The nucleotide composition of key enzymes involved in medium-chain-length polyhydroxyalkanoates (mcl-PHA) synthesis was analyzed in two newly isolated strains of Pseudomonas. The isolated strains were tested for their abilities to synthesize polyhydroxyalkanoates using three different substrates as a carbon source: sodium octanoate, oleic acid, and sodium gluconate. Both analyzed strains were able to accumulate mcl-PHA in a range from 2.07 to 21.40%, which depended on the substrate used. Potential nitrogen-dependent regulation of mcl-PHA synthesis was analyzed by cell cultivation in nitrogen-limiting and non-limiting conditions. The analyzed strains demonstrated an incremental increase of mcl-PHAs in response to nitrogen starvation when oleic acid and sodium gluconate were applied as the carbon source. The transcriptional analysis showed that the induction of gene coding for PHA synthases was correlated with an increment in mcl-PHAs content. Both analyzed strains revealed differences in terms of the studied gene's expression, showing a dependence on the carbon source used.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are biodegradable, biocompatible polyesters produced by many bacteria as intracellular storage compounds of energy and carbon. They have gained great attention as a new green alternative to petrochemical plastic such as polypropylene. The present study was focused on mcl-PHA production by two Pseudomonas strains (Gl01 and Gl06). To make this process more economical, waste rapeseed oil was used as a carbon source. The levels of PHAs synthesized by Pseudomonas sp. Gl01 strain and Gl06 strain were 21.0%, and 19.3% of CDW, respectively. The polyester accumulation increased until nitrogen and oxygen were depleted from the medium. Reverse transcription real-time PCR approach was applied to quantitatively evaluate phaC1, phaC2, and phaZ gene expression. A positive correlation was found between the cellular PHA content and the gene expression of the PHA synthases. The transcriptional analysis revealed that the phaC1 and phaZ genes could be co-transcribed. The overexpressed phaC2 gene was only observed in the Gl06 strain. The monomeric composition of the obtained mcl-PHA was dependent on the strain: 3-hydroxyoctanoic acid (3HO) and 3-hydroxydecanoic acid (3HD) were dominant in strain Gl01, whereas 3-hydroxyhexanoic (3HHx) was present in significant amounts in strain Gl06.Practical applications: This article presents the procedure of medium-chain-length polyhydroxyalkanoates (mcl-PHAs) synthesis by the Pseudomonas Gl01 and Gl06 strains from waste rapeseed oil as a feedstock. Beside the microbial production of these environmentally friendly plastics, this approach also represents a new way of utilizing waste oils. The knowledge of the key enzymes involved in mcl-PHAs accumulation is necessary in order to understand the mechanisms of their synthesis, and to use them effectively in biotechnological applications.

The present study describes medium-chain-length polyhydroxyalkanoates (mcl-PHAs) production by the Pseudomonas Gl01 strain isolated from mixed microbial communities utilized for PHAs synthesis. A two-step fedbatch fermentation was conducted with glucose and waste rapeseed oil as the main carbon source for obtaining cell growth and mcl-PHAs accumulation, respectively. The results show that the Pseudomonas Gl01 strain is capable of growing and accumulating mcl-PHAs using a waste oily carbon source. The biomass value reached 3.0 g/l of CDW with 20% of PHAs content within 48 h of cultivation. The polymer was purified from lyophilized cells and analyzed by gas chromatography (GC). The results revealed that the monomeric composition of the obtained polyesters depended on the available substrate. When glucose was used in the growth phase, 3-hydroxyundecanoate and 3hydroxydodecanoate were found in the polymer composition, whereas in the PHAs-accumulating stage, the Pseudomonas Gl01 strain synthesized mcl-PHAs consisting mainly of 3hydroxyoctanoate and 3-hydroxydecanoate. The transcriptional analysis using reverse-transcription real-time PCR reaction revealed that the phaC1 gene could be transcribed simultaneously to the phaZ gene.

Polyhydroxyalkanoates (PHAs) can be produced by microorganisms and are a biodegradable alternative to fossil-fuel based plastics. Currently, the focus is on reducing production costs by exploring alternative substrates for PHAs production, and on producing copolymers which are less brittle than monomers. Accordingly, this study used a substrate consisting of wastewater from waste-glycerol fermentation, supplemented with different amounts of acetic and propionic acids. These substrates were used to feed mixed microbial communities enriched from activated sludge in a sequencing batch reactor. A reactor supplemented with 2 mL of acetic acid produced 227.8 mg/L of a homopolymer of hydroxybutyrate (3HB); 4 mL of acetic acid produced 279.8 mg/L 3HB; whereas 4 mL of propionic acid produced 673.0 mg/L of a copolymer of 3HB and 3HV (hydroxyvalerate). Ribosomal Intergenic Spacer Analysis (RISA) was used to show the differences between the communities created in the reactors. Thauera species predominated in biomass that produced 3HB; Paracoccus denitrificans in the biomass that produced 3HB-co-3HV. Because P. denitrificans produced the more desirable copolymer, it may be advantageous to promote its growth in PHAs-producing reactors by adding propionate.

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