Polyhydroxyalkanoate involvement in stress-survival of two psychrophilic bacterial strains from the High Arctic.

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.

Genomic and phenotypic comparison of polyhydroxyalkanoates producing strains of genus Caldimonas/Schlegelella.

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.

Genetic engineering of low-temperature polyhydroxyalkanoate production by Acidovorax sp. A1169, a psychrophile isolated from a subglacial outflow.

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.

Urany-Less Low Voltage Transmission Electron Microscopy: A Powerful Tool for Ultrastructural Studying of Cyanobacterial Cells.

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.

On the bioprotective effects of 3-hydroxybutyrate: Thermodynamic study of binary 3HB-water systems.

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.

Biosynthesis of versatile PHA copolymers by thermophilic members of the genus Aneurinibacillus.

Thermophilic members of the genus Aneurinibacillus constitute a remarkable group of microorganisms that exhibit extraordinary flexibility in terms of polyhydroxyalkanoates (PHA) synthesis. In this study, we demonstrate that these Gram-positive bacteria are capable of the utilization of selected lactones, namely, γ-valerolactone (GVL), γ-hexalactone (GHL), and δ-valerolactone (DVL) as the structural precursors of related PHA monomers. In the presence of GVL, a PHA copolymer consisting of 3-hydroxybutyrate, 3-hydroxyvalerate, and also 4-hydroxyvalerate was synthesized, with a 4 HV fraction as high as 53.1 mol%. Similarly, the application of GHL resulted in the synthesis of PHA copolymer containing 4-hydroxyhexanaote (4HHx) (4HHx fraction reached up to 11.5 mol%) and DVL was incorporated into PHA in form of 5-hydroxyvalerate (5 HV) (maximal 5 HV content was 44.2 mol%). The produced materials were characterized by thermoanalytical and spectroscopic methods; the results confirmed extremely appealing material properties of produced copolymers. Further, due to their unique metabolic features and capability of incorporating various PHA monomers into the PHA chain, thermophilic Aneurinibacillus spp. can be considered not only promising chassis for PHA production but also potential donors of PHA-relevant genes to improve PHA production in other thermophiles by using approaches of synthetic biology.

Use of Flavin-Related Cellular Autofluorescence to Monitor Processes in Microbial Biotechnology.

Cellular autofluorescence is usually considered to be a negative phenomenon because it can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with the signal of various fluorescent probes. Nevertheless, in our work, we adopted a different approach, and green autofluorescence induced by flavins was used as a tool to monitor fermentation employing the bacterium Cupriavidus necator. The autofluorescence was used to distinguish microbial cells from abiotic particles in flow cytometry assays, and it was also used for the determination of viability or metabolic characteristics of the microbial cells. The analyses using two complementary techniques, namely fluorescence microscopy and flow cytometry, are simple and do not require labor sample preparation. Flavins and their autofluorescence can also be used in a combination with other fluorophores when the need for multi-parametrical analyses arises, but it is wise to use dyes that do not emit a green light in order to not interfere with flavins' emission band (500-550 nm).

Novel thermophilic polyhydroxyalkanoates producing strain Aneurinibacillus thermoaerophilus CCM 8960.

Aneurinibacillus thermoaerophilus CCM 8960 is a thermophilic bacterium isolated from compost in Brno. The bacterium accumulates polyhydroxyalkanoates (PHAs), a biodegradable and renewable alternative to petrochemical polymers. The bacterium reveals several features that make it a very interesting candidate for the industrial production of PHA. At first, due to its thermophilic character, the bacterium can be utilized in agreement with the concept of next-generation industrial biotechnology (NGIB), which relies on extremophiles. Second, the bacterium is capable of producing PHA copolymers containing a very high portion of 4-hydroxybutyrate (4HB). Such materials possess unique properties and can be advantageously used in multiple applications, including but not limited to medicine and healthcare. Therefore, this work focuses on the in-depth characterization of A. thermoaerophilus CCM 8960. In particular, we sequenced and assembled the genome of the bacterium and identified its most important genetic features, such as the presence of plasmids, prophages, CRISPR arrays, antibiotic-resistant genes, and restriction-modification (R-M) systems, which might be crucial for the development of genome editing tools. Furthermore, we focused on genes directly involved in PHA metabolism. We also experimentally studied the kinetics of glycerol and 1,4-butanediol (1,4BD) utilization as well as biomass growth and PHA production during cultivation. Based on these data, we constructed a metabolic model to reveal metabolic fluxes and nodes of glycerol and 1,4BD concerning their incorporation into the poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) structure. KEY POINTS: • Aneurinibacillus sp. H1 was identified as Aneurinibacillus thermoaerophilus. • PHA metabolism pathway with associated genes was presented. • Unique monomer composition of produced PHAs was reported.

Combination of Hypotonic Lysis and Application of Detergent for Isolation of Polyhydroxyalkanoates from Extremophiles.

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.

Degradation of P(3HB-co-4HB) Films in Simulated Body Fluids.

A novel model of biodegradable PHA copolymer films preparation was applied to evaluate the biodegradability of various PHA copolymers and to discuss its biomedical applicability. In this study, we illustrate the potential biomaterial degradation rate affectability by manipulation of monomer composition via controlling the biosynthetic strategies. Within the experimental investigation, we have prepared two different copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate-P(3HB-co-36 mol.% 4HB) and P(3HB-co-66 mol.% 4HB), by cultivating the thermophilic bacterial strain Aneurinibacillus sp. H1 and further investigated its degradability in simulated body fluids (SBFs). Both copolymers revealed faster weight reduction in synthetic gastric juice (SGJ) and artificial colonic fluid (ACF) than simple homopolymer P3HB. In addition, degradation mechanisms differed across tested polymers, according to SEM micrographs. While incubated in SGJ, samples were fragmented due to fast hydrolysis sourcing from substantially low pH, which suggest abiotic degradation as the major degradation mechanism. On the contrary, ACF incubation indicated obvious enzymatic hydrolysis. Further, no cytotoxicity of the waste fluids was observed on CaCO-2 cell line. Based on these results in combination with high production flexibility, we suggest P(3HB-co-4HB) copolymers produced by Aneurinibacillus sp. H1 as being very auspicious polymers for intestinal in vivo treatments.

Effects of Differing Monomer Compositions on Properties of P(3HB-co-4HB) Synthesized by Aneurinibacillus sp. H1 for Various Applications.

Films prepared from poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymers produced by Aneurinibacillus sp. H1 using an automatic film applicator were homogeneous and had a defined thickness, which allowed a detailed study of physicochemical properties. Their properties were compared with those of a poly (3-hydroxybutyrate) homopolymer film prepared by the same procedure, which proved to be significantly more crystalline by DSC and XRD. Structural differences between samples had a major impact on their properties. With increasing 4-hydroxybutyrate content, the ductility and release rate of the model hydrophilic active ingredient increased significantly. Other observed properties, such as the release of the hydrophobic active substance, the contact angle with water and ethylene glycol, or the surface morphology and roughness, were also affected by the composition. The identified properties predetermine these copolymers for wide use in areas such as biomedicine or smart biodegradable packaging for food or cosmetics. The big advantage is the possibility of fine-tuning properties simply by changing the fermentation conditions.

The role of polyhydroxyalkanoates in adaptation of Cupriavidus necator to osmotic pressure and high concentration of copper ions.

Polyhydroxyalkanoates (PHA) are abundant microbial polyesters accumulated in the form of intracellular granules by numerous prokaryotes primarily as storage of carbon and energy. Apart from their storage function, the presence of PHA also enhances the robustness of the microbial cells against various stressors. In this work, we investigated the role of PHA in Cupriavidus necator, a model organism concerning PHA metabolism, for adaptation to osmotic pressure and copper ions. In long-term laboratory evolution experiments, the bacterial culture was cultivated in presence of elevated doses of sodium chloride or copper ions (incubations lasted 78 passages for Cu2+ and 68 passages for NaCl) and the evolved strains were compared with the wild-type strain in terms of growth and PHA production capacity, cell morphology (investigated by various electron microscopy techniques), activities of selected enzymes involved in PHA metabolism and other crucial metabolic pathways, the chemical composition of bacterial biomass (determined by infrared and Raman spectroscopy) and also considering robustness against various stressors. The results confirmed the important role of PHA metabolism for adaptation to both tested stressors.

Polyhydroxyalkanoates synthesis by halophiles and thermophiles: towards sustainable production of microbial bioplastics.

Polyhydroxyalkanoates (PHA) are microbial polyesters produced by numerous prokaryotes. These materials are generally considered to be renewable and biodegradable alternatives to petrochemical polymers in numerous applications. PHA are accumulated by microbial cells in form of intracellular granules primarily as storage compounds; nevertheless, numerous recent reports also highlight the importance of PHA for the stress robustness of bacteria. Therefore, in this review, we focus on summarizing current knowledge on PHA accumulation in halophiles and thermophiles - prokaryotic microorganisms adapted to high salinity and high temperature, respectively. Utilization of extremophiles for PHA production brings numerous benefits stemming especially from the enhanced robustness of the process against contamination by common mesophilic microflora as a basement of the Next-Generation Industrial Biotechnology concept. Further, recent advances and future perspectives in metabolic engineering and synthetic biology of halophiles and thermophiles for PHA production improvement are also summarized and suggested. Facts and ideas gathered in this review hold a promise that biotechnological production of PHA by extremophiles can be sustainable and economically feasible enabling PHA to enter the market massively and compete with non-biodegradable petrochemical polymers in suitable applications.

Complete Genome Sequence of the Type Strain Tepidimonas taiwanensis LMG 22826T, a Thermophilic Alkaline Protease and Polyhydroxyalkanoate Producer.

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.

Biotechnological Conversion of Grape Pomace to Poly(3-hydroxybutyrate) by Moderately Thermophilic Bacterium Tepidimonas taiwanensis.

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.

The Multiple Roles of Polyphosphate in Ralstonia eutropha and Other Bacteria.

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that - with the exception of a P-storage-function - most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

The First Insight into Polyhydroxyalkanoates Accumulation in Multi-Extremophilic Rubrobacter xylanophilus and Rubrobacter spartanus.

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.

The underexplored role of diverse stress factors in microbial biopolymer synthesis.

Polyhydroxyalkanoates (PHA) are microbial polyesters which, apart from their primary storage role, enhance the stress robustness of PHA accumulating cells against various stressors. PHA also represent interesting alternatives to petrochemical polymers, which can be produced from renewable resources employing approaches of microbial biotechnology. During biotechnological processes, bacterial cells are exposed to various stressor factors such as fluctuations in temperature, osmolarity, pH-value, elevated pressure or the presence of microbial inhibitors. This review summarizes how PHA helps microbial cells to cope with biotechnological process-relevant stressors and, vice versa, how various stress conditions can affect PHA production processes. The review suggests a fundamentally new strategy for PHA production: the fine-tuned exposure to selected stressors, which might be used to boost PHA production and even to tailor their structure.

Evaluation of mesophilic Burkholderia sacchari, thermophilic Schlegelella thermodepolymerans and halophilic Halomonas halophila for polyhydroxyalkanoates production on model media mimicking lignocellulose hydrolysates.

In this work, the mesophilic bacterium Burkholderia sacchari, the halophilic bacterium Halomonas halophila, and the thermophilic bacterium Schlegelella thermodepolymerans were evaluated with regards to their suitability for polyhydroxyalkanoates (PHA) production from model media mimicking lignocellulose hydrolysates. B. sacchari was capable of utilizing all the tested "model hydrolysates", yielding comparable PHA titers and turning out as very robust against lignocellulose-derived microbial inhibitors. On the contrary, H. halophila reached substantially higher PHA titers on hexoses-rich media, while S. thermodepolymerans preferred media rich in pentoses. Both extremophiles were more sensitive to microbial inhibitors than B. sacchari. Nevertheless, considering substantially higher PHA productivity of both extremophiles even in the presence of microbial inhibitors and also other positive factors associated with utilization of extremophiles, such as the reduced risk of microbial contamination, both H. halophila and S. thermodepolymerans are auspicious candidates for sustainable PHA production from abundantly available, inexpensive lignocelluloses.

First Complete Genome of the Thermophilic Polyhydroxyalkanoates-Producing Bacterium Schlegelella thermodepolymerans DSM 15344.

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.