Biodegradation of PHB/PBAT films and isolation of novel PBAT biodegraders from soil microbiomes.

Polyhydroxyalkanoates (PHAs) are important candidates for replacing petroleum-based plastics. This transition is urgent for the development of a biobased economy and to protect human health and natural ecosystems. PHAs are biobased and biodegradable polyesters that when blended with other polymers, such as poly(butylene adipate-co-terephthalate) (PBAT), acquire remarkable improvements in their properties, which allow them to comply with the requirements of packaging applications. However, the biodegradation of such blends should be tested to evaluate the impact of those polymers in the environment. For instance, PBAT is a compostable aliphatic-aromatic copolyester, and its biodegradation in natural environments, such as soil, is poorly studied. In this work, we evaluated the biodegradation of a bilayer film composed of PHB and PBAT, by a soil microbiome. The bilayer film reached 47 ± 1 % mineralization in 180 days and PHB was no longer detected after this period. The increased crystallinity of the PBAT residue was a clear sign of biodegradation, indicating that the amorphous regions were preferentially biodegraded. Seven microorganisms were isolated, from which 4 were closely related to microorganisms already known as PHB degraders, but the other 3 species, closely related to Streptomyces coelicoflavus, Clonostachys rosea and Aspergillus insuetus, were found for the first time as PHB degraders. Most remarkably, two fungi closely related to Purpureocillium lilacinum and Aspergillus pseudodeflectus (99.83 % and 100 % identity by ITS sequencing) were isolated and identified as PBAT degraders. This is very interesting due to the rarity of isolating PBAT-degrading microorganisms. These results show that the bilayer film can be biodegraded in soil, at mesophilic temperatures, showing its potential to replace synthetic plastics in food packaging.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Evaluation of the effects of decellularized extracellular matrix nanoparticles incorporation on the polyhydroxybutyrate/nano chitosan electrospun scaffold for cartilage tissue engineering.

Recent research focuses on fabricating scaffolds imitating the extracellular matrix (ECM) in texture, composition, and functionality. Moreover, specific nano-bio-particles can enhance cell differentiation. Decellularized ECM nanoparticles possess all of the mentioned properties. In this research, cartilage ECM, extracted from the cow's femur condyle, was decellularized, and ECM nanoparticles were synthesized. Finally, nanocomposite electrospun fibers containing polyhydroxybutyrate (PHB), chitosan (Cs) nanoparticles, and ECM nanoparticles were fabricated and characterized. TEM and DLS results revealed ECM nanoparticle sizes of 17.51 and 21.6 nm, respectively. Optimal performance was observed in the scaffold with 0.75 wt% ECM nanoparticles (PHB-Cs/0.75E). By adding 0.75 wt% ECM, the ultimate tensile strength and elongation at break increased by about 29 % and 21 %, respectively, while the water contact angle and crystallinity decreased by about 36° and 2 %, respectively. Uneven and rougher surfaces of the PHB-Cs/0.75E were determined by FESEM and AFM images, respectively. TEM images verified the uniform dispersion of nanoparticles within the fibers. After 70 days of degradation in PBS, the PHB-Cs/0.75E and PHB-Cs scaffolds demonstrated insignificant weight loss differences. Eventually, enhanced viability, attachment, and proliferation of the human costal chondrocytes on the PHB-Cs/0.75E scaffold, concluded from MTT, SEM, and DAPI staining, confirmed its potential for cartilage tissue engineering.

Stability and release mechanism of double emulsification (W1/O/W2) for biodegradable pH-responsive polyhydroxybutyrate/cellulose acetate phthalate microbeads loaded with the water-soluble bioactive compound niacinamide.

Microbeads of biodegradable polyhydroxybutyrate (PHB) offer environmental benefits and economic competitiveness. The aim of this study was to encapsulate a water-soluble bioactive compound, niacinamide (NIA), in a pH-responsive natural matrix composed of PHB and cellulose acetate phthalate (CAP) by double emulsification (W1/O/W2) to improve the encapsulation efficiency (%EE) and loading capacity (%LC). PHB was produced in-house by Escherichia coli JM109 pUC19-23119phaCABA-04 without the inducing agent isopropyl ß-D-1-thiogalactopyranoside (IPTG). The influences of PHB and polyvinyl alcohol (PVA) concentrations, stirring rate, PHB/CAP ratio and initial NIA concentration on the properties of NIA-loaded pH-responsive microbeads were studied. The NIA-loaded pH-responsive PHB/CAP microbeads exhibited a spherical core-shell structure. The average size of the NIA-loaded pH-responsive microbeads was 1243.3 ± 11.5 µm. The EE and LC were 33.3 ± 0.5 % and 28.5 ± 0.4 %, respectively. The release profiles of NIA showed pH-responsive properties, as 94.2 ± 3.5 % of NIA was released at pH 5.5, whereas 99.3 ± 2.4 % of NIA was released at pH 7.0. The NIA-loaded pH-responsive PHB/CAP microbeads were stable for >90 days at 4 °C under darkness, with NIA remaining at 73.65 ± 1.86 %. A cytotoxicity assay in PSVK1 cells confirmed that the NIA-loaded pH-responsive PHB/CAP microbeads were nontoxic at concentrations lower than 31.3 µg/mL, in accordance with ISO 10993-5.

A new strategy for the treatment of middle ear infection using ciprofloxacin/amoxicillin-loaded ethyl cellulose/polyhydroxybutyrate nanofibers.

A middle ear infection occurs due to the presence of several microorganisms behind the eardrum (tympanic membrane) and is very challenging to treat due to its unique location and requires a well-designed treatment. If not treated properly, the infection can result in severe symptoms and unavoidable side effects. In this study, excellent biocompatible ethyl cellulose (EC) and biodegradable polyhydroxybutyrate (PHB) biopolymer were used to fabricate drug-loaded nanofiber scaffolds using an electrospinning technique to overcome antibiotic overdose and insufficient efficacy of drug release during treatment. PHB polymer was produced from Halomonas sp., and the purity of PHB was found to around be 90 %. Additionally, ciprofloxacin (CIP) and amoxicillin (AMX) are highly preferable since both drugs are highly effective against gram-negative and gram-positive bacteria to treat several infections. Obtained smooth nanofibers were between 116.24 and 171.82 nm in diameter and the addition of PHB polymer and antibiotics improved the morphology of the nanofiber scaffolds. Thermal properties of the nanofiber scaffolds were tested and the highest Tg temperature resulted at 229 °C. The mechanical properties of the scaffolds were tested, and the highest tensile strength resulted in 4.65 ± 6.33 MPa. Also, drug-loaded scaffolds were treated against the most common microorganisms that cause the infection, such as S.aureus, E.coli, and P.aeruginosa, and resulted in inhibition zones between 10 and 21 mm. MTT assay was performed by culturing human adipose-derived mesenchymal stem cells (hAD MSCs) on the scaffolds. The morphology of the hAD MSCs' attachment was tested with SEM analysis and hAD MSCs were able to attach, spread, and live on each scaffold even on the day of 7. The cumulative drug release kinetics of CIP and AMX from drug-loaded scaffolds were analysed in phosphate-buffered saline (pH: 7.4) within different time intervals of up to 14 days using a UV spectrophotometer. Furthermore, the drug release showed that the First-Order and Korsmeyer-Peppas models were the most suitable kinetic models. Animal testing was performed on SD rats, matrix and collagen deposition occurred on days 5 and 10, which were observed using Hematoxylin-eosin and Masson's trichrome staining. At the highest drug concentration, a better repair effect was observed. Results were promising and showed potential for novel treatment.

Deciphering heterogeneous enzymatic surface reactions on xylan using surface plasmon resonance spectroscopy.

Xylans' unique properties make it attractive for a variety of industries, including paper, food, and biochemical production. While for some applications the preservation of its natural structure is crucial, for others the degradation into monosaccharides is essential. For the complete breakdown, the use of several enzymes is required, due to its structural complexity. In fact, the specificity of enzymatically-catalyzed reactions is guided by the surface, limiting or regulating accessibility and serving structurally encoded input guiding the actions of the enzymes. Here, we investigate enzymes at surfaces rich in xylan using surface plasmon resonance spectroscopy. The influence of diffusion and changes in substrate morphology is studied via enzyme surface kinetics simulations, yielding reaction rates and constants. We propose kinetic models, which can be applied to the degradation of multilayer biopolymer films. The most advanced model was verified by its successful application to the degradation of a thin film of polyhydroxybutyrate treated with a polyhydroxybutyrate-depolymerase. The herein derived models can be employed to quantify the degradation kinetics of various enzymes on biopolymers in heterogeneous environments, often prevalent in industrial processes. The identification of key factors influencing reaction rates such as inhibition will contribute to the quantification of intricate dynamics in complex systems.

Alkaline-Tolerant Bacillus cereus 12GS: A Promising Polyhydroxybutyrate (PHB) Producer Isolated from the North of Mexico.

Environmental pollution caused by petroleum-derived plastics continues to increase annually. Consequently, current research is interested in the search for eco-friendly bacterial polymers. The importance of Bacillus bacteria as producers of polyhydroxyalkanoates (PHAs) has been recognized because of their physiological and genetic qualities. In this study, twenty strains of Bacillus genus PHA producers were isolated. Production was initially evaluated qualitatively to screen the strains, and subsequently, the strain B12 or Bacillus sp. 12GS, with the highest production, was selected through liquid fermentation. Biochemical and molecular identification revealed it as a novel isolate of Bacillus cereus. Production optimization was carried out using the Taguchi methodology, determining the optimal parameters as 30 °C, pH 8, 150 rpm, and 4% inoculum, resulting in 87% and 1.91 g/L of polyhydroxybutyrate (PHB). Kinetic studies demonstrated a higher production within 48 h. The produced biopolymer was analyzed using Fourier-transform infrared spectroscopy (FTIR), confirming the production of short-chain-length (scl) polyhydroxyalkanoate, named PHB, and differential scanning calorimetry (DSC) analysis revealed thermal properties, making it a promising material for various applications. The novel B. cereus isolate exhibited a high %PHB, emphasizing the importance of bioprospecting, study, and characterization for strains with biotechnological potential.

Effects of methane to oxygen ratio on cell growth and polyhydroxybutyrate synthesis in high cell density cultivation of Methylocystis sp. MJC1.

Polyhydroxybutyrate (PHB) production through CH4 conversion by methanotrophs offers a solution for greenhouse gas emissions and plastic waste concerns. In this study, we aimed to achieve high cell density cultivation of Methylocystis sp. MJC1 for efficient PHB production. Cultivating MJC1 using CH4 and air (3:7, v/v) yielded a final cell density of 52.9 g/L with a 53.7% (28.4 g/L) PHB content after 210 h, showcasing PHB mass production potential. However, long-term cultivation led to a low volumetric productivity of 0.200 g/L/h. To address this, we conducted cultivation at various O2/CH4 ratios using O2 instead of air, which significantly improved the PHB productivity. Under high O2 conditions (O2/CH4 ratio of 1.5), biomass productivity increased 1.51-fold compared to that under low O2 conditions in the same time frame; however, PHB accumulation was delayed. Using an equal ratio of CH4 and O2 induced active cell growth and selective PHB production, achieving the highest PHB productivity (0.365 g/L/h) with a final cell density of 55.9 g/L and PHB content of 61.7% (34.5 g/L) in 162 h. This study highlighted the significance of the O2/CH4 ratio in CH4 conversion and PHB production by M. sp. MJC1.

Preparation and Preliminary Analysis of Several Nanoformulations Based on Plant Extracts and Biodegradable Polymers as a Possible Application for Chronic Venous Disease Therapy.

Nanotechnology is one of the newest directions for plant-based therapies. Chronic venous disease often predisposes to long-term and invasive treatment. This research focused on the inclusion of vegetal extracts from Sophorae flos (SE), Calendulae flos (CE), and Ginkgo bilobae folium (GE) in formulations with PHB and PLGA polymers and their physicochemical characterization as a preliminary stage for possible use in the development of a complex therapeutic product. The samples were prepared by an oil-water emulsification and solvent evaporation technique, resulting in suspensions with high spreadability and a pH of 5.5. ATR-FTIR analysis revealed bands for stretching vibrations (O-H, C=O, and C-H in symmetric and asymmetric methyl and methylene) in the same regions as the base components, but switched to high or low wavenumbers and absorbance, highlighting the formation of adducts/complexes between the extracts and polymers. The obtained formulations were in the amorphous phase, as confirmed by XRD analysis. AFM analysis emphasized the morphological peculiarities of the extract-polymer nanoformulations. It could be noticed that, in the case of SE-based formulations, the dominant characteristics for SE-PHB and SE-PLGA composition were the formation of random large (SE-PHB) and smaller uniform (SE-PLGA) particles; further on, these particles tended to aggregate in the case of SE-PHB-PLGA. For the CE- and GE-based formulations, the dominant surface morphology was their porosity, generally with small pores, but larger cavities were observed in some cases (CE- and GE-PHB). The highest roughness values at the (8 µm × 8 µm) scale were found for the following samples and succession: CE-PHB < SE-PLGA < SE-PHB-PLGA. In addition, by thermogravimetric analysis, impregnation in the matrix of compression stockings was evaluated, which varied in the following order: CE-polymer > SE-polymer > GE-polymer. In conclusion, nine vegetal extract-polymer nanoformulations were prepared and preliminarily characterized (by advanced physicochemical methods) as a starting point for further optimization, stability studies, and possible use in complex pharmaceutical products.

Methylosinus trichosporium OB3b bioaugmentation unleashes polyhydroxybutyrate-accumulating potential in waste-activated sludge.

BACKGROUND: Wastewater treatment plants contribute approximately 6% of anthropogenic methane emissions. Methanotrophs, capable of converting methane into polyhydroxybutyrate (PHB), offer a promising solution for utilizing methane as a carbon source, using activated sludge as a seed culture for PHB production. However, maintaining and enriching PHB-accumulating methanotrophic communities poses challenges. RESULTS: This study investigated the potential of Methylosinus trichosporium OB3b to bioaugment PHB-accumulating methanotrophic consortium within activated sludge to enhance PHB production. Waste-activated sludges with varying ratios of M. trichosporium OB3b (1:0, 1:1, 1:4, and 0:1) were cultivated. The results revealed substantial growth and methane consumption in waste-activated sludge with M. trichosporium OB3b-amended cultures, particularly in a 1:1 ratio. Enhanced PHB accumulation, reaching 37.1% in the same ratio culture, indicates the dominance of Type II methanotrophs. Quantification of methanotrophs by digital polymerase chain reaction showed gradual increases in Type II methanotrophs, correlating with increased PHB production. However, while initial bioaugmentation of M. trichosporium OB3b was observed, its presence decreased in subsequent cycles, indicating the dominance of other Type II methanotrophs. Microbial community analysis highlighted the successful enrichment of Type II methanotrophs-dominated cultures due to the addition of M. trichosporium OB3b, outcompeting Type I methanotrophs. Methylocystis and Methylophilus spp. were the most abundant in M. trichosporium OB3b-amended cultures. CONCLUSIONS: Bioaugmentation strategies, leveraging M. trichosporium OB3b could significantly enhance PHB production and foster the enrichment of PHB-accumulating methanotrophs in activated sludge. These findings contribute to integrating PHB production in wastewater treatment plants, providing a sustainable solution for resource recovery.

Oceanimonas pelagia sp. nov., a novel biosurfactant-producing and plastic-degrading potential bacterium isolated from marine coastal sediment.

A marine bacterial strain, named NTOU-MSR1T, was isolated from marine sediment of northern coast of Taiwan. This bacterium was Gram-stain-negative, aerobic, and motile, with a single flagellum. Its rod-shaped cells measured approximately 0.5-0.6 µm in width and 1.8-2.0 µm in length. NTOU-MSR1T grew at temperatures ranging from 10 to 45 °C, optimally at 30 °C. The pH range for growth was 7.0-10.0, with optimal growth at pH 7.0-8.0. It tolerated NaCl concentrations up to 12%. The cell membrane predominantly contained fatty acids such C16:1ω7c, C18:1ω7c, and C16:0. The overall genome relatedness indices indicated that strain NTOU-MSR1T had an average nucleotide identity (ANI) of 87.88% and a digital DNA-DNA hybridization (dDDH) value of 35.40% compared to its closest related species, O. marisflavi 102-Na3T. These values fell below the 95% and 70% threshold for species delineation, respectively. These findings suggested that the strain NTOU-MSR1T was a new member of the Oceanimonas genus. Its genomic DNA had a G + C content of 61.0 mol%. Genomic analysis revealed genes associated with the catechol branch of ß- ketoadipate pathway for degrading polycyclic aromatic hydrocarbons, resistance to heavy metal, biosynthesis of polyhydroxybutyrate and the production of glycoside hydrolases (GH19, GH23, and GH103) for chitin and glycan digestion. Additionally, NTOU-MSR1T was capable of synthesizing biosurfactants and potentially degrading plastic. The proposed name for this new species is Oceanimonas pelagia, with the type strain designated as NTOU-MSR1T (= BCRC 81403T = JCM 36023T).

A dynamic model of growth phase of bio-conversion of methane to polyhydroxybutyrate using dynamic flux balance analysis.

Biological conversion of waste methane to biodegradable plastics is a way of reducing their production cost. This study addresses the computational modeling of the growth phase reactor of the process of polyhydroxybutyrate production. The model was used for investigating the effect of gas recycling and inlet gas retention time on the reactor performance. The model was run by the use of a genome-scale metabolic network of Methylocystis hirsuta in a dynamic flux balance analysis framework. The reactor has been modeled for two separate feeding scenarios: a pure methane feed and a biogas feed. The mass transfer coefficient parameter was predicted as a function of superficial gas velocities by the regression of data from published experiments. The results show an increase of removal efficiency by 38% and biomass concentration by 2.8 g/L with the increase of gas recycle ratio from 0 to 30 at the empty bed residence time of 60  min .

The production, recovery, and valorization of polyhydroxybutyrate (PHB) based on circular bioeconomy.

As an energy-storage substance of microorganisms, polyhydroxybutyrate (PHB) is a promising alternative to petrochemical polymers. Under appropriate fermentation conditions, PHB-producing strains with metabolic diversity can efficiently synthesize PHB using various carbon sources. Carbon-rich wastes may serve as alternatives to pure sugar substrates to reduce the cost of PHB production. Genetic engineering strategies can further improve the efficiency of substrate assimilation and PHB synthesis. In the downstream link, PHB recycling strategies based on green chemistry concepts can replace PHB extraction using chlorinated solvents to enhance the economics of PHB production and reduce the potential risks of environmental pollution and health damage. To avoid carbon loss caused by biodegradation in the traditional sense, various strategies have been developed to degrade PHB waste into monomers. These monomers can serve as platform chemicals to synthesize other functional compounds or as substrates for PHB reproduction. The sustainable potential and cycling value of PHB are thus reflected. This review summarized the recent progress of strains, substrates, and fermentation approaches for microbial PHB production. Analyses of available strategies for sustainable PHB recycling were also included. Furthermore, it discussed feasible pathways for PHB waste valorization. These contents may provide insights for constructing PHB-based comprehensive biorefinery systems.

Polyhydroxybutyrate-based osteoinductive mineralized electrospun structures that mimic components and tissue interfaces of the osteon for bone tissue engineering.

Scaffolds for bone tissue engineering should enable regeneration of bone tissues with its native hierarchically organized extracellular matrix (ECM) and multiple tissue interfaces. To achieve this, inspired by the structure and properties of bone osteon, we fabricated polyhydroxybutyrate (PHB)-based mineralized electrospun fibrous scaffolds. After studying multiple PHB-based fibers, we chose 7%PHB/1%Gelatin fibers (PG) to fabricate mineralized fibers that mimic mineralized collagen fibers in bone. The mineralized PG (mPG) surface had a rough, hydrophilic layer of low crystalline calcium phosphate which was biocompatible to bone marrow stromal cells (BMSCs), induced their proliferation and was osteoinductive. Subsequently, by modulating the electrospinning process, we fabricated mPG-based novel higher order fibrous scaffolds that mimic the macroscale geometries of osteons of bone ECM. Inspired by the aligned collagen fibers in bone lamellae, we fabricated mPG scaffolds with aligned fibers that could direct anisotropic elongation of mouse BMSC (mBMSCs). Further, we fabricated electrospun mPG-based osteoinductive tubular constructs which can mimic cylindrical bone components like osteons or lamellae or be used as long bone analogues based on their dimensions. Finally, to regenerate tissue interfaces in bone, we introduced a novel bi-layered scaffold-based approach. An electrospun bi-layered tubular construct that had PG in the outer layer and 7%PHB/0.5%Polypyrrole fibers (PPy) in the inner layer was fabricated. The bi-layered tubular construct underwent preferential surface mineralization only on its outer layer. This outer mineralized layer supported osteogenesis while the inner PPy layer could support neural cell growth. Thus, the bi-layered tubular construct may be used to regenerate haversian canal in the osteons which hosts nerve fibers. Overall, the study introduced novel techniques to fabricate biomimetic structures that can regenerate components of bone osteon and its multiple tissue interfaces. The study lays foundation for the fabrication of a modular scaffold that can regenerate bone with its hierarchical structure and complex tissue interfaces.

Cellulose/Polyhydroxybutyrate (PHB) Composites as a Sustainable Bio-Based Feedstock to 3D-Printing Applications.

In this work, we have studied the potential application for 3D-printing of a polymer made from combining a biodegradable and biocompatible polymer (i.e., polyhydroxybutyrate, PHB) with natural bio-based fiber (i.e., cellulose). To this end, a masterbatch at 15 wt.% in filler content was prepared by melt-blending, and then this system was "diluted" with pure PHB in a second extrusion phase in order to produce filaments at 1.5 and 3 wt.% of cellulose. For comparison, a filament made of 100% virgin PHB pellets was prepared under the same conditions. All the systems were then processed in the 3D-printer apparatus, and specimens were mainly characterized by static (tensile and flexural testing) and dynamic mechanical analysis. Thermogravimetric analysis, differential scanning calorimetry, spectroscopic measurements, and morphological aspects of PHB polymer and composites were also discussed. The results showed a significant negative impact of the process on the mechanical properties of the basic PHB with a reduction in both tensile and flexural mechanical properties. The PHB-cellulose composites showed a good dispersion filler in the matrix but a poor interfacial adhesion between the two phases. Furthermore, the cellulose had no effect on the melting behavior and the crystallinity of the polymer. The addition of cellulose improved the thermal stability of the polymer and minimized the negative impact of extrusion. The mechanical performance of the composites was found to be higher compared to the corresponding (processed) polymer.

Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Intranasal Epitope-Polymer Vaccine Lodges Resident Memory T Cells Protecting Against Influenza Virus.

Intranasal vaccines, unlike injectable vaccines, boost immunity along the respiratory tract; this can significantly limit respiratory virus replication and shedding. There remains a need to develop mucosal adjuvants and vaccine delivery systems that are both safe and effective following intranasal administration. Here, biopolymer particles (BP) densely coated with repeats of MHC class I restricted immunodominant epitopes derived from influenza A virus namely NP366, a nucleoprotein-derived epitope and PA224, a polymerase acidic subunit derived epitope, are bioengineered. These BP-NP366/PA224 can be manufactured at a high yield and are obtained at ≈93% purity, exhibiting ambient-temperature stability. Immunological characterization includes comparing systemic and mucosal immune responses mounted following intramuscular or intranasal immunization. Immunization with BP-NP366/PA224 without adjuvant triggers influenza-specific CD8+ T cell priming and memory CD8+ T cell development. Co-delivery with the adjuvant poly(I:C) significantly boosts the size and functionality of the influenza-specific pulmonary resident memory CD8+ T cell pool. Intranasal, but not intramuscular delivery of BP-NP366/PA224 with poly(I:C), provides protection against influenza virus challenge. Overall, the BP approach demonstrates as a suitable antigen formulation for intranasal delivery toward induction of systemic protective T cell responses against influenza virus.

Production and characterization of polyhydroxybutyrate bioplastic precursor from Parageobacillus toebii using low-cost substrates and its potential antiviral activity.

There is an increasing desire for bioplastics produced from renewable resources as an alternative to their petrochemical counterparts. These biopolymers have long-unnoticed antiviral properties. This study aimed to produce and characterize bioplastics by Parageobacillus toebii using low-cost substrates and determine their antiviral activity against coxsackievirus B4. Seven low-cost substrates (bagasse, water hyacinth, rice straw, rice water, sesame husks, molasses, and corn syrup) were compared with glucose for bioplastic precursor production. The highest bioplastic produced was from water hyacinth and glucose, followed by molasses, rice straw, rice water, sesame husks, and bagasse. Water hyacinth and glucose media were further optimized to increase the bioplastic precursor yield. The optimization of the media leads to increases in bioplastic precursor yields of 1.8-fold (3.456 g/L) and 1.496-fold (2.768 g/L), respectively. These bioplastics were further characterized by thermogravimetric analysis (TGA), Fourier-transformed infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H NMR), and gas chromatography-mass spectrometry (GC-MS). They are thermostable, and their characterizations confirm the presence of polyhydroxybutyrate. The antiviral assay showed reasonable antiviral effects for bioplastics from water hyacinth (80.33 %) and glucose (55.47 %) media at 250 µg/mL maximum non-toxic concentrations (MNTC). The present investigation demonstrates a low-cost model for producing polyhydroxybutyrate bioplastic precursor for antiviral applications.

Genetic characterization of a novel Salinicola salarius isolate applied for the bioconversion of agro-industrial wastes into polyhydroxybutyrate.

BACKGROUND: Polyhydroxybutyrate (PHB) has emerged as a promising eco-friendly alternative to traditional petrochemical-based plastics. In the present study, we isolated and characterized a new strain of Salinicola salarius, a halophilic bacterium, from the New Suez Canal in Egypt and characterized exclusively as a potential PHB producer. Further genome analysis of the isolated strain, ES021, was conducted to identify and elucidate the genes involved in PHB production. RESULTS: Different PHB-producing marine bacteria were isolated from the New Suez Canal and characterized as PHB producers. Among the 17 bacterial isolates, Salinicola salarius ES021 strain showed the capability to accumulate the highest amount of PHB. Whole genome analysis was implemented to identify the PHB-related genes in Salinicola salarius ES021 strain. Putative genes were identified that can function as phaCAB genes to produce PHB in this strain. These genes include fadA, fabG, and P3W43_16340 (encoding acyl-CoA thioesterase II) for PHB production from glucose. Additionally, phaJ and fadB were identified as key genes involved in PHB production from fatty acids. Optimization of environmental factors such as shaking rate and incubation temperature, resulted in the highest PHB productivity when growing Salinicola salarius ES021 strain at 30°C on a shaker incubator (110 rpm) for 48 h. To maximize PHB production economically, different raw materials i.e., salted whey and sugarcane molasses were examined as cost-effective carbon sources. The PHB productivity increased two-fold (13.34 g/L) when using molasses (5% sucrose) as a fermentation media. This molasses medium was used to upscale PHB production in a 20 L stirred-tank bioreactor yielding a biomass of 25.12 g/L, and PHB of 12.88 g/L. Furthermore, the produced polymer was confirmed as PHB using Fourier-transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC-MS), and nuclear magnetic resonance spectroscopy (NMR) analyses. CONCLUSIONS: Herein, Salinicola salarius ES021 strain was demonstrated as a robust natural producer of PHB from agro-industrial wastes. The detailed genome characterization of the ES021 strain presented in this study identifies potential PHB-related genes. However, further metabolic engineering is warranted to confirm the gene networks required for PHB production in this strain. Overall, this study contributes to the development of sustainable and cost-effective PHB production strategies.

Physicochemical characterization of polyhydroxybutyrate (PHB) produced by the rare halophile Brachybacterium paraconglomeratum MTCC 13074.

BACKGROUND: Polyhydroxybutyrate is a biopolymer produced by bacteria and archaea under nitrogen-limiting conditions. PHB is an essential polymer in the bioplastic sector because of its biodegradability, eco-friendliness, and adaptability. The characterization of PHB is a multifaceted process for studying the structure and its properties. This entire aspect can assure the long-term viability and performance attributes of the PHB. The characteristics of PHB extracted from the halophile Brachybacterium paraconglomeratum were investigated with the objective of making films for application in healthcare. RESULTS: This was the first characterization study on PHB produced by a rare halophile, Brachybacterium paraconglomeratum (MTCC 13074). In this study, the strain produced 2.72 g/l of PHB for.5.1 g/l of biomass under optimal conditions. Methods are described for the determination of the physicochemical properties of PHB. The prominent functional groups CH3 and C = O were observed by FT-IR and the actual chemical structure of the PHB was deduced by NMR. GCMS detects the confirmation of four methyl ester derivatives of the extracted PHB in the sample. Mass spectrometry revealed the molecular weight of methyl 3-hydroxybutyric acid (3HB) present in the extract. The air-dried PHB films were exposed to TGA, DSC and a universal testing machine to determine the thermal profile and mechanical stability. Additionally, the essential property of biopolymers like viscosity was also assessed for the extracted PHB. CONCLUSIONS: The current study demonstrated the consistency and quality of B. paraconglomeratum PHB. Therefore, Brachybacterium sps are also a considerable source of PHB with desired characteristics for industrial production.