Davide Marchetto

Microtox solid phase test: Effect of diluent used in toxicity test.

Microtox solid phase test is an acute toxicity test for solid matrices based on inhibition of natural bioluminescence of the marine bacterium Vibrio fischeri. Protocols developed to overcome the effects of confounding factors are proposed in the literature that differs by important practical and methodological issues. This work focused on diluents used for sediment resuspension and dilution. Two artificial seawaters, one natural seawater and two phosphate buffer solutions, were compared. The results showed that toxicity data obtained using different diluents were not comparable and that test sensitivity is highest using 0.1M phosphate buffer solution. The effects of medium on test organism were also investigated, in order to verify the capacity of media to maintain bioluminescence. The results underlined the importance of pH and Eh to explain the observed differences in toxicity.

Iron-binding characterization and polysaccharide production by Klebsiella oxytoca strain isolated from mine acid drainage

Aims:  To investigate Klebsiella oxytoca strain BAS‐10 growth on ferric citrate under anaerobic conditions for exopolysaccharide (EPS) production and localization on cell followed by the purification and the EPS determination of the iron‐binding stability constant to EPS or biotechnological applications.

A bio-generated Fe(III)-binding exopolysaccharide used as new catalyst for phenol hydroxylation

The hydroxylation of phenol with aqueous H2O2 to afford catechol and hydroquinone was studied in a biphasic reaction medium, as well as in pure water, in the presence of a bio-generated iron(III) catalyst. This catalyst was purified from a culture of Klebsiella oxytoca BAS-10 growing under anaerobic conditions, with Fe(III)-citrate as the energy and carbon source. The overproduction of an exopolymer (EPS) encrusted bacterial cells. The EPS, binding Fe3+, (Fe-EPS), was extracted and studied before and after a reaction with phenol. Some of the reaction’s parameters, such as temperature, pH, and molar ratio between reagents and catalyst, were investigated to identify the best compromise between conversion and selectivity. The results could be useful either from a synthetic point of view or to support the biodegradation of aromatic substrates.

Assessment of phenolic herbicide toxicity and mode of action by different assays

A phytotoxicity assay based on seed germination/root elongation has been optimized and used to evaluate the toxic effects of some phenolic herbicides. The method has been improved by investigating the influence of experimental conditions. Lepidium sativum was chosen as the most suitable species, showing high germinability, good repeatability of root length measurements, and low sensitivity to seed pretreatment. DMSO was the most appropriate solvent carrier for less water-soluble compounds. Three dinitrophenols and three hydroxybenzonitriles were tested: dinoterb, DNOC, 2,4-dinitrophenol, chloroxynil, bromoxynil, and ioxynil. Toxicity was also determined using the Vibrio fischeri Microtox® test, and a highly significant correlation was found between EC50 values obtained by the two assays. Dinoterb was the most toxic compound. The toxicity of hydroxybenzonitriles followed the order: ioxynil >bromoxynil >chloroxynil; L. sativum exhibited a slightly higher sensitivity than V. fischeri to these compounds. A QSAR analysis highlighted the importance of hydrophobic, electronic, and hydrogen-bonding interactions, in accordance with a mechanism of toxic action based on protonophoric uncoupling of oxidative phosphorylation. The results suggest that the seed germination/root elongation assay with L. sativum is a valid tool for the assessment of xenobiotic toxicity and can be recommended as part of a test battery.

Bio-generated metal binding polysaccharides as catalysts for synthetic applications and organic pollutant transformations

Iron and palladium binding an exopolysaccharide (EPS) were obtained and purified from cultures of bacterial cells of Klebsiella oxytoca BAS-10. The strain BAS-10 was able to grow under anaerobic conditions with Fe(III)-citrate as energy and carbon source, producing Fe(III)-EPS that was extracted and used as catalyst in the oxidation reaction of phenol with H(2)O(2). The same bacterial strain was cultivated anaerobically with Na-citrate and Pd(2)(NO)(3) was added during the exponential growth to afford a Pd-EPS, named Bio-Pd (A), that, after isolation and purification, was used as catalyst in the reductive dehalogenation of chlorobenzene as model reaction. For comparison other two palladium binding polysaccharides were prepared: (a) a second type Pd-EPS, named Bio-Pd (B), was obtained by an exchange reaction with Pd acetate starting from an iron-free EPS produced by strain BAS-10 growing on Na-citrate medium; (b) a third type of palladium, named Bio-Pd (C), bound to a different polysaccharide, was recovered after the same exchange reaction applied on glycolipid emulsan obtained from an aerobic culture of Acinetobacter venetianus RAG 1. The superiority of Bio-Pd (A), as catalyst, vs Bio-Pd (B) and (C) was demonstrated. This approach to use microorganisms to prepare metal bound polysaccharides is novel and permits to prepare metal species, sequestrated in aqueous phase that can be useful either as catalysts for synthetic applications or to support the microbial biotransformation of pollutants.

A Rapid Electrochemical Procedure for the Detection of Hg(0) Produced by Mercuric-Reductase: Application for Monitoring Hg- resistant Bacteria Activity.

In this work, gold microelectrodes are employed as traps for the detection of volatilized metallic mercury produced by mercuric reductase (MerA) extracted from an Hg-resistant Pseudomonas putida strain FB1. The enzymatic reduction of Hg (II) to Hg (0) was induced by NADPH cofactor added to the samples. The amount of Hg(0) accumulated on the gold microelectrode surface was determined by anodic stripping voltammetry (ASV) after transferring the gold microelectrode in an aqueous solution containing 0.1 M HNO(3) + 1 M KNO(3). Electrochemical measurements were combined with spectrofluorometric assays of NADPH consumption to derive an analytical expression for the detection of a relative MerA activity of different samples with respect to that of P. putida. The method developed here was employed for the rapid determination of MerA produced by bacteria harbored in soft tissues of clams (Ruditapes philippinarum), collected in high Hg polluted sediments of Northern Adriatic Sea in Italy.

Manila clams from Hg polluted sediments of Marano and Grado lagoons (Italy) harbor detoxifying Hg resistant bacteria in soft tissues.

A mechanism of mercury detoxification has been suggested by a previous study on Hg bioaccumulation in Manila clams (Ruditapes philippinarum) in the polluted Marano and Grado lagoons and in this study we demonstrate that this event could be partly related to the detoxifying activities of Hg-resistant bacteria (MRB) harbored in clam soft tissues. Therefore, natural clams were collected in six stations during two different periods (winter and spring) from Marano and Grado Lagoons. Siphons, gills and hepatopancreas from acclimatized clams were sterile dissected to isolate MRB. These anatomical parts were glass homogenized or used for whole, and they were lying on a solid medium containing 5mgl(-1) HgCl2 and incubated at 30°C. A total of fourteen bacterial strains were isolated and were identified by 16S rDNA sequencing and analysis, revealing that strains were representative of eight bacterial genera, four of which were Gram-positive (Enterococcus, Bacillus, Jeotgalicoccus and Staphylococcus) and other four were Gram-negative (Stenotrophomonas, Vibrio, Raoultella and Enterobacter). Plasmids and merA genes were found and their sequences determined. Fluorescence in situ hybridization (FISH) technique shows the presence of Firmicutes, Actinobacteria and Gammaproteobacteria by using different molecular probes in siphon and gills. Bacterial clumps inside clam flesh were observed and even a Gram-negative endosymbiont was disclosed by transmission electronic microscope inside clam cells. Bacteria harbored in cavities of soft tissue have mercury detoxifying activity. This feature was confirmed by the determination of mercuric reductase in glass-homogenized siphons and gills.

Diatom quantification and their distribution with salinity brines in coastal sediments of Terra Nova Bay (Antarctica).

Benthic diatoms represent an important element of global nutritional productivity; to raise attention on their role, which is often neglected due to analytical difficulties, surface (1 cm top layer) coastal sediments from Gerlache inlet to Penguin Bay at Terra Nova Bay were collected and stored at -20 °C. DNA amplification by real-time PCR, based on diatom-specific oligonucleotide primers designed on small-subunit rRNA (SSU rRNA), was performed in addition to diatom conventional cell counting and spectrophotometric determination of photo-pigments. Moreover, cations and anions were determined in sediments with the aim to identify factors involved in the control of diatom abundance. Diatom distribution was found quite heterogeneous displaying significant differences from site to site. The salinity in sediments ranged from 45.1 at Gerlache inlet to 76.2 at Penguin Bay and it was correlated with cell abundance, biodiversity, amount of pigments and amplified DNA. The dominant species, Fragilariopsis curta, was associated to sediment salinity brines.

Biogeochemical, Isotopic and Bacterial Distributions Trace Oceanic Abyssal Circulation.

We explore the possibility of tracing routes of dense waters toward and within the ocean abyss by the use of an extended set of observed physical and biochemical parameters. To this purpose, we employ mercury, isotopic oxygen, biopolymeric carbon and its constituents, together with indicators of microbial activity and bacterial diversity found in bottom waters of the Eastern Mediterranean. In this basin, which has been considered as a miniature global ocean, two competing sources of bottom water (one in the Adriatic and one in the Aegean seas) contribute to the ventilation of the local abyss. However, due to a recent substantial reduction of the differences in the physical characteristics of these two water masses it has become increasingly complex a water classification using the traditional approach with temperature, salinity and dissolved oxygen alone. Here, we show that an extended set of observed physical and biochemical parameters allows recognizing the existence of two different abyssal routes from the Adriatic source and one abyssal route from the Aegean source despite temperature and salinity of such two competing sources of abyssal water being virtually indistinguishable. Moreover, as the near-bottom development of exogenous bacterial communities transported by convectively-generated water masses in the abyss can provide a persistent trace of episodic events, intermittent flows like those generating abyssal waters in the Eastern Mediterranean basin may become detectable beyond the availability of concomitant measurements.

Biochemical characterization of some cyanobacterial strains from salt marshes of the Venice Lagoon

Three different strains of filamentous cyanobacteria, Tychonema, Limnothrix, and Pseudoanabaena, were selected among the fastest growing taxa collected in the salt marshes of Venice Lagoon and were grown in laboratory for growth rate determination and biochemical characterization of chlorophyll-a, total proteins, total carbohydrates, and exopolysaccharides. Experiments were carried out both in liquid medium and two different substrates: artificial plant protection fabric and ground indigenous shells. Cyanobacterial behavior was recorded to better understand colonization of natural and new artificial marshes.