Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable polymer with properties comparable to polypropylene and therefore has the potential to replace conventional plastics. PHB is intracellularly accumulated by prokaryotic organisms. For the cells PHB functions manly as carbon and energy source, but all possible functions of PHB are still not known. Synechocystis (cyanobacteria) accumulates PHB using light as energy and CO2 as carbon source. The main trigger for PHB accumulation in cyanobacteria is nitrogen and phosphorous depletion with simultaneous surplus of carbon and energy. For the above reasons, obtaining knowledge about external factors influencing PHB accumulation is of highest interest. This study compares the effect of continuous light exposure and day/night (16/8 h) cycles on selected physiology parameters of three Synechocystis strains. We show that continuous illumination at moderate light intensities leads to an increased PHB accumulation in Synechocystis salina CCALA 192 (max. 14.2% CDW - cell dry weight) compared to day/night cycles (3.7% CDW). In addition to PHB content, glycogen and cell size increased, while cell density and cell viability decreased. The results offer new approaches for further studies to gain deeper insights into the role of PHB in cyanobacteria to obtain bioplastics in a more sustainable and environmentally friendly way.

• Keywords
• PHB, Synechocystis, cell size, continuous illumination, day/night cycle, glycogen,
• Publication type
• Journal Article MeSH

An ever-growing body of literature evidences the protective role of polyhydroxyalkanoates (PHAs) against a plethora of mostly physical stressors in prokaryotic cells. To date, most of the research done involved bacterial strains isolated from habitats not considered to be life-challenging or extremely impacted by abiotic environmental factors. Polar region microorganisms experience a multitude of damaging factors in combinations rarely seen in other of Earth's environments. Therefore, the main objective of this investigation was to examine the role of PHAs in the adaptation of psychrophilic, Arctic-derived bacteria to stress conditions. Arctic PHA producers: Acidovorax sp. A1169 and Collimonas sp. A2191, were chosen and their genes involved in PHB metabolism were deactivated making them unable to accumulate PHAs (ΔphaC) or to utilize them (Δi-phaZ) as a carbon source. Varying stressors were applied to the wild-type and the prepared mutant strains and their survival rates were assessed based on CFU count. Wild-type strains with a functional PHA metabolism were best suited to survive the freeze-thaw cycle - a common feature of polar region habitats. However, the majority of stresses were best survived by the ΔphaC mutants, suggesting that the biochemical imbalance caused by the lack of PHAs induced a permanent cell-wide stress response thus causing them to better withstand the stressor application. Δi-phaZ mutants were superior in surviving UV irradiation, hinting that PHA granule presence in bacterial cells is beneficial despite it being biologically inaccessible. Obtained data suggests that the ability to metabolize PHA although important for survival, probably is not the most crucial mechanism in the stress-resistance strategies arsenal of cold-loving bacteria. KEY POINTS: • PHA metabolism helps psychrophiles survive freezing • PHA-lacking psychrophile mutants cope better with oxidative and heat stresses • PHA granule presence enhances the UV resistance of psychrophiles.

• Keywords
• Arctic bacteria, Environmental stressors, Polyhydroxyalkanoates, Survival,
• MeSH
• Bacteria metabolism   MeSH
• Polyhydroxyalkanoates * metabolism   MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polyhydroxyalkanoates * MeSH
• Carbon MeSH

Polyhydroxyalkanoates (PHAs) have emerged as an environmentally friendly alternative to conventional polyesters. In this study, we present a comprehensive analysis of the genomic and phenotypic characteristics of three non-model thermophilic bacteria known for their ability to produce PHAs: Schlegelella aquatica LMG 23380T, Caldimonas thermodepolymerans DSM 15264, and C. thermodepolymerans LMG 21645 and the results were compared with the type strain C. thermodepolymerans DSM 15344T. We have assembled the first complete genomes of these three bacteria and performed the structural and functional annotation. This analysis has provided valuable insights into the biosynthesis of PHAs and has allowed us to propose a comprehensive scheme of carbohydrate metabolism in the studied bacteria. Through phylogenomic analysis, we have confirmed the synonymity between Caldimonas and Schlegelella genera, and further demonstrated that S. aquatica and S. koreensis, currently classified as orphan species, belong to the Caldimonas genus.

• Keywords
• Caldimonas, DSM 15264, DSM 15344T, LMG 21645, LMG 23380T, Next-Generation Industrial Biotechnology, PHAs, Schlegelella, de novo assembly,
• Publication type
• Journal Article MeSH

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

Tepidimonas taiwanensis is a moderately thermophilic, Gram-negative, rod-shaped, chemoorganoheterotrophic, motile bacterium. The alkaline protease producing type strain T. taiwanensis LMG 22826T was recently reported to also be a promising producer of polyhydroxyalkanoates (PHAs)-renewable and biodegradable polymers representing an alternative to conventional plastics. Here, we present its first complete genome sequence which is also the first complete genome sequence of the whole species. The genome consists of a single 2,915,587-bp-long circular chromosome with GC content of 68.75%. Genome annotation identified 2,764 genes in total while 2,634 open reading frames belonged to protein-coding genes. Although functional annotation of the genome and division of genes into Clusters of Orthologous Groups (COGs) revealed a relatively high number of 694 genes with unknown function or unknown COG, the majority of genes were assigned a function. Most of the genes, 406 in total, were involved in energy production and conversion, and amino acid transport and metabolism. Moreover, particular key genes involved in the metabolism of PHA were identified. Knowledge of the genome in connection with the recently reported ability to produce bioplastics from the waste stream of wine production makes T. taiwanensis LMG 22826T, an ideal candidate for further genome engineering as a bacterium with high biotechnological potential.

• Keywords
• Tepidimonas taiwanensis, Oxford Nanopore Technologies, alkaline protease, functional annotation, hybrid assembly, polyhydroxyalkanoates,
• MeSH
• Bacterial Proteins MeSH
• Burkholderiales * genetics   MeSH
• Endopeptidases MeSH
• Polyhydroxyalkanoates * genetics   MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• alkaline protease MeSH Browser
• Bacterial Proteins MeSH
• Endopeptidases MeSH
• Polyhydroxyalkanoates * MeSH

Actinobacteria belonging to the genus Rubrobacter are known for their multi-extremophilic growth conditions-they are highly radiation-resistant, halotolerant, thermotolerant or even thermophilic. This work demonstrates that the members of the genus are capable of accumulating polyhydroxyalkanoates (PHA) since PHA-related genes are widely distributed among Rubrobacter spp. whose complete genome sequences are available in public databases. Interestingly, all Rubrobacter strains possess both class I and class III synthases (PhaC). We have experimentally investigated the PHA accumulation in two thermophilic species, R. xylanophilus and R. spartanus. The PHA content in both strains reached up to 50% of the cell dry mass, both bacteria were able to accumulate PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units, none other monomers were incorporated into the polymer chain. The capability of PHA accumulation likely contributes to the multi-extremophilic characteristics since it is known that PHA substantially enhances the stress robustness of bacteria. Hence, PHA can be considered as extremolytes enabling adaptation to extreme conditions. Furthermore, due to the high PHA content in biomass, a wide range of utilizable substrates, Gram-stain positivity, and thermophilic features, the Rubrobacter species, in particular Rubrobacter xylanophilus, could be also interesting candidates for industrial production of PHA within the concept of Next-Generation Industrial Biotechnology.

• Keywords
• Rubrobacter spartanus, Rubrobacter xylanophilus, extremophiles, polyhydroxyalkanoates, stress conditions,
• Publication type
• Journal Article MeSH

Schlegelella thermodepolymerans is a moderately thermophilic bacterium capable of producing polyhydroxyalkanoates-biodegradable polymers representing an alternative to conventional plastics. Here, we present the first complete genome of the type strain S. thermodepolymerans DSM 15344 that was assembled by hybrid approach using both long (Oxford Nanopore) and short (Illumina) reads. The genome consists of a single 3,858,501-bp-long circular chromosome with GC content of 70.3%. Genome annotation identified 3,650 genes in total, whereas 3,598 open reading frames belonged to protein-coding genes. Functional annotation of the genome and division of genes into clusters of orthologous groups revealed a relatively high number of 1,013 genes with unknown function or unknown clusters of orthologous groups, which reflects the fact that only a little is known about thermophilic polyhydroxyalkanoates-producing bacteria on a genome level. On the other hand, 270 genes involved in energy conversion and production were detected. This group covers genes involved in catabolic processes, which suggests capability of S. thermodepolymerans DSM 15344 to utilize and biotechnologically convert various substrates such as lignocellulose-based saccharides, glycerol, or lipids. Based on the knowledge of its genome, it can be stated that S. thermodepolymerans DSM 15344 is a very interesting, metabolically versatile bacterium with great biotechnological potential.

• Keywords
• PHA, de novo assembly, functional annotation, hybrid assembly,
• MeSH
• Molecular Sequence Annotation MeSH
• Comamonadaceae genetics   MeSH
• Genome, Bacterial * MeSH
• Sequence Analysis, DNA MeSH
• Whole Genome Sequencing MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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

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