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
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.
Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation.
- MeSH
- Cupriavidus necator růst a vývoj metabolismus ultrastruktura MeSH
- cytoplazmatická granula metabolismus ultrastruktura MeSH
- fluorescenční mikroskopie MeSH
- konfokální mikroskopie MeSH
- polyhydroxyalkanoáty chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Coffee is one of the world's most popular beverages and has been growing steadily in commercial importance. Nowadays, coffee is the second largest traded commodity in the world, after petroleum. Hence, coffee industry is responsible for the generation of large amounts of waste, especially spent coffee grounds (SCG). Various attempts to valorize this waste stream of coffee industry were made. This article summarizes our research and publications aiming at the conversion of SCG into valuable products - polyhydroxyalkanoates (PHAs) and carotenoids. At first, oil extracted from SCG (approx. 15 wt% oil in SCG) can be efficiently (YP/S=0.82 g/g) converted into PHA employing Cupriavidus necator H16. Further, the solid residues after oil extraction can be hydrolyzed (by the combination of chemical and enzymatic hydrolysis) yielding fermentable sugars, which can be further used as a substrate for the production of PHAs employing Bacillus megaterium (YP/S=0.04 g/g) or Burkholderia cepacia (YP/S=0.24 g/g). Alternatively, SCG hydrolysate can be used as a substrate for biotechnological production of carotenoids by carotenogenic yeast Sporobolomyces roseus. Solid residues after either oil extraction or hydrolysis can be used as fuel in industrial boilers to generate heat and energy. Therefore, entire biomass of SCG can be used for sustainable production of PHAs and/or carotenoids employing bio-refinery approach.
- MeSH
- biopaliva mikrobiologie MeSH
- biotechnologie metody MeSH
- extrakce kapalina-kapalina metody MeSH
- karotenoidy biosyntéza chemie izolace a purifikace MeSH
- káva chemie mikrobiologie MeSH
- odpadky - odstraňování metody MeSH
- polyhydroxyalkanoáty biosyntéza chemie izolace a purifikace MeSH
- zachování přírodních zdrojů metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R (2) = 0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process.
Whole whey hydrolyzed by Alcalase (WWH) was tested as a complex nitrogen source for the production of poly(3-hydroxybutyrate) (PHB) from waste frying oils by Cupriavidus necator H16. Addition of WWH (10 % (v/v) of cultivation media) supported the growth and PHB accumulation; PHB yields in Erlenmeyer flasks were more than 3.5-fold higher than in control cultivations. The positive influence of WWH on PHB production was confirmed in experiments performed in laboratory fermentor. C. necator cultivated with WWH produced 28.1 g PHB l(-1) resulting in a very high product yield coefficient of 0.94 g PHB per g oil. Since PHB yields were ~40 % higher than in the control cultivation, WWH can be considered as an excellent inexpensive nitrogen source for PHB production by C. necator.
- MeSH
- Cupriavidus necator růst a vývoj metabolismus MeSH
- dusík metabolismus MeSH
- hydrolýza MeSH
- hydroxybutyráty metabolismus MeSH
- kultivační média chemie MeSH
- mléčné bílkoviny metabolismus MeSH
- polyestery metabolismus MeSH
- subtilisiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The article summarizes the examination methods in ophthalmology that are currently available and useful in diagnosing an eye disease.
- MeSH
- akupunkturní terapie využití MeSH
- bolest MeSH
- dospělí MeSH
- extrakce zubů škodlivé účinky MeSH
- lidé MeSH
- management bolesti MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
