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Lignocellulose is the only feasible carbohydrates feedstock for commercial scale and carbon neutral production of poly(3-hydroxybutyrate) (PHB) biopolymer by its great abundance and availability. Microbial cell factories for fermentative PHB synthesis are highly restricted by the growth suppression of inhibitors from lignocellulose pretreatment. This study targeted a potential PHB-producing cell factory Corynebacterium glutamicum owing to its strong inhibitors tolerance. A systematic metabolic engineering was conducted starting with the stable PHB synthesis pathway construction from glucose and xylose, followed by the enhancement of PHB synthesis on PHA synthase activity and stability, cell morphology modification, and growth factors regulation. The relocation of the PHA synthase on the cell membrane guided by secrete signal peptides and cell membrane display motifs increased the PHB content by 2.4 folds. Excessive nitrogen preferentially promoted the PHB synthesis capacity and resulted in the PHB content increased by 13.3 folds. Modification of the genes responsible for cell division changed the cell morphology but the cell size was not enlarged to a PHB accumulation favorable environment. The metabolic engineering of C. glutamicum resulted in a high fermentative production of PHB using wheat straw as feedstock. This study provided an important microbial cell factory choice for PHB production using lignocellulose feedstock.
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Corynebacterium glutamicum , Ácido 3-Hidroxibutírico/metabolismo , Biomasa , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Hidroxibutiratos/metabolismo , Lignina , Ingeniería Metabólica , Poliésteres/metabolismoRESUMEN
Poly(3-hydroxybutyrate) (PHB) is a microbially produced biopolymer that is emerging as a propitious alternative to petroleum-based plastics owing to its biodegradable and biocompatible properties. However, to date, the relatively high costs related to the PHB production process are hampering its widespread commercialization. Since feedstock costs add up to half of the total production costs, ample research has been focusing on the use of inexpensive industrial side streams as carbon sources. While various industrial side streams such as second-generation carbohydrates, lignocellulose, lipids, and glycerol have been extensively investigated in liquid fermentation processes, also gaseous sources, including carbon dioxide, carbon monoxide, and methane, are gaining attention as substrates for gas fermentation. In addition, recent studies have investigated two-stage processes to convert waste gases into PHB via organic acids or alcohols. In this review, a variety of different industrial side streams are discussed as more sustainable and economical carbon sources for microbial PHB production. In particular, a comprehensive overview of recent developments and remaining challenges in fermentation strategies using these feedstocks is provided, considering technical, environmental, and economic aspects to shed light on their industrial feasibility. As such, this review aims to contribute to the global shift towards a zero-waste bio-economy and more sustainable materials.
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Glicerol , Petróleo , Ácido 3-Hidroxibutírico , Ríos , Monóxido de Carbono , Dióxido de Carbono , Biopolímeros , Plásticos , MetanoRESUMEN
BACKGROUND: Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin proteins (PhaPs) stabilizing the PHB granules. The transcription factor PhaR controls the phaP genes. RESULTS: Inactivation of phaR led to decreases in PHB accumulation, less cell yield, increases in exopolysaccharide (EPS) production, some improvement in heat stress tolerance, and slightly better growth under microaerobic conditions. Changes in the transcriptome upon phaR inactivation were analyzed. PhaR appeared to be involved in the repression of various target genes, including some PHB-degrading enzymes and others involved in EPS production. Furthermore, in vitro gel shift analysis demonstrated that PhaR bound to the promoter regions of representative targets. For the phaP1 and phaP4 promoter regions, PhaR-binding sites were determined by DNase I footprinting, allowing us to deduce a consensus sequence for PhaR-binding as TGCRNYGCASMA (R: A or G, Y: C or T, S: C or G, M: A or C). We searched for additional genes associated with a PhaR-binding sequence and found that some genes involved in central carbon metabolism, such as pdhA for pyruvate dehydrogenase and pckA for phosphoenolpyruvate carboxykinase, may be regulated positively and directly by PhaR. CONCLUSIONS: These results suggest that PhaR could regulate various genes not only negatively but also positively to coordinate metabolism holistically in response to PHB accumulation.
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Proteínas Bacterianas/genética , Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Regulación Bacteriana de la Expresión Génica , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Sitios de Unión , Carbono/metabolismo , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
Cationic cyclopentadienyliron (CpFeâº) is one of the most fruitful organometallic moieties that has been utilized to mediate the facile synthesis of a massive number of macromolecules. However, the ability of this compound to function as a nucleating agent to improve other macromolecule properties has not been explored. This report scrutinizes the influence of the cationic complex as a novel nucleating agent on the spherulitic morphology, crystal structure, and isothermal and non-isothermal crystallization behavior of the Poly(3-hydroxybutyrate) (PHB) bacterial origin. The incorporation of the CpFe⺠into the PHB materials caused a significant increase in its spherulitic numbers with a remarkable reduction in the spherulitic sizes. Unlike other nucleating agents, the SEM imageries exhibited a good dispersion without forming agglomerates of the CpFe⺠moieties in the PHB matrix. Moreover, according to the FTIR analysis, the cationic organoiron complex has a strong interaction with the PHB polymeric chains via the coordination with its ester carbonyl. Yet, the XRD results revealed that this incorporation had no significant effect on the PHB crystalline structure. Though the CpFe⺠had no effect on the polymer's crystal structure, it accelerated outstandingly the melt crystallization of the PHB. Meanwhile, the crystallization half-times (t0.5) of the PHB decreased dramatically with the addition of the CpFeâº. The isothermal and non-isothermal crystallization processes were successfully described using the Avrami model and a modified Avrami model, as well as a combination of the Avrami and Ozawa methods. Finally, the effective activation energy of the PHB/CpFe⺠nanocomposites was much lower than those of their pure counterparts, which supported the heterogeneous nucleation mechanism with the organometallic moieties, indicating that the CpFe⺠is a superior nucleating agent for this class of polymer.
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Plásticos Biodegradables/química , Ciclopentanos/química , Hidroxibutiratos/química , Compuestos Organometálicos/química , Poliésteres/química , Plásticos Biodegradables/síntesis química , Rastreo Diferencial de Calorimetría , Cationes/química , Cristalización , Ciclopentanos/síntesis química , Hidroxibutiratos/síntesis química , Nanocompuestos/química , Compuestos Organometálicos/síntesis química , Poliésteres/síntesis química , Polímeros/síntesis química , Polímeros/química , TemperaturaRESUMEN
BACKGROUND: Over the last 10-15 years, a technology has been developed to engineer bacterial poly(3-hydroxybutyrate) (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli, which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals. RESULTS: In this study, we have bioengineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding ß-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled PHB production in B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40-50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. CONCLUSIONS: This study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.
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Bacillus megaterium/química , Ingeniería Genética/métodos , ProhibitinasRESUMEN
PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for ß-ketothiolase (phaA Cma , 1179 bp), acetoacetyl-CoA reductase (phaB Cma , 738 bp), and PHA synthase (phaC Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD600, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.
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Comamonadaceae/genética , Escherichia coli/genética , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Vías Biosintéticas/genética , Clonación Molecular , Escherichia coli/metabolismo , Sistemas de Lectura Abierta , Alineación de SecuenciaRESUMEN
Several composites based on poly(3-hydroxybutyrate) (PHB) and mesoporous SBA-15 silica were prepared by solvent-casting followed by a further stage of compression molding. The thermal stability, phase transitions and crystalline details of these composites were studied, paying special attention to the confinement of the PHB polymeric chains into the mesopores of the silica. For that, differential scanning calorimetry (DSC) and real-time variable-temperature X-ray scattering at small angles (SAXS) were performed. Confinement was stated first by the existence of a small endotherm at temperatures around 20 °C below the main melting or crystallization peak, being later confirmed by a notable discontinuity in the intensity of the main (100) diffraction from the mesoporous silica observed through SAXS experiments, which is related to the change in the scattering contrast before and after the crystallization or melting of the polymer chains. Furthermore, the usual α modification of PHB was developed in all samples. Finally, a preliminary investigation of mechanical and relaxation parameters was carried out through dynamic-mechanical thermal analysis (DMTA). The results show, in the temperature interval analyzed, two relaxations, named α and ß (the latest related to the glass transition) in order of decreasing temperatures, in all specimens. The role of silica as a filler is mainly observed at temperatures higher than the glass transition. In such cases, stiffness is dependent on SBA-15 content.
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In this investigation, the electrospun nanocomposite scaffolds were developed utilizing poly-3-hydroxybutyrate (PHB), zein, and multiwalled carbon nanotubes (MWCNTs) at varying concentrations of MWCNTs including 0.5 and 1 wt%. Based on the SEM evaluations, the scaffold containing 1 wt% MWCNTs (PZ-1C) exhibited the lowest fiber diameter (384 ± 99 nm) alongside a suitable porosity percentage. The presence of zein and MWCNT in the chemical structure of the scaffold was evaluated by FTIR. Furthermore, TEM images revealed the alignment of MWCNTs with the fibers. Adding 1 % MWCNTs to the PHB-zein scaffold significantly enhanced tensile strength by about 69 % and reduced elongation by about 31 %. Hydrophilicity, surface roughness, crystallinity, and biomineralization were increased by incorporating 1 wt% MWCNTs, while weight loss after in vitro degradation was decreased. The MG-63 cells exhibited enhanced attachment, viability, ALP secretion, calcium deposition, and gene expression (COLI, RUNX2, and OCN) when cultivated on the scaffold containing MWCNTs compared to the scaffolds lacking MWCNTs. Moreover, the study found that MWCNTs significantly reduced platelet adhesion and hemolysis rates below 4 %, indicating their favorable anti-hemolysis properties. Regarding the aforementioned results, the PZ-1C electrospun composite scaffold is a promising scaffold with osteogenic properties for bone tissue engineering applications.
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Hidroxibutiratos , Nanotubos de Carbono , Osteogénesis , Poliésteres , Ingeniería de Tejidos , Andamios del Tejido , Zeína , Nanotubos de Carbono/química , Zeína/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Osteogénesis/efectos de los fármacos , Humanos , Poliésteres/química , Hidroxibutiratos/química , Hidroxibutiratos/farmacología , Huesos/efectos de los fármacos , Huesos/metabolismo , Hemólisis/efectos de los fármacos , Prohibitinas , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Nanocompuestos/química , Adhesión Celular/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Resistencia a la Tracción , Osteoblastos/efectos de los fármacos , Osteoblastos/citología , Porosidad , PolihidroxibutiratosRESUMEN
In the present scenario, rising environmental concerns of non-biodegradable plastic pollution and depletion of petroleum based raw materials lead to the development of biopolymers. The biodegradability of biopolymers gives them a specific advantage for the environmental concerns. Polyhydroxyalkanoates (PHAs) are a type of biopolymers which are synthesized by microorganisms. Although there are different substrates available in pure forms which are currently used in the production of PHA, 40% of production cost depends on the expensive substrate which is a major disadvantage and make it far from many applications. The use of an inexpensive carbon source which is high in organic matter content such as waste streams of process industries can make this process viable and diminish PHA production cost. This study explores the current research initiatives on various agricultural and industrial waste feedstocks, formulations and processing conditions for producing PHA in a way that is both inexpensive and beneficial to the environment. The creation of fermentation conditions and metabolic engineering techniques for promoting microbial growth and PHA synthesis were also discussed in the review.
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For tissue engineering applications, cell adhesion and proliferation are crucial factors, and blending polymers is one of the most effective ways to create a biocompatible scaffold with desired properties. In order to create new potential porous, biodegradable scaffolds using salt leaching technique, poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were blended in different ratios. SEM, BET, FTIR, and water contact angle measurements were used to analyze the scaffolds' porous surface, surface area, and roughness, chemical interaction, and hydrophilicity. Additionally, a hemolysis assay revealed that the mixtures were hemocompatible and had no impact on red blood cells. Different cells- Vero, Hela and MDBK cell lines cultured on the porous mats of these biopolymeric scaffolds exhibited significant increase in cell viability and attachment over time. The overall finding was that blended scaffolds exhibited reduced crystallinity, diverse porosity, higher surface area and hydrophilicity, and better cell viability, proliferation and adhesion. Our findings imply that a blended scaffold could be more suitable for use in tissue engineering applications.
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Polihidroxialcanoatos , Andamios del Tejido , Andamios del Tejido/química , Poliésteres/farmacología , Poliésteres/química , Supervivencia Celular , Ingeniería de Tejidos/métodos , Proliferación Celular , Materiales Biocompatibles/químicaRESUMEN
Piezoelectric materials are promising for biomedical applications because they can provide mechanical or electrical stimulations via converse or direct piezoelectric effects. The stimulations have been proven to be beneficial for cell proliferation and tissue regeneration. Recent reports showed that doping different contents of reduced graphene oxide (rGO) or polyaniline (PANi) into biodegradable polyhydroxybutyrate (PHB) enhanced their piezoelectric response, showing potential for biomedical applications. In this study, we aim to determine the correlation between physiochemical properties and the in vitro cell response to the PHB-based composite scaffolds with rGO or PANi. Specifically, we characterized the surface morphology, wetting behavior, electrochemical impedance, and piezoelectric properties of the composites and controls. The addition of rGO and PANi resulted in decreased fiber diameters and hydrophobicity of PHB. The increased surface energy of PHB after doping nanofillers led to a reduced water contact angle (WCA) from 101.84 ± 2.18° (for PHB) to 88.43 ± 0.83° after the addition of 3 wt % PANi, whereas doping 1 wt % rGO decreased the WCA value to 92.56 ± 2.43°. Meanwhile, doping 0.2 wt % rGO into PHB improved the piezoelectric properties compared to the PHB control and other composites. Adding up to 1 wt % rGO or 3 wt % PANi nanofillers in PHB did not affect the adhesion densities of bone marrow-derived mesenchymal stem cells (BMSCs) on the scaffolds. The aspect ratios of attached BMSCs on the composite scaffolds increased compared to the PHB control. The study indicated that the PHB-based composites are promising for potential applications such as regenerative medicine, tissue stimulation, and bio-sensing, which should be further studied.
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Grafito , Células Madre Mesenquimatosas , Polímeros/farmacología , Polímeros/química , Grafito/farmacología , Grafito/químicaRESUMEN
Poly(3-hydroxybutyrate) (PHB) was accumulated in full-scale municipal waste activated sludge at pilot scale. After accumulation, the fate of the PHB-rich biomass was evaluated over two weeks as a function of initial pH (5.5, 7.0 and 10), and incubation temperature (25, 37 and 55°C), with or without aeration. PHB became consumed under aerobic conditions as expected with first order rate constants in the range of 0.19 to 0.55 d-1. Under anaerobic conditions, up to 63 percent of the PHB became consumed within the first day (initial pH 7, 55°C). Subsequently, with continued anaerobic conditions, the polymer content remained stable in the biomass. Degradation rates were lower for acidic anaerobic incubation conditions at a lower temperature (25°C). Polymer thermal properties were measured in the dried PHB-rich biomass and for the polymer recovered by solvent extraction using dimethyl carbonate. PHB quality changes in dried biomass, indicated by differences in polymer melt enthalpy, correlated to differences in the extent of PHB extractability. Differences in the expressed PHB-in-biomass melt enthalpy that correlated to the polymer extractability suggested that yields of polymer recovery by extraction can be influenced by the state or quality of the polymer generated during downstream processing. Different post-accumulation process biomass management environments were found to influence the polymer quality and can also influence the extraction of non-polymer biomass. An acidic post-accumulation environment resulted in higher melt enthalpies in the biomass and, consequently, higher extraction efficiencies. Overall, acidic environmental conditions were found to be favourable for preserving both quantity and quality after PHB accumulation in activated sludge.
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Polímeros , Aguas del Alcantarillado , Aguas del Alcantarillado/química , Ácido 3-Hidroxibutírico , Biomasa , Hidroxibutiratos/química , Hidroxibutiratos/metabolismo , Poliésteres/química , Poliésteres/metabolismoRESUMEN
One of the important directions in the development of modern medical devices is the search and creation of new materials, both synthetic and natural, which can be more effective in their properties than previously used materials. Traditional materials such as metals, ceramics, and synthetic polymers used in medicine have certain drawbacks, such as insufficient biocompatibility and the emergence of an immune response from the body. Natural biopolymers have found applications in various fields of biology and medicine because they demonstrate a wide range of biological activity, biodegradability, and accessibility. This review first described the properties of the two most promising biopolymers belonging to the classes of polyhydroxyalkanoates and polysaccharides-polyhydroxybutyrate and chitosan. However, homopolymers also have some disadvantages, overcome which becomes possible by creating polymer composites. The article presents the existing methods of creating a composite of two polymers: copolymerization, electrospinning, and different ways of mixing, with a description of the properties of the resulting compositions. The development of polymer composites is a promising field of material sciences, which allows, based on the combination of existing substances, to develop of materials with significantly improved properties or to modify of the properties of each of their constituent components.
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Poly(3-hydroxybutyrate) (PHB) belongs to the family of polyhydroxyalkanoates, biopolymers used for agricultural, industrial, or even medical applications. However, scaling up the production is still an issue due to the myriad of parameters involved in the fermentation processes. The present work seeks, firstly, to scale up poly(3-hydroxybutyrate) (PHB) production by wild type C. necator ATCC 17697 from shaken flasks to a stirred-tank bioreactor with the optimized media and fructose as carbon source. The second purpose is to improve the production of PHB by applying both the batch and fed-batch fermentation strategies in comparison with previous works of wild type C. necator with fructose. Furthermore, thinking of biomedical applications, physicochemical, and cytotoxicity analyses of the produced biopolymer, are presented. Fed-batch fermentation with an exponential feeding strategy enabled us to achieve the highest values of PHB concentration and productivity, 25.7 g/l and 0.43 g/(l h), respectively. The PHB productivity was 3.3 and 7.2 times higher than the one in batch strategy and shaken flask cultures, respectively. DSC, FTIR, 1H, and 13C NMR analysis led to determine that the biopolymer produced by C. necator ATCC 17697 has a molecular structure and characteristics in agreement with the commercial PHB. Additionally, the biopolymer does not induce cytotoxic effects on the NIH/3T3 cell culture. Due to the improved fermentation strategies, PHB concentration resulted in 40 % higher of the already reported one for wild type C. necator using other fed-batch modes and fructose as a carbon source. Thus the produced PHB could be attractive for biomedical applications, which generate a rising interest in polyhydroxyalkanoates during recent years.
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In this study, a phaCR gene encoding PHA synthase was identified in Rhodoligotrophos defluvii which was adjacent to ß-ketothiolase encoded by phaAR gene and acetoacetyl-CoA reductase encoded by phaBR gene. Amino acid comparison of PhaCR showed the highest homology of 65.98% with PhaC of R. appendicifer, while its homology with typical class I PHA synthase in Cupriavidus necator was only 42.54%. PHA synthesis genes were then transformed into E. coli harboring phaCABR and phaCRABC which were cultured with 15 g/L glucose respectively, and 20.46 wt% and 16.95 wt% of CDW for poly(3-hydroxybutyrate) (PHB) were accumulated respectively. To further explore the effect of substrate specificity for PHA production, the ptsG gene was then deleted and 15 g/L glucose and 1.5 g/L propionate were co-employed as carbon sources, which enabled the synthesis of poly(3HB-co-3HV) copolymer. As a result, poly(3HB-co-3HV) was accumulated up to 24.74 wt% of CDW, and the highest content of 3-hydroxyvalerate (3HV) was 10.86 mol%. The Td5 was 260 °C, which implied that it possessed good thermal stability, and the Mw of GPC in recombinant strains were between 22 and 26 × 104 g/mol, and the highest PDI was 3.771. The structure of poly (3HB-co-3HV) copolymer was determined through 1H NMR analysis.
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Escherichia coli/genética , Ingeniería Metabólica/métodos , Poliésteres/metabolismoRESUMEN
Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).
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Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.
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Bacterias/enzimología , Hidrolasas de Éster Carboxílico/genética , Bacterias/genética , Bacterias/metabolismo , Reactores Biológicos/microbiología , Comamonas testosteroni/enzimología , Comamonas testosteroni/genética , Comamonas testosteroni/metabolismo , Cupriavidus/enzimología , Cupriavidus/genética , Cupriavidus/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Marinobacter/enzimología , Marinobacter/genética , Marinobacter/metabolismo , Pseudomonas stutzeri/enzimología , Pseudomonas stutzeri/genética , Pseudomonas stutzeri/metabolismo , Ralstonia/enzimología , Ralstonia/genética , Ralstonia/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
Actually, in order to replace traditional fossil-based polymers, many efforts are devoted to the design and development of new and high-performance bioplastics materials. Poly(hydroxy alkanoates) (PHAS) as well as polylactides are the main candidates as naturally derived polymers. The intention of the present study is to manufacture fully bio-based blends based on two polyesters: poly (3-hydroxybutyrate) (PHB) and polylactic acid (PLA) as real competitors that could be used to replace petrol polymers in packaging industry. Blends in the shape of films have been prepared by chloroform solvent cast solution methodology, at different PHB/PLA ratios: 1/0, 1/9, 3/7, 5/5, 0/1. A series of dynamic explorations have been performed in order to characterize them from a different point of view. Gas permeability to N2, O2, and CO2 gases and probe (TEMPO) electron spin resonance (ESR) analyses were performed. Blend surface morphology has been evaluated by Scanning Electron Microscopy (SEM) while their thermal behavior was analyzed by Differential Scanning Calorimetry (DSC) technique. Special attention was devoted to color and transparency estimation. Both probe rotation mobility and N2, O2, and CO2 permeation have monotonically decreased during the transition from PLA to PHB, for all contents of bio-blends, namely because of transferring from PLA with lower crystallinity to PHB with a higher one. Consequently, the role of the crystallinity was elucidated. The temperature dependences for CO2 permeability and diffusivity as well as for probe correlation time allowed the authors to evaluate the activation energy of both processes. The values of gas transport energy activation and TEMPO rotation mobility are substantially close to each other, which should testify that polymer segmental mobility determines the gas permeability modality.
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A series of samples based on poly(3-hydroxybutyrate) (PHB) containing five different additives were prepared and their thermal stability and flammability were discussed. The samples first underwent flammability screening by using Pyrolysis Combustion Flow Calorimeter (PCFC) analyses. Then, four samples were selected for further investigations. PHB composites containing sepiolite (Sep.) inorganic nanofiller, and also organic ammonium polyphosphate (APP) were examined for flammability and thermal behavior using PCFC, thermogravimetric analysis (TGA), flame test, and Differential Scanning Calorimetry (DSC) analyses. Moreover, burning behavior of samples were captured on a digital camera to give a deeper sense of their flammability character for comparison. The results revealed a significant improvement of flammability and thermal stability of composites, particularly in the presence of sepiolite with respect to the value obtained for unfilled PHB. Regarding TGA results, the char residue yield was increased to ca. 20.0 wt.% in the presence of sepiolite, while 0.0 wt.% was observed for PHB. PCFC measurements uncovered higher performance of PHB-Sep. sample as signaled by 40% reduction in the peak of heat release rate with respect to PHB. According to observations, PHB-Sep. sample showed non-dripping behavior with high capacity of charring in the presence of Sep. in a vertical flame test.
RESUMEN
Syngas from gasified organic waste materials is a promising feedstock for the biotechnological synthesis of the bioplastic poly([R]-3-hydroxybutyrate) (PHB) with Rhodospirillum rubrum. In a first approach, growth studies were carried out with this strain in gas-tight serum vials. When syngas (40% CO, 40% H2, 10% CO2, and 10% N2 v/v) was diluted with N2 to 60%, a 4-fold higher biomass production was detected compared to samples grown on 100% syngas, thus indicating a growth inhibitory effect. The best performing syngas-mixture was then used for C-, C,N-, and C,P-limited fed-batch fermentations in a bioreactor with continuous syngas and acetate supply. It was found that C,P-limited PHB productivity was 5 times higher than for only C-limited growth and reached a maximal PHB content of 30% w/w. Surprisingly, growth and PHB production stopped when N, as a second nutrient, became growth-limiting. Finally, it was concluded that a minimal supply of 0.2 g CO g-1 biomass h-1 has to be guaranteed in order to cover the cellular maintenance energy.