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Population explosion in recent years has driven the environment to overuse nondegradable substances. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are generated and retained as cytoplasmic granules in microorganisms with restricted nutritional availability and can be used to manufacture bioplastics. The current study attempts to screen soil isolates for PHA production and optimize their media parameters. Among all the isolates, 17 were identified and confirmed by Sudan black staining, as they are screening for PHA production and are identified by their colony characteristics. The isolation of the most promising strain, GS-14, was achieved through the sodium hypochlorite method, and subsequent quantification involved establishing a standard curve of crotonic acid. Notably, isolate GS-14 presented the highest yield, which was determined by extrapolating its data onto the standard curve. Characterization of the PHA polymer was subsequently performed, and the results were used to discern its properties. FTIR confirmed characteristic PHA absorption bands, with a prominent C = O stretching peak at 1732 cm⁻¹. LC-MS detected a molecular mass of 641.6 g/mol, indicative of an oligomeric species, while the actual polymer molecular weight is estimated between 5,000 and 20,000 Da. DSC revealed an exothermic peak at 174 °C, allowing the calculation of crystallinity, a key determinant of mechanical properties. Furthermore, the PHA-producing organism was identified as Bacillus australimaris through the sequencing of 16 S ribosomal RNA. The media optimization was performed via Minitab software, with statistical analyses employed to interpret the resulting data comprehensively.

The study addresses degradation of polyhydroxyalkanoates (PHA) with different chemical compositions—the polymer of 3-hydroxybutyric acid [P(3HB)] and copolymers of P(3HB) with 3-hydroxyvalerate [P(3HB/3HV)], 4-hydroxybutyrate [P(3HB/4HB)], and 3-hydroxyhexanoate [P(3HB/3HHx)] (10–12 mol%)—in the agro-transformed field soil of the temperate zone. Based on their degradation rates at 21 and 28 °C, polymers can be ranked as follows: P(3HB/4HB) > P(3HB/3HHx) > P (3HB/3HV) > P(3HB). The microbial community on the surface of the polymers differs from the microbial community of the soil with PHA specimens in the composition and percentages of species. Thirty-five isolates of bacteria of 16 genera were identified as PHA degraders by the clear zone technique, and each of the PHA had both specific and common degraders. P(3HB) was degraded by bacteria of the genera Mitsuaria, Chitinophaga, and Acidovorax, which were not among the degraders of the three other PHA types. Roseateles depolymerans, Streptomyces gardneri, and Cupriavidus sp. were specific degraders of P(3HB/4HB). Roseomonas massiliae and Delftia acidovorans degraded P(3HB/3HV), and Pseudoxanthomonas sp., Pseudomonas fluorescens, Ensifer adhaerens, and Bacillus pumilus were specific P(3HB/3HHx) degraders. All four PHA types were degraded by Streptomyces.

This study delves into the exploration of polyhydroxyalkanoate (PHA) biosynthesis genes within wild-type yeast strains, spotlighting the exceptional capabilities of isolate DMG-2. Through meticulous screening, DMG-2 emerged as a standout candidate, showcasing vivid red fluorescence indicative of prolific intracellular PHA granules. Characterization via FTIR spectroscopy unveiled a diverse biopolymer composition within DMG-2, featuring distinct functional groups associated with PHA and polyphosphate. Phylogenetic analysis placed DMG-2 within the Hanseniaspora valbyensis NRRL Y-1626 group, highlighting its distinct taxonomic classification. Subsequent investigation into DMG-2’s PHA biosynthesis genes yielded promising outcomes, with successful cloning and efficient PHA accumulation confirmed in transgenic E. coli cells. Protein analysis of ORF1 revealed its involvement in sugar metabolism, supported by its cellular localization and identification of functional motifs. Genomic analysis revealed regulatory elements within ORF1, shedding light on potential splice junctions and transcriptional networks influencing PHA synthesis pathways. Spectroscopic analysis of the biopolymer extracted from transgenic E. coli DMG2-1 provided insights into its co-polymer nature, comprising segments of PHB, PHV, and polyphosphate. GC-MS analysis further elucidated the intricate molecular architecture of the polymer. In conclusion, this study represents a pioneering endeavor in exploring PHA biosynthesis genes within yeast cells, with isolate DMG-2 demonstrating remarkable potential. The findings offer valuable insights for advancing sustainable bioplastic production and hold significant implications for biotechnological applications.

Polyhydroxyalkanoates (PHA) are bio-based microbial biopolyesters; their stiffness, elasticity, crystallinity and degradability are tunable by the monomeric composition, selection of microbial production strain, substrates, process parameters during production, and post-synthetic processing; they display biological alternatives for diverse technomers of petrochemical origin. This, together with the fact that their monomeric and oligomeric in vivo degradation products do not exert any toxic or elsewhere negative effect to living cells or tissue of humans or animals, makes them highly stimulating for various applications in the medical field. This article provides an overview of PHA application in the therapeutic, surgical and tissue engineering area, and reviews strategies to produce PHA at purity levels high enough to be used in vivo. Tested applications of differently composed PHA and advanced follow-up products as carrier materials for controlled in vivo release of anti-cancer drugs or antibiotics, as scaffolds for tissue engineering, as guidance conduits for nerve repair or as enhanced sutures, implants or meshes are discussed from both a biotechnological and a material-scientific perspective. The article also describes the use of traditional processing techniques for production of PHA-based medical devices, such as melt-spinning, melt extrusion, or solvent evaporation, and emerging processing techniques like 3D-printing, computer-aided wet-spinning, laser perforation, and electrospinning.

Microbial polyhydroxyalkanoates (PHA) are proteinaceous storage granules ranging from 100 nm to 500 nm. Bacillus sp. serve as unique bioplastic sources of short-chain length and medium-chain length PHA showcasing properties such as biodegradability, thermostability, and appreciable mechanical strength. The PHA can be enhanced by adding functional groups to make it a more industrially useful biomaterial. PHA blends with hydroxyapatite to form nanocomposites with desirable features of compressibility. The reinforced matrices result in nanocomposites that possess significantly improved mechanical and thermal properties both in solid and melt states along with enhanced gas barrier properties compared to conventional filler composites. These superior qualities extend the polymeric composites’ applications to aggressive environments where the neat polymers are likely to fail. This nanocomposite can be used in different industries as nanofillers, drug carriers for packaging essential hormones and microcapsules, etc. For fabricating a bone scaffold, electrospun nanofibrils made from biocomposite of hydroxyapatite and polyhydroxy butyrate, a form of PHA, can be incorporated with the targeted tissue. The other methods for making a polymer scaffold, includes gas foaming, lyophilization, sol–gel, and solvent casting method. In this review, PHA as a sustainable eco-friendly NextGen biomaterial from bacterial sources especially Bacillus cereus, and its application for fabricating bone scaffold using different strategies for bone regeneration have been discussed.

Background The bacterial production of polyhydroxyalkanoates (PHAs), a class of non-toxic, biodegradable, and bio-based polymers, has gained increasing attention as a sustainable alternative to petrochemical plastics. Among PHA producers, Cupriavidus necator H16 and Pseudomonas putida KT2440 are used for their ability to synthesise short- and medium-chain-length PHAs, respectively. While PHAs have been produced from simple hexoses like glucose and fructose, there remains a lack of systematic and integrated analysis linking carbon source, strain selection, monomer composition, and polymer crystallinity to blend behavior in ultrathin films. Results PHB and mcl-PHA production using Cupriavidus necator H16 and Pseudomonas putida KT2440 on glucose and fructose were compared herein. C. necator accumulated PHB up to 60 wt% on fructose and 45 wt% on glucose, with high molecular weight (0.7–1.3 MDa), while P. putida produced mcl-PHA up to 22 wt% on fructose and 18 wt% on glucose, with lower molecular weight (46–47 kDa) and a C6 – C12 monomer profile. Notably, C. necator exhibited extreme cell elongation (up to 30 μm) during PHB accumulation on fructose. Extracted polymers were systematically solvent-blended at defined ratios (100:0, 80:20, 60:40, 40:60, and 20:80 PHB:mcl-PHA) and cast into ultrathin films (~ 20 μm) with varying composition. Crystallinity was modelled using a Gaussian fitting approach on FTIR spectra via custom MATLAB code, enabling localised phase analysis and offering a rapid alternative to DSC for thin film crystallinity estimation. While film blends exhibited tunable crystallinity and multiple melting transitions, elongation at break was consistent across compositions, with increases observed at higher mcl-PHA content. Conclusions This study provides a systematic comparison of PHAs from C. necator H16 and P. putida KT2440 grown on common hexoses, with full characterisation of monomer composition, molecular weight, and thermal behaviour to guide thin film bioplastic design. Blending PHB and mcl-PHA in ultrathin films revealed reduced melting points and crystallinity, likely due to reduced crystal size from film thickness constraints. This work offers a comparative reference for microbial PHA production and presents a strategy to design bioplastics with tunable properties for temperature-responsive packaging and drug delivery applications. Graphical abstract

Adenosine-5’-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics’ impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida , respectively. We identify carbon sources (acetate for E. coli , oleate for P. putida ) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species. ATP dynamics influence bioproduction yet are largely unexplored in this context. Here, authors unravel ATP dynamics across various conditions, identify carbon sources which boost ATP levels and bioproduction, and uncover metabolic bottlenecks, shedding light on how ATP dynamics can be used to enhance bioproduction.

A homo-aromatic polyester was produced from glucose for the first time.Aromatic polyhydroxyalkanoate (PHA) was produced to the highest titer yet achieved.3D model-based rational engineering of PHA synthase was used to enhance aromatic PHA production.Polymer production was enhanced by utilizing heterologous phasins.Pilot-scale fermentative production of aromatic PHAs was successful. We report the development of a metabolically engineered bacterium for the fermentative production of polyesters containing aromatic side chains, serving as sustainable alternatives to petroleum-based plastics. A metabolic pathway was constructed in an Escherichia coli strain to produce poly[d-phenyllactate(PhLA)], followed by three strategies to enhance polymer production. First, polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were introduced to increase the polymer accumulation. Next, metabolic engineering was performed to redirect the metabolic flux toward PhLA. Furthermore, PHA synthase was engineered based on in silico simulation results to enhance the polymerization of PhLA. The final strain was capable of producing 12.3 g/l of poly(PhLA), marking it the first bio-based process for producing an aromatic homopolyester. Additional heterologous gene introductions led to the high level production of poly(3-hydroxybutyrate-co-11.7 mol% PhLA) copolymer (61.4 g/l). The strategies described here will be useful for the bio-based production of aromatic polyesters from renewable resources. Graphical abstract [Display omitted] The production of polyhydroxyalkanoates (PHAs) containing aromatic repeating units has been studied for decades, but has yet to be demonstrated in large-scale fermentation. This study reports the successful production of aromatic polyesters from glucose in high titers that was performed both in lab-scale (5 l) and pilot-scale (30 l) fermenters. Therefore, the current Technology Readiness Level (TRL) of this technology lies between 4 and 5. To advance this technology for industrialization, demonstration in industry-scale fermenters and the development of large-scale downstream processes will be necessary. Nevertheless, given the ongoing development of various fermentation technologies and purification processes for other PHAs, we anticipate that the industrialization of aromatic PHAs is feasible, potentially replacing petroleum-based aromatic polymers for use as commodity, engineering, and biomedical plastics. Lee et al. report the fermentative production of aromatic polyesters, which can serve as sustainable alternatives to petroleum-based plastics. This represents the first instance of homo-aromatic polyester production using glucose. Further pathway engineering facilitated the production of poly(3-hydroxybutyrate-co-d-phenyllactate), achieving the highest titer of aromatic polyester reported using microorganisms thus far.

The production of biodegradable and biobased polymers is one way to overcome the present plastic pollution while using cheap and abundant feedstocks. Polyhydroxyalkanoates are a promising class of biopolymers that can be produced by various microorganisms. Within the production process, batch-to-batch variation occurs due to changing feedstock composition when using waste streams, slightly different starting conditions, or biological variance of the microorganisms. Therefore, reliable and online-capable measurement methods of the product concentration are required to monitor and eventually react to those fluctuations. In this work, we present a flexible approach to determine polyhydroxyalkanoate concentrations based on a simple mathematical model. The data-driven model correlates polyhydroxyalkanoate concentrations with optical densities measured at 600 nm, a widespread, fast, and cheap lab measurement. We found that with different cultivation conditions, the correlation needs to be updated for a reasonable PHA determination during the process. We test this approach for the production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Cupriavidus necator using fructose and propionic acid as carbon sources. This flexible approach allows a simple estimation of PHA concentrations and offers the possibility to further enhance biopolymer production when combined with advanced control strategies. Key points ∙ Development of a simple mathematical model to estimate polyhydroxyalkanoate concentrations. ∙ Optical density measurement is used to determine polyhydroxyalkanoate concentration. ∙ The approach is tested for the production of PHB and PHBV with C. necator.