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

Purple photosynthetic bacteria (PPB) are versatile microorganisms capable of producing various value-added chemicals, e.g., biopolymers and biofuels. They employ diverse metabolic pathways, allowing them to adapt to various growth conditions and even extreme environments. Thus, they are ideal organisms for the Next Generation Industrial Biotechnology concept of reducing the risk of contamination by using naturally robust extremophiles. Unfortunately, the potential of PPB for use in biotechnology is hampered by missing knowledge on regulations of their metabolism. Although Rhodospirillum rubrum represents a model purple bacterium studied for polyhydroxyalkanoate and hydrogen production, light/chemical energy conversion, and nitrogen fixation, little is known regarding the regulation of its metabolism at the transcriptomic level. Using RNA sequencing, we compared gene expression during the cultivation utilizing fructose and acetate as substrates in case of the wild-type strain R. rubrum DSM 467T and its knock-out mutant strain that is missing two polyhydroxyalkanoate synthases PhaC1 and PhaC2. During this first genome-wide expression study of R. rubrum, we were able to characterize cultivation-driven transcriptomic changes and to annotate non-coding elements as small RNAs.

• Klíčová slova
• Acetate, Depolymerase knock-out, Fructose, Gene ontology, Genome, Metabolism, Polyhydroxyalkanoates, RNA-Seq, Rhodospirillum rubrum, Transcriptome,
• Publikační typ
• časopisecké články 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

Sample preparation protocols for conventional high voltage transmission electron microscopy (TEM) heavily rely on the usage of staining agents containing various heavy metals, most commonly uranyl acetate and lead citrate. However high toxicity, rising legal regulations, and problematic waste disposal of uranyl acetate have increased calls for the reduction or even complete replacement of this staining agent. One of the strategies for uranyless imaging is the employment of low-voltage transmission electron microscopy. To investigate the influence of different imaging and staining strategies on the final image of cyanobacterial cells, samples stained by uranyl acetate with lead citrate, as well as unstained samples, were observed using TEM and accelerating voltages of 200 kV or 25 kV. Moreover, to examine the possibilities of reducing chromatic aberration, which often causes issues when imaging using electrons of lower energies, samples were also imaged using a scanning transmission electron microscopy at 15 kV accelerating voltages. The results of this study demonstrate that low-voltage electron microscopy offers great potential for uranyless electron microscopy.

• Klíčová slova
• Synechocystis, contrasting agents, low voltage electron microscopy, polyhydroxyalkanoates, transmission electron microscopy, uranyl acetate,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Halomonas halophila, PHA isolation, Schlegelella thermodepolymerans, extremophiles, halophiles, polyhydroxyalkanoate (PHA), sodium dodecyl sulfate (SDS), thermophiles,
• Publikační typ
• časopisecké články 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