Investigating the anti-bioadhesion properties of short, medium chain length, and amphiphilic polyhydroxyalkanoate films.

Silicone materials are widely used in fouling release coatings, but developing eco-friendly protection via biosourced coatings, such as polyhydroxyalcanoates (PHA) presents a major challenge. Anti-bioadhesion properties of medium chain length PHA and short chain length PHA films are studied and compared with a reference Polydimethylsiloxane coating. The results highlight the best capability of the soft and low-roughness PHA-mcl films to resist bacteria or diatoms adsorption as compared to neat PDMS and PHBHV coatings. These parameters are insufficient to explain all the results and other properties related to PHA crystallinity are discussed. Moreover, the addition of a low amount of PEG copolymers within the coatings, to create amphiphilic coatings, boosts their anti-adhesive properties. This work reveals the importance of the physical or chemical ambiguity of surfaces in their anti-adhesive effectiveness and highlights the potential of PHA-mcl film to resist the primary adhesion of microorganisms.

Synthesis of Fluorinated Polyhydroxyalkanoates from Marine Bioresources as a Promising Biomaterial Coating.

By successive enzymatic and chemical modifications, novel fluorinated polyhydroxyalkanoates were synthesized and characterized. Unsaturated polyhydroxyalkanoate, PHAU, was first produced by fermentation using marine bacteria Pseudomonas raguenesii, and a graft copolymer PHAU-g-C8F17 was further prepared by controlled thiol-ene reaction in the presence of perfluorodecanethiol (PFDT). The PFDT grafting is realized by two different processes. In the first method, PHAU was previously solubilized in toluene. The grafting in solution is more efficient than the direct heterogeneous grafting onto a PHAU film. The degrees of grafting were determined by 1H NMR. The characterization of the microstructure by SEM-EDX and modulated and conventional DSC showed the formation of microdomains due to the organization of the hydrophobic segments of graft PFDT. Biomaterials prepared by 3D printing and coated by PHAU-g-C8F17 have the potential to be used as novel contrast agents as shown by Hahn echo experiments.

Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov., Two Acidophilic Bacteria Isolated from Decaying Wood, Hydrolyzing Cellulose and Producing Poly-3-hydroxybutyrate.

Two novel strains, HW T2.11T and HW T5.17T, were isolated from decaying wood (forest of Champenoux, France). Study of the 16S rRNA sequence similarity indicated that the novel strains belong to the genus Acidisoma. The sequence similarity of the 16S rRNA gene of HW T2.11T with the corresponding sequences of A. tundrae and A. sibiricum was 97.30% and 97.25%, while for HW T5.17T it was 96.85% and 97.14%, respectively. The DNA G+C contents of the strains were 62.32-62.50%. Cells were Gram-negative coccobacilli that had intracellular storage granules (poly-3-hydroxybutyrate (P3HB)) that confer resistance to environmental stress conditions. They were mesophilic and acidophilic organisms growing at 8-25 °C, at a pH of 2.0-6.5, and were capable of using a wide range of organic compounds and complex biopolymers such as starch, fucoidan, laminarin, pectin and cellulose, the latter two being involved in wood composition. The major cellular fatty acid was cyclo C19:0ω8c and the major quinone was Q-10. Overall, genome relatedness indices between genomes of strains HW T2.11T and HW T5.17T (Orthologous Average Nucleotide Identity (OrthoANI) value = 83.73% and digital DNA-DNA hybridization score = 27.5%) confirmed that they belonged to two different species. Genetic predictions indicate that the cyclopropane fatty acid (CFA) pathway is present, conferring acid-resistance properties to the cells. The two novel strains might possess a class IV polyhydroxyalcanoate (PHA) synthase operon involved in the P3HB production pathway. Overall, the polyphasic taxonomic analysis shows that these two novel strains are adapted to harsh environments such as decaying wood where the organic matter is difficult to access, and can contribute to the degradation of dead wood. These strains represent novel species of the genus Acidisoma, for which the names Acidisoma silvae sp. nov. and Acidisomacellulosilytica sp. nov. are proposed. The type strains of Acidisoma silvae and Acidisomacellulosilytica are, respectively, HW T2.11T (DSM 111006T; UBOCC-M-3364T) and HW T5.17T (DSM 111007T; UBOCC-M-3365T).

Amphiphilic and Perfluorinated Poly(3-Hydroxyalkanoate) Nanocapsules for 19F Magnetic Resonance Imaging.

Nanoparticles have recently emerged as valuable tools in biomedical imaging techniques. Here PEGylated and fluorinated nanocapsules based on poly(3-hydroxyalkanoate) containing a liquid core of perfluorooctyl bromide PFOB were formulated by an emulsion-evaporation process as potential 19F MRI imaging agents. Unsaturated poly(hydroxyalkanoate), PHAU, was produced by marine bacteria using coprah oil and undecenoic acid as substrates. PHA-g-(F; PEG) was prepared by two successive controlled thiol-ene reactions from PHAU with firstly three fluorinated thiols having from 3 up to 17 fluorine atoms and secondly with PEG-SH. The resulting PHA-g-(F; PEG)-based PFOB nanocapsules, with a diameter close to 250-300 nm, are shown to be visible in 19F MRI with an acquisition time of 15 min. The results showed that PFOB-nanocapsules based on PHA-g-(F; PEG) have the potential to be used as novel contrast agents for 19F MRI.

Water-Soluble Poly(3-hydroxyalkanoate) Sulfonate: Versatile Biomaterials Used as Coatings for Highly Porous Nano-Metal Organic Framework.

Water-soluble poly(3-hydroxyalkanoate) containing ionic groups were designed by two successive photoactivated thiol-ene reactions. Sodium-3-mercapto-1-ethanesulfonate (SO3-) and poly(ethylene glycol) (PEG) methyl ether thiol were grafted onto poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) PHO(67)U(33) to introduce both ionic groups and hydrophilic moieties. The grafted copolymers PHO(67)SO3-(20)PEG(13) were then used as biocompatible coatings of nano-metal organic frameworks (nanoMOFs) surfaces. Scanning electron microscopy and scanning transmission electron microscopy coupled with energy dispersive X-ray characterizations have clearly demonstrated the presence of the copolymer on the MOF surface. These coated nanoMOFs are stable in aqueous and physiological fluids. Cell proliferation and cytotoxicity tests performed on murine macrophages J774.A1 revealed no cytotoxic side effect. Thus, biocompatibility and stability of these novel hybrid porous MOF structures encourage their use in the development of effective therapeutic nanoparticles.

Use of Natural Antimicrobial Peptides and Bacterial Biopolymers for Cultured Pearl Production.

Cultured pearls are the product of grafting and rearing of Pinctada margaritifera pearl oysters in their natural environment. Nucleus rejections and oyster mortality appear to result from bacterial infections or from an inappropriate grafting practice. To reduce the impact of bacterial infections, synthetic antibiotics have been applied during the grafting practice. However, the use of such antibiotics presents a number of problems associated with their incomplete biodegradability, limited efficacy in some cases, and an increased risk of selecting for antimicrobial resistant bacteria. We investigated the application of a marine antimicrobial peptide, tachyplesin, which is present in the Japanese horseshoe crab Tachypleus tridentatus, in combination with two marine bacterial exopolymers as alternative treatment agents. In field studies, the combination treatment resulted in a significant reduction in graft failures vs. untreated controls. The combination of tachyplesin (73 mg/L) with two bacterial exopolysaccharides (0.5% w/w) acting as filming agents, reduces graft-associated bacterial contamination. The survival data were similar to that reported for antibiotic treatments. These data suggest that non-antibiotic treatments of pearl oysters may provide an effective means of improving oyster survival following grafting procedures.

Biosynthesis and characterization of polyhydroxyalkanoates by Pseudomonas guezennei from alkanoates and glucose.

The biosynthesis of medium chain length poly(3-hydroxyalkanoates) mcl PHAs by Pseudomonas guezennei using glucose, sodium octanoate, and 10-undecenoic acid as sole or mixed carbon sources was investigated. Chemical composition of polyesters was analyzed by GCMS and NMR. The copolyester produced by P. guezennei from glucose mainly consisted of 3-hydroxyoctanoate and 3-hydroxydecanoate, and the presence of 3-hydroxydodec-5-enoate was demonstrated. Using sodium octanoate as the sole nutrient, the microorganism produced a poly(3-hydroxyoctanoate) (PHO) polymer containing up to 94 mol% 3-hydroxyoctanoate. Biosynthesis of poly[(3-hydroxyoctanoate)-co-(3-hydroxyundecenoate)] (PHOU) copolymers bearing terminal reactive double bonds on its side chains with unsaturation degree ranging from 8.8% to 78.2% was obtained by tuning the ratio of sodium octanoate/10-undecenoic acid in the medium. Thermal analysis indicated semi-crystalline polymers with melting temperatures (T(m)) ranging from 46 to 55°C, fusion enthalpy (ΔH) comprised between 3 and 35 J/g and glass transition temperature (T(g)) from -36 to -44°C, except for the highly amorphous 78.2% unsaturated PHOU with a low T(g) (-50°C). Molecular weights determined by GPC ranged from 119000 and 530000 g/mol. The biosynthesis of natural polyesters with controlled ratio of vinyl-terminated side chains is of great interest for further chemical modifications.

Development of a three-steps derivatization assay for the localization of double bond in monounsaturated monomers of poly-beta-hydroxyalkanoates by GC-MS.

A new gas chromatography-mass spectrometry (GC-MS) method for the localization of double bond in monounsaturated 3-hydroxyalkenoic acids monomers has been developed. A three steps derivation assay was used including a methanolysis, then acetylation and dimethyldisulfide (DMDS) addition to alkene groups. Electron impact GC-MS analysis of such derivatives offers characteristic fragments allowing the unambiguous determination of double bond position in side chain. This novel method is well-suited for the routine analysis of poly-beta-hydroxyalkanoates (PHAs), and was used to characterize monounsaturated monomers in both 3-hydroxyalkenoic acids standards as well as in mcl-PHAs and poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) produced by bacterial strain Pseudomonas guezennei from glucose or a mixture of sodium octanoate plus 10-undecenoic acid, respectively.

Investigations into the uptake of copper, iron and selenium by a highly sulphated bacterial exopolysaccharide isolated from microbial mats.

A bacterium isolated from microbial mats located on a polynesian atoll produced a high molecular weight (3,000 kDa) and highly sulphated exopolysaccharide. Previous studies showed that the chemical structure of this EPS consisted of neutral sugars, uronic acids, and high proportions of acetate and sulphate groups. The copper- and iron-binding ability of the purified pre-treated native EPS was investigated. Results showed that this EPS had a very high affinity for both copper (9.84 mmol g(-1) EPS) and ferrous iron (6.9 mmol g(-1) EPS). Amazingly, this EPS did not show any affinity for either ferric ions or selenium salts. This finding is one of the first steps in assessing the biotechnological potential of this polysaccharide.

Floridoside extracted from the red alga Mastocarpus stellatus is a potent activator of the classical complement pathway.

Many biological properties of algae have been found to have useful applications in human health, particularly in the fields of oncology and immunology. Floridoside, extracted from the red alga Mastocarpus stellatus, has a structure similar to the xenoantigen Gal alpha 1-3 Gal. This xenoantigen has been described to induce a high immune response in human xenografts and is mediated by natural anti-gal antibodies that activate the classical complement pathway. Based on this property, we analyzed the potential activities of floridoside on the immune system. We demonstrated that floridoside activates a complement cascade via the classical complement pathway, through the recruitment and activation of natural IgM. This algal molecule could represent an important step in the development of a potent new anticomplementary agent for use in therapeutic complement depletion.

A novel mcl-PHA produced on coprah oil by Pseudomonas guezennei biovar. tikehau, isolated from a "kopara" mat of French Polynesia.

Pseudomonas guezennei biovar. tikehau was isolated from a microbial mat on the atoll of Tikehau in French Polynesia, and is able to synthesize medium chain length poly-beta-hydroxyalkanaote copolymers when grown on coprah oil. A two-step cultivation process was used and the biosynthesis of PHAs was followed along 52h by regular culture sampling. The polyester was purified from freeze-dried cells and analysed by nuclear magnetic resonance (NMR), Fourier transform infra red (FTIR), and gas chromatography mass spectrometries. The copolyester produced by P. guezennei biovar. tikehau from coprah oil mainly consisted of saturated monomers, i.e. 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), and the monomeric composition of the polyester did not change during the fermentation process. However, yield of PHAs production varied from 4% of the cellular dry weight (CDW) to 63% obtained after 36h. Scan electron microscopy was used to study the morphology and organization of PHAs granules within the cells and revealed the presence of several granules occupying almost the entire cell volume.

Isethionic acid and floridoside isolated from the red alga, Grateloupia turuturu, inhibit settlement of Balanus amphitrite cyprid larvae.

Isethionic acid (2-hydroxyethane sulfonic acid) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga, Grateloupia turuturu, and tested for anti-settlement activity against cyprid larvae of the tropical barnacle, Balanus amphitrite and for their toxicity to nauplius larvae. Isethionic acid was active for anti-settlement but had the disadvantage of being toxic to nauplius larvae. Floridoside was a potent inhibitor of cyprid settlement at non-toxic concentrations to nauplii (0.01 mg ml(-1)).

NMR 13C-isotopic enrichment experiments to study carbon-partitioning into organic solutes in the red alga Grateloupia doryphora.

The red alga Grateloupia doryphora Montagne (Howe) (Cryptonemiales, Halymeniaceae) was used as a model to investigate the effects of changes in seawater salinity on the intracellular low-molecular-weight organic compounds. Carbon-partitioning into major organic solutes was followed by 13C nuclear magnetic resonance (NMR) spectroscopy on living algae incubated in NaH13CO3-enriched seawater, and by high resolution 1H and 13C NMR experiments performed on 13C-enriched algal extracts. NMR and high performance liquid chromatography (HPLC) analyses both demonstrated that floridoside level was the most affected by changes in salinity: it rose under the hypersaline treatment and decreased under hyposaline one. Moreover, at low salinity, the high labeling of floridoside (45.3% 13C-enrichment for C1) together with its low concentrations both provided evidence of great increase in the de novo biosynthesis and turnover rate. Our experiments also demonstrated a high incorporation of photosynthetic carbon into amino acids, especially glutamate, under hypoosmotic conditions. On the other hand, isethionic acid and N-methyl-methionine sulfoxide were only partly labeled, which indicates they do not directly derive from carbon photoassimilation. In algae exposed to high salinity, elevated concentrations of floridoside coupled to a low labeling (9.4%) were observed. These results suggest that hyperosmotic conditions stimulated floridoside biosynthesis from endogen storage products rather than from carbon assimilation through photosynthesis.

Crystal structure and chirality of natural floridoside.

The crystal structure and absolute configuration of natural floridoside (2-O-alpha-D-galactopyranosylglycerol) were determined by single-crystal X-ray diffraction analysis. The space group is orthorhombic P2(1)2(1)2(1) with Z=4, a=4.885(1), b=9.734(1), c=23.886(2) A at 296 +/- 2 K. The structure was solved by a direct method and refined to R=0.0351 from 1914 reflections of Cu Kalpha radiation.

Complete 1H and 13C spectral assignment of floridoside.

Floridoside (2-O-alpha-D-galactopyranosylglycerol) was extracted from the red marine alga Rhodymenia palmata, and purified by ion-exchange chromatography: 1D and 2D NMR spectroscopy experiments were used to unambiguously assign the complete 1H and 13C spectra.