Enhanced microbial nitrogen transformations in association with intertidal macrobiota

Microbial nitrogen processing in direct association with marine animals and seaweeds is poorly understood. Macrobiota supply a substrate for microbes to reside, and a source of excreted nitrogen and dissolved organic carbon (DOC). We tested the role of a mussel (Mytilus 15 californianus), a red alga (Prionitis sternbergii) and an inert substrate for microbial activity using enclosed chambers and enriched ammonium and nitrate. Chambers with seawater from the same environment served as a control. We found that mussels and Prionitis elevated ammonium oxidation and nitrate reduction two orders of magnitude over that of seawater, while the effect of simply an inert substrate had relatively little effect. Extrapolating to a square meter of shoreline, 20 microbial activity associated with mussels could oxidize 2.5 mmol of ammonium and reduce per 1.2 mmol of nitrate per day. A square meter of seaweed could produce even higher rates, at 135.2 and 320.5 mmol per day for nitrification and nitrate reduction, respectively. Seawater collected from the shore versus 2-5 km offshore showed no difference in ammonium oxidation or nitrate reduction. Microbial nitrogen metabolism associated with mussels did not change whether 25 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-198 Manuscript under review for journal Biogeosciences Discussion started: 17 May 2018 c © Author(s) 2018. CC BY 4.0 License.


Introduction 35
Anthropogenic doubling of the supply of biologically available nitrogen (Galloway et al., 2008) (Fowler et al., 2013) has increased the importance of understanding the multiple components of the nitrogen cycle.In marine ecosystems, microbial activity has been shown to be a key driver in the nitrogen cycle, and while phototrophs can dominate uptake in the water column (Flombaum et al., 2013), chemolithotrophs and chemoheterotrophs have also been 40 shown to be quantitatively significant to nitrogen cycling (Capone et al., 2008;Francis et al., 2007;Zehr and Ward, 2002).In coastal marine areas, the large biomass of macrofauna and macrophytes presents the opportunity for microbial taxa to form associations where microbes have habitat as well as a predictable nitrogen supply (Moulton et al., 2016).Many of these macrobiota are restricted in movement, making them reliable substrates for microbial 45 populations.Further, animals and plants create strong gradients in oxygen and inorganic and organic nutrients such that processes that vary over hundreds of meters or kilometers in the open ocean can change over scales of mm in proximity to an animal (de Goeij et al., 2013) or over a scale of meters relative to species aggregations (Clasen and Shurin, 2015).There are many quantitative estimates of microbial nitrogen fluxes, including ammonium oxidation 50 (nitrification), in seawater from disparate marine locales (Beman, J. Michael et al., 2011;Ward and Bouskill, 2011).Comparatively, there is little knowledge of the microbially-mediated nitrogen fluxes associated with nearshore species, including whether the presence of animal and plant hosts enhance the diversity and/or intensity of microbial functions.With the harvest and loss of many marine species (Maranger et al., 2008;Worm et al., 2006), the importance of 55 determining the biogeochemical role of microbes associated with macrobiota becomes more urgent.Here, we quantify microbial nitrogen processing in coastal and offshore water and in association with two key coastal species.Because dissolved organic matter is one of the microbial resources supplied by macrobiota in aquatic systems (Hansell and Carlson, 2015), we also manipulated dissolved organic carbon (DOC) to examine the effect of carbon availability on 60 microbial nitrogen processing.
Across diverse aquatic ecosystems, the metabolic activities of animals and plants can generate the environmental niches necessary for a variety of microbial metabolisms (Allgeier et al., 2014;Croll, 2005;Layman et al., 2011;Schindler et al., 2001;Subalusky et al., 2015;Vanni, 2002).In marine systems, there is an increasing appreciation that animals, primarily through their excreta, 65 contribute significantly to nitrogen supply (Moulton et al., 2016;Pather et al., 2014) which can help relieve nitrogen limitation of microbial carbon fixation.Microbial nitrogen processing, including nitrification and nitrate reduction, is enhanced in proximity to animals in marine systems (Heisterkamp et al., 2013;Pfister et al., 2014Pfister et al., , 2016a;;Stief, 2013;Welsh and Castadelli, 2004).These enhanced nitrogen metabolisms also contribute to nitrous oxide production 70 (Heisterkamp et al., 2010(Heisterkamp et al., , 2013)), as well as retention of nitrogen (Pfister et al., 2016a).The rocky shores of the northeast Pacific are characterized by high levels of dissolved inorganic nitrogen, both from upwelling and from ammonium excretion by animals., including mammals (Roman and McCarthy, 2010), birds (Wootton, 1991), and invertebrates (Aquilino et al., 2009;75 Bracken, 2004;Pfister et al., 2016a).Open ocean areas are characterized by relatively consistent gradients in oxygen that generate predictable areas of oxidizing and reducing processes that might be limited by nutrient concentrations (Bristow et al., 2017).In contrast, high nutrients and the photosynthetic and respiratory processes of coastal biota can drive wide fluctuations in oxygen, possibly leading to both oxidizing and reducing microbial metabolisms in the same 80 location over a diel cycle (e.g,(de Goeij et al., 2013;Pfister et al., 2016b).
A further effect of macrobiota beyond nitrogen regeneration and the production of oxygen gradients is the production of dissolved organic matter.In addition to ammonium excretion and dissolved organic nitrogen (DON) production by macrobiota, macroalgae also produce DOC, likely enhancing select microbial metabolisms.DOC and DON can support different types of 85 microbial metabolisms in aquatic ecosystems, resulting in divergent outcomes for coastal productivity and nutrient cycling.DOC release by macroalgae may stimulate heterotrophic nitrate reduction, where microbes respire DOC with nitrate (NO 3 -) or nitrite (NO 2 -) as alternative electron acceptors.DOC can also stimulate the oxidation of NH 4 through heterotrophic nitrification.In addition to promoting microbial transformations between NH 4 and NO 2 /NO 3 , 90 enhancing the DOC supply can result in competition between different microbial metabolisms for DIN.Work in streams suggests heterotrophic bacteria may compete with chemolithotrophs for DIN (Butturini and Sabater, 2000), a result that may depend upon the ratio of C:N, where increasing DOC increases C:N and promotes nitrogen competition (Strauss and Lamberti, 2000).In sum, increasing the supply of DOC to marine microbes could have counteracting effects on 95 nitrification rates.While an increase in NH 4 oxidation would indicate stimulation of heterotrophic nitrifiers, a decrease in NH 4 oxidation rate would be consistent with increased competition for NH 4 with heterotrophic microbes.While the precise role of DOC in nitrogen metabolisms is likely varied and still not fully described, DOC contributes greatly to heterotrophy in microbes and fuels the quantitatively significant 'microbial loop' (Azam, 1998).100 The effects that macrobiota have on both nitrogen excretion and DOC release are poorly understood.We tested how the presence of the California mussel (Mytilus californianus), a red alga (Prionitis sternbergii), and the proximity to shore affected microbial nitrogen transformations during both daylight and nighttime periods.We hypothesized that macrobiota (both mussels and algae) would enhance microbial nitrogen cycling, and nearshore seawater 105 would have greater microbial activity compared with offshore because it was in close proximity to the macrobiota.We used gas-tight chambers and added enriched ammonium ( 15 NH 4 ) or nitrate ( 15 NO 3 ) to estimate the flux of ammonium and nitrate.Further, gas-tight chambers allowed us to test whether microbial denitrification resulted in loss of nitrogen via N 2 gas.We then manipulated DOC to test its specific effects on nitrogen transformations.In sum, we asked: 1) 110 how does the presence of mussel, red algal tissue or inert substrates affect microbial nitrogen cycling?, 2) does the microbial activity in seawater differ between from the nearshore versus 1-5 km offshore?, 3) are there diel cycles in these microbial nitrogen transformations, and 4) does the experimental addition of dissolved organic carbon (DOC) alter microbial nitrogen metabolism?

Chambers for assaying microbial metabolisms
In order to quantify the microbial nitrogen transformations that both retain and lose dissolved 120 nitrogen we enclosed seawater and some components of the rocky shore environment within 2 gas-tight Plexiglas chambers.Each 2.26 L chamber measured approximately 15 cm in diameter, 30 cm in height, and contained 2 ports at the top: one for an o-ring sealed connection to an oxygen probe and the other with a septate lid for gas-tight sampling of seawater.From 29 Jun to 22 Aug 2012, 54 assays were done in the chambers either in situ in tidepools 2 km east of Neah 125 Bay, WA, USA at Second Beach, WA (n=19) (48.23°N, 124.40°WW), at the shore at Tatoosh Island, WA (n=26) 48.39°N, 124.74°W) or onboard the R/V Clifford Barnes using seawater 2-3 km from each of these shore-based sites (n=9).The Second Beach site is described in Pather et al., (2014) and has tidepools at a height of 1.2 to 1.5 m above Mean Lower Low Water (MLLW), with a diversity of species (described in (Pfister, 2007;Pfister et al., 2016b).The chambers were 130 anchored into a number of these tidepools for 3-5 hours at a time during periods of low tides when the tidepools were emergent.Thus, the chambers contained tidepool water and were incubated under natural light and temperature conditions.Experimental trials included tidepool seawater only (n= 5), seawater with the California mussel Mytilus californianus (n=9), or seawater with bioballs and ceramic rings (n=5).Bioballs are topographically complex 26 mm 135 plastic balls used in commercial aquaria to provide substrate for microbes, while Filstar™ ceramic rings (1 cm diam) are also used in filtration (Aquatic Eco-systems™); both had been anchored in the tidepools for one month to initiate a microbial community in which to query function.At Tatoosh Island, where wave action was more significant, the chambers were placed at 140 the shore in a water bath within a shaded styrofoam cooler rather than in tidepools.The chambers were filled with seawater at the shore of Tatoosh Island and contained seawater only (n=8), seawater with the California mussel Mytilus californianus (n=12), seawater with the red alga Prionitis sternbergii (n=3), or seawater with bioballs (n=3).Bioballs had incubated at the lower edge of the mussel bed for one month prior to use in the chambers.For all experiments, the 145 wet mass of Prionitis was weighed with a Pesola™ spring scale, while the mussel dry mass was estimated from individual length measurements of the mussels (Wootton, 2004).
Microbial nitrogen metabolisms were compared in shore-based seawater collections versus seawater collected offshore in 2012.The offshore samples were collected with a CTD rosette system with 10 L Niskin bottles on the R/V Clifford Barnes 2-5 km offshore from Tatoosh Island 150 (48.432°N, 124.73°W) or Second Beach (48.37°N, 124.57°W) at a depth of 1m.The offshore assays were done with the chambers in a cooler with a water bath onboard the ship deck.We compared 4 replicates of each shore and offshore chambers during Jun and Jul of 2012.
We initiated each run by filling the chamber with seawater and any macrobiota or bioballs.
Oxygen and temperature were immediately recorded by a probe that remained in the chamber 155 through the duration of the experiment.We added an enrichment of either 10000 ‰ of d 15 NH 4 (as 0.05M ammonium chloride, 15 NH 4 Cl) or 10000 ‰ of d 15 NO 3 (as 0.05M sodium nitrate, Na 15 NO 3 , Cambridge Isotopes).We thus increased 15 N-NH 4 + or 15 N-NO 3 -by a factor of ten with the intention of maximizing our ability to detect the enriched signal in N 2 gas.Both ammonium and nitrate concentrations in seawater in this region is typically high (>2 and >10 µmolL -1 , 160 respectively), minimizing any concentration-related effects from tracer addition.The chamber was agitated to mix the tracer and then agitated 3-4 more times during the 3-to 5-hour incubation period.No samples were taken during the incubation so that we did not compromise the gas-tight nature of the chambers.At the end of the incubation, we inserted a needle attached to a gas-tight syringe through a rubber septa, drew out seawater and injected into a 30 ml serum 165 vial with a rubber stopper that had been evacuated to 160 mtorr with a Welsh 8905 Vacuum Pump.

Testing the effects of adding DOC
A further set of experiments in 2014 tested whether DOC additions enhanced microbial 170 nitrogen processing by increasing the concentration of DOC approximately 6 times above the ambient nearshore concentration to 1000 uM DOC.We added 1.0 ml of a 1.96 M glucose solution to one chamber at the beginning of the experiment while the other served as a control across all paired experiments.All paired experimental runs were performed at Tatoosh Island and resulted in 8 paired runs with seawater, and 4 paired experiments with either bioballs or trying to detect an enriched signal in N 2 gas.This tripling of 15 N-NH 4 + allowed us to test whether ammonium oxidation changed with added DOC; an increase in NH 4 oxidation would indicate stimulation of heterotrophic nitrifiers, while a decrease would be consistent with increased 180 competition for nitrogen by heterotrophs.

Quantifying enrichment results
In all experiments, a water sample prior to tracer addition (T o ) was collected to quantify concentrations of ammonium, nitrate, nitrite, phosphorus, and silica, as well as natural these initial measures to calculate the exact initial enrichment which could deviate from the target enrichment due to natural variation in nutrient and 15 N concentration.We collected the T o sample by filtering ~180 ml of source water through a syringe-filter (Whatman GF/F) into HDPE bottles, which we kept frozen until analysis.For the final sample (T f ) after 3-5 hours of incubation, we filtered directly from the individual chamber.All nutrient concentrations were 190 analyzed at the University of Washington Marine Chemistry lab (methods from UNESCO, 1994), while isotope determinations were done at University of Massachusetts, Dartmouth using methodology for isotopic composition reported previously (Pather et al., 2014;Pfister et al., 2014Pfister et al., , 2016b)).Briefly, nitrogen stable isotopes of ammonium were measured according to a modified version of the NH 4 oxidation method detailed in Zhang et al., (2007).NH 4 is oxidized 195 to nitrite using a hypobromite solution and then reduced to N 2 O using a sodium azide-acetic acid reagent before analysis on an IRMS (isotope ratio mass spectrometer).The stable isotope ratios of nitrate were measured by cadmium reduction to nitrite, followed by reaction with azide to N 2 O (McIlvin and Altabet, 2005).For the DOC analysis, an additional 25 ml were filtered into a 40 ml VOA vial (Shimadzu Inc).We tested for the presence of enriched N 2 gas in the chambers 200 deployed in 2012 using sample collection and analytical procedures described in (Charoenpong et al., 2014).Chamber oxygen and temperature were recorded with a Hach™ HQ4D and a LDO probe.

Stable isotope enrichment experiments can quantify nitrogen processing in marine 205
environments by tracking the transfer of the tracer between its source and product pools (Glibert, Pamela M. et al., 1982;Lipschultz, 2008).The traditional isotope tracer transfer model generally involves estimating a single rate parameter from time 0 to time t (Lipschultz, 2008) and has the general form: Equation ( 1) 210 where k is the sink or product at time t (or the average ), s is the source and R designates the atom % ( 15 N/( 15 N + 14 N )x100) of either the source or sink component.The source-product model (Equation 1), is thus used to estimate individual nitrogen transformation rates.We estimated ammonium oxidation to nitrite with a 15 NH 4 tracer experiment, while monitoring the 15 N enrichment in nitrite.Nitrate reduction to nitrite was estimated with a 15 NO 3 tracer 215 experiment, while monitoring the 15 N enrichment in nitrite.
A previous study of enrichment in tidepools showed substantial flux in inorganic nitrogen pools that was best described with differential equation models fit to multiple time points, and underestimated with source-product models (Pfister et al., 2016b).Source-product models likely underestimated the oxidation of ammonium here too because remineralization by species within 220 the chamber diluted the 15 NH 4 tracer.We still used the simpler source-product models because we had only a two-sampling protocol, at the beginning and the end of the experiment, to prevent gas escape.Isotope dilution is important and indicates ammonium remineralization by species within the chamber.We quantified ammonium remineralization in chambers with 15 NH 4 tracer using the methods of (Pather et al., 2014)

Dynamics of nutrients and isotopes in chambers
The presence of either the California mussel or the red alga Prionitis amplified net changes

Nitrogen transformation rates
Microbial nitrogen processing rates increased when either the California mussel or the red alga Prionitis was present.Ammonium oxidation rates with the mussel (14.1 nmol L -1 h -1 ) or red alga (32.8 nmol L -1 h -1 ) were two orders of magnitude greater than ammonium oxidation in 250 seawater only or with bioball surfaces which were less than 1 nmol L -1 h -1 (Fig 1d, F  1 ) compared with bioballs and seawater (Figure 1e, F 5,19 =17.64, p<0.001, logged values).For all these estimates of microbial nitrogen processing, we found high overlap in the rates estimated 255 with living, intact mussels compared with mussel shells only, indicating that the responsible microbes reside on the shell surface, rather than the mussel tissues (Figure 1d, e).The presence of mussels was further associated with increased ammonium remineralization, remineralization with mussels was twice that with bioballs and the red alga Prionitis, and an order of magnitude more that seawater alone (Figure 1f).260 Our estimates of mussel or algal mass within each chamber resulted in per gram estimates of the effect of these macrobiota on nitrogen transformation rates.For every gram of mussel dry mass, 3.21 nmol (se=0.64) of ammonium were oxidized per L per hour, while 1.60 nmol of nitrate were reduced (se=0.41).A comparable contribution is made per g of Prionitis wet mass with 1.50 nmol ammonium oxidized (se=0.27)and 1.56 nmol nitrate reduced (se=0.72).265 DIN uptake in chambers could be due to both microbial transformations or seaweed uptake.
We thus estimated what percentage of total DIN uptake was attributed to microbial activity based on our tracer enrichment and found that nitrate reduction accounted for as little as 4.2% of the decrease in nitrate concentration during the day, but as much as 87.2% at night.Estimates of ammonium oxidation revealed that ammonium oxidation made up 5.2 -7.4 % of total 270 ammonium uptake during the day.

Day versus night nitrogen transformations
Nitrification in association with mussels or in seawater alone did not differ between night and day hours (F 1,11 =0.583, p=0.461), but chambers with mussels again showed ammonium oxidation rates two orders of magnitude higher than seawater only (Fig 2a).Nitrate reduction 275 was ten times higher when mussels were present compared to the microbial activity of seawater only (mussels>seawater, F 1,14 =68.1, p<0.001), with greater daytime rates (day>night, F 1,14 =5.83, p=0.030), suggested only for seawater (e.g a near interaction, F 1,14 =3.76, p=0.073).Seawater nitrate reduction rates during the day (1.15 nmol L -1 h -1 ) were four times greater than those at night (0.26 nmol L -1 h -1 , Fig 2b), though these rates were still small relative to mussels.280

Onshore versus offshore microbial nitrogen transformations
The seawater only chambers showed no difference in ammonium oxidation rates whether collected at the shore (mean=0.23 nmol L -1 h -1 ) or offshore (mean=1.12,Fig 3a, t 3.8 =1.65, p=0.177), although there was low sample size (n=4).Overall, there was little change in nutrient 285 concentration when seawater from either offshore or nearshore was isolated; the overall mean change in DIN was less than 1 uM for both nearshore (-0.70) and offshore (0.63).There was also no difference in silica uptake between the two regions either (t=-0.679,p=0.525), indicating that diatom activity did not differ in the two regions.290

Nitrogen transformation rates with added DOC
On average, the coastal seawater that was used in the chambers contained 145 uM DOC; replicates with the addition of DOC were increased approximately 6 times that amount to1000 uM.Prionitis enhanced the DOC in the chambers also, with a mean increase in DOC of 9.31 mmol C per hour over the course of the experiment (n=4).Nitrification rates did not change 295 significantly when glucose was added (Fig 4a), although we acknowledge that our sample size was small and nitrification was not detected in some instances across both treatments, perhaps impeding a strong test of glucose effects.However, DOC addition did change nutrient uptake rates.The addition of DOC to experimental chambers generally resulted in greater uptake of The greater uptake of DIN in chambers with supplemental DOC could be due to increased microbial respiration with DOC.The effect of glucose on the uptake of DIN or phosphorus did not differ based on whether seawater, bioballs or Prionitis was in the chamber (F 2.31 =0.645, p=0.531 and F 2.31 =0.264, p=0.770, respectively), suggesting that the background metabolism of heterotrophic bacteria was the same regardless of the substrate available and even though the 310 DIN concentration declined a further 6.5 uM on average when Prionitis was present.If microbial respiration increased with DOC, we were unable to detect it by measuring oxygen concentrations.Whether we pooled treatments for seawater, bioballs and Prionitis or examined them separately, dissolved oxygen measurements did not differ (t= 1.125, p=0.277, df=16).315 4 Discussion

Seascape Scale Importance of Macrobiota for Microbial N Metabolism
The per mass estimates of microbial nitrogen transformations that we measured reveal significant microbial processing rates along coastal shorelines.Studies from (Wootton, 2004) estimate that a square meter of mussel bed can contain 32,425 g dry mass of mussel.320 Extrapolating from our measurements on both day and night, microbial nitrification in a square meter of mussel bed would amount to 2.5 mmol per day, with an additional 1.2 mmol of nitrate A similar calculation can be done for macroalgae using (Paine, 2002) control plots in the intertidal at Tatoosh Island where macroalgal mass was estimated to be 8.6 kg per square meter.
If Prionitis has any functional similarity to other seaweeds sampled by (Paine, 2002), then ammonium oxidation could reach 135.2 mm per day for a square meter of seaweed, while nitrate reduction would be 320.5 mmol.The macroalga contribution is thus comparable to water column 330 nitrification only when we consider a volume in excess of 50 million liters, or an area approximately 71 m on a side to a depth of 10 m.Even if Prionitis is exceptional with respect to microbial function when compared with other seaweeds, the potential contribution of macroalgae to microbial function could be substantial.Thus, independent of macroalgal effects on DIN uptake (Fig 1a, b, c) or via ammonium remineralization by mussels (Fig 1f), the microbiome of 335 each of these species makes distinct contributions to nitrogen cycling.
We demonstrated that seawater isolated from the immediate vicinity of benthic substrates had similar rates of nitrogen metabolism offshore (Fig 3).As this measurement indicates no difference in the activity of suspended microbes, we conclude that microbial metabolism was elevated due to microbes directly associated with the mussel and the red alga.Previous analyses 340 of 16s rRNA sequencing of mussels and Prionitis (Pfister et al., 2014) and metagenomic analysis of mussel shell microbes (Pfister et al., 2010)  processing.However, the nearshore results likely reflect benthic-associated activity influencing adjacent seawater.

Microbial Metabolism and Dissolved Organic Carbon
When considering the effect of DOC in microbial assemblages, there are 3 groups of microbes that might be affected.There are nitrifiers that are either heterotrophic or 350 chemolithotrophic (Ward, 2008), as well as heterotrophic bacteria that might consume DOC and assimilate ammonium, but not nitrify (e.g.Kirchman, 1994).Thus, added DOC might be expected to increase heterotrophic nitrification if DOC was limiting nitrifier growth.
Alternatively, added DOC could decrease nitrification if generalist heterotrophic bacteria were stimulated and then outcompete chemolithotrophs for ammonium (Butturini and Sabater, 2000), 355 although we do not know if ammonium was ever limiting.A third possibility is that heterotrophic nitrifiers are such a small percentage of nitrification activity that there is no detectable effect of elevated DOC.We found mixed evidence for the effects of DOC on nitrification.Ammonium oxidation was never stimulated by DOC (Figure 4); if anything, there was a nonsignificant trend of decreased ammonium oxidation with glucose, suggesting that 360 general heterotrophic bacteria were consuming the elevated DOC.Our DOC additions were accompanied by decreased dissolved inorganic nitrogen and phosphorus in the surrounding seawater, suggesting that heterotrophic microbial metabolism increased, a result consistent with other glucose addition studies with microbes (Zhang et al., 2013).Bacterial production in seawater has been shown to increase with glucose addition (Caron et al., 2000;Jacquet et al., 365 2002), with heterotrophic bacteria released from carbon limitation when DOC is added (Jacquet et al., 2002;Joint et al., 2002).In streams, glucose additions have shown decreased nitrification with DOC (Strauss and Lamberti, 2000), a result attributed to heterotrophic bacteria in direct competition with nitrifiers.While (Strauss and Lamberti, 2000) documented decreased oxygen and increased respiration with added DOC, we detected no effect of DOC on the change in 370 oxygen within chambers (Fig. 4h).The unknown contribution of photosynthesis to oxygen concentrations, as well as the relatively high oxygen content of the seawater in these locales could have masked oxygen differences.Nonetheless, DOC stimulated nutrient uptake, presumably by heterotrophic microbes and the effect of DOC was the same whether seawater, bioballs or Prionitis was in the chamber (Fig 4b-i).Thus, the background metabolism of 375 heterotrophic bacteria was unchanged even when Prionitis was present and reduced chamber DIN concentrations 6.5 uM over the course of the experimental runs.
A final explanation to explain the increased DIN uptake with added DOC is that bacteria are able to compete with any phototrophs for nitrate when an organic carbon source is increased (e.g.(Diner et al., 2016).Nitrate reduction rates are high with Prionitis and this alga also 380 provisions DOC, perhaps promoting the coupling of heterotrophy and nitrate reduction.Whether any of the decreased nitrate concentration associated with Prionitis in chambers could be attributed to heterotrophic nitrate reduction is unknown at this time, because our experiments with added DOC did not assay nitrate reduction, only ammonium oxidation.
In sum, while DOC concentrations can be elevated in nearshore areas compared with 385 offshore, there was little evidence that enhanced DOC changed nitrification rates, even in the chambers with Prionitis, where DIN levels were lower due to seaweed uptake.Whether heterotrophic nitrifiers are present remains unknown, though previous analysis of microbes at these sites suggest the presence of taxa associated with heterotrophic nitrification (e.g. Arthrobacter (Hynes and Knowles, 1982), Crenarchaeota (Offre et al., 2013), and Alcaligenes 390 faecalis, (Joo et al., 2005), though they were detected in only a small fraction of samples (Pfister  et al., 2014).Analysis of 16s rRNA of seawater, mussels and Prionitis do show sharp distinctions in b-diversity, with some taxa unique to each (Pfister et al., 2014).
Taken together our data suggest that chemolithotrophic nitrifiers are dominating nitrification in this area.Other heterotrophic bacteria can noticeably depress DIN and phosphate 395 concentrations when DOC is supplemented, suggesting there may be some carbon limitation for heterotrophic microbial metabolisms.If, as suggested by (Strauss and Lamberti, 2000), the C:N ratio in the water column determines the relative fitness of heterotrophic bacteria versus chemolithotrophic nitrifiers, then the many regions where DIN concentrations in seawater are lower than they are at our Washington coastal sites may show a different result.400 Of note is that many seaweeds produce detectable amounts of DOC in coastal areas (Wada and Hama, 2013), with as much as 14% of net primary production being released as DOC in a kelp species (Reed et al., 2015).Among other seaweeds, 20 to 30% of that DOC can be taken up within 2 hours (Brylinsky, 1977), suggesting an active heterotrophic assemblage in proximity.
Seaweeds also have a diverse assemblage of microbial associates (Lemay et al., 2018;Marzinelli 405 et al., 2018;Michelou et al., 2013;Pfister et al., 2014).Which of these associated microbes benefit from this DOC and whether others are inhibited is unknown.While we tested the effect of elevated glucose on nitrification with enriched ammonium, a next step is to test if those microbes involved in the nitrate reduction pathways are affected by glucose addition.
Macrobiota that serve as hosts for microbes provide a predictable substrate for attachment 410 in a fluid environment and provide dissolved organic matter in many forms (Carlson and Hansell, 2015).The mussels studied here also excrete ammonium and likely DON (Bayne and Scullard, 1977;Pather et al., 2014).Their filter feeding activities release DOC in many forms, and continually process organic matter that can be utilized by microbes (Jacobs et al., 2015).Through filter feeding and mucus production, there is increasing evidence that marine 415 invertebrates and microbes are connected through their production and use of dissolved organic matter (Rädecker et al., 2015;Rix et al., 2016).

The multiple factors influencing nitrogen availability
Our experiments provide insight into the fate of nitrogen in coastal systems.While 420 ammonium oxidation and nitrate reduction rates were two orders of magnitude higher than any water column estimates, we have no evidence that nitrate reduction continued through to denitrification and the release of N 2 gas as we never detected enriched 15 N in N 2 gas (e.g.(Jensen et al., 2011).Thus, nitrogen was being retained in our experimental system.If ammonium oxidation and nitrate reduction are occurring relatively constantly, as suggested by our 425 experiments, then a diversity of microbially-mediated DIN dynamics may take place across microenvironments that differ in oxygen levels.The net result could be continued microbial use of ammonium and nitrate and the ability for the microenvironment surrounding the animal or seaweed to sustain a range of microbial metabolisms, a result obtained for other marine invertebrates (de Goeij et al., 2013;Heisterkamp et al., 2013).Research in tidepools containing 430 these same species has also shown both nitrogen oxidation and reduction processes (Pfister et al., 2016b).In all instances to date, the metabolism of the host macrobiota results in a daily range of oxygen levels, thus providing a diversity of environmental niches that favor different microbial transformations through time.

Conclusions 435
The marine mussel and alga species studied here were loci for microbial nitrogen metabolism, elevating ammonium oxidation and nitrate reduction two orders of magnitude over that of seawater alone.For mussels, microbial nitrogen processing did not differ between daylight and nighttime hours.While the addition of DOC did not increase ammonium oxidation, it resulted in greater uptake of DIN, suggesting that DOC stimulated heterotrophic microbial 440 activity.In addition to providing a template for a diverse set of ecological interactions, the marine macrobiota studied here hosted a diverse set of microbial metabolisms and enhanced rates of carbon and nitrogen cycling in coastal ecosystems.
Code and data availability.Available from the authors upon request.445 Competing interests.The authors declare that they have no competing interests.between day and night hours for mussels or seawater at the shore.Data are log-transformed (from nmol L -1 h -1 ) to facilitate comparison.Rates with mussels were always greater (for a. 660 F 1,11 =52.59, p<0.001, and b.F 1,14 =68.14, p<0.001).Ammonium oxidation rates in association with mussels or in seawater alone did not differ between day and night (F 1,11 =0.58, p=0.461), while nitrate reduction in seawater was greater during the day (F 1,14 =5.83, p=0.030).Changes to the isotopes of inorganic nitrogen are shown in Figure A1.
. Briefly by fitting an exponential model decline in 225 d 15 NH 4 from the beginning to the end of the experiment y=ae -bx .The parameters a and b were fitted where b was the exponential decay constant in the d 15 N NH4 enrichment.Remineralization rates were thus calculated as:  4 5  = − * [4] (Equation 2) in nmol L -1 h -1 , where [4] was the mean concentration of ammonium in nM.230 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-198Manuscript under review for journal Biogeosciences Discussion started: 17 May 2018 c Author(s) 2018.CC BY 4.0 License.
to ammonium and nitrate concentration in the experimental chambers compared with chambers 235 that contained bioballs or only coastal seawater (Fig 1).Chambers during daylight hours with Prionitis and mussels had increased ammonium over the course of the experiment compared with the relatively unchanged coastal seawater and bioball treatments (Fig 1a, F 5,51 =6.150, p<0.001), while nitrate decreased with Prionitis and increased with mussels (Fig 1b, F 5,51 =3.512, p=0.008).Changes in nitrite did not differ among treatments (Fig 1c, F 5,51 =0.66, p=0.659).240 The dynamics of d 15 N NH4 , d 15 N NO2, and d 15 N NO3 within the chambers revealed transfer of 15 N isotope and thus microbial transformations.When 15 N-NH 4 + was added, enrichment in d 15 N NO2, and d 15 N NO3 and any dilution in the d 15 N NH4 signal was measured (Figure A1 a, b).Similarly, enrichment in d 15 N NH4 and d 15 N NO2 followed the addition of 15 N-NO 3 -(Figure A1 c, d).Deviations in our target of initial enrichment (10000 and 2000‰) occurred due to natural 245 variation in nutrient concentrations at the time of tracer addition.
Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-198Manuscript under review for journal Biogeosciences Discussion started: 17 May 2018 c Author(s) 2018.CC BY 4.0 License.nitrite and nitrate with Prionitis, bioballs or in seawater alone while ammonium showed a trend 300 toward greater uptake only with Prionitis, otherwise there was little overall change in ammonium concentration (Fig 4 b,c,d).DOC addition was also associated with an increased uptake of DIN and phosphorus, regardless of the composition of the chamber (Fig 4 e,f).Silica was unchanged with bioballs or seawater alone, while there was greater uptake of silica with Prionitis, suggesting Prionitis hosts diatoms (Fig 4g).305 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-198Manuscript under review for journal Biogeosciences Discussion started: 17 May 2018 c Author(s) 2018.CC BY 4.0 License.reduction.As a comparison, at this site it would take a volume of seawater of 1 million liters to host the same microbial nitrogen metabolism, the equivalent of a 10 m by 10 m area of the ocean to 10 m depth.325 both indicate DNA sequences associated with a diversity of nitrogen metabolisms.The similarity of nearshore to offshore microbial function would appear at odds with our previous work showing that natural isotopes of ammonium and nitrate (d 15 N NH4 and d 15 N NO3 ) are enriched near the shore, indicating increased microbial 345 Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-198Manuscript under review for journal Biogeosciences Discussion started: 17 May 2018 c Author(s) 2018.CC BY 4.0 License.

FIGURE CAPTIONS Figure 1 .
FIGURE CAPTIONS Figure 1.The change in the concentrations of DIN (a.ammonium, b. nitrate, c. nitrite) in uM over the course of the experiment when only seawater was present, versus the addition of bioballs or mussel or Prionitis.Nitrogen transformation rates (in nmol L -1 h -1 ) for d. ammonium 655 oxidation, e. nitrate reduction, and f. the ammonium remineralization rate.Letters indicate statistical differences with ANOVA and Tukey HSD.

Figure 2 .
Figure 2. The ammonium oxidation rate (a.) and the nitrate reduction rate (b.) contrasted

Figure 3
Figure 3. a.The ammonium oxidation rate in surface seawater collected at the shore versus 665 offshore 2-5 km, based on 4 trials in each locale in Jun and Jul of 2012.The rates did not differ

Figure 4 .
Figure4.The effect of supplemental DOC on a. the rate of ammonium oxidation (in nmol 670 L -1 h -1 ), b-g the change in nutrient concentrations (uM), and h. the oxygen concentration (in mg L -1 ).An * indicates a significant difference (p<0.05) between the control and the DOC addition for each of seawater alone, or seawater with bioballs or the red alga Prionitis.‡ indicates 0.10>p<0.05.