Microbial assemblages for environmental quality assessment: Knowledge, gaps and usefulness in the European Marine Strategy Framework Directive.

The EU Marine Strategy Framework Directive 2008/56/EC (MSFD) defines a framework for Community actions in the field of marine environmental policy in order to achieve and/or maintain the Good Environmental Status (GES) of the European seas by 2020. Microbial assemblages (from viruses to microbial-sized metazoa) provide a major contribution to global biodiversity and play a crucial role in the functioning of marine ecosystems, but are largely ignored by the MSFD. Prokaryotes are only seen as "microbial pathogens," without defining their role in GES indicators. However, structural or functional prokaryotic variables (abundance, biodiversity and metabolism) can be easily incorporated into several MSFD descriptors (i.e. D1. biodiversity, D4. food webs, D5. eutrophication, D8. contaminants and D9. contaminants in seafood) with beneficial effects. This review provides a critical analysis of the current MSFD descriptors and illustrates the reliability and advantages of the potential incorporation of some prokaryotic variables within the set of indicators of marine environmental quality. Following a cost/benefit analysis against scientific and economic criteria, we conclude that marine microbial components, and particularly prokaryotes, are highly effective for detecting the effects of anthropogenic pressures on marine environments and for assessing changes in the environmental health status. Thus, we recommend the inclusion of these components in future implementations of the MSFD.

Disentangling natural and anthropogenic impacts on groundwater by hydrogeochemical, isotopic and microbiological data: Hints from a municipal solid waste landfill.

Within human-impacted areas, high levels of inorganic compounds in groundwater are broadly and preventively attributed to local anthropogenic pollution, thoroughly disregarding geogenic natural background levels. Particularly in landfills, a proper evaluation of the significant adverse environmental effects should be completed through a detailed groundwater characterization, and appropriate reference values established prior to landfill onset. However, the monitoring network may lack a full hydrogeological representativeness of the site and of the background conditions of groundwater. This study aimed at disentangling natural and anthropogenic impacts through a synoptic analysis of hydrogeochemical, isotopic and microbiological characteristics of groundwaters from a municipal solid waste landfill area in Central Italy. Samples were collected during four seasonal monitoring surveys from the mostly anoxic aquifer underlying the target area. Field parameters, inorganic and organic compounds, environmental isotopes, faecal contamination, and microbial community characteristics were determined, along with a detailed hydrogeological conceptual model. Key inorganic contaminants (As, Fe and Mn) exceeded the local threshold values in most of the sampling points, while organic contamination was generally very low. Stable isotopes suggested that groundwater originated mainly from local rainfall, except at one monitoring points where tritium levels might indicate moderate impact. Microbiological data and the microbial community characterization, assessed by flow cytometry and BIOLOG assays, provided further supportive information, also highlighting fundamental effects of groundwater quality alterations. Overall, an integrated multi-parametric approach proved suitable to distinguish geogenic and anthropogenic impacts, thus improving strategies and schemes for protection and management of groundwaters in landfills and waste related industrial areas.

Colonization of overlaying water by bacteria from dry river sediments.

We studied the diversity, community composition and activity of the primary microbial colonizers of the water above freshly re-wetted sediments from a temporary river. Dried sediments, collected from Mulargia River (Sardinia, Italy), were covered with sterile freshwater in triplicate microcosms, and changes of the planktonic microbial assemblage were monitored over a 48 h period. During the first 9 h bacterial abundance was low (1.5 x 10(4) cells ml(-1)); it increased to 3.4 x 10(6) cells ml(-1) after 28 h and did not change thereafter. Approximately 20% of bacteria exhibited DNA de novo synthesis already after 9 h of incubation. Changes of the ratios of (3)H-leucine to (3)H-thymidine incorporation rates indicated a shift of growth patterns during the experiment. Extracellular enzyme activity showed a maximum at 48 h with aminopeptidase activity (430.8 +/- 22.6 nmol MCA l(-1) h(-1)) significantly higher than alkaline phosphatase (98.6 +/- 4.3 nmol MUF l(-1) h(-1)). The primary microbial colonizers of the overlaying water - as determined by 16S rRNA gene sequence analysis - were related to at least six different phylogenetic lineages of Bacilli and to Alphaproteobacteria (Brevundimonas spp. and Caulobacter spp.). Large bacterial cells affiliated to one clade of Bacillus sp. were rare in the dried sediments, but constituted the majority of the planktonic microbial assemblage and of cells with detectable DNA-synthesis until 28 h after re-wetting. Their community contribution decreased in parallel with a rise of flagellated and ciliated protists. Estimates based on cell production rates suggested that the rapidly enriched Bacillus sp. suffered disproportionally high loss rates from selective predation, thus favouring the establishment of a more heterogenic assemblage of microbes (consisting of Proteobacteria, Actinobacteria and Cytophaga-Flavobacteria). Our results suggest that the primary microbial colonizers of the water above dried sediments are passively released into the plankton and that their high growth potential is counteracted by the activity of bacterivorous protists.

Microbial responses to polycyclic aromatic hydrocarbon contamination in temporary river sediments: Experimental insights.

Temporary rivers are characterized by dry-wet phases and represent an important water resource in semi-arid regions worldwide. The fate and effect of contaminants have not been firmly established in temporary rivers such as in other aquatic environments. In this study, we assessed the effects of sediment amendment with Polycyclic Aromatic Hydrocarbons (PAHs) on benthic microbial communities. Experimental microcosms containing natural (Control) and amended sediments (2 and 20 mg PAHs kg(-1) were incubated for 28 days. The PAH concentrations in sediments were monitored weekly together with microbial community structural (biomass and phylogenetic composition by TGGE and CARD-FISH) and functional parameters (ATP concentration, community respiration rate, bacterial carbon production rate, extracellular enzyme activities). The concentration of the PAH isomers did not change significantly with the exception of phenanthrene. No changes were observed in the TGGE profiles, whereas the occurrence of Alpha- and Beta-Proteobacteria was significantly affected by the treatments. In the amended sediments, the rates of carbon production were stimulated together with aminopeptidase enzyme activity. The community respiration rates showed values significantly lower than the Control after 1 day from the amendment then recovering the Control values during the incubation. A negative trend between the respiration rates and ATP concentration was observed only in the amended sediments. This result indicates a potential toxic effect on the oxidative phosphorylation processes. The impoverishment of the energetic resources that follows the PAH impact may act as a domino on the flux of energy from prokaryotes to the upper level of the trophic chain, with the potential to alter the temporary river functioning.

Extracellular enzyme activity and dynamics of bacterial community in mucilaginous aggregates of the northern Adriatic Sea.

Bacterial degradation of mucilaginous aggregates (creamy layers, stringers and macroflocs) collected during two summer events (2001-2002) was tested. The objective was to describe the temporal trend of the bacterial activity, abundance and composition in the aggregated and dissolved organic matter under different trophic conditions. In the native aggregates proteins and organic phosphorous were actively hydrolyzed as aminopeptidase and alkaline phosphatase activities represented up to 87% and 25% of total activity, respectively; polysaccharides were less hydrolyzed and the highest activities were observed for beta-glucosidase (5% of the total). This hydrolysation pattern tends to a progressive accumulation of long persistent polysaccharides. During short term incubations nutrient addition (P, N and Glucose) differently stimulated bacterial growth in the seawater: P played the main role in stimulating bacterial production from 3 to 6 folds higher than in the control, whereas a secondary C-limitation was observed only for bacteria growing on seawater from macroflocs. This scarce dissolved organic carbon (DOC) bioavailability was confirmed by the lower DOC removal (13% macroflocs, 36% stringers). The total amount of carbon incorporated by bacteria living on aggregates was similar (0.58 mg C L(-1)) both in the control and under P enrichments showing a more balanced condition with respect to the seawater. Hence the well-known P limitation in the Northern Adriatic Sea affects only dissolved organic carbon uptake without influencing the uptake of aggregated organic matter. Organic matter limitation was observed only on stringers--total C incorporated raised to 0.96 mg C L(-1) after PNG addition. Macroflocs release of refractory compounds leads to DOC accumulation (73 microM DOC) contributing to inflate the pool of refractory DOC in the surrounding waters. Several evidences, including different monosaccharide composition of stringers and macroflocs (glucose 15% and 56% on stringers and macroflocs, respectively), bring to the conclusion that stringers are in an older stage in comparison with macroflocs. Community composition described by fluorescence in situ hybridization did not show significant differences between free-living and attached bacteria but it was modified by the different enrichment conditions: Cytophaga-Flavobacteria increased after inorganic nutrients enrichments while organics advantaged gamma-Proteobacteria.

Organic priority substances and microbial processes in river sediments subject to contrasting hydrological conditions.

Flood and drought events of higher intensity and frequency are expected to increase in arid and semi-arid regions, in which temporary rivers represent both a water resource and an aquatic ecosystem to be preserved. In this study, we explored the variation of two classes of hazardous substances (Polycyclic Aromatic Hydrocarbons and Nonylphenols) and the functioning of the microbial community in river sediments subject to hydrological fluctuations (Candelaro river basin, Italy). Overall, the concentration of pollutants (∑PAHs range 8-275ngg(-1); ∑NPs range 299-4858ngg(-1)) suggests a moderate degree of contamination. The conditions in which the sediments were tested, flow (high/low) and no flow (wet/dry/arid), were associated to significant differences in the chemical and microbial properties. The total organic carbon contribution decreased together with the stream flow reduction, while the contribution of C-PAHs and C-NPs tended to increase. NPs were relatively more concentrated in sediments under high flow, while the more hydrophobic PAHs accumulated under low and no flow conditions. Passing from high to no flow conditions, a gradual reduction of microbial processes was observed, to reach the lowest specific bacterial carbon production rates (0.06fmolCh(-1)cell(-1)), extracellular enzyme activities, and the highest doubling time (40h) in arid sediments. In conclusion, different scenarios for the mobilization of pollutants and microbial processes can be identified under contrasting hydrological conditions: (i) the mobilization of pollutants under high flow and a relatively higher probability for biodegradation; (ii) the accumulation of pollutants during low flow and lower probability for biodegradation; (iii) the drastic reduction of pollutant concentrations under dry and arid conditions, probably independently from the microbial activity (abiotic processes). Our findings let us infer that a multiple approach has to be considered for an appropriate water resource exploitation and a more realistic prevision of the impact of pollutants in temporary waters.

Microbial communities in streambed sediments recovering from desiccation.

Climate change affects running waters not only by increasing temperatures but also by increasing discharge variability as more frequent and severe floods and more frequent and longer droughts occur, especially in upper reaches. Mediterranean streams are known to experience droughts, but Central European headwaters are also beginning to be affected. The development of bacterial communities (abundance, composition) and the recovery of microbial functions (bacterial production, extracellular enzyme activity) were explored after rewetting desiccated streambed sediments via a sediment core perfusion technique. The bacterial community composition changed only slightly in the sediments from the Central European stream Breitenbach (Germany), but distinctly in the Mediterranean Mulargia River (Sardinia, Italy) during 4 days of experimental rewetting. Breitenbach sediments probably enabled survival of bacterial communities more similar to indigenous streambed communities, because they were less dry. High activity of enzymes involved in polymer degradation at the beginning of rewetting in both sediments indicated the persistence of extracellular enzymes during drought. After 4 days, nearly all microbial activities reached a level similar to unaffected sediments for the Breitenbach, but not for Mulargia. Here, much more intense drying resulted in a more distinct change and reduction of the microbial community, responsible for slower recovery of structure and functions.

Responses of benthic bacteria to experimental drying in sediments from Mediterranean temporary rivers.

In the semiarid Mediterranean regions, water scarcity represents a common physiological stress for microbial communities residing in river sediments. However, the effect of drying has not yet adequately been evaluated when analyzing riverine microbiological processes. The bacterial community structure (abundance, biomass, composition) and functioning (carbon production, live cell percentage) were assessed during experimental desiccation in microcosms with sediments from different Mediterranean temporary rivers (Tagliamento, Krathis, Mulargia, Pardiela). Our results showed that the overall responses to drying of the bacterial community were independent from sediment origin and strictly related to water content. During desiccation, a prompt decline (up to 100%) of the initial bacterial carbon production was followed by a slower decrease in abundance and biomass, with an overall reduction of 74% and 78%, respectively. By the end of the experiment, live cells were still abundant but depressed in their main metabolic functions, thus resulting in a drastic increase in the community turnover time. Only 14% of the initial live cell biomass was available in dry sediments to immediately start the reactivation of the aquatic microbial food web after the arrival of new water. Community composition analysis showed a relative increase in alpha- and beta-Proteobacteria, when passing from wet to dry conditions. Our results suggest that the occurrence of drought events could affect carbon cycling through the freshwater microbial compartment, by temporarily limiting microbial mineralization and altering bacterial community structure.

Bacterial uptake of DOM released from P-limited phytoplankton.

The growth and the structure of a coastal bacterioplankton community were monitored in short-term bottle experiments in order to investigate the bacterial uptake of extracellular organic carbon released by the diatom Cylindrotheca closterium grown under P-balanced and P-depleted conditions. Bacterial specific growth rates and carbon demand were significantly lower in the exudates from P-depleted algae (24% and 30% reduction, respectively). The origin of the extracellular carbon appeared also to affect the taxonomic composition of the bacterioplankton assemblage, mainly reducing the development of gamma-Proteobacteria. This pattern of bacterial carbon uptake could contribute to a longer persistence of the exudates released in P-depleted conditions affecting the dynamics of the carbon cycle in marine environments.