Ionic Liquid Gating Induces Anomalous Permeation through Membrane Channel Proteins.

Membrane channel proteins (MCPs) play key roles in matter transport through cell membranes and act as major targets for vaccines and drugs. For emerging ionic liquid (IL) drugs, a rational understanding of how ILs affect the structure and transport function of MCP is crucial to their design. In this work, GPU-accelerated microsecond-long molecular dynamics simulations were employed to investigate the modulating mechanism of ILs on MCP. Interestingly, ILs prefer to insert into the lipid bilayer and channel of aquaporin-2 (AQP2) but adsorb on the entrance of voltage-gated sodium channels (Nav). Molecular trajectory and free energy analysis reflect that ILs have a minimal impact on the structure of MCPs but significantly influence MCP functions. It demonstrates that ILs can decrease the overall energy barrier for water through AQP2 by 1.88 kcal/mol, whereas that for Na+ through Nav is increased by 1.70 kcal/mol. Consequently, the permeation rates of water and Na+ can be enhanced and reduced by at least 1 order of magnitude, respectively. Furthermore, an abnormal IL gating mechanism was proposed by combining the hydrophobic nature of MCP and confined water/ion coordination effects. More importantly, we performed experiments to confirm the influence of ILs on AQP2 in human cells and found that treatment with ILs significantly accelerated the changes in cell volume in response to altered external osmotic pressure. Overall, these quantitative results will not only deepen the understanding of IL-cell interactions but may also shed light on the rational design of drugs and disease diagnosis.

Recent Developments in Ionic Liquid-Assisted Topical and Transdermal Drug Delivery.

Ionic liquids (ILs) have attracted growing interest as designer solvents/materials for exploring unrealized functions in many areas of research including drug formulations and delivery owing to their inherent tunable physicochemical and biological properties. The use of ILs in the pharmaceutical industry can address challenges related to the use of conventional organic solvent-based chemical permeation enhancers. Their tunability in forming ion pairs with a diverse range of ions enables the task-specific optimization of ILs at the molecular level. In particular, ILs comprising second- and third-generation cations and anions have been extensively used to design biocompatible drug delivery systems to address the challenges related to conventional topical and transdermal drug delivery, including limited permeability, high cytotoxicity, and skin irritation. This review highlights the progress in IL-related research with particular emphasis on the very recent conceptual developments in transdermal drug delivery. Technological advancement and approaches for the formation of IL-based topical and transdermal delivery systems, as well as their promising application in drug delivery, are also discussed.

Converting Tretinoin into Ionic Liquids for Improving Aqueous Solubility and Permeability across Skin.

PURPOSE: The aim of this study is to convert tretinoin (Tr), an active pharmaceutical ingredient (API), into ionic liquid for improving aqueous solubility and permeability of Tr in transdermal drug delivery applications. METHODS: Three ionic liquids of Tr (TrILs) were synthesized through neutralization reactions, which were characterized to confirm the compositions and ionic interactions. The in vitro drug release studies and skin penetration tests were carried out to assess the performance of formulations containing TrILs. RESULTS: The TrIL formed by choline and Tr at the molar ratio of 2:1 (2[Ch][Tr]), was found to have prominent solubility, stability as well as permeability. In contrast with the insoluble Tr, 2[Ch][Tr] presented as clear and transparent aqueous solution even after diluted to 14%. The aqueous solution of 2[Ch][Tr] demonstrated better permeation effect, of which the solution with 20% of 2[Ch][Tr] showed the optimal delivery efficiency in both epidermis (2.09 ± 0.18‰) and dermis (3.31 ± 0.48‰), realizing the improvement on the permeability of API. Meanwhile, TrILs can be easily fabricated as o/w emulsions as transdermal formulation. The emulsions are also able to improve the skin permeability of Tr, though the enhanced effect is inferior to TrILs solutions. CONCLUSIONS: Ionic liquid technology can be used to improve solubility and permeability of Tr, providing a high potential strategy for the development of topical formulations and the desired transdermal application of drugs.

Transdermal Delivery of Metformin Utilizing Ionic Liquid Technology: Insight Into the Relationship Between Counterion Structures and Properties.

PURPOSE: The purpose of the present study was to explore the feasibility of transdermal delivery of metformin, a commonly used oral antidiabetic drug, by ionic liquid (IL) technology. METHODS: Metformin hydrochloride (MetHCl) was first transformed into three kinds of ILs with different counterions. The physicochemical properties of the obtained ILs were characterized in depth. The simulation of stable configuration and calculation of interaction energies were conducted based on density functional theory (DFT). Skin-PAMPA was used to evaluate the intrinsic transdermal permeation properties. The cytotoxicity assay of these ILs was conducted using HaCaT cells to evaluate the toxicity to skin. These metformin ILs were then formulated into transdermal patch, and the transdermal potential was further evaluated using in vitro dissolution test and skin permeation assay. Finally, the pharmacokinetic profiles of these metformin IL-containing patches were determined. RESULTS: Among all the three Met ILs, metformin dihexyl sulfosuccinate (MetDH) with proper overall physiochemical and biological properties demonstrated the highest relative bioavailability. Metformin docusate (MetD) with the highest lipophilicity and intrinsic transdermal permeability exhibited the most significant sustained release profile in vivo. Both MetDH and MetD were the promising candidates for further clinical investigations. CONCLUSIONS: Overall, the properties of ILs were closely related to the structures of counterion. IL technology provided the opportunities to finely tune the solid-state and biological properties of Metformin and facilitated the successful delivery by transdermal route.

Pressurized Natural Deep Eutectic Solvent Extraction of Galanthamine and Related Alkaloids from Narcissus pseudonarcissus.

The isolation of a compound from a natural source involves many organic and mostly toxic solvents for extraction and purification. Natural deep eutectic solvents have been shown to be efficient options for the extraction of natural products. They have the advantage of being composed of abundantly available common primary metabolites, being nontoxic and environmentally safe solvents. The aim of this study was to develop a natural deep eutectic solvent-based extraction method for galanthamine, an important therapeutic agent for the treatment of Alzheimer's disease. This alkaloid can be produced by synthesis or by extraction from Narcissus bulbs. To develop an efficient extraction method, a number of different natural deep eutectic solvents was first tested for their solubilization capacity of galanthamine bromide salt. Promising results were obtained for ionic liquids, as well as some amphoteric and acidic natural deep eutectic solvents. In a two-cycle extraction process, the best solvents were tested for the extraction of galanthamine from bulbs. The ionic liquids produced poor yields, and the best results were obtained with some acid and sugar mixtures, among which malic acid-sucrose-water (1 : 1 : 5) proved to be the best, showing similar yields to that of the exhaustive Soxhlet extraction with methanol. Furthermore, the natural deep eutectic solvent was more selective for galanthamine.

Simultaneous Visualization of Wet Cells and Nanostructured Biomaterials in SEM using Ionic Liquids.

This work presents a successful methodology to image mammalian cells adhered to nanostructured titanium by using scanning electron microscopy (SEM) operating in low-vacuum mode following ionic liquid treatment. Human osteoblast-like Saos-2 cells were treated with a room-temperature ionic liquid, 1-ethyl-3-methylimidazolium tetrafluoroborate, and subsequently imaged on titanium by SEM. Titanium substrates were modified to create laser-induced periodic surface structures (LIPSS) for visualization at the submicron scale. By using a combination of fluorescence-based cell metabolism along with light microscopy and SEM image analysis, the shape and location of irradiated cells were confirmed to be unchanged after multiple irradiation sessions; the viability of minimally irradiated cells was also unaltered. The wet imaging conditions combined with a rapid facile protocol using ionic liquid allows this technique to fulfill a niche in examining cellular behavior on biomaterials with submicron surface features. The demonstrated method to track observed cell adhesion to submicron surface features by SEM has great implications for understanding cell migration on nanostructured surfaces as well as the exploration of simpler SEM preparation methods for cellular imaging.

Computational analysis of the effect of [Tea][Ms] and [Tea][H2PO4] ionic liquids on the structure and stability of Aß(17-42) amyloid fibrils.

Experimental studies have reported the possibility of affecting the growth/dissolution of amyloid fibres by the addition of organic salts of the room-temperature ionic-liquid family, raising the tantalizing prospect of controlling these processes under physiological conditions. The effect of [Tea][Ms] and [Tea][H2PO4] at various concentrations on the structure and stability of a simple model of Aß42 fibrils has been investigated by computational means. Free energy computations show that both [Tea][Ms] and [Tea][H2PO4] decrease the stability of fibrils with respect to isolated peptides in solution, and the effect is significantly stronger for [Tea][Ms]. The secondary structure of fibrils is not much affected, but single peptides in solution show a marked decrease in their ß-strand character and an increase in α-propensity, again especially for [Tea][Ms]. These observations, consistent with the experimental picture, can be traced to two primary effects, i.e., the difference in the ionicity of the [Tea][Ms] and [Tea][H2PO4] water solutions and the remarkable affinity of peptides for [Ms]- anions, due to the multiplicity of H-bonds.

Catalytic Conversion of Glucose into Levulinic Acid Using 2-Phenyl-2-Imidazoline Based Ionic Liquid Catalyst.

Levulinic acid (LA) is an industrially important product that can be catalytically valorized into important value-added chemicals. In this study, hydrothermal conversion of glucose into levulinic acid was attempted using Brønsted acidic ionic liquid catalyst synthesized using 2-phenyl-2-imidazoline, and 2-phenyl-2-imidazoline-based ionic liquid catalyst used in this study was synthesized in the laboratory using different anions (NO3, H2PO4, and Cl) and characterized using 1H NMR, TGA, and FT-IR spectroscopic techniques. The activity trend of the Brønsted acidic ionic liquid catalysts synthesized in the laboratory was found in the following order: [C4SO3HPhim][Cl] > [C4SO3HPhim][NO3] > [C4SO3HPhim][H2PO4]. A maximum 63% yield of the levulinic acid was obtained with 98% glucose conversion at 180 °C and 3 h reaction time using [C4SO3HPhim][Cl] ionic liquid catalyst. The effect of different reaction conditions such as reaction time, temperature, ionic liquid catalyst structures, catalyst amount, and solvents on the LA yield were investigated. Reusability of [C4SO3HPhim][Cl] catalyst up to four cycles was observed. This study demonstrates the potential of the 2-phenyl-2-imidazoline-based ionic liquid for the conversion of glucose into the important platform chemical levulinic acid.

Implication of Threonine-Based Ionic Liquids on the Structural Stability, Binding and Activity of Cytochrome†c.

Ionic liquids (ILs) are useful in pharmaceutical industries and biotechnology as alternative solvents or sources for protein extraction and purification, preservation of biomolecules and for regulating the catalytic activity of enzymes. However, the binding mechanism, the non-covalent forces responsible for protein-IL interactions and dynamics of proteins in IL need to be investigated in depth for the effective use of ILs as alternatives. Herein, we disclose the molecular level understanding of the structural intactness and reactivity of a model protein cytochrome c (Cyt c) in biocompatible threonine-based ILs with the help of experimental techniques such as isothermal titration calorimetry (ITC), fluorescence spectroscopy, transmission electron microscopy (TEM) as well as molecular docking. Hydrophobic and electrostatic forces are responsible for the structural and conformational integrity of Cyt c in IL. The ITC experiments revealed the Cyt c-IL binding free energies are in the range of 10-14 kJ/mol and the molecular docking studies demonstrated that ILs interact at the surfaces of Cyt c. The results look promising as the ILs used here are non-toxic and biocompatible, and thus may find potential applications in structural biology and biotechnology.

Neat Ionic liquid and α-Chymotrypsin-Polymer Surfactant Conjugate-Based Biocatalytic Solvent.

Performing biocatalysis in nonaqueous solvents is advantageous as it imparts enhanced solubility to hydrophobic substrates and an ability to increase the temperature for shifting reaction equilibrium in the forward direction. In this work, we show the design and development of another class of nonaqueous composite solvent obtained by mixing surface modified enzyme and neat ionic liquid (IL). We systematically probe the interaction and solubility of industrially relevant α-chymotrypsin in its native or surface-bound polymer-surfactant bioconjugated form, with neat protic (N-methyl-2-pyrrolidonium trifluoromethanesulfonate; [NMP][OTf]), or aprotic (1-methyl-3-(4-sulfobutyl)-1H-imidazol-3-ium trifluoromethanesulfonate; [HO3S(CH2)4MIm][OTf]), ILs. Polarized optical micrographs show that the lyophilized powder of native α-chymotrypsin, nCT, does not disperse in either of the neat ILs, however, its polymer surfactant (PS)-coated bioconjugate counterparts, PScCT, in the waterless state, can be well-dispersed and solubilized in the neat [HO3S(CH2)4MIm][OTf]. The solubilization of waterless bioconjugates of PScCT in neat aprotic IL provides a composite liquid, WL-ImPScCT (WL: waterless, Im: [HO3S(CH2)4MIm][OTf]), having a viscosity of 69.6 Pa·s at 25 °C with a shear-thinning behavior, ≈ 15 w/w % α-chymotrypsin, and ≈ 1.2 w/w % residual water content. Detailed secondary structural analysis using circular dichroism and Fourier self-deconvolution on the ATR-FTIR data of WL-ImPScCT liquid reveals retention of the near native secondary structure of α-chymotrypsin. Further, using a combination of fluorescence spectroscopy and electron spray ionization mass spectrometry, we show that scattering of dry and powdered bovine serum albumin (BSA) protein on the WL-ImPScCT composite liquid results in the solubilization of the former, followed by limited proteolysis of BSA by the α-chymotrypsin. Our results, therefore, show the stabilization of α-chymotrypsin in a neat aprotic IL environment to yield a composite liquid, which not only acts as a nonaqueous, nonvolatile, and environmentally benign solvent, but also provides a biocatalytic platform capable of carrying out reactions relevant for biotransformations, food processing, drug delivery, and various other applications.

Magnetic ionic liquids: interactions with bacterial cells, behavior in aqueous suspension, and broader applications.

Previously, we demonstrated capture and concentration of Salmonella enterica subspecies enterica ser. Typhimurium using magnetic ionic liquids (MILs), followed by rapid isothermal detection of captured cells via recombinase polymerase amplification (RPA). Here, we report work intended to explore the broader potential of MILs as novel pre-analytical capture reagents in food safety and related applications. Specifically, we evaluated the capacity of the ([P66614+][Ni(hfacac)3-]) ("Ni(II)") MIL to bind a wider range of human pathogens using a panel of Salmonella and Escherichia coli O157:H7 isolates, including a "deep rough" strain of S. Minnesota. We extended this exploration further to include other members of the family Enterobacteriaceae of food safety and clinical or agricultural significance. Both the Ni(II) MIL and the ([P66614+][Dy(hfacac)4-]) ("Dy(III)") MIL were evaluated for their effects on cell viability and structure-function relationships behind observed antimicrobial activities of the Dy(III) MIL were determined. Next, we used flow imaging microscopy (FIM) of Ni(II) MIL dispersions made in model liquid media to examine the impact of increasing ionic complexity on MIL droplet properties as a first step towards understanding the impact of suspension medium properties on MIL dispersion behavior. Finally, we used FIM to examine interactions between the Ni(II) MIL and Serratia marcescens, providing insights into how the MIL may act to capture and concentrate Gram-negative bacteria in aqueous samples, including food suspensions. Together, our results provide further characterization of bacteria-MIL interactions and support the broader utility of the Ni(II) MIL as a cell-friendly capture reagent for sample preparation prior to cultural or molecular analyses. Graphical abstract.

The Role of Ionic Liquids in the Pharmaceutical Field: An Overview of Relevant Applications.

Solubility, bioavailability, permeation, polymorphism, and stability concerns associated to solid-state pharmaceuticals demand for effective solutions. To overcome some of these drawbacks, ionic liquids (ILs) have been investigated as solvents, reagents, and anti-solvents in the synthesis and crystallization of active pharmaceutical ingredients (APIs), as solvents, co-solvents and emulsifiers in drug formulations, as pharmaceuticals (API-ILs) aiming liquid therapeutics, and in the development and/or improvement of drug-delivery-based systems. The present review focuses on the use of ILs in the pharmaceutical field, covering their multiple applications from pharmaceutical synthesis to drug delivery. The most relevant research conducted up to date is presented and discussed, together with a critical analysis of the most significant IL-based strategies in order to improve the performance of therapeutics and drug delivery systems.

Synthesis and Characterization of Surface-Active Ionic Liquids Used in the Disruption of Escherichia Coli Cells.

Twelve surface-active ionic liquids (SAILs) and surface-active derivatives, based on imidazolium, ammonium, and phosphonium cations and containing one, or more, long alkyl chains in the cation and/or the anion, were synthetized and characterized. The aggregation behavior of these SAILs in water, as well as their adsorption at solution/air interface, were studied by assessing surface tension and conductivity. The CMC values obtained (0.03-6.0 mM) show a high propensity of these compounds to self-aggregate in aqueous media. Their thermal properties were also characterized, namely the melting point and decomposition temperature by using DSC and TGA, respectively. Furthermore, the toxicity of these SAILs was evaluated using the marine bacteria Aliivibrio fischeri (Gram-negative). According to the EC50 values obtained (0.3-2.7 mg L-1 ), the surface-active compounds tested should be considered "toxic" or "highly toxic". Their ability to induce cell disruption of Escherichia coli cells (also Gram-negative), releasing the intracellular green fluorescent protein (GFP) produced, was investigated. The results clearly evidence the capability of these SAILs to act as cell disruption agents.

Ionic Liquids as Tool to Improve Enzymatic Organic Synthesis.

Ionic liquids (ILs) have now been acknowledged as reaction media for biotransformations. The first three examples were reported in this field in 2000, and since then, numerous applications have been reported for biocatalytic reactions using ILs. Two topics using ILs for enzymatic reactions have been reviewed from the standpoint of biocatalyst mediating organic synthesis; the first is "Biocatalysis in Ionic Liquids" which includes various types of biocatalytic reactions in ILs (section 2): (1) recent examples of lipase-mediated reactions using ILs as reaction media for biodiesel oil production and for sugar ester production, (2) oxidase-catalyzed reactions in ILs, (3) laccase-catalyzed reactions, (4) peroxidase-catalyzed reactions, (4) cytochrome-mediated reactions, (5) microbe-mediated hydrations, (6) protease-catalyzed reactions, (8) whole cell mediated asymmetric reduction of ketones, (10) acylase-catalyzed reactions, (11) glycosylation or cellulase-mediated hydrolysis of polysaccharides, (12) hydroxynitrile lyase-catalyzed reaction, (13) fluorinase or haloalkane dehydrogenase-catalyzed reaction, (14) luciferase-catalyzed reactions, and (15) biocatalytic promiscuity of enzymatic reactions for organic synthesis using ILs. The second is "Enzymes Activated by Ionic Liquids for Organic Synthesis", particularly describing the finding story of activation of lipases by the coating with a PEG-substituted IL (section 3). The author's opinion toward "Future Perspectives of Using ILs for Enzymatic Reactions" has also been discussed in section 4.

Hybrid electrochemical and biological treatment of herbicidal ionic liquids comprising the MCPA anion.

The present study was focused on the application of an electrochemical oxidation process combined with biodegradation for the removal of novel Herbicidal Ionic Liquids (HILs) -promising protection plant products which incorporate herbicidal anions and ammonium cations. The influence of carbon chain length (n = 8, 10, 12, 14, 16, 18) in the dialkyldimethylammonium cations on electrochemical oxidation kinetics, degradation efficiency and biodegradation by activated sludge was investigated. It was established that the applied cation influenced the heterogeneous rate constant and diffusion coefficient of electrochemical oxidation. The oxidation efficiency ranged from 17% in case of HILs with C8 alkyl chain to approx. 60% in case of HILs comprising C14 and C16 alkyl chains after 3 h of electrochemical treatment. Subsequent biodegradation studies revealed that electrochemical oxidation improved the mineralization efficiency of the studied HILs. The mineralization efficiency of electrochemically-treated HILs ranged from 28% in case of HILs comprising the C8 alkyl chain to 57% in case of HILs with C14 and C16 alkyl chains after 28 days. In case of untreated HILs, the corresponding mineralization efficiency ranged from 0 to 8%, respectively. This confirms the feasibility of a hybrid electrochemical-biological approach for treatment of herbicidal ionic liquids based on MCPA.

Ketoprofen-Based Ionic Liquids: Synthesis and Interactions with Bovine Serum Albumin.

The development of ionic liquids based on active pharmaceutical ingredients (API-ILs) is a possible solution to some of the problems of solid and/or hydrophobic drugs such as low solubility and bioavailability, polymorphism and an alternative route of administration could be suggested as compared to the classical drug. Here, we report for the first time the synthesis and detailed characterization of a series of ILs containing a cation amino acid esters and anion ketoprofen (KETO-ILs). The affinity and the binding mode of the KETO-ILs to bovine serum albumin (BSA) were assessed using fluorescence spectroscopy. All compounds bind in a distance not longer than 6.14 nm to the BSA fluorophores. The estimated binding constants (KA) are in order of 105 L mol-1, which is indicative of strong drug or IL-BSA interactions. With respect to the ketoprofen-BSA system, a stronger affinity of the ILs containing l-LeuOEt, l-ValOBu, and l-ValOEt cation towards BSA is clearly seen. Fourier transformed infrared spectroscopy experiments have shown that all studied compounds induced a rearrangement of the protein molecule upon binding, which is consistent with the suggested static mechanism of BSA fluorescence quenching and formation of complexes between BSA and the drugs. All tested compounds were safe for macrophages.

The ionic liquid 1-octyl-3-methylimidazolium (M8OI) is an activator of the human estrogen receptor alpha.

Recent environmental sampling around a landfill site in the UK demonstrated that unidentified xenoestrogens were present at higher levels than control sites; that these xenoestrogens were capable of super-activating (resisting ligand-dependent antagonism) the murine variant 2 ERß and that the ionic liquid 1-octyl-3-methylimidazolium chloride (M8OI) was present in some samples. To determine whether M8OI was a contributor to the xenoestrogen pool in the soils, activation of human estrogen receptors by M8OI was examined. M8OI activated the human ERα in MCF7 cells in a dose-response manner. These effects were inhibited by the ER antagonist ICI182780; occurred in the absence of any metabolism of M8OI and were confirmed on examination of ER-dependent induction of trefoil factor 1 mRNA in MCF7 cells. M8OI also super-activated the murine variant 2 ERß in a murine hepatopancreatobiliary cell line. The human ERß was not activated by M8OI when expressed in HEK293 cells. These data demonstrate that M8OI is a xenoestrogen capable of activating the human ERα and super-activating the murine variant 2 ERß.

A Multiperspective Approach to Solvent Regulation of Enzymatic Activity: HMG-CoA Reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC.

BACKGROUND: Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated sugar source can be inhibitory to microbial growth and impair expression of the desired biosynthetic pathway. RESULTS: We utilized laboratory evolution to select Escherichia coli strains capable of robust growth in the presence of the IL, 1-ethyl-3-methyl-imidizolium acetate ([EMIM]OAc). Whole genome sequencing of the evolved strain identified a point mutation in an essential gene, cydC, which confers tolerance to two different classes of ILs at concentrations that are otherwise growth inhibitory. This mutation, cydC-D86G, fully restores the specific production of the bio-jet fuel candidate D-limonene, as well as the biogasoline and platform chemical isopentenol, in growth medium containing ILs. Similar amino acids at this position in cydC, such as cydC-D86V, also confer tolerance to [EMIM]OAc. We show that this [EMIM]OAc tolerance phenotype of cydC-D86G strains is independent of its wild-type function in activating the cytochrome bd-I respiratory complex. Using shotgun proteomics, we characterized the underlying differential cellular responses altered in this mutant. While wild-type E. coli cannot produce detectable amounts of either product in the presence of ILs at levels expected to be residual in sugars from pretreated biomass, the engineered cydC-D86G strains produce over 200 mg/L D-limonene and 350 mg/L isopentenol, which are among the highest reported titers in the presence of [EMIM]OAc. CONCLUSIONS: The optimized strains in this study produce high titers of two candidate biofuels and bioproducts under IL stress. Both sets of production strains surpass production titers from other IL tolerant mutants in the literature. Our application of laboratory evolution identified a gain of function mutation in an essential gene, which is unusual in comparison to other published IL tolerant mutants.

Effect of pretreatment severity on the cellulose and lignin isolated from Salix using ionoSolv pretreatment.

The ionoSolv pretreatment is a new technique employing protic low-cost ionic liquids and has previously been applied to successfully fractionate switchgrass and the grass Miscanthus giganteus. This study investigates the effect of using the protic ionic liquid solution [N2220][HSO4]80% with two different acid/base ratios (1.02 and 0.98) at 120, 150 and 170 °C on the pretreatment outcome of the hardwood willow. The ionic liquid solution was able to fractionate willow, and a pulp and lignin fraction were recovered after treatment. The pretreatment success was determined via enzymatic hydrolysis of the pulp, which showed that the ionoSolv pretreatment was able to increase enzymatic glucose yields compared to untreated willow biomass. The pretreatment produced a cellulose-rich pulp with high hemicellulose and lignin removal. The pulp composition and glucose yield after saccharification were greatly influenced by the acidity of the ionic liquid solution, temperature and pretreatment time. The extracted lignin was analysed via 2-D HSQC NMR spectroscopy and GPC to investigate the changes in the lignin structure induced by the pretreatment severity. The lignin structure (in terms of inter-unit linkages and S/G ratio) and molecular weight varied significantly depending on the pretreatment conditions used.