R. Danovaro

Assessing the trophic state and eutrophication of coastal marine systems: a new approach based on the biochemical composition of sediment organic matter.

We used a biochemical approach based on the analysis of the quality and quantity of sedimentary organic matter for identifying new descriptors of the trophic state and environmental quality of coastal marine systems. A large-scale study, including 99 stations, belonging to 33 transects, was carried out along 250 km of the Apulian coasts (Mediterranean Sea) in March and September 2000. The investigated area covered a wide range of anthropogenic impacts (industrial ports, tourist harbours, areas affected by power plants and industrial wastes, mariculture areas). Other sites, including marine protected areas (i.e., without any apparent impact), were used as control. Water column and benthic parameters provided different indications and classifications of the trophic state of coastal marine systems. We found that phytopigment content of the sediments changed in response to all different sources of anthropogenic impact and resulted in a useful descriptor of the trophic state and environmental quality. Highest sediment chlorophyll-a concentrations, indicating conditions of increasing eutrophication, were found in areas impacted by the discharge of heated waters from a power plant. In particular, the contribution of the autotrophic biomass to the biopolymeric carbon pool appeared to be a good descriptor of the decreasing environmental quality. Independently from the sampling period or the pollution source such contribution was significantly lower in transects subjected to anthropogenic impact than in control areas. Differences in trophic conditions were evident both in terms of quantity (i.e., total organic matter content) and quality (i.e., biochemical composition) of sediment organic matter. In particular, sediment protein concentration appeared to be a good descriptor of the trophic state of the benthic systems at different spatial scales. Multivariate (MDS) analysis allowed identifying areas characterised by hypertrophic, eutrophic and meso-oligotrophic conditions and to define relative threshold levels. A classification of the trophic state of coastal systems based on protein and carbohydrate concentrations is proposed.

Benthic response to particulate fluxes in different trophic environments : a comparison between the Gulf of Lions-Catalan Sea (western-Mediterranean) and the Cretan Sea (eastern-Mediterranean)

Quantitative information on particle fluxes, sedimentary OM composition, microbial and meiofaunal parameters is summarised from various stations in the Gulf of Lions and Catalan Sea (North-Western Mediterranean) and in the Cretan Sea (Eastern Mediterranean), investigated between 1993 and 1996. Benthic responses in relation to the different trophic conditions in the two areas were compared in terms of: (1) temporal and spatial variability of the mass fluxes; (2) pelagic–benthic coupling in organic matter composition (such as proteins, lipids, soluble carbohydrates and CPE); (3) microbial response to changes in organic matter composition and POC fluxes; (4) meiofaunal response to changes in organic matter composition and particle fluxes; (5) relative significance of bacteria and meiofauna. The two areas were also compared to identify ratios between productivity, vertical fluxes and benthic standing stocks. Mass fluxes at equal depths were up to two orders of magnitude higher in the Western than in the Eastern Mediterranean. Clear seasonal changes were reported in both areas, although mass fluxes and variability were consistently higher in the North-Western Mediterranean. From primary production estimates in the Western and Eastern Mediterranean (140–160 vs. ≈19–60 mgC m−2 d−1, respectively) it has been calculated that a carbon export from the euphotic layer to 1000 m depth is equivalent to about 10% in the Gulf of Lions and 2–3% in the Cretan Sea. Chlorophyll-a concentrations, at similar depths, were 2–3 times higher in the Western basin. Carbohydrates were the dominant biochemical component in the Cretan Sea sediments while total amino-acids represented an important fraction of the biodegradable material in the Gulf of Lions–Catalan Sea. In the Western Mediterranean, bacterial densities (annual mean 7.9×108 cells g−1) were about 4 times higher than in the Cretan Sea (annual mean 2.1×108 cells g−1), indicating that, in deep-sea sediments, different trophic conditions are influencing bacterial densities. Meiofaunal abundance and biomass were similar on the continental shelves of both areas (940–2558 ind./10 cm2) but, at bathyal depths, densities in the Cretan Sea (range: 60–120 ind./10 cm2) were 4–25 times lower than those in the NW-Mediterranean (500–1500 ind./10 cm2). In contrast to what was observed in the North-Western Mediterranean, deep-sea meiofaunal assemblages of the Cretan Sea did not react (in terms of density or biomass) to the seasonal variations in food inputs. Here, the bacterial to meiofaunal biomass ratio displayed much higher values (up to >20), possibly causing competition for food sources with small metazoans. The efficiency with which the POC fluxes were being exploited was estimated. In the NW-Mediterranean about 0.14 mgC/d are available to each meiofaunal individual compared to the 0.07 mgC ind.−1 of the Eastern Mediterranean. Similarly, 3.3 mgC were provided daily per 1 mgC of bacterial biomass in the Western Mediterranean, compared to 0.07 in the Eastern Mediterranean. These data indicate that the benthic components in the Cretan Sea, are subject to more limiting trophic conditions, and so might have a higher efficiency in exploiting the particulate organic fluxes.

Large Fraction of Dead and Inactive Bacteria in Coastal Marine Sediments: Comparison of Protocols for Determination and Ecological Significance

ABSTRACT It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 ± 0.2) × 108 cells g−1 and (53.1 ± 16.0) × 108 cells g−1 in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was “reactivated.” Bacterial turnover rates estimated ranged from 0.01 to 0.1 day−1 but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day−1). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.

Determination of virus abundance in marine sediments

ABSTRACT In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.

Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments

ABSTRACT We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.

Labile organic matter and microbial biomasses in deep-sea sediments (Eastern Mediterranean Sea)

Abstract The distribution and the biochemical composition of organic matter were analysed in surface sediments (0–15 cm) of 22 continental and bathyal stations (110–2401 m; Ionian and Aegean Seas). Variations of labile organic matter (LOM), total organic matter (TOM), bacterial density and biomass were related to the benthic bacterial populations and to the environmental conditions. TOM was significantly correlated with water depth, but not with LOM, which was mostly composed of carbohydrates (89.6) followed by proteins and lipids (5.4 and 5.0.%). LOM ranged between 1.5 adn 2.7 mg g −1 sediment dry weight, declining in concentration from the sediment surface of 15 cm depth. Bacterial counts ranged betweem 0.16 and 9.42 × 10 8 cells g t-1 sediment dry weight. Bacterial density and biomasses in surface sediments (0–1 cm) were significantly correlated with carbohydrates and DNA concentrations. Although the overlying waters are oligotrophic and low concentrations of TOM are present, the relatively high percentage of LOM and bacterial density suggest that organic matter in deep-sea sediments of the Eastern Mediterranean Sea is not totally depleted of nutritional value.

Microbial and meiofaunal response to intensive mussel-farm biodeposition in coastal sediments of the Western Mediterranean

We studied the impact of organic loads due to the biodeposition of a mussel farm in a coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry, microbial and meiofaunal assemblages were investigated from March 1997 to February 1998 on monthly basis at two stations: the first was located under the mussel farm, while the second was at about 1-km distance and served as control. Benthic response to changes in the biodepositional regime was investigated in terms of biochemical composition of the sedimentary organic matter, phytopigment content, bacterial abundance and composition and meiofaunal community structure. A large accumulation of chloroplastic pigments, proteins and lipids was observed under the mussel farm. Such changes in the sedimentary conditions reflected the accumulation of faeces and pseudo-faeces and led to the creation of reducing conditions. Microbial assemblages beneath the mussel cultures increased their densities and displayed, when compared to the control, a larger cyanobacterial importance associated to a strong decrease of the picoeukaryotic cell density. Farm sediments displayed significant changes in meiofaunal density: turbellarian, ostracod and kinorhynch densities decreased significantly, while copepods remained constant or increased possibly profiting of the enrichment in microphytobenthic biomass associated to mussel biodeposits. The comparative analysis of the mussel biodeposition and fish-farm impact on sediments beneath the cultures revealed that mussel farms induced a considerably lower disturbance on benthic community structure.

Differential responses of benthic microbes and meiofauna to fish-farm disturbance in coastal sediments

Bacterial and meiofaunal abundance and biomass and their response to the disturbance induced by fish-farm biodeposition were investigated from March to October 1997 on a monthly basis at two stations of the Gaeta Gulf (Tyrrhenian Sea, Mediterranean Sea). The biopolymeric fraction of the organic matter was characterized by high concentrations which was similar at both fish-farming-impacted and control stations. Similarly, bacteria accounted for a small fraction of the biopolymeric organic carbon (< 1%), while the contribution due to auto-fluorescent cell biomass (i.e. prokaryotic and eukaryotic cells displaying auto-fluorescence) to the total biopolymeric carbon was quantitatively negligible (< 0.1%). Benthic bacteria appear to be sensitive to organic enrichment as their abundance increased significantly beneath the cage, whilst numbers of meiofauna was lower than in the control. Changes occurred also in terms of individual nematode biomass that increased as result of the biodeposition. A particularly useful tool appeared to be represented by the ratio of benthic auto-fluorescent cells to bacterial abundance, bacteria to meiofaunal biomass and auto-fluorescent cells to meiofauna biomass. All these parameters described well the impact due to biodeposition on the benthic environment as their ratios displayed significantly higher values in farm sediments, but recovered rapidly (15 days) to values observed in the control (i.e. undisturbed conditions) immediately after cage removal. Changes observed in the present study highlight that the increased organic loading determined a shift of the relative contribution of the different benthic components to the total biopolymeric carbon, so that in highly impacted systems total benthic biomass becomes increasingly dominated by microbial components.

Initial Fish-Farm Impact on Meiofaunal Assemblages in Coastal Sediments of the Western Mediterranean

Abstract We studied the initial impact of organic loads due to the biodeposition of a new fish farm in a non-impacted coastal area of the Tyrrhenian Sea (Western Mediterranean). Sediment chemistry and meiofaunal assemblages were investigated from July 1997 to February 1998 on a monthly basis at two stations: the first was located under the fish farm, while the second was at about 1 km distance, and served as control. Variations in the biochemical composition of the sedimentary organic matter and meiofaunal community structure were also related to changes in reared fish biomass. The presence of the cage-induced rapid changes in the benthic compartment: the sediments reached rapidly (i.e., after only 6 weeks) reducing conditions. A significant accumulation of biopolymeric carbon was observed beneath the cage both at the beginning of this study and 5–7 months after cage disposal and appeared to be related to the fish-farm production cycle. Sedimentary proteins and lipids resulted to be good descriptors of medium-term fish-farm impact. Changes in the sediment conditions beneath the cage determined a significant reduction of the total meiofaunal density (on average for 70%). Most meiofaunal taxa, including nematodes, copepods and ostracods displayed a significant decrease in the farm sediments within 3 months after cage installation. Kinorhynchs appeared extremely sensitive to reducing conditions of the sediments and disappeared almost completely, whereas polychaete densities remained unvaried. After initial impact meiofaunal assemblages responded to organic enrichment recovering, at least partially, their apparent structural characteristics. These data indicate that studies carried out several months after cage deployment might underestimate the actual impact on benthic assemblages.

Nematode community response to fish-farm impact in the western Mediterranean

A previous investigation on fish-farm biodeposition effects on benthos, carried out in the Gaeta Gulf (northwestern Mediterranean Sea), revealed a strong impact on meiofaunal assemblages. This study implements these findings by examining in detail the nematode assemblage and its response to organic enrichment from the start of a fish farm activity to the conclusion of the fish rearing cycle. Density, community structure and individual size were utilised for univariate (genus, trophic diversity and abundance patterns) and multivariate analyses (MDS) in order to identify the best descriptors of impact and the response of the nematode assemblages. Nematodes displayed significantly reduced densities, diversity and richness in sediments beneath fish farms. The impact of biodeposition was evident not only from structural community parameters but also in terms of functional indices. Forty-five days after starting fish farming, an increase of the nematode individual biomass was observed. MDS ordination pointed out the presence of two different nematode communities in disturbed sediments and in the control site. These results were substantiated by the analysis of the temporal changes of k-dominance curves, the maturity index and, to a lesser extent, by the index of trophic diversity. Some nematode genera were highly sensitive to biodeposition (Setosabatieria, Latronema and Elzalia) and disappeared almost completely in farm sediments, whereas other genera largely increased their dominance (Sabatieria, Dorylaimopsis and Oxystomina). This study indicates that nematodes are very sensitive to this kind of environmental disturbance. The use of simple tools, such as the k-dominance analysis and maturity index, are recommended for monitoring of aquaculture impact.