Antecedent precipitation influences the bacterial processing of terrestrial dissolved organic matter in a North Carolina estuary

Accepted Manuscript Antecedent precipitation influences the bacterial processing of terrestrial dissolved organic matter in a North Carolina estuary C.L. Osburn, J.N. Atar, T.J. Boyd, M.T. Montgomery PII:

S0272-7714(18)30689-9

DOI:

https://doi.org/10.1016/j.ecss.2019.03.016

Reference:

YECSS 6159

To appear in:

Estuarine, Coastal and Shelf Science

Received Date: 25 August 2018 Revised Date:

4 February 2019

Accepted Date: 24 March 2019

Please cite this article as: Osburn, C.L., Atar, J.N., Boyd, T.J., Montgomery, M.T., Antecedent precipitation influences the bacterial processing of terrestrial dissolved organic matter in a North Carolina estuary, Estuarine, Coastal and Shelf Science (2019), doi: https://doi.org/10.1016/ j.ecss.2019.03.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Antecedent precipitation influences the bacterial processing of terrestrial dissolved organic matter in a North Carolina estuary

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Osburn, C. L.1*, Atar, J. N.1#, Boyd, T. J.2, and Montgomery, M. T.2

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1. Department of Marine, Earth, and Atmospheric Sciences, North Carolina State University, Raleigh, NC 27695; [email protected]; +1-919-515-0382

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2. Chemistry Division, Naval Research Laboratory, 4555 Overlook Ave SW, Washington, DC 20375; [email protected]; +1-202-404-6424; [email protected]; +1-202-404-6419

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* corresponding author

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# now at Antea Group, Charlotte, North Carolina

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version 27-Mar-19

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Abstract

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Estuaries and coastal waters ultimately receive the terrestrial dissolved organic matter (DOM) exported from coastal watersheds, more directly during extreme precipitation events. Recent work suggests DOM’s degradation in coastal waters varies with its quality, which also might vary as a function of precipitation, activating contributions from different sources within a watershed. The aim of this study was to determine the extent to which microbial degradation of terrestrial DOM in the Newport River Estuary, eastern North Carolina, was influenced by precipitation events occurring within the preceding seven days from sampling. We hypothesized that DOM stored in forested wetlands (e.g., pocosins and Cypress swamps) that become connected to the main channel of the Newport River during high precipitation events was more labile than DOM flowing into the estuary under low precipitation events. DOM quality was assessed with optical and stable C isotope (δ13C) measurements, while DOM lability was assessed by measurements of bacterial production (BP) and mineralization of 14C-labeled phenanthrene (Pmin), a polyaromatic tracer compound. Aromatic content of DOM, assessed by specific ultraviolet absorbance at 254 nm (SUVA254) was highest in the river with values well over 5.0 L mg C-1 m1 , and decreased with salinity. Antecedent precipitation (AP) of at least 100 mm in the seven days prior to sampling resulted in dissolved organic carbon (DOC) concentrations >20 mg L-1, at salinities <10. Similarly, fluorescence humification index (HIX) values were highest in the estuary after the highest AP. Generally depleted δ13C-DOC values (-26 to -28‰) in the estuary up to a salinity of 30 indicated a substantial source of DOM likely originating from the forested swamps and tidal wetlands fringing the estuary. BP exhibited wide variability yet declined with salinity, while median values after higher AP (40 µg C L-1 d-1) were double that under lower AP. By contrast, aromatic mineralization (Pmin) rates increased as both DOC and CDOM concentrations, and SUVA254 and HIX values, declined with salinity. However, Pmin rates were highest after the highest AP for the three events sampled. Results indicate that flooding of coastal wetlands mobilizes a large pool of labile DOM which have a large impact on the carbon cycle in estuaries. By altering the quality, as well as quantity of terrestrial organic matter inputs to estuarine systems, extreme events may also affect utilization of aromatic organics by estuarine microbial assemblages, an intriguing research question worthy of further study.

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1. Introduction

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There is good evidence to suggest that coastal ecosystems along the southeastern Atlantic and Gulf coasts of the United States are experiencing increasing extreme weather events delivering heavy precipitation, especially those associated with tropical storms, which are increasing in intensity (Walsh et al., 2016). One of the consequences of such climatic change is higher freshwater discharges into estuaries and coastal waters, as well as regional flooding along low lying areas such as riparian wetlands. Along with the larger volume flux of freshwater into these terrestrial-aquatic interfaces (TAIs), solute fluxes also are dramatically larger during extreme events. The “Pulse-Shunt Concept” provides a watershed-based conceptualization of the effect of extreme events on solute fluxes, focused specially on dissolved organic matter (DOM) (Raymond et al., 2016). Pulse-shunt suggests a framework whereby extreme precipitation events trigger pulses of DOM which are then shunted away from source of origin downstream. In coastal watersheds, pulse-shunt suggests that estuaries and coastal waters ultimately receive the DOM exported during extreme precipitation events. Terrestrial DOM in coastal waters is subject to photochemical and microbial degradation, but recent work suggests much of its degradation in coastal waters varies with its quality. For example, in the Cape Fear River Estuary, DOM bioavailability to bacteria increased after floodwaters from Hurricanes Fran (1996) and Floyd (1999), each of which delivered ca. 30 and 50% of annual DOC loadings to the coastal Atlantic Ocean, respectively (Avery et al., 2004). Bianchi et al., (2013) showed that substantial terrestrial DOM was remineralized to CO2 in the shelf waters of the northern Gulf of Mexico after spring rain-induced flooding in the lower Mississippi River basin in 2011. An important outcome of that study was that biological degradation of terrestrial molecules such as lignin was consistent with molecular characterization of resident microbial communities and geochemical properties of DOM. In a survey of 20-years of data from eastern North Carolina’s Pamlico Sound, it was found that this coastal ecosystem metabolized much of the DOM delivered to it after two 500-year floods (Paerl et al., 2018). These studies suggest that flood-related loading of terrestrial DOM to coastal waters enhances carbon degradation in coastal environments.

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DOM is fundamentally important to ecosystem function, being the base of microbial food webs, influencing the underwater light field through its absorption of sunlight, and undergoing photochemical and/or microbial mineralization back to carbon dioxide (CO2). In estuaries, much work has been done to characterize the quality of DOM, which, unlike other bio-active solutes such as nitrate, ammonium, or phosphate, varies considerably depending upon its source. DOM diversity in estuaries is linked partially to mixing between river water and seawater but also in situ production by planktonic communities, as determined by a variety of optical and chemical measurements (Bauer and Bianchi, 2012). Mixing dynamics of estuaries, affecting the residence time of water within them, exerts a major control on the quality of DOM beyond simple binary mixing between river water and seawater (Bauer and Bianchi, 2012; Ward et al., 2017). Extreme precipitation events may exacerbate these dynamics. In the Neuse River-Pamlico Sound, such events increased DOM loads by 20 to 50% (Paerl et al., 2018). The fate of this material is suspected to be metabolized by bacteria or degraded by sunlight, but few studies have examined the ultimate fate of DOM in estuaries and coastal waters resulting from extreme precipitation events.

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Salinity gradients in estuaries and coastal waters represent mixing of terrestrial and marine DOM but also can include inputs from coastal wetlands (e.g., salt marsh) that can be important. The stable isotopes of carbon (δ13C) provide an assessment of DOM origin related to plant source and biogeochemical cycling that can partly resolve this uncertainty (Bauer and Bianchi, 2012). Moreover,

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terrestrial DOM loading into estuaries can be studied using measurements of chromophoric dissolved organic matter (CDOM), which absorbs sunlight in the ultraviolet and visible ranges of the electromagnetic spectrum, and is abundant in terrestrial DOM. DOC-specific UV absorption at 254 nm (SUVA254) corresponds to aromatic ring content of DOM; a metric which has been correlated to terrestrial DOM sources across many riverine environments and is an important means of quantifying terrestrial loads of DOC (Spencer et al., 2012; Weishaar et al., 2003). CDOM fluorescence also can be used a metric of DOM quality distinguishing freshwater and marine sources in estuaries and coastal waters (Murphy et al., 2008; Osburn et al., 2016, 2012; Stedmon et al., 2003). Recent work coupling δ13C-DOC values and CDOM absorbance and fluorescence measurements have provided analytical frameworks to evaluate DOM sources and biogeochemical cycling in many aquatic systems across gradients of salinity and climate (Osburn et al., 2016; Osburn and Stedmon, 2011).

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Current paradigms of microbially-mediated DOM reactivity consider compositionally-complex terrestrially-derived DOM (originating from degradation of plant material into organic matter stored in watersheds) to be refractory—resistant to microbial degradation—compared to compositionally less complex planktonic derived DOM. However, given the right environmental conditions, most organic carbon substrates are labile; in rivers and estuaries, substrates that are reactive on timescales faster than fluvial transport will be removed, with the remaining material persistent until conditions are optimum for its degradation. In coastal marine environments, DOM removal could be caused by combinations of photochemistry (Osburn et al., 2009; Stubbins et al., 2010; Vodacek et al., 1997), microbial degradation (Medeiros et al., 2017; Rowe et al., 2018), or combinations thereof (Moran et al., 2000). Based on experiments conducted in coastal watersheds, wetland DOM biodegradation was greater than stream DOM (Fellman et al., 2009). Moreover, Ward et al. (2013) found that the terrestrial biomarker, lignin, was indeed microbially reactive when released into fluvial systems under high water periods and transits downstream in the Amazon River system. Thus, we suspect similar processing of more complex organic matter occurs in estuaries.

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The aim of this study was to determine the extent to which microbial degradation of terrestrial DOM from the Newport River watershed in its estuary was influenced by precipitation events occurring within the preceding seven days. We hypothesized that DOM stored in forested wetlands (e.g., pocosins and Cypress swamps) that become connected to the main channel of the Newport River during high precipitation events is more labile than DOM flowing into the estuary under low precipitation events. We utilized optical (absorbance and fluorescence) and geochemical (δ13C values) properties of DOM to assess the terrestrial nature of the DOM as a means of explaining this increased lability. Further, we used mineralization of phenanthrene, a polycyclic aromatic hydrocarbon, as a proxy for terrestrial DOC reactivity due to aromatic C degradation. We anticipated that DOM stored in wetlands adjacent to the main channel of coastal rivers and estuaries are key pulses of reactive carbon which are shunted into estuaries and coastal waters after substantive precipitation, consistent with the “pulse-shunt” and “river floodplain” concepts (Junk et al., 2004; Raymond et al., 2016), highlighting the importance of lateral exchanges between wetlands and coastal rivers in the export of DOC across TAIs.

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2. Methods

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2.1. Study site

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The Newport River is a small (31 km2) estuary located along the Lower Outer Banks of North Carolina on the landward side in between Bogue and Back Sounds, the latter which also receives outflow of the

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North River Estuary (Fig. 1; Google Maps and 2016 North Carolina Digital Orthoimagery through NC OneMap Geospatial Portal). The region is an important shellfish harvesting though it has been placed under restriction due to above-threshold counts for fecal coliforms (Coulliette and Noble, 2008). The basin land use was about half silviculture (concentrated around the lower half of the Newport) with the other half about evenly split between agriculture and urban (1998 estimates) (Mattheus et al., 2009) and includes two wastewater discharges. Most of the land cover is coastal plain forest (45%) and wetlands (~40%) including Cypress swamps and pocosins: palustrine wetlands having mineral soil and large accumulations of organic carbon (Coulliette and Noble, 2008; Kirby-Smith and Costlow, 1989). The tidal freshwater Newport River flows through an extensive Spartina-dominated brackish marsh in a narrow channel until it opens up into the estuary proper where it mixes with both Harlowe and Core Creeks. Ditching occurs throughout the marsh for local drainage. This shallow (ca. 1 m mean depth) estuary is well mixed with microtidal flow caused by communication with Onslow Bay in the South Atlantic Bight, through Beaufort Inlet, with an average residence time of about 6 days (Kirby-Smith and Costlow, 1989). The watershed area of the estuary is approximately 310 km2, providing a land:water ratio of 10:1 (Ensign et al., 2008).

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Antecedent precipitation (AP) was quantified as the cumulative rainfall (inches, converted to mm) in the seven days prior to each sampling recorded at Morehead City, NC (https://www.usclimatedata.com/climate/morehead-city/north-carolina/unitedstates/usnc0464/2015/8). Based on historic averages, the total monthly rainfall for the region is 125 mm on average with a low of 80 mm (Apr) and high of 190 mm (Aug). On average, the area received 116 mm of precipitation in November. Historical data for the headwaters (Newport, NC) or mid-estuary at Core Creek were unavailable from US Climate Data so they were obtained from the same community reported gauges at each of the two sites in Carteret County, NC from the CoCoRaHS Network (https://www.cocorahs.org).

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2.2. Sample collection and processing

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Sampling occurred during two-day periods in August 2014, November 2014, and August 2015, using a small boat. Sampling stations were determined by salinity rather than by geographic location, in order to cover a freshwater to seawater gradient irrespective of tidal stage, which influenced navigation in this shallow estuary. This meant that specific stations were not revisited per se and resulted in an unequal number of stations sampled during the three sampling events. Water samples were collected at approximately 0.5 m below the surface in 1 L brown HDPE bottles stored at ambient temperature in a cooler until returned to the lab for processing. Each bottle was triple rinsed with sample water prior to filling. Temperature (°C) and salinity (unitless) were measured at 0.5 m depth using a hand-held YSI Pro Plus meter and a Quattro sensor array. The instrument was calibrated prior to each sampling trip.

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Upon return to the laboratory, a known volume of whole water sample was filtered through a precombusted (450 °C, 6 hours) 0.7 μm mesh size Whatman glass fiber filter (GF/F). The GF/F filtrate was stored at 4 °C in a 60 mL polycarbonate bottle for roughly 1 week until measurements on optical properties (fluorescence and absorbance) were performed. A second aliquot of filtrate was acidified with 85% H3PO4 to a pH of 2 and stored at 4°C until measurement for dissolved organic carbon (DOC) concentration and its stable carbon isotope ratio (δ13C-DOC).

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2.3. DOC concentrations and δ13C values

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DOC concentration measurements ([DOC]) were made on an OI 1030 TOC analyzer operating in wet chemical oxidation mode and calibrated using standards of caffeine over the range of 0 to 40 mg C L-1 (Osburn and St-Jean, 2007). Stable carbon isotope values were corrected for reagent blanks and normalized to the Vienna Pee Dee Belemnite (VPDB) scale using IAEA standards of sucrose (-10.8‰) and caffeine (-27.77‰). Routine analysis of the University of Miami Deep Sea Reference DOC (DSR) for DOC concentrations averaged (± 1 standard deviation) 45 ± 3μM, with corresponding δ13C-DOC values of −20.5 ± 0.4‰ (N = 11). Error on DOC concentrations by this method was <3% and standard deviation on δ13C-DOC values was ±0.4‰.

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2.4. Absorbance and fluorescence

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Spectral absorbance (200-800 nm) of CDOM was measured using a Varian 300UV spectrophotometer using ultrapure laboratory water as a blank. After blank subtraction, Napierian absorption coefficients were calculated from absorbance: aλ = 2.303×Aλ/L

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(1)

where aλ is the absorption (m-1) at wavelength, λ, Aλ is the absorbance from the spectrophotometer, and L is the pathlength of the absorbance cell, in meters (m). CDOM was quantified as the absorption at 254 nm (a254). Specific ultraviolet absorption at 254 nm (SUVA254) was computed as an index of DOM aromaticity (Weishaar et al., 2003). SUVA254 was calculated as decadal absorption (A254/L) divided by DOC concentration, giving units of L mg C-1 m-1. Values for natural waters typically range between 1-5 L mg C-1 m-1 (Spencer et al., 2012); with higher values indicating influence of dissolved iron (Poulin et al., 2014). Spectral slope coefficients from 275 to 295 and from 350 to 400 nm were fit on natural logtransformed absorption spectra. The ratio of these slopes, SR, was used as index of molecular weight of DOM and its source (terrestrial vs. marine) (Helms et al., 2008). SR values of <1.0 are common for rivers and other sources of terrestrial DOM while values >2.0 are common for seawater. Values between 1.0 and 2.0 are common in estuaries.

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Excitation-emission matrix (EEM) fluorescence was measured on GF/F filtrates using a Varian Eclipse spectrofluorometer. Excitation (Ex) wavelengths were sampled from 240 to 450 nm at 5 nm intervals and emission (Em) wavelengths were sampled from 300 to 600 nm at 2 nm intervals. Ultrapure laboratory water was used as a blank. Corrections were made for excitation lamp and emission detector responses, as well as for inner filtering effect, which required dilution of sample with ultrapure laboratory water. Final fluorescence values were rescaled for dilution as necessary. The biological index (BIX) was calculated using the ratio of emission intensities at 380 nm and 430 nm at an excitation of 310 nm (Huguet et al., 2009). BIX values >1.0 correspond to freshly produced DOM of algal or bacterial origin, whereas values <0.6 indicate little freshly produced material. Humification generally increases the aromaticity of DOM, and this was estimated with a humification index, HIX, calculated from the ratio of two integrated emission wavebands: 435–480 nm to 300–345 nm, at 255 nm (Zsolnay et al., 1999). HIX values <10 correspond to relatively non-humified DOM and generally decrease as BIX increases (Birdwell and Engel, 2010). Optical data processing was performed using in-house codes written in Matlab (Mathworks, Inc.).

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2.5. Bacterial production and mineralization

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Bacterial production (BP) of the heterotrophic bacterial assemblage was measured by the leucine incorporation microcentrifugation method of (Smith and Azam, 1992) for water (1.0 mL) as adapted by (Montgomery et al., 2010). Unfiltered aliquots of surface water samples from each station were added to 2.0 mL microcentrifuge tubes (three experimental and one killed control) pre-charged with [3H-4,5]-Lleucine (120 mCi mmoL-1, final concentration = 20 nM; American Radiochemical Corporation, St. Louis, MO). All samples were incubated at in situ temperature for 30 min. Incubations were ended by adding 57 µL of 100 % trichloroacetic acid (5 % final concentration; TCA, Fisher Scientific) and frozen for storage prior to processing (Smith and Azam, 1992). A constant isotope dilution factor of two was used for all samples and was estimated from sediment dissolved free amino acids measurements (Burdige and Martens, 1990) and saturation experiments (Tuominen, 1995). Leucine incorporation rate was then converted to bacterial carbon (Simon and Azam, 1989). Assay detection limit was 1.0 µg C kg-1 d-1, though average values that were below one standard deviation were considered below detect (0, BD).

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Rates of bacterial aromatic metabolism was measured by mineralization of 14C-phenanthrene to 14CO2 (Pmin) using a modification of Boyd et al. (1996) and Pohlman et al. (2002). Radiolabeled 9-14Cphenanthrene (55.7 mCi mmol-1, American Radiochemical Corporation, St. Louis, MO) was added to 100 x 16 mm polycarbonate test tubes (ca. 0.04 µg mL-1, depending on specific activity) prior to the addition of water samples (5 mL; triplicate live and one kill). Unfiltered whole water samples were incubated for ca. 48 hr at in situ temperature in the dark and evolved 14CO2 captured on NaOH-soaked filter papers. H2SO4 (2 mL, 2 N; Fisher Scientific) was added to end live incubations (or at T0 for kills) to partition any remaining CO2 into headspace of the tube and to the filter paper trap. Filter paper traps containing metabolized 14CO2 were removed, radioassayed using Ecolite+, MP Biomedicals, Solon, OH, on a Beckman LS6500 Scintillation Counter and subsequently used to calculate substrate mineralization using the specific activity of the isotope. Detection limit of the assay was typically 0.01 µg CO2 L-1 d-1 (abbreviated µg C L-1 d-1) though average values that were below one standard deviation were considered below detect (0, BD).

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2.6. Statistics

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Results often were not normally-distributed or had unequal variances; hence, non-parametric tests Kruskal-Wallis tests were conducted to determine significance of differences DOM properties and microbial activity for the three samplings. Bonferroni corrections were applied and Dunn’s post-test allowed for comparing differences in the mean values of DOM properties and microbial activity metrics between specific samplings, based on different amounts of AP preceding each sampling.

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Principle components analysis (PCA) was conducted on DOM and bacterial activity variables from a correlation matrix. In Euclidean PC1-PC2 space, convex hulls quantify the areal distribution of scores as a means to describe the homogeneity among groups. Location of the convex hull is related to loadings of the variables provides an indication of the variables most strongly influencing the group of sample scores. Convex hull areas were calculated for each group (based on sampling and related to AP) to estimate its homogeneity within each group, with smaller hull areas indicating more homogenous samples. We used this approach to hypothesize that DOM and bacterial response would be more homogenous under higher amounts of AP.

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3. Results

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3.1. Antecedent precipitation and hydrography during each sampling

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AP measured in the 7 d prior to each sampling was different for each sampling event, with August of 2014 and 2015 being higher than November 2014 (Table 1). Most rainfall occurred in August 2014 and of the 200.4 mm of total precipitation that fell during that time, 54% (109 mm) fell on August 1, 2014; this amount was >50% of the 10-year average monthly total precipitation for August. The headwaters in Newport, NC recorded 158 mm total precipitation while mid-estuary across from Morehead City at Core Creek similarly recorded 154 mm.

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August 2015 received about half the total rainfall of August 2014, again about 50% on one day (04 AUG 2015). August 2014 only had 2 days without precipitation (8 and 9 d prior to sampling), preceded by 6 mm (day -10) and 119 mm on 10 and 11 days prior to sampling. August 2015 had 117 mm of precipitation spread out over 8 to 14 d prior to that sampling event. Core Creek precipitation was similar at 91 mm over the seven days preceding the sampling whereas slightly lower totals of 37 mm were recorded at the headwaters in Newport, NC.

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In contrast, the November 2014 sampling were preceded by a very dry period of 8 d without precipitation and only 12 mm over the previous 25 d. November 2014 had the lowest AP prior to sampling: 34 mm, about 16% of the AP for the August 2014 sampling and about 30% the AP for the August 2015 sampling. Likewise, the least amount of total precipitation was during the seven days preceding the November 2014 sampling in both Newport (21 mm) and Core Creek (37 mm). We illustrate AP in detail to demonstrate the variability in the gradient of AP over which we examined DOM’s properties and its microbial reactivity. For context, there were five rain events similar to that of August 2014 (>150 mm within a week) during the two years of these samplings; two in 2014 and three in 2015.

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Surface water temperatures in the estuary were primarily influenced by time of year during which each sampling occurred (Table 1). Warmer temperatures (26 – 28 ° C) occurred in August, while cooler temperatures (10 – 13 °C) occurred in November. Salinity ranged from 0 at the tidal freshwater endmember of the estuary near Newport, NC, to 35.92, full strength seawater collected outside Beaufort Inlet in Onslow Bay. During Aug 2014, Onslow Bay was markedly fresher (S = 30) as a result of outflow of the Newport River occurring after the large AP (Table 1). Sea-state prohibited sampling outside of Beaufort Inlet in Nov 2014. In Aug 2015, a full salinity gradient from freshwater to seawater was sampled, which included salinity values >30 outside Beaufort Inlet on the shelf waters of Onslow Bay.

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3.2. DOM trends across the estuarine gradient

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DOC concentrations decreased approximately linearly with salinity during each sampling event (Fig. 2A). The overall regression was significant (DOC = -0.83×Salinity+28.63; r2 = 0.86; p < 0.001; N = 29). Zero salinity DOC values were highest in Aug 2014 (36.89 mg L-1) followed by Aug 2015 (27.38 mg L-1) and Nov 2014 (20.58 mg L-1). Highest DOC concentrations were consistent with previous values presented for the river at and above head of tide (Ensign et al., 2012). CDOM concentrations, measured as a254 values, also decreased with increasing salinity, with very consistent trends for Nov 2014 and Aug 2015, both of which were lower than low salinity (<10) a254 values for Aug 2014 (Fig. 2B). Aug 2014 has a cluster of observations that were higher in DOC and in CDOM in the oligohaline region of the estuary.

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DOC and a254 were highly correlated (r = 0.98; p < 0.001; N = 29). Aromatic DOC was estimated by SUVA254 values, which also decreased with increasing salinity (Fig. 2C). However, it was clear that the higher AP in Aug 2014 resulted in a flush of more aromatic DOC as high SUVA254 values >4.0 L mg C-1 m-1

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were maintained across the salinity gradient, even to salinity of 30. Nov 2014 SUVA254 values were also rather high ranging between 3.0 and 5.0 L mg C-1 m-1. By contrast, SUVA254 values in Aug 2015 declined from ca. 5 to 1.6 L mg C-1 m-1 as salinity increased and were comparatively lower than in 2014 at mid salinities.

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Consistent with SUVA254 values, δ13C-DOC values indicated mostly terrestrial sources in the estuary, with the most depleted values (ca. -28.5‰) at lower salinities and most enriched values at higher salinities (e.g., -23 to -22‰). Aug 2014 and Nov 2014 exhibited very narrow ranges in these values throughout the estuary, from -28.6 to -26.1‰. In somewhat stark contrast, δ13C-DOC values in Aug 2015 ranged from -28 to -25‰ across most stations <30, gradually increasing with salinity, but then increased dramatically at salinity >30. At these higher salinities in Aug 2015, δ13C-DOC values were consistent with a marine origin: -23.7 to -22.4‰ (Fig. 2C).

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Other optical indicators of DOM quality followed the aforementioned trends of DOM with salinity (Table 1). Spectral slope ratio (SR) values increased with salinity and ranged from a low of 0.704 at zero salinity in Aug 2014 to a high of 2.989 at S = 35.92 in Aug 2015. BIX values indicative of recent fresh autochthonous DOM were about 0.4 in the freshwaters of the Newport River and increased to values ranging from 0.6 to 0.9 in coastal waters at salinities >30. HIX values trended opposite BIX values, decreasing along the salinity gradient. Highest values of HIX were found in freshwaters and ranged from 10-25. At salinities >30, HIX was generally <5. These results are generally supportive of the SUVA254 and δ13C-DOC results which suggested that DOM in the NPRE was strongly dominated by terrestrial sources.

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3.3. Trends in DOM properties with increasing AP

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AP was different for each sampling trip and provided a gradient over which to examine DOM properties in NPRE linked to rainfall. No significant difference in DOC concentration (P = 0.388) or CDOM abundance (P = 0.258) was found among sampling trips, which was expected given the extent of the estuary sampled during each event. However, it was notable that median DOC values increased with increasing AP and that the lowest AP (34 mm in Nov 2014) had a median DOC value just under 40% of that in Aug 2014 and 52% of that in Aug 2015 (Fig. 3A). By contrast, median a254 values during the highest AP (200 mm in Aug 2014) was nearly twice as much as in Nov 2014 or Aug 2015, when AP was lower (34 and 101 mm, respectively) (Fig. 3B). Thus, higher AP clearly mobilized into the estuary a substantial amount of DOC that had very strong CDOM absorption at 254 nm.

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Higher AP in Aug 2014 also resulted in a larger amount of aromatic carbon compared to lower AP in Nov 2014 and Aug 2015, as evidenced by SUVA254 values (Fig. 3C). Highest SUVA254 values (4.80±0.59 L mg C-1 m-1) occurred during Aug 2014; though, Aug 2015 had lower values (3.18±1.25 L mg C-1 m-1) than did Nov 2014 (4.38±0.97 L mg C-1 m-1). SR values, which are inversely correlated with increasing molecular weight, supported the SUVA254 results and suggested that molecular weight was highest in Aug 2014 under the highest amount of AP (Fig. 3D). SR mean (±SD) values were 0.79±0.09, 0.89±0.10, and 1.19±0.73 for the three months, respectively.

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The preceding optical measurements of DOM quality were consistent with stable C isotope values of the bulk DOM pool. Results from δ13C-DOC measurements during each sampling event were consistent with terrestrial sources of DOM dominating in the Newport River Estuary (Fig. 3E). Values for δ13C-DOC ranged from a minimum of -28.56‰ for Aug 2014 to a maximum of -22.64‰ for Aug 2015, covering nearly the entire range of stable carbon isotope values for DOM along river to ocean transects

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(Raymond and Bauer, 2001). Although the minimum value of δ13C-DOC for Aug 2015, -26.32‰ was roughly 2‰ enriched relative to Aug and Nov 2014, this value remains in the range for terrestrial DOC in rivers.

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Non-parametric MANOVA test on SUVA254 and δ13C-DOC values showed that despite the dramatic difference in AP, Aug and Nov 2014 had qualitatively similar DOM (P = 0.607). In contrast, DOM in the estuary in Aug 2015 was significantly different from DOM in Aug and Nov 2014 (P = 0.011 and P = 0.030, respectively. A notable difference in DOM quality was observed in SR values for Aug 2015, which were higher than for Nov 2014, when AP was lower. However, the range of δ13C-DOC values, reaching -22‰, suggested that Aug 2015 sampling incorporated more observations of the seawater end member, despite having higher AP than Nov 2014.

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Fluorescence biological (BIX) and humification (HIX) indices confirm qualitative differences among the sampling events but also demonstrate the overlap due to estuarine mixing (Fig. 3F). Aug 2014 has the highest HIX values and the lowest BIX values, while just the opposite was observed for Aug 2015. Nov 2014 BIX and HIX values fell in between the freshwater end member for the Newport River and the marine end members of Onslow Bay.

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3.4. Trends in bacterial production and aromatic mineralization

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BP and Pmin showed opposite patterns with increasing salinity (Fig. 2). Across the estuarine gradient for each sampling event, BP decreased with salinity and, hence, also with DOC and a254 (Fig. 2A). However, Pmin generally increased with salinity (Fig. 2B). Substantial variability among samplings indicated that both BP and Pmin responded differently to AP. Like the DOM concentrations, sufficient variability in values across the estuarine gradient during each sampling event meant that no significant differences were determined, yet it was clear that higher AP were correlated with higher median values for BP (Fig. 4A) and higher median values of Pmin for Aug 2014, compared to Nov 14 and Aug 15 (Fig. 4B).

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Pairwise comparisons using a Bonferroni correction revealed that the mean rate of BP during Nov 2014 was significantly less (by about 10 µg C L-1 d-1) than during Aug 2014 (P = 0.026) or Aug 2015 (P = 0.026), the latter events each having much higher antecedent precipitation than the former event (Table 1). Temperature is known to strongly influence bacterial production (e.g., White et al., 1991) though this can be more significant at lower temperature (<16oC; Šolić et al., 2017) as was seen in the Nov 2014 sampling. However, there did not seem to be a strong temperature dependence on rates of bacterial production measured for the combined data sets in this study (Figure S1).

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The Kruskal-Wallis test was also ran on Pmin results and pairwise comparisons (with Bonferroni correction) likewise revealed that the Aug 2014 event resulted in higher rates of Pmin in the NRPE (also about 10 µg C L-1 d-1) than Nov 2014 (P < 0.001). However, a key difference was that Aug 2014 also was significantly greater (14 µg C L-1 d-1) than Aug 2015 (P = 0.006). Nov 2014 and Aug 2015 were not significantly different with respect to Pmin despite Aug 2014 having higher BP in the estuary than Nov 2014.

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3.5. PCA results show relatedness between AP, DOM quality, and microbial activity

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Ordination of DOM and bacterial activity results revealed strong correlations between DOM variables and microbial activity (Fig. 5A). PC1 captured most of the variance in the data set (78%), while PC2 capture 22%. Positive correlations with PC1 were found for AP as well as SUVA254 and HIX indicating this

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axis represented more humified, aromatic DOC, which was present in the Newport River Estuary as a consequence of higher runoff flushing terrestrial DOM from the watershed into the estuary. Both BP and Pmin were positively correlated with PC1. While δ13C-DOC values were negatively correlated on PC1, this result was expected because the more negative (13C depleted) values indicate terrestrial sources of organic matter. SR was negative on PC1 due to the lower values of slope ratio observed in terrestrial DOM sources. Negative correlation of salinity and BIX with PC1 were sensible in light of mixing between river water and seawater, the latter having a higher BIX value than freshwaters flowing into the Newport River Estuary.

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Positive correlations were found between PC2 and salinity, as well as SR, and δ13C-DOC, while negative correlation was found between PC2 and SUVA254 (Fig. 5B). This result suggested these DOM properties trended toward a seawater source that typically has lower SUVA254, higher SR, and enriched δ13C-DOC values (Helms et al. 2008; Bouillon et al. 2011). Interestingly, AP, BP, and Pmin also were positively correlated to PC2. Thus, PC2 appeared to represent dilution of terrestrial DOM with seawater DOM upon mixing.

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Convex hull areas showed a difference in the homogeneity within each group, with a larger convex hull area indicating the more widely dispersed the DOM quality and reactivity as defined by our component loadings. Conversely, smaller convex areas indicate a more homogenous quality. The convex area for Aug 2015 was 7.16, larger than the areas for Aug 2014 (2.76) or Nov 2014 (2.94; Fig. 5C). This result underscored the more similar DOM quality between Aug and Nov 2014 compared to Aug 2015, despite the differences in AP.

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4.1. High levels of AP increase transfer of aromatic C into estuaries

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A major finding of this study was that heavy precipitation events in a small, coastal watershed correlated with not only the flux but also the metabolism of terrestrial DOC in its downstream estuary. Small estuaries such as the Newport, much like small inland rivers, are strongly affected by local precipitation in terms of hydrology and, from our results, also in terms of lateral C fluxes. Precipitation need not be from a tropical storm; August 2014 sampling was not associated with such an extreme weather event but did result from substantial AP (200 mm). This amount was 13% of the 30-year mean annual rainfall at Morehead City, NC, and 5% greater than the 30-year mean monthly rainfall for August (U.S. Climate Data, 2018). The significant increases in freshwater DOC concentrations were found after the highest AP, which indicated that DOC loads to the Newport Estuary also were higher, though the effect appeared to be maximized in the oligohaline region of the estuary (salinities <10). It is expected that inundation of forested Cypress wetlands adjacent to the river was the source of significant amounts of aromatic, terrestrial DOM flushed into the estuary proper, as evidenced by the SUVA254 values in Aug of 2014 and 2015 (Table 2 and Fig. 2). Stable C isotope and fluorescence-based HIX values confirm the quality of DOM was complex, aromatic-rich material consistent with that accumulating in forested swamps in the lower Atlantic Coastal Plain (Hackney et al., 2007).

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A key question in coastal carbon cycling is the manner to which DOM quality is modulated by linkages across TAIs, especially those caused by major storm events (Ward et al. 2017; Raymond et al. 2016). Using fluorescence, Osburn et al. (2012) identified distinct changes in DOM and POM quality in the Neuse River Estuary (just north of the Newport) resulting from Hurricane Irene in August 2011 that

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exemplified a shift towards terrestrial dominance of both carbon pools. In the much larger Congo River watershed, the spring flush period brought on by rainfall resulted in higher loads of CDOM and the terrestrial biomarker, lignin (Spencer et al., 2010). However, like in the Neuse Estuary, CDOM properties suggested substantial DOC photodegradation downstream in the Congo (Lambert et al., 2016). This effect has been notable in the Neuse River Estuary and Pamlico Sound over the past 20 years and the implication is that extreme precipitation events can result in shifting the mode of estuaries into CO2 sources to the atmosphere (Paerl et al., 2018). Microbial degradation of terrestrial DOM was implicated; however, although a roughly 3-fold increase in DOC was modeled in the Neuse Estuary after Irene, CDOM properties indicated lower molecular weight DOM as the flood pulse migrated down estuary, likely resulting from its substantial photodegradation after the flood (Miller et al., 2016). We find the same results here, albeit on the smaller Newport River Estuary, wherein the effects of precipitation on DOM loading and its reactivity (discussed below) were apparent.

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Convex hull area for Aug 2014 was comparable to Nov 2014 yet less than the area for Aug 2015, indicating DOM was least homogeneous for the latter sampling event (Fig. 5C). Convex hull areas also demonstrated how the Aug 2015 sampling incorporated more coastal water from Onslow Bay. Negative PC1 scores and positive PC2 scores for Aug 2015 were highly correlated with corresponding loadings for salinity, δ13C-DOC, and SR. This meant that as salinity increased (i.e., more offshore water), δ13C-DOC values increased reflecting a shift towards marine DOM sources and away from the depleted bog water source that strongly influenced the Aug 2014 sampling. Likewise, increases in SR also reflected a shift from high molecular weight, terrestrial DOM to low molecular weight, marine DOM (Helms et al., 2008).

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We observed nearly 30-fold decreases in DOC concentration and 300-fold decreases in CDOM concentrations along the salinity gradient of the estuary (Figs. 2A-B). Given the proportionally higher amounts of CDOM, high SUVA254, and depleted δ13C-DOC values, it appears these changes may be due to loss of terrestrial DOM from the Newport River’s watershed into its estuary. A four-fold decrease in SUVA254 between the highest values in the freshwater endmember during Aug 2014 (near 5.5 L mg C-1 m1 ) and the lowest value (1.37 L mg C-1 m-1) from Onslow Bay sampled in Aug 2015 suggests that most of the DOC in the estuary was aromatic. At a salinity of 18, the mid-salinity point of the freshwaterseawater mixing in the estuary, this translated into about 20 mg L-1 DOC.

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4.2. AP and aromatic degrading bacteria into estuaries

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Precipitation is known as a major dispersant of pathogenic bacteria into the adjacent estuary and coastal waters of Core Sound, as well as, exchange with Onslow Bay via the Beaufort Inlet (Coulliette and Noble, 2008). Roughly 10 years prior to the present study, work on the Newport River Estuary indicated a substantial role in “wet weather” events in the distribution fecal coliforms in shellfish harvesting areas, such that both space-time and Bayesian (probabilistic) models were developed to link weather factors to water quality decision making (Coulliette et al., 2009). It is possible that resident bacterial communities capable of aromatic DOC degradation also were dispersed into the Newport River Estuary under the larger precipitation events. This supposition could explain why median BP values were very similar during August, yet Pmin was nearly 3x higher in Aug 2014 than in Aug 2015, coincident with double the precipitation between these sampling periods (Fig. 4). Dispersal of aromatic-degrading bacterial populations from the watershed into the estuary may have “seeded” the estuary with a microbial capacity to degrade aromatic carbon.

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On the other hand, median SUVA254 values suggested that, while DOC in Aug 2015 was less aromatic than in Aug 2014, it still was terrestrial (Fig. 3A). Moreover, δ13C-DOC values in Aug 2015 were nearly 3‰ enriched relative to median δ13C-DOC values in Aug 2014 (Fig. 3E). A plot of δ13C-DOC values vs. SUVA254 values revealed two trends (Fig. 6A); a concave pattern characterized both Aug 2014 and 2015 while a convex pattern was apparent in Nov 2014. The concavity could be explained by the August DOM samples containing a larger amount of DOM derived from C4 vegetation (e.g., Spartina) from adjacent salt marshes along the tidal portion of the river mixed with C3 vegetation in terrestrial DOM sourced from swamp forests (Bouillon et al., 2011). High AP during this time may also have increased runoff from these adjacent salt marshes. The narrower range in δ13C-DOC values in Nov 2014 indicate a dominant terrestrial influence in the estuary from the swamp forests, even under lower AP. Marine DOC values at higher salinities (>34) suggested that Aug 2015 simply had a higher degree of mixing of these multiple sources. The overlap of these source contributions explains the relatively consistent δ13C-DOC values along the oligo- to meso-haline regions of the estuary (0 – 25 salinity) (Fig. 2C).

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Likewise, heavy precipitation events caused major impacts for estuarine biogeochemistry in terms of accelerating the degradation of terrestrial C in coastal waters. In part, this could be due to nutrients being flushed into estuaries from watersheds along with DOM. Bacterial growth efficiencies (BGE) are typically highest in estuaries, nearly double that in rivers and oceans (del Giorgio and Cole, 1998). The supposition from this paradigm is that estuaries and coastal waters receiving large inputs of terrestrial organic matter are sites of intense carbon cycling and processing (Apple and del Giorgio, 2007). BP decreased as a function of salinity and increased as a function of SUVA254 value, supporting prior results in a tropical estuary that terrestrial DOC promotes BP after recent precipitation (Montgomery et al. 2013).

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Our BP values were consistent with measurements from other estuarine ecosystems over a range of sizes. Median BP for the Newport River Estuary was 40 µg C L-1 d-1, whereas BP was 14-179 µg C L-1 d-1 in Mobile Bay, the second largest estuary in the northern Gulf of Mexico, and a much larger system than the Newport River Estuary (McManus et al., 2004). In the Neuse River Estuary, BP had a median value of 60 µg C L-1 d-1, with a high value of 484 µg C L-1 d-1 (Peierls and Paerl, 2010). While temperature was a driving factor in BP results for the Neuse River Estuary, the authors did find that DOC was strongly correlated to temperature-corrected BP. Their results suggest that temperature was a second order effect on DOC degradation, which supports our finding that BP did not vary significantly with temperature (Fig. S1). An important outcome of that study also was that the correlations were stronger at freshwater stations than at brackish water stations. Our BP results were consistent with these trends. Aug 2014 and 2015 both had similar temperatures which were about 10 °C warmer than temperatures in Nov 2014. The mean BP were not significantly different in Aug between years, but BP results were significantly different between Aug and Nov (p = 0.016) (Table 1).

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The fact that rates of Pmin increased with salinity while rates of BP decreased with salinity suggested that local drivers other than carbon quality could be at play in terms of providing an optimized environment for aromatic DOC to be remineralized in this small estuary. While labile carbon substrates such as sugars and amino acids support rapid production (Kirchman, 2003), experimental evidence has shown that water soluble petroleum products also correlated with increased BP over a 5-day period while a control mesocosm saw a decline in BP after two days (Koshikawa et al., 2007). The increase in BP as a result of oil-enrichment was consistent with the increase in BP with SUVA254 that we determined for the Newport River Estuary (Fig. 5A) but counterintuitive to a decrease in Pmin with increasing SUVA254.

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One possibility is that high SUVA254 values are coincident with enough carbon sufficient for the higher growth rates measured by BP, which occurred under comparatively large (>10 mg L-1) DOC concentrations. The DOC was not entirely aromatic carbon; up to 10% of the DOC in freshwater environments is itself bioavailable substrates such as low molecular weight carbohydrates and amino acids (Benner, 2003). The mean DOC concentration for all three samplings was 16.57 mg L-1, thus roughly 1.5 mg L-1 could comprise substrates that support nearly all bacterial carbon demand (Kirchman 2003). In other words, given the very high DOC concentrations in the estuary, aromatic carbon likely was not needed to support bacteria until more readily available substrate concentrations (e.g., sugars, amino acids) became limiting, thus forcing bacteria to switch to other substrates.

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Nutrients (N, P) were not measured for this study, but prior work suggests they could differently affect BP and aromatic mineralization. N concentrations in the Newport River Estuary have been historically low and N:P ratios were <8, suggesting N limitation (Kirby-Smith and Costlow, 1989). Development in the watershed since that study may be changing water quality (Coulliette and Noble, 2008). Hence, increased nutrients in runoff coincident with increased DOM from forested wetlands and pocosins may be facilitating higher BP.

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After normalizing Pmin to BP to remove variations associated solely with heterotrophic growth rates, aromatic mineralization increased with salinity in a tropical estuary (Montgomery et al., 2013). That study noted a potential decrease in growth efficiency for a substrate along estuarine transects (c.f. del Giorgio et al., (1997)). Thus, demand for aromatic carbon may be increasing as DOC concentrations decrease, especially for bacterial communities adapted to aromatic C sources such as lignin and PAHs which can support heterotrophy in seawater (del Giorgio and Cole, 1998).

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Another possibility driving these results is that the residence time in the estuary increases with mixing of river water and seawater in the open region of the eastern and southeastern Newport River Estuary (Fig. 1). Here, wind-driven advective mixing is important, and we noted several front lines setting up and dispersing. In the Choptank River estuary, Bouvier and del Giorgio, (2002) found that phylogenetic succession was rather sharp and oriented around rapid transitions of salinity such as would occur at frontal boundaries. This effect may explain the dramatic decrease in BP in Nov 2014 during comparatively lower AP in the Newport River’s watershed. Likewise, this could explain the increase in Pmin/BP which occurred at salinities >27 during the higher AP events in Aug of 2014 and 2015.

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4.3. Aromatic DOM supports microbial metabolism down estuary

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Aromatic characteristics of OM decreased with migration down the Newport River Estuary to Beaufort Inlet during all three sampling events as is typical in estuaries (Helms et al., 2008). However, this decrease in SUVA254 values also coincided with an increase in phenanthrene mineralization rate by the heterotrophic natural assemblage. The steepness of the change with salinity in SUVA254 versus that for Pmin appeared to increase with higher level of precipitation prior to the sampling event (Fig. 6B).

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This was a remarkable outcome of our study in that the higher median rates of BP and Pmin that occurred when antecedent precipitation was high provided strong evidence that the DOC in terrestrial runoff was more bioreactive than at conditions closer to base flow. High correlations of terrestrial DOM qualitative variables with PC1 indicated this axis represented the strength of the terrestrial influence of DOM in the Newport River Estuary. Most notable of these results were that the highest scores on PC1 occurred in Aug 2014 which was preceded by the largest amount of precipitation (Table 1).

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Effects on BP were consistent with findings from similarly small headwater forested catchment in New England that showed increases in bioavailable DOC (“BDOC”) following storm events (Wilson et al., 2016) as well as findings of increased bioavailability in the much larger Cape Fear River after hurricanes (Avery et al., 2004). However, the phenanthrene results were somewhat surprising because they suggest depressed aromatic bioreactivity. We hypothesize that our results are indicative of a subtle difference in relating distinct measurements of DOM quality (e.g., SUVA254, fluorescence properties) to rates of its reactivity (e.g., phenanthrene mineralization). BP results are consistent with these prior studies in that bacterial consumption and protein synthesis are increased by a larger supply of terrestrial DOC. However, phenanthrene results showed that aromatic C is not immediately utilized until transported further down-estuary. One explanation is that increased light penetration in more marine waters promotes combined photochemical and bacterial degradation as observed in experimental treatments (Moran et al., 2000).

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Indeed, Pearl et al. (2018) implied that extreme precipitation events can result in shifting the mode of estuaries into CO2 sources to the atmosphere after passage of these events. Microbial degradation of terrestrial DOM in Pamlico Sound, transported via the Neuse River Estuary, was posited as the key driver of this process. Increased rates of Pmin measured for the Newport under estuarine mixing, as SUVA254 and HIX values decreased and δ13C-DOC values increased, support this supposition. In the Newport River Estuary, rates of community respiration during August were 285 µg C L-1 d-1 and during November were 199 µg C L-1 d-1 (Kenney et al., 1988). The majority of this respiration is likely bacteria (Robinson, 2008). Our Pmin rates are <0.5% of these bulk rates but emphasize the importance of individual compounds. Results from the present study thus provide direct evidence of enhanced microbial mineralization of terrestrial DOC in estuaries.

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4.4. Pulsing and shunting of terrestrial DOC in watersheds leads to its consumption in estuaries

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Our results were consistent with both the pulse shunt and flood pulse concepts that have shaped fluvial biogeochemistry over the past 30 years and are only now beginning to provide a framework to understand the effects of large storm events on estuaries and coastal waters (Paerl et al. 2018). The Pulse-Shunt Concept fits our study surprisingly well which may be a function of the smaller size of the Newport compared to larger systems with multiple riverine inputs and/or extensive tidal wetlands. Pulses of DOM were mobilized during large rain events and were extended well down the salinity gradient, as evidenced by SUVA254 values (Fig. 2D). Indeed, δ13C values of DOC remained depleted at higher salinities under the largest precipitation event in our study (Aug 2014). Combining these two parameters provide a clear gradient to identify the influence of adjacent wetlands on DOM shunted in to the Newport River Estuary (Fig. 6A).

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Finally, we make some rough estimates of steady-state aromatic DOC mineralization in the estuary. Mean removal rate of aromatic C was estimated from Pmin at 0.35 µg C L-1 d-1 (Table 2). Upscaling this value to the estuary equates to an annual CO2 release rate of 0.13 g C m-2 yr-1. This value is <1% of CO2 release rates recently summarized for estuaries in the SE United States (Najjar et al., 2018), but likely represents an underestimate of actual terrestrial DOC mineralization. Nonetheless, estuaries are prime sources of CO2 degassing to the atmosphere, offsetting about 50% of the CO2 uptake by tidal wetlands and shelf waters across the coastal zone (Najjar et al., 2018). Our results emphasize the role of extreme weather events in modulating these rates by supplying bioreactive terrestrial DOC to estuaries.

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5. Conclusions

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Extreme weather events are likely dramatically changing estuarine carbon cycling in ways we only just beginning to understand. Emerging concepts of hydrological connectivity of rivers to their floodplains and dynamics across multiple TAIs that represent the land-ocean continuum provide a context for understanding organic matter transport and processing within climatic regions as these regions undergo climate change (Ward et al., 2017). Localized events such as thunderstorms are episodic, but have important effects on regional carbon cycling via increased connectivity of wetland, fluvial, and estuarine systems. These connections likely alter estuarine and coastal carbon cycling, shifting ecosystem states between net autotrophy and net heterotrophy (Crosswell et al., 2014; Paerl et al., 2018).

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Extending the Pulse Shunt Concept to estuarine waters, we discovered that antecedent precipitation was an important driver for mobilizing bioreactive DOM from forested wetlands in the watershed of the Newport River Estuary. A heavy rain event enhanced mean rates of bacterial production and aromatic carbon mineralization in the estuary by 20% and 76%, respectively, above relatively lower periods of antecedent precipitation. These rates are comparable to losses of DOC in larger estuarine systems such as the Hudson River (del Giorgio and Pace, 2008). Optical and stable carbon isotope indicators of DOM quality revealed that relatively fresh terrestrial DOC, possibly originating in forested wetlands in the tidal freshwater reach of the Newport River, promoted these higher rates of bacterial processing of terrestrial DOC in its estuary. Winds and tides complicated the estuarine responses by increasing advective mixing, even in microtidal systems such as the Newport, as seen in larger regional estuaries (e.g., Dixon et al., 2014).

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This study has shown that extreme precipitation events need not originate as tropical storms or hurricanes to have an episodic, weather-driven effect on the carbon cycle in estuaries. Precipitation events can have a dramatic impact on terrestrial organic matter inputs to estuarine systems but may also affect the utilization of aromatic organics by estuarine microbial assemblages, an intriguing research questions generated by this study which warrants further investigation. There might also be importance in the microbial processing of precipitation-driven inputs of anthropogenic compounds in coastal systems. Heterotrophic bacterial assemblages may respond to episodic elevated input by increasing metabolism of runoff-related aromatic contaminants (e.g., PAHs, pesticides) and thus decrease the negative impact of contaminants on local food chains and reduce the likelihood of export from the estuary (Montgomery et al., 2013, 2008).

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Quantification of C fluxes in coastal waters remains an active area of research maintained by greater uncertainties of C dynamics as the (sometimes not-so) subtleties are brought into focus via intensive study of carbon biogeochemistry such as we have done for the Newport River Estuary. Despite this relatively small size of this estuary, its ephemeral yet dramatic response in carbon loading and carbon processing indicates that response of small coastal systems to extreme weather events are of key importance yet remain poorly represented in time and space in coastal carbon models (Laruelle et al., 2017; Najjar et al., 2018).

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6. Acknowledgements

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The authors thank Hans Paerl, Ben Peierls, Karen Rossignol, for extensive site and laboratory support and valuable discussions at IMS UNC-Morehead City. Emily Barnett provided sampling support. Jami Montgomery helped with revisions of figures and text. This work was supported by the Strategic Environmental Research and Development Program ER-2124 (POC: Andrea Leeson). This manuscript was improved with the comments of two anonymous reviewers.

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Author’s Roles: All authors participated in field sampling including small boat operation, sample collecting and processing, and writing of the manuscript. MTM processed samples and analyzed data for BP and Pmin. JA processed samples for CDOM. CLO processed samples for DOC and δ13C-DOC, and analyzed data of OM character and quality. TJB oversaw field sampling and collection including boat operation.

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Keywords: Precipitation, Newport River Estuary, dissolved organic carbon, bacterial production, biodegradation, estuaries.

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Table 1. Antecedent precipitation (AP), temperature, salinity, and DOM properties of the NPRE during three sampling events. Blanks indicate missing values.

14-Nov

13.4 13 12.6 12.1 10.7 10.9 11 12.7 11.7 13.2 101.9 24.2 26.3 26.5 27.4 27.4 27.3 28 28 28.1 28.3 28.4 27.8 27.4

δ C-DOC BIX (‰)

HIX

0.01 0.7 2.5 11.5 21 27.5 29 30

36.89 30.27 32.23 29.76 8.48 8.21 2.46 2.51

451.31 367.54 353.43 328.91 98.54 67.58 27.41 26.42

5.31 5.27 4.76 4.8 5.05 3.57 4.83 4.57

0.7 0.72 0.76 0.77 0.79 0.85 0.95 0.88

-28.56 -28.56 -28.43 -28.5 -27.81 -28.11 -26.08 -26.38

0.39 0.41 0.39 0.4 0.45 0.5 0.56 0.58

25.76 22.32 20.33 19.33 16.47 14.33 11.72 11.69

0.11 1.02 5.59 10.21 18.32 20.25 24.73 25.64 27.43 27.67 0.07 2.76 4.9 7 10.99 12.77 17.3 21.22 23.84 26.59 31.09 33.6 35.92

20.58 21.13 n.d. 17.57 n.d. 8.56 6.09 3.83 4.18 4.28 27.38 26.09 26.55 21.55 15.98 17.85 13.97 11.43 9.19 7.3 3.91 3.39 0.72

269.98 259.08 211.55 166.91 100.2 86.24 55.69 28.31 34.33 28.78 308.31 268.19 200.6 196.23 166.93 147.66 112.94 82.71 63.94 44.65 14.33 10.68 2.66

5.7 5.32 n.d. 4.12 n.d. 4.37 3.97 3.21 3.57 2.92 4.89 4.46 3.28 3.95 4.54 3.59 3.51 3.14 3.02 2.65 1.59 1.37 1.61

0.79 0.81 0.83 0.85 0.88 0.88 0.9 1.03 0.97 1.04 0.74 0.8 0.82 0.79 0.86 0.85 0.9 0.88 0.91 0.97 1.13 2.25 2.99

-28.51 -28.16 n.d. -27.99 n.d. -28.05 -27.89 -27.14 -27.41 -27.54 -27.32 -25.44 -27.81 -27.58 -28.19 -27.29 -27.02 -27.2 -27.6 -23.58 -22.66 -22.75 -23.42

0.44 0.44 0.43 0.46 0.48 0.51 0.54 0.62 0.6 0.61 0.4 0.45 0.45 0.47 0.49 0.47 0.55 0.53 0.54 0.58 0.61 0.73 0.91

16.15 18.01 18.34 17.34 14.25 13.43 11.98 7.5 9.49 8.12 9.97 15.34 14.06 13.27 14.57 13.37 10.42 12.27 11.89 8.95 7.17 5.55 2.25

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SUVA254 -1 -1 S (L mg C m ) R

EP

15-Aug

34

DOC a254 -1 -1 (mg L ) (m )

SC

26.3 26.3

13

Salinity

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Water Sampling AP Temp (Year-Month) (mm) o ( C) 14-Aug 200.4 19.4 26.3 27.8 26.2 26.2

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Table 2. Bacterial production (BP) and phenanthrene mineralization (Pmin) during three sampling events in the Newport River Estuary.

200.4

14-Nov

34

15-Aug

101.9

Pmin -1 -1 (mg C L d ) AVG SD 0.22 0.05 0.45 0.04 0.4 0.18 0.66 0.21 0.55 0.27 0.83 0.5 0.72 0.15 n.d. n.d. 0.04 0 0.17 0.04 0.17 0.09 0.07 0.03 0.15 0.07 0.1 0.01 n.d. n.d. 0.17 0.07 0.21 0.02 n.d. n.d. 0.14 0.01 0.21 0.03 0.23 0.02 0.18 0.03 0.14 0.04 n.d. n.d. 0.1 0.01 0.16 0.28 n.d. n.d. 0.34 0.37 0.36 0.44 0.53 0.34 0.08 0.03

EP 843

AC C

842

Pmin/BP 0.011 0.007 0.005 0.011 0.011 0.024 0.033 n.d. 0 0.01 0.009 0.004 0.007 0.005 n.d. 0.01 0.011 n.d. 0.002 0.004 0.005 0.002 0.003 n.d. 0.002 0.003 n.d. 0.011 0.009 0.021 0.012

RI PT

14-Aug

BP -1 -1 Salinity (mg C L d ) AVG SD 0.01 20 2 0.7 65 2 2.5 81 9 11.5 59 5 21 51 4 27.5 34 2 29 22 1 30 21 0 0.11 159 13 1.02 17 1 5.59 19 1 10.21 18 0 18.32 21 2 20.25 20 1 24.73 n.d. n.d. 25.64 18 2 27.43 19 3 27.67 n.d. n.d. 0.07 72 3 2.76 48 6 4.9 47 3 7 72 2 10.99 42 4 12.77 47 3 17.3 41 2 21.22 47 3 23.84 44 1 26.59 31 1 31.09 40 2 33.6 26 1 35.92 7 1

SC

AP (mm)

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Figure Captions.

845 846 847

Figure 1. The Newport River Estuary with rain gauges (hollow circles) and sampling locations indicated and coded by sampling event: circles (Aug 2014, A14-salinity), squares (Nov 2014, N14-salinity), triangles (Aug 2015, A15-salinity) (Google maps and 2016 North Carolina Digital Orthoimagery).

848 849

Figure 2. A-D: Scatterplots of DOM characteristics vs. salinity; E-F: scatter plots of bacteria activity vs. salinity.

850 851 852

Figure 3: Boxplots for concentrations of A) DOC and B) CDOM absorption (a254). Boxplots for DOM quality parameters: C) SUVA254; D) SR; and E) δ13C-DOC. Panel F shows the relationship between BIX and HIX for the three samplings.

853

Figure 4: Boxplots for A) BP and B) Pmin for the three samplings.

854 855 856 857

Figure 5: Results of PCA for DOM characteristics and bacterial activity in the NPRE. A) Correlations between variables and PC1; B) correlations between variables and PC2; C) scores plot with convex hulls drawn for each sampling event. Convex hull areas were 2.76, 2.94, and 7.16 for Aug-14, Nov-14, and Aug-15, respectively.

858 859

Figure 6: A) Relationship between δ13C-DOC values and SUVA254 values; B) relationship between Pmin normalized by BP to SUVA254.

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N14-1, A15-2 A15-5

A14-2, A15-21 A15-23 N14-25,27.6 A14-0.7 A15-26 N14-5, A15-7 N14-18,-A15-17 N14-20 N14-24 N14-27.4 N14-10, A15-12

A15-36 A15-33 A14-21

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A15-31 A14-11

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AUG 2014 NOV 2014 AUG 2015 GAUGE

A14-29

2 km A14-30

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Figure 2. 

B

A 400 a254 (m-1)

30

20

10

300 200 100 0

0 0

10

20

-26

-28 -29 0

10

20

30

D

4 3 2 1

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Salinity 1.0 F

E Pmin (g C L-1 d-1)

EP

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40

5

40

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BP (g C L-1 d-1)

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Salinity

Salinity 180

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Aug 2014 Nov 2014 Aug 2015

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120 100

80 60 40

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Figure 3. 

A

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a254 (m )

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20

300 200

SC

100

0

0 Aug-14 Nov-14 Aug-15

6

4

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SR

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2.0

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Aug-14 Nov-14 Aug-15

Sampling date

Sampling date

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AC C

-24 -25 -26 -27

F

Aug-14 Nov-14 Aug-15

25 20 HIX

-1

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SUVA254 (L mg C m )

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-22

13

D

5

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 C-DOC (‰)

Sampling date

3.5

C

15 10 5

-28 -29

0 Aug-14 Nov-14 Aug-15 Sampling date

 

Aug-14 Nov-14 Aug-15

M AN U

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-23

 

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30 DOC (mg L )

 

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Figure 4.  1.0 B 0.8

60 40

0.4 0.2

20 0.0

0

Aug-14 Nov-14 Aug-15

 

AC C

EP

TE D

 

M AN U

Aug-14 Nov-14 Aug-15 Sampling date

 

SC

-1

-1

80

0.6

RI PT

-1

Pmin (g C L d )

160

-1

BP (g C L d )

A

Sampling date

 

Figure 5.      A   

SC

 

RI PT

ACCEPTED MANUSCRIPT

   

M AN U

   

B         

TE D

 

3

Aug-14 Nov-14 Aug-15

2

0

AC C

-1

-3 -6

-4

-2

0

PC1

 

C     

EP

1

-2

 

2

4

6

 

ACCEPTED MANUSCRIPT

Figure 6.  -20 Aug-14 Nov-14 Aug-15 -1

Pmin (g C L d )

-26

0.4

0.2

-28 -30

0.0 1

2

3

4

SUVA254 (L mg C-1 m-1)

 

6

AC C

EP

TE D

 

5

0

1

M AN U

0

 

0.6

-1

-24

SC

13

 C-DOC (‰)

-22

B

0.8

RI PT

A

2

3

4

SUVA254 (L mg C-1 m-1)

5

6