Daniela Chmelová

Polyhydroxyalkanoates (PHAs) are microbial polyesters that belong to a group of bioplastics with the potential to replace petroleum-derived plastics. Their main drawback is the high production cost, which puts them at a disadvantage compared to conventional plastics. A significant part of these costs arises from the isolation of PHAs from the cellular biomass of producing microorganisms. This review summarizes the main approaches used to recover both scl- and mcl-PHAs from native or dried (lyophilized) biomass, with attention to physical, chemical, and biological methods. Key parameters influencing extraction efficiency, polymer purity, and the final material properties are discussed, including pretreatment steps that often determine the overall outcome. The review also compares traditional halogenated solvent extraction with more environmentally acceptable alternatives and considers how different strategies can be combined to improve recovery. The current literature highlights the need for sustainable and economically acceptable processes that would make large-scale PHA production more feasible.

Today, PHB and its copolymers—potential plastic substitutes—are produced by fermenting sugar, which is not scalable to the volumes of plastic consumption. PHB from CH₄ can offer a sustainable process route, with CH₄ potentially produced from a variety of waste biomass streams through anaerobic digestion, gasification, and methanation. The high molar mass (M_w) of PHB is a key determinant of its mechanical properties, and strain, culture conditions and downstream processing influence it. In this work, the strain Methylocystis sp. GB 25 (DSMZ 7674) was grown on natural gas as the sole carbon and energy source and air (1:1) in a loop reactor with 350 L active fermentation volume, at 35 °C and ambient pressure. After two days of continuous growth, the bacteria were limited in P and N for 1, 2, and 2.5 days to determine the optimal conditions for PHB accumulation and the highest Mw as the target. The biomass was then centrifuged and spray-dried. For downstream processing, chloroform solvent extraction and selected enzymatic treatment were deployed, yielding ~40% PHB from the biomass. The PHB obtained by solvent extraction exhibited high average weight molar masses of M_w ~1.1–1.5 × 10⁶ g mol⁻¹. The highest M_w was obtained after one day of limitation, whereas enzyme treatment resulted in partially degraded PHB. Cold chloroform maceration, interesting due to energy savings, did not achieve sufficient extraction efficiency because it was unable to extract high-molar-mass PHB fractions. The extracted PHB has a high molar mass, more than double that of standard commercial PHB, and was characterized by DSC, which showed a high degree of crystallinity of up to 70% with a melting temperature of close to 180 °C. Mechanical tensile properties measurements, as well as dynamic mechanical thermal analysis (DMTA), were performed. Degradation of the PHB by enzymes was also determined. Methanotrophic PHB is a promising bioplastics material. The high M_w can limit and delay polymer degradation in practical processing steps, making the material more versatile and robust.

Polyhydroxyalkanoates (PHAs) offer a sustainable substitute for conventional petroleum-based plastics. Halophiles possess a remarkable ability to produce PHAs from low-cost by-products like crude whey or molasses, offering a sustainable alternative to traditional plastics. This study highlights the efficient production of PHAs by Halomonas halophila CCM 3662 using a basic culture medium designed to simultaneously enhance biomass and polyhydroxybutyrate (PHB) production. Through optimization via response surface methodology (RSM), the optimal medium composition was identified as 22.85 g/l glucose, 4.36 g/l ammonium sulfate, and 13.22 g/l sodium hydrogen. Under these conditions, biomass production reached 1.44±0.07 g/l, while PHB content achieved 50.16±0.84%. This represented a 2.8-fold increase in biomass production and a 2.5-fold increase in PHB production compared to a medium without optimized composition. This study provides valuable insights into the optimal medium composition for the growth of the halophilic H. halophila and its potential application in PHB production for various uses.

This review aims to provide a comprehensive overview of the application of bacterial and fungal laccases for the removal of pharmaceuticals from the environment. Laccases were evaluated for their efficacy in degrading pharmaceutical substances across various categories, including analgesics, antibiotics, antiepileptics, antirheumatic drugs, cytostatics, hormones, anxiolytics, and sympatholytics. The capability of laccases to degrade or biotransform these drugs was found to be dependent on their structural characteristics. The formation of di-, oligo- and polymers of the parent compound has been observed using the laccase mediator system (LMS), which is advantageous in terms of their removal via commonly used processes in wastewater treatment plants (WWTPs). Notably, certain pharmaceuticals such as tetracycline antibiotics or estrogen hormones exhibited degradation or even mineralization when subjected to laccase treatment. Employing enzyme pretreatment mitigated the toxic effects of degradation products compared to the parent drug. However, when utilizing the LMS, careful mediator selection is essential to prevent potential increases in environment toxicity. Laccases demonstrate efficiency in pharmaceutical removal within WWTPs, operating efficiently under WWTP conditions without necessitating isolation.

The accumulation of polyhydroxyalkanoates (PHAs) by microorganisms usually occurs in response to environmental stress conditions. Therefore, it is advantageous to choose two-step cultivation. The first phase is aimed at maximizing biomass production, and only in the second phase, after setting the suitable conditions, PHA production starts. The aim of this work was to optimize the composition of the minimal propagation medium used for biomass production of Cupriavidus necator DSM 545 using the response surface methodology (RSM). Based on the results from the search for optimization limits, the glucose concentration, the ammonium sulfate concentration and the phosphate buffer molarity were chosen as independent variables. The optimal values were found as follows: the glucose concentration 10.8 g/L; the ammonium sulfate concentration 0.95 g/L; and the phosphate buffer molarity 60.2 mmol/L. The predicted biomass concentration was 4.54 g/L, and the verified value was at 4.84 g/L. Although this work was primarily focused on determining the optimal composition of the propagation medium, we also evaluated the optimal composition of the production medium and found that the optimal glucose concentration was 6.7 g/L; the ammonium sulfate concentration 0.60 g/L; and the phosphate buffer molarity 20 mmol/L. The predicted PHB yield was 54.7% (w/w) of dry biomass, and the verified value was 49.1%.

Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.

Polyhydroxyalkanoates (PHAs) represent a promising alternative to commercially used petroleum-based plastics. Pseudomonas oleovorans is a natural producer of medium-chain-length PHA (mcl-PHA) under cultivation conditions with nitrogen limitation and carbon excess. Two-step cultivation appears to be an efficient but more expensive method of PHA production. Therefore, the aim of this work was to prepare a minimal synthetic medium for maximum biomass yield and to optimize selected independent variables by response surface methodology (RSM). The highest biomass yield (1.71 ± 0.04 g/L) was achieved in the optimized medium containing 8.4 g/L glucose, 5.7 g/L sodium ammonium phosphate and 35.4 mM phosphate buffer. Under these conditions, both carbon and nitrogen sources were completely consumed after 48 h of the cultivation and the biomass yield was 1.7-fold higher than in the conventional medium recommended by the literature. This approach demonstrates the possibility of using two-stage PHA cultivation to obtain the maximum amount of biomass and PHA.