Dan Kucera

Biotechnological conversion of spent coffee grounds into polyhydroxyalkanoates and carotenoids

Coffee is one of the worlds 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.

Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing

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.

Characterization of the promising poly(3-hydroxybutyrate) producing halophilic bacterium Halomonas halophila

This work explores molecular, morphological as well as biotechnological features of the highly promising polyhydroxyalkanoates (PHA) producer Halomonas halophila. Unlike many other halophiles, this bacterium does not require expensive complex media components and it is capable to accumulate high intracellular poly(3-hydroxybutyrate) (PHB) fractions up to 82% of cell dry mass. Most remarkably, regulating the concentration of NaCl apart from PHB yields influences also the polymers molecular mass and polydispersity. The bacterium metabolizes various carbohydrates including sugars predominant in lignocelluloses and other inexpensive substrates. Therefore, the bacterium was employed for PHB production on hydrolysates of cheese whey, spent coffee grounds, sawdust and corn stover, which were hydrolyzed by HCl; required salinity of cultivation media was set up during neutralization by NaOH. The bacterium was capable to use all the tested hydrolysates as well as sugar beet molasses for PHB biosynthesis, indicating its potential for industrial PHB production.

Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

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.

Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation

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.

Production of Polyhydroxyalkanoates Using Hydrolyzates of Spruce Sawdust: Comparison of Hydrolyzates Detoxification by Application of Overliming, Active Carbon, and Lignite

Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the price of the carbon source used in the fermentation. Burkholderia cepacia and Burkholderia sacchari are generally considered promising candidates for PHA production from lignocellulosic hydrolyzates. The wood waste biomass has been subjected to hydrolysis. The resulting hydrolyzate contained a sufficient amount of fermentable sugars. Growth experiments indicated a strong inhibition by the wood hydrolyzate. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification. All methods of detoxification had a positive influence on the growth of biomass and PHB production. Furthermore, lignite was identified as a promising alternative sorbent which can be used for detoxification of lignocellulose hydrolyzates. Detoxification using lignite instead of activated carbon had lower inhibitor removal efficiency, but greater positive impact on growth of the bacterial culture and overall PHA productivity. Moreover, lignite is a significantly less expensive adsorbent in comparison with activated charcoal and; moreover, used lignite can be simply utilized as a fuel to, at least partially, cover heat and energetic demands of fermentation, which should improve the economic feasibility of the process.

Quantitative Raman Spectroscopy Analysis of Polyhydroxyalkanoates Produced by Cupriavidus necator H16

We report herein on the application of Raman spectroscopy to the rapid quantitative analysis of polyhydroxyalkanoates (PHAs), biodegradable polyesters accumulated by various bacteria. This theme was exemplified for quantitative detection of the most common member of PHAs, poly(3-hydroxybutyrate) (PHB) in Cupriavidus necator H16. We have identified the relevant spectral region (800–1800 cm−1) incorporating the Raman emission lines exploited for the calibration of PHB (PHB line at 1736 cm−1) and for the selection of the two internal standards (DNA at 786 cm−1 and Amide I at 1662 cm−1). In order to obtain quantitative data for calibration of intracellular content of PHB in bacterial cells reference samples containing PHB amounts—determined by gas chromatography—from 12% to 90% (w/w) were used. Consequently, analytical results based on this calibration can be used for fast and reliable determination of intracellular PHB content during biotechnological production of PHB since the whole procedure—from bacteria sampling, centrifugation, and sample preparation to Raman analysis—can take about 12 min. In contrast, gas chromatography analysis takes approximately 8 h.

Chicken feather hydrolysate as inexpensive complex nitrogen source for PHA production by Cupriavidus necator on waste frying oils

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.

Polyhydroxyalkanoates in Bacterial Cells - More Than just Storage Materials

Since polyhydroxyalkanoates (PHAs) belong among the most widespread storage materials within bacteria genus, it can be assumed that the ability of PHAs accumulation represent significant advantage in the natural environments. Of course, probably the most obvious is the possibility to utilize PHAs when external carbon sources are depleted. Nevertheless, it is likely that PHAs play much more complex role in the stress response of bacteria. Therefore, the aim of this work was to investigate possible influence of PHA accumulation in bacteria on physical properties of the cells and their cytoplasm with respect to possible stress survival. Cells of bacteria Cupriavidus necator H16 with various poly(3-hydroxybutyrate) (PHB) content (25 – 91 % of cell dry weight) were subjected to analytical centrifugation employing LUMiSizer. We observed that slope of index instability of bacterial culture increased with rise of PHB content in cells. It indicates that accumulation of PHB granules in bacterial cells affects overall physico-mechanical properties of the cells in particular sedimentation potential and density which might influence their behavior in natural environment such as sedimentation, surface absorption and subsequent biofilm formation. Furthermore, when investigated by Cryo-SEM, the PHB containing cells showed needle-type plastic deformations while these structures were absent in the cells without polymer. This suggests that native intracellular PHB granules reveal completely different mechanical and physico-chemical properties than any other component of bacterial cytoplasm and their flexibility even in deeply-frozen state is significantly higher than that of PHB isolated from bacterial cells. Based on these observations, it can be expected that, aside from their involvement in metabolism, presence of PHB granules dramatically changes physico-mechanical properties of cytoplasm and overall properties of cells which might represent important advantage when cells are exposed to stress conditions.