Production of polyhydroxyalkanoates (PHA), microbial biopolyesters, employing extremophilic microorganisms is a very promising concept relying on robustness of such organisms against microbial contamination, which provides numerous economic and technological benefits. In this work, we took advantage of the natural susceptibility of halophilic and thermophilic PHA producers to hypotonic lysis and we developed a simple and robust approach enabling effective isolation of PHA materials from microbial cells. The method is based on the exposition of microbial cells to hypotonic conditions induced by the diluted solution of sodium dodecyl sulfate (SDS) at elevated temperatures. Such conditions lead to disruption of the cells and release of PHA granules. Moreover, SDS, apart from its cell-disruptive function, also solubilizes hydrophobic components, which would otherwise contaminate PHA materials. The purity of obtained materials, as well as the yields of recovery, reach high values (values of purity higher than 99 wt.%, yields close to 1). Furthermore, we also focused on the removal of SDS from wastewater. The simple, inexpensive, and safe technique is based on the precipitation of SDS in the presence of KCl. The precipitate can be simply removed by decantation or centrifugation. Moreover, there is also the possibility to regenerate the SDS, which would substantially improve the economic feasibility of the process.

• Keywords
• Halomonas halophila, PHA isolation, Schlegelella thermodepolymerans, extremophiles, halophiles, polyhydroxyalkanoate (PHA), sodium dodecyl sulfate (SDS), thermophiles,
• Publication type
• Journal Article MeSH

Polyhydroxyalkanoates (PHA) are microbial polyesters that have recently come to the forefront of interest due to their biodegradability and production from renewable sources. A potential increase in competitiveness of PHA production process comes with a combination of the use of thermophilic bacteria with the mutual use of waste substrates. In this work, the thermophilic bacterium Tepidimonas taiwanensis LMG 22826 was identified as a promising PHA producer. The ability to produce PHA in T. taiwanensis was studied both on genotype and phenotype levels. The gene encoding the Class I PHA synthase, a crucial enzyme in PHA synthesis, was detected both by genome database search and by PCR. The microbial culture of T. taiwanensis was capable of efficient utilization of glucose and fructose. When cultivated on glucose as the only carbon source at 50 °C, the PHA titers reached up to 3.55 g/L, and PHA content in cell dry mass was 65%. The preference of fructose and glucose opens the possibility to employ T. taiwanensis for PHA production on various food wastes rich in these abundant sugars. In this work, PHA production on grape pomace extracts was successfully tested.

• Keywords
• Tepidimonas taiwanensis, grape pomace, polyhydroxyalkanoates, thermophiles,
• Publication type
• Journal Article MeSH

Extremophilic microorganisms are considered being very promising candidates for biotechnological production of various products including polyhydroxyalkanoates (PHA). The aim of this work was to evaluate the PHA production potential of a novel PHA-producing thermophilic Gram-positive isolate Aneurinibacillus sp. H1. This organism was capable of efficient conversion of glycerol into poly(3-hydroxybutyrate) (P3HB), the homopolyester of 3-hydroxybutyrate (3HB). In flasks experiment, under optimal cultivation temperature of 45 °C, the P3HB content in biomass and P3HB titers reached 55.31% of cell dry mass and 2.03 g/L, respectively. Further, the isolate was capable of biosynthesis of PHA copolymers and terpolymers containing high molar fractions of 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB). Especially 4HB contents in PHA were very high (up to 91 mol %) when 1,4-butanediol was used as a substrate. Based on these results, it can be stated that Aneurinibacillus sp. H1 is a very promising candidate for production of PHA with tailored material properties.

• Keywords
• Aneurinibacillus sp., P(3HB-co-3HV-co-4HB), P(3HB-co-4HB), polyhydroxyalkanoates, thermophiles,
• Publication type
• Journal Article MeSH

Carotenogenic yeasts are non-conventional oleaginous microorganisms capable of utilizing various waste substrates. In this work, four red yeast strains (Rhodotorula, Cystofilobasidium, and Sporobolomyces sp.) were cultivated in media containing crude, emulsified, and enzymatically hydrolyzed animal waste fat, compared with glucose and glycerol, as single C-sources. Cell morphology (cryo-SEM (cryo-scanning electron microscopy), TEM (transmission electron microscopy)), production of biomass, lipase, biosurfactants, lipids (gas chromatography/flame ionization detection, GC/FID) carotenoids, ubiquinone, and ergosterol (high performance liquid chromatography, HPLC/PDA) in yeast cells was studied depending on the medium composition, the C source, and the carbon/nitrogen (C/N) ratio. All studied strains are able to utilize solid and processed fat. Biomass production at C/N = 13 was higher on emulsified/hydrolyzed fat than on glucose/glycerol. The production of lipids and lipidic metabolites was enhanced for several times on fat; the highest yields of carotenoids (24.8 mg/L) and lipids (54.5%/CDW (cell dry weight)) were found in S. pararoseus. Simultaneous induction of lipase and biosurfactants was observed on crude fat substrate. An increased C/N ratio (13-100) led to higher biomass production in fat media. The production of total lipids increased in all strains to C/N = 50. Oppositely, the production of carotenoids, ubiquinone, and ergosterol dramatically decreased with increased C/N in all strains. Compounds accumulated in stressed red yeasts have a great application potential and can be produced efficiently during the valorization of animal waste fat under the biorefinery concept.

• Keywords
• biosurfactants, carotenogenic yeasts, carotenoids, ergosterol, lipase, lipids, ubiquinone,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Cupriavidus malaysiensis, P(3HB-co-3HV-co-4HB), polyhydroxyalkanoates, terpolymer,
• Publication type
• Journal Article MeSH