Presence Of Polyhydroxybutyrate Research Articles

The effects of PHB-g-MA types on the mechanical, thermal, morphological, structural, and rheological properties of polyhydroxybutyrate biopolymers

This study investigates the grafting of polyhydroxybutyrate (PHB) chains with maleic anhydride (MA) in concentrations ranging from 5 % to 10 % by weight. This process was conducted during microwave treatment and using a reactive extruder, employing benzoyl peroxide (BPO) as the initiator. The impact of these methods on PHB's overall properties was thoroughly investigated. In the study, PHB–g–MA was incorporated into neat PHB via the extrusion process at a 5 % loading rate. Notably, the mechanical properties exhibited an increase in the presence of PHB–g–MA, likely due to morphological improvements in the neat PHB, as indicated by morphological characterization. X-ray diffraction results indicated crystallinity percentages increase with the addition of MA. Differential scanning calorimetry revealed minimal variation in melting and crystallization temperatures when PHB–g–MA was included. Both storage and loss moduli were enhanced by the incorporation of PHB–g–MA, and the blends exhibited consistent tan delta values. Regarding rheological properties, the storage and loss moduli of PHB blends containing PHB–g–MA blends were observed to rise with rising frequency values. Based on these results, the microwave process was identified as the most effective method for grafting.

Valorization of Waste from Argan Seeds for Polyhydroxybutyrate Production Using Bacterial Strains Isolated from Argan Soils.

The aim of this work was to study the valorization of argan seed pulp, a waste material obtained from argan oil extraction, for the biosynthesis of polyhydroxybutyrate (PHB). A new species that showed the metabolic capacity for the conversion of argan waste into the bio-based polymer was isolated from an argan crop located in Teroudant, a southwestern region of Morocco, where the arid soil is exploited for goat grazing. The PHB accumulation efficiency of this new species was compared to the previously identified species 1B belonging to the genus Sphingomonas, and results were reported as dry cell weight residual biomass and PHB final yield measured. Temperature, incubation time, pH, NaCl concentration, nitrogen sources, residue concentrations, and culture medium volumes were analyzed with the aim of obtaining a maximum accumulation of PHB. UV-visible spectrophotometry and FTIR analysis confirmed that PHB was present in the material extracted from the bacterial culture. The results of this wide investigation indicated that the new isolated species 2D1 had a higher efficiency in PHB production compared to the previously identified strain 1B, which was isolated from a contaminated argan soil in Teroudant. PHB final yield of the two bacterial species, i.e., the new isolated and 1B, cultivated under optimal culture conditions, in 500 mL MSM enriched with 3% argan waste, were 21.40% (5.91 ± 0.16 g/L) and 8.16% (1.92 ± 0.23 g/L), respectively. For the new isolated strain, the result of the UV-visible spectrum indicates the absorbance at 248 nm, while the FTIR spectrum showed peaks at 1726 cm-1 and 1270 cm-1: these characteristic peaks indicated the presence of PHB in the extract. The data from the species 1B UV-visible and FTIR spectra were previously reported and were used in this study for a correlation analysis. Furthermore, additional peaks, uncharacteristic of standard PHB, suggest the presence of impurities (e.g., cell debris, solvent residues, biomass residues) that persisted after extraction. Therefore, a further enhancement of the sample purification during extraction is recommended for more accuracy in the chemical characterization. If 470,000 tons of argan fruit waste can be produced annually, and 3% of waste is consumed in 500 mL culture by 2D1 to produce 5.91 g/L (21.40%) of the bio-based polymer PHB, it can be estimated that the amount of putative PHB that can be extracted annually from the total argan fruit waste is about 2300 tons.

Potential use of <i>Bacillus paramycoides</i> for the production of the biopolymer polyhydroxybutyrate from leftover carob fruit agro-waste

This study was designed to investigate, at a laboratory scale, the possibility of valorizing the leftover carob fruits to produce the eco-friendly biopolymer polyhydroxybutyrate (PHB) by using the bacterial strain Bacillus paramycoides, which has been isolated from the botanical garden of Skikda University in Algeria. The PHB production was tested under various conditions: a pH of 3–8, temperature range of 30–44 °C, carob extracted molasses concentration of 2–8% v/v, an incubation time of 24–96 h and an agitation speed of 150–300 rpm. The effects of different nitrogen sources and carob extracted molasses treatment types were also investigated. The PHB concentration was determined quantitatively as crotonic acid by measuring the absorbance at 300 nm. Cell growth was quantified by measuring the density of the culture at 600 nm. The presence of PHB was confirmed by applying high-performance liquid chromatography (HPLC) using an Aminex HPX-87H and implementing gas chromatography analysis. The best yield of PHB synthesis was obtained by using 6% v/v of 5 M H2SO4 treated with carob molasses as a carbon source, with peptone as a nitrogen source; incubation was conducted at 37 °C for 96 h at an agitation speed of 300 rpm (114.95 mg/L). The HPLC analysis confirmed the synthesis of PHB by B. paramycoides to have a chromatogram retention time of 22.5 min. Carob waste was successfully valorized to PHB.

Dissolved oxygen as a propulsive parameter for polyhydroxyalkanoate production using Bacillus endophyticus cultures

Conventional biopolymers resembling synthetic polymers produced from microorganisms, polyhydroxyalkanoate (PHA) synthesized utilizing renewable resources have gained supreme attention recently. PHA accumulation within the microbial cell is an innate capability of bacteria to store carbon and energy when nutrient imbalance pertains. Gram positive Bacillus endophyticus capable of synthesizing PHA was focused in this study. Study focuses on the possibility of attaining high PHA yield in relation to the varying dissolved oxygen levels induced during production phase. There was a gradual increment in PHA production from 34.5 to 53.03% when cultivated in bioreactor that maintained least dissolved oxygen of 0.4 mg/L at 32 °C. The metabolic flux of organism was altered during oxygen stress brought by varying agitation rate and volume resulting in the accumulation of Nicotinamide Adenine Dinucleotide Hydrogen (NADH), which led to increase in the overall PHA production. PHA yield was found to be favored by decreasing the oxygen supply thereby inducing an oxygen stress environment. This report was the first one that was correlating the hypothesis that links PHA yield and oxygen stress condition during production phase. PHA produced was characterized by FTIR and 1HNMR spectra in which the presence of Polyhydroxybutyrate was confirmed.Graphical abstract

Screening of Chaetomorpha linum Lipidic Extract as A New Potential Source of Bioactive Compounds.

Recent studies have shown that marine algae represent a great source of natural compounds with several properties. The lipidic extract of the seaweed Chaetomorpha linum (Chlorophyta, Cladophorales), one of the dominant species in the Mar Piccolo of Taranto (Mediterranean, Ionian Sea), revealed an antibacterial activity against Vibrio ordalii and Vibrio vulnificus, common pathogens in aquaculture, suggesting its potential employment to control fish and shellfish diseases due to vibriosis and to reduce the public health hazards related to antibiotic use in aquaculture. This extract showed also an antioxidant activity, corresponding to 170.960 ± 16. mmol Trolox equivalent/g (oxygen radical absorbance capacity assay—ORAC) and to 30.554 ± 2.30 mmol Trolox equivalent/g (Trolox equivalent antioxidant capacity assay—TEAC). The chemical characterization of the extract, performed by 1D and 2D NMR spectroscopy, highlighted the presence of free, saturated (SAFAs), unsaturated (UFAs) and polyunsaturated (PUFAs) fatty acids. The high content of ω-6 and ω-3 PUFAs confirmed also by gas chromatography indicates the potentiality of this algal species in the production of fortified food. The antibacterial activity seems related to the presence of linolenic acid present at high density, while the antioxidant activity could be likely ascribable to molecules such as carotenoids and chlorophylls (characterized also by thin-layer chromatography), known for this property. The presence of polyhydroxybutyrate, a biopolymer with potentiality in the field of biodegradable bioplastics was also detected. The exploitation of C. linum for a future biotechnological application is also encouraged by the results from a first attempt of cultivating this species in an integrated multi-trophic aquaculture (IMTA) system.

Re-evaluation of gellan gum (E418) as food additive.

The Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re‐evaluating the safety of gellan gum (E 418) as a food additive. Following the conceptual framework for the risk assessment of certain food additives re‐evaluated under Commission Regulation (EU) No 257/2010, the Panel considered that adequate exposure and toxicity data were available. Based on the reported use levels, a refined exposure of up to 72.4 mg/kg body weight (bw) per day in toddlers at the 95th percentile was estimated. Gellan gum is unlikely to be absorbed intact and would not be fermented by human intestinal microbiota. There is no concern with respect to carcinogenicity and genotoxicity. No adverse effects were reported in chronic studies at the highest doses tested in mice and rats (3,627 and 1,460 mg gellan gum/kg bw per day, respectively). Repeated oral intake up to 200 mg/kg bw per day for 3 weeks had no adverse effects in humans. The Panel concluded that there is no need for a numerical acceptable daily intake (ADI) for gellan gum (E 418), and that there is no safety concern at the refined exposure assessment for the reported uses and use levels of gellan gum (E 418) as a food additive. The Panel recommended to better define the specifications of gellan gum including the absence of viable cells of the microbial source and the presence of polyhydroxybutyrate (PHB), protein and residual bacterial enzymatic activities.

Acceleration of the crystal growth rate of low molecular weight organic compounds in supercooled liquids in the presence of polyhydroxybutyrate

Modification of the crystallization kinetics of amorphous systems is an important topic, in particular in the area of drug delivery. Herein, we studied the impact of polyhydroxybutyrate (PHB), a biocompatible polymer with a low glass transition temperature (Tg), on crystal growth rates from the supercooled liquid phase of a variety of organic compounds. Low levels of PHB were mixed with the supercooled liquid and growth rates were monitored using optical microscopy at several temperatures above the Tg of the mixture. Growth rates were determined from a plot of crystal size as a function of time. It was found that PHB inhibited the growth of low Tg compounds, while accelerated crystal growth was observed for compounds that had a much higher Tg (>50 °C) than the polymer. The observations were rationalized based on the extent of mobility differences between the crystallizing compound and the polymer. This is the first reported example of a polymer having compound specific inhibitory or accelerator effects on crystal growth.

Production and characterization of polyhydroxybutyrate fromVibrio harveyiMCCB 284 utilizing glycerol as carbon source

Production and characterization of polyhydroxybutyrate (PHB) from moderately halophilic bacterium Vibrio harveyi MCCB 284 isolated from tunicate Phallusia nigra. Twenty-five bacterial isolates were obtained from tunicate samples and three among them exhibited an orange fluorescence in Nile red staining indicating the presence of PHB. One of the isolates, MCCB 284, which showed rapid growth and good polymer yield, was identified as V. harveyi. The optimum conditions of the isolate for the PHB production were pH 8·0, sodium chloride concentration 20gl-1 , inoculum size 0·5% (v/v), glycerol 20gl-1 and 72h of incubation at 30°C. Cell dry weight (CDW) of 3·2gl-1 , PHB content of 2·3gl-1 and final PHB yield of 1·2gl-1 were achieved. The extracted PHB was characterized by FTIR, NMR and DSC-TGA techniques. An isolate of V. harveyi that could effectively utilize glycerol for growth and PHB accumulation was obtained from tunicate P. nigra. PHB produced was up to 72% based on CDW. This is the first report of an isolate of V. harveyi which utilizes glycerol as the sole carbon source for PHB production with high biomass yield. This isolate could be of use as candidate species for commercial PHB production using glycerol as the feed stock or as source of genes for recombinant PHB production or for synthetic biology.

Optimization of Polyhydroxybutyrate Production by Bacillus safensis EBT1

The present study deals with the enhanced production of polyhydroxybutyrate (PHB) using a novel potent strain. On the basis of morphological, biochemical, and 16S rRNA analysis, the strain was identified as Bacillus safensis EBT1. The presence of PHB in the cells was identified and affirmed using fluorescent microscopy and Fourier transform infrared spectroscopy. The molecular weight of PHB was ascertained by gel permeation chromatography. Under optimized conditions, B. safensis EBT1 showed maximum PHB production and growth with glucose as the carbon source, yeast extract as the nitrogen source, and sugarcane bagasse as the substrate. The process parameters were optimized using central composite design: pH 7, 45 °C and 48 h incubation time. At these conditions, highest biomass concentration of 7.54 g/L and PHB production of 6.41 g/L were observed.

Role of polyhydroxybutyrate in mitochondrial calcium uptake

Polyhydroxybutyrate (PHB) is a biological polymer which belongs to the class of polyesters and is ubiquitously present in all living organisms. Mammalian mitochondrial membranes contain PHB consisting of up to 120 hydroxybutyrate residues. Roles played by PHB in mammalian mitochondria remain obscure. It was previously demonstrated that PHB of the size similar to one found in mitochondria mediates calcium transport in lipid bilayer membranes. We hypothesized that the presence of PHB in mitochondrial membrane might play a significant role in mitochondrial calcium transport. To test this, we investigated how the induction of PHB hydrolysis affects mitochondrial calcium transport. Mitochondrial PHB was altered enzymatically by targeted expression of bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. The expression of PhaZ7 induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential in HepG2 but not in U87 and HeLa cells. Furthermore, it significantly inhibited mitochondrial calcium uptake in intact HepG2, U87 and HeLa cells stimulated by the ATP or by the application of increased concentrations of calcium to the digitonin permeabilized cells. Calcium uptake in PhaZ7 expressing cells was restored by mimicking calcium uniporter properties with natural electrogenic calcium ionophore – ferutinin. We propose that PHB is a previously unrecognized important component of the mitochondrial calcium uptake system.

The effect of 3-hydroxybutyrate methyl ester on learning and memory in mice

Learning and memory requires energy-demanding cellular processes and can be enhanced when the brain is supplemented with metabolic substrates. In this study, we found that neuroglial cell metabolic activity was significantly elevated when cultured in the presence of polyhydroxybutyrate (PHB) degradation product 3-hydroxybutyrate (3-HB) and derivatives. We demonstrated that the receptor for 3-HB, namely, protein upregulated in macrophages by IFN-γ (PUMA-G), was expressed in brain and upregulated in mice treated with 3-hydroxybutyrate methyl ester (3-HBME). We also affirmed increased expression of connexin 36 protein and phosphorylated ERK2 (extracellular signal-regulated kinase 2) in brain tissues following 3-HBME treatment, although these differences were not statistically significant. Mice treated with 3-HBME performed significantly ( p < 0.05) better in the Morris water maze than either the negative controls (no treatment) or positive controls (acetyl- l-carnitine treatment). Moreover, we found that 3-HBME enhanced gap junctional intercellular communication between neurons. Thus, 3-HB and derivatives enhance learning and memory, possibly through a signaling pathway requiring PUMA-G that increases protein synthesis and gap junctional intercellular communication.

Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells.

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.