Benthic bacterial response to variable estuarine water inputs

Abstract

Estuarine waters are known to enhance productivity in coastal environments, but little is known about the impact that nutrient-rich waters can have on benthic microbial processes. After intensive surface sediment sampling in a wide coastal area impacted by river plume waters, we performed on-site microcosm experiments in which we added estuarine waters, rich in both organic and inorganic N and P, to pristine offshore sediments. This experimental approach has been conducted in different coastal areas for two consecutive years, in which the river-plume waters differed in both inorganic and organic N and P content. Benthic bacterial response (total bacterial abundance and biomass, aminopeptidase, β-d-glucosidase and bacterial C production) was investigated in treated and non-treated sediments. All structural and functional microbial variables increased significantly after plume-water supply (within 12–24 h bacterial abundance in the sediment doubled and enzymatic activities increased up to >50%). Results indicate that inorganic N supply from plume waters was sufficient to induce a significant response in benthic bacterial abundance, independently from the presence of high and/or variable N:P ratios. However, bacterial carbon production and exo-enzymatic activities increased significantly when the supply of organic P from plume waters was associated with a decrease of organic N:P ratios. We conclude that plume waters have important effects on benthic bacterial dynamics, but the extent of their biogeochemical implications is largely dependent upon their organic P availability and on stoichiometric ratios of organic nutrients supplied by plume waters.

Benthic bacteria, Estuarine waters, Nutrients

1 Introduction

“Nutrient-fertilisation” associated with freshwater plumes is known to influence primary productivity [1] and to stimulate bacterioplankton production and respiration in wide sectors of continental shelves [2–5]; this, together with the consequent increase of organic matter fluxes to sediments (pelagic–benthic coupling) is expected to cause a significant benthic response [6].

Freshwater plumes are highly dynamic and their areal extension and intensity can vary widely over short time scales [7]. This highly dynamic behaviour can have important ecological and biogeochemical consequences [8], as previous field studies reported that areas influenced by river waters displayed different accumulation and biochemical composition of sediment organic matter [9]. Changes in trophic conditions of the sediment surface were found to affect the distribution and composition of meiofaunal and macrofaunal assemblages [10,11], and to be associated to changes in exo-enzymatic activities and benthic bacterial biomass [9,12]. However, although it is clear that estuarine waters can affect the benthic compartment, factors and mechanisms responsible for such changes have not yet been identified.

Freshwater plumes can play an important role in microbial dynamics at least in two ways: (i) by modifying the quantity and availability of organic nutrients to heterotrophic bacteria and/or (ii) by supplying inorganic nutrients, which can be used by heterotrophic bacteria as nutritional supplements. In this regard, bacterioplankton assemblages are known to compete with autotrophic components for inorganic nutrient uptake, which is used to supplement organic sources poor in N and P, especially in oligotrophic systems [13–17]. Nonetheless, if heterotrophic bacterial growth is significantly stimulated by a large inorganic nutrient supply, this could have important consequences for organic matter cycling and overall ecosystem functioning [15]. This applies also to benthic bacteria, which are expected to respond to both organic and inorganic nutrient addition, but the extent of such response, especially in field conditions, has not been investigated yet. Recent studies provide conflicting results, as field investigations demonstrated a higher bacterial carbon production in coastal areas influenced by river plume waters [11], whereas studies conducted in laboratory microcosms reported that benthic bacteria did not increase in abundance after addition of inorganic N and P, thus leading to hypothesize that inorganic nutrient alone are not always sufficient to induce a numerical benthic bacterial response [18].

To better understand factors influencing benthic bacterial metabolism and activity in coastal sediments, it is important to clarify whether the benthic bacterial response to a freshwater plume is stimulated by the organic or by the inorganic nutrient supply. Moreover, it is not clear how benthic bacteria respond to changes in N:P ratio. In order to elucidate the impact of estuarine waters with different nutrient conditions on benthic bacteria, we carried out on-site microcosm experiments, in which we added estuarine waters (rich in both organic and inorganic nutrients and collected at the sediment–water interface) to pristine offshore sediments. This experimental approach was adopted to mimic the spread of a plume over offshore benthic system. The experiment was conducted in different coastal areas of the Northern Adriatic Sea (Mediterranean Sea) for two consecutive years, in which the river-plume waters were characterised by different N:P ratios and dissolved organic nutrient concentrations (DOC, DON, DOP). Bacterial abundance, biomass, exo-enzymatic activities and C production were investigated in order to provide new insights on bacterial mediated biogeochemical consequences of estuarine water inputs.

2 Materials and methods

2.1 Study site

The Po river represents one of the major freshwater inputs of the entire Mediterranean basin (accounting for 30–50% of the total river inputs; Fig. 1) and displaying an annual mean discharge rate of 1600 m³ s⁻¹, with peaks up to 4000 m³ s⁻¹ (in spring and autumn; [19]). The Adriatic Sea is a semi-enclosed basin characterised, especially in the northern area, by high concentrations of inorganic nutrients (especially N because of the peculiarity of the Po river outflow) and high values of primary production, which strongly influence biological productivity of the whole basin [20]. The river plume is extremely dynamic and, due to the high variability of the river inputs, its extension and location are characterised by wide spatial–temporal changes. Generally the plume extends southeastwards for 10 to 100 km, depending on season and precipitation, causing variable gradients in salinity and nutrient supply [19].

1

Study area: reported are location of sampling stations, the areas selected for the seawater and sediment sampling in 1997 and 1998. Dotted line indicates the plume area.

2.2 Sampling strategy and experimental design

All experiments were carried out on board R/V Urania in the Adriatic Sea (Fig. 1) in February 1997 (EXP1), when a high rate of river flow was recorded (1429 m³ s⁻¹), and repeated in February 1998 (EXP2), when a much lower rate of river flow was recorded (937 m³ s⁻¹; data from Authority for River Po-Parma). For each sampling period, the experimental design included six microcosms: three contained offshore sediments supplemented with natural plume water (“treated” microcosms), and three contained offshore sediments and were incubated with their original overlying water (“control” microcosms).

To do this, we initially identified the river plume using a Tow-Fish SARAGO system, which allowed a fast horizontal survey of the extension of the low-salinity waters [9,11]. After identification and mapping of the extent of the plume area, we identified sites used for plume water sampling. These sites were located along the Italian coast south of the Po river delta in 1997 (43°65′04′ N, 13°18′06′ E) and in front of the Po delta in 1998 (44°41′12′ N, 12°31′05′ E; Fig. 1). Bottom depth of these sites ranged from 14.9 to 26.4 m. Water sampling was carried out using a multi-corer (Mod Midi, inner corer diameter 9.5 cm; [11]), which allowed collecting the top 20 cm water overlying the bottom. This water was carefully siphoned out from each core, prefiltered onto 0.2 μm sterile Millipore filters and used for our experiments.

Then we selected the areas for sediment sampling out of the influence of the river plume: at 30 miles offshore (43°83′41′ N, 13°43′07′ E) in 1997, and at ca. 50 miles east of the river delta (44°38′86′ N, 13°28′06′ E) in 1998. In this case, stations depth ranged from 40.1 to 49.8 m. Undisturbed sediment samples were collected using the same multi-corer. Each replicate (n= 3) was sampled from independent deployments. “Control” microcosms were set keeping sediment cores (sediment layer: 0–2 cm, equivalent to a volume of ca. 250 cm³) with their overlying water (250 cm³) in sterilised glass containers (1000 cm³). “Treated” microcosms were made of sediments in which the overlying water was removed immediately after recovery and carefully replaced with 250 cm³ of plume water collected as previously described. Time between plume water sampling and sediment sampling was always less than 6 h, during which water was stored in the dark at in situ temperature. All microcosms were incubated onboard at in situ temperature for 36 h and sampled at time 0 and after 8, 12, 24, and 36 h.

Nutrient rich water supplemented to pristine sediments was collected in coastal stations where the plume extended down to the sediment–water interface. We selected areas closer to the offshore pristine sediments. This was done to mimic the actual behaviour of plume waters, which when rich offshore sediments are largely diluted with high salinity marine waters but still rich in organic nutrients and these conditions are known to be those stimulating biotic response in frontal areas [6,9–11].

2.3 Analysis of dissolved nutrients and DOC

Inorganic nutrient concentrations (NO₂, NO₃, NH₄, PO₄) were determined before starting the incubations both in nutrient-rich (estuarine) and control (i.e., overlying the sampled sediments) waters. Analyses were carried spectrophotometrically using an auto-analyser [21,22]. Total dissolved nitrogen (TDN) and total dissolved phosphorus (TDP) were measured according to Walsh [23] and Koroleff [24]. Dissolved organic nitrogen (DON) and dissolved organic phosphorus (DOP) were calculated as the difference between TDN and inorganic nitrogen (NO₂+ NO₃+ NH₄) and between TDP and inorganic phosphorus (PO₄), respectively [25,26]. Dissolved organic carbon (DOC) was determined by filtration of seawater with a 0.45 μm filter followed by high temperature catalytic oxidation (HTCO) using a Carlo Erba TOC analyser (model 480).

2.4 Bacterial abundance, biomass and production

Total counts of bacteria were performed using acridine orange according to standard protocols [9]. For each of the six microcosms (three treated microcosms and three control microcosms), sediment subsamples (ca. 1 ml) were collected in three replicates, transferred into sterile test tubes and fixed with 4 ml of 0.2 μm pre-filtered formalin (2% final concentration), previously buffered with Na₂B₄O₇.10H₂O. Samples were sonicated three times (Branson Sonifier 2200; 60W for 1 min), diluted 250–1000 times with sterile, 0.2 μm prefiltered formalin (2% final concentration), stained for 5 min with acridine orange (final concentration 0.01%) and then filtered onto black Nuclepore polycarbonate 0.2 μm pore size filters at less than or equal to 100 mm Hg. For each slide at least 10 microscope fields were observed and at least 400 cells were counted per filter. Bacterial size was measured (as maximum length and width) using a micrometer on all cells counted. Bacterial biovolume was estimated assigning bacteria into three different size classes [9]: small (<0.065 μm³), medium (0.065–0.320 μm³) and large (0.320–0.574 μm³), and then converted into carbon content assuming 310 fg C μm³ [27]. Average cell size was calculated as the ratio of total bacterial biomass to total bacterial abundance. Data were normalised to sediment dry weight after desiccation (60 °C, 24 h).

Bacterial carbon production was measured by incorporation of ³H-leucine [28]. Each sediment sample (0.2 ml of slurry, 3 replicates for each microcosm) was added to 6 μCi of l-[4,5-³H] leucine (Amersham; leucine added: 0.5 μM final concentration) and incubated in the dark for 1 h at in situ temperature. After incubation, bacterial incorporation was stopped by adding 80% ethanol. After two washes of the samples with ethanol (80%) by mixing, centrifuging and decanting the supernatant, the sediment was transferred with ethanol (80%) onto a polycarbonate filter (0.2 μm mesh size). Subsequently, the filters were washed four times with 5% TCA (tricloroacetic acid). Samples were heated in 2N NaOH for 2 h in a water bath at 100 °C. One ml of supernatant was transferred to scintillation vials and 10 ml of scintillation fluid was added. Measurements of radioactivity were run using a liquid scintillation counter (Packard, Tric-Carb 2100 TR). For each sample, three replicates and two blanks were analysed. Data of ³H-leucine incorporation were converted into bacterial carbon production using the formula (described in [29]):  

where nmol LEU_inc are nanomoles of leucine incorporated into bacterial proteins, 100/7.3 is the mol% of leucine into the total bacterial aminoacid pool [30], M is the molar weight of leucine, 0.86 is the conversion factor of bacterial protein production to bacterial C production [30], R is the correction for radioactive decay of the ³H-leucine, 2 is the intracellular isotope dilution (estimated as in Simon and Azam [30]) and g is sediment dry weight.

2.5 Exo-enzymatic activities

Analyses of extracellular enzymatic activities (β-d-glucosidase, MUF-β-glucopyranoside [MUF-glu] and aminopeptidase, l-leucine-4-methylcoumarinyl-7-amide [Leu-MCA]) were performed as described by Danovaro et al. [9]. Incubations were done by adding pre-filtered (0.2 μm) seawater. Exo-enzymatic activity measurements were carried out in triplicate for each microcosm by adding 150 μl of MUF-glu and Leu-MCA (final concentration 100 μM). After 1 h of incubation in the dark at in situ temperature [31], samples were centrifuged (3000 rpm, 5 min). The release of fluorescent dye was measured with a Perkin–Elmer spectrofluorometer at 380 nm excitation, 440 nm emission for MUF-glu; [31] and 365 nm excitation, 455 nm emission for Leu-MCA, [32]. Solutions of 7-amino-4-methylcoumarin and 4-methylumbelliferone were used as standards for Leu-MCA and MUF-glu, respectively (freshly prepared with pre-filtered and sterilized sea water). Each analysis was carried out on three replicates. Data were normalised to sediment dry weight after desiccation (60 °C, 24 h) and reported as nmol released g⁻¹ sediment h⁻¹.

2.6 Statistical analyses

In both years we identify differences among treated and untreated sediments (“control”) of all microbial parameters (bacterial abundance and biomass, bacterial C production and exo-enzymatic activities) by a two-ways ANOVA analysis (p level 0.05).

3 Results

3.1 Environmental variables and nutrient concentrations

Temperature ranged from 10.5 to 11.0 °C at all sites and sampling periods investigated. Salinity, oxygen content, inorganic and organic nutrient concentrations in offshore and estuarine waters used for our experiments are shown in Table 1. In both 1997 and 1998, plume and offshore waters displayed a salinity delta of ca. 2 PSU (35.7 vs 37.7 PSU in 1997 and 36.3 and 38.0 PSU in 1998, plume and offshore water, respectively).

1

Environmental parameters and nutrient concentrations during time course experiments carried out during cruises EXP1 and EXP2

Sampling area	Salinity	Oxygen	NO2		NO3		NH4		DIN		PO4		DON		DOP		TDN		TDP		DOC	N:P	DON:DOP	DIN:DIP
	μM	μM	SD	μM	SD	μM	SD	μM	SD	μM	SD	μM	SD	μM	SD	μM	SD	μM	SD	μM
EXP1 1997
Plume waters	35.7	314	1.05	0.30	6.64	2.52	0.39	0.17	8.08	2.99	0.02	0.01	7.87	2.00	0.21	0.05	15.95	3.66	0.23	0.01	84.2	69	37	420
Offshore waters (control)	37.7	269.9	0.39	0.10	1.20	1.05	0.17	0.15	1.76	1.30	0.03	0.01	6.22	0.89	0.08	0.03	7.99	2.04	0.11	0.06	96.7	71	73	61
Ratio (plume to offshore)			2.7		5.5		2.3		4.6		0.7		1.3		2.5		2.0		2.1		0.9	1.0	0.5	6.9
Statistical significance			∗		∗		ns		∗		ns		ns		∗		∗		∗		ns
EXP2 1998
Plume waters	36.3	305.4	0.46	0.24	4.70	2.32	0.26	0.09	5.42	2.65	0.01	0.01	10.47	2.70	0.31	0.08	15.90	2.11	0.32	0.09	138.3	49	33	543
Offshore waters (control)	38.0	273.4	0.29	0.09	0.54	0.12	0.11	0.07	0.93	0.29	0.01	0.01	8.98	2.53	0.30	0.22	9.92	2.62	0.31	0.14	154.2	31	29	108
Ratio (plume to offshore)			1.6		8.8		2.4		5.8∗		1.2		1.2		1.0		1.6		1.0		0.9	1.6	1.1	5.0
Statistical significance			ns		∗		ns		∗		ns		ns		ns		∗		ns		ns

TDN, total dissolved nitrogen; TDP, total dissolved phosphorus; DON, dissolved organic nitrogen; DOP, dissolved organic phosphorus. SD, standard deviation. ^∗ Indicates p < 0.05; ns, not significant.

NO⁻₂ concentrations were significantly higher in plume waters than in offshore control waters in 1997 (p < 0.05), but not in 1998 (Table 1; ANOVA, ns); NO⁻₃ concentrations were significantly higher in plume waters then in offshore control waters in both the two experiments (p < 0.05), whereas NO⁻₄ concentrations did not differ among plume and offshore waters (Table 1; ANOVA, ns). As a result, dissolved inorganic nitrogen was significantly higher (about 5 times; Table 1) in plume then in offshore waters in both experiments (p < 0.05).

Phosphates, whose concentrations were always close to their analytical detection limit, did not differ among treated and control waters in the two years (Table 1; ANOVA, ns). Organic N and organic P concentrations were not significantly different between plume and offshore waters (Table 1; ANOVA, ns), with the exception of DOP in 1997, which was double in plume compared to offshore waters (p < 0.05).

Total dissolved N concentrations were significantly higher (roughly double) in estuarine waters as compared to offshore waters (TDN ranging from 15.90–15.95 μM in estuarine waters to 7.99–9.92 μM in offshore waters; ANOVA p < 0.05). Total dissolved P concentrations in plume waters were about double compared to the offshore waters in 1997 (p < 0.05), but no significant differences were observed in 1998 (Table 1; ANOVA, ns).

DOC concentrations had similar values in plume and offshore waters in both experiments (range 84.2–96.7 μM in 1997 and 138.3–154.2 μM in 1998; Table 1).

N:P ratios in plume vs control seawater were similar during 1997 (69 vs 71), but not during 1998, when they decreased from 49 to 31. DIN:DIP ratios were seven- and five times higher in plume waters rather than in control waters, in 1997 and 1998, respectively.

DOC, DON and DOP concentrations in plume and control waters were lower in 1997 and higher in 1998 (p < 0.05), a year in which a lower flow rate of the river Po was observed. In both years, DIN supply from estuarine waters was always extremely high, leading to an increase of inorganic N:P ratio (up to 420–543 vs 61–108 for DIN:DIP ratio). The N:P ratios were extremely high, especially when compared with stoichiometric ratios typical for bacterial biomass (N:P ca. 5.5–7.9), thus leading to potentially P-limited conditions. The supply of total dissolved N was almost identical in the two years (15.95 and 15.90 μM in 1997 and 1998, respectively). The main difference in estuarine nutrient supply between 1997 and 1998 was related to organic P load (DOP). In 1998, organic P concentrations were, in both plume and control waters, higher than in 1997 (Table 1), but not significantly different among treated and control microcosms (see text above). Conversely, in 1997, plume waters contained an amount of organic P ca. 2.5 times higher than control waters; thus, plume waters were an important input of dissolved organic phosphorus, which resulted in a decrease of DON:DOP ratio (from 73 to 37).

3.2 Bacterial parameters

The results of the two time-course experiments are shown in Figs. 2–7. The comparisons between treated and control samples revealed that, in both years, treated samples showed a rapid and significant (p < 0.05) increase in bacterial direct counts (by 50% within the first 12 h, which roughly doubled after 24–36 h; Fig. 2). Bacterial biomass displayed similar patterns, but the increase in bacterial biomass was different in the two years (mean increase: 14% vs 52% in 1997 and 1998, respectively; Fig. 3), whereas bacterial cell size (despite a tendency to barely decrease in amended microcosms) did not change significantly with time or treatment (Fig. 4).

2

Total bacterial abundance in control and treated microcosms measured during the time course experiments. (a) In 1997 (EXP1) and (b) in 1998 (EXP2). Error bars were calculated considering an average value derived from triplicate determinations carried out for each microcosm. na, Not available.

3

Bacterial biomass in control and treated microcosms measured during the time course experiments. (a) In 1997 (EXP1) and (b) in 1998 (EXP2). Error bars were calculated considering an average value derived from triplicate determinations carried out for each microcosm. na, Not available.

4

Bacterial cell size in control and treated microcosms measured during the time course experiments. (a) In 1997 (EXP1) and (b) in 1998 (EXP2). Error bars were calculated considering an average value derived from triplicate determinations carried out for each microcosm. na, Not available.

5

Bacterial carbon production in control and treated microcosms measured during the time course experiments. (a) In 1997 (EXP1) and (b) in 1998 (EXP2). Error bars were calculated considering an average value derived from triplicate determinations carried out for each microcosm.

6

l-Aminopeptidase activities in control and treated microcosms measured during the time course experiments. (a) In 1997 (EXP1) and (b) in 1998 (EXP2). Error bars were calculated considering an average value derived from triplicate determinations carried out for each microcosm.

7

β-d-glucosidase activities in control and treated microcosms, measured during the time course experiments in 1998 (EXP2). Error bars were calculated considering an average value derived from triplicate determinations carried out for each microcosm.

Bacterial C production (BCP; Fig. 5) was significantly enhanced after treatment with nutrient-rich plume waters in both years (p < 0.05). During both experiments, the highest increase of bacterial carbon production was observed after 8 h (103% in 1997 and 52% in 1998). Bacterial carbon production in treated microcosms increased on average by 22–38%, when compared with the control (in 1998 and 1997, respectively).

Bacterial exo-enzymatic activities generally displayed a positive response to the addition of nutrient-rich plume waters, but with clear differences in the two years (Figs. 6, 7). In 1997, the addition of nutrient-rich waters did not result in a significant increase of aminopeptidase activity (on average the increase was ca. 3% compared with the control). However, in 1998, aminopeptidase activity increased significantly (on average by 57%; p < 0.05). In both years, the highest percentage increase was observed after 8 h, in correspondence with the highest increase in BCP. In 1998, β-d-glucosidase activity (which was not measured in 1997) displayed a positive response to enrichment with estuarine-waters (again highest after 8 h), with an average increase of 37%.

4 Discussion

In coastal systems, a benthic response to estuarine waters is expected, but reports on the response of benthic bacterial assemblages to plumes of nutrient-rich estuarine water are scarce. We investigated the response of offshore benthic bacterial assemblages to the addition of estuarine nutrient-rich waters in northern Adriatic Sea. To do this, we simulated the spread of a plume of lower salinity waters on offshore surface sediments (which are only occasionally impacted by the arrival of the plume). The comparison between two subsequent years (1997 and 1998) enabled us to better understand the effects of variable river-flow rates, which produced notable differences in the extension of sediments covered by the plume water, as well as qualitative and quantitative differences in the bacterial response due to varying nutrient conditions in the estuarine plume waters.

The advantage of the present experimental approach is dual: (i) it allows experimental manipulation with natural plume waters, and therefore with natural sources of inorganic and organic nutrients (i.e., without the addition of N and P in chemical forms that are commonly used for laboratory experiments). This makes it possible to investigate the bacterial response to N and P sources as they occur in the environment; (ii) it allows direct examination of the effects of actual shifts in nutrient concentrations. This is a novel approach because most available studies involving nutrient enrichments are conducted by adding artificial mixtures of nutrients (arranged to Redfield ratios [33]; or to values arbitrarily selected [25,34–38]).

The DIN supply from estuarine waters was always extremely high, leading to an increase of the inorganic N:P ratio, indicative of potential P-limited conditions. Although the supply of total dissolved N was almost identical in the two years, clear differences were observed in terms of organic P load, which were higher in 1998 than in 1997.

Heterotrophic bacterial assemblages are expected to respond to increased availability in inorganic nutrients in aquatic and terrestrial environments [16,25,39]. Previous studies carried out in coastal sediments of the Adriatic Sea suggest that enzymatic activities and rates of degradation of organic matter are also stimulated in sediments beneath the estuarine water plume [9].

Results presented here indicate that benthic bacterial assemblages were stimulated by the arrival of dissolved nutrients brought by the estuarine waters, causing significant increases in abundance, biomass and carbon production. The supply of estuarine water to offshore sediments induced rapid microbial response: benthic bacterial abundance increased in both 1997 and 1998 by ca. 50% within 8–12 h. This finding also suggests that the influence of the river plume on benthic microbial assemblages can be consistent, relevant and widespread (reaching an extension of several miles).

A positive benthic bacterial response might be caused by several factors (including temperature, organic nutrients, etc.), but the present study suggests that the factor, which characterised consistently the two sampling years and all experimental conditions, was increased DIN concentrations. In fact, in both years DIN concentrations in plume waters were roughly double than that in offshore sites at the sediment–water interface, whereas temperature, DOC and inorganic P concentrations did not change. Our results suggest that, as previously reported for bacterioplankton [14], the supply of dissolved inorganic nitrogen might induce a significant and positive numerical response also in bacteria from coastal sediments.

Changes in DIN concentration apparently had no effect on bacterial size and this result is also consistent with field observations in a large scale study on the whole Adriatic basin, in which bacterioplankton from estuarine and off-shore areas displayed similar size [40]. Our results therefore suggest that the simple addition of inorganic N is sufficient to induce a significant numerical response, independently from the presence of high and/or variable N:P ratios. Moreover, bacterial response to nutrient supply was so fast that the arrival of a freshwater plume (a temporarily and highly dynamic phenomenon) can have an impact over wide sea-bottom areas.

Heterotrophic bacteria tend to have high P requirements relative to phytoplankton and, particularly in diluted estuarine waters, primary production might be limited by P concentrations and/or high N:P [5,37]. Bacterioplankton in the Mediterranean basin is generally assumed to be P-limited [41]. Phosphorus limitation of heterotrophic bacterial activity has also been reported for other coastal marine waters [25]. Conversely, it generally was assumed that most of the P pools are accumulated and recycled in surface sediments, so that sediments are not expected to display P-limited conditions [42]. Our experiments indicate that also in marine sediments, organic P can limit benthic bacterial growth. Based on our data, it is possible to hypothesize that the supply of organic P with estuarine waters stimulated bacterial carbon production, however the extent of stimulation was higher when the supply of organic P from plume waters was associated with the decrease of the organic N:P ratio. It is thus possible to conclude that benthic bacterial carbon production was significantly stimulated by the arrival of plume waters that are rich in organic P, and particularly when the supply of organic P caused a decrease of the organic N:P ratio. Moreover, since in 1997 plume waters contained about 30% less inorganic P than offshore waters, we can hypothesise that organic P could represent one of the main limiting factors for bacterial metabolism in the investigated coastal sediments.

In marine systems, nutrient concentrations and cycling greatly influence the dynamics and structure of the food webs. Several studies hypothesised that limiting nutrient conditions can reduce bacterial growth rates, biomass and carbon demand thus leading to the “malfunctioning” of the microbial loop [37,43]. This, in turn, might have significant implications for organic carbon storage or degradation and subsequent export of DOC.

Our results suggest that the supply of nutrient-rich waters, which enhanced rates of bacterial production by roughly 50%, might accelerate sediment organic carbon consumption and therefore the diagenesis of organic matter. This was also confirmed by the analysis of exo-enzymatic activities in the sediment. Benthic bacterial uptake is mediated by enzymatic activities (such as aminopeptidase and glucosidase), which degrade proteins and carbohydrates into low molecular weight substrates (i.e., amino acids and glucose) directly available to bacteria. Enzymatic activities increased (i.e., by 37–57% for glucosidase and aminopeptidase) immediately after the addition of estuarine nutrient-rich waters to offshore sediments (i.e., within 8 h) in both years, indicating that also enzymatic activities responded to nutrient inputs. However, such increase was of different relevance in the two years. These results suggest that plume waters can stimulate enzymatic activities, but such effect is significant only in conditions of lower N:P ratios (in our case, N:P < 50). Since increased exo-enzymatic activities mean increased rates of organic matter mobilisation, with consequent release of dissolved organic matter from the sediment, we suggest that the arrival of a plume on offshore sediments can have important implications for organic matter storage and/or burial in the sediment. However, results show that the extent of these biogeochemical implications is largely dependent on organic P availability and on stoichiometric ratios of organic nutrients supplied by plume waters.

Finally, the selection of different sedimentary systems for comparing the effects of plume waters in the two subsequent years had the advantage of allowing the identification of consistent microbial responses to nutrient rich water addition, but had the limit of investigating habitats with likely different microbial assemblages. Future studies should take into account the taxonomic composition of benthic microbial consortia in order to highlight the presence of species-specific effects.

Acknowledgements

The authors are indebted to Dr G. Catalano and Dr S. Cozzi (Institute of Marine Science –Trieste; National Research Council – Italy) for help in nutrient analyses and to Dr A. Russo (Department of Marine Science – Polytechnic University of Marche, Italy) and Dr A. Puddu (CNR–IRSA) for providing information on river discharge and DOC measurements. This work was financially supported by the Italian Government MURST (PRISMA Research Project) and by the EU within the frame of the programme INTERPOL (EU contract: EVK3-2000-00526) and MEDVEG (EU contract QLRT-2000-02456).