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.

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.

Isolation of poly(3-hydroxybutyrate) from bacterial biomass using soap made of waste cooking oil.

The aim of this work was to develop a soap-based method for the isolation of poly(3-hydroxybutyrate) from bacterial biomass. The method consisted of adding soap derived from waste cooking oil to a concentrated (25%) biomass suspension, heating and centrifugal separation. Purity above 95% could be achieved with soap:cell dry mass ratios at least 0.125 g/g, making the method comparable to other surfactant-based protocols. Molecular weights Mw of products from all experiments were between 350 and 450 kDa, being high enough for future material applications. Addition of hydrochloric acid to the wastewater led to the precipitation of soap and part of non-P3HB cell mass. The resulting precipitate was utilized as a carbon source in biomass production and increased substrate-to-P3HB conversion.

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.

Introducing the Newly Isolated Bacterium Aneurinibacillus sp. H1 as an Auspicious Thermophilic Producer of Various Polyhydroxyalkanoates (PHA) Copolymers-2. Material Study on the Produced Copolymers.

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.

Introducing the Newly Isolated Bacterium Aneurinibacillus sp. H1 as an Auspicious Thermophilic Producer of Various Polyhydroxyalkanoates (PHA) Copolymers-1. Isolation and Characterization of the Bacterium.

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.

Application of osmotic challenge for enrichment of microbial consortia in polyhydroxyalkanoates producing thermophilic and thermotolerant bacteria and their subsequent isolation.

Polyhydroxyalkanoates are microbial polyesters which are considered being biological alternatives to petrochemical polymers. Extremophiles, such as thermophilic PHA producers, hold promise to improve competitiveness of PHA production process. Therefore, this work aimed at isolation of new strains, which could produce PHA under elevated temperature. Since traditional Nile red staining of colonies provided false positive results in thermophiles, we developed a novel strategy of enriching microbial consortia by PHA producers. This so called "osmoselective strategy" is based on application of osmotic challenge by sudden exposition of the mixed microbial culture to hypertonic and subsequently to hypotonic conditions; moreover, this strategy relies on the fact that PHA protect bacteria from negative effects of rapid fluctuations in osmotic pressure. In combination with fast and reliable ATR-FTIR inspection of selected colonies for presence of PHA, we were able to isolate several promising thermophilic or thermotolerant PHA producing strains belonging to the genera Bacillus, Aneurinibacillus and Chelatococcus, which indeed deserves further investigation to evaluate their potential for industrial production of PHA.

Production of polyhydroxyalkanoates on waste frying oil employing selected Halomonas strains.

The aim of this work was to study the potential of selected Halomonas species for conversion of waste frying oil into polyhydroxyalkanoates (PHA). In total nine Halomonas strains were experimentally screened for their capability of PHA production. Among them, Halomonas neptunia and Halomonas hydrothermalis were identified as potent PHA producers. Initial concentration of NaCl was identified as parameter influencing PHA yields as well as molecular weight of the polymer. In addition, H. hydrothermalis was capable of biosynthesis of a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate P(3HB-co-3HV). When valerate was utilized as a precursor, the 3HV fraction in the copolymer reached high values of 50.15 mol.%. PHA production on lipid substrates by Halomonas has not been reported so far. Bearing in mind all the positive aspects of employing extremophiles in industrial biotechnology, H. hydrothermalis seems to be a very interesting halophilic strain for production of PHA using lipid substrates.

Biotechnological Production of Poly(3-Hydroxybutyrate-co-4-Hydroxybutyrate-co-3-Hydroxyvalerate) Terpolymer by Cupriavidus sp. DSM 19379.

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.