ouyang et al., 2021) while their reaction rates vary under different environmental settings (Calvo-Martin et al., 2021). Net NO3−production in our experiments (group A) reveals the dominance of nitrification, probably fed by organic Frontiers in Marine Science | www.frontiersin.org 9June 2021 | Volume 8 | Article 642143