Brewer's spent grain as a no-cost substrate for polyhydroxyalkanoates production: Assessment of pretreatment strategies and different bacterial strains.

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 ±â€¯0.02 g PHA per g cell dried weight, corresponding to 1.13 ±â€¯0.06 g L-1 and a PHA yield of 23 ±â€¯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.

Production of polyhydroxyalkanoates on waste frying oil employing selected Halomonas strains.

The aim of this work was to study the potential of selected Halomonas species for conversion of waste frying oil into polyhydroxyalkanoates (PHA). In total nine Halomonas strains were experimentally screened for their capability of PHA production. Among them, Halomonas neptunia and Halomonas hydrothermalis were identified as potent PHA producers. Initial concentration of NaCl was identified as parameter influencing PHA yields as well as molecular weight of the polymer. In addition, H. hydrothermalis was capable of biosynthesis of a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate P(3HB-co-3HV). When valerate was utilized as a precursor, the 3HV fraction in the copolymer reached high values of 50.15 mol.%. PHA production on lipid substrates by Halomonas has not been reported so far. Bearing in mind all the positive aspects of employing extremophiles in industrial biotechnology, H. hydrothermalis seems to be a very interesting halophilic strain for production of PHA using lipid substrates.

Biotechnological Production of Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate-co-3-Hydroxyvalerate) Terpolymer by Cupriavidus sp. DSM 19379.

The terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 4-hydroxybutyrate (4HB) was produced employing Cupriavidus sp. DSM 19379. Growth in the presence of γ-butyrolactone, ε-caprolactone, 1,4-butanediol, and 1,6-hexanediol resulted in the synthesis of a polymer consisting of 3HB and 4HB monomers. Single and two-stage terpolymer production strategies were utilized to incorporate the 3HV subunit into the polymer structure. At the single-stage cultivation mode, γ-butyrolactone or 1,4-butanediol served as the primary substrate and propionic and valeric acid as the precursor of 3HV. In the two-stage production, glycerol was used in the growth phase, and precursors for the formation of the terpolymer in combination with the nitrogen limitation in the medium were used in the second phase. The aim of this work was to maximize the Polyhydroxyalkanoates (PHA) yields with a high proportion of 3HV and 4HB using different culture strategies. The obtained polymers contained 0-29 mol% of 3HV and 16-32 mol% of 4HB. Selected polymers were subjected to a material properties analysis such as differential scanning calorimetry (DSC), thermogravimetry, and size exclusion chromatography coupled with multi angle light scattering (SEC-MALS) for determination of the molecular weight. The number of polymers in the biomass, as well as the monomer composition of the polymer were determined by gas chromatography.

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 polymer's 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.

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.

Involvement of polyhydroxyalkanoates in stress resistance of microbial cells: Biotechnological consequences and applications.

Polyhydroxyalkanoates (PHA) are polyesters accumulated by numerous prokaryotes as storage materials; they attract attention as "green" alternatives to petrochemical plastics. Recent research has demonstrated that their biological role goes beyong their storage function, since they presence in cytoplasm enhances stress resistance of microorganisms. To address these complex functions, this review summarizes the protective effects of PHA for microrganisms; the involvement of PHA in stress resistance is discussed also from a praxis-oriented perspective. The review discourses the controlled application of stress to improve PHA productivity. Also the manifold advantages of using stress adapted microbes - extremophiles as PHA producers are discussed.

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.

The presence of PHB granules in cytoplasm protects non-halophilic bacterial cells against the harmful impact of hypertonic 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.

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.

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.

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.

Biotechnological conversion of spent coffee grounds into polyhydroxyalkanoates and carotenoids.

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.