Polyhydroxybutyrate Research Articles

Optimized Polyhydroxybutyrate Production by Neobacillus niacini GS1 Utilizing Corn Flour, Wheat Bran, and Peptone: A Sustainable Approach

Plastic pollution is a pressing environmental challenge, necessitating the development of biodegradable alternatives like polyhydroxybutyrate (PHB). This study focuses on optimizing PHB production by Neobacillus niacini GS1, a bacterium isolated from a municipal dumping site. By utilizing agricultural residues such as corn flour, wheat bran, and peptone as substrates, we aimed to establish an eco-friendly method for biopolymer production, contributing to sustainable agricultural residue management and bioplastic innovation. The bacterium was identified using morphological, biochemical, and molecular techniques. The optimization process involved adjusting variables such as inoculum age, inoculum size, incubation time, agitation rate, incubation temperature, pH of the medium, carbon sources, and nitrogen sources. Response surface methodology (RSM) was employed to identify optimal conditions, with the highest PHB yield of 61.1% achieved under specific conditions: 37 °C, pH 7, and an agitation rate of 150 rpm. These findings underscore the potential of Neobacillus niacini GS1 in converting agro-industrial residues into valuable biopolymers, promoting sustainable bioplastic production, and advancing agricultural residue valorization efforts through the use of eco-friendly materials.

Light Intensity Effects on Bioproduct Recovery from Fuel Wastewater Using Purple Phototrophic Bacteria in a Hybrid Biofilm-Suspended Growth System.

This research looked at how three different light intensities (1600, 4300, and 7200 lx) affect the biomass development, treatment of fuel synthesis wastewater and the recovery of valuable bioproducts between biofilm and suspended growth in a purple-bacteria enriched photobioreactor. Each condition was run in duplicate using an agricultural shade cloth as the biofilm support media in a continuously mixed batch reactor. The results showed that the highest chemical oxygen demand (COD) removal rate (56.8 ± 0.9%) was found under the highest light intensity (7200 lx), which also led to the most biofilm formation and highest biofilm biomass production (1225 ± 95.7 mg). The maximum carotenoids (Crts) and bacteriochlorophylls (BChls) content occurred in the suspended growth of the 7200 lx reactor. BChls decreased with light intensity in suspended growth, while in biofilm both Crts and BChls were relatively stable between light conditions, likely due to an averaging effect as biofilm thickened at higher light intensity. Light intensity did not affect protein content of the biomass, however, biofilm showed a lower average (41.2% to 43.7%) than suspended biomass (45.4% to 47.7%). For polyhydroxybutyrate (PHB) the highest cell concentration in biofilm occurred at 1600 lx (11.4 ± 2.4%), while for suspended growth it occurred at 7200 lx (22.7 ± 0.3%), though total PHB productivity remained similar between reactors. Shading effects from the externally located biofilm could explain most variations in bioproduct distribution. Overall, these findings suggest that controlling light intensity can effectively influence the treatment of fuel synthesis wastewater and the recovery of valuable bioproducts in a biofilm photobioreactor.

Multifunctional hybrid films made from CoT3OTx4 and CoFeT2OT4 nanoparticles inside a poly 3-hydroxybutyrate matrix and study of their impact in methyl orange photodegradation.

This work aims to produce hybrid materials with potential applications in dye photodegradation. Therefore, hybrid films were obtained by incorporating cobalt (II, III) oxide (Co3O4) or cobalt ferrite (CoFe2O4) nanoparticles (NPs) with 18 ± 1.6 nm and 26 ± 1.3 nm, respectively, into a poly 3-hydroxybutyrate (P3HB) polymeric matrix. The Co3O4@P3HB and CoFe2O4@P3HB hybrid films were fabricated by solvent casting in a ratio of 85 mg to 15 mg (P3HB-NPs). Different spectroscopic and microscopy techniques characterized the Co3O4 and CoFe2O4 NPs and the P3HB, Co3O4@P3HB and CoFe2O4@P3HB films. The optical band gap for Co3O4 and CoFe2O4 NPs was estimated from their diffuse reflectance spectra (DRS) around 2.5 eV. X-ray diffraction (XRD) of the hybrid films revealed that the nanometric sizes of the Co3O4 and CoFe2O4 nanoparticles incorporated into the P3HB are preserved. The magnetic hysteresis curve of CoFe2O4 nanoparticles and CoFe2O4@P3HB film showed a ferromagnetic behaviour at 300 K. Transmission electron microscopy (TEM) confirmed the formation of nanocrystals, and scanning electron microscopy (SEM) provided evidence for the successful incorporation of the NPs into the P3HB matrix. The surface roughness and hydrophilicity of the hybrid films are increased compared to the P3HB film. The impact of the nanoparticles and the hybrid films on the photodegradation of methyl orange (MO) in its acidic form was studied. The photodegradation tests were carried out by direct sunlight exposure. The CoFe2O4@P3HB hybrid film achieved 85% photodegradation efficiency of a methyl orange solution of 20 ppm after 15 minutes of exposure to sunlight. After 30 minutes of exposure to sunlight, the nanoparticles and the hybrid films reached about 90% of the MO degradation. The results suggest that combining nanoparticles with the polymer significantly enhances photodegradation compared to isolated nanoparticles.

Engineering Halomonas bluephagenesis for synthesis of polyhydroxybutyrate (PHB) in the presence of high nitrogen containing media

The trade-offs exist between microbial growth and bioproduct synthesis including intracellular polyester polyhydroxybutyrate (PHB). Under nitrogen limitation, more carbon flux is directed to PHB synthesis while growth is inhibited with diminishing overall carbon utilization, similar to the suboptimal carbon utilization during glycolysis-derived pyruvate decarboxylation. This study reconfigured the central carbon network of Halomonas bluephagenesis to improve PHB yield theoretically and practically. It was found that the downregulation of glutamine synthetase (GS) activity led to a synchronous improvement on PHB accumulation and cell growth under nitrogen non-limitation condition, increasing the PHB yield from glucose (g/g) to 85% of theoretical yield, PHB titer from 7.6 g/L to 12.9 g/L, and from 51 g/L to 65 g/L when grown in shake flasks containing a rich N-source, and grown in a fed-batch cultivation conducted in a 7-L bioreactor also containing a rich N-source, respectively. Results offer better metabolic balance between glucose conversion efficiency and microbial growth for economic PHB production.

Lignin valorization to bioplastics with an aromatic hub metabolite-based autoregulation system

Exploring microorganisms with downstream synthetic advantages in lignin valorization is an effective strategy to increase target product diversity and yield. This study ingeniously engineers the non-lignin-degrading bacterium Ralstonia eutropha H16 (also known as Cupriavidus necator H16) to convert lignin, a typically underutilized by-product of biorefinery, into valuable bioplastic polyhydroxybutyrate (PHB). The aromatic metabolism capacities of R. eutropha H16 for different lignin-derived aromatics (LDAs) are systematically characterized and complemented by integrating robust functional modules including O-demethylation, aromatic aldehyde metabolism and the mitigation of by-product inhibition. A pivotal discovery is the regulatory element PcaQ, which is highly responsive to the aromatic hub metabolite protocatechuic acid during lignin degradation. Based on the computer-aided design of PcaQ, we develop a hub metabolite-based autoregulation (HMA) system. This system can control the functional genes expression in response to heterologous LDAs and enhance metabolism efficiency. Multi-module genome integration and directed evolution further fortify the strain’s stability and lignin conversion capacities, leading to PHB production titer of 2.38 g/L using heterologous LDAs as sole carbon source. This work not only marks a leap in bioplastic production from lignin components but also provides a strategy to redesign the non-LDAs-degrading microbes for efficient lignin valorization.

Enhancement of H2-water mass transfer using methyl-modified hollow mesoporous silica nanoparticles for efficient microbial CO2 reduction

Inorganic-microbial hybrid catalysis is an emerging technology that uses electrical energy to drive microorganisms to reduce CO2 into high value-added compounds, and it has broad application prospects in CO2 reduction. However, the low current density (production yield) limits its practical application. Hydrogen-mediated inorganic-microbial hybrid catalysis system can achieve higher current density, but it is limited by low H2 mass transfer. Here, silica nanoparticles were used to enhance the hydrogen mass transfer for highly efficient CO2 reduction. Solid silica (SN), mesoporous silica (MSN), hollow mesoporous silica (HMSN), and methyl-modified hollow mesoporous silica (MHMSN) were firstly prepared and tested for the enhancement of hydrogen mass transfer. Of these, MHMSN nanoparticles at a concentration of 0.3 wt% were the best at enhancing gas-liquid mass transfer, the volumetric mass transfer coefficient (KLa) and saturated dissolved hydrogen concentration of H2 are 0.53 min-1 and 1.81 mg l-1, respectively. Compared with the control group without added nanoparticles, MHMSN significantly increased the solubility and KLa of H2. This can be attributed that the addition of MHMSN promoted the detached process of hydrogen bubbles from the electrode surface, which made the diameter of hydrogen bubbles smaller, increased the gas-liquid mass transfer area, and strengthened the mass transfer process of H2. Furthermore, it was added to the inorganic-microbial hybrid catalysis system to effectively promote the microbial carbon reduction process, achieving a polyhydroxybutyrate (PHB) yield of up to 700 mg l-1, and the electron utilization rate and CO2 conversion rate were 51 % and 58 % higher than the control group, respectively. These results demonstrated that the addition of MHMSN is an effective approach to enhancing the performance of H2-mediated inorganic-microbial hybrid catalysis system.

Influence of Oligomeric Lactic Acid and Structural Design on Biodegradation and Absorption of PLA-PHB Blends for Tissue Engineering

The advancing development in biomaterials and biology has enabled the extension of 3D printing technology to the bioadditive manufacturing of degradable hard tissue substitutes. One of the key advantages of bioadditive manufacturing is that it has much smaller design limitations than conventional manufacturing and is therefore capable of producing implants with complex geometries. In this study, three distinct blends of polylactic acid (PLA) and polyhydroxybutyrate (PHB) were produced using Fused Deposition Modeling (FDM) technology. Two of these blends were plasticized with oligomeric lactic acid (OLA) at concentrations of 5 wt% and 10 wt%, while the third blend remained unplasticized. Each blend was fabricated in two structural modifications: solid and porous. The biodegradation behavior of the produced specimens was examined through an in vitro experiment using three different immersion solutions: saline solution, Hank’s balanced salt solution (HBSS), and phosphate-buffered saline (PBS). All examined samples were also subjected to chemical analysis: atomic absorption spectroscopy (AAS), scanning electron microscopy (SEM), and energy-dispersive spectrometry (EDS). The results of the degradation experiments indicated a predominantly better absorption capacity of the samples with a porous structure compared to the full structure. At the same time, the blend containing a higher concentration of OLA exhibited enhanced pH stability over the evaluation period, maintaining relatively constant pH values before experiencing a minor decline at the end of the study. This observation indicates that the increased presence of the plasticizer may provide a buffering effect, effectively mitigating the acidification associated with material degradation.

Dual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in Escherichia coli.

The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into Escherichia coli's genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10-3 per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the egfp gene in the RecA+ strain MG1655 and the RecA- strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into E. coli MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various E. coli strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology.

The Absence of Phasins PhbP2 and PhbP3 in Azotobacter vinelandii Determines the Growth and Poly-3-hydroxybutyrate Synthesis.

Phasins are proteins located on the surface of poly-3-hydroxybutyrate (P3HB) granules that affect the metabolism of the polymer, the size and number of the granules, and some also have stress-protecting and growth-promoting effects. This study evaluated the effect of inactivating two new phasins (PhbP2 or PhbP3) on the cellular growth, production, and molecular mass of P3HB in cultures under low or high oxygen transfer rates (OTR). The results revealed that under high OTRₘₐₓ conditions (between 8.1 and 8.9 mmol L-1 h-1), the absence of phasins PhbP2 and PhbP3 resulted in a strong negative effect on the growth rate; in contrast, the rates of specific oxygen consumption increased in both cases. This behavior was not observed under a low oxygen transfer rate (3.9 ± 0.71 mol L-1 h-1), where cellular growth and oxygen consumption were the same for the different strains evaluated. It was observed that at high OTR, the absence of PhbP3 affected the production of P3HB, decreasing it by 30% at the end of cultivation. In contrast, the molecular weight remained constant over time. In summary, the absence of phasin PhbP3 significantly impacted the growth rate and polymer synthesis, particularly at high maximum oxygen transfer rates (OTRₘₐₓ).

Valorization of biodiesel-derived crude glycerol for simultaneous biosynthesis of biodegradable polyhydroxybutyrate and exopolysaccharide by the newly isolated Burkholderia sp. SCN-KJ

This study demonstrated that Burkholderia sp. SCN-KJ is a promising novel species for the biovalorization of crude glycerol to polyhydroxybutyrate (PHB) and galactose-rich heteroexopolysaccharide (EPS). Whole-genome and genetic evolution analyses revealed separation of the different clades according to the ANIb and dDDH analyses, which confirmed that Burkholderia sp. SCN-KJ is a novel species. The highest PHB production from crude glycerol was 12.9 ± 0.4 g/L (72.9 ± 2.1 % w/w), with a productivity of 0.46 g/L/h and YP/S of 0.3 g/g at 28 h in a 10 L fermenter. The galactose-rich hetero-EPS began to be produced after nitrogen depletion, resulting in a concentration of 22.4 ± 0.2 g/L at 38 h. Examination of the carbon-to‑nitrogen ratio (C/N) showed that nitrogen-rich condition (C/N 20) was optimal for PHB production, whereas nitrogen-depleted condition promoted EPS production, showing two different extrema. The findings showed that Burkholderia sp. SCN-KJ has the potential to transform the landscape of biovalorization for sustainable production.

Unlocking sustainable solutions: wood waste as a novel substrate for polyhydroxybutyrate (PHB) production to combat plastic pollution

ABSTRACT Polyhydroxybutyrate (PHB) is considered as a hope for bioplastic production, which can serve as a sustainable alternative. Utilizing feedstock as substrate is widely explored for the production but wood waste, which is abundant in cellulose, hemicellulose and lignocellulose, has limited studies for PHB production. Herein, wood waste is used as a biobased feedstock Hydrolyses of wood waste was done using sulphuric acid (H2SO4) to break down of cellulose and hemicellulose into simple carbon forms. The hydrolysed product was analysed for sugar presence by quantitative and qualitative methods. Pseudomonas fluorescens bacterial strain was used for the production purpose using hydrolysed wood waste as substrate media. The Plackett-Burman design (PBD) and response surface methodology (RSM) were applied to optimize the growth media. The results of PBD were used to identify significant factors influencing PHB production, which were then further optimized using RSM. The work’s results conclusively demonstrated that P. fluorescens possesses the capability to effectively utilize wood waste and wastewater as substrate media up to production rate of 13–14 mg mL−1 of PHB. Fourier Transformed Infra-Red (FTIR) spectroscopic peaks confirm the produced product is PHB, which is a type of polyhydroxyalkanoate (PHA), classified within the polyester family highlighting wood waste potential as a sustainable solution to address plastic pollution.

Metabolic engineering of Halomonas bluephagenesis for the production of ethylene glycol and glycolate from xylose

Halophilic Halomonas bluephagenesis, a natural producer of poly-3-hydroxybutyrate (PHB), was metabolically engineered to synthesize ethylene glycol and glycolate from xylose. Xylose utilization was achieved by overexpressing either the xylonate pathway or the ribulose-1-phosphate pathway. The key genes encoding for xylonate dehydratase and 2-keto-3-deoxy-xylonate aldolase in the xylonate pathway were screened. With further overexpressing aldehyde reductase gene yjgB, ethylene glycol accumulation was improved to 0.91g/L, accompanied with 1.48g/L of PHB accumulation. The disruption of native glycolate oxidase was found to be essential for glycolate production, and the defective recombinant strain produced 0.80g/L glycolate with 1.14g/L PHB in shake flask cultures. These results indicated that H. bluephagenesis has the potential to produce diverse metabolic chemicals from xylose.

Extension of shelf life of tomato (Solanum lycopersicum L.) by using a coating of polyhydroxybutyrate-carboxymethyl cellulose-pectin-thymol conjugate.

This study targets explicitly finding an alternative to petroleum-based plastic films that burden the environment, which is a high priority. Hence, polymeric films were prepared with carboxymethyl cellulose (CMC) (4%), pectin (2%), and polyhydroxybutyrate (PHB) (0.5%) with different concentrations of thymol (0.3%, 0.9%, 1.8%, 3%, and 5%) and glycerol as a plasticizer by solution casting technique. The prepared films were tested for mechanical, optical, antimicrobial, and antioxidant properties. Film F5 (CMC+P+PHB+0.9%thymol) showed an excellent tensile strength of 15MPa, Young's modulus of 395MPa, antioxidant activity (AA) (92%), rapid soil biodegradation (21 days), and strong antimicrobial activity against bacterial and fungal cultures such as Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Aspergillus niger, and Aspergillus flavus. The thymol content increase in films F6 (1.8%), F7 (3%), and F8 (5%) displayed a decrease in mechanical properties due to thymol's hydrophobicity. For shelf life studies on tomatoes, F2, a film without thymol (poor antimicrobial and antioxidant activities), F5 (film with superior mechanical, optical, antimicrobial, and antioxidant properties), and F7 (film with low mechanical properties) were selected. Film F5 coatings on tomato fruit enhanced the shelf life of up to 15 days by preventing weight loss, preserving firmness, and delaying changes in biochemical constituents like lycopene, phenols, and AA. Based on the mechanical, optical, antimicrobial, antioxidant, and shelf life results, the film F5 is suitable for active food packaging and preservation. PRACTICAL APPLICATION: The developed active biodegradable composite can be utilized as a coating to extend the shelf life of fruits and vegetables. These coatings are easy to produce and apply, offering a sustainable solution to reduce food waste. On an industrial scale, they can be applied to food products, ensuring longer freshness without any technical challenges.

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

Encapsulation of Bacillus subtilis in Electrospun Poly(3-hydroxybutyrate) Fibers Coated with Cellulose Derivatives for Sustainable Agricultural Applications.

One of the latest trends in sustainable agriculture is the use of beneficial microorganisms to stimulate plant growth and biologically control phytopathogens. Bacillus subtilis, a Gram-positive soil bacterium, is recognized for its valuable properties in various biotechnological and agricultural applications. This study presents, for the first time, the successful encapsulation of B. subtilis within electrospun poly(3-hydroxybutyrate) (PHB) fibers, which are dip-coated with cellulose derivatives. In that way, the obtained fibrous biohybrid materials actively ensure the viability of the encapsulated biocontrol agent during storage and promote its normal growth when exposed to moisture. Aqueous solutions of the cellulose derivatives-sodium carboxymethyl cellulose and 2-hydroxyethyl cellulose, were used to dip-coat the electrospun PHB fibers. The study examined the effects of the type and molecular weight of these cellulose derivatives on film formation, mechanical properties, bacterial encapsulation, and growth. Scanning electron microscopy (SEM) was utilized to observe the morphology of the biohybrid materials and the encapsulated B. subtilis. Additionally, ATR-FTIR spectroscopy confirmed the surface chemical composition of the biohybrid materials and verified the successful coating of PHB fibers. Mechanical testing revealed that the coating enhanced the mechanical properties of the fibrous materials and depends on the molecular weight of the used cellulose derivatives. Viability tests demonstrated that the encapsulated B. subtilis exhibited normal growth from the prepared materials. These findings suggest that the developed fibrous biohybrid materials hold significant promise as biocontrol formulations for plant protection and growth promotion in sustainable agriculture.

Enhancing nitrogen removal by simultaneous nitritation and denitritation in a multi-cycle SBR with supplementation of solid carbon sources

Simultaneous nitritation and denitritation have the potential to significantly improve nitrogen removal in sewage treatment processes. However, their application in low-strength sewage treatment systems presents challenges. This study explored the impact of four solid carbon sources (SCSs) on N-removal via nitrite in a multi-cycle SBR with biocarriers. Results showed that both N-removal efficiencies and nitrite accumulation rates increased with higher COD/N ratios, indicating that high COD/N ratios can improve the competition between denitrifiers and nitrite-oxidizing bacteria for nitrite, leading to stable simultaneous nitritation and denitritation. The supplementation of SCSs further enhanced this high-efficiency N-removal process, with polybutylene succinate (PBS) and polycaprolactone (PCL) showing greater increases in N-removal via nitrite than poly-hydroxybutyrate (PHB) and poly-hydroxyalkanoate (PHA). Moreover, PBS showed the most significant increase in denitrification efficiency in anoxic conditions, while PHA was the most effective external SCS at a moderate level of dissolved oxygen. These findings suggest that the incorporation of external SCSs can facilitate the simultaneous nitritation and denitrification process in multi-cycle SBRs, underscoring the importance of selecting an appropriate SCS for optimizing nitrogen removal in sewage treatment projects.

All-organic transistors printed on a biodegradable and bioderived substrate for sustainable bioelectronics

Biodegradable electronics is an incipient need in order to mitigate the alarming increase of electronic waste worldwide caused by capillary penetration of electronic devices and sensors. Flexibility, solution processability, low capital expenditure, and energy-efficient processes, which are distinctive features of organic printed electronics, have to be complemented by a sustainable sourcing and end-of-life of materials employed. This requirement calls for solutions where materials, especially substrates that typically represent the largest volume, can be biodegraded in the environment with no harm, yet assuring that no precious resources are dispersed. In this work, the bioderived and biodegradable biopolymer polyhydroxybutyrate (PHB) was used as a substrate, cast from an acetic acid solution, for all-organic field effect transistors (OFETs) based on an inkjet printed polymer semiconductor. The OFETs showed small device-to-device variation, a proper current modulation with ION/IOFF of about 1.2·103, mobility values as high as 0.07 cm2/Vs in saturation regime and channel length/width normalized leakage currents in the order of nA, which remained almost unaltered also after intensive mechanical stresses upon bending and rolling. Such mechanical stability and flexibility, together with the biodegradability and bioderivation, make PHB an appealing candidate for the development of sustainable printed bioelectronics, with widespread future applications in the biomedical and food packaging sector.

Effect of Different Sucrose Concentrations on the Biosynthesis of Poly(3-Hydroxybutyrate) and Alginate by the Bacterial Strain Azotobacter Vinelandii 12 under Different Aeration Conditions

Effect of Different Sucrose Concentrations on the Biosynthesis of Poly(3-Hydroxybutyrate) and Alginate by the Bacterial Strain Azotobacter Vinelandii 12 under Different Aeration Conditions

Characterization of Bacillus pacificus KUMBNGBT-39-Derived Polyhydroxybutyrate and Impact of Physicochemical Factors on its Production.

The extensive use of various chemicals in synthetic plastics is toxic and threatens the biosphere. To address this, the study aimed to isolate, screen, characterize, optimize, and quantify polyhydroxybutyrate (PHB)-producing bacteria using cost-effective residues. Isolated from a landfill site, the Gram-positive, rod-shaped, spore-forming, motile bacterium with intracellular PHB granules was identified as Bacillus pacificus based on phenotypic and genotypic characteristics. Optimal PHB production parameters included a nutrient broth medium, 72h of incubation, a temperature of 37° C, a pH of 7.0, glucose as the carbon source, ammonium chloride as the nitrogen source, and a carbon-to-nitrogen ratio of 4:1, resulting in a 1.42-fold PHB production increase. B. pacificus was also cultured on various low-cost substrates. Among the oil wastes, feedstock showed the highest PHB production (1.983 ± 0.005g/L) and among agricultural residues, the maximum PHB was obtained from rice bran (1.626 ± 0.01g/L). UV-visible spectrophotometric, FT-IR, and HR-LCMS analysis of extracted PHB confirmed characteristics of PHB molecules (ʎ-max at 210nm, functional groups between 1152 and 2925cm-1). The 1H NMR analysis revealed distinct signals for protons resonating at aliphatic CH3 proton groups, bridged CH protons, and shielding CH2 proton regions that matched PHBs. Thermal gravimetric analysis (TGA) and direct scanning colorimetric (DSC) analysis revealed 89.4% degradation and melting temperature at 124.1°C for the extracted PHB compound.

Effect of Amorphous Halomonas-PHB on Growth, Body Composition, Immune-Related Gene Expression and Vibrio anguillarum Resistance of Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatu ♂) Juveniles.

Poly-β-hydroxybutyrate (PHB) is a bacterial metabolite produced by bacteria such as Halomonas sp. that serves as a carbon and energy storage compound for bacteria under nutrient-limited conditions. Two experiments were conducted to investigate the effects of dietary supplementation with Halomonas-PHB on hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatu ♂). In experiment I, juvenile groupers were fed basal diets supplemented with 3% Halomonas-PHB (3% HM-PHB) containing 1.4% PHB and 3% Halomonas (3% HM) without PHB, as well as a control diet, for seven weeks. The results showed no significant difference in survival rate, weight gain, and crude fat content between the 3% HM-PHB group and the control group; however, the crude protein of the 3% HM-PHB group was significantly lower than that of the control group. Furthermore, supplementation with 3% HM-PHB increased the fatty acids content in fish muscles, including long-chain unsaturated fatty acids C18:1n9, EPA, and DHA. In experiment II, groupers were fed a basal diet supplemented with 6.5% Halomonas-PHB (6.5% HM-PHB) containing 3% PHB and 6.5% Halomonas (6.5% HM) containing no PHB, as well as a basal diet (Control). After seven weeks of rearing, the fish were challenged with Vibrio anguillarum for 48 h. Although no significant difference in survival rate and growth was observed among different groups, the dietary supplement of 6.5% Halomonas-PHB improved the survival rate of V. anguillarum challenged grouper and significantly increased the gene expressions of catalase (CAT) and superoxide dismutase (SOD) in blood, interleukin 1 (IL1) and interleukin 10 (IL10) in the liver, spleen, head kidney, and blood (p < 0.05). In conclusion, dietary supplementation of Halomonas-PHB had no significantly positive effect on fish growth performance but increased the content of fatty acids, including long-chain unsaturated fatty acids C18:1n9, EPA, and DHA in fish muscle; it also improved the V. anguillarum resistance, possibly through increasing immune-related gene expression in different tissues and organs. Our findings offer compelling evidence that Halomonas-PHB can be utilized as a feed additive in intensive grouper farming to enhance the groupers' resistance to Vibrio.