In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles.
- MeSH
- Comamonadaceae * genetics MeSH
- Genetic Engineering MeSH
- Polyhydroxyalkanoates * MeSH
- Temperature MeSH
- Carbon MeSH
- Publication type
- Journal Article MeSH
The polymeric cytisine-enriched fibers based on poly(3-hydroxybutyrate) were obtained using electrospinning method. The biocompatibility study, advanced thermal analysis and release of cytisine from the poly(3-hydroxybutyrate) fibers were carried out. The nanofibers' morphology was evaluated by scanning electron microscopy. The formation and description of phases during the thermal processes of fibers by the advanced thermal analysis were examined. The new quantitative thermal analysis of polymeric fibers with cytisine phases based on vibrational, solid and liquid heat capacities was presented. The apparent heat capacity of fibers was measured using the standard differential scanning calorimetry. The quantitative analysis allowed for the study of the glass transition and melting/crystallization process. The mobile amorphous fraction, degree of crystallinity and rigid amorphous fraction were determined depending on the thermal history of semicrystalline polymeric fibers. Furthermore, the cytisine dissolution behaviour was studied. It was observed that the kinetic of the release from polymeric nanofiber is delayed than for the marketed product. The immunosafety of the tested polymeric nanofibers with cytisine was confirmed by the Food and Drug Agency Guidance as well as the European Medicines Agency. The polymeric matrix with cytisine seems to be a promising candidate for the prolonged release formulation.
A novel method for the extraction of river water contaminants as model analytes of ranging polarities, including bisphenols A, C, S, Z, fenoxycarb, kadethrin, and deltamethrin, using small compact fibrous disks has been developed and validated. Polymer nanofibers and microfibers prepared from poly(3-hydroxybutyrate), polypropylene, polyurethane, polyacrylonitrile, poly(lactic acid), and polycaprolactone doped with graphene were evaluated in terms of extraction efficiency, selectivity, and stability in organic solutions. Our novel extraction procedure comprised preconcentration of analytes from 150 mL river water to 1 mL of eluent using a compact nanofibrous disk freely vortexed in the sample. Small nanofibrous disks with a diameter of 10 mm were cut from a compact and mechanically stable 1-2 mm thick micro/nanofibrous sheet. After 60 min extraction in a magnetically stirred sample located in a beaker, the disk was removed from the liquid and washed with water. Then, the disk was inserted into a 1.5 mL HPLC vial and extracted with 1.0 ml methanol upon short intensive shaking. Our approach avoided the undesired problems related to the manual handling typical of "classical" SPE procedure since the extraction was carried out directly in the HPLC vial. No sample evaporation, reconstitution, or pipetting was required. The nanofibrous disk is affordable, needs no support or holder, and its use avoids creation of plastic waste originating from disposable materials. Recovery of compounds from the disks was 47.2-141.4% depending on the type of polymer used and the relative standard deviations calculated from 5 extractions ranged from 6.1 to 11.8% for poly(3-hydroxybutyrate), 6.3-14.8% for polyurethane, and 1.7-16.2% for polycaprolactone doped with graphene. A small enrichment factor was obtained for polar bisphenol S using all sorbents. A higher preconcentration reaching up to 40-fold was achieved for lipophilic compounds such as deltamethrin when using poly(3-hydroxybutyrate) and graphene-doped polycaprolactone.
- Publication type
- Journal Article MeSH
Sensitive electrophoretic determination of 3-hydroxybutyrate (3HB) as an indicator of human ketogenesis is performed in fused silica capillary covalently coated by an anionic copolymer of poly(acrylamide-co-sodium-2-acrylamido-2-methylpropanesulphonate) (PAMAMPS). Baseline separation of 3HB from other components of human serum is achieved in a 20 μm capillary with an effective length of 17 cm covered by 4% PAMAMPS, which generates a cathodic EOF with a mobility of 8.30 ± 0.00 · 10-9 m2/V.s in 80 mM MES/His as background electrolyte. 3HB migrates in counter-current electrophoretic mode against EOF, that effectively improving electrophoretic resolution. Sample pre-treatment is based on adding of 45 μL acetonitrile to 15 μL serum and, after shaking, a 28 mm long zone of supernatant is injected into the capillary, and sharpened after turning on a separation voltage of 20 kV using the technique of large volume sample stacking, where the EOF forces the residual acetonitrile from the capillary. When combined with universal contactless conductivity detection, the achieved LOD and LOQ are 0.43 μM and 1.44 μM, respectively, that are sufficiently low for monitoring the physiological 3HB level. The performed clinical study subsequently showed that serum 3HB increases from a concentration of 71 μM, corresponding to normal food, to level of 1924 μM after 60 h of fasting and returns to the normal physiological concentration 48 h after commencing consumption of high-saccharide food.
- MeSH
- Acetonitriles MeSH
- Acrylic Resins MeSH
- Alkanesulfonates MeSH
- Electrophoresis, Capillary * methods MeSH
- 3-Hydroxybutyric Acid MeSH
- Humans MeSH
- Fasting * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The cyanobacterial genus Synechocystis is of particular interest to science and industry because of its efficient phototrophic metabolism, its accumulation of the polymer poly(3-hydroxybutyrate) (PHB) and its ability to withstand or adapt to adverse growing conditions. One such condition is the increased salinity that can be caused by recycled or brackish water used in cultivation. While overall reduced growth is expected in response to salt stress, other metabolic responses relevant to the efficiency of phototrophic production of biomass or PHB (or both) have been experimentally observed in three Synechocystis strains at stepwise increasing salt concentrations. In response to recent reports on metabolic strategies to increase stress tolerance of heterotrophic and phototrophic bacteria, we focused particularly on the stress-induced response of Synechocystis strains in terms of PHB, glycogen and photoactive pigment dynamics. Of the three strains studied, the strain Synechocystis cf. salina CCALA192 proved to be the most tolerant to salt stress. In addition, this strain showed the highest PHB accumulation. All the three strains accumulated more PHB with increasing salinity, to the point where their photosystems were strongly inhibited and they could no longer produce enough energy to synthesize more PHB.
- Publication type
- Journal Article MeSH
Fused deposition modelling (FDM) is a process of additive manufacturing allowing creating of highly precise complex three-dimensional objects for a large range of applications. The principle of FDM is an extrusion of the molten filament and gradual deposition of layers and their solidification. Potential applications in pharmaceutical and medical fields require the development of biodegradable and biocompatible thermoplastics for the processing of filaments. In this work, the potential of production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) filaments for FDM was investigated in respect to its thermal stability. Copolymer P(3HB-co-4HB) was biosynthesised by Cupriavidus malaysiensis. Rheological and mechanical properties of the copolymer were modified by the addition of plasticizers or blending with poly(lactic acid). Thermal stability of mixtures was studied employing thermogravimetric analysis and rheological analyses by monitoring the time-dependent changes in the complex viscosity of melt samples. The plasticization of P(3HB-co-4HB) slightly hindered its thermal degradation but the best stabilization effect was found in case of the copolymer blended with poly(lactic acid). Overall, rheological, thermal and mechanical properties demonstrated that the plasticized P(3HB-co-4HB) is a potential candidate of biodegradable polymer for FDM processes.
Effect of physicochemical properties including dissociation constant (pKa) and partition coefficient (log P) of the compounds on their extraction efficiency in sample preparation using fibrous polymer sorbents has been demonstrated. Poly-ε-caprolactone as meltblown/electrospun composite fibers, and polypropylene, polyethylene, poly(3-hydroxybutyrate), poly(lactic acid), and polyamide 6 in the meltblown fiber format were used as sorbents in solid-phase extraction. In addition, the polycaprolactone fibers were coated with dopamine, dopamine combined with heparin, and tannin, respectively, to modify their extraction properties. These fibers that were not yet used for extractions and the unique combination of sorbents and analytes significantly extends the scope of nanofibrous extraction. The extraction efficiency was determined using model pharmaceuticals including acetylsalicylic acid, moxonidine, metoprolol, propranolol, propafenone, diltiazem, atorvastatin, and amiodarone. These model compounds displayed the widest differences in both pKa and log P values. The extraction efficiency of some of the fibers reached 96.64%. Coating of polycaprolactone fibers with dopamine significantly improved extraction efficiency of slightly retained metoprolol while moxonidine was not retained on any sorbent. The fibrous sorbents were also tested for extraction of pharmaceuticals in bovine serum albumin and human serum, respectively, to demonstrate their capability to extract them from a complex protein-containing matrix. The clean-up efficiency of our fibers was compared with that of a commercial restricted access media (RAM) C-18 alkyl-diol silica column. Our technique is in accordance with the requirements of modern sample preparation techniques.
- MeSH
- Solid Phase Extraction MeSH
- Humans MeSH
- Nanofibers * MeSH
- Polymers MeSH
- Proteins MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The aim of this work was to develop a soap-based method for the isolation of poly(3-hydroxybutyrate) from bacterial biomass. The method consisted of adding soap derived from waste cooking oil to a concentrated (25%) biomass suspension, heating and centrifugal separation. Purity above 95% could be achieved with soap:cell dry mass ratios at least 0.125 g/g, making the method comparable to other surfactant-based protocols. Molecular weights Mw of products from all experiments were between 350 and 450 kDa, being high enough for future material applications. Addition of hydrochloric acid to the wastewater led to the precipitation of soap and part of non-P3HB cell mass. The resulting precipitate was utilized as a carbon source in biomass production and increased substrate-to-P3HB conversion.
- MeSH
- Biomass MeSH
- Bioreactors * MeSH
- Hydroxybutyrates MeSH
- 3-Hydroxybutyric Acid MeSH
- Soaps * MeSH
- Polyesters MeSH
- Cooking MeSH
- Publication type
- Journal Article MeSH
Complex in vitro characterization of a blended material based on Poly(Lactic Acid), Poly(Hydroxybutyrate), and Thermoplastic Starch (PLA/PHB/TPS) was performed in order to evaluate its potential for application in the field of tissue engineering. We focused on the biological behavior of the material as well as its mechanical and morphological properties. We also focused on the potential of the blend to be processed by the 3D printer which would allow the fabrication of the custom-made scaffold. Several blends recipes were prepared and characterized. This material was then studied in the context of scaffold fabrication. Scaffold porosity, wettability, and cell-scaffold interaction were evaluated as well. MTT test and the direct contact cytotoxicity test were applied in order to evaluate the toxic potential of the blended material. Biocompatibility studies were performed on the human chondrocytes. According to our results, we assume that material had no toxic effect on the cell culture and therefore could be considered as biocompatible. Moreover, PLA/PHB/TPS blend is applicable for 3D printing. Printed scaffolds had highly porous morphology and were able to absorb water as well. In addition, cells could adhere and proliferate on the scaffold surface. We conclude that this blend has potential for scaffold engineering.
The aim of this work was to investigate the thermophilic bacterium Schelegelella thermodepolymerans DSM 15344 in terms of its polyhydroxyalkanoates (PHA) biosynthesis capacity. The bacterium is capable of converting various sugars into PHA with the optimal growth temperature of 55 °C; therefore, the process of PHA biosynthesis could be robust against contamination. Surprisingly, the highest yield was gained on xylose. Results suggested that S. thermodepolymerans possess unique xylose metabolism since xylose is utilized preferentially with the highest consumption rate as compared to other sugars. In the genome of S. thermodepolymerans DSM 15344, a unique putative xyl operon consisting of genes responsible for xylose utilization and also for its transport was identified, which is a unique feature among PHA producers. The bacterium is capable of biosynthesis of copolymers containing 3-hydroxybutyrate and also 3-hydroxyvalerate subunits. Hence, S.thermodepolymerans seems to be promising candidate for PHA production from xylose rich substrates.
- MeSH
- Comamonadaceae * MeSH
- 3-Hydroxybutyric Acid MeSH
- Polyhydroxyalkanoates * MeSH
- Xylose MeSH
- Publication type
- Journal Article MeSH
