Sugarcane bagasse pretreatment using three imidazolium-based ionic liquids; mass balances and enzyme kinetics

ABSTRACT: BACKGROUND: Effective pretreatment is key to achieving high enzymatic saccharification efficiency in processing lignocellulosic biomass to fermentable sugars, biofuels and value-added products. Ionic liquids (ILs), still relatively new class of solvents, are attractive for biomass pretreatment because some demonstrate the rare ability to dissolve all components of lignocellulosic biomass including highly ordered (crystalline) cellulose. In the present study, three ILs, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc) are used to dissolve/pretreat and fractionate sugarcane bagasse. In these IL-based pretreatments the biomass is completely or partially dissolved in ILs at temperatures greater than 130[DEGREE SIGN]C and then precipitated by the addition of an antisolvent to the IL biomass mixture. For the first time mass balances of IL-based pretreatments are reported. Such mass balances, along with kinetics data, can be used in process modelling and design. RESULTS: Lignin removals of 10% mass of lignin in bagasse with [C4mim]Cl, 50% mass with [C2mim]Cl and 60% mass with [C2mim]OAc, are achieved by limiting the amount of water added as antisolvent to 0.5 water:IL mass ratio thus minimising lignin precipitation. Enzyme saccharification (24 h, 15FPU) yields (% cellulose mass in starting bagasse) from the recovered solids rank as: [C2mim]OAc(83%)>[C2mim]Cl(53%) = [C4mim]Cl(53%). Composition of [C2mim]OAc-treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are characteristic of aqueous alkali pretreatments while those of chloride IL-treated solids resemble aqueous acid pretreatments. All ILs are fully recovered after use (100% mass as determined by ion chromatography). CONCLUSIONS: In all three ILs regulated addition of water as an antisolvent effected a polysaccharide enriched precipitate since some of the lignin remained dissolved in the aqueous IL solution. Of the three IL studied [C2mim]OAc gave the best saccharification yield, material recovery and delignification. The effects of [C2mim]OAc pretreatment resemble those of aqueous alkali pretreatments while those of [C2mim]Cl and [C4mim]Cl resemble aqueous acid pretreatments. The use of imidazolium IL solvents with shorter alkyl chains results in accelerated dissolution, pretreatment and degradation.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic liquids

A novel double-step reduction procedure for the synthesis of gold nanoparticles (AuNPs) using amino acid ionic liquids has been employed. 1-Dodecyl-3-methyl imidazolium tryptophan ([C(12)mim]Trp) and 1-ethyl-3-methyl imidazolium tryptophan ([C(2)mim]Trp) were used for this synthesis. The synthesized AuNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. The behavior of these AuNPs were also probed in a biological media. It was proven that AuNPs synthesized at [C(12)mim]Trp have more stability than AuNPs synthesized at [C(2)mim]Trp due to the longer alkyl chain of the imidazolium moiety. The solubility test shows that the resultant AuNPs have a hydrophilic nature. Finally, it was seen that due to the presence of a biomolecule, namely Trp, in the structure of AuNPs protecting shell, higher stability and biocompatibility was achieved in the biological media.     

咪唑离子液体对四尾栅藻的毒性

按照藻类生长抑制试验标准方法,以四尾栅藻为受试生物,研究了离子液体1-丁基-3-甲基咪唑氯盐([C4mim][Cl])和1-丁基-3-甲基咪唑四氟硼酸盐([C4mim][BF4])对四尾栅藻的影响,测定了半数有效浓度(EC50)和叶绿素含量。结果表明:[C4mim][Cl]和[C4mim][BF4]对四尾栅藻的生长和叶绿素的产生均具有抑制作用,毒性效应随着浓度的增大和培养时间的增长而增加,96 h的EC50分别为57.8和38.8 mg/L。离子液体质量浓度为200mg/L时,四尾栅藻的叶绿素总量仅为对照组的32%和26%。此结果可以说明离子液体对生态系统存在潜在的负面效应。     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Oligonucleotide synthesis using ionic liquids as soluble supports

The continuing evolution of the methodology for the solution-phase synthesis of oligonucleotides using soluble ionic tags as handles for easy purification is described. This methodology may provide a more cost efficient route for the large scale synthesis of oligonucleotides.     

A profile of the in vitro anti-tumor activity of imidazolium-based ionic liquids

The anti-cancer activity and cytotoxicity of imidazolium-based ionic liquids has been determined for the first time via NCI’s in vitro 60 human tumor cell lines. The preliminary SAR showed that the chain length of alkyl substitution at N-3 position of imidazole ring plays crucial role towards anti-tumor activity and cytotoxicity of these ionic liquids. The ionic liquids with alkyl substitution of C-12 chain length were found to be effective against all 60 tumor cell lines and show very low cytotoxicity in most of the cases. Further increase in chain length resulted in enhanced growth inhibition of tumor cell lines as well as high cytotoxicity. Interestingly, active compounds 1-dodecyl-3-methylimidazolium chloride (8), 1-dodecyl-3-methylimidazolium tetrafluoroborate (9), 1-hexadecyl-3-methylimidazoium chloride (10), 1-octadecyl-3-methylimidazolium chloride (11), 1-octadecyl-3-methylimidazolium hexafluorophosphate (12), 1-octadecyl-3-methylimidazolium bis(triflic)imide (13) and 1-octadecyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate (14) were highly active against leukemia cell lines, especially compounds 13 and 14 where the cytotoxicity was also very low as given by LC₅₀ >100 μM in all six leukemia cell lines.     

Theoretical study on the interactions between methanol and imidazolium-based ionic liquids

To better understand the property of the binary systems composing of imidazolium salt, [emim]⁺Aˉ (A=Clˉ, Brˉ, BF₄ˉ, and PF₆ˉ) and methanol, we have investigated in detail the interactions of methanol molecule with anions Aˉ, cation [emim]⁺, and ion pair [emim]⁺Aˉ of several ionic liquids (ILs) based on 1-ethyl-3-methylimidazolium cation by performing density functional theory calculations. It is found that H-bonds are universally involved in these systems, which may play an important role for the miscibility of methanol with imidazolium-based ILs. The interaction mechanisms of methanol molecule with anion and cation are found to be different in nature: the former mainly involves LP_X-s_{O - H}^* \sigma_{{O - H}}^{*} interaction, while the latter relates with the decisive orbital overlap of the type of LP_O-s_{C - H}^* \sigma_{{C - H}}^{*} . Based on the present calculations, we have provided some reasonable interpretations for properties of the binary mixtures of ILs and alcohol and revealed valuable information for the interaction details between ILs and alcohols, which is expected to be useful for the design of more efficient ILs to form superior solvent system with alcohol.     

Scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters

The scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters have been studied. Herein, we report the effects of modifying the structure of the imidazolium cation and how important the choice of counter ion becomes on model glycosylation reactions of thioglycosides at room temperature in the presence of N-iodosuccinimide (NIS).     

Feruloyl esterase-catalysed synthesis of glycerol sinapate using ionic liquids mixtures

The ability of a feruloyl esterase (AnFaeA), either in free or immobilised (cross-linked enzyme aggregates) form, to catalyse the esterification of glycerol, a major by-product of the biodiesel industry, with sinapic acid was studied in four hexafluorophosphate anion-containing ionic liquids: ([Bmim][PF(6)], [Omim][PF(6)], [C(2)OHmim][PF(6)] and [C(5)O(2)mim][PF(6)]). Such ionic liquids are considered 'green' reaction systems. The synthetic reaction was optimised in [C(2)OHmim][PF(6)] and the highest conversion yield was 72.5+/-2.1%, while, at the same reaction conditions in [C(5)O(2)mim] [PF(6)], a similar conversion yield was obtained (76.7+/-1.5%). AnFaeA was active in its free and immobilised form, with the latter retaining a part of its synthetic activity after 5 consecutive 24h-period reaction cycles. Sinapic acid was esterified to one of the primary hydroxyl groups of glycerol and retained, after esterification, 63.1+/-0.3% and 89.5+/-1.1% of its antioxidant activity against low-density lipoprotein oxidation, when added at concentrations of 10 and 60muM, respectively, in the assay mixture.     

Probing anion-cellulose interactions in imidazolium-based room temperature ionic liquids: a density functional study

The interactions of the cellulose molecule with several anions, including acetate , alkyl phosphate, tetrafluoroborate and hexafluorophosphate anions which are most commonly involved in the imidazolium ionic liquids (ILs), have been studied by performing density functional theory calculations. Based on calculated geometries, energies, IR characteristics, and electronic properties of the cellulose-anion complexes, it is found that the strength of interactions of anions with cellulose follows the order: acetate anion > alkyl phosphate anion > tetrafluoroborate anion > hexafluorophosphate anion, which is consistent with the experimentally observed solubility trend of cellulose in the corresponding imidazolium-based ILs. The present study may provide basic aids to some extent for understanding the dissolution behavior of cellulose in the imidazolium-based ILs.     

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C₄mim][PF₆], [C₆mim][PF₆], [C₈mim][PF₆], [C₆mim][BF₄] and [C₈mim][BF₄] as extracting solvents were examined. [C₆mim][BF₄] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.     

Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquids mixtures

Novozym 435-catalyzed synthesis of 6-O-lauroyl-d-glucose in ionic liquids (ILs) was investigated. The highest lipase activity was obtained in water-miscible [Bmim][TfO] which can dissolve high concentration of glucose, while the highest stability of lipase was shown in hydrophobic [Bmim][Tf(2)N]. The optimal activity and stability of lipase could be obtained in [Bmim][TfO] and [Bmim][Tf(2)N] mixture (1:1, v/v). Specifically, the activity of lipase was increased from 1.1 to 2.9 micromolmin(-1)g(-1) by using supersaturated glucose solution in this mixture, compared with reaction using saturated solution. After 5 times reuse of lipase, 86% of initial activity was remained in this mixture, while the residual activity in pure [Bmim][TfO] was 36%. Therefore, the productivity obtained by using ILs mixtures was higher than those in pure ILs.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.     

Green microwave-assisted synthesis of cellulose/calcium silicate nanocomposites in ionic liquids and recycled ionic liquids

Fabrication of biomass materials by a microwave-assisted method in ionic liquids allows the high value-added applications of biomass by combining three major green chemistry principles: using environmentally preferable solvents, using an environmentally friendly method, and making use of renewable biomass materials. Herein, we report a rapid and green microwave-assisted method for the synthesis of the cellulose/calcium silicate nanocomposites in ionic liquids and recycled ionic liquids. These calcium silicate nanoparticles or nanosheets as prepared were homogeneously dispersed in the cellulose matrix. The experimental results confirm that the ionic liquids can be used repeatedly. Of course, the slight differences were also observed using ionic liquids and recycled ionic liquids. Compared with other conventional methods, the rapid, green, and environmentally friendly microwave-assisted method in ionic liquids opens a new window to the high value-added applications of biomass.     

Chiral ionic liquids: synthesis and applications

Over the last ten years, interest and publications involving ionic liquids have expanded exponentially. Thus far, they have predominantly been used in organic synthesis and separations. However, their use is rapidly expanding into other areas of science and technology. Research involving chiral ionic liquids has been much more limited and only recently has come to the forefront. In this work, we review the synthesis of chiral ionic liquids and their use. Today, this is an area of research that is poised for rapid development and expansion.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Lipase-catalyzed glycerolysis in ionic liquids directed towards diglyceride synthesis

This work examined the lipase-catalyzed glycerolysis of triglycerides (TG) in a list of commercially available ionic liquids (ILs) with varied cations and anions for the purpose of developing an efficient reaction protocol for diglyceride (DG) production and to understand whether ILs could assist the reaction systems. The reaction performances (reaction rate, TG conversion and DG yield) were found to be greatly dependent on the structure and property of ILs. The reactions in [TOMA·Tf₂N] and Ammoeng 120 produced comparable yield of DG to those most efficient conventional systems but with less by-products. Temperature enhancement generally yields positive effect on the conversion of TG, which was much more significant for the ILs with high viscosity. Unusually, increasing substrate concentration in many types of ILs led to enhanced conversion and yield; whereas the increase of glycerol/TG ratio resulted in a dramatic improvement of the reactions in the ILs with strong acidic anions. This work also sorted out some promising IL candidates, namely the ILs with good DG formation selectivity and the ones being able to achieve high TG conversion, which offered possibility to design binary IL systems. Overall, this study presented the first attempt concerning evaluation and characterization of lipase-catalyzed glycerolysis of TG for DG production in IL-based systems.     

Lipase-catalyzed synthesis of fatty acid sugar ester using extremely supersaturated sugar solution in ionic liquids

Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin-coated microspheres move toward the plus end of microtubules, whereas dynein-coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin- and dynein-coated microspheres on microtubules oriented and glutaraldehyde-immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87-98% of microspheres move in the designated direction at a mean velocity of 0.22-0.28 microm/s for kinesin-coated microspheres and 0.34-0.39 microm/s for dynein-coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro.