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Poly-(3-hydroxybutyrate), PHB, is a bacterial polyester in industrial demand as a biodegradable alternative to fossil-derived nondegradable plastics. Moreover, apart from being used directly as a bioplastic, valorization of PHB to its monomer building blocks and other value-added chemicals is feasible but less explored. In this study, Brønsted acid ionic liquid (BAIL) catalyzed depolymerization of PHB was investigated as a highly selective route to 3-hydroxybutyric acid, 3-HBA. The hydrolysis of PHB to 3-HBA was performed in a biphasic solvent medium composed of methyl isobutyl ketone (MIBK) and water, where the organic phase had dual roles as an efficient medium for dissolution of the polymer and as solvent for the monomeric products, which were enriched in this phase after cooling, with the Brønsted acid ionic liquid (BAIL) catalyst partitioned into the aqueous phase for facile recycling. The effects of reaction parameters, including the temperature, types of IL in terms of cations and anions, and the amount of water and IL, were studied to assess the yield of 3-HBA. Furthermore, protic acids such as sulfuric acid, methanesulfonic acid, and p-toluenesulfonic acid (p-TsOH) were also applied for comparison as acid catalysts for the hydrolysis of PHB to 3-HBA. Among the tested catalysts, the ILs containing the p-TsO- as anion as well as p-TsOH alone were found to be highly selective in promoting hydrolysis to 3-HBA, with complete depolymerization of PHB at >90% yield of 3-HBA in 4 h at 120 °C using a BAIL with sulfobutylated 1-methylimidazolium as the cation component and p-TsO- as the anion ([ImSO3H+][p-TsO-]). Although the use of p-TsOH as the sole catalyst also yielded efficient PHB hydrolysis with high reaction rates, it had a disturbing effect on the biphasic MIBK-water system by forming a single-phase reaction mixture at high 3-HBA yields, obstructing the recoveries of the products as well as the catalyst. In contrast, the biphasic reaction mixture remained intact when using IL as catalyst, which allowed facile and efficient separation of the product from the catalyst. Both the 3-HBA and the [ImSO3H+][p-TsO-] IL were recovered in high purity, the latter after applying a solvent extraction scheme based on ethyl acetate, whereby the recoveries of 3-HBA and IL reached ≈90%. The compositions of the synthesized ILs and the progress of the hydrolysis process, as well as the purity of the recovered product, were confirmed by NMR analysis. This sustainable approach to selective hydrolytic transformation of PHB into 3-HBA using a recoverable acidic IL catalyst in a biphasic solvent media of aqueous methyl isobutyl ketone hence resulted in efficient product separation and catalyst recovery.

Biomass from four different Nordic microalgal species, grown in BG-11 medium or synthetic wastewater (SWW), was explored as inexpensive carbohydrate-rich feedstock for polyhydroxybutyrate (PHB) production via microbial fermentation. Thermochemical pre-treatment (acid treatment followed by autoclavation) with 2% hydrochloric acid or 1% sulphuric acid (v/v) was used to maximize sugar yield prior to fermentation. Pre-treatment resulted in ∼5-fold higher sugar yield compared to the control. The sugar-rich hydrolysate was used as carbon source for the PHB-producing extremophilic bacterium Halomonas halophila. Maximal PHB production was achieved with hydrolysate of Chlorococcum sp. (MC-1) grown on BG-11 medium (0.27 ± 0.05 g PHB/ g DW), followed by hydrolysate derived from Desmodesmus sp. (RUC-2) grown on SWW (0.24 ± 0.05 g PHB/ g DW). Nordic microalgal biomass grown on wastewater therefore can be used as cheap feedstock for sustainable bioplastic production. This research highlights the potential of Nordic microalgae to develop a biobased economy.

The replacement of fossil-based plastics with biobased and biodegradable alternatives has become an important research challenge in recent years, aiming to eliminate the negative environmental impact of persistent plastics in nature. In this report, design of experiments was successfully exploited to develop an efficient and sustainable method for extracting intracellular PHA from Photobacterium ganghwense C2.2 using dihydrolevoglucosenone (Cyrene) and ethanol as biobased solvents obtainable from sustainable sources. The extraction conditions were studied and optimized against the yield and molecular weight. The temperature range for the extraction was scouted by using differential scanning calorimetry, while size exclusion chromatography coupled to refractive index and multiangle light scattering detectors was used to assess the molecular weights of the extracted polymers. The examined ranges in the model were, respectively, 1.6–8.4% (w/v) of lyophilized cells content per 10 mL of solvent, 3–17 min extraction time, and temperatures from 116 to 144 °C. Time and temperature strongly affected the extraction yields and molecular weights of the obtained polymers while the concentration of bacterial biomass only effected the molecular weight. Several quadratic and interaction coefficients were significant in the well-fit partial least-squares regression models (R² > 0.8, Q² > 0.6) indicating that nonlinear effects and interacting parameter contributed to the optimization targets. The optimized extraction should be performed at 130 °C for 15 min with 2% loading of bacterial biomass. The predicted yield and molecular weight of the polymer matched the values obtained from the real experiment under the optimized conditions. The method setup provided similar yield and higher molecular weight in much shorter time compared to overnight Soxhlet extraction with CHCl₃. The clean ¹H nuclear magnetic resonance spectra of polymers extracted from bacteria indicate that high purity materials can be obtained using an optimized extraction scheme. Additionally, the Cyrene solvent could be recycled at least five times and still performed the extraction equally well as the fresh solvent. Finally, the current method demonstrated a high potential for scalability using a HP4750 stirred filtration cell. Three different filtration conditions were tested, achieving up to 97.4% recovery at 80 °C using a 0.3 μm glass fiber membrane, with a flux of 312.5 LMH.

High-throughput screening (HTS) methods for characterization of microbial production of polyhydroxyalkanoates (PHA) are currently under investigated, despite the advent of such systems in related fields. In this study, phenotypic microarray by Biolog PM1 screening of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99 identified 49 and 54 carbon substrates to be metabolized by these bacteria, respectively. Growth on 15 (Halomonas sp. R5-57) and 14 (Pseudomonas sp. MR4-99) carbon substrates was subsequently characterized in 96-well plates using medium with low nitrogen concentration. Bacterial cells were then harvested and analyzed for putative PHA production using two different Fourier transform infrared spectroscopy (FTIR) systems. The FTIR spectra obtained from both strains contained carbonyl-ester peaks indicative of PHA production. Strain specific differences in the carbonyl-ester peak wavenumber indicated that the PHA side chain configuration differed between the two strains. Confirmation of short chain length PHA (scl-PHA) accumulation in Halomonas sp. R5-57 and medium chain length PHA (mcl-PHA) in Pseudomonas sp. MR4-99 was done using Gas Chromatography-Flame Ionization Detector (GC-FID) analysis after upscaling to 50 mL cultures supplemented with glycerol and gluconate. The strain specific PHA side chain configurations were also found in FTIR spectra of the 50 mL cultures. This supports the hypothesis that PHA was also produced in the cells cultivated in 96-well plates, and that the HTS approach is suitable for analysis of PHA production in bacteria. However, the carbonyl-ester peaks detected by FTIR are only indicative of PHA production in the small-scale cultures, and appropriate calibration and prediction models based on combining FTIR and GC-FID data needs to be developed and optimized by performing more extensive screenings and multivariate analyses.

In this report, 1-ethyl-3-methylimidazolium acetate, [EMIM][AcO] ionic liquid (IL) and acetic acid (AA) comprised solvents were used for the thermal treatment of poly (vinylidene difluoride), PVDF. Here, besides the various combinations of IL and AA in solvents, the pure IL and AA were also applied as a solvent upon thermal treatments. The samples obtained after the treatment were analysed for structural and crystalline phase changes, porosity, and molecular weight distribution with various analytical techniques. The Kamlet-Taft parameters measurement of the IL and AA containing solvents with different solvatochromic dyes was also performed to examine their solvent properties and correlate with the properties of the treated PVDF materials. The treatment of PVDF with pure IL results in the formation of highly carbonaceous material due to extensive dehydroflurination (DHF) as well as possibly successive cross-linking in the polymer chains. Upon IL and AA combined solvent treatment, the neat PVDF which composed of both α- and β crystalline phases was transformed to porous and β-phase rich material whereas in case of pure AA the non-porous and pure α-phase polymeric entity was obtained. A combined mixture of IL and AA resulted in a limited the DHF process and subsequent cross-linking in the polymer chains of PVDF allowed the formation of a porous material. It was observed that the porosity of the thermally treated materials was steadily decreasing with increase in the amount of AA in solvents composition and solvent with an AA:IL mole ratio of 2:1 resulted in a PVDF material with the highest porosity amongst the applied solvents. A recovery method for the IL and AA combined solvent after the thermal treatment of PVDF was also established. Hence, with varying the type of solvents in terms of composition, the highly carbonaceous materials as well as materials with different porosities as well as crystalline phases can be obtained. Most importantly here, we introduced new IL and AA containing recoverable solvents for the synthesis of porous PVDF material with the electroactive β-phase.

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain’s metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain’s complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.

Valorization of renewable and biodegradable biopolymers to value added chemicals and green fuels is currently considered as an important research topic aiming at reducing the dependency on fossil derived feedstocks as well as their negative consequences on the environment. In this report, we are introducing an ionic liquid (IL) mediated, sustainable, and green synthesis of crotonic acid (CA) from poly-(3-hydroxybutyrate, PHB), a biopolymer derived from microbial fermentation. In this actual case, imidazolium cation comprising ILs have been used in the synthesis, where the influence of various reaction parameters such as reaction temperature and types of ILs as well as the amount of polymer, water, and IL in the reaction mixture were examined. The conversion of PHB to CA in IL took place by a base catalyzed depolymerization with formation of crotonyl terminated polymeric entities as intermediates, a mechanism that was confirmed by NMR analysis of the reaction mixtures sampled when the reactions were carried out at various temperatures. The rate of CA formation via the IL mediated base catalyzed depolymerization increased with increasing temperature in the tested interval, and 97% yield of CA was obtained after 90 min at 140 °C. The [EMIM][AcO] IL applied as solvent and catalyst is capable of completely depolymerizing PHB to CA in 5 h at 120 °C up to a polymer loading of 40 wt%. At higher loadings the depolymerization became incomplete, which is attributed to a deactivation of the IL due to hydrogen bonding interactions with the in situ formed CA, confirmed by NMR and DSC techniques. Since the depolymerization is base catalyzed, the only tested ILs that were able to form CA were based on acetate anions, whereas the less basic or neutral [EMIM][Cl] IL was found to be inactive. Finally, more than 90% of CA as well as [EMIM][AcO] IL were recovered in high purity by solvent extraction with brine (saturated aqueous NaCl) and 2-methyl tetrahydrofuran (2-Me-THF). Most importantly, here we introduce a sustainable, metal free, and single solvent based reaction approach for selective depolymerization of PHB to industrially valuable CA in basic and recoverable ILs.

Background: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains.

Results: Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains.

Conclusions: Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.