PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003.

Among the different tools which can be studied and managed to tailor-make polyhydroxyalkanoates (PHAs) and enhance their production, bacterial strain and carbon substrates are essential. The assimilation of carbon sources is dependent on bacterial strain's metabolism and consequently cannot be dissociated. Both must wisely be studied and well selected to ensure the highest production yield of PHAs. Halomonas sp. SF2003 is a marine bacterium already identified as a PHA-producing strain and especially of poly-3-hydroxybutyrate (P-3HB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P-3HB-co-3HV). Previous studies have identified different genes potentially involved in PHA production by Halomonas sp. SF2003, including two phaC genes with atypical characteristics, phaC1 and phaC2. At the same time, an interesting adaptability of the strain in front of various growth conditions was highlighted, making it a good candidate for biotechnological applications. To continue the characterization of Halomonas sp. SF2003, the screening of carbon substrates exploitable for PHA production was performed as well as production tests. Additionally, the functionality of both PHA synthases PhaC1 and PhaC2 was investigated, with an in silico study and the production of transformant strains, in order to confirm and to understand the role of each one on PHA production. The results of this study confirm the adaptability of the strain and its ability to exploit various carbon substrates, in pure or mixed form, for PHA production. Individual expression of PhaC1 and PhaC2 synthases in a non-PHA-producing strain, Cupriavidus necator H16 PHB¯4 (DSM 541), allows obtaining PHA production, demonstrating at the same time, functionality and differences between both PHA synthases. All the results of this study confirm the biotechnological interest in Halomonas sp. SF2003.

Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.

A halophilic Gram-negative eubacterium was isolated from the Iroise Sea and identified as an efficient producer of polyhydroxyalkanoates (PHA). The strain, designated SF2003, was found to belong to the Halomonas genus on the basis of 16S rRNA gene sequence similarity. Previous biochemical tests indicated that the Halomonas sp. strain SF2003 is capable of supporting various culture conditions which sometimes can be constraining for marine strains. This versatility could be of great interest for biotechnological applications. Therefore, a complete bacterial genome sequencing and de novo assembly were performed using a PacBio RSII sequencer and Hierarchical Genome Assembly Process software in order to predict Halomonas sp. SF2003 metabolisms, and to identify genes involved in PHA production and stress tolerance. This study demonstrates the complete genome sequence of Halomonas sp. SF2003 which contains a circular 4,36 Mbp chromosome, and replaces the strain in a phylogenetic tree. Genes related to PHA metabolism, carbohydrate metabolism, fatty acid metabolism and stress tolerance were identified and a comparison was made with metabolisms of relative species. Genes annotation highlighted the presence of typical genes involved in PHA biosynthesis such as phaA, phaB and phaC and enabled a preliminary analysis of their organization and characteristics. Several genes of carbohydrates and fatty acid metabolisms were also identified which provided helpful insights into both a better knowledge of the intricacies of PHA biosynthetic pathways and of production purposes. Results show the strong versatility of Halomonas sp. SF2003 to adapt to various temperatures and salinity which can subsequently be exploited for industrial applications such as PHA production.

In Vitro and In Vivo Evaluation of Pectin/Copper Exchanged Faujasite Composite Membranes.

The biocompatibility and excellent ion exchange capacity make faujasites ideal candidates for tissue engineering applications. A novel pectin/copper exchanged faujasite hybrid membrane was synthesized by solvent casting technique, using calcium chloride as the crosslinking agent. AFM images revealed the egg-box model organization of calcium cross-linked pectin chains used as a matrix. The morphology of composite membranes was characterized by SEM and their elemental composition was determined using EDX. The higher contact angle of P (1%) when compared to that of native pectin figured out an enhanced hydrophobicity of hybrid material. The embedded faujasite particles maintained their crystalline structure as revealed by XRD and their interactions with the polymer matrix was evaluated by FTIR. The composite membrane with 1% (w/w) of copper exchanged faujasite, P(1%), exhibited better thermal stability, excellent antibacterial activity, controlled swelling and degradation. Finally, it displayed cell viability of 89% on NIH3T3 fibroblast cell lines and aided in improving wound healing and re-epithelialisation in Sprague Dawley rats. The obtained data suggested their potential as ideal matrices for efficient treatment of burn wounds.

Rapid and qualitative fluorescence-based method for the assessment of PHA production in marine bacteria during batch culture.

The expansion of polyhydroxyalkanoates (PHAs) into the biodegradable polymers market is mainly prevented by their production process which is still complicated with a low efficiency, resulting in relatively expensive products. In this study, we developed a method that used the lipophilic fluorescent probe Nile Red (1 mg l(-1) solution in DMSO) directly into the culture broth to stain the PHA inclusions inside bacterial cells followed by detection of the emitted fluorescence by both microscopic and spectrometric techniques. Epifluorescence microscopy provides a rapid tool to distinguish producing from non-producing bacterial species and the relative fluorescence intensity (FI) determined at the maximum of emission spectra in the wavelength region of 560-710 nm (λ(ex): 543 nm), allows a fast assessment of the cultural conditions that may enhance PHA production yield. During two-step cultivation in 500-ml flasks with glucose as the sole carbon source, the method aimed to select bacterial strains efficient for PHA synthesis among a marine collection. Subsequently, the NR assay was used to determine the C0/N0 ratio of the producing media that may improve the polymer yield as well as to follow the time course of fermentation. Characterization by GC-MS and DSC confirmed the production of the P(3-HB) homopolymer.

Antibacterial and wound healing analysis of gelatin/zeolite scaffolds.

In this article, gelatin/copper activated faujasites (CAF) composite scaffolds were fabricated by lyophilisation technique for promoting partial thickness wound healing. The optimised scaffold with 0.5% (w/w) of CAF, G (0.5%), demonstrated pore size in the range of 10-350 µm. Agar disc diffusion tests verified the antibacterial role of G (0.5%) and further supported that bacterial lysis was due to copper released from the core of CAF embedded in the gelatin matrix. The change in morphology of bacteria as a function of CAF content in gelatin scaffold was studied using SEM analysis. The confocal images revealed the increase in mortality rate of bacteria with increase in concentration of incorporated CAF in gelatin matrix. Proficient oxygen supply to needy cells is a continuing hurdle faced by tissue engineering scaffolds. The dissolved oxygen measurements revealed that CAF embedded in the scaffold were capable of increasing oxygen supply and thereby promote cell proliferation. Also, G (0.5%) exhibited highest cell viability on NIH 3T3 fibroblast cells which was mainly attributed to the highly porous architecture and its ability to enhance oxygen supply to cells. In vivo studies conducted on Sprague Dawley rats revealed the ability of G (0.5%) to promote skin regeneration in 20 days. Thus, the obtained data suggest that G (0.5%) is an ideal candidate for wound healing applications.

Faujasites incorporated tissue engineering scaffolds for wound healing: in vitro and in vivo analysis.

Exploring the possibility of using inorganic faujasites in tissue engineering scaffolds is a prospective approach in regenerative medicine. Novel gelatin/hyaluronic acid (HA)/faujasite porous scaffolds with low surface energy were fabricated by lyophilization. The pore size of gelatin/HA scaffold was 50-2000 µm, whereas it was greatly reduced to 10-250 µm after incorporation of 2.4% (w/w) of faujasites in polymer matrix, GH(2.4%). Micro computed tomography analysis showed that the porosity of GH(2.4%) was 90.6%. The summative effect was ideal for growth of dermal fibroblasts and cellular attachment. XRD analysis revealed that the embedded faujasites maintained their crystallinity in the polymer matrix even though they interacted with the polymers as indicated by FT-IR analysis. Coupling with effective reinforcement of faujasites, GH(2.4%) demonstrated compression modulus of 929 ± 7 Pa and glass transition temperature of 31 ± 0.05 °C. It exhibited controlled swelling and degradation, allowing sufficient space for tissue regrowth. The latter is further supported by capability of faujasites to provide efficient oxygen supply to fibroblast cells. GH(2.4%) showed a cell viability of 91 ± 8% on NIH 3T3 fibroblast cell lines. The in vivo studies on Sprague-Dawley rats revealed its ability to enhance wound healing by accelerating re-epithelization and collagen deposition. These findings indicated its potential as excellent wound dressing material.

Pectin/carboxymethyl cellulose/microfibrillated cellulose composite scaffolds for tissue engineering.

Highly porous three-dimensional scaffolds made of biopolymers are of great interest in tissue engineering applications. A novel scaffold composed of pectin, carboxymethyl cellulose (CMC) and microfibrillated cellulose (MFC) were synthesised using lyophilisation technique. The optimised scaffold with 0.1% MFC, C(0.1%), showed highest compression modulus (~3.987 MPa) and glass transition temperature (~103 °C). The pore size for the control scaffold, C(0%), was in the range of 30-300 µm while it was significantly reduced to 10-250 µm in case of C(0.1%). Using micro computed tomography, the porosity of C(0.1%) was estimated to be 88%. C(0.1%) showed excellent thermal stability and lower degradation rate compared to C(0%). The prepared samples were also characterised using XRD and FTIR. C(0.1%) showed controlled water uptake ability and in vitro degradation in PBS. It exhibited highest cell viability on NIH3T3 fibroblast cell line. These results suggest that these biocompatible composite scaffolds can be used for tissue engineering applications.