Shining light on priming in euphotic sediments: Nutrient enrichment stimulates export of stored

Estuarine sediments are important sites for the interception, processing and retention of organic matter, prior to its export to the coastal oceans. Stimulated microbial co-metabolism (priming) potentially increases export of refractory organic matter through increased production of hydrolytic enzymes. By using the microphytobenthos community to directly introduce a pulse of labile carbon into sediment, we traced a priming effect and assessed the decomposition and export of pre-existing organic matter. We show enhanced efflux of pre-existing carbon from intertidal sediments enriched with water column nutrients. Nutrient enrichment increased production of labile microphytobenthos-carbon which stimulated degradation of previously unavailable organic matter and led to increased liberation of "old" (6855 ± 120 years BP) refractory carbon as dissolved organic carbon. These enhanced DOC effluxes occurred at a scale that decreases estimates for global organic carbon burial in coastal systems and should be considered as an impact of eutrophication on estuarine carbon budgets.


Introduction
Estuaries, and particularly shallow photic estuarine sediments (<40 m) 1 , are hotspots for 35 organic matter (OM) processing, altering terrestrial OM received from rivers prior to its export to 36 the coastal ocean [2][3][4] . The extent of terrestrial OM processing that occurs along the estuarine 37 continuum largely determines whether estuaries function as carbon (C) sources or sinks 4 . The 38 priming effect (PE) describes the additional release of C from a refractory source of OM (pre-39 existing sediment OM in this study, or added refractory material in others) stimulated by addition 40 of a labile form of C. In terrestrial environments, increased C release from soils is usually 41 measured as evolution of additional CO 2 into a headspace from amended treatments (with labile 42 C added) when compared to non-amended controls 5 . Although PE has been well-described and 43 explored within soils 6 , PE has only recently gained recognition in aquatic systems. Within 44 aquatic sciences, PE has primarily been investigated as a potential pathway for additional OM 45 processing within settings where heterotrophy dominates (e.g., riverine dissolved OM, hyporheic 46 zone, deep sediment; Fig. 1) 7-10 and has not been consistently demonstrated to occur 11 . 47 Occurrence of PE is highly dependent on substrate composition, sediment structure, and/or 48 microbial community composition 11 . Studies examining priming within coastal benthos are 49 limited 8,12,13 , but have found positive PEs within their limited scope (i.e., vial incubations of 50 sediment slurries). 51 PE studies in aquatic environments have thus far relied on the evolution of 13 CO 2 from 52 dissolved inorganic carbon (DI 13 C) derived from either labile or refractory C sources (study 53 dependent) to quantify the relative contributions from microbial processing of the 13 C addition. A 54 number of approaches have been used in various environments in an attempt to identify PEs, i.e., 55 to demonstrate that microbial degradation of refractory terrestrial organic C has been stimulated 56 This is a non-peer reviewed EarthArXiv preprint of a manuscript submitted to Environmental Science and Technology 5 following the addition of labile C 10,14-16 . These approaches use additions of both refractory OM 57 and labile C to stimulate mineralization of added OM 13,17 or pre-existing sediment OM (Fig. 58 2A) 8,9,12,18 . Addition of unlabeled C (refractory or labile) into the sediment confounds 59 partitioning of export pathways by introducing new OM. Any exported C derived from this 60 newly added OM is indistinguishable from that derived from pre-existing sediment OM. In this 61 study, we used the in situ MPB community to inject a pulse of labile MPB 13 C into coastal 62 sediments (Fig. 2B). This approach was intended to preserve both the production (loading rate) 63 and composition (proportion of relative sugars) of priming additions produced daily by diatoms 64 within highly productive shallow coastal environments 12, 19 . This method preserves the microbial 65 community, as boundary layers and sediment structure are maintained during label addition with 66 minimal disturbance. This differs from all other PE studies, which have directly added single 67 labile and/or refractory compounds to homogenized sediment ( Fig. 2A) 8,9,12,13,20 .

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Some PE studies account for both dissolved inorganic C (DIC) and dissolved organic 69 carbon (DOC) pools when identifying additional stimulated breakdown and release of C 8,14-16 , 70 but it remains common to solely measure the evolution of DI 13 C and DIC 9,10,13,18 . This approach 71 works well for systems where heterotrophic evolution of DIC is the only or major pathway for C 72 loss. However, relying on DIC effluxes alone to identify PE becomes problematic in systems 73 where primary production during light exposure utilizes DIC at rates exceeding the evolution of 74 respired DIC (Fig. 1B). This scenario occurs in shallow coastal benthic sediments, where there is 75 considerable DIC demand by MPB during light periods, and can result in the re-capture and 76 recycling of previously respired carbon. Strong uptake of DIC in euphotic settings could 77 potentially be wrongly interpreted as a negative priming effect as labeled DIC is recycled and 78 reincorporated into biomass instead of being evolved as 13 CO 2 . This is especially the case in 79 This is a non-peer reviewed EarthArXiv preprint of a manuscript submitted to Environmental Science and Technology 6 systems that are DIC-limited or have elevated rates of primary productivity due to 80 eutrophication. We argue that recycling of DIC in euphotic situations can be partially offset by 81 refining the definition of priming to encompass all C remineralized from refractory OM (i.e., 82 including DOC effluxes from sediment OM). In productive systems, heterotrophic bacteria are 83 provided with rich algal-derived organic matter that can fuel breakdown of otherwise refractory 84 pre-existing sediment OM as DOC. Measuring PEs using the evolution of DOC in addition to 85 DIC from both labile and refractory OM sources will account for all substrates produced by the 86 microbial community during remineralization. We applied 13 C to MPB in situ to track the production of algal carbon that occurred during a 112 single tidal minimum within the intertidal setting in order to track production and processing of 113 MPB derived C. Application of stable isotope (SI) tracer material (99% NaH 13 CO 3 ) during a tidal 114 low allowed for incorporation across ~ 4 hours of 1549±140 µmol 13 C m -2 into sediment OC 115 followed by significant flushing of non-incorporated 13 C from the sediment during tidal 116 inundation of the site as confirmed by loss of 99.0% of the material in the label application based 117 on measured incorporation in the sediment within the initial cores 32 . Of the 13 C incorporated into 118 sediment OC, ~46% or 716 µmol 13 C is expected to be in the form of carbohydrates as 119 calculated from uptake rates for 13 C presented in Oakes, et al. 19 for mannose, fucose, rhamnose, 120 galactose, glucose, xylose, and OC. Bare sediment within two experimental plots (1 m 2 ) was 121 labeled with 99% NaH 13 CO 3 when sediments were first exposed at low tide, following the 122 method outlined in Oakes and Eyre 33 . Label applications were prepared using NaCl-amended

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The initial pulse of nutrients was added to incubation tanks and bags holding replacement water for sampling shortly prior to cores being randomly allocated to the two incubation tanks. An 148 additional pulse of NH 4 + was applied to the elevated treatment tank at the end of 1.5 d in an effort 149 to mimic the nutrient availability that occurs with regular inundation of tidal sediments. An  Flooding within the Richmond River occurs at regular intervals 27,28 and dating of basal core 297 organic matter just upstream from our study site showed an age of 5,312 -5,583 y BP 29 , which is 298 similar to the age of the effluxed DOC. Given the tendency for material composed of older Δ 14 C 299 to be less photo-reactive and bioavailable, and the refractory nature of the characterized compounds, the exported material is likely directly transported to the coastal shelf with minimal 301 reworking after hydrolysis by heterotrophic bacteria in the sediment.

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Is this priming?

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The high C:N ratio, small molecular size, and radiocarbon age of effluxed DOC provide 304 compelling evidence that PE occurred within the intertidal sediments in this study. Microbial 305 processing of MPB-C under elevated nutrient loads resulted in carbon released from breakdown 306 of older sediment OM via hydrolysis 30 that was largely exported via DOC effluxes (Fig.1B). The 307 combination of a labile pulse of C, enhanced by increased nutrient availability, stimulated 308 microbial degradation of older refractory OM, likely through increased bacterial production of 309 hydrolytic extracellular enzymes 31 . Although we did not measure enzyme activity, increased 310 breakdown of sediment OM was indicated by the old radiocarbon age and increased aromaticity 311 of the increased DOC effluxes produced in the elevated treatment ( Fig. 5A & B). The pulse of 312 labile MPB-C was strongly retained within sediment OM in both treatments across 3.5 d (Fig. 3), produced labile 13 C and subsequently recycled respired DI 13 C to support productivity.

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Respiration of older sediment OM provided increased DIC to support MPB productivity 318 (Fig. 3A) within a system that has been previously found to be DIC-limited 33 . Algal production 319 supported by recycled DIC was captured by oxygen fluxes and production to respiration 320 measurements, as increased bacterial respiration of OM increasingly offset initial productivity in 321 the elevated treatment (Supplemental Fig. 1). However, these dynamics are not supported by consideration of DIC fluxes alone, as the considerable primary productivity that occurred during 323 light periods offset the respired carbon that would have been exported in a less productive 324 system. A potential solution to this problem could be to include DOC exports from sediment 325 OM, a byproduct of remineralization that has not previously been considered in evaluation of 326 PEs (Fig. 3B & 4B). It is important to acknowledge that DOC exports can also consist of MPB highly productive benthic environment. 333 We posit that some of the difficulty identifying positive PEs in aquatic systems 11 may be 334 due to the examination of solely heterotrophic relationships during the processing of OM.

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Exclusion of any interactions with primary producers misses potential co-metabolism or 336 processes that occur in situ ( Fig. 1 bottom), including the recapture and recycling of the products 337 of PE (CO 2 /DIC) during high productivity. This is largely an artefact of PE studies having been 338 developed in soils 5,34 where remineralization is the sole process affecting the respiratory CO 2 339 evolution, primary producers (MPB) are absent, and CO 2 is easily monitored as a production 340 only function (Fig. 1 top). In aquatic systems, the evolution of CO 2 is likely to be at least 341 partially offset by primary productivity in many settings where priming is likely to occur (e.g.

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shallow benthic microbial communities, suspended estuarine microbial communities). Therefore  This is a non-peer reviewed EarthArXiv preprint of a manuscript submitted to Environmental Science and