Thermal Reprocessing and Closed-Loop Chemical Recycling of Styrene-Butadiene Rubber Enabled by Exchangeable and Cleavable Acetal Linkages.

The covalent cross-linking is an essential prerequisite for achieving the unique entropic elasticity of rubber products; however, the formation of a 3D cross-linked network and permanent cross-links makes thermosetting rubbers difficult to be recycled, causing serious environmental pollution at the end of their life. Herein, a facile, green, and promising strategy to introduce the exchangeable and cleavable acetal bonds into the chemically cross-linked networks of diene-typed rubbers is reported. For the first time, the hydroxyl-functionalized styrene-butadiene rubber (ESBR-HEMA) is prepared by introducing 2-hydroxyethyl methacrylate (HEMA) during the emulsion polymerization of styrene-butadiene rubber (ESBR). Then, based on hydroxyl-vinyl ether addition reactions, divinyl ether (DVE) could serve as a cross-linking agent to facilely and effectively cross-link hydroxyl-functionalized rubbers without additional additives, producing exchangeable and hydrolyzable acetal linkages. What's more, the acetal-containing cross-linked network in ESBR-HEMA vulcanizates could rearrange their topologies at elevated temperatures, endowing them with malleable and thermal reprocessing abilities. Moreover, the hydrolyzable acetal bonds could be selectively cleaved into hydroxyl and aldehyde groups in acidic conditions, resulting in a closed-loop chemical recycling of the ESBR-HEMA rubber. Hence, this work provides a facile and green cross-linking strategy for hydroxyl-functionalized rubbers to address the inherent problems brought from the covalent cross-linking of rubbers.

Thermoresponsive hydrophobic copolymer grafted on agar microspheres for all-aqueous bioseparations.

Agar microspheres were prepared by water-oil emulsification and cross-linked under alkaline condition. The thermoresponsive hydrophobic copolymer, poly(N-isopropylacrylamide-co-lauryl methacrylate-co-acrylamide), was grafted on the agar microspheres via atom transfer radical polymerization. The agar microspheres grafted with copolymers were characterized by light microphotography, elemental analysis, Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The chain lengths and hydrophobic monomer ratio of the grafting linear polymer had significant effects on the hydrophobicity and adsorption capacity of agar microspheres at different temperatures. The thermoresponsive microspheres were used for separation of proteins and showed binding and release behavior by change of temperatures without change in mobile phase composition. Thus, we suggest thermoresponsive agar microspheres as an alternative separation media for all-aqueous bioseparations.

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

BACKGROUND: High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically. RESULTS: Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively. CONCLUSIONS: The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.

Photo-Modulated Therapeutic Protein Release from a Hydrogel Depot Using Visible Light.

The use of biomacromolecular therapeutics has revolutionized disease treatment, but frequent injections are required owing to their short half-life in vivo. Thus there is a need for a drug delivery system that acts as a reservoir and releases the drug remotely "on demand". Here we demonstrate a simple light-triggered local drug delivery system through photo-thermal interactions of polymer-coated gold nanoparticles (AuNPs) inside an agarose hydrogel as therapeutic depot. Localized temperature increase induced by the visible light exposure caused reversible softening of the hydrogel matrix to release the pre-loaded therapeutics. The release profile can be adjusted by AuNPs and agarose concentrations, light intensity and exposure time. Importantly, the biological activity of the released bevacizumab was highly retained. In this study we demonstrate the potential application of this facile AuNPs/hydrogel system for ocular therapeutics delivery through its versatility to release multiple biologics, compatibility to ocular cells and spatiotemporal control using visible light.

Site-Specific and High-Loading Immobilization of Proteins by Using Cohesin-Dockerin and CBM-Cellulose Interactions.

Immobilization of enzymes enhances their properties for application in industrial processes as reusable and robust biocatalysts. Here, we developed a new immobilization method by mimicking the natural cellulosome system. A group of cohesin and carbohydrate-binding module (CBM)-containing scaffoldins were genetically engineered, and their length was controlled by cohesin number. To use green fluorescent protein (GFP) as an immobilization model, its C-terminus was fused with a dockerin domain. GFP was able to specifically bind to scaffoldin via cohesin-dockerin interaction, while the scaffoldin could attach to cellulose by CBM-cellulose interaction. Our results showed that this mild and convenient approach was able to achieve site-specific immobilization, and the maximum GFP loading capacity reached ∼0.508 µmol/g cellulose.

Layer-by-Layer Assembly of Metal-Organic Frameworks in Macroporous Polymer Monolith and Their Use for Enzyme Immobilization.

New monolithic materials comprising zeolitic imidazolate framework (ZIF-8) located on the pore surface of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith previously functionalized with N-(3-aminopropyl)-imidazole have been prepared via a layer-by-layer self-assembly strategy. These new ZIF-8@monolith hybrids are used as solid-phase carriers for enzyme immobilization. Their performance is demonstrated with immobilization of a model proteolytic enzyme trypsin. The best of the conjugates enable very efficient digestion of proteins that can be achieved in mere 43 s.

Separation of capsaicin from capsaicinoids by macroporous resin adsorption chromatography.

The aim of present study is to develop an efficient and low-cost method for capsaicin production isolated from capsaicinoids by macroporous resin adsorption chromatography. HZ816 resin has shown the best adsorption and desorption capacities for capsaicin among other resins. To optimize the operating parameters for separation, initial concentration, diameter-to-height ratio, mobile phase ratio, and crystallization method were investigated. When capsaicinoids solution (5 g/L) was loaded onto the column (diameter-to-height ratio = 1:12) with ethanol/1% w/w NaOH (4:6, v/v) as the mobile phase, capsaicin was purified most effectively. By using acid neutralization as the crystallization method, the purity of capsaicin improved from 90.3 to 99.5% with 82.3% yield. In conclusion, this study provides a simple and low-cost method for the industrial-scale production of high-purity capsaicin.

Enhanced production of poly-3-hydroxybutyrate by Escherichia coli over-expressing multiple copies of NAD kinase integrated in the host genome.

OBJECTIVES: With the help of attB-attP recombination technique, multiple copies of yfjB gene encoding the NAD kinase of Escherichia coli were inserted into the host chromosome to promote NADPH-dependent poly-3-hydroxybutyrate (PHB) production. RESULTS: The yfjB integration mutant E. coli T2 harbored a similar metabolic profile to that of the wild type control. When PHB biosynthesis operon was introduced, the yfjB integration mutant produced 3 g PHB l(-1) from 18.2 g glucose l(-1), while the wild type consumed 15.7 g glucose l(-1) to afford 2.34 g PHB l(-1) in 48 h of shake-flask cultivation. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by six to eightfold with yfj Bover-expression, which may be the primary reason for the improved PHB production. CONCLUSION: A practical method is demonstrated to construct genetically-stable strains harboring extra copies of NAD kinase to enhance NADPH-dependent reactions.

Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production.

Yeast to directly convert cellulose and, especially, the microcrystalline cellulose into bioethanol, was engineered through display of minicellulosomes on the cell surface of Saccharomyces cerevisiae. The construction and cell surface attachment of cellulosomes were accomplished with two individual miniscaffoldins to increase the display level. All of the cellulases including a celCCA (endoglucanase), a celCCE (cellobiohydrolase), and a Ccel_2454 (ß-glucosidase) were cloned from Clostridium cellulolyticum, ensuring the thermal compatibility between cellulose hydrolysis and yeast fermentation. Cellulases and one of miniscaffoldins were secreted by α-factor; thus, the assembly and attachment to anchoring miniscaffoldin were accomplished extracellularly. Immunofluorescence microscopy, flow cytometric analysis (FACS), and cellulosic ethanol fermentation confirmed the successful display of such complex on the yeast surface. Enzyme-enzyme synergy, enzyme-proximity synergy, and cellulose-enzyme-cell synergy were analyzed, and the length of anchoring miniscaffoldin was optimized. The engineered S. cerevisiae was applied in fermentation of carboxymethyl cellulose (CMC), phosphoric acid-swollen cellulose (PASC), or Avicel. It showed a significant hydrolytic activity toward microcrystalline cellulose, with an ethanol titer of 1,412 mg/L. This indicates that simultaneous saccharification and fermentation of crystalline cellulose to ethanol can be accomplished by the yeast, engineered with minicellulosome.

New agar microspheres for the separation and purification of natural products.

A new type of agar chromatography media has been prepared with a yield over 80% using a water-in-oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40-165 µm (average ∼90 µm). Cross-linking of the resulting agar microspheres with epichlorohydrin and 1,4-butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross-linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross-linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion-exchange mode and (ii) with poly-ß-cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.

Bioethanol from lignocellulosic biomass: current findings determine research priorities.

"Second generation" bioethanol, with lignocellulose material as feedstock, is a promising alternative for first generation bioethanol. This paper provides an overview of the current status and reveals the bottlenecks that hamper its implementation. The current literature specifies a conversion of biomass to bioethanol of 30 to ~50% only. Novel processes increase the conversion yield to about 92% of the theoretical yield. New combined processes reduce both the number of operational steps and the production of inhibitors. Recent advances in genetically engineered microorganisms are promising for higher alcohol tolerance and conversion efficiency. By combining advanced systems and by intensive additional research to eliminate current bottlenecks, second generation bioethanol could surpass the traditional first generation processes.

Surfactant-assisted dilute ethylenediamine fractionation of corn stover for technical lignin valorization and biobutanol production.

In this study, a surfactant-assisted diluted ethylenediamine (EDA) fractionation process was investigated for co-generation of technical lignin and biobutanol from corn stover. The results showed that the addition of PEG 8000 significantly enhanced cellulose recovery (88.9 %) and lignin removal (68.9 %) in the solid fraction. Moreover, the pulp achieved 86.5 % glucose yield and 82.6 % xylose yield in enzymatic hydrolysis. Structural characterization confirmed that the fractionation process promoted the preservation of active ß-O-4 bonds (35.8/100R) in isolated lignin and functionalized the lignin through structural modification using EDA and surfactant grafting. The enzymatic hydrolysate of the pulps yielded a sugar solution for acetone-butanol-ethanol (ABE) fermentation, resulting in an ABE concentration of 15.4 g/L and an overall yield of 137.2 g/Kg of dried corn stalk. Thus, the surfactant-assisted diluted EDA fractionation has the potential to enhance the overall economic feasibility of second-generation biofuels production within the framework of biorefinery.

Interacting polymer-modification enzymes in heparan sulfate biosynthesis.

Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcAß1-4GlcNSO3α1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluorescence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a functional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O-sulfated glucosamine residues. The inability to achieve simultaneous 2-O- and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme interactions in heparan sulfate biosynthesis.

Preparation of monomeric and polymeric ß-cyclodextrin functionalized monoliths for rapid isolation and purification of puerarin from Radix puerariae.

Monomeric and epichlorohydrin polymerized ß-CD functionalized monoliths were prepared for the rapid isolation and purification of the isoflavonoid puerarin, a well-known traditional Chinese drug, from a crude extract of Radix puerariae (root of the plant Pueraria lobata). Two copolymers poly(isocyanatoethyl methacrylate-co-methyl methacrylate-co-ethylene dimethacrylate) (poly(IEM-co-MMA-co-EDMA)) and poly(glycidyl methacrylate-co-EDMA) (poly(GMA-co-EDMA)) were developed as facile, highly reactive and versatile monolithic matrix. SEM characterization demonstrated that the modified monoliths had homogenous porous structure and morphology. The success of the chemical modification of the monolithic matrix was confirmed by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), solid-state (13) C NMR and elemental analysis. It was demonstrated that polymeric ß-CD modified monoliths had better separation and selectivity for puerarin, recovering puerarin with a purity of 96% (m%) and a yield of 93% (m%). Compared with poly(glycidyl methacrylate-co-EDMA), poly(isocyanatoethyl methacrylate-co-methyl methacrylate-co-EDMA) monolithic matrix had higher reactivity, which significantly improved the ß-CD ligand density and thus the selectivity of the monoliths. Puerarin with a purity of 96% (m%) and with a yield of 89% (m%) was recovered on the monolith.

Molecularly imprinted polymers for the selective recognition of microorganisms.

Molecularly imprinted polymers (MIPs) emerged half a century ago have now attracted tremendous attention as artificial receptors or plastic antibodies. Although the preparation of MIPs targeting small molecules, peptides, or even proteins is straightforward and well-developed, the molecular imprinting of microorganisms still remains a big challenge. This review highlights the preparation of MIPs that reveal biomimetic specificity and selectivity towards microorganisms by creating the well-defined cell recognition sites. We present the state-of-the-art strategies for the expeditious synthesis of MIPs targeting microorganism including surface components imprinting, cell mediated lithography, and microcontact stamping. These receptor-like biomimetic materials have garnered increasing attention in different fields. In this review, we also describe the diverse applications of microorganism-imprinted polymers such as microbial activation, microbial fuel cells, and microorganism detection and sensing. The major challenges and further prospects on the design of microorganism-imprinted polymers is also outlined.

Selective binding of heparin oligosaccharides in a magnetic thermoresponsive molecularly imprinted polymer.

Heparin is a highly sulfated polysaccharide, applied in clinic for treatment of thrombotic diseases. The biological activity is closely related to its molecular structure e.g. compositions of disaccharides and oligosaccharides units. The classical method to isolate the oligosaccharides after depolymerization by heparinases or nitrous acid I s by size exclusion chromatography which is a time-consuming process. In this study, we explored the possibility for rapid separation of oligosaccharides using a novel polymer material. The magnetic thermoresponsive molecularly imprinted polymers (MIPs) were synthesized using heparin disaccharide as a template, AEM, NIPAAm, and AAm as functional monomer, and MBAA as crosslinker by surface radical polymerization in an aqueous media. Incubation of the MIP with hepairn oligosaccharides demonstrated specific binding to the template molecule. This binding to the targeted molecule was affected by reaction temperature with regard to binding capacity and specificity. The recognition specificity and selectivity can be modulated by varying the compositions of multi-functional monomers. The pseudo-second-order kinetic model and Langmuir isotherm model provide the best fit to the equilibrium adsorption of heparin disaccharides by MIPs. The results suggest that the new material can be used for rapid separation of di- and tetra-saccharides of heparin, which can also be adapted to the applications for isolation of oligosaccharides from other polysaccharides, e.g. heparan sulfate and chondoriting sulfate.

An novel immobilization method of Saccharomyces cerevisiae to sorghum bagasse for ethanol production.

Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6+/-0.2g dry cell weight (DCW)/g dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200g/L, nearly 100% total sugar was consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(Lh), respectively. The immobilized cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(Lh) appeared at the dilution rate of 0.3h(-1).

Preparation and characterization of polyethyleneimine modified ion-exchanger based on poly(methacrylate-co-ethylene dimethacrylate) monolith.

A polyethyleneimine (PEI) modified ion-exchanger was prepared based on poly(methacrylate-co-ethylene dimethacrylate) monolith cast in 100 mm x 4.6 mm I.D. stainless steel tube with heptane as the porogenic solvent at 65 degrees C for 12 h. The pores larger than 500 nm presented 85% of total pore volume of PEI monolith and provided the better permeability for separation. Bovine serum albumin (BSA) binding capacity on the column was enhanced with increasing the molecular weight of PEI, indicated that the brush ligand emanated from the surface and captured more protein by multiple binding sites. Titration experiment as well as BSA retention versus the pH of mobile phase showed that the monolith exhibited weak ion-exchange property, and recovered BSA on the monolith reached 97% when NaCl content in mobile phase was higher than 0.5 M. Frontal analysis and gradient elution of BSA indicated that PEI monolith provided the rapid mass transfer in chromatographic procedure, which made the dynamic binding capacities as well as column efficiency keep as constants at high operating flow rate. Fast separation of three mode proteins mixture (lysozyme, hemoglobin and BSA) on the monolith was achieved within 3 min at velocity of 1445 cm/h. This demonstrated the potential of PEI monolith for the rapid analysis and separation of proteins.

Application and appreciation of chemical sand fixing agent-poly (aspartic acid) and its composites.

The sand fixing agent-poly (aspartic acid) (PASP) and its composites were applied in the field by two forms (spraying around by PASP solution and PASP powder directly). It was found that the sand fixing effect in powder form was not as good as in solution form, but it was more practical in dry region. It needed 9, 6 and 7 days for PASP, xanthan gum-PASP (X2) and ethyl cellulose-PASP (E3) to attain the maximal mechanical strength after they were applied, respectively. The sand fixing effect decreased when the material was subjected to repeated hydration-dehydration cycles and the material had no negative influence on plant growth. The PASP and its composites had water-retaining ability and could reduce the water evaporation.

Synthesis, characterization and application of a novel chemical sand-fixing agent-poly(aspartic acid) and its composites.

A novel sand-fixing agent-poly(aspartic acid) and its composites were synthesized to improve sand particles compressive strength and anti-wind erosion properties. The relationship between the concentration of sand-fixing agent and the sand-fixing properties was studied by three kinds of aging tests. Some composites were choose to improve the sand-fixing property and the composition of 40% xanthan gum and 60% ethyl cellulose were chosen to compare sand-fixing property with lignosulfonate. The results showed that the sand-fixing and water-retaining properties of xanthan gum and ethyl cellulose composites were better than that of lignosulfonate. The biodegradability experiment showed that the PASP and its composites were environment-friendly products and the field test showed that the PASP composites could improve wind erosion disturbance.