Cyanobacteria are prokaryotic organisms characterised by their complex structures and a wide range of pigments. With their ability to fix CO2, cyanobacteria are interesting for white biotechnology as cell factories to produce various high-value metabolites such as polyhydroxyalkanoates, pigments, or proteins. White biotechnology is the industrial production and processing of chemicals, materials, and energy using microorganisms. It is known that exposing cyanobacteria to low levels of stressors can induce the production of secondary metabolites. Understanding of this phenomenon, known as hormesis, can involve the strategic application of controlled stressors to enhance the production of specific metabolites. Consequently, precise measurement of cyanobacterial viability becomes crucial for process control. However, there is no established reliable and quick viability assay protocol for cyanobacteria since the task is challenging due to strong interferences of autofluorescence signals of intercellular pigments and fluorescent viability probes when flow cytometry is used. We performed the screening of selected fluorescent viability probes used frequently in bacteria viability assays. The results of our investigation demonstrated the efficacy and reliability of three widely utilised types of viability probes for the assessment of the viability of Synechocystis strains. The developed technique can be possibly utilised for the evaluation of the importance of polyhydroxyalkanoates for cyanobacterial cultures with respect to selected stressor-repeated freezing and thawing. The results indicated that the presence of polyhydroxyalkanoate granules in cyanobacterial cells could hypothetically contribute to the survival of repeated freezing and thawing.

• Klíčová slova
• Biotechnology, Cyanobacteria, Flow cytometry, Fluorescent viability probes, Stress resistance, Viability assessment,
• MeSH
• fluorescenční barviva * MeSH
• fyziologický stres * MeSH
• mikrobiální viabilita * MeSH
• polyhydroxyalkanoáty metabolismus   MeSH
• průtoková cytometrie * metody   MeSH
• sinice * fyziologie   MeSH
• Synechocystis * fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva * MeSH
• polyhydroxyalkanoáty 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.

• Klíčová slova
• Arctic bacteria, Environmental stressors, Polyhydroxyalkanoates, Survival,
• MeSH
• Bacteria metabolismus   MeSH
• polyhydroxyalkanoáty * metabolismus   MeSH
• uhlík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyhydroxyalkanoáty * MeSH
• uhlík MeSH

In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles.

• Klíčová slova
• Arctic, Bioplastics, Extremophile, Low-temperature biotechnology, Oligotrophy,
• MeSH
• Comamonadaceae * genetika   MeSH
• genetické inženýrství MeSH
• polyhydroxyalkanoáty * MeSH
• teplota MeSH
• uhlík MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyhydroxyalkanoáty * MeSH
• uhlík MeSH

Microorganisms must face various inconvenient conditions; therefore, they developed several approaches for protection. Such a strategy also involves the accumulation of compatible solutes, also called osmolytes. It has been proved that the monomer unit 3-hydroxybutyrate (3HB), which is present in sufficient concentration in poly(3-hydroxybutyrate) (PHB)-accumulating cells, serves as a chemical chaperone protecting enzymes against heat and oxidative stress and as a cryoprotectant for enzymes, bacterial cells, and yeast. The stress robustness of the cells is also strongly dependent on the behavior and state of intracellular water, especially during stress exposure. For a better understanding of the protective mechanism and effect of strongly hydrophilic 3HB in solutions at a wide range of temperatures, a binary phase diagram of system sodium 3HB (Na3HB)-water in equilibrium and the state diagrams showing the glass transitions in the system were constructed. To investigate the activity of water in various compositions of the Na3HB/water system, three experimental techniques have been used (dynamic water sorption analysis, water activity measurements, and sorption calorimetry). First, Na3HB proved its hydrophilic nature, which is very comparable with known compatible solutes (trehalose). Results of differential scanning calorimetry demonstrated that Na3HB is also highly effective in depressing the freezing point and generating a large amount of nonfrozen water (1.35 g of water per gram of Na3HB). Therefore, Na3HB represents a very effective cryoprotectant that can be widely used for numerous applications.

• MeSH
• hydroxybutyráty * chemie   farmakologie   MeSH
• kyselina 3-hydroxymáselná MeSH
• polyestery * chemie   MeSH
• Saccharomyces cerevisiae MeSH
• teplota MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydroxybutyráty * MeSH
• kyselina 3-hydroxymáselná MeSH
• polyestery * MeSH

The cyanobacterial genus Synechocystis is of particular interest to science and industry because of its efficient phototrophic metabolism, its accumulation of the polymer poly(3-hydroxybutyrate) (PHB) and its ability to withstand or adapt to adverse growing conditions. One such condition is the increased salinity that can be caused by recycled or brackish water used in cultivation. While overall reduced growth is expected in response to salt stress, other metabolic responses relevant to the efficiency of phototrophic production of biomass or PHB (or both) have been experimentally observed in three Synechocystis strains at stepwise increasing salt concentrations. In response to recent reports on metabolic strategies to increase stress tolerance of heterotrophic and phototrophic bacteria, we focused particularly on the stress-induced response of Synechocystis strains in terms of PHB, glycogen and photoactive pigment dynamics. Of the three strains studied, the strain Synechocystis cf. salina CCALA192 proved to be the most tolerant to salt stress. In addition, this strain showed the highest PHB accumulation. All the three strains accumulated more PHB with increasing salinity, to the point where their photosystems were strongly inhibited and they could no longer produce enough energy to synthesize more PHB.

• Klíčová slova
• Glycogen, Pigments, Poly(3-hydroxybutyrate), Salt stress, Synechocystis sp.,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Tepidimonas taiwanensis, grape pomace, polyhydroxyalkanoates, thermophiles,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Rubrobacter spartanus, Rubrobacter xylanophilus, extremophiles, polyhydroxyalkanoates, stress conditions,
• Publikační typ
• časopisecké články MeSH

Aneurinibacillus sp. H1 is a promising, moderately thermophilic, novel Gram-positive bacterium capable of the biosynthesis of polyhydroxyalkanoates (PHA) with tunable monomer composition. In particular, the strain is able to synthesize copolymers of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV) with remarkably high 4HB and 3HV fractions. In this study we performed an in-depth material analysis of PHA polymers produced by Aneurinibacillus sp. H1 in order to describe how the monomer composition affects fundamental structural and physicochemical parameters of the materials in the form of solvent-casted films. Results of infrared spectroscopy, X-ray diffractometry and thermal analysis clearly show that controlling the monomer composition enables optimization of PHA crystallinity both qualitatively (the type of the crystalline lattice) and quantitatively (the overall degree of crystallinity). Furthermore, resistance of the films against thermal and/or enzymatic degradation can also be manipulated by the monomer composition. Results of this study hence confirm Aneurinibacillus sp. H1 as an auspicious candidate for thermophilic production of PHA polymers with material properties that can be tuned together with their chemical composition by the corresponding adjustment of the cultivation process.

• Klíčová slova
• Aneurinibacillus sp., P(3HB-co-3HV-co-4HB), P(3HB-co-4HB), crystallinity, polyhydroxyalkanoates, thermophiles,
• Publikační typ
• časopisecké články MeSH