Nutrient regeneration mediated by extracellular enzymes in water column and interstitial water through a microcosm experiment.

In coastal lakes the role of microorganisms in driving nutrients regeneration at different water depths and in sediments is not yet fully understood. The dynamics of microbial (algal and bacterial) abundance and bacterial activities involved in organic matter transformation were measured, together with nutrient concentrations, through a microcosm experiment set up using the oligotrophic Faro lake as a study model over a total period of 15 days and with a four-day frequency. Water column at different depths (surface, middle and bottom) and interstitial water obtained by sediment centrifugation were used in appropriate ratios (mixed 1:1 with surface waters) to fill 21-Litre plastic aquaria in order to simulate processes occurring in natural conditions. At early experimental period, the sharp decrease of dissolved organic nutrients and the abundant production of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) in correspondence with high phytoplankton abundance in bottom and interstitial water reflected the relevance of organic nutrients for inorganic nutrients regeneration and phytoplankton growth. Size fractionation of LAP and AP as well as the positive relationship between microbial compartments suggested that bacteria and phytoplankton worked in close reciprocal synergy, and coupling of nitrogen and phosphorus regeneration, especially in bottom and interstitial waters, was observed. At later experimental period, the change in bacterial community, especially the increase of filamentous shaped cells, together with a simultaneous increase of protozoan abundance indicated that nutrient replenishment made the microbial loop structure more competitive. In oligotrophic conditions, such as those in Faro lake, organic nutrient enrichment of bottom and interstitial waters was associated with changes in the bacterial community, with consequent stimulation of extracellular enzymes to support phytoplankton growth. Nutrient availability from microbial regeneration resulted in an increased complexity of the microbial loop structure, with bacteria and phytoplankton adopting specific strategies to respond to the changing environment.

Different pathways of nitrogen and phosphorus regeneration mediated by extracellular enzymes in temperate lakes under various trophic state.

Several Italian and Chinese temperate lakes with soluble reactive phosphorus concentrations < 0.015 mg L-1 were studied to estimate nitrogen and phosphorus regeneration mediated by microbial decomposition and possible different mechanisms driven by prevailing oligo- or eutrophic conditions. Leucine aminopeptidase (LAP), beta-glucosidase (GLU) and alkaline phosphatase (AP), algal, and bacterial biomass were related to trophic and environmental variables. In the eutrophic lakes, high algal and particulate organic carbon concentrations stimulated bacterial respiration (> 20 µg C L-1 h-1) and could favor the release of inorganic phosphorus. High extracellular enzyme activities and phosphorus solubilizing bacteria abundance in sediments accelerated nutrient regeneration. In these conditions, the positive GLU-AP relationship suggested the coupling of carbon and phosphorus regeneration; an efficient phosphorus regeneration and high nitrogen levels (up to 0.067 and 0.059 mg L-1 NH4 and NO3 in Italy; 0.631 and 1.496 mg L-1 NH4 and NO3 in China) led to chlorophyll a peaks of 14.9 and 258.4 µg L-1 in Italy and China, respectively, and a typical algal composition. Conversely, in the oligo-mesotrophic lakes, very low nitrogen levels (in Italy, 0.001 and 0.005 mg L-1 NH4 and NO3, respectively, versus 0.053 and 0.371 mg L-1 in China) induced high LAP, while low phosphorus (33.6 and 46.3 µg L-1 total P in Italy and China, respectively) led to high AP. In these lakes, nitrogen and phosphorus regeneration were coupled, as shown by positive LAP-AP relationship; however, the nutrient demand could not be completely met without the supply from sediments, due to low enzymatic activity and phosphorus solubilizing bacteria found in this compartment.

Contribution of Bicarbonate Assimilation to Carbon Pool Dynamics in the Deep Mediterranean Sea and Cultivation of Actively Nitrifying and CO2-Fixing Bathypelagic Prokaryotic Consortia.

Covering two-thirds of our planet, the global deep ocean plays a central role in supporting life on Earth. Among other processes, this biggest ecosystem buffers the rise of atmospheric CO2. Despite carbon sequestration in the deep ocean has been known for a long time, microbial activity in the meso- and bathypelagic realm via the "assimilation of bicarbonate in the dark" (ABD) has only recently been described in more details. Based on recent findings, this process seems primarily the result of chemosynthetic and anaplerotic reactions driven by different groups of deep-sea prokaryoplankton. We quantified bicarbonate assimilation in relation to total prokaryotic abundance, prokaryotic heterotrophic production and respiration in the meso- and bathypelagic Mediterranean Sea. The measured ABD values, ranging from 133 to 370 µg C m-3 d-1, were among the highest ones reported worldwide for similar depths, likely due to the elevated temperature of the deep Mediterranean Sea (13-14°C also at abyssal depths). Integrated over the dark water column (≥200 m depth), bicarbonate assimilation in the deep-sea ranged from 396 to 873 mg C m-2 d-1. This quantity of produced de novo organic carbon amounts to about 85-424% of the phytoplankton primary production and covers up to 62% of deep-sea prokaryotic total carbon demand. Hence, the ABD process in the meso- and bathypelagic Mediterranean Sea might substantially contribute to the inorganic and organic pool and significantly sustain the deep-sea microbial food web. To elucidate the ABD key-players, we established three actively nitrifying and CO2-fixing prokaryotic enrichments. Consortia were characterized by the co-occurrence of chemolithoautotrophic Thaumarchaeota and chemoheterotrophic proteobacteria. One of the enrichments, originated from Ionian bathypelagic waters (3,000 m depth) and supplemented with low concentrations of ammonia, was dominated by the Thaumarchaeota "low-ammonia-concentration" deep-sea ecotype, an enigmatic and ecologically important group of organisms, uncultured until this study.

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.

Microbial life in the Lake Medee, the largest deep-sea salt-saturated formation.

Deep-sea hypersaline anoxic lakes (DHALs) of the Eastern Mediterranean represent some of the most hostile environments on our planet. We investigated microbial life in the recently discovered Lake Medee, the largest DHAL found to-date. Medee has two unique features: a complex geobiochemical stratification and an absence of chemolithoautotrophic Epsilonproteobacteria, which usually play the primary role in dark bicarbonate assimilation in DHALs interfaces. Presumably because of these features, Medee is less productive and exhibits reduced diversity of autochthonous prokaryotes in its interior. Indeed, the brine community almost exclusively consists of the members of euryarchaeal MSBL1 and bacterial KB1 candidate divisions. Our experiments utilizing cultivation and [(14)C]-assimilation, showed that these organisms at least partially rely on reductive cleavage of osmoprotectant glycine betaine and are engaged in trophic cooperation. These findings provide novel insights into how prokaryotic communities can adapt to salt-saturated conditions and sustain active metabolism at the thermodynamic edge of life.

Metagenomic analysis of hadopelagic microbial assemblages thriving at the deepest part of Mediterranean Sea, Matapan-Vavilov Deep.

The marine pelagic zone situated > 200 m below the sea level (bls) is the largest marine subsystem, comprising more than two-thirds of the oceanic volume. At the same time, it is one of the least explored ecosystems on Earth. Few large-scale environmental genomics studies have been undertaken to examine the phylogenetic diversity and functional gene repertoire of planktonic microbes present in mesopelagic and bathypelagic environments. Here, we present the description of the deep-sea microbial community thriving at > 4900 m depth in Matapan-Vavilov Deep (MVD). This canyon is the deepest site of Mediterranean Sea, with a deepest point located at approximately 5270 m, 56 km SW of city Pylos (Greece) in the Ionian Sea (36°34.00N, 21°07.44E). Comparative analysis of whole-metagenomic data revealed that unlike other deep-sea metagenomes, the prokaryotic diversity in MVD was extremely poor. The decline in the dark primary production rates, measured at 4908 m depth, was coincident with overwhelming dominance of copiotrophic Alteromonas macleodii'deep-ecotype' AltDE at the expense of other prokaryotes including those potentially involved in both autotrophic and anaplerotic CO(2) fixation. We also demonstrate the occurrence in deep-sea metagenomes of several clustered regularly interspaced short palindromic repeats systems.

Metagenomics of the deep Mediterranean, a warm bathypelagic habitat.

BACKGROUND: Metagenomics is emerging as a powerful method to study the function and physiology of the unexplored microbial biosphere, and is causing us to re-evaluate basic precepts of microbial ecology and evolution. Most marine metagenomic analyses have been nearly exclusively devoted to photic waters. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a metagenomic fosmid library from 3,000 m-deep Mediterranean plankton, which is much warmer (approximately 14 degrees C) than waters of similar depth in open oceans (approximately 2 degrees C). We analyzed the library both by phylogenetic screening based on 16S rRNA gene amplification from clone pools and by sequencing both insert extremities of ca. 5,000 fosmids. Genome recruitment strategies showed that the majority of high scoring pairs corresponded to genomes from Rhizobiales within the Alphaproteobacteria, Cenarchaeum symbiosum, Planctomycetes, Acidobacteria, Chloroflexi and Gammaproteobacteria. We have found a community structure similar to that found in the aphotic zone of the Pacific. However, the similarities were significantly higher to the mesopelagic (500-700 m deep) in the Pacific than to the single 4000 m deep sample studied at this location. Metabolic genes were mostly related to catabolism, transport and degradation of complex organic molecules, in agreement with a prevalent heterotrophic lifestyle for deep-sea microbes. However, we observed a high percentage of genes encoding dehydrogenases and, among them, cox genes, suggesting that aerobic carbon monoxide oxidation may be important in the deep ocean as an additional energy source. CONCLUSIONS/SIGNIFICANCE: The comparison of metagenomic libraries from the deep Mediterranean and the Pacific ALOHA water column showed that bathypelagic Mediterranean communities resemble more mesopelagic communities in the Pacific, and suggests that, in the absence of light, temperature is a major stratifying factor in the oceanic water column, overriding pressure at least over 4000 m deep. Several chemolithotrophic metabolic pathways could supplement organic matter degradation in this most depleted habitat.

Uptake of Escherichia coli, Vibrio cholerae non-O1 and Enterococcus durans by, and depuration of mussels (Mytilus galloprovincialis).

The uptakes of Escherichia coli, Vibrio cholerae non-O1 and Enterococcus durans by mussels (Mytilus galloprovincialis) and the times for depuration were investigated in order to determine the most useful indicator of vibrio contamination. The mussels were maintained in tanks of static seawater contaminated with bacteria at 5 log10 CFU/ml for bioaccumulation. Depuration was carried out by circulating fresh seawater through the tanks. Each organism was presented alone and with others to mussels, at temperatures of 14 and 21 degrees C. In water contaminated with either single or mixed organisms, the bacteria accumulated rapidly in the mussels reaching high concentrations after 1 h. With both single and mixed organisms, the maximum numbers of E. coli in mussels were 6.6 log10 CFU/g at 14 degrees C and 5.4 log10 CFU/g at 21 degrees C. Both V. cholerae non-O1 and E. durans alone or with other organisms reached a number ranging from 6.5 to 7 log10 CFU/g at both temperatures. During depuration the numbers of all the organisms slowly decreased, with E. coli alone, numbers ranged from 2.8 to 2 log10 CFU/g after 72 h at both 14 and 21 degrees C, and the organisms were undetectable after 144 h. With mixed organisms at 14 degrees C E. coli became undetectable after 168 h but at 21 degrees C no E. coli were recovered after 72 h. At 14 degrees C V. cholerae non-O1 alone also was undetectable after 168 h, but at 21 degrees C and with mixed organisms at both temperatures. V. cholerae was recovered after 168 h at numbers about 1 log10 CFU/g. After 168 h numbers of E. durans alone ranged from 2.6 log10 CFU/g at 14 degrees C to 1.5 log10 CFU/g at 21 degrees C, and with mixed organisms the numbers ranged from 2.3 to 2.0 log10 CFU/g at both temperatures. Of the three bacteria of faecal origin, E. durans is quickly acquired by mussels and released more slowly than the others, while E. coli quickly becomes undetectable. The results suggest that, for this kind of seafood, enterococci may be a more appropriate indicator than E. coli of risks to consumers from vibrios.