Characterization of the exopolymer-producing Pseudoalteromonas sp. S8-8 from Antarctic sediment.

A synergistic approach using cultivation methods, chemical, and bioinformatic analyses was applied to explore the potential of Pseudoalteromonas sp. S8-8 in the production of extracellular polymeric substances (EPSs) and the possible physiological traits related to heavy metal and/or antibiotic resistance. The effects of different parameters (carbon source, carbon source concentration, temperature, pH and NaCl supplement) were tested to ensure the optimization of growth conditions for EPS production by the strain S8-8. The highest yield of EPS was obtained during growth in culture medium supplemented with glucose (final concentration 2%) and NaCl (final concentration 3%), at 15 °C and pH 7. The EPS was mainly composed of carbohydrates (35%), followed by proteins and uronic acids (2.5 and 2.77%, respectively) and showed a monosaccharidic composition of glucose: mannose: galactosamine: galactose in the relative molar proportions of 1:0.7:0.5:0.4, as showed by the HPAE-PAD analysis. The detection of specific molecular groups (sulfates and uronic acid content) supported the interesting properties of EPSs, i.e. the emulsifying and cryoprotective action, heavy metal chelation, with interesting implication in bioremediation and biomedical fields. The analysis of the genome allowed to identify a cluster of genes involved in cellulose biosynthesis, and two additional gene clusters putatively involved in EPS biosynthesis. KEY POINTS: • A cold-adapted Pseudoalteromonas strain was investigated for EPS production. • The EPS showed emulsifying, cryoprotective, and heavy metal chelation functions. • Three gene clusters putatively involved in EPS biosynthesis were evidenced by genomic insights.

New Insights into the Culture Method and Antibacterial Potential of Gracilaria gracilis.

Enormous marine biodiversity offers an endless reservoir of chemicals for many applications. In this scenario, the extraction of seaweeds represents an interesting source of compounds displaying antimicrobial activity. In particular, among the different red algae, Gracilaria gracilis plays an important role due to the presence of important bioactives in its composition. In spite of these features, an efficient culture system is still absent. In the present study, a novel algal culture method was developed and compared to another more common cultural practice, widely reported in literature. A higher efficiency of the new method, both for daily growth rate and biomass, was assessed. Furthermore, the growth inhibitory activity of five extracts, obtained using ethanol, methanol, acetone, chloroform or diethyl ether as a solvent, from the cultured G. gracilis was tested against Gram-positive and Gram-negative pathogens. Algal extracts exhibited a considerable inhibitory activity against B. subtilis strains, while a slight inhibition was observed against V. fischeri. The different extracts showed significant differences in bacterial growth inhibition, with the highest activity that was recorded for the ethanol extract, followed by that of methanol. Based on the chemical characterization, these findings could be related to the antimicrobial activity played by the combination of total carbohydrates and polyphenols, which were determined at high levels in ethanol and methanol extracts, as well as by the highest number and levels of single polyphenols. Conversely, the lower growth inhibitory activities found in chloroform and diethyl ether extracts could be related to the isolation of minor lipid classes (e.g., neutral and medium polar lipids) composed by fatty acids, such as stearic, oleic and arachidonic acids, typically characterized by antimicrobial activity. In consideration of the results obtained, the present study has a double implication, involving both the field of cultural practices and the exploitation of natural sources for the isolation of antimicrobial agents useful both in pharmaceutical and food applications.

Efficiency in hydrocarbon degradation and biosurfactant production by Joostella sp. A8 when grown in pure culture and consortia.

Joostella strains are emerging candidates for biosurfactant production. Here such ability was analyzed for Joostella strain A8 in comparison with Alcanivorax strain A53 and Pseudomonas strain A6, all previously isolated from hydrocarbon enrichment cultures made of polychaete homogenates. In pure cultures Joostella sp. A8 showed the highest stable emulsion percentage (78.33%), hydrophobicity rate (62.67%), and an optimal surface tension reduction during growth in mineral medium supplemented with diesel oil (reduction of about 12mN/m), thus proving to be highly competitive with Alcanivorax and Pseudomonas strains. During growth in pure culture different level of biodegradation were detected for Alcanivorax strain A53 (52.7%), Pseudomonas strain A6 (38.2%) and Joostella strain A8 (26.8%). When growing in consortia, isolates achieved similar abundance values, with the best efficiency that was observed for the Joostella-Pseudomonas co-culture. Gas-chromatographic analysis revealed an increase in the biodegradation efficiency in co-cultures (about 90%), suggesting that the contemporary action of different bacterial species could improve the process. Results were useful to compare the efficiencies of well-known biosurfactant producers (i.e. Pseudomonas and Alcanivorax representatives) with a still unknown biosurfactant producer, i.e. Joostella, and to confirm them as optimal biosurfactant-producing candidates.

Biosurfactant production by hydrocarbon-degrading Brevibacterium and Vibrio isolates from the sea pen Pteroeides spinosum (Ellis, 1764).

Among filter-feeders, pennatulids are the most complex and polymorphic members of the cnidarian class Anthozoa. They display a wide distribution throughout all the oceans, constituting a significant component of the sessile megafauna from intertidal to abyssal depths. In this study, a total of 118 bacterial isolates from enrichment cultures, carried out with homogenates of the sea pen Pteroeides spinosum (Ellis, 1764), were screened for hydrocarbon utilization by using the 2,6-dichlorophenol indophenol assay. Among them, 83 hydrocarbon-oxidizing isolates were analyzed for biosurfactant production by standard screening tests (i.e., emulsifying activity, E24 detection, surface tension measurement, microplate assay). The 16S rRNA gene sequencing revealed the affiliation of the most promising isolates to the genera Brevibacterium and Vibrio. Biosurfactant production resulted strongly affected by salinity and temperature conditions, and occurred in the presence of diesel oil and/or crude oil, whereas no production was observed when isolates were grown on tetradecane. The strains resulted able to create stable emulsions, thus suggesting the production of biosurfactants. Further analyses revealed a glycolipidic nature of the biosurfactant extracted from Vibrio sp. PBN295, a genus that has been only recently reported as biosurfactant producer. Results suggest that pennatulids could represent a novel source for the isolation of hydrocarbon-oxidizing bacteria with potential in biosurfactant production.

Biosurfactant activity, heavy metal tolerance and characterization of Joostella strain A8 from the Mediterranean polychaete Megalomma claparedei (Gravier, 1906).

The effect of heavy metals on the activity of biosurfactants produced by Joostella strain A8 from the polychaete Megalomma claparedei was investigated. Biosurfactant activity was first improved by evaluating the influence of abiotic parameters. Higher E(24) indices were achieved at 25 °C in mineral salt medium supplemented with 2 % glucose, 3 % sodium chloride (w/v) and 0.1 % ammonium chloride (w/v). Considerable surface tension reduction was never recorded. Heavy metal tolerance was preliminarily assayed by plate diffusion method resulting in the order of toxicity Cd > Cu > Zn. The activity of biosurfactants was then evaluated in the presence of heavy metals at different concentrations in liquid cultures that were incubated under optimal conditions for biosurfactant activity. The production of stable emulsions resulted generally higher in the presence of metals. These findings suggest that biosurfactant production could represent a bacterial adaptive strategy to defend cells from a stress condition derived from heavy metals in the bulk environment.

Influence of salinity and temperature on the activity of biosurfactants by polychaete-associated isolates.

Influence of different parameters on biosurfactant (BS) activity was carried out on strains that were isolated from the polychaetes Megalomma claparedei, Sabella spallanzanii and Branchiomma luctuosum and additional 30 strains that were previously identified as potential BS producers from crude oil enrichments of the same polychaete specimens. The selection of BS-producing strains from polychaete natural samples was carried out by using standard screening tests. The BS activity by each isolate was evaluated for the effect of salinity and temperature on emulsion production and surface tension reduction, during incubation in mineral medium supplemented with tetradecane or diesel oil. All isolates showed a similar time course of BS activity, and the latter was more influenced by salinity rather than temperature. Some of the BS producers belonged to genera that have not (i.e. Citricoccus, Cellulophaga, Tenacibaculum and Maribacter) or have poorly been (Psychrobacter, Vibrio, and Pseudoalteromonas) reported as able to produce BSs. This is remarkable as some of them have previously been detected in hydrocarbon-enriched samples. Results confirm that filter-feeding polychaetes are an efficient source for the isolation of BS producers.

The biodegradation efficiency on diesel oil by two psychrotrophic Antarctic marine bacteria during a two-month-long experiment.

Two psychrotrophic bacterial strains isolated from Antarctic seawaters were investigated for their capability to degrade commercial diesel oil. The efficiency of hydrocarbon utilization was studied at 4 and 20 degrees C over a period of two-months. Strains were cultured in a mineral liquid medium supplemented with diesel oil as the sole source of carbon and energy. The viable counts for the bacterial abundance estimation and the culture extractions for the subsequent gas-chromatographic analysis were carried out simultaneously. The biodegradation efficiency was higher at 20 degrees C than at 4 degrees C for both strains and the decrease in hydrocarbon concentrations reached more than 85% after 60 days of incubation at 20 degrees C. Our results suggest the possible exploitation of these two bacterial strains in future biotechnological processes, directly as field-released micro-organisms both in cold and temperate contaminated marine environments.