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Polyhydroxyalkanoates (PHAs) are synthesized by a variety of microorganisms as intracellular storage granules under imbalanced nutrition and excess carbon. Carbon sources are key contributors to the high cost of PHA production, hence the need for exploration of cheap and sustainable raw materials for bacteria fermentation. In the present study, Halomonas alkalicola strain Ext isolated from a hypersaline lake in Kenya was assessed for its ability to utilize fruit peels hydrolysates (FPH) as sole carbon sources for PHA production. Sugars were extracted from dried peels of banana, mango, orange, and pineapple fruits through mechanical pretreatment and dilute acid hydrolysis. Fruit peels pretreated with 3 % H2SO4 at 121℃ were utilized for shake flask fermentation to produce PHAs by Halomonas alkalicola Ext. At optimal C:N ratios of between 20:1 and 30:1, the bacterium could produce up to 0.45±0.03, 0.394±0.12, 0.39±0.05, and 0.28±0.0 g/L of PHAs from hydrolysates of orange peels, mango peels, banana peels, and pineapple peels, respectively. A maximum PHA content of 16.92 % was achieved on orange peels hydrolysates with 4 % substrate loading. Monomer analysis with gas chromatography-mass spectrometry (GC-MS) revealed that the bacterium produced a copolymer Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 3-hydroxyvalerate (3HV) contents of 5.77 %, 6.08 %, 6.79 %, 6.845 %, for orange peels, pineapple peels, mango peels and banana peels substrates, respectively. The findings of this study suggest that fruit peels waste is a potential feedstock for sustainable production of PHAs by Halomonas alkalicola.

The search for cost-effective substrates for the manufacturing of valuable products has led to the use of agrowastes as alternative sources of reducing sugar. Numerous bacteria build up polyhydroxyalkanoates (PHAs) as storage materials. This research aimed to produce PHA from Pseudomonas aeruginosa using agrowastes as carbon sources. The agrowastes (corncob, plantain peduncle and sugarcane bagasse) were treated with 1 % NaOH and analysed. The agrowastes were hydrolysed using cellulase produced by Aspergillus niger isolated from agrowastes dumpsite. The agrowaste hydrolysate was used in place of glucose for PHA production in a submerged fermentation. Nile blue A test and Sudan black test showed positive results for the isolate with a bright orange fluorescence on irradiation with UV light and was identified as Pseudomonas aeruginosa (accession number 0L405443). Sugarcane bagasse gave the highest potential for PHA production with PHA values of 5.86 mg/mL, followed by corn cob (5.29 mg/mL) and the least was obtained in plantain peduncle with a yield of 3.58 mg/mL. The findings using response surface methodology (RSM) for optimization show that all the four factors (carbon source, pH, temperature and incubation time) were statistically significant (P

Recently, polyhydroxyalkanoates (PHAs) have been produced using raw sewage in our laboratory; however, the production concentrations are low. Therefore, this study aimed to enhance PHA production by applying different strategies. PHA production was higher in sewage-containing medium than in mineral salt medium and was enhanced 22-fold after glucose supplementation. A relatively high degree of glucose consumption (83.6 ± 1.59 %) was also achieved. Bacteria incubated with cheese whey diluted with sewage showed higher PHA production than bacteria incubated with cheese whey diluted with distilled water did. The expression of the PHA synthase gene (phaC) was evaluated via real-time polymerase chain reaction using low- and high-carbon-containing sewage. Relatively higher phaC expression levels were observed in high-carbon-containing sewage but at lower nitrogen concentrations. The characteristics of the produced PHA were comparable to those of standard PHA. Therefore, this study revealed that the bacterium Bacillus sp. CYR1 can produce PHA from low- or high-carbon-containing wastewater.

Priestia is a genus of biotechnologically important bacteria adapted to thrive in a wide range of environmental conditions including the marine sediments. Here, we screened and isolated a strain from the Bagamoyo marine mangrove-inhabited sediments and then employed whole genome sequencing to recover and define its full genome. De novo-assembly with Unicycler (v. 0.4.8) and annotation with Prokaryotic Genome Annotation Pipeline (PGAP) revealed that that its genome contains one chromosome (5,549,131 bp), with a GC content of 37.62%. Further analysis showed that the genome contains 5,687 coding sequences (CDS), 4 rRNAs, 84 tRNAs, 12 ncRNAs, and at least 2 plasmids (1,142 bp and 6,490 bp). On the other hand, antiSMASH-based secondary metabolite analysis revealed that the novel strain (MARUCO02) contains gene clusters for biosynthesis of MEP-DOXP-dependent versatile isoprenoids (eg. carotenoids), siderophores (synechobactin and schizokinen) and polyhydroxyalkanoates (PHA). The genome dataset also informs about the presence genes encoding enzymes required for generation of hopanoids, compounds that confer adaption to harsh environmental conditions including industrial cultivation recipes. Our data from this novel Priestia megaterium strain MARUCO02 can be used for reference and in genome-guided selection of strains for production of isoprenoids as well as industrially useful siderophores and polymers, amenable for biosynthetic manipulations in a biotechnological process.

Background: Polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV) are the most common polyhydroxyalkanoates (PHAs). They are important candidates for biodegradable plastics that accumulate in the cytoplasm in the form of intracellular granules under unbalanced growth conditions. The present study focused on the production and optimization of PHAs from Paraburkholderia sp. PFN29 for the first time under submerged fermentation. Results: Two hundred bacterial isolates were collected from soil samples in Thailand, six of which were found to accumulate PHB using the Nile red staining method. Six isolates were identified by 16S rDNA gene sequencing and were shown to be closely related to the genera Azotobacter, Sphingobium and Paraburkholderia. The best strain, Paraburkholderia sp. PFN29, showed the highest PHB concentration (5 ± 0.17 g/L). Optimization of PHB accumulation was achieved in modified medium containing 3% (w/v) glucose, and 0.1% (w/v) (NH4)2Fe(SO4) with incubation at pH 7.0 and 35°C for 96 h with shaking at 200 rpm. In preliminary PHBV synthesis, PFN29 was utilized to synthesize PHBV from glucose and organic acids (levulinic acid and propionic acid). 1H nuclear magnetic resonance (1H NMR) spectroscopy and gas chromatography-mass spectrometry (GC/MS) analyses confirmed the extracted PHAs compared to standard PHB and PHBV. Conclusions: PFN29 can be used for feasible PHB production with a PHB content of up to 97.3% of 5.14 ± 0.17 g/L CDW. The PHBV concentration can be further improved by optimizing the production parameters as cosubstrates. The present study provides useful data on PHA production by Paraburkholderia, which may be used as a candidate species for commercial PHA production. How to cite: Sriyapai T, Chuarung T, Kimbara K, et al. Production and optimization of polyhydroxyalkanoates (PHAs) from Paraburkholderia sp. PFN 29 under submerged fermentation. Electron J Biotechnol 2022;56. https://doi.org/10.1016/j.ejbt.2021.12.003

A key step for the production of polyhydroxyalkanoates (PHAs) from organic waste streams is the selection of a biomass with a high PHA-storage capacity (selection-step), which is usually performed in sequencing batch reactors (SBR). A major advancement would be to perform such selection in continuous reactors to facilitate the full-scale implementation of PHA production from municipal wastewater (MWW)-derived feedstock. The present study therefore investigates to what extent a simple continuous-flow stirred-tank reactor (CSTR) represents a relevant alternative to anSBR. To this end, we operated two selection reactors (CSTR vs. SBR) on filtered primary sludge fermentate while performing a detailed analysis of the microbial communities, and monitoring PHA-storage over long-term (∼150 days) and during accumulation batches. Our study demonstrates that a simple CSTR is as effective as an SBR in selecting biomass with high PHA-storage capacity (up to 0.65 gPHA gVSS−1) while being 50% more efficient in terms of substrate to biomass conversion yields. We also show that such selection can occur on VFA-rich feedstock containing nitrogen (N) and phosphorus (P) in excess, whereas previously, selection of PHA-storing organisms in a single CSTR has only been studied under P limitation. We further found that microbial competition was mostly affected by nutrient availability (N and P) rather than by the reactor operation mode (CSTR vs. SBR). Similar microbial communities therefore developed in both selection reactors, while microbial communities were very different depending on N availability. Rhodobacteraceae gen. were most abundant when growth conditions were stable and N-limited, whereas dynamic N- (and P-) excess conditions favoured the selection of the known PHA-storer Comamonas, and led to the highest observed PHA-storage capacity. Overall, we demonstrate that biomass with high storage capacity can be selected in a simple CSTR on a wider range of feedstock than just P-limited ones.

Microplastics (MPs) has been concerned as emerging vectors for spreading antibiotic resistance and pathogenicity in aquatic environments, but the role of biodegradable MPs remains largely unknown. Herein, field in-situ incubation method combined with metagenomic sequencing were employed to reveal the dispersal characteristics of microbial community, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and virulence factors (VFs) enriched by MPs biofilms. Results showed that planktonic microbes were more prone to enrich on biodegradable MPs (i.e., polyhydroxyalkanoate and polylactic acid) than non-biodegradable MPs (i.e., polystyrene, polypropylene and polyethylene). Distinctive microbial communities were assembled on biodegradable MPs, and the abundances of ARGs, MGEs, and VFs on biofilms of biodegradable MPs were much higher than that of non-biodegradable MPs. Notably, network analysis showed that the biodegradable MPs selectively enriched pathogens carrying ARGs, VFs and MGEs concurrently, suggesting a strong potential risks of co-spreading antibiotic resistance and pathogenicity through horizontal gene transfer. According to WHO priority list of Antibiotic Resistant Pathogens (ARPs) and ARGs health risk assessment framework, the highest abundances of Priority 1 ARPs and Rank I risk ARGs were found on polylactic acid and polyhydroxyalkanoate, respectively. These findings elucidate the unique and critical role of biodegradable MPs for selective enrichment of high-risk ARGs and priority pathogens in freshwater environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are biopolymers produced by microorganisms under nutrient limiting conditions and in the presence of excess carbon source. PHAs have gained popularity as a sustainable alternative to traditional plastics. However, large scale production of PHAs is economically challenging due to the relatively high costs of organic carbon. Alternative options include using organisms capable of phototrophic or mixotrophic growth. This study aimed at the production of poly(3-hydroxybutyrate) P(3HB), a type of PHA, at pilot scale using the freshwater cyanobacterium Synechocystis sp. PCC6803. First, to identify optimal conditions for P(3HB) production from Synechocystis sp. PCC6803, different supplemental carbon source concentrations and salinity levels were tested at laboratory scale. The addition of 4 g/L acetate with no added NaCl led to P(3HB) accumulation of 10.7 % dry cell weight on the 28th day of cultivation. Although acetate additions were replicated in an outdoor 400 L serpentine photobioreactor, P(3HB) content was lower, implying uncontrolled conditions impact on biopolymer production efficiency. An optimized P(3HB) extraction methodology was developed to remove pigments, and the biopolymer was characterized and subjected to 3D printing (fused deposition modelling) to confirm its processability. This study thus successfully led to the large-scale production of P(3HB) using sustainable and environmentally friendly cyanobacterial fermentation., Competing Interests: Declaration of Competing Interest None., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Plastic pollution presents a significant environmental problem contributing to increased CO 2 emissions and persistently accumulation in ecosystems. Biobased polymers, like polyhydroxyalkanoates (PHAs), offer a part of a solution with their biodegradability and reduced carbon footprint. However, effective end-of-life strategies, such as controlled enzymatic depolymerization, are crucial for sustainability, relying on efficient PHA depolymerases (PHAases). Here we describe the synthesis of two new chromogenic compounds derived from polyhydroxyoctanoate (PHO) and their application in a continuous, quantitative spectrophotometric assay for PHO depolymerase and other medium chain lengths PHAase activity within 10 min. These substrates allow activity measurement at temperatures above 45 °C, simplifying the comparison of PHAases and aiding enzymatic degradation progress. The study also explores enzyme specificity and identifies key amino acids involved in PHO recognition by PfPHOase. The 3-hydroxyoctanoyl moieties of both compounds were found to bind specifically to a groove formed by the amino acids Phe96, Phe125, Ile171, and Val230, which are highly conserved in known mcl-PHA depolymerases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Polyhydroxybutyrate (PHB) is a biodegradable and biocompatible biopolyester, naturally produced and self-assembled as spherical inclusions inside bacteria. These PHB particles contain a hydrophobic PHB core covalently coated with PHB synthase (PhaC), which serves as an anchoring linker for foreign proteins of interest. Protein engineering of PhaC enables the display of biologically active protein functions on the surface of PHB particles suitable for different applications. Many biomolecules, such as e.g. antigens, enzymes, fluorescent proteins were immobilized to PHB particles and exhibited superior functionalities when compared to their respective soluble counterparts. Recently, PHB particles have been successfully applied for various diagnostics applications. This mini review provides an overview of the unique design space of PHB particles towards the development of safe and cost-effective diagnostic tools, and highlights the important research progresses of manufacturing PHB particles-based diagnostics., Competing Interests: Declaration of Competing Interest B.H.A. Rehm is co-founder and shareholder of PolyBatics Ltd that commercializes veterinary TB diagnostic products related to the PHA bead technology. The other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

As environmental pollution intensifies, the interest in bioplastics is growing. The bioplastic polyhydroxyalkanoates (PHAs), which are produced and degraded by microorganisms, have received considerable attention. However, the production cost of PHA is still high, and several ways to increase economy of PHA production have been studied. Therefore, as one way of solution, Halomonas species were screened and evaluated with cheap substrates such as molasses and soybean flour. Among tested strains, Halomonas cerina YK44 was selected and used for polyhydroxybutyrate (PHB) production with molasses and soybean flour together, whose combination was not evaluated well before, in tap water. The medium composition optimization showed maximum PHB production at 4 % sugarcane molasses, 2 % NaCl, 0.05 % soybean flour, and pH 8 in tap water (9.2 g/L DCW, 7.3 g/L PHB, and 79.7 % PHB contents). However, cell growth of halotolerant H. cerina YK44 was disturbed by 0.2 % furfural, which existed in biomass based sugars as inhibitors. Physical and thermal analyses revealed that PHB film started from sugarcane molasses and soybean flour was no different from that initiated from simple sugars (Tm was 175.8 °C and 176.2 °C, PDI was 1.29, and 1.31, respectively)., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may influence the work reported in this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that otherwise exhibit many properties similar to that of conventional, non-biodegradable plastics. Hence, they are considered as potential substitutes for non-biodegradable plastics. But industrial production and commercialization of PHA have been hindered greatly due to the high cost requirements, especially those involved in the provision of suitable carbon sources. In this study, sugarcane molasses was used as a cheap carbon source for PHA production using a wild strain of Enterobacter cloacae isolated from sugarcane extract. This isolate was selected for PHA production based on Sudan Black B staining, and it was subsequently identified by performing BLASTn using its 16S rRNA sequence (GenBank accession number: ON314993). The PHA yield was studied under varying conditions of initial pH, molasses concentration and inoculum concentration. The highest yield of 4.13 - 4.98 g/L or 48 - 56% PHA was obtained after 48-60 h incubation at initial pH 7, molasses concentration 4% and inoculum concentration 2%. After downstream processing, the extracted PHA was characterized by FTIR spectroscopy. It is concluded that E. cloacae has the potential to utilize sugarcane molasses as a cheap carbon source to yield impressive amounts of PHA, and that it should be considered a candidate for further studies and for the cheaper industrial production of PHA.

Polyhydroxyalkanoates (PHA) are prokaryotic macromolecules accumulated within the cytoplasm as granules. Due to their suitable mechanical properties, biocompatibility, degradation time, ability to be blended, surface modified, and form copolymers, it is widely used in medical devices and as scaffolds in bone tissue engineering. This review describes in brief the production and extraction sources, physico-chemical characteristic, mechanical properties, degradation rate and applications of various PHAs and its copolymers with special emphasis to its role as scaffolds in bone tissue engineering.

Extremophilic microbes show a unique metabolism due to the adaptations they display to deal with extreme environmental parameters characterizing the extreme ecosystems that they inhabit (high salt concentration, high temperatures, and extreme pH values, high exposure to solar radiation etc.). Halophilic microorganisms characterised and isolated from saltmarshes, brines, salted ponds, salty lagoons etc. have recently attracted attention due to their potential biotechnological applications (as whole cells used for different purposes like wastewater treatments, or their biomolecules: enzymes, antibiotics, carotenoids, bioplastics). Alicante county (southeast of Spain) accounts for a significant number of salty environments like coastal or inland salty ponds from where sodium chloride (NaCl)is obtained, marshes, salty lagoons, etc. The best system characterised so far from a microbiological point of view is “Salinas de Santa Pola”, also termed “Salinas Bras del Port”. However, there are many other salty environments to be explored, like the natural park of Torrevieja and la Mata lagoons, salty lagoon located in Calpe city or inland salted ponds like those located in the northwest of the county. This review summarises the most relevant biotechnological applications of halophilic microbes described up to now. In addition, special attention is focused on ecosystems such as the lagoons of Torrevieja or inland salt marshes as natural environments whose microbial biodiversity is worthy of being studied in search of new strains and species with the aim to analyze their potential biotechnological applications (pharmaceutical, food industry, biomedicine, etc.).

Biodegradation in marine medium of PHBV films with or without 5 % wt. of phenolic compounds (catechin, ferulic acid, and vanillin) was assessed at laboratory scale. Respirometric analyses and film disintegration kinetics were used to monitor the process over a period of 162 days. Structural changes in the films were analyzed throughout the exposure period using FESEM, DSC, Thermogravimetric analyses, XRD, and FTIR spectra. Respirometric tests showed complete biodegradation of all materials during the exposure period (the biodegradation half-time ranged between 63 and 79 days) but at different rates, depending on the phenolic compound incorporated. Ferulic acid and vanillin accelerate the PHBV biodegradation, whereas catechin delayed the process. Disintegration kinetics confirmed these results and showed that degradation occurred from the surface to the interior of the films. This was controlled by the degradation rate of the polymer amorphous phase and the formation of a biomass coating on the film surface. This is the result of the compounds generated by polymer degradation in combination with excretions from microorganisms. This coating has the potential to affect the enzyme diffusion to the polymer substrate. Moreover, the cohesion forces of the amorphous phase (reflected in its glass transition temperature) affected its degradation rate, while the slower degrading crystalline fragments were released, thus contributing to the disintegration process on the film's surface. Ferulic acid, with its hydrolytic effect, enhanced degradation, as did vanillin for its plasticizing and weakening effect in the amorphous phase of polymer matrix. In contrast, catechin with cross-linking effect hindered the progress of the material degradation, considerably slowing down the process rate., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Carla Ivonne La Fuente Arias reports financial support was provided by Government of Valencia. Amparo Chiralt reports financial support was provided by Government of Valencia. Chelo Gonzalez Martinez reports financial support was provided by State Agency of Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are a well-known group of biodegradable and biocompatible bioplastics that are synthesised and stored by microorganisms as carbon and energy reservoirs. Extracellular PHA depolymerases (ePhaZs), secreted by a limited range of microorganisms, are the main hydrolytic enzymes responsible for their environmental degradation. Pseudomonas sp. GK13, initially identified as P. fluorescens GK13, produces PHA and a prototypic ePhaZ that specifically degrades mcl-PHA. In this study, a comprehensive characterization of strain GK13 was performed. The whole genomic sequence of GK13 was consolidated into one complete chromosome, leading to its reclassification as P. solani GK13. We conducted a detailed in silico examination of the bacteria genomic sequence, specifically targeting PHA metabolic functions. From the different growth conditions explored, PHA accumulation occurred only under carbon/nitrogen (C/N) imbalance, whereas ePhaZ production was induced even at balanced C/N ratios in mineral media. We extend our study to other bacteria belonging to the Pseudomonas genus revealing that the ePhaZ production capacity is closely associated with mcl-PHA synthesis capacity, as also suggested by metagenomic samples. This finding suggests that these types of microorganisms could contribute to the carbon economy of the microbial community, by storing PHA in carbon-rich times, and sharing it with the rest of the population during times of carbon scarcity through PHA hydrolysis. The conclusion pointed that carbon cycle metabolism performed by P. solani GK13 may contribute to the environmental circular economy at a microscopic scale., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Crystalline bio-based composites exhibit discontinuous feeding and warpage of printed parts in traditional filament printing. Therefore, an additive manufacturing (AM) system based on micro-screw extrusion was designed, and wood flour/polyhydroxyalkanoates (WF/PHA) composites without additives were manufactured. The differential scanning calorimetry test showed that the crystallization temperature and melting temperature of the WF/PHA composites increased by 35.7 °C and 46.1 °C, respectively. The evaluation of the extrusion stability of the micro-screw extrusion system showed that the micro-screw speed had a greater influence on extrusion output than the printing temperature. The forming process under pressure and the WF/PHA blend prevented the warpage of printed samples. The tensile and flexural strengths of the WF/PHA composites reached 38.7 MPa and 77.3 MPa, respectively, and the modulus of the WF/PHA composites significantly improved with the increase in WF content. The rheological, thermal, and mechanical properties of the printed samples were used to determine the reason for the decrease in warpage. Finally, the test printing of thin and large-area sheets provides the conditions for the application of AM in fully bio-based composites.

The global consumption of plastics generates accelerated environmental pollution in landfills and marine ecosystems. Biopolymers are the materials with the greatest potential to replace synthetic polymers in the market due to their good biodegradability, however, there are still several disadvantages, mainly related to their production cost. Considering the above, the generation of biodegradable and biocompatible bioplastics stands out as an alternative solution, some of which are made from renewable raw materials, including polyhydroxyalkanoates PHAs. Although much research has been done on bacteria with the capacity for intracellular accumulation of PHAs, among others, it is also possible to produce PHAs using mixed microbial cultures instead of a single microorganism, using natural microbial consortia that have the capacity to store high amounts of PHAs. In this contribution, three methods for the extraction and purification of PHAs produced by fermentation using volatile fatty acids as a carbon source at different concentrations were evaluated, using the pure strain Burkholderia cepacia 2G-57 and the mixed cultures of the activated sludge from the El Salitre WWTP, in order to select the best method from the point of view of environmental sustainability as this will contribute to the scalability of the process. The mixed cultures were identified by sequencing of the 16S gene. A yield of 89% was obtained from the extraction and purification of PHA using acetic acid as a solvent, which according to its properties is "greener" than chloroform. The polymer obtained was identified as polyhydroxybutylated PHB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study involved creating oligomeric conjugates of 3-hydroxy fatty acids and diclofenac, named Dic-oligo(3HAs). Advanced NMR techniques confirmed no free diclofenac in the mix. We tested diclofenac release under conditions resembling healthy and chronic wound skin. These oligomers were used to make P(3HO) blends, forming patches for drug delivery. Their preparation used the solvent casting/porogen leaching (SCPL) method. The patches' properties like porosity, roughness, and wettability were thoroughly analysed. Antimicrobial assays showed that Dic-oligo(3HAs) exhibited antimicrobial activity against reference (S. aureus, S. epidermis, S. faecalis) and clinical (Staphylococcus spp.) strains. Human keratinocytes (HaCaT) cell line tests, as per ISO 10993-5, showed no toxicity. A clear link between material roughness and HaCaT cell adhesion was found. Deep cell infiltration was verified using DAPI and phalloidin staining, observed under confocal microscopy. SEM also confirmed HaCaT cell growth on these scaffolds. The strong adhesion and proliferation of HaCaT cells on these materials indicate their potential as wound dressing layers. Additionally, the successful diclofenac release tests point to their applicability in treating both normal and chronic wounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB -4 harboring the gene phaC BP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The scope of the current study was to investigate the efficiency of a two-stage anaerobic-aerobic process for the simultaneous treatment and valorization of selective wastewater streams from a confectionary industry. The specific wastewater (confectionary industry wastewater, CIW) was a mixture of the rinsing eluting during washing of the cauldrons in which jellies and syrups were produced, and contained mainly readily fermentable sugars, being thus of high organic load. The first stage of the process was the dark fermentation (DF) of the CIW in continuous, attached-biomass systems, in which the effect on hydrogen yields and distribution of metabolites were studied for different packing materials (ceramic or plastic), hydraulic retention times, HRTs (12 h-30 h) and feed substrate concentration (20 g COD/L- 50 g COD/L). In the second stage, the effectiveness of the aerobic treatment of the DF effluents was evaluated in terms of the reduction of the organic load and the production of polyhydroxyalkanoates (PHAs) through an enriched mixed microbial culture (MMC). The MMC was developed in a continuous draw and fill system, in which the accumulation potential of PHAs was studied. It was shown that the hydrogen production rates decreased for increasing substrate concentration and HRTs, with a maximum of 12.70 ± 0.35 m 3 H 2 /m 3 initial CIW achieved for the lowest HRT and feed concentration and using ceramic beads as packing material. Butyrate, acetate and lactate were the main metabolites generated in all cases, in different ratios. The distribution of metabolites during DF was shown to highly affect the efficiency of the second process in terms of both the reduction of organic load and the PHAs yields. The highest removal of organic load achieved after 48 h of aerobic treatment was 84.0 ± 0.9 %, whereas the maximum PHAs yield was 21.46 ± 0.13 kg PHAs/m 3 initial CIW., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bone Morphogenetic Protein 4 (BMP4) is crucial for bone and cartilage tissue regeneration, essential in medical tissue engineering, cosmetology, and aerospace. However, its cost and degradation susceptibility pose significant clinical challenges. To enhance its osteogenic activity while reducing dosage and administration frequency, we developed a novel long-acting BMP4 delivery system using poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PBVHx) nanoparticles with soybean lecithin-modified BMP4 (sBP-NPs). These nanoparticles promote directed osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through sustained BMP4 release. sBP-NPs exhibited uniform size (100-200 nm) and surface charges, with higher BMP4 entrapment efficiency (82.63 %) compared to controls. After an initial burst release within 24 h, sBP-NPs achieved 80 % cumulative BMP4 release within 20 days, maintaining levels better than control BP-NPs with unmodified BMP4. Co-incubation and nanoparticle uptake experiments confirmed excellent biocompatibility of sBP-NPs, promoting hBMSC differentiation towards osteogenic lineage with increased expression of type I collagen, calcium deposition, and ALP activity (> 20,000 U/g protein) compared to controls. Moreover, hBMSCs treated with sBP-NPs exhibited heightened expression of osteogenic genetic markers, surpassing control groups. Hence, this innovative strategy of sustained BMP4 release from sBP-NPs holds potential to revolutionize bone regeneration in minimally invasive surgery, medical cosmetology or space environments., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHAs) are a group of natural polyesters that are synthesised by microorganisms. In general, their thermoplasticity and (in some forms) their elasticity makes them attractive alternatives to petrochemical-derived polymers. However, the high crystallinity of some PHAs - such as poly(3-hydroxybutyrate) (P3HB) - results in brittleness and a narrow processing window for applications such as packaging. The production of copolymeric PHA materials is one approach to improving the mechanical and thermal properties of PHAs. Another solution is the manufacture of PHA-based block copolymers. The incorporation of different polymer and copolymer blocks coupled to PHA, and the resulting tailorable microstructure of these block copolymers, can result in a step-change improvement in PHA-based material properties. A range of production strategies for PHA-based block copolymers has been reported in the literature, including biological production and chemical synthesis. Biological production is typically less controllable, with products of a broad molecular weight and compositional distribution, unless finely controlled using genetically modified organisms. By contrast, chemical synthesis delivers relatively controllable block structures and narrowly defined compositions. This paper reviews current knowledge in the areas of the production and properties of PHA-based block copolymers, and highlights knowledge gaps and future potential areas of research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Plastic pollution is the biggest environmental concern of our time. Breakdown products like micro- and nano-plastics inevitably enter the food chain and pose unprecedented health risks. In this scenario, bio-based and biodegradable plastic alternatives have been given a momentum aiming to bridge a transition towards a more sustainable future. Polyhydroxyalkanoates (PHAs) are one of the few thermoplastic polymers synthesized 100 % via biotechnological routes which fully biodegrade in common natural environments. Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] is a PHA copolymer with great potential for the commodity polymers industry, as its mechanical properties can be tailored through fine-tuning of its molar HHx content. We have recently developed a strategy that enables for reliable tailoring of the monomer content of P(HB-co-HHx). Nevertheless, there is often a lack of comprehensive investigation of the material properties of PHAs to evaluate whether they actually mimic the functionalities of conventional plastics. We present a detailed study of P(HB-co-HHx) copolymers with low to moderate hydroxyhexanoate content to understand how the HHx monomer content influences the thermal and mechanical properties and to link those to their abiotic degradation. By increasing the HHx fractions in the range of 2 - 14 mol%, we impart an extension of the processing window and application range as the melting temperature (T m ) and glass temperature (T g ) of the copolymers decrease from T m 165 °C to 126 °C, T g 4 °C to -5.9 °C, accompanied by reduced crystallinity from 54 % to 20 %. Elongation at break was increased from 5.7 % up to 703 % at 14 mol% HHx content, confirming that the range examined was sufficiently large to obtain ductile and brittle copolymers, while tensile strength was maintained throughout the studied range. Finally, accelerated abiotic degradation was shown to be slowed down with an increasing HHx fraction decreasing from 70 % to 55 % in 12 h., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO 2 ), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO 2 . The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Numerous studies have been conducted to develop a rapid protocol for the quantification of poly(3-hydroxybutyrate) during bacterial fermentation as an alternative to time-consuming gravimetric or analytical methods. Fluorescence spectroscopy is one of the most promising approaches. In this study, it could be demonstrated that the novel fluorescent probe LipidGreen2 is able to stain selectively poly(3-hydroxybutyrate) in Cupriavidus necator. Optimal excitation and emission wavelengths were evaluated using 3D-Excitation-Emission-Matrix, displaying the best intensities between 440-460 nm and 490–520 nm for excitation and emission, respectively. The lipophilic fluorophore LipidGreen2 showed a high long-term stability even when incubated under ambient lighting. Due to a strong linear relationship between side scatter and biomass concentration, the influence of the inner filter effects could be incorporated, and adjusting the sample to a specific OD is thus superfluous. The developed method allows a very accurate quantification of poly(3-hydroxybutyrate) in just 15 min, following a comprehensible and simple protocol. It is also excellently suited for bioimaging of intracellular poly(3-hydroxybutyrate) granules.

Polyhydroxyalkanoate (PHA) is a fully biodegradable bioplastic. To foster a circular economy, the integration of PHA production into wastewater treatment facilities can be accomplished using mixed microbial consortia. The effectiveness of this approach relies greatly on the enrichment of PHA-accumulating microorganisms. Hence, our study focused on bioaugmenting Thauera mechernichensis TL1 into mixed microbial consortia with the aim of enriching PHA-accumulating microorganisms and enhancing PHA production. Three sequencing batch reactors-SBRctrl, SBR2.5%, and SBR25%-were operated under feast/famine conditions. SBR2.5% and SBR25% were bioaugmented with T. mechernichensis TL1 at 2.5%w/w of mixed liquor volatile suspended solids (MLVSS) and 25%w/w MLVSS, respectively, while SBRctrl was not bioaugmented. SBR2.5% and SBR25% achieved maximum PHA accumulation capacities of 56.3 %gPHA/g mixed liquor suspended solids (MLSS) and 50.2 %gPHA/gMLSS, respectively, which were higher than the 25.4 %gPHA/gMLSS achieved by SBRctrl. The results of quantitative polymerase chain reaction targeting the 16S rRNA gene specific to T. mechernichensis showed higher abundances of T. mechernichensis in SBR2.5% and SBR25% compared with SBRctrl in the 3rd, 17th, and 31st cycles. Fluorescence in situ hybridization, together with fluorescent staining of PHA with Nile blue A, confirmed PHA accumulation in Thauera spp. The study demonstrated that bioaugmentation of T. mechernichensis TL1 at 2.5%w/w MLVSS is an effective strategy to enhance PHA accumulation and facilitate the enrichment of PHA-accumulating microorganisms in mixed microbial consortia. The findings could contribute to the advancement of PHA production from wastewater, enabling the transformation of wastewater treatment plants into water and resource recovery facilities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Methanotrophic bacteria can use atmospheric methane (CH 4 ) as a sole carbon source for the growth and production of polyhydroxyalkanoates (PHA). The development of CH 4 bioconversion processes relies heavily on the selection of an efficient methanotrophic culture. This research assessed the effect of selected growth conditions, such as nitrogen sources on the enrichment of methanotrophic cultures from various environments for PHA accumulation. Nitrate-based medium favoured the culture growth and selection for PHA-producing methanotrophic cultures with Methylocystis sp. as a major genus and accumulation of up to 27 % polyhydroxybutyrate (PHB) in the biomass. Three PHB-producing cultures: enriched from waste activated sludge (AS), peat bog soil (PB) and landfill biocover soil (LB) were then tested for their ability to produce PHA copolymer at different CH 4 :O 2 ratios. All enriched cultures were able to utilise valeric acid as a cosubstrate for the accumulation of PHA with a 3-hydroxyvaleric (3HV) fraction of 21-41 mol% depending on the inoculum source and CH 4 concentration. The process performance of selected cultures was evaluated and compared to the culture of reference strain Methylocystis hirsuta DSM 18500. All mixed cultures irrespective of their inoculum source had similar levels of 3HV fraction in the PHA (38 ± 2 mol%). The highest poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production was observed for AS culture at 10 % CH 4 with an accumulation of 27 ± 3 % of dry cell weight (DCW), 3HV fraction of 39 ± 2 mol% and yield of 0.42 ± 0.02 g-PHA/g-substrate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The versatile capacity of purple phototrophic bacteria (PPB) for producing valuable bioproducts has gathered renewed interest in the field of resource recovery and waste valorisation. However, greater knowledge regarding the viability of applying PPB technologies in outdoor, large-scale systems is required. This study assessed, for the first time, the upscaling of the phototrophic polyhydroxyalkanoate (PHA) production technology in a pilot-scale system operated in outdoor conditions. An integrated system composed of two up-flow anaerobic sludge blanket (UASB) reactors (for fermentation of wastewater with molasses), and two high-rate algal ponds retrofitted into PPB ponds, was operated in a wastewater treatment plant under outdoor conditions. UASB's adaptation to the outdoor temperatures involved testing different operational settings, namely hydraulic retention times (HRT) of 48 and 72 h, and molasses fermentation in one or two UASBs. Results have shown that the fermentation of molasses in both UASBs with an increased HRT of 72 h was able to ensure a suitable operation during colder conditions, achieving 3.83 ± 0.63 g COD Fermentative Products /L, compared to the 3.73 ± 0.85 g COD Fermentative Products /L achieved during warmer conditions (molasses fermentation in one UASB; HRT 48 h). Furthermore, the PPB ponds were operated under a light-feast/dark-aerated-famine strategy and fed with the fermented wastewater and molasses from the two UASBs. The best PHA production was obtained during the summer of 2018 and spring of 2019, attaining 34.7 % gPHA/gVSS with a productivity of 0.11 gPHA L -1  day -1 and 36 % gPHA/gVSS with a productivity of 0.14 gPHA L -1  day -1 , respectively. Overall, this study showcases the first translation of phototrophic PHA production technology from an artificially illuminated laboratory scale system into a naturally illuminated, outdoor, pilot-scale system. It also addresses relevant process integration aspects with UASBs for pre-fermenting wastewater with molasses, providing a novel operational strategy to achieve photosynthetic PHA production in outdoor full-scale systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Synthetic biodegradable and bio-based polymers have emerged as sustainable alternatives to nonrenewable petroleum-derived polymers which cause serious environmental issues. In particular, polyhydroxyalkanoates (PHA) are promising biopolymers owing to their outstanding biodegradability and biocompatibility. The production of the homopolymer poly(3-hydroxybutyrate) (PHB) and copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from type II methanotrophs via microbial fermentation was presented. For the efficient extraction and recovery of intracellular PHA from methanotrophs, different extraction approaches were investigated including solvent extraction using 1,3-dioxolane as a green solvent, integrated cell lysis and solvent extraction, and cell digestion without the use of organic solvents. Among various extraction approaches, the integrated method exhibited the highest extraction performance, with PHA recovery and purity exceeding 91 % and 93 %, respectively, even when the PHA content of the cells was low. Furthermore, the molecular weight, thermal stability, and mechanical properties of the recovered PHA were comprehensively analyzed to suggest its suitable practical applications. The obtained properties were comparable to that of the commercial PHA products and PHA produced from other microbial species, indicating an efficient recovery of high-quality PHA produced from methanotrophs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Polyhydroxyalkanoates (PHA) recovery from industrial wastewater has been highlighted as a promising strategy for a circular bioeconomy. However, the high and varying level of nitrogen in wastewater makes enrichment of mixed microbial culture (MMC) low efficiency. In this study, spatial separation of nitrifiers and denitrifiers was adopted by adding biocarriers in MMC and decreasing the sludge retention time (SRT) to accelerate the enrichment of PHA-storing MMC fed by mixed wastewater containing glycerol and propionate. Nitrifiers and denitrifiers were sustained on biocarriers, obtaining a high total inorganic nitrogen removal and allowing a more efficient selective pressure of a high carbon and nitrogen ratio (C/N) under low SRT conditions. The maximum PHA cell content and relative abundance of PHA-storing bacteria were increased to 60.51 % (SRT 6 d) and 49.62 % (SRT 6 d) with the decrease of SRT, respectively. This study demonstrates an efficient way to highly enrich PHA-storing MMC from crude glycerol, which provide a relevant technical support for high-efficiency enrichment of PHA-storing bacteria in low C/N wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biopolymers produced as microbial carbon storage systems, such as polyhydroxyalkanoates (PHAs), offer potential to be used in place of petrochemically derived plastics. Low-value organic feedstocks, such as food waste, have been explored as a potential substrate for the microbial production of PHAs. In this review, we discuss the biosynthesis, composition and producers of PHAs, with a particular focus on the genetic and process engineering efforts to utilise non-native substrates, derived from food waste from across the entire supply chain, for microbial growth and PHA production. We highlight a series of studies that have achieved impressive advances and discuss the challenges of producing PHAs with consistent composition and properties from mixed and variable food waste and by-products., Competing Interests: Declaration of Competing Interest No conflict of interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

As polyhydroxybutyrate (P(3HB)) was struggling with mechanical properties, efforts have been directed towards increasing mole fraction of 3-hydroxyhexanoate (3HHx) in P(3HB-co-3HHx) to improve the properties of polyhydroxyalkanoates (PHAs). Although genetic modification had significant results, there were several issues related to cell growth and PHA production by deletion of PHA synthetic genes. To find out easier strategy for high 3HHx mole fraction without gene deletion, Cupriavidus necator H16 containing phaC2 Ra -phaA Cn -phaJ1 Pa was examined with various oils resulting that coconut oil gave the highest 3HHx mole fraction. When fatty acid composition analysis with GC-MS was applied, coconut oil was found to have very different composition from other vegetable oil containing very high lauric acid (C12) content. To find out specific fatty acid affecting 3HHx fraction, different fatty acids from caproic acid (C6) to stearic acid (C18) was evaluated and the 3HHx mole fraction was increased to 26.5 ± 1.6 % using lauric acid. Moreover, the 3HHx mole fraction could be controlled from 9 % to 31.1 % by mixing bean oil and lauric acid with different ratios. Produced P(3HB-co-3HHx) exhibited higher molecular than P(3HB-co-3HHx) from phaB-deletion mutant. This study proposes another strategy to increase 3HHx mole fraction with easier way by modifying substrate composition without applying deletion tools., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD 600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g -x . H -1 , where the rate of PHA production was 0.28 g L -1 . H -1 and PHA concentration was 4.94 g. L -1 . This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Further it is stated that no conflict of interest is present in this research article., (Copyright © 2023. Published by Elsevier B.V.)

Pseudomonas sp. LFM693 is a 2-methylisocitrate lyase (prpB) disrupted mutant. This enzyme catalyzes a step in the 2-methylcitrate cycle, the only known and described pathway for propionate oxidation in this organism. The affected mutants can efficiently produce PHA containing even and odd-chain length hydroxyalkanoates (HA even/odd ) in the presence of propionate and glucose. In this study, a constant fed-batch configuration was utilized to control the composition of PHA and decrease the toxicity of propionate. The incorporation of HA odd into the copolymer was linear, ranging from 7 to approximately 30 %, and correlated directly with the propionate/glucose molar ratio in the feeding solution. This allowed for the molecular composition of the mclPHA to be fine-tuned with minimum process monitoring and control. The average PHA content was 52 % cell dry weight with a molar composition that favored 3-hydroxyalkanoates containing C8, C9, and C10. The conversion factor of propionate to HA odd varied between 0.36 and 0.53 mol·mol -1 (Y HAodd/prop .), which are significantly lower than the theoretical maximum efficiency (1.0 mol·mol -1 ). These results along with the lack of 2-methylisocitrate as a byproduct provides further support for the evidence that the mutant prpB - is still capable of oxidizing propionate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production., Competing Interests: Declaration of competing interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript entitled, “Enhancing polyhydroxyalkanoate production in Cupriavidus sp. L7L through wcaJ gene deletion”., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The current processes for producing polyhydroxyalkanoates (PHAs) are costly, owing to the high cost of cultivation feedstocks, and the need to sterilise the growth medium, which is energy-intensive. PHA has been identified as a promising biomaterial with a wide range of potential applications and its functionalization from waste streams has made significant advances recently, which can help foster the growth of a circular economy and waste reduction. Recent developments and novel approaches in the functionalization of PHAs derived from various waste streams offer opportunities for addressing these issues. This study focuses on the development of sustainable, efficient, and cutting-edge methods, such as advanced bioprocess engineering, novel catalysts, and advances in materials science. Chemical techniques, such as epoxidation, oxidation, and esterification, have been employed for PHA functionalization, while enzymatic and microbial methods have indicated promise. PHB/polylactic acid blends with cellulose fibers showed improved tensile strength by 24.45-32.08 % and decreased water vapor and oxygen transmission rates while PHB/Polycaprolactone blends with a 1:1 ratio demonstrated an elongation at break four to six times higher than pure PHB, without altering tensile strength or elastic modulus. Moreover, PHB films blended with both polyethylene glycol and esterified sodium alginate showed improvements in crystallinity and decreased hydrophobicity., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest related to this research paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-P tac -tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biobased materials with promising properties for environmentally friendly applications. Due to high production costs, which are related to the cost of the carbon sources combined with conversion insufficiencies, currently only small quantities are produced. This results in a lack of reliable data on properties and application potential for the variety of polymers from different types of production strains. This study investigated the potential for the production of mcl-PHA from volatile fatty acids (VFA) at a larger scale, given their potential as low-cost and sustainable raw material within a carboxylate-platform based biorefinery. Pseudomonas citronellolis (DSMZ 50332) was chosen as the production strain, and acetic acid was selected as the main carbon and energy source. Nitrogen was limited to trigger polymer production, and a fed-batch process using a pH-stat feeding regime with concentrated acid was established. We report successful production, extraction, and characterization of mcl PHA, obtaining a total of 1.76 kg from two 500-litre scale fermentations. The produced polymer was identified as a copolymer of 3-hydroxydecanoate (60.7%), 3-hydroxyoctanoate (37.3%), and 3-hydroxyhexanoate (2.0%) with a weight average molecular weight (M w ) of 536 kDa. NMR analysis indicates the presence of unsaturated side chains, which may offer additional possibilities for modification. The results confirm that there is a potential to produce significant amounts of mcl-PHA with interesting rubber-like properties from waste-derived VFA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Substituting synthetic plastics with bioplastics, primarily due to their inherent biodegradable properties, represents a highly effective strategy to address the current global issue of plastic waste accumulation in the environment. Advances in bioplastic research have led to the development of materials with improved properties, enabling their use in a wide range of applications in major commercial sectors. Bioplastics are derived from various natural sources such as plants, animals, and microorganisms. Polyhydroxyalkanoate (PHA), a biopolymer synthesized by bacteria through microbial fermentation, exhibits physicochemical and mechanical characteristics comparable to those of synthetic plastics. In response to the growing demand for these environmentally friendly plastics, researchers are actively investigating various cleaner production methods, including modification or derivatization of existing molecules for enhanced properties and new-generation applications to expand their market share in the coming decades. By 2026, the commercial manufacturing capacity of bioplastics is projected to reach 7.6 million tonnes, with Europe currently holding a significant market share of 43.5 %. Bioplastics are predominantly utilized in the packaging industry, indicating a strong focus of their application in the sector. With the anticipated rise in bioplastic waste volume over the next few decades, it is crucial to comprehend their fate in various environments to evaluate the overall environmental impact. Ensuring their complete biodegradation involves optimizing waste management strategies and appropriate disposal within these facilities. Future research efforts should prioritize exploration of their end-of-life management and toxicity assessment of degradation products. These efforts are crucial to ensure the economic viability and environmental sustainability of bioplastics as alternatives to synthetic plastics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Glucose and xylose are fermentable sugars readily available from lignocellulosic biomass, and are a sustainable carbon substrate supporting industrial biotechnology. Three strains were assessed in this work - Paraburkholderia sacchari, Hydrogenophaga pseudoflava, and Bacillus megaterium - for their ability to uptake both C 5 and C 6 sugars contained in a hardwood hydrolysate produced via a thermomechanical pulping-based process with concomitant production of poly(3-hydroxyalkanoate) (PHA) biopolymers. In batch conditions, B. megaterium showed poor growth after 12 h, minimal uptake of xylose throughout the cultivation, and accumulated a maximum of only 25 % of the dry biomass as PHA. The other strains simultaneously utilized both sugars, although glucose uptake was faster than xylose. From hardwood hydrolysate, P. sacchari accumulated 57 % of its biomass as PHA within 24 h, whereas H. pseudoflava achieved an intracellular PHA content of 84 % by 72 h. The molecular weight of the PHA synthesized by H. pseudoflava (520.2 kDa) was higher than that of P. sacchari (265.5 kDa). When the medium was supplemented with propionic acid, the latter was rapidly consumed by both strains and incorporated as 3-hydroxyvalerate subunits into the polymer, demonstrating the potential for production of polymers with improved properties and value. H. pseudoflava incorporated 3-hydroxyvalerate subunits with at least a 3-fold higher yield, and produced polymers with higher 3-hydroxyvalerate content than P. sacchari. Overall, this work has shown that H. pseudoflava can be an excellent candidate for bioconversion of lignocellulosic sugars to PHA polymers or copolymers as part of an integrated biorefinery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

The use of biodegradable plastics is constantly raising, increasing the likeliness for these polymers to end up in the environment. Environmental applications foreseeing the intentional release of biodegradable plastics have been also recently proposed, e.g., for polyhydroxyalkanoates (PHAs) acting as slow hydrogen releasing compounds to stimulate microbial reductive dehalogenation processes. However, the effects of their release into the environment on the ecosystems still need to be thoroughly explored. In this work, the use of PHAs to enhance the microbial reductive dechlorination of polychlorobiphenyls (PCBs) and their impact on the metabolic and compositional features of the resident microbial community have been investigated in laboratory microcosms of a polluted marine sediment from Mar Piccolo (Taranto, Italy), and compared with recent findings on a different contaminated marine sediment from Pialassa della Baiona (Ravenna, Italy). A decreased biostimulation efficiency of PHAs on PCBs reductive dechlorination was observed in the sediment from Mar Piccolo, with respect to the sediment from Pialassa della Baiona, suggesting that the sediments' physical-chemical characteristics and/or the biodiversity and composition of its microbial community might play a key role in determining the outcome of this biostimulation strategy. Regardless of the sediment origin, PHAs were found to have a specific and pervasive effect on the sediment microbial community, reducing its biodiversity, defining a newly arranged microbial core of primary degraders and consequently affecting, in a site-specific way, the abundance of subdominant bacteria, possibly cross-feeders. Such potential to dramatically change the structure of autochthonous microbial communities should be carefully considered, since it might have secondary effects, e.g., on the natural biogeochemical cycles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)