Anti-Bacterial Adhesion on Abiotic and Biotic Surfaces of the Exopolysaccharide from the Marine Bacillus licheniformis B3-15.

The eradication of bacterial biofilm represents a crucial strategy to prevent a clinical problem associated with microbial persistent infection. In this study we evaluated the ability of the exopolysaccharide (EPS) B3-15, produced by the marine Bacillus licheniformis B3-15, to prevent the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 on polystyrene and polyvinyl chloride surfaces. The EPS was added at different times (0, 2, 4 and 8 h), corresponding to the initial, reversible and irreversible attachment, and after the biofilm development (24 or 48 h). The EPS (300 µg/mL) impaired the initial phase, preventing bacterial adhesion even when added after 2 h of incubation, but had no effects on mature biofilms. Without exerting any antibiotic activity, the antibiofilm mechanisms of the EPS were related to the modification of the (i) abiotic surface properties, (ii) cell-surface charges and hydrophobicity, and iii) cell-to-cell aggregation. The addition of EPS downregulated the expression of genes (lecA and pslA of P. aeruginosa and clfA of S. aureus) involved in the bacterial adhesion. Moreover, the EPS reduced the adhesion of P. aeruginosa (five logs-scale) and S. aureus (one log) on human nasal epithelial cells. The EPS could represent a promising tool for the prevention of biofilm-related infections.

Orange Peel Waste as Feedstock for the Production of Glycerol-Free Biodiesel by the Microalgae Nannochloropsis oculata.

The bioconversion of agri-food waste into high-value products is gaining growing interest worldwide. Orange peel waste (OPW) is the main by-product of orange juice production and contains high levels of moisture and carbohydrates. In this study, the orange waste extract (OWE) obtained through acid hydrolysis of OPW was used as a substrate in the cultivation of the marine microalgae Nannochloropsis oculata. Photoheterotrophic (PH) and Photoautotrophic (PA) cultivations were performed in OWE medium and f/2 medium (obtained by supplementing OWE with macro- and micronutrients of f/2 medium), respectively, for 14 days. The biomass yields in PA and PH cultures were 390 mg L-1 and 450 mg L-1, while oil yields were 15% and 28%, respectively. The fatty acid (FA) profiles of PA cultures were mostly represented by saturated (43%) and monounsaturated (46%) FAs, whereas polyunsaturated FAs accounted for about 10% of the FAs. In PH cultures, FA profiles changed remarkably, with a strong increase in monounsaturated FAs (77.49%) and reduced levels of saturated (19.79%) and polyunsaturated (2.72%) FAs. Lipids obtained from PH cultures were simultaneously extracted and converted into glycerol-free biodiesel using an innovative microwave-assisted one-pot tandem protocol. FA methyl esters were then analyzed, and the absence of glycerol was confirmed. The FA profile was highly suitable for biodiesel production and the microwave-assisted one-pot tandem protocol was more effective than traditional extraction techniques. In conclusion, N. oculata used OWE photoheterotrophically, resulting in increased biomass and oil yield. Additionally, a more efficient procedure for simultaneous oil extraction and conversion into glycerol-free biodiesel is proposed.

Nutritional conditions of the novel freshwater Coccomyxa AP01 for versatile fatty acids composition.

AIMS: This study was to analyse the biomass production and fatty acids (FAs) profiles in a newly isolated chlorophyte, namely Coccomyxa AP01, under nutritionally balanced (NB) conditions (comparing nitrate and urea as nitrogen sources) and nitrogen or phosphate deprivation. METHODS AND RESULTS: Lipid yields was about 30%-40% of dried biomasses in all examined nutritional conditions. Under NB conditions, lipids were principally constituted by monounsaturated FAs, mainly represented by oleic acid, and saturated and polyunsaturated FAs at similar concentrations. Nutrients deprivation induced remarkable changes in FAs profiles, with the highest amounts of saturated (42%-46%), followed by similar amounts of monounsaturated and polyunsaturated, and the emergence of rare long-chain FAs. Under phosphate deprivation, biomass yield was similar to NB conditions, with the highest yield of saturated (mainly palmitic acid) and of polyunsaturated FAs (33%) (mainly linoleic and linolenic acids). CONCLUSIONS: Balanced or deprived nutritional conditions in Coccomyxa AP01 induced a selective production and composition of FAs. The phosphate-deprivation condition concomitantly provided high biomass yield and the production of high value saturated and polyunsaturated FAs with industrial interest. SIGNIFICANCE AND IMPACT OF THE STUDY: Coccomyxa AP01 could be considered a promising source of different FAs, including also docosapentaenoic acid, for several commercial purposes spanning from biodiesel production, pharmaceutical and cosmetic applications to innovative aquaculture fish feeds.

Role of Phage Capsid in the Resistance to UV-C Radiations.

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H2O2) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H2O2. These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.

Glutamine-induced filamentous cells of Pseudomonas mediterranea CFBP-5447T as producers of PHAs.

Polyhydroxyalkanoates (PHAs) are considerable biopolymers that have gained an increasing biotechnological interest in different applications, although their industrial production presents several limitations. Filamentous bacterial cells could represent a possible strategy to increase PHA yield, since more abundant PHA inclusions can be stored in elongated than in rod-shaped cells. At first, we determined the optimal batch culture conditions to induce filamentation in Pseudomonas mediterranea CFBP-5447T, using glutamine, glycerol, glucose, and sodium octanoate, as the sole carbon source, at low- (100 rpm) or high- (250 rpm) shaking speeds. Successively, a fermentative process was set up using glutamine in a co-metabolic strategy with glycerol, and the PHAs production was compared in rod-shaped and filamentous cells. High glutamine concentrations (from 28 to 56 mM) were able to induce alone filamentation, whereas at lower glutamine concentrations (5-10 mM), the shaking speeds became critical to allow or not filamentous phenotype. PHA granule production was higher in filamentous than in rod-shaped cells, when glycerol (46.6 mM) was added to glutamine (5 mM) in co-metabolism, and fermentation was performed at a low-shaking speed. After extraction and precipitation, PHA yield was about two times higher in filamentous than that rod-shaped cells. Our results provide new insights into filament-inducing conditions and indicate a potential use of filamentous P. mediterranea CFBP-5447T cells to increase PHA yield. These findings could have great advantages in PHAs recovering during downstream processes, since the harvesting of elongated cells is much less time-consuming and energy expensive than required with rod-shaped cells.

Anti-herpes simplex virus 1 and immunomodulatory activities of a poly-γ- glutamic acid from Bacillus horneckiae strain APA of shallow vent origin.

Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe, clinical syndromes in the central nervous system. The emergence of resistant strains to drugs actually in use encourages the searching for novel antiviral compounds, including those of natural origin. In this study, the recently described poly-γ-glutamic acid (γ-PGA-APA), produced by the marine thermotolerant Bacillus horneckiae strain APA, and previously shown to possess biological and antiviral activity, was evaluated for its anti-HSV-1 and immunomodulatory properties. The biopolymer hindered the HSV-1 infection in the very early phase of virus replication. In addition, the γ-PGA-APA was shown to exert low cytotoxicity and noticeable immunomodulatory activities towards TNF-α and IL-1ß gene expression. Moreover, the capacity to positively modulate the transcriptional activity of the cytokine genes was paired with increased level of activation of the transcription factor NF-kB by γ-PGA-APA. Overall, as non-cytotoxic biopolymer able to contribute in the antiviral defense against HSV-1, γ-PGA-APA could lead to the development of novel natural drugs for alternative therapies.

Comparison of Prokaryotic Diversity in Cold, Oligotrophic Remote Lakes of Chilean Patagonia.

The prokaryotic abundance and diversity in three cold, oligotrophic Patagonian lakes (Témpanos, Las Torres and Mercedes) in the northern region Aysén (Chile) were compared in winter and summer using 16S rRNA fluorescence in situ hybridization and PCR-denaturing gradient gel electrophoresis technique. Prokaryotic abundances, numerically dominated by Bacteria, were quite similar in the three lakes, but higher in sediments than in waters, and they were also higher in summer than in winter. The relative contribution of Archaea was greater in waters than in sediments, and in winter rather than in summer. Despite the phylogenetic analysis indicated that most sequences were affiliated to a few taxonomic groups, mainly referred to Proteobacteria (consisting of Beta-, Alpha- and Gammaproteobacteria) and Euryarchaeota (mainly related to uncultured methanogens), their relative abundances differed in each sample, resulting in different bacterial and archaeal assemblages. In winter, the abundance of the dominant bacterial phylotypes were mainly regulated by the increasing levels of total organic carbon in waters. Archaeal abundance and richness appeared mostly influenced by pH in winter and total nitrogen content in summer. The prokaryotic community composition at Témpanos lake, located most northerly and closer to a glacier, greatly differed in respect to the other two lakes. In this lake was detected the highest bacterial diversity, being Betaproteobacteria the most abundant group, whereas Alphaproteobacteria were distinctive of Mercedes. Archaeal community associated with sediments was mainly represent by members related to the order of Methanosarcinales at Mercedes and Las Torres lakes, and by Crenarchaeota at Témpanos lake. Our results indicate that the proximity to the glacier and the seasonality shape the composition of the prokaryotic communities in these remote lakes. These results may be used as baseline information to follow the microbial community responses to potential global changes and to anthropogenic impacts.

Prokaryotic Community in Lacustrine Sediments of Byers Peninsula (Livingston Island, Maritime Antarctica).

Byers Peninsula (Livingston Island, Antarctica), the largest seasonally ice-free region of the Maritime Antarctica, holds a large number of lakes, ponds, and streams. The prokaryotic structure and bacterial diversity in sediment samples collected during the 2008-2009 austral summer from five inland lakes, two coastal lakes, and an estuarine site were analyzed by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) and 16S rRNA 454 tag pyrosequencing techniques, respectively. Differently from inland lakes, which range around the oligotrophic status, coastal lakes are eutrophic environments, enriched by nutrient inputs from marine animals. Although the prokaryotic abundances (estimated as DAPI stained cells) in sediment samples were quite similar among inland and coastal lakes, Bacteria always far dominated over Archaea. Despite the phylogenetic analysis indicated that most of sequences were affiliated to a few taxonomic groups, mainly referred to Proteobacteria, Bacteroidetes, and Actinobacteria, their relative abundances greatly differed from each site. Differences in bacterial composition showed that lacustrine sediments were more phyla rich than the estuarine sediment. Proteobacterial classes in lacustrine samples were dominated by Betaproteobacteria (followed by Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria), while in the estuarine sample, they were mainly related to Gammaproteobacteria (followed by Deltaproteobacteria, Epsilonproteobacteria, Alphaproteobacteria, and Betaproteobacteria). Higher number of sequences of Alphaproteobacteria, Cyanobacteria, Verrucomicrobia, and Planctomycetes were observed in sediments of inland lakes compared to those of coastal lakes, whereas Chloroflexi were relatively more abundant in the sediments of coastal eutrophic lakes. As demonstrated by the great number of dominant bacterial genera, bacterial diversity was higher in the sediments of inland lakes than that in coastal lakes. Ilumatobacter (Actinobacteria), Gp16 (Acidobacteria), and Gemmatimonas (Gemmatimonadetes) were recovered as dominant genera in both inland and coastal lakes, but not in the estuarine sample, indicating that they may be useful markers of Antarctic lakes. The proximity to the sea, the different lake depths and the external or internal origin of the nutrient sources shape the bacterial communities composition in lacustrine sediments of Byers Peninsula.

In Vitro Antibiofilm Activity of an Exopolysaccharide from the Marine Thermophilic Bacillus licheniformis T14.

The development of antibiofilm strategies is of major interest in contrasting bacterial pathogenic biofilms. A novel fructose and fucose rich exopolysaccharide (EPS1-T14) produced by the recently described thermophilic Bacillus licheniformis T14, isolated from a shallow hydrothermal vent of Panarea Island (Eolian Island, Italy), was evaluated for its effects on biofilm formation by multiresistant clinical strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The antibiofilm activity of EPS1-T14 was assessed by microtiter plate assays and visualized by confocal laser scanning microscopic images. EPS1-T14, with molecular weight of 1000 kDa, reduced biofilm formation on abiotic surfaces without affecting bacterial vitality. The novel EPS1-T14 is a water-soluble, noncytotoxic exopolymer able to prevent biofilm formation and its use may represent a promising therapeutic strategy for combating bacterial biofilm-associated infections. EPS1-T14 as antiadhesive biomolecule could be useful for novel prospective in medical and nonmedical applications.

Changes in prokaryotic community composition accompanying a pronounced temperature shift of a shallow marine thermal brine pool (Panarea Island, Italy).

Hot Lake is a recently described thermal brine pool off Panarea Island (Eolian Islands, Italy) where emitted fluids are highly saline and rich in CO2 and H2S. The prokaryotic community composition in surface sediment samples was analyzed by high-throughput Illumina sequencing targeting the V3 region of the 16S rRNA at two time points that differed mainly with respect to temperature conditions, high-temperature (94 °C, HT09) and low-temperature (28.5 °C, LT10). Bacterial richness and diversity were greater than those of Archaea under both temperature conditions. In contrast to Bacteria, diversity and evenness of Archaea greatly increased at LT10. While the phylogenetic analysis indicated the presence of members mostly affiliated with the same taxonomic groups, their relative abundances differed from HT09 to LT10, resulting in different bacterial and archaeal assemblages. Both HT09 and LT10 were dominated by members of the Epsilonproteobacteria. Within this subphylum, bacteria of the genus Sulfurimonas were most frequently detected at HT09, while Arcobacter prevailed at LT10. The abundance of other dominant taxonomic groups (≥1 % of Illumina reads) also correlated with temperature conditions. Members assigned to hyperthermophilic Euryarchaeota (Thermococci) or to thermophilic (Caldiserica) and thermoresistant (Firmicutes) bacterial taxa were dominant at HT09, while those related to non-thermophilic Bacteroidetes, Fusobacteria and Actinobacteria were dominant at LT10. Several, probably photosynthetic, members of the Alphaproteobacteria, Chlorobi, Cyanobacteria and Chloroflexi were recovered under both temperature conditions. The co-occurrence of photosynthetic and chemolithotrophic microorganisms represents a unique feature of shallow vents such as Hot Lake.

Diversity of prokaryotic community at a shallow marine hydrothermal site elucidated by Illumina sequencing technology.

To investigate the prokaryotic community structure and composition in an active hydrothermal site, named Black Point, off Panarea Island (Eolian Islands, Italy), we examined sediment and fluid samples, differing in temperature, by a massive parallel sequencing (Illumina) technique targeting the V3 region of the 16S rRNA gene. The used technique enabled us to detect a greater prokaryotic diversity than that until now observed and to reveal also microorganisms occurring at very low abundance (≤0.01 %). Most of sequences were assigned to Bacteria while Archaea were a minor component of the microbial community in both low- and high-temperature samples. Proteobacteria (mainly consisting of Alpha-, Gamma-, and Epsilonproteobacteria) dominated among all samples followed by Actinobacteria and Bacteroidetes. Analyzed DNA obtained from samples taken at different temperatures indicated the presence of members of different dominant genera. The main differences were observed between sediment samples where Rhodovulum and Thiohalospira prevailed at high temperature, while Thalassomonas and Sulfurimonas at low temperature. Chlorobium, Acinetobacter, Sulfurimonas, and Brevundimonas were abundant in both low- and high-temperature fluid samples. Euryarchaeota dominated the archaeal community in all samples. Classes of Euryarchaeota embracing hyperthermophilic members (Thermococci and Thermoplasmata) and of Crenarchaeota (Thermoprotei) were more abundant in high-temperature samples. A great number of sequences referred to Bacteria and Archaea still remained unaffiliated, indicating that Black Point site represents a rich source of so-far uncharted prokaryotic diversity.

A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy).

A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13(T) and Bacillus sonorensis DSM 13779(T). Further DNA-DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13(T) and 39.12 % with B. sonorensis DSM 13779(T). Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l(-1)) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a ß-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.

Lichenysin-like Polypeptide Production by Bacillus licheniformis B3-15 and Its Antiadhesive and Antibiofilm Properties.

We report the ability of the crude biosurfactant (BS B3-15), produced by the marine, thermotolerant Bacillus licheniformis B3-15, to hinder the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 to polystyrene and human cells. First, we attempted to increase the BS yield, optimizing the culture conditions, and evaluated the surface-active properties of cell-free supernatants. Under phosphate deprivation (0.06 mM) and 5% saccharose, the yield of BS (1.5 g/L) increased by 37%, which could be explained by the earlier (12 h) increase in lchAA expression compared to the non-optimized condition (48 h). Without exerting any anti-bacterial activity, BS (300 µg/mL) prevented the adhesion of P. aeruginosa and S. aureus to polystyrene (47% and 36%, respectively) and disrupted the preformed biofilms, being more efficient against S. aureus (47%) than P. aeruginosa (26%). When added to human cells, the BS reduced the adhesion of P. aeruginosa and S. aureus (10× and 100,000× CFU/mL, respectively) without altering the epithelial cells' viability. As it is not cytotoxic, BS B3-15 could be useful to prevent or remove bacterial biofilms in several medical and non-medical applications.

Vertical distribution of Archaea and Bacteria in a meromictic lake as determined by fluorescent in situ hybridization.

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A "red-water" layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem.

Hydrating Capabilities of the Biopolymers Produced by the Marine Thermophilic Bacillus horneckiae SBP3 as Evaluated by ATR-FTIR Spectroscopy.

The surfactin-like lipopeptide (BS-SBP3) and the exopolysaccharide (EPS-SBP3) produced by the polyextremophilic Bacillus horneckiae SBP3 (DSM 103063) have been recently described as valuable biopolymers useful in biotechnological applications. To investigate the hydrating capabilities of BS-SBP3 and EPS-SBP3, here we evaluated (i) their wetting properties, measuring the contact angle; (ii) their moisture uptake abilities using the gravimetric method; and (iii) their hydrating states (from 0 to 160% w/w of water content) using ATR-FTIR spectroscopy. BS-SBP3 reduced the water contact angle on a hydrophobic surface from 81.7° to 51.3°, whereas the contact angle in the presence of EPS-SBP3 was 72.9°, indicating that BS-SBP3 improved the wettability of the hydrophobic surface. In the moisture uptake tests, EPS-SBP3 absorbed more water than BS-SBP3, increasing its weight from 10 mg to 30.1 mg after 36 h of 100% humidity exposure. Spectral distance and cross-correlation analyses were used to evaluate the molecular changes of the two biopolymers during the hydration process. As the water concentration increased, BS-SBP3 spectra changed in intensity in the two contributions of the OH-stretching band named "closed" and "open" (3247 and 3336 cm-1, respectively). Differently, the spectra of EPS-SBP3 exhibited a broader peak (3257 cm-1), which shifted at higher water concentrations. As evaluated by the spectral distance and the wavelet cross-correlation analysis, the OH-stretching bands of the BS-SBP3 and EPS-SBP3 changed as a function of water content, with two different sigmoidal trends having the inflection points at 80% and 48%, respectively, indicating peculiar water-properties of each biopolymer. As wetting agents, these biopolymers might replace industrially manufactured additives in agriculture and the food and cosmetic industries.

Evaluation of Betacoronavirus OC43 and SARS-CoV-2 Elimination by Zefero Air Sanitizer Device in a Novel Laboratory Recirculation System.

Indoor air sanitizers contrast airborne diseases and particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/Coronavirus disease 2019 (COVID-19). The commercial air sanitizer Zefero (Cf7 S.r.l., San Giovanni La Punta, Italy) works alternatively using a set of integrated disinfecting technologies (namely Photocatalysis/UV mode) or by generating ozone (Ozone mode). Here we evaluated the virucidal efficacy of Zefero setup modes against human Betacoronavirus OC43 and SARS-CoV-2. For this purpose, we designed a laboratory test system in which each virus, as aerosol, was treated with Photocatalysis/UV or Ozone mode and returned into a recirculation plexiglass chamber. Aerosol samples were collected after different times of exposure, corresponding to different volumes of air treated. The viral RNA concentration was determined by qRT-PCR. In Photocatalysis/UV mode, viral RNA of OC43 or SARS-CoV-2 was not detected after 120 or 90 min treatment, respectively, whereas in Ozone mode, viruses were eliminated after 30 or 45 min, respectively. Our results indicated that the integrated technologies used in the air sanitizer Zefero are effective in eliminating both viruses. As a reliable experimental system, the recirculation chamber developed in this study represents a suitable apparatus for effectively comparing the disinfection capacity of different air sanitizers.

Distribution and diversity of bacteria in a saline meromictic lake as determined by PCR-DGGE of 16S rRNA gene fragments.

The variations in vertical distribution and composition of bacteria in the meromictic Lake Faro (Messina, Italy) were analysed by culture-independent methods in two different mixing conditions. Water samples were collected from a central station from the surface to the bottom (30 m depth) on two different sampling dates--the first characterised by a well-mixed water mass and the second by a marked stratification. A 'red-water' layer, caused by a dense growth of photosynthetic sulphur bacteria, was present at a depth of 25 m in December 2005 and at 15 m in August 2006, defining two different zones in terms of their physicochemical properties. The vertical distribution of bacterioplankton showed that the interface zones were more densely populated than others. In both sampling periods, the highest numbers of live cells were observed within 'red water' layers. The dominant phylotypes of the bacterial community were determined by sequencing the Denaturing Gradient Gel Electrophoresis (DGGE) bands resulting from PCR amplification of 16S rRNA gene fragments. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably across the two different sampling periods. Proteobacteria (α-, γ-, δ- and ε subclass members), Cytophaga-Flavobacterium-Bacteroides, green sulphur bacteria and Cyanobacteria were retrieved from Lake Faro. Most of the bands showed DNA sequences that did not match with other previously described organisms, suggesting the presence of new indigenous bacterial phylotypes.

Antiproliferative activity of carotenoid pigments produced by extremophile bacteria.

Various microorganisms are able to synthesize pigments, which usually present antioxidant properties. The aim of this work was to evaluate the antiproliferative activity of bacterial pigments against cancer cells Neuro-2a, Saos-2 and MCF-7. Pigments were obtained from Deinococcus sp. UDEC-P1 and Arthrobacter sp. UDEC-A13. Both bacterial strains were isolated from cold environments (Patagonia and Antarctica, respectively). Pigments were purified and analyzed by HPLC. Antiproliferative activity was evaluated by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) assay. Deinoxanthin carotenoid obtained from Deinococcus sp. UDEC-P1 was able to reduce significatively the viability of Saos-2 (37.1%), while no effect was observed against MCF-7 and Neuro-2a. Pigments obtained from Arthrobacter sp. UDEC-A13 showed a significant viability reduction of three tumour cells (20.6% Neuro-2a, 26.3% Saos-2 and 13.2% MCF-7). Therefore, carotenoid pigments produced by extremophilic bacteria Deinococcus sp. UDEC-P1 and Arthrobacter sp. UDEC-A13 could be proposed as novel complementary compounds in anticancer chemotherapy.

Thermal properties of an exopolysaccharide produced by a marine thermotolerant Bacillus licheniformis by ATR-FTIR spectroscopy.

The novel exopolysaccharide (EPS-B3-15) produced by the marine thermotolerant Bacillus licheniformis strain B3-15, constituted by mannose and glucose, has been recently reported as a valuable biopolymer in pharmaceutical applications. To dynamically characterize the thermal behavior of the whole EPS-B3-15 system, the Attenuated Total Reflectance Fourier Transform Infra-Red spectroscopy technique was used over a temperature range from ambient to 78.5 °C. The molecular changes of EPS-B3-15 during the heating process were evaluated by the spectral distance (SD) and wavelet cross-correlation (XWT) analysis. The thermal analysis revealed that the EPS-B3-15 possessed high stability until 78.5 °C. As evaluated by SD and XWT, the molecular structure of EPS-B3-15 at 45 °C was partially modified, due to -OH groups and the -COOH and -OH interactions, conferring a new conformational structure to the biopolymer. The thermal characterization provides novel information about the molecular conformations of the whole EPS-B3-15 system at different temperatures. The thermostable EPS-B3-15 can be successfully employed for biotechnological, nanotechnological and material science applications even at high temperatures.

Effects of Heavy Ion Particle Irradiation on Spore Germination of Bacillus spp. from Extremely Hot and Cold Environments.

Extremophiles are optimal models in experimentally addressing questions about the effects of cosmic radiation on biological systems. The resistance to high charge energy (HZE) particles, and helium (He) ions and iron (Fe) ions (LET at 2.2 and 200 keV/µm, respectively, until 1000 Gy), of spores from two thermophiles, Bacillushorneckiae SBP3 and Bacilluslicheniformis T14, and two psychrotolerants, Bacillus sp. A34 and A43, was investigated. Spores survived He irradiation better, whereas they were more sensitive to Fe irradiation (until 500 Gy), with spores from thermophiles being more resistant to irradiations than psychrotolerants. The survived spores showed different germination kinetics, depending on the type/dose of irradiation and the germinant used. After exposure to He 1000 Gy, D-glucose increased the lag time of thermophilic spores and induced germination of psychrotolerants, whereas L-alanine and L-valine increased the germination efficiency, except alanine for A43. FTIR spectra showed important modifications to the structural components of spores after Fe irradiation at 250 Gy, which could explain the block in spore germination, whereas minor changes were observed after He radiation that could be related to the increased permeability of the inner membranes and alterations of receptor complex structures. Our results give new insights on HZE resistance of extremophiles that are useful in different contexts, including astrobiology.