Pablo Domínguez de María

2-Methyltetrahydrofuran (2-MeTHF): A Biomass-Derived Solvent with Broad Application in Organic Chemistry

2-Methyl-tetrahydrofuran (2-MeTHF) can be derived from renewable resources (e.g., furfural or levulinic acid) and is a promising alternative solvent in the search for environmentally benign synthesis strategies. Its physical and chemical properties, such as its low miscibility with water, boiling point, remarkable stability compared to other cyclic-based solvents such as THF, and others make it appealing for applications in syntheses involving organometallics, organocatalysis, and biotransformations or for processing lignocellulosic materials. Interestingly, a significant number of industries have also started to assess 2-MeTHF in several synthetic procedures, often with excellent results and prospects. Likewise, preliminary toxicology assessments suggest that the use of 2-MeTHF might even be extended to more processes in pharmaceutical chemistry. This Minireview describes the properties of 2-MeTHF, the state-of-the-art of its use in synthesis, and covers several outstanding examples of its application from both industry and academia.

Novel choline-chloride-based deep-eutectic-solvents with renewable hydrogen bond donors: levulinic acid and sugar-based polyols

Choline chloride (ChCl) has been combined with renewable hydrogen bond donors (HBD) to form novel deep eutectic solvents (DES) with melting points lower than 100 °C. In some cases (e.g. sorbitol or isosorbide as HBDs) chiral DES can be easily produced. Moreover, the addition of glycerol decreases the viscosity of DES significantly. In the ChCl : levulinic acid DES case, singular properties were observed. Thus, equimolecular mixtures of ChCl : levulinic acid (1 : 1) formed a crystal at room temperature, rapidly melting in contact with air humidity. Addition of excess of acetone to ChCl : levulinic acid (1 : 2) leads to the almost quantitative precipitation of choline chloride, which can be used again to form a DES.

From biomass to feedstock: one-step fractionation of lignocellulose components by the selective organic acid-catalyzed depolymerization of hemicellulose in a biphasic system

A concept for a highly integrated fractionation of lignocellulose in its main components (cellulose-pulp, soluble hemicellulose sugars and lignin) is described, based on the selective catalytic depolymerization of hemicellulose in a biphasic solvent system. This leads to an effective disentanglement of the compact lignocellulose structure, liberating and separating the main components in a single step. At mild temperatures (80–140 °C), oxalic acid catalyzes selectively the depolymerization of hemicellulose to soluble sugars in aqueous solution, whereas the more crystalline cellulose-pulp remains solid and inaccessible to the acid catalysis. In the presence of a second organic phase consisting of bio-based 2-methyltetrahydrofuran (2-MTHF), lignin is directly separated from the pulp and the soluble carbohydrates by in situextraction. The oxalic acid catalyst can be crystallized from the aqueous solution, recovered and re-used. The delignified cellulose-pulp obtained from this biphasic system can be directly subjected to enzymatic depolymerization, affording soluble oligomers and glucose at rates almost comparable to those observed for the hydrolysis of commercial microcrystalline Avicel®. Overall, the concept may offer a promising approach for an efficient and selective pre-treatment of lignocellulosic materials under mild and environmentally-friendly conditions.

Enzymatic Synthesis of Optically Active Tertiary Alcohols: Expanding the Biocatalysis Toolbox

Enantiopure tertiary alcohols are very valuable building blocks for the synthesis of many different natural products and pharmaceuticals. As a consequence, several chemical and enzymatic strategies to afford such chiral structures have been described. Promising enzymatic approaches with agents such as epoxide hydrolases, dehalogenases and hydroxynitrile lyases have been reported, as well as dihydroxylation by microorganisms. Apart from those valuable options, the hydrolase‐based kinetic resolution of tertiary alcohols has been known for the last three decades, as several wild‐type enzymes have been reported to be able to accept these sterically hindered molecules. More recently, the existence of an amino acid motif within an enzyme’s active site has been identified as highly relevant for the acceptance of such bulky structures. This discovery clearly facilitates the identification of novel biocatalysts for this application. Although several tertiary alcohols have been successfully resolved with wild‐type biocatalysts, enantioselectivities have often been too low for synthetic purposes. These limitations have recently been overcome by accessing enzymes from the metagenome through directed evolution or by rational protein design. This minireview describes the state of the art in this area, highlighting aspects of basic academic research into the practical application of biocatalysts for the synthesis of optically active tertiary alcohols.

Iron-Catalyzed Furfural Production in Biobased Biphasic Systems: From Pure Sugars to Direct Use of Crude Xylose Effluents as Feedstock

Selective catalytic routes for processing the carbohydrate fractions of lignocellulose to deliver valuable platform chemicals are important and challenging paths for biomass valorization. A key step in this value chain is the dehydration of monomeric sugars to afford furan derivatives as valuable materials for numerous applications. Furfural can be derived from the C5sugar xylose, which is the most abundant sugar of the hemicellulose fraction in lignocellulose. Chemical approaches for xylose dehydration usually involve acidic conditions, using either mineral acids 5, 8] or acidic heterogeneous catalysts such as zeolites, MCM-41 materials, and heteropolyacids. To overcome humin formation in furfural dehydration, 13] the application of aqueous biphasic systems (using organic solvents such as methyl isobutyl ketone or toluene) for the in situ extraction of furfural has recently been proposed. 14–16] For sugar dehydration, different catalysts (e.g. , CrCl2, ZnCl2, FeCl3) have been assessed in non-aqueous deep-eutectic solvents such as choline chloride fructose mixtures as well as in monophasic aqueous media. 19] In this Communication a biphasic approach for xylose dehydration to afford furfural is reported. The approach is based on aqueous solutions of FeCl3·6 H2O and NaCl, combined with a second 2-methyltetrahydrofuran (2-MTHF) phase as biomass-derived solvent (Figure 1). After proof-of-concept experiments using pure commercially available crystalline xylose, the dehydration strategy is also assessed by directly using the aqueous, nonpurified xylose effluent obtained from pretreatment of lignocellulose with oxalic acid. In preliminary experiments, aqueous solutions of xylose were treated with catalytic amounts of different catalysts [i.e. , Fe(acac)3, FeCl3·6 H2O, FeSO4·7 H2O, FeCl2·4 H2O, MnCl2, Cu(OAc)2, and CuCl2·2 H2O] and subsequently layered with 2MTHF as organic phase. Among the tested catalysts, FeCl3·6 H2O displayed superior results and hence was selected for further assessments. After conducting the reaction at 140 8C for up to 6 h, the resulting furfural concentration in the 2-MTHF phase was determined by gas chromatography (GC). Initial kinetic measurements were taken with FeCl3·6 H2O loadings of 40 mol %. The furfural yield increased linearly up to 40 % furfural yield after 6 h. Hence the furfural production rate kfurfural was determined, based on the slope of the data from kinetic experiments conducted on 1 mmol scale. Further studies were done to optimize the efficiency. Thus, different amounts of NaCl were added to the aqueous phase (Table 1). The furfural production rate kfurfural improved considerably with increasing NaCl loading (Table 1, entries 1, 3–6). The rate could be increased by a factor of more than two by adding 20 wt % NaCl (entries 1 and 5). The effect of salt has been suggested to enhance the partitioning coefficient of furfural to organic phase. Consequently, running the reaction with 20 wt % NaCl (entry 5) for 4 h afforded a 70 % yield of furfural. However, the yield did not increase at longer reaction times (6 h) due to humin formation, which was avoided by applying shorter residence times. Increasing the amount of catalyst (up to 0.6 mmol) at 20 wt % NaCl loading afforded high furfural yields, of 65 to 70 %, after 2 h reaction time at 140 8C. A further increase of the NaCl loading to 30 wt % did not result in a better furfural production rate (entry 6), presumably due to furfural degradation. Finally, previous studies on biomass processing showed the potential of using seawater as solvent. 23] Gratifyingly, in this case the direct use of seawater (comprising different salts) with FeCl3·6 H2O also resulted in an improved furfural production rate (entry 8). Aqueous solutions of FeCl3 (0.08 m) are acidic (pH 1.4). To assess whether or not the sugar dehydration in these solutions was dominated by Brønsted acidity, we ran control experiments in aqueous HCl with an identical proton concentration, c(H) = 0.04 m. Table 1, entries 1 and 2 show that the dehydration rate with FeCl3·6 H2O is significantly higher than that with HCl at the same pH. Consistently, the addition of NaCl improves the performance of both, HCl and FeCl3·6 H2O, but with largely superior outcomes in the case of FeCl3·6 H2O (entries 6 and 7). This demonstrates that the activity of FeCl3·6 H2O in xylose dehydration is not solely governed by its Brønsted acidity. Figure 1. Iron-catalyzed xylose dehydration. 98 % of the furfural was extracted into the 2-MTHF phase.

Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater

Cellulases are promising catalysts for the depolymerization of cellulose under mild conditions. Reengineered cellulases are required to match application demands in biorefineries and to avoid cost-intensive downstream processing. This manuscript provides a novel fluorescence-based high throughput screening method for directed evolution of cellulases, based on 4-methylumbelliferyl-β-D-cellobioside (4-MUC). The 4-MUC high throughput screening system was successfully employed to identify CelA2 variants with enhanced stability and activity in mixtures of water with deep eutectic solvents like choline chloride : glycerol (ChCl : Gly), and seawater. The cellulase variant 4D1 (L21P; L184Q; H288R; K299I; D330G; N442D) was isolated and showed, compared to wild type, an increase in specific activity in 30% (v/v) ChCl : Gly (7.5-fold; 0.4 to 3.0 U mg−1) and in concentrated seawater (1.6-fold; 5.5 to 9.3 U mg−1). In addition, the residual activity of 4D1 in the presence of 3-fold concentrated seawater is unaffected whereas CelA2 wild type loses >50% of its activity. Furthermore, the position H288 was identified as a key position for activity and resistance in 4D1.

Biomass pretreatment affects Ustilago maydis in producing itaconic acid

BackgroundIn the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.ResultsU. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.ConclusionThe fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.

Fractionation of lignocellulosic biomass using the OrganoCat process

The fractionation of lignocellulose in its three main components, hemicellulose, lignin and cellulose pulp can be achieved in a biphasic system comprising water and bio-based 2-methyltetrahydrofuran (2-MeTHF) as solvents and oxalic acid as catalyst at mild temperatures (up to 140 °C). This so-called OrganoCat concept relies on selective hemicellulose depolymerization to form an aqueous stream of the corresponding carbohydrates, whereas solid cellulose pulp remains suspended and the disentangled lignin is to a large extent extracted in situ with the organic phase. In the present paper, it is demonstrated that biomass loadings of 100 g L−1 can be efficiently fractionated within 3 h whereby the mild conditions assure that no significant amounts of by-products (e.g. furans) are formed. Removing the solid pulp by filtration allows to re-use the water and organic phase without product separation in repetitive batch mode. In this way, (at least) 400 g L−1 biomass can be processed in 4 cycles, leading to greatly improved biomass-to-catalyst and biomass-to-solvent ratios. Economic analysis of the process reveals that the improved biomass loading significantly reduces capital and energy costs in the solvent recycle, indicating the importance of process integration for potential implementation. The procedure was successfully scaled-up from the screening on bench scale to 3 L reactor. The feedstock flexibility was assessed for biomasses containing moderate-to-high hemicellulose content.

Recent trends in industrial microbiology

The majority of current biotechnological applications are of microbial origin, and it is widely appreciated that the microbial world contains by far the greatest fraction of biodiversity in the biosphere. Because of their biotech impact, numerous efforts are being undertaken worldwide, with an ultimate goal to deliver new usable substances of microbial origin to the marketplace. However, the direct isolation of microbes always revealed that the majority are not amenable to be cultured and no representatives for many major microbial phyla have been thus far characterized. Therefore, the knowledge on new microbes and/or genomic information thereof, or from their communities, will pose an enormous potential to provide industry with novel products and processes based on the use of microbial resources, and contribute to and extend the basic mechanistic knowledge on the functioning of organisms. The present review highlights some examples and advances in the exploration of the genetic reservoir of (un)cultured microbes for industrial applications.

Lipase-Catalyzed (Trans)esterification of 5-Hydroxy- methylfurfural and Separation from HMF Esters using Deep-Eutectic Solvents

5-Hydroxymethylfurfural (HMF) is a valuable biomass-derived building block. Among possible HMF valorization products, a broad range of HMF esters can be synthesized. These HMF esters have found some promising applications, such as monomers, fuels, additives, surfactants, and fungicides, and thus several catalytic approaches for HMF (trans)esterifications have been reported. The intrinsic reactivity of HMF is challenging, forcing the use of mild reaction conditions to avoid by-product formation. This paper explores the lipase-catalyzed (trans)esterification of HMF with different acyl donors (carboxylic acids and methyl- and ethyl esters) mostly in solvent-free conditions. The results demonstrate that lipases may be promising alternatives for the synthesis of HMF esters-with high productivities and reactions at high substrate loadings-provided that robust systems for lipase immobilization are applied to assure an adequate reusability of the enzymes. Once (trans)esterifications have been conducted, the separation of unreacted HMF and HMF esters is performed by using deep-eutectic solvents (DES) as separation agents. DES are able to dissolve hydrogen-bond donors (e.g., HMF), whereas non-hydrogen-bond donors (in this case HMF esters) form a second phase. By using this approach, high ester purities (>99 %) and efficiencies (up to >90 % HMF ester recovery) in separations were obtained by using choline chloride-based DES.