The impact of the polychaete Nereis diversicolor and enrichment with macroalgal (Chaetomorpha linum) detritus on benthic metabolism and nutrient dynamics in organic-poor and organic-rich sediment

Journal of Experimental Marine Biology and Ecology, 231 (1998) 201–223

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The impact of the polychaete Nereis diversicolor and enrichment with macroalgal (Chaetomorpha linum) detritus on benthic metabolism and nutrient dynamics in organic-poor and organic-rich sediment Kim Hansen, Erik Kristensen* Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Received 8 January 1997; received in revised form 1 April 1998; accepted 5 April 1998

Abstract The combined impact of the burrow-dwelling polychaete, Nereis diversicolor, and organic enrichment with macroalgal detritus, Chaetomorpha linum, on sediment metabolism (CO 2 production) and nutrient (NH 41 and NO 32 ) dynamics was assessed in laboratory microcosms over 35 days with homogenized sandy (0.5% LOI) and muddy (8.6% LOI) sediment from a shallow estuary (Kertinge Nor, Denmark). In addition, the gross C and net N mineralization ( 1 DOC evolution) was followed in 4 time series using closed anaerobic jars containing the two sediment types with and without organic addition. The unenriched treatments showed that the sandy sediment exhibited higher C and N mineralization than the muddy, due to a more reactive indigenous organic pool (fresh diatoms vs. degraded macroalgal detritus). A higher excess CO 2 production due to enrichment in the muddy than the sandy sediment indicates a higher instant capacity for carbon mineralization in the former sediment. In contrast to the sandy, the muddy sediment had previously been exposed to extensive input of filamentous algae and may therefore be better adapted to this particular detritus type. The excess NH 1 4 production due to enrichment was highest in sandy sediment, suggesting that N was recycled rapidly by the microbial community of the relatively nitrogen-poor muddy sediment, resulting in a low net N mineralization. It also appears that C and N mineralization was uncoupled in sandy sediment, as indicated by the simultaneous initial production of DOC and NH 1 4 with no concomitant CO 2 production. The presence of animals enhanced C mineralization in all treatments; highest in the enriched treatments. The enhancement in absolute rates was similar when both sediment types were enriched, indicating that the faunal enhancement with added detritus is independent of sediment type. The faunal enhancement of N mineralization, on the other hand, was highest in sandy sediment irrespective of organic enrichment. Denitrification estimates, based on mass balance, also appeared to be stimulated significantly by the presence of animals, whereas organic

*Corresponding author. E-mail: [email protected] 0022-0981 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0022-0981( 98 )00070-7

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enrichment had a minor influence on this particular process.  1998 Elsevier Science B.V. All rights reserved. Keywords: Benthic fluxes; Bioturbation; C and N mineralization; Chaetomorpha linum; Filamentous macroalgae; Macrofauna; Marine sediment; Nereis diversicolor

1. Introduction Most organic input to coastal sediments is usually derived from autochthonously produced detritus originating from benthic micro- and macrophytes (Kemp et al., 1997). A common symptom of eutrophication in shallow coastal and estuarine areas is the massive occurrence of free-floating ephemeral macroalgae (e.g. Rosenberg et al., 1990; ¨ et al., 1996). The presence of large macroalgal mats must Sfriso et al., 1992; Sundback have an important impact on nutrient dynamics of the ecosystem by acting as an effective ‘filter’ for the flux of nutrients from the sediment to the water column (Owens and Stewart, 1983; Lavery and McComb, 1991; Krause-Jensen et al., 1996). Collapse and subsequent decomposition of macroalgal mats, on the other hand, may create anoxic bottom conditions even in shallow waters, and result in extinction of the benthic fauna ´ 1992). (e.g. Jørgensen, 1980; Llanso, The response of coastal sediment systems to a massive organic input has been described in a number of studies (e.g. Kelly and Nixon, 1984; Hansen and Blackburn, 1992; Enoksson, 1993). Increased organic input to sediments will usually enhance the overall community metabolism, and stimulate the recycling of nutrients (e.g. Andersen and Hargrave, 1984; van Duyl et al., 1992). However, recent studies have shown that very high detritus supplies may in fact hamper bacterial activity (Holmer and Kristensen, 1994; Kristensen and Hansen, 1995). The degradability of organic matter is principally a function of the chemical composition and thus the origin and age of the substrate (e.g. Westrich and Berner, 1984; Kristensen and Blackburn, 1987; Burdige, 1991). The microbial response to an organic matter supply may depend on the bacterial population composition and physiological state, which in turn is controlled by the size and chemical structure of the indigenous organic pool in the sediment (Aller and Yingst, 1980; Parkes et al., 1993; Kristensen and Hansen, 1995). Several studies have stressed the important role of benthic macrofauna on biogeochemical processes and nutrient exchange in coastal marine sediments (Henriksen et al., 1983; Aller, 1988; Kristensen, 1988; Andersen and Kristensen, 1991). Reworking activities, such as burrowing, may translocate fresh reactive organic matter from the sediment surface to deeper sediment strata. Ventilatory activities of burrow-dwellers may enhance the exchange of solutes (electron acceptors and metabolites) across the sediment–water interface. Macrofaunal-induced enhanced release of metabolites such as CO 2 and NH 41 result from: (1) direct metabolic exchange by the macrofauna itself; (2) stimulation of mineralization processes in the burrow wall environment; and (3) enhanced transport processes across the burrow wall associated with ventilation activities and radial diffusion from the surrounding sediment (Kristensen and Blackburn, 1987; Aller, 1988; Kristensen, 1988; Andersen and Kristensen, 1991).

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In the present study, we quantified the impact of the common burrow-dwelling polychaete, Nereis diversicolor, on organic matter decay and nutrient dynamics in two coastal sediment types, an organic-poor and an organic-rich, supplemented with detritus of the filamentous macroalga Chaetomorpha linum. N. diversicolor and C. linum were chosen for the experiments because they are dominant features in the study area. The mineralization of algal detritus was studied by monitoring fluxes of CO 2 and dissolved inorganic nitrogen across the sediment–water interface over a 35-day period. Furthermore, the microbial decay of added C. linum material was studied by the use of closed, anaerobic microcosms (jar experiment). The results provided information on the combined action of macrofaunal activities and organic enrichment on rates and stoichiometry of mineralization processes in sediments of different composition.

2. Materials and methods

2.1. Study site Sediment was obtained in November 1993 from two locations in the inner regions of the shallow cove, Kertinge Nor, on the east coast of Fyn, Denmark (Hansen and Kristensen, 1997). The two locations, ‘sandy’ and ‘muddy’, were situated about 30 and 130 m from the shore at 30 and 130 cm water depth, respectively. The sediment from the inner station (sandy) consisted of well-sorted low-organic sand. Benthic microalgae were the dominant primary producers, as no macroalgae were observed at the sampling date. The benthic macrofauna was relatively rich and dominated by the polychaete Nereis diversicolor (900–1000 m 22 ) and the crustacean Corophium volutator (7000– 12000 m 22 ). Small gastropods (Hydrobia spp.) and a number of unidentified polychaetes were found in variable densities. The sediment of the outer station (muddy) appeared organic-rich mainly from recent mass occurrence of the filamentous macroalgae Chaetomorpha linum and Cladophora sericea. The sediment surface was uncovered at the sampling date, but only few macrobenthic organisms were observed. For a more ˚ et al. (1995). detailed description of the study area consult Riisgard

2.2. Sediment collection and handling To reduce the natural heterogeneity, and to obtain equal starting conditions, sediment used in the experiment was homogenized before use. The upper 5–8 cm of the sediment was sampled and sieved through a 1.5-mm mesh to remove macrofauna and larger particles (i.e. shells and gravel). The sandy sediment was sieved and homogenized on location, whereas the muddy sediment was brought to the laboratory, sieved in a 80-l container, and allowed to settle for 24 h. Sampling of the two sediment types, however, was displaced 3 weeks of practical reasons. Intact specimens of Nereis diversicolor were collected from the sandy location. Fresh Chaetomorpha linum had been collected from another location in Kertinge Nor during September 1993. The algal material was rinsed in seawater to remove adherent fauna and epiphytes before being dried (12 h at 1108C).

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2.3. Core experiments After homogenization, both sandy and muddy sediment was split into two portions. One portion of each was enriched with dried and blended ( , 500 mm) C. linum detritus (SA- and MA-sediment). An amount of | 1.33 mg dw cm 23 algal material was added and mixed homogeneously into the sediment. This is equivalent to a deposition and burial of 186 g dw C. linum m 22 , which is comparable to the observed standing crop of ˚ et al., 1995). The remaining this algae in Kertinge Nor (up to 250 g dw m 22 , Riisgard sediment was maintained as unenriched control (SC- and MC-sediment). Sediment microcosms were established by transferring the homogenised sediment into 25-cm long and 8-cm I.D. plexiglass core liners to a depth of | 14 cm. Eight cores of each sediment treatment (32 cores total) were established and placed in a darkened, continuously aerated seawater tank (200 l) at 158C. Subsequently, 4 cores of each treatment were added, 6 medium-sized (185–419 mg wet wt) individuals of N. diversicolor, equivalent to a density of 1194 m 22 and a biomass of | 338 g wet wt m 22 . Unenriched sandy and muddy Nereis-inhabited sediment cores are denoted SN- and MN-cores, respectively, while those enriched with algae are denoted SAN- and MAN-cores, respectively. Seawater ( | 17‰) overlying the cores was continuously exchanged and stirred at a rate below the resuspension limit. Stirring was performed by 30 mm long magnet bars placed 2 cm under the water surface in each core and driven by an external rotating magnet ( ¯ 40 rpm). A few nereids that appeared dead on the sediment surface were replaced during the experiment. The exchange of total CO 2 (TCO 2 5 H 2 CO 3 1 HCO 32 1 CO 322 ) and DIN (dissolved inorganic nitrogen; NO 22 1 NO 32 and NH 41 ) across the sediment–water interface was measured regularly (3–5 days interval) during a 35-day experimental period. Cores were sealed with gas-tight plastic lids during flux measurements, while stirring was maintained. Flux rates were calculated from the change in concentration between initial and final samples during incubation periods of 3 to 6 h, depending on treatment. Tests showed that oxygen never decreased below 60% of air saturation. Samples for TCO 2 were transferred to gas-tight bottles with no head-space and analyzed within 8 h of sampling. Samples for DIN were filtered (GF / C) and frozen immediately for later analysis. At the end of the experiment (i.e. after 35 days), cores were sectioned into 0.5- to 2-cm intervals to 10 cm depth. Subsamples of the sediment slices and the initial sediment mixtures were examined for water content (weight loss after drying at 1308C for 6 h), specific density (weight of a known sediment volume) and organic content (loss-on-ignition; LOI) at 5208C for 6 h, and as particulate organic carbon (POC) and 1 nitrogen (PON). Exchangeable NH 1 4 (exch-NH 4 ) was analyzed on sediment subsamples ( | 2 g) from each depth interval by extracting with 5 ml of 2 M KCl for 30 min at 58C followed by centrifugation for 10 min at 3000 rpm. The supernatant was frozen for later NH 1 4 analysis. The remaining sediment was used for porewater extraction by centrifugation in double-chambered centrifuge tubes at 1500 rpm for 8 min (sandy sediment) or by squeezing through a GF / C filter under N 2 pressure (muddy sediment). Samples for TCO 2 and NH 1 4 were treated as mentioned above.

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2.4. Jar experiment Sediment for use in the jar experiment was enriched with Na 2 SO 4 to obtain an initial porewater concentration of approximately 50 mM in order to avoid SO 22 depletion. 4 Samples of the various sediment types were transferred to 50-ml polyethylene centrifuge tubes (‘jars’) allowing no head space. Fifty jars were established for each sediment type (SC-, SA-, MC- and MA-jars, respectively). The jars were sealed with screwcaps, taped and incubated in anaerobic sandy sediment at 158C. The temporal pattern and rates of anaerobic decomposition processes were followed for 59 days. At regular intervals (decreasing frequency from once a day initially to | twice a week at the end) two jars of each type were processed and analyzed for solid phase parameters and porewater solutes as described above for the core experiment. In addition, porewater was analyzed for dissolved organic carbon (DOC).

2.5. Chemical analysis TCO 2 was analyzed by the flow injection / diffusion cell technique of Hall and Aller (1992) using a Kontron Ion Chromatograph. Samples for porewater TCO 2 were added HgCl 2 before analysis to precipitate interfering sulfides. The concentrations of NO 2 2 and NO 32 were determined on a flow injection analyzer (Tecator FIAstar 5010) using the method of Armstrong et al. (1967). NO 22 is included in the presented NO 32 data. NH 41 concentration was measured manually by the salicylate–hypochlorite method (Bower and Holm-Hansen, 1980). Particulate organic carbon (POC) and nitrogen (PON) of predried sediment subsamples were analyzed on a Carlo Erba EA1108 CHN Elemental Analyzer according to the methods of Kristensen and Andersen (1987). Porewater DOC was determined on 0.45-mm filtered samples by a Shimadzu TOC-5000 Total Organic Carbon Analyzer after acidification with 2 M H 2 PO 4 (to pH , 2) to remove dissolved inorganic carbon.

3. Results

3.1. Visual observations during core sectioning A brownish colored, oxidized surface zone of 10–14 mm and 10–12 mm depth was evident throughout the experiment in sandy (SC-cores) and muddy (MC-cores) control sediment, respectively, while the addition of algal material diminished the depth of this zone to 2–3 mm in both SA-cores and MA-cores. Below the oxidized zone, the sandy sediment was grey and greyish-black and the muddy sediment was uniformly brownishgrey. In N. diversicolor-bioturbated cores, burrow structures extended the oxidized zone into the otherwise reduced sediment in the form of a 1 to 4-mm thick oxidized wall lining around the burrows. Burrows were observed to 5–6 cm and 10–12 cm depth during sectioning of unenriched sandy (SN-cores) and muddy (MN-cores) sediment, respectively. In the enriched sandy (SAN-cores) and muddy (MAN-cores), on the other

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hand, burrows were only evident in the upper 4 cm and 6–8 cm, respectively. The recovery of worms was 58% in the sandy sediments and 73% in the muddy sediments.

3.2. Sediment characteristics The initial characteristics of the homogenized sediments before algal addition showed that the muddy sediment contained 16 times more organic matter (LOI) on weight basis than the sandy sediment (Table 1). However, due to the higher porosity in the former, the difference was only a factor of 4–5 (POC and PON) on volume basis. The molar C:N ratio of the sediments (Table 1) reflected the origin of the organic matter; i.e. diatoms (C:N 5 7–8) in the sandy and C. linum (C:N 5 14.8) in the muddy sediment. The addition of algal material corresponded to a concentration in the sediment of 36.2 mmol C cm 23 and 2.44 mmol N cm 23 (0–14 cm depth integrated amount: 5222 mmol C m 22 and 352 mmol N m 22 ); increasing the POC and PON content by | 13% and | 7%, respectively, in the sandy sediment, and | 2.5% and | 1.9%, respectively, in the muddy sediment. There was no measureable difference in sediment POC and PON content from the start to the end of the experiment.

3.3. Sediment–water fluxes 3.3.1. TCO2 flux While TCO 2 release in control sediments remained more or less constant throughout the experiment at a mean rate of 2163 (SC-cores) and 1462 (MC-cores) mmol m 22 d 21 , the algal addition in SA- and MA-cores was evident as initially increasing TCO 2 fluxes (Fig. 1). The fluxes in the enriched sediments peaked after 10 days at rates of 2.2 (sandy) and 6.8 (muddy) times higher than the control sediments followed by gradually decreasing rates. At the end of the experiment, TCO 2 fluxes in enriched sandy and muddy sediment remained 1.7 and 4.0 times higher than the controls. The presence of N. diversicolor caused an immediate and dramatic increase in the release of TCO 2 with a peak after 2–3 days which was 3.2 and 3.6 times higher than the control in unenriched, and 3.1 and 5.3 times higher than the defaunated control in enriched sandy and muddy sediments, respectively. Fluxes remained at a constant high Table 1 Initial (day 0) values of sediment porosity, organic content and molar C:N ratios in ‘sandy’ and ‘muddy’ sediments before addition of algal material

21

Porosity (vol vol ) LOI (% of dw) POC (mmol C cm 23 ) PON (mmol N cm 23 ) C:N ratio (molar)

Sandy sediment

Muddy sediment

0.36 0.53 269 35 7.7

0.82 8.57 1467 126 11.6

Organic content is presented as loss-on-ignition (LOI), particulate organic carbon (POC) and nitrogen (PON). Values for POC and PON are expressed on a volume basis.

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Fig. 1. TCO 2 release from sandy and muddy sediment cores during the 35-day experimental period. Sediment abbreviations: SC: sandy control sediment; SA: sandy enriched sediment; SN: sandy Nereis-inhabited sediment; SAN: sandy enriched Nereis-inhabited sediment; MC: muddy control sediment; MA: muddy enriched sediment; MN: muddy Nereis-inhabited sediment; MAN: muddy enriched Nereis-inhabited sediment. Values are presented as the mean of 3 cores (6SE).

level in all bioturbated sediments except for a gradual decrease after day 15 in enriched cores (SAN- and MAN-cores). Although the CO 2 fluxes in these cores approached those of unenriched bioturbated cores (SN- and MN-cores) at the end of experiment, the fluxes

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became only similar at the end in the sandy sediment whereas there was still a difference factor of about 2 in the muddy sediment. The time-integrated (35 days) TCO 2 efflux was 1.6 times higher in sandy (SC-cores) than muddy (MC-cores) control sediment (Table 2), whereas the opposite pattern (1.5 times) was evident for the enriched sediment. The faunal-induced excess TCO 2 release was most pronounced in the muddy sediment. However, the stimulation of TCO 2 release in the bioturbated relative to the uninhabited situation was 2.7 and 4.3 times higher for unenriched sandy and muddy sediment, respectively, but only 2.2 times higher when both sediment types were enriched (Table 2).

3.3.2. DIN flux The flux of NH 1 4 was directed out of the sediment in all sediment types except for MC-cores where a slight net uptake occurred throughout most of the experiment (Fig. 2). NH 1 4 release was increased more by the presence of Nereis diversicolor than by algal addition, and was markedly higher in sandy than in muddy sediment. The NH 1 4 efflux in bioturbated sediments rapidly reached a maximum of 10–12 and 5–6 mmol m 22 d 22 irrespective of algal addition in sandy and muddy cores, respectively. The similarity of NH 1 4 release in the two types of bioturbated sandy sediments continued with a gradual

2 Fig. 2. NH 1 4 and NO 3 exchange in sandy and muddy sediment cores during the 35-day experimental period. Positive values indicate release from the sediment and negative values uptake by the sediment. Values are presented as mean of 3 cores (6SE).

Sandy sediment

Muddy sediment

Unenriched

22

Enriched

Unenriched

Enriched

SC

SN

SA

SAN

MC

MN

MA

MAN

C

TCO 2 flux (mmol m ) pw TCO 2 pool (mmol m 22 ) gross C mineral. (mmol m 22 ) excess C (% of added POC) gross jar C mineral (mmol m 22 ) excess jar C (% of added POC)

755 775 1530 – 1455 –

2074 424 2497 – – –

1455 878 2332 15.4 3092 31.3

3232 721 3953 27.9 – –

454 319 773 – 519 –

1940 20 1960 – – –

2205 2052 4257 66.7 2989 47.3

4875 1176 6051 78.3 – –

N

22 NH 1 ) 4 flux (mmol m 22 NO 2 flux (mmol m ) 3 pw NH 41 (mmol m 22 ) net N mineral (mmol m 22 ) excess N (% of added PON) gross jar N mineral (mmol m 22 ) excess jar N (% of added PON)

55 15 167 237 – 279 –

224 8 87 319 – – –

115 29 293 399 46.0 392 32.1

235 216 228 447 36.4 – –

–3 14 43 54 – 78 –

107 27 24 96 – – –

14 230 118 102 13.6 137 16.8

132 285 63 110 4.0 – –

Negative data denote uptake by the sediment. Porewater pools (pw pool) of accumulated TCO 2 and NH 1 4 in sediment cores are calculated as the difference between final and initial concentrations in the upper 10 cm (extrapolated to total sediment depth, 14 cm). Total gross C and net N mineralization were calculated as the sum of integrated fluxes and accumulated porewater pools. Excess C and N denotes the surplus production of dissolved inorganic carbon and nitrogen in algal enriched sediments relative to unenriched sediments, given as percentage of the POC and PON content in the added algal material. The 35-day gross C and N mineralization from jars (extrapolated to a 14-cm sediment column) are presented for comparison. The N mineralization is here denoted ‘gross’ although bacterial assimilation is included.

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Table 2 Carbon (C) and nitrogen (N) mineralization integrated over the 35-day experimental period

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decrease in both throughout the experiment. In the bioturbated muddy sediment, the enriched treatments generally exhibited higher NH 1 4 release than the unenriched after day 14. Addition of algal material to the sandy sediment increased the time-integrated efflux of NH 1 4 by a factor of 2.1 when no animals were present, but only 5% in the bioturbated treatments (Table 2). In the defaunated muddy sediment, the algal addition changed the NH 1 4 flux from slightly negative to slightly positive, whereas in the bioturbated treatments, the efflux was enhanced 23% by algal additions. The timeintegrated effluxes of NH 1 4 were several-fold higher in the defaunated sandy than the muddy treatments, but only about a factor 2 higher in the bioturbated treatments (Table 2). The NO 2 3 flux pattern varied considerably between sediment types and treatments (Fig. 2). NO 2 3 was released from the sediment in both control sediments (SC and MC). NO 32 fluxes in sandy control sediment was initially close to zero, but increased gradually to | 1.3 mmol m 22 d 21 after day 17. The muddy control sediment showed a more fluctuating pattern, but with the same time-integrated rate as SC-sediment (Table 2). Algal enrichment of sandy and muddy sediments caused a shift towards uptake of NO 2 3 with gradually increasing rates through time (Fig. 2). The integrated NO 2 3 uptake in MA-cores over the experimental period was | 3 times higher than in SA-cores (Table 2). The presence of N. diversicolor in sandy unenriched sediment only reduced the NO 2 3 efflux after day 22 relative to SC-cores (Fig. 2). The presence of animals in enriched sandy sediment only affected NO 2 3 during the last incubation (day 35). The most pronounced effect of N. diversicolor was observed in enriched muddy sediment, where NO 2 3 uptake on average increased almost 3-fold relative to MA-cores (Fig. 2 Table 2). In unenriched muddy sediment, nereids diminished the NO 32 efflux relative to MC-cores, resulting in a slight net uptake over the 35 day period. Unfortunately, the experiments were exposed to varying NO 2 3 concentrations in the overlying water. The sandy sediment was exposed to concentrations ranging from 2–4 mM initially to 12–15 mM at the end, whereas NO 2 3 concentrations overlying muddy sediment increased from 12–18 mM initially to 29–36 mM at the end.

3.4. Porewater solutes in cores Final profiles of porewater TCO 2 and NH 1 were affected markedly by algal 4 enrichment and animals additions; the former treatment generally increased and the latter decreased concentrations (Fig. 3). The impact of N. diversicolor on porewater profiles was largest in muddy sediment, consistent with the visual observation that worms buried deeper and appeared more active here. Likewise, deflections in the porewater profiles indicated deeper burrowing depth in unenriched than enriched treatments of both sediment types. The presence of N. diversicolor in sandy sediment reduced the total depth integrated TCO 2 pool in the entire 14-cm sediment column to | 61% (unenriched) and | 84% (enriched) of the amount in the equivalent uninhabited sediments (Fig. 3). In the muddy sediment, the bioturbation impact was more pronounced with reductions of porewater CO 2 to | 54% (unenriched) and | 63% (enriched) of the amount in the uninhabited sediments. Profiles of porewater NH 1 4 were generally similar to those of TCO 2 (Fig. 3),

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Fig. 3. Vertical profiles (0–10 cm) of porewater TCO 2 and NH 1 4 in sandy and muddy sediment cores. Values are presented as mean of 3 cores (6SE).

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although concentrations were considerably higher in sandy than muddy sediment. The total NH 1 4 pool in bioturbated sandy sediments was reduced to | 57% (unenriched) and | 79% (enriched) of the amount in uninhabited sediments, whereas the values in muddy sediment were | 27% and | 61%, respectively.

3.5. Porewater solutes in jars The temporal pattern of porewater solutes in the various jar types showed several distinct phases (Figs. 4 and 5). Production rates of TCO 2 and NH 41 were generally enhanced in enriched sediment, but the rates varied temporally, being highest during the initial phase and decreasing gradually toward the end of the experiment. The initial (day 0–6) accumulation of TCO 2 in sandy sediment, however, was somewhat lower in enriched than in control jars; 508 and 726 nmol cm 23 d 21 , respectively (Fig. 4). TCO 2 production then increased rapidly in SA-jars, reaching maximum rates of 1939 nmol cm 23 d 21 from day 10 to 15. Subsequently, TCO 2 production rates ceased to 43 and 110 nmol cm 23 d 21 in SC- and SA-jars, respectively, during the last period (day 35–59). In muddy sediment, the initial (day 0–7) TCO 2 production in control and enriched jars were 184 and 1326 nmol cm 23 d 21 , respectively. During the last period (day 35–59) rates in MA-jars were only about twice those in MC-jars (130 and 60 nmol cm 23 d 21 , respectively). The temporal pattern of SO 422 in the various jars was generally a mirror image of TCO 2 (data not shown). The overall C:S stoichiometry was | 2.1 and | 2.0 in MC- and MA-jars, respectively, corresponding to the expected ratio of 2 for the stoichiometry of sulfate reduction. In sandy jars, however, the C:S stoichiometry was somewhat higher; | 2.6 and | 2.5 in SC- and SA-jars, respectively. Dissolved organic carbon (DOC) was detected in all jar types (Fig. 4), showing higher concentrations in sandy than in muddy sediment, and with highest concentrations in enriched jars. An accumulation of DOC occurred in enriched SA-jars within the first 10 days (12 mmol cm 23 ), but subsequently DOC decreased rapidly concurrent with the steepest increase in CO 2 and reached constant values in the range of 2.7–3.7 mmol cm 23 during the remaining period. In control jars, DOC only accumulated during the first 3 days (2.8 mmol cm 23 ) but decreased again within a few days to concentrations less than half the concentrations in SA-jars (range: 0.9–1.6 mmol cm 23 ). DOC was almost constant throughout the experiment in muddy sediment with generally lower absolute concentrations, but, due to higher porosity, had similar volume specific concentrations as in sandy sediment (range: 0.7–2.1 mmol cm 23 in MC-jars and 1.4–2.9 mmol cm 23 in MA-jars). NH 1 4 accumulated faster in sandy than in muddy jars with only limited initial impact of algal additions (Fig. 5). Initial rates (when corrected for adsorption) were 303 and 411 nmol cm 23 d 21 in SC- and SA-jars, respectively (day 0–3). A small depression was observed in SA-jars from day 8 to 13, which coincided with the period of rapid TCO 2 accumulation. The final rates (day 35–59) were 11 and 26 nmol cm 23 d 21 in SC- and SA-jars, respectively. The temporal pattern of NH 1 4 accumulation in the muddy jars differed markedly from that in sandy jars. NH 41 accumulation was more or less linear in MC-jars (14 nmol cm 23 d 21 , averaged over the entire experimental period). After an

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Fig. 4. Temporal pattern of TCO 2 and DOC (dissolved organic carbon) accumulation in sandy and muddy sediment jars over 59 days. Values are presented as mean6range of duplicates.

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Fig. 5. Temporal pattern of porewater NH 1 4 accumulation in sandy and muddy sediment jars over 59 days. Values are presented as mean6range of duplicates.

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initial increase in MA-jars (day 0–1) NH 1 4 concentrations decreased from day 1 to 3. This was followed by an almost linear NH 1 4 production during the remaining period (mean rate 27 nmol cm 23 d 21 ).

4. Discussion The two sediment types used in the present study had basically different characteristics (e.g. porosity and organic content; Table 1) even though they were obtained within a relatively short distance and contained largely the same particle sizes of the sandy mineral phase (unpubl. results). In order to minimize heterogeneity and to ascertain equal starting conditions, we have chosen to use sieved and homogenized sediment microcosms. As these procedures destroy the original chemical, physical and biological structure of the sediment (e.g. Kristensen and Blackburn, 1987; van Duyl et al., 1992), the results obtained from our laboratory measurements should not directly be extrapolated to in situ conditions.

4.1. Carbon dynamics The higher carbon mineralization in sandy than muddy control sediment despite a | 5-fold higher organic content (volume basis) in the latter (Fig. 1 Table 2), suggests that mineralization processes are more dependent on the degradability than the quantity of the organic pool (e.g. Westrich and Berner, 1984; Middelburg, 1989; Boudreau, 1992). The sandy sediment contained a relatively small, but highly reactive and nitrogen-rich organic pool (as indicated by a low C:N ratio of 7.7) derived from benthic microalgae, which are the dominating primary producers at the sandy site. The muddy sediment, on the other hand, contained a large, but relatively nitrogen-poor (C:N ratio 11.6) detritus pool derived mostly from deposited filamentous macroalgal debris (primarily Chaetomorpha linum and Cladophora sericea). The once so dominating filamentous macroalgal mats, however, disappeared from the muddy site | 1 ]12 years before the ˚ sampling date (Riisgard et al., 1995). The more labile fractions of the deposited macroalgae must have been mineralized shortly after the disappearance of the mats, leaving the more recalcitrant components in the sediment. An experiment performed 8 months earlier with sediment from the same location showed significantly higher specific carbon and nitrogen mineralization rates (Hansen, unpublished data), substantiating the progressive accumulation of refractory components in the sedimentary organic pool when no fresh substrate is being supplied. Carbon mineralization increased significantly as a result of algal enrichment (Table 2). This is in accordance with several other studies on microbial responses following additions of organic substrates to sediments (e.g. Kelly and Nixon, 1984; Hansen and Blackburn, 1992; Enoksson, 1993). The different patterns of DOC and TCO 2 accumulation in sandy and muddy sediment jars (Fig. 4) suggest different capacities of the anaerobic respirers (i.e. sulfate reducers) present initially. The instantaneous TCO 2 production and relatively limited DOC accumulation in muddy sediment implies that the

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anaerobic respirers present could keep pace of the primary microbial attack by populations of hydrolyzers and fermenters. In the organic-poor sandy sediment, on the other hand, TCO 2 production in enriched sediment was similar to control sediment during the initial phase (|8 days), while the extensive accumulation of DOC indicates enhanced activity by hydrolyzers and fermenters. The subsequent simultaneous steep increase in TCO 2 production and DOC consumption suggests that the population of sulfate reducers initially had a low capacity for mineralization of the produced DOC substrates, and needed a lag phase before reaching an optimum population size and metabolic capacity. A similar lag phase between DOC accumulation and TCO 2 production after addition of organic matter to anoxic sediments has previously been reported (Westrich and Berner, 1984; Kristensen and Hansen, 1995). Microbial populations in sandy sediments where benthic microalgae are the primary source of organic matter are generally poorly adapted and need time to adjust to an added substrate (C. linum) of different chemical composition. Anaerobic respirers in muddy sediments previously exposed to extensive input of filamentous algae may therefore be better adapted to this particular detritus type. The larger excess TCO 2 production due to enrichment in muddy sediment (Table 2) also indicates a higher instant capacity for carbon mineralization in this sediment type. When the excess carbon mineralization from enriched control cores is assumed to be derived from the added C. linum detritus, |67% of the added pool in muddy sediment was mineralized after 35 days, but only |15% in sandy sediment. In the jar experiment, however, excess TCO 2 production in enriched muddy and sandy sediment corresponded to |47 and |31% of the added carbon after 35 days. The higher production in enriched muddy cores than in jars is probably caused by exposure of the sediment surface of cores to oxygen. The gradually decreasing CO 2 evolution in enriched muddy jars (Fig. 4) suggests a decreasing reactivity of the added algal material under anaerobic conditions (Westrich and Berner, 1984). Kristensen et al. (1995) found that fresh and labile organic material is degraded at similar rates under aerobic and anaerobic conditions, whereas decomposition of old and refractory material is faster under aerobic than anaerobic conditions. So, the presence of more aggressive aerobic respiration processes in the oxic surface layer of enriched muddy cores appeared sufficient to maintain a more or less constant and high carbon mineralization throughout the experiment (Fig. 1). The lower carbon mineralization in enriched sandy cores than in jars may be explained by efflux of DOC across the sediment–water interface due to (1) the initial inability of sulfate reducers to exploit the new substrate and (2) subsequent SO 22 depletion in the lower sediment strata, which, in contrast to ‘jar sediment’, was not 4 22 enriched with SO 22 was measured in the core 4 . Unfortunately neither DOC nor SO 4 experiment. The extensive DOC accumulation in sediment jars (Fig. 4), however, suggest that initial efflux of DOC from sediment cores may have occurred. This hypothesis is supported by the relatively low C:N ratio (3.0) of accumulated porewater TCO 2 and NH 1 4 in these cores (Fig. 3 Table 3). Although the relative animal enhancement of carbon mineralization in unenriched was similar to or higher than enriched sediments, the enhancement in absolute terms was 1.5–1.7 times higher in the latter, indicating that N. diversicolor stimulated the mineralization of added algal carbon in both sediment types (Table 2). The absolute

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217

Table 3 Stoichiometry of dissolved inorganic carbon and nitrogen Sandy sediment

Core (a) C:N in fluxes (b) C:N in porewater (c) Total C:N Jar (d) C:N in jars

Muddy sediment

Unenriched

Enriched

12.6 4.6 6.5

13.7 3.0 5.8 7.9

5.2

8.9 4.9 7.8 –

Unenriched

Enriched

14.8 3.2 8.8

41.3 7.4 14.3

19.4 neg 20.4

neg 17.4 41.7

103.7 18.7 55.0

–

6.7

–

21.8

–

(a) fluxes across the sediment–water interface in sediment cores (C:N in fluxes), (b) accumulation in porewaters of cores (C:N in porewater), (c) the C:N ratio of total production in cores (total C:N), determined as the sum of fluxes and porewater accumulation, and (d) accumulation in jars (C:N in jars). neg–denotes negative DIN flux and consequentially negative C:N ratios.

fauna-induced excess TCO 2 production in enriched sediments was about the same in both types: 1621 and 1794 mmol C m 22 , respectively, corresponding to 31 and 34% of the added algal carbon in sandy and muddy sediment. This suggests that the faunalinduced enhancement of carbon mineralization when algal material is deposited and buried in sediments quantitatively is independent of the actual sediment type. Compared with the corresponding unenriched treatments, on the other hand, the excess carbon mineralization from enriched bioturbated cores is equivalent to 28 and 78% of the C. linum detritus added to sandy and muddy sediment, respectively. Enhanced TCO 2 production in bioturbated sediment is a combination of a direct metabolic contribution by the fauna and an indirect stimulation of microbial activity. The infauna contributes directly to organic matter decomposition via ingestion and assimilation of detritus and associated microorganisms, which may remove substrates otherwise available to the microbial decomposers (e.g. Cammen, 1980; Tenore et al., 1982). The respiratory TCO 2 production of N. diversicolor was not measured here, but has under similar conditions been estimated to be |27 mmol TCO 2 m 22 d 22 (Kristensen, 1989; Banta et al., in prep.) which is equivalent to 15–46% of the carbon mineralization. Moreover, the direct animal contribution accounts for 53–58% (enriched) and 80–97% (unenriched) of the bioturbation-induced excess TCO 2 production; highest in sandy sediment. These figures suggest that the stimulatory effect of burrowing infauna on microbial decomposition processes increases when the sediment is supplied with fresh organic matter. Studies have shown that enhanced microbial activity in bioturbated sediments of medium organic content generally accounts for more than half of the increased metabolism (O 2 uptake or TCO 2 production) relative to a defaunated sediment (e.g. Kristensen, 1985; Andersen and Kristensen, 1988). In accordance with the present study, however, Kristensen et al. (1992) found that most or all of the enhanced TCO 2 production caused by N. diversicolor in an organic-poor (,0.5% LOI) sediment originated from worm respiration alone. Accordingly, the enhanced supply of O 2 into the sediment due to bioturbation, predominantly stimulates degradation of old and refractory material. Thus, benthic macrofauna should primarily stimulate microbial mineralization in sediments with a large and refractory indigenous detritus pool (i.e.

218

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muddy sediment), while the macrofaunal impact on the microbial decay of fresh and relatively labile detritus is low (Andersen and Kristensen, 1992; Kristensen et al., 1992).

4.2. Nitrogen dynamics The present experiments demonstrated distinctly different temporal patterns of nitrogen transformations in sandy and muddy sediment (Fig. 2), and it is remarkable to observe that despite a much lower organic content, net rates of nitrogen mineralization were 3–4 times higher in the organic-poor sandy sediment (Table 2). The excess NH 1 4 production in enriched sandy sediment relative to the controls corresponded to a larger proportion (36–46%) of the added organic nitrogen than in muddy sediment (4–14%). Despite the high production rates, some NH 1 4 nitrogen must have been assimilated in enriched sandy sediment, as seen in jars (Fig. 5) during the phase of rapid TCO 2 production (day 8–15). NH 41 accumulation (denoted gross N mineralization in Table 2) in enriched muddy jars only exceeded the level in control jars after 12–14 days of incubation, suggesting that nitrogen was recycled rapidly by the microbial community resulting in a low apparent NH 1 4 production (Lancelot and Billen, 1985; Kristensen and Hansen, 1995) as also reflected in the much higher C:N ratio (21.8) of mineralization products relative to control sediment (Table 3). From the jar experiment it appears, that the initial carbon and nitrogen mineralization in both enriched sandy and muddy sediment was uncoupled (Figs. 4 and 5). The initial accumulation of DOC in sandy jars was associated with relatively low carbon and high nitrogen mineralization. Kristensen and Hansen (1995) described a similar pattern in organic-poor sediments enriched with either seagrass detritus or yeast. They proposed that hydrolyzing and fermenting bacteria transformed the added organic carbon to small organic molecules, while organic nitrogen was mineralized directly to NH 1 4 . The role of dissolved organic nitrogen (DON) was not addressed in the present study. However, DON may be quantitatively important in sedimentary nitrogen cycling (e.g. Lund and Blackburn, 1989; Hansen and Blackburn, 1992; Enoksson, 1993). The C:N stoichiometry of mineralization products in the porewater of cores and jars agrees reasonably well (Table 3). The low C:N ratios of 5.2 in sandy and 6.7 in muddy control jars indicate that nitrogen was mineralized preferentially to carbon in both sediment types since the C:N ratio of the bulk particulate organic pool was 7.7 and 11.6 in sandy and muddy sediment, respectively. Similar patterns of preferential nitrogen mineralization in marine sediments have been shown by several investigators (e.g. Klump and Martens, 1987; Boudreau et al., 1992; Kristensen and Hansen, 1995). The C:N ratios for the efflux of TCO 2 and DIN in all core types were higher than the C:N ratios of both the particulate organic pool and of the accumulated porewater pools, indicating loss of mineralized nitrogen by nitrification–denitrification. The bioturbation-enhanced net nitrogen mineralization was higher in sandy than muddy sediment (Table 2). NH 1 4 excretion rates from N. diversicolor added to sediment cores was estimated to be |2.1 mmol m 22 d 21 (B. Christensen, unpubl. data) and implies that worm excretion was a major source of NH 1 4 production by the sediment community. Estimated NH 41 excretion integrated over the entire experiment (|73 mmol), however, accounted for 89 (unenriched sandy), 152 (enriched sandy), 174

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219

(unenriched muddy) and 913% (enriched muddy) of the measured excess net nitrogen mineralization in bioturbated sediments (Table 2). This suggests that a part of the worm NH 1 4 excretion was nitrified and subsequently denitrified in the burrow walls or immobilized by the microbial community (Kristensen et al., 1992). A similar contribution of Nereis spp., accounting for a substantial fraction of or even more than the enhanced sediment NH 1 4 efflux has been observed in a number of studies (Henriksen et al., 1983; Kristensen, 1985). The gradually decreasing efflux of NH 1 4 in sandy control sediment, coinciding with a increasing NO 32 efflux towards the end of the experiment (Fig. 2) indicates a progressively growing population of nitrifying bacteria. Net influx of NH 1 4 in muddy control sediment and efflux of NO 2 3 also suggests nitrification activity in the upper oxidized sediment layer, and indicates that NH 41 produced in the lower sediment strata was insufficient to meet the NH 1 4 demand for nitrification processes. Enrichment of the 2 sediments resulted in a change in direction of the NO 2 3 flux from release to NO 3 uptake 2 in both sandy and muddy sediments (Fig. 2). A similar effect on NO 3 flux following input of algal material has been observed by others (e.g. Jensen et al., 1990; Hansen and Blackburn, 1992; Enoksson, 1993). The balance between nitrification and denitrification is an important determinant of sedimentary DIN exchange. The source of NO 2 3 for denitrification may be provided either from NO 2 3 diffusing from the overlying water into the sediment or from NO 32 produced within the sediment by nitrification (Seitzinger, 1988). The shift toward uptake of NO 32 may indicate that denitrification was stimulated and / or that nitrification was hampered. The nitrifying bacteria were probably limited due to narrowing of the oxic surface layer or inhibited by increased sulfide concentrations in the enriched sediments (Henriksen and Kemp, 1988; Sloth et al., 1995). The effect of organic loading on denitrification is largely determined by the NO 2 3 concentration in the overlying water (Caffrey et al., 1993; Sloth et al., 1995). When denitrification is considered insignificant in the anaerobically incubated jars due 1 to the lack of NO 2 3 supplies, the C:N ratio of mineralization products (CO 2 and NH 4 ) in jars (Table 3) should be representative for the ratio by which these compounds were produced in the anoxic sediment of the similarly treated cores. Based on measured gross C mineralization in cores and the C:N ratio of mineralization products in jars, an estimate of gross N mineralization can be obtained for all core types. By relating this estimate to the measured net N mineralization, a rough estimate of denitrification can be derived (Table 4). The estimated rates of denitrification ranged between 23.0 and 5.4 mmol m 22 d 21 with highest rates in bioturbated cores. The negative denitrification in enriched and defaunated sandy cores must be an artefact, possibly due to an initial loss of unaccounted DOC in these cores (se above). Accordingly no reliable denitrification estimates can be made from any of the enriched sandy sediments. The estimated denitrification rates in the other cores (1.5–5.4 mmol m 22 d 21 ), where DOC is of minor importance, are comparable to rates obtained in other coastal environments (Koike and Sørensen, 1988; Binnerup et al., 1992; Caffrey et al., 1993). The most pronounced trend among treatments was the high denitrification rates in bioturbated cores; a factor of 2–3 higher than in defaunated cores. A number of studies have shown that burrow-dwelling infauna may enhance both nitrification and denitrification rates up to 5 times (e.g. Kristensen et al., 1985, 1991; Pelegrı´ et al., 1994). The enhancement of denitrification is

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220

Table 4 Estimated mean daily nitrification and denitrification in the various core treatments Sandy sediment

mmol m

22

d

21

Measured gross C mineralization Estimated gross N mineralization Measured net N mineralization Estimated denitrification Estimated nitrification

Muddy sediment

Unenriched

Enriched

Unenriched

Enriched

SC

SN

SA

SAN

MC

MN

MA

MAN

43.7

71.3

66.6

112.9

22.1

56.0

121.6

172.9

8.4

13.7

8.4

14.3

3.3

8.4

5.6

7.9

6.8

9.1

11.4

12.8

1.5

2.7

2.9

3.1

1.6

4.6

23.0

1.5

1.8

5.4

2.7

4.8

2.0

4.8

23.3

1.0

2.2

5.2

1.8

2.4

Measured gross C mineralization is derived as the sum of TCO 2 fluxes and accumulated porewater TCO 2 (Table 2, normalized to daily rates). Estimated gross N mineralization is obtained from measured gross C mineralization divided by the C:N ratio of mineralization products in jars (Table 3). Measured net N mineralization is derived as the sum of DIN fluxes and accumulated porewater NH 1 4 (Table 2, normalized to daily rates). Denitrification is calculated as the difference between estimated gross and measured net N mineralization. Nitrification is calculated as denitrification plus measured efflux of NO 2 3 .

promoted by higher NO 2 3 availability caused by enhanced nitrification in the oxidized burrow walls (enhanced coupled nitrification–denitrification) and by increased down2 ward transport of NO 2 3 by ventilation activities. The high NO 3 uptake in enriched 2 muddy cores may partly be explained by the higher NO 3 concentrations in the overlying water, but also by lower rates of nitrification in these highly reduced sediments (Table 4).

Acknowledgements We thank Hanne Brandt for assistance in the laboratory. Thanks are also due to Uffe Thomsen and Mikael Hjorth Jensen for valuable discussions. This work was supported by the Centre for Strategic Environmental Research in Marine Areas, Grant no. 422 and SNF Grant no. 9601423.

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