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.
Polyhydroxyalkanoates (PHAs) are polyesters of significant interest due to their biodegradability and properties similar to petroleum-derived plastics, as well as the fact that they can be produced from renewable sources such as by-product streams. In this study, brewer's spent grain (BSG), the main by-product of the brewing industry, was subjected to a set of physicochemical pretreatments and their effect on the release of reducing sugars (RS) was evaluated. The RS obtained were used as a substrate for further PHA production in Burkholderia cepacia, Bacillus cereus, and Cupriavidus necator in liquid cultures. Although some pretreatments proved efficient in releasing RS (acid-thermal pretreatment up to 42.1 gRS L-1 and 0.77 gRS g-1 dried BSG), the generation of inhibitors in such scenarios likely affected PHA production compared with the process run without pretreatment (direct enzymatic hydrolysis of BSG). Thus, the maximum PHA accumulation from BSG hydrolysates was found in the reference case with 0.31 ± 0.02 g PHA per g cell dried weight, corresponding to 1.13 ± 0.06 g L-1 and a PHA yield of 23 ± 1 mg g-1 BSG. It was also found that C. necator presented the highest PHA accumulation of the tested strains followed closely by B. cepacia, reaching their maxima at 48 h. Although BSG has been used as a source for other bioproducts, these results show the potential of this by-product as a no-cost raw material for producing PHAs in a waste valorization and circular economy scheme.
Polyhydroxyalkanoates (PHA) are microbial polyesters which accumulate as intracellular granules in numerous prokaryotes and mainly serve as storage materials; beyond this primary function, PHA also enhance the robustness of bacteria against various stress factors. We have observed that the presence of PHA in bacterial cells substantially enhances their ability to maintain cell integrity when suddenly exposed to osmotic imbalances. In the case of the non-halophilic bacterium Cupriavidus necator, the presence of PHA decreased plasmolysis-induced cytoplasmic membrane damage during osmotic up-shock, which subsequently enabled the cells to withstand subsequent osmotic downshock. In contrast, sudden induction of osmotic up- and subsequent down-shock resulted in massive hypotonic lysis of non-PHA containing cells as determined by Transmission Electron Microscopy and Thermogravimetrical Analysis. Furthermore, a protective effect of PHA against hypotonic lysis was also observed in the case of the halophilic bacterium Halomonas halophila; here, challenged PHA-rich cells were capable of retaining cell integrity more effectively than their PHA-poor counterparts. Hence, it appears that the fact that PHA granules, as an added value to their primary storage function, protect halophiles from the harmful effect of osmotic down-shock might explain why PHA accumulation is such a common feature among halophilic prokaryotes. The results of this study, apart from their fundamental importance, are also of practical biotechnological significance: because PHA-rich bacterial cells are resistant to osmotic imbalances, they could be utilized in in-situ bioremediation technologies or during enrichment of mixed microbial consortia in PHA producers under conditions of fluctuating salinity.
- MeSH
- Bacteria cytologie účinky léků metabolismus MeSH
- Cupriavidus necator cytologie účinky léků metabolismus ultrastruktura MeSH
- Halomonas cytologie účinky léků metabolismus ultrastruktura MeSH
- mikrobiální viabilita účinky léků MeSH
- osmóza * MeSH
- polyhydroxyalkanoáty farmakologie MeSH
- teplota MeSH
- termogravimetrie MeSH
- Publikační typ
- časopisecké články MeSH
This study evaluates a biorefinery concept for producing poly(3-hydroxybutyrate) (PHB) with the cyanobacterial strain Synechocystis salina. Due to this reason, pigment extraction and cell disruption were investigated as pre-treatment steps for the harvested cyanobacterial biomass. The results demonstrated that at least pigment removal was necessary to obtain PHB with processable quality (weight average molecular weight: 569-988kgmol-1, melting temperature: 177-182°C), which was comparable to heterotrophically produced PHB. The removed pigments could be utilised as additional by-products (chlorophylls 0.27-1.98mgg-1 TS, carotenoids 0.21-1.51mgg-1 TS, phycocyanin 0-127mgg-1 TS), whose concentration depended on the used nutrient source. Since the residual biomass still contained proteins (242mgg-1 TS), carbohydrates (6.1mgg-1 TS) and lipids (14mgg-1 TS), it could be used as animal feed or converted to biomethane (348 mn3 t-1VS) and fertiliser. The obtained results indicate that the combination of photoautotrophic PHB production with pigment extraction and utilisation of residual biomass offer the highest potential, since it contributes to decrease the environmental footprint of the process and because biomass could be used in a cascading way and the nutrient cycle could be closed.
Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L-1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L-1 contained 42% PHB.
Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.
Numerous prokaryotes accumulate polyhydroxybutyrate (PHB) intracellularly as a storage material. It has also been proposed that PHB accumulation improves bacterial stress resistance. Cupriavidus necator and its PHB non-accumulating mutant were employed to investigate the protective role of PHB under hypertonic conditions. The presence of PHB granules enhanced survival of the bacteria after exposure to hypertonic conditions. Surprisingly, when coping with such conditions, the bacteria did not utilize PHB to harvest carbon or energy, suggesting that, in the osmotic upshock of C. necator, the protective mechanism of PHB granules is not associated with their hydrolysis. The presence of PHB granules influenced the overall properties of the cells, since challenged PHB-free cells underwent massive plasmolysis accompanied by damage to the cell membrane and the leakage of cytoplasm content, while no such effects were observed in PHB containing bacteria. Moreover, PHB granules demonstrated "liquid-like" properties indicating that they can partially repair and stabilize cell membranes by plugging small gaps formed during plasmolysis. In addition, the level of dehydration and changes in intracellular pH in osmotically challenged cells were less pronounced for PHB-containing cultures, demonstrating the important role of PHB for bacterial survival under hyperosmotic conditions.
- MeSH
- časové faktory MeSH
- Cupriavidus necator cytologie účinky léků metabolismus ultrastruktura MeSH
- cytoplazmatická granula účinky léků metabolismus ultrastruktura MeSH
- elektronová kryomikroskopie MeSH
- fluoresceiny metabolismus MeSH
- fluorescenční mikroskopie MeSH
- hydroxybutyráty metabolismus MeSH
- hypertonické roztoky farmakologie MeSH
- krystalizace MeSH
- mikrobiální viabilita účinky léků MeSH
- osmotický tlak účinky léků MeSH
- termogravimetrie MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
The chicken feather hydrolysate (FH) has been tested as a potential complex nitrogen source for the production of polyhydroxyalkanoates by Cupriavidus necator H16 when waste frying oil was used as a carbon source. The addition of FH into the mineral salt media with decreased inorganic nitrogen source concentration improved the yields of biomass and polyhydrohyalkanoates. The highest yields were achieved when 10 vol.% of FH prepared by microwave-assisted alkaline hydrolysis of 60 g l-1 feather was added. In this case, the poly(3-hydroxybutyrate) (PHB) yields were improved by more than about 50% as compared with control cultivation. A positive impact of FH was also observed for accumulation of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when sodium propionate was used as a precursor. The copolymer has superior processing and mechanical properties in comparison with PHB homopolymer. The application of FH eliminated the inhibitory effect of propionate and resulted in altered content of 3-hydroxyvalerate (3HV) in copolymer. Therefore, the hydrolysed feather can serve as an excellent complex source of nitrogen for the polyhydroxyalkanoates (PHA) production. Moreover, by the combination of two inexpensive types of waste, such as waste frying oil and feather hydrolysate, it is possible to produce PHA with substantially improved efficiency and sustainability. SIGNIFICANCE AND IMPACT THE STUDY: Millions of tons of feathers, important waste product of poultry-processing industry, are disposed off annually without any further benefits. Thus, there is an inevitable need for new technologies that enable ecologically and economically sensible processing of this waste. Herein, we report that alkali-hydrolysed feathers can be used as a complex nitrogen source considerably improving polyhydroxyalkanoates production on waste frying oil employing Cupriavidus necator.
- MeSH
- aminokyseliny analýza MeSH
- biomasa MeSH
- Cupriavidus necator metabolismus MeSH
- dusík metabolismus MeSH
- hydrolýza MeSH
- hydroxybutyráty metabolismus MeSH
- kur domácí MeSH
- kyseliny pentanové metabolismus MeSH
- odpad tekutý - odstraňování MeSH
- oleje rostlin metabolismus MeSH
- peří chemie MeSH
- polyestery metabolismus MeSH
- polyhydroxyalkanoáty biosyntéza MeSH
- uhlík metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Poly(3-hydroxybutyrate) (PHB) is a common carbon- and energy-storage compound simultaneously produced and degraded into its monomer 3-hydroxybutyrate (3HB) by numerous bacteria and Archae in a metabolic pathway called the PHB cycle. We investigated 3HB as a chemical chaperone capable of protecting model enzymes, namely lipase and lysozyme, from adverse effects of high temperature and oxidation. Heat-mediated denaturation of lipase in the presence or absence of 3HB was monitored by dynamic light scattering (DLS) revealing a significant protective effect of 3HB which increased as its concentration rose. Furthermore, when compared at the same molar concentration, 3HB showed a greater protective effect than the well-known chemical chaperones trehalose and hydroxyectoine. The higher protective effect of 3HB was also confirmed when employing differential scanning calorimetry (DSC) and lysozyme as a model enzyme. Furthermore, 3HB was capable of protecting lipase not only against thermal-mediated denaturation but also against oxidative damage by Cu(2+) and H2O2; its protection was higher than that of trehalose and comparable to that of hydroxyectoine. Taking into account that the PHB-producing strain Cupriavidus necator H16 reveals a 16.5-fold higher intracellular concentration than the PHB non-producing mutant C. necator PHB(-4), it might be expected that the functional PHB cycle might be responsible for maintaining a higher intracellular level of 3HB which, aside from other positive aspects of functional PHB metabolism, enhances stress resistance of bacterial strains capable of simultaneous PHB synthesis and mobilization. In addition, 3HB can be used in various applications and formulations as an efficient enzyme-stabilizing and enzyme-protecting additive.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- Cupriavidus necator chemie enzymologie metabolismus MeSH
- hydroxybutyráty metabolismus MeSH
- kyselina 3-hydroxymáselná chemie metabolismus MeSH
- muramidasa chemie metabolismus MeSH
- ochranné látky chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- polyestery metabolismus MeSH
- stabilita enzymů MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.
