Extraction Of Polyhydroxybutyrate Research Articles

Exploring 1,3-Dioxolane Extraction of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from Methylocystis hirsuta and Mixed Methanotrophic Strain: Effect of Biomass-to-Solvent Ratio and Extraction Time.

The increasing need for biodegradable polymers demands efficient and environmentally friendly extraction methods. In this study, a simple and sustainable method for extracting polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV) from Methylocystis hirsuta and a mixed methanotrophic consortium with different biopolymer contents was presented. The extraction of biopolymers with 1,3-dioxolane was initially investigated by varying the biomass-to-solvent ratio (i.e., 1:2 w v-1, 1:4 w v-1, 1:6 w v-1, 1:8 w v-1 and 1:10 w v-1) and extraction time (6, 8 and 10 h) at the boiling point of the solvent and atmospheric pressure. Based on the results of the preliminary tests, and only for the most efficient biomass-to-solvent ratio, the extraction kinetics were also studied over a time interval ranging from 30 min to 6 h. For Methylocystis hirsuta, the investigation of the extraction time showed that the maximum extraction was reached after 30 min, with recovery yields of 87% and 75% and purities of 98.7% and 94% for PHB and PHB-co-HV, respectively. Similarly, the extraction of PHB and PHB-co-HV from a mixed methanotrophic strain yielded 88% w w-1 and 70% w w-1 recovery, respectively, with 98% w w-1 purity, at a biomass-to-solvent ratio of 6 in 30 min.

Extraction of Environment-Friendly Biodegradable Poly-Hydroxy Butyrate Using Novel Hydrodynamic Cavitation Method

Polyhydroxy butyrate (PHB) is one of the best environment-friendly bioplastic alternatives for petroleum-based plastic due to its biodegradability. However, it has less commercial popularity owing to the high cost of downstream processing that involves repeated centrifugation and the use of costly harmful solvents, as well as a labor-intensive process. Hydrodynamic Cavitation (HC) offers easy and simple mechanisms for downstream processing. Also, biopolymer extracted for haloarchea show an advantage of least contamination under the halophilic condition on an industrial level. In this paper, a haloarchaeal consortium producing biopolymer isolated from commercial rock salt has been subjected to HC as well as distilled water lysis. A maximum of 23 g.L-1 PHB was extracted in 40 min run with 50 passes and 0.10 cavitation number at 3.9 bar pressure. The extracted biopolymer was characterized and was found to be PHB. Comparative analysis shows that HC results in a substantial reduction in the downstream processing time. Moreover, it has double the efficiency of PHB extraction as compared to the distilled water lysis method. This paper reports the HC process as a techno-commercial alternative to industrial PHB extraction.

Evaluation of the production and extraction of polyhydroxybutyrate from volatile fatty acids by means of mixed cultures and B. cepacia

The global consumption of plastics generates accelerated environmental pollution in landfills and marine ecosystems. Biopolymers are the materials with the greatest potential to replace synthetic polymers in the market due to their good biodegradability, however, there are still several disadvantages, mainly related to their production cost. Considering the above, the generation of biodegradable and biocompatible bioplastics stands out as an alternative solution, some of which are made from renewable raw materials, including polyhydroxyalkanoates PHAs. Although much research has been done on bacteria with the capacity for intracellular accumulation of PHAs, among others, it is also possible to produce PHAs using mixed microbial cultures instead of a single microorganism, using natural microbial consortia that have the capacity to store high amounts of PHAs. In this contribution, three methods for the extraction and purification of PHAs produced by fermentation using volatile fatty acids as a carbon source at different concentrations were evaluated, using the pure strain Burkholderia cepacia 2G-57 and the mixed cultures of the activated sludge from the El Salitre WWTP, in order to select the best method from the point of view of environmental sustainability as this will contribute to the scalability of the process. The mixed cultures were identified by sequencing of the 16S gene. A yield of 89% was obtained from the extraction and purification of PHA using acetic acid as a solvent, which according to its properties is "greener" than chloroform. The polymer obtained was identified as polyhydroxybutylated PHB.

An efficient and reusable N,N-dimethylacetamide/LiCl solvent system for the extraction of high-purity polyhydroxybutyrate from bacterial biomass

Multiple methods have been developed for the extraction of the bioplastic polyhydroxybutyrate (PHB) from bacteria. Many of these approaches suffer from low efficiency, are toxic, non-reusable, complex, and costly, which prompts the development of alternative processes. The N,N-dimethylacetamide/LiCl (DMAc/LiCl) solvent system is a widely employed, reusable, and simple method for the solubilization of cellulose and other biological macromolecules. Since bacteria are mostly constituted of polysaccharides, proteins, and other polymers, it may be possible to employ DMAc/LiCl to dissolve them and extract bioplastics. Here, the DMAc/LiCl system was adapted for the breakage of PHB-producing Cupriavidus necator cells combined with the extraction of the polyester. After screening for optimized conditions, the extraction was carried out by incubating 200 mg of dry biomass at 110 ℃ for three hours in DMAc/LiCl. With these parameters, the recovery rate was 95.3% with an excellent PHB purity of 99.0%. Furthermore, the solvent could be reused five times with a minimal loss of efficiency. Extracted PHB characterization indicated that it exhibited physicochemical properties comparable to commercial PHB including its molecular weight, thermal stability, and crystallinity. Thus, the DMAc/LiCl-based extraction of high-purity PHB has the potential to become a widespread method because of its efficiency, recyclability, and convenience.

Polyhydroxybutyrate (PHB) production by Halomonas boliviensis isolated from waste water

Plastic materials are often utilized as plastic packaging and have various uses since their longevity and consistency. Plastic products are generally not biodegradable or persist in our surroundings, posing major harm to the ecology. Biopolymers, or bioplastics, are developing as a blessing in the fight against waste buildup. Polyhydroxybutyrate (PHB) is a biopolymer that can be used instead of manufactured polymers. PHB is a fatty storage substance that accumulates inside microbes' cell walls when stressed. Halophilic microbes can be highly useful in manufacturing PHB since they are cheap, and PHB extraction is considerably easy in halo-tolerant species. As a result, our research emphasizes the identification of PHB-generating halotolerant species of microbes using untreated wastewater. Nile blue A and Sudan Black B staining were used to identify PHB favourable strains. The effective microbe generated PHB on a large scale utilizing sewage as the medium.

Isolation and Extraction of PHB from Soil Isolate: Determining its Physio- Chemical characteristics with an application study in Biomedical

Biopolymer Poly Hydroxy Butyrate (PHB) is gaining importance based on its physiochemical and biological characteristics. Hence, the characteristics of the PHB extracted from soil bacterial isolate as studied with an aim of developing a 2D films for biomedical applications. The isolates from soil samples were screened for the ability to produce PHB and confirmed using Sudan black staining methods. The physio-chemical characteristics were studied using FTIR, FESEM and GCMS analysis. FTIR spectrum obtained for the extracted polymer shows peaks at 1723.51 cm-1and 1287.63 cm-1 which confirms that the extracted polymer is PHB. FESEM image reveals the presence of pores of different size throughout the sample. The surface consists of multiple pores with different sizes. GC-MS analysis revealed the presence of various compounds attributing for the PHB production from the isolate. As a biological characteristic, a novel wound dressing 2D laminate bio composite films were developed using PHB extracts. The antibacterial activity of the developed film showed significant inhibitory zones against Escherichia coli and Klebsiella pneumoniae. The present research work showed promising results and proved that the PHB obtained from bacterial sp. can be used as an alternative to non-degradable plastics with a wide range of applications in biomedical industries.

Chemical Recycling of Polyhydroxybutyrate (PHB) into Bio-Based Solvents and Their Use in a Circular PHB Extraction

Two novel protocols for the chemical valorization of polyhydroxybutyrate (PHB) were developed, aiming at the production of two bio-based molecules: methyl 3-hydroxybutyrate (MHB) and methyl 3-methoxybutyrate (MMB). Optimized reaction conditions were applied to pure PHB and PHB inclusions inside bacterial cells as starting materials. MHB was synthesized through a single-step catalytic methanolysis, while MMB was synthesized through a three-step process: thermolytic distillation to give crotonic acid (CA), esterification to give methyl crotonate (MC), and oxa-Michael addition of MeOH. The obtained MHB and MMB were tested as solvents for the recovery of PHB itself both from freeze-dried single strain cultures (SSC) and mixed microbial cultures (MMC) with low to medium contents of PHB (22–57 wt %). High PHB recovery was achieved: up to 96 ± 1% through MHB and up to 98 ± 1% through MMB. Extraction from MMC slurry (with a PHB content of 39% on dry weight) was also performed, recovering 77 ± 2% using MHB and 92 ± 2% using MMB. High purities and excellent molecular weights and polydispersity indexes of extracted PHB were obtained with both MHB and MMB. Solubility in water, octanol/water partition coefficients (log Kow), and aerobic ready biodegradability of both solvents were also evaluated.

Novel insights in dimethyl carbonate-based extraction of polyhydroxybutyrate (PHB)

BackgroundPlastic plays a crucial role in everyday life of human living, nevertheless it represents an undeniable source of land and water pollution. Polyhydroxybutyrate (PHB) is a bio-based and biodegradable polyester, which can be naturally produced by microorganisms capable of converting and accumulating carbon as intracellular granules. Hence, PHB-producing strains stand out as an alternative source to fossil-derived counterparts. However, the extraction strategy affects the recovery efficiency and the quality of PHB. In this study, PHB was produced by a genetically modified Escherichia coli strain and successively extracted using dimethyl carbonate (DMC) and ethanol as alternative solvent and polishing agent to chloroform and hexane. Eventually, a Life Cycle Assessment (LCA) study was performed for evaluating the environmental and health impact of using DMC.ResultsExtraction yield and purity of PHB obtained via DMC, were quantified, and compared with those obtained via chloroform-based extraction. PHB yield values from DMC-based extraction were similar to or higher than those achieved by using chloroform (≥ 67%). To optimize the performance of extraction via DMC, different experimental conditions were tested, varying the biomass state (dry or wet) and the mixing time, in presence or in absence of a paper filter. Among 60, 90, 120 min, the mid-value allowed to achieve high extraction yield, both for dry and wet biomass. Physical and molecular dependence on the biomass state and solvent/antisolvent choice was established. The comparative LCA analysis promoted the application of DMC/ethanol rather than chloroform/hexane, as the best choice in terms of health prevention. However, an elevated impact score was achieved by DMC in the environmental-like categories in contrast with a minor contribution by its counterpart.ConclusionThe multifaceted exploration of DMC-based PHB extraction herein reported extends the knowledge of the variables affecting PHB purification process. This work offers novel and valuable insights into PHB extraction process, including environmental aspects not discussed so far. The findings of our research question the DMC as a green solvent, though also the choice of the antisolvent can influence the impact on the examined categories.

Production of blue-colored polyhydroxybutyrate (PHB) by one-pot production and coextraction of indigo and PHB from recombinant Escherichia coli

Polyhydroxyalkanoate (PHA) is a promising substitute for petroleum-based plastics. For economically feasible and sustainable production of PHA, various strains and feedstocks have been reported till date, using which, polyhydroxybutyrate (PHB) could be produced on a large scale. However, the sensory characteristics of PHA, such as color, limit its further application in industries related to packaging, textiles, and medical materials. In this study, we investigated the feasibility of simultaneous production of indigo and PHB in recombinant Escherichia coli to generate colored bioplastics. With that objective, one-pot production and extraction of both indigo and PHB were successfully performed, and biotransformation of indole to indigo was reinforced by the introduction of PHA synthetic genes. Upon further investigation of the factors responsible for the increased indigo production, groEL and grpE, related to heat-shock proteins, and CYP102G4, were found to be up-regulated when analyzed by semi-quantitative reverse transcription-PCR. In the thermograms analyzed by thermal gravimetric analysis and differential scanning calorimetry, there was no notable difference between PHB and PHB-indigo film, implying that indigo did not affect the thermal properties of PHB itself. Overall, this study demonstrates the production of blue-colored PHB and enhanced production of indigo by the introduction of PHA synthetic genes.

The bioextraction of bioplastics with focus on polyhydroxybutyrate: a review

Use of bioplastics such as polyhydroxybutyrate (PHB) is increasing steadily. PHB extraction methods including solvent extraction and chemical digestion surfactant require the harmful reagents and result in severe quantitative and qualitative environmental and economic losses. Due to the friendly properties, biological extraction methods (called bioextraction methods) are gaining more attention to PHB extraction. Bioextraction methods can reduce the serious concerns in environmental pollution and cost by green tools such as genetic and metabolic engineering of PHB-accumulating cells in order to self-disrupt, induce, and modify predatory bacteria, and the utilization of mealworm digestive system. Through biotechnology mechanisms, a purified PHB biopolymer can be extracted by manufacturers in a eco-friendly system, while water and acetic acid can then be used for pre-treatment other than SDS and chloroform. As in many aspects of bioextraction, the cell engineering is accompanied by the modification of culture condition and feedstocks in order to bypass conventional approaches and their drawbacks, although these problems are yet to be solved, it can, however, be reduced by increasing the use of biotechnological approaches in bioextraction methods. The integration of biological with conventional approaches can decrease environment limitations and costs through genetically modified producers and culture optimization, and therefore establishing suitable and commercially viable extraction methods. However, PHB bioextraction through green technology may be advantageous over conventional methods with maximum impact on human health and environment. This review discusses about using bioextraction methods as green technologies for PHB extraction from bacterial producer cells with less environmental limitation.

Isolation of Polyhydroxybutyrate (PHB) Producing Bacteria, Optimization of Culture Conditions for PHB production, Extraction and Characterization of PHB

Polyhydroxybutyrates (PHBs) are energy reserves synthesized by different micro-organisms such as Alcaligenes, Pseudomonas, Staphylococcus, Algae, in excess of carbon and limitation of nutrients like nitrogen. These biopolymers are suitable alternate to synthetic carbon-based polymers. However, the high production cost limits their commercialization. The aim of this study was thus, focused on optimization of culture condition for maximum PHB production in an attempt to reduce the production cost. The micro-organisms for this purpose were isolated from 4 different soil samples and screened for PHB production. Culture conditions for these organisms were optimized by changing the parameters, viz., incubation time, pH, carbon source and NaCl concentration. Thus, optimized culture condition was used to culture the isolates for extraction of PHB and its analysis. The extracted compounds on FTIR-analysis gave characteristic C=O peak of PHB, thus, confirming the seven isolates to be PHB producers. Results for optimized parameters for the isolated PHB positive species showed that synthesis of PHB was maximum at 48 hours i.e. during the early stages of stationary phase. However, different isolates favored different culture conditions. Highest PHB accumulation and growth of isolates were seen at pH 7 and 9. Similarly, it was observed that glucose was favored by 4 isolates and sucrose was favored by 3 isolates. Interestingly, NaCl concentration did not cause significant effect on neither the bacterial growth nor the PHB production. During the extraction of PHB from the optimized culture conditions, extraction of PHB from broth gave significant yield than that from agar. A good PHB yield from broth amounting to 36.41% and 34.59% was observed for Bacillus pasteurii and Micrococcus luteus respectively, showing a potential for their exploitation in industrial PHB production. At optimized conditions, 7 isolates exhibited significant PHB yields, thus showing a potential for further exploitation.

Polyhydroxybutyrate (PHB) Synthesis by Spirulina sp. LEB 18 Using Biopolymer Extraction Waste.

The reuse of waste as well as the production of biodegradable compounds has for years been the object of studies and of global interest as a way to reduce the environmental impact generated by unsustainable exploratory processes. The conversion of linear processes into cyclical processes has environmental and economic advantages, reducing waste deposition and reducing costs. The objective of this work was to use biopolymer extraction waste in the cultivation of Spirulina sp. LEB 18, for the cyclic process of polyhydroxybutyrate (PHB) synthesis. Concentrations of 10, 15, 20, 25, and 30% (v/v) of biopolymer extraction waste were tested. For comparison, two assays were used without addition of waste, Zarrouk (SZ) and modified Zarrouk (ZM), with reduction of nitrogen. The assays were carried out in triplicate and evaluated for the production of microalgal biomass and PHB. The tests with addition of waste presented a biomass production statistically equal to ZM (0.79gL-1) (p < 0.1). The production of PHB in the assay containing 25% of waste was higher when compared to the other cultivations, obtaining 10.6% (w/w) of biopolymer. From the results obtained, it is affirmed that the use of PHB extraction waste in the microalgal cultivation, aiming at the synthesis of biopolymers, can occur in a cyclic process, reducing process costs and the deposition of waste, thus favoring the preservation of the environment.

Comparison of different solvents for extraction of polyhydroxybutyrate from Cupriavidus necator.

Polyhydroxybutyrate (PHBs) have attracted much attention due to their biodegradability and biocompatibility properties. The main drawback to the commercial production of them is their high cost. The recovery of PHB from bacterial cytoplasm significantly increases total processing costs. Efficient, economical, and environment-friendly extraction of PHB from cells is required for its industrial production. In the present study, several nonhalogenated organic solvents (ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, and acetic acid) were examined for their efficacy regarding recovery at different temperatures from culture broth containing Cupriavidus necator cells. The highest recovery percentage (98.6%) and product purity (up to 98%) were seen to be those of ethylene carbonate-assisted extraction at 150°C within 60min of incubation time. Average molecular weight of the recovered PHB (1.3×106) was not significantly affected by the extraction solvent and conditions. The melting point of PHB extracted using ethylene carbonate was measured to be 176.2°C with an enthalpy of fusion of 16.8% and the corresponding degree of crystallinity of 59.2%. NMR and GC analyses confirmed that the extracted biopolymer was PHB. The presented strategy can help researchers to reduce the cost to obtain the final product.

Characterization of Polyhydroxybutyrate (PHB) Produced by Novel Bacterium Lysinibacillus sphaericus BBKGBS6 Isolated From Soil

The aim of the study was to characterization of polyhydroxybutyrate (PHB) produced by novel bacterium Lysinibacillus sphaericus BBKGBS6 isolated from soil. The present study reports that the strain L. sphaericus BBKGBS6, which was isolated from agricultural soil and is capable of producing PHB. Extraction of PHB was done by solvent extraction method. The results indicated the presence of crotonic acid and confirmed the presence of polyhydroxybutyrate in the sample. The FTIR spectra were observed characteristic absorption bonds for ester and the presence of C=O and C–O were obtained. GCMS results showed the major molecular fragmentation were, 117 m/z (C5H9O3 +), 104 m/z (C4H7O3 +), 74 m/z (C3H6O2 +), 61 m/z (C2H3O2 +), 43 m/z (C2H3O), 59 m/z (C2H3O). 1H and 13C NMR spectra were recorded using purified samples. The molecular weight of PHB (5.64 × 105) was estimated based on viscosity measurement. Films were prepared by the solvent casting method. The structure of crystalline polymers can be determined or refined through best fitting of X-ray powder diffraction profiles. The procedures provide results grossly in agreement with respect to the confirmation of the chain in the powder but differing significantly on a more detailed scale. Such differences represent an additional reason of interest in performing a new structural study. Differential scanning calorimetric experiments was performed using Universal V4.5A TA Instruments, (m.p. 156.61 °C; ΔH = 28.54 J/g) USIC Dharwad. The crystallinity (Xc) of PHB is calculated as per equation given below $${{X}_{c}}\,=\,{D}{{H}_{f}} \times {1}00/{D}{{H}_0} \times {W}$$ . The PHB extracted, PHB Sigma and PHB–TS showed two endothermal peaks in between 140 and 200 °C. The enthalpy of melting (ΔHf) was 28.09 J/g for standard PHB and for extracted one is 56.42 J/g. The glass transition temperature of the sample was 140 °C and amorphous temperature was 176.08 °C. Thermo gravimetric analysis (TGA) is a method of thermal analysis in which changes in physical and chemical properties of materials are measured as a function of increasing temperature (with constant heating rate), or as a function of time (with constant temperature and/or constant mass loss). The decomposition temperature at a 10% level determined by TGA for pure PHB in ScCO2 at 70 °C and 22 MPa was 293.32 °C. Tensile strength of PHB film (28.23 Mpa) was carried out according to ASTMD 882 using universal testing machine (Model Lx 5, LYOD ISNT). Water vapor transmission rate of PHB film (29 g/m2/day) was measured as per ASTM E96-95 and carried out according to the desiccant method. Oxygen transmission rate of PHB film 472.36 (cc/m2/day/atm 65% Rh and 27 °C) was measured as per the method of ASTM D-1434-66.

A Novel Affinity Chromatographic Material for the Purification of Extracellular Polyhydroxybutyrate Depolymerases

A novel affinity chromatographic material, which is composed of silica matrix, coated with polyhydroxybutyrate (PHB) powder, suitable for the purification of PHB depolymerases, was developed. The surface morphology of the PHB-silica coated particles (silica-PHB composite particles) was examined by scanning electron microscopy and revealed a successful uniform coating of silica particles with PHB. Moreover, the complex of these materials retained its homogeneity even after incubation at 80 °C for 6 h, whereas the strong binding of PHB on silica surface was further verified by thermal gravimetric analysis and by PHB extraction- from silica surface- experiments. This novel material was demonstrated to be suitable for both, the one-step on-batch and on-column purification of Thermus thermophilus extracellular PHB depolymerase. The enzyme exhibited higher affinity against the composite of silica-PHB particles than PHB powder, since the one-step purification-fold and the overall recovery of the enzyme were 2.8 and 4 times higher respectively, in the first case. Reusability of the silica-PHB composites particles was examined by determining the recoveries of PHB depolymerase. The enzyme recoveries were ranged from 30 to 35% for the first five uses, whereas for further uses recoveries gradually dropped to 15–18% indicating that the particles could be used repeatedly for five times. This material could be also a suitable support for lipases or other proteins that exhibit strong affinity to hydrophobic materials.

Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by solvent extraction with 1,2‐propylene carbonate

AbstractAn integrated procedure for the recovery of polyhydroxybutyrate (PHB) produced by Cupriavidus necator based on the extraction with 1,2‐propylene carbonate was evaluated. The effect of temperature (100–145°C) and contact time (15–45 min), precipitation period, and biomass pretreatments (pH shock and/or thermal treatments) on PHB extraction efficiency and polymer properties was evaluated. The highest yield (95%) and purity (84%) were obtained with the combination of a temperature of 130°C and a contact time of 30 min, with a precipitation period of 48 h. Under these conditions, PHB had a molecular weight of 7.4×105, which was the highest value obtained. Lower values (2.2×105) were obtained for higher temperatures (145°C), while lower temperatures resulted in incomplete extraction yields (45–54%). No further yield improvement was achieved with the pH/heat pretreatments, but the polymer's molecular weight was increased to 1.3×106. The PHB physical properties were not significantly affected by any of the tested procedures, as shown by the narrow ranges obtained for the glass transition temperature (4.8–5.0°C), melting temperature (170.1–180.1°C), melting enthalpy (77.8–88.5 J/g) and crystallinity (55–62%). 1,2‐Propylene carbonate was shown to be an efficient solvent for the extraction of PHB from biomass. The precipitation procedure was found to highly influence the polymer recovery and its molecular weight. Although polymer molecular weight and purity were improved by applying pH/heat pretreatment to the biomass, the procedure involves the use of large amounts of chemicals, which increases the recovery costs and makes the process environmentally unfriendly.