Charles M. Cai

Co‐solvent Pretreatment Reduces Costly Enzyme Requirements for High Sugar and Ethanol Yields from Lignocellulosic Biomass

We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover.

THF co-solvent enhances hydrocarbon fuel precursor yields from lignocellulosic biomass

A novel single phase co-solvent system using tetrahydrofuran (THF) promotes hydrolysis of maple wood to sugars, sugar dehydration, and lignin extraction simultaneously and achieves higher overall yields of the fuel precursors furfural, 5-hydroxymethylfurfural (HMF), and levulinic acid (LA) than previously reported from biomass. In a one-pot reaction, we obtained yields of 86% furfural, 21% HMF, and 40% LA in the liquid phase and over 90% extraction of lignin as a solid powder. The co-solvent reaction also produced a glucan-rich residue that is highly digestible by enzymes for biological conversion to ethanol or further thermochemical reaction to additional HMF and levulinic acid. These findings enable an integrated conversion platform in which THF is both a co-solvent and final co-product to enhance production of fuel precursors for catalytic upgrading to renewable liquid hydrocarbons fuels.

Coupling metal halides with a co-solvent to produce furfural and 5-HMF at high yields directly from lignocellulosic biomass as an integrated biofuels strategy

Metal halides are selective catalysts suitable for production of the fuel precursors furfural and 5-HMF from sugars derived from lignocellulosic biomass. However, they do not perform nearly as well when applied to biomass even in combination with immiscible extracting solvents or expensive ionic co-solvents. Here, we couple metal halides with a highly tunable co-solvent system employing renewable tetrahydrofuran (THF) to significantly enhance co-production of furfural and 5-HMF from biomass in a single phase reaction strategy capable of integrating biomass deconstruction with catalytic dehydration of sugars. Screening of several promising metal halide species at 170 °C in pH-controlled reactions with sugar solutions and larger 1 L reactions with maple wood and corn stover revealed how the interplay between relative Bronsted and Lewis acidities was responsible for enhancing catalytic performance in THF co-solvent. Combining FeCl3 with THF co-solvent was particularly effective, achieving one of the highest reported simultaneous yields of furfural (95%) and 5-HMF (51%) directly from biomass with minimal levulinic acid formation (6%). Furthermore, over 90% of the lignin from biomass was extracted by THF and recovered as a fine lignin powder. Tuning the volume ratio of THF to water from 4 : 1 to 1 : 1 preserved 10% to 31% of the reacted biomass as a glucan-rich solid suitable for further catalytic reaction, enzymatic digestion, or possible pulp and paper production.

CELF pretreatment of corn stover boosts ethanol titers and yields from high solids SSF with low enzyme loadings

A major challenge to economically produce ethanol from lignocellulosic biomass is to achieve industrially relevant ethanol titers (>50 g L−1) to control operating and capital costs for downstream ethanol operations while maintaining high ethanol yields. However, due to reduced fermentation effectiveness at high biomass solids loadings, excessive amounts of enzymes are typically required to obtain reasonable ethanol titers, thereby trading off reduced operating and capital costs with high enzyme costs. In this study, we applied our newly developed Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment to produce highly digestible glucan-rich solids from corn stover. Simultaneous saccharification and fermentation (SSF) was then applied to pretreated solids from CELF at 15.5 wt% solids loadings (corresponding to 11 wt% glucan loadings) in modified shake flasks to achieve an ethanol titer of 58.8 g L−1 at 89.2% yield with an enzyme loading of 15 mg-protein per g-glucan-in-raw-corn-stover (-RCS) in only 5 days. By comparison, SSF of corn stover solids from dilute acid pretreatment at 18.3 wt% solids loading (or 10 wt% glucan loading) only achieved an ethanol titer and a yield of 47.8 g L−1 and 73.0%, respectively, despite needing longer fermentation times (∼20 days) and an additional 18 h of prehydrolysis at 50 °C. Remarkably, although longer fermentation times were required at more economical enzyme loadings of 5 and 2 mg-protein per g-glucan-in-RCS, high solids SSF of CELF pretreated corn stover realized final ethanol titers consistently above 50 g L−1 and yields over 80%.

Cosolvent pretreatment in cellulosic biofuel production: effect of tetrahydrofuran-water on lignin structure and dynamics

The deconstruction of cellulose is an essential step in the production of ethanol from lignocellulosic biomass. However, the presence of lignin hinders this process. Recently, a novel cosolvent based biomass pretreatment method called CELF (Cosolvent Enhanced Lignocellulosic Fractionation) which employs tetrahydrofuran (THF) in a single phase mixture with water, was found to be highly effective at solubilizing and extracting lignin from lignocellulosic biomass and achieving high yields of fermentable sugars. Here, using all-atom molecular-dynamics simulation, we find that THF preferentially solvates lignin, and in doing so, shifts the equilibrium configurational distribution of the biopolymer from a crumpled globule to coil, independent of temperature. Whereas pure water is a bad solvent for lignin, the THF : water cosolvent acts as a “theta” solvent, in which solvent : lignin and lignin : lignin interactions are approximately equivalent in strength. Under these conditions, polymers do not aggregate, thus providing a mechanism for the observed lignin solubilization that facilitates unfettered access of celluloytic enzymes to cellulose.

Local Phase Separation of Co-solvents Enhances Pretreatment of Biomass for Bioenergy Applications.

Pretreatment facilitates more complete deconstruction of plant biomass to enable more economic production of lignocellulosic biofuels and byproducts. Various co-solvent pretreatments have demonstrated advantages relative to aqueous-only methods by enhancing lignin removal to allow unfettered access to cellulose. However, there is a limited mechanistic understanding of the interactions between the co-solvents and cellulose that impedes further improvement of such pretreatment methods. Recently, tetrahydrofuran (THF) has been identified as a highly effective co-solvent for the pretreatment and fractionation of biomass. To elucidate the mechanism of the THF-water interactions with cellulose, we pair simulation and experimental data demonstrating that enhanced solubilization of cellulose can be achieved by the THF-water co-solvent system at equivolume mixtures and moderate temperatures (≤445 K). The simulations show that THF and water spontaneously phase separate on the local surface of a cellulose fiber, owing to hydrogen bonding of water molecules with the hydrophilic cellulose faces and stacking of THF molecules on the hydrophobic faces. Furthermore, a single fully solvated cellulose chain is shown to be preferentially bound by water molecules in the THF-water mixture. In light of these findings, co-solvent reactions were performed on microcrystalline cellulose and maple wood to show that THF significantly enhanced cellulose deconstruction and lignocellulose solubilization at simulation conditions, enabling a highly versatile and efficient biomass pretreatment and fractionation method.

Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol

Significance Future production of renewable transportation fuels such as ethanol must rely on abundant nonfood plant sources also known as lignocellulosic biomass. However, a major historical barrier to low-cost production of ethanol from biomass is the low ethanol yields and titers that result from fermentation of biomass solids at high solids when compared with simple sugar fermentations. Here, we show that combining a cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment process with subsequent simultaneous saccharification and fermentation (SSF) can achieve similar high ethanol yields and titers that match that of separate pure glucose fermentations. We demonstrate a strategy whereby direct fermentation of biomass to ethanol is now limited by the microbe rather than by the process. Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a long-standing limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery. Here, we illustrate how competing factors that limit ethanol yields during high-solids fermentations are overcome by integrating newly developed cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment with SSF. First, fed-batch glucose fermentations by Saccharomyces cerevisiae D5A revealed that this strain, which has been favored for SSF, can produce ethanol at titers of up to 86 g⋅L−1. Then, optimizing SSF of CELF-pretreated corn stover achieved unprecedented ethanol titers of 79.2, 81.3, and 85.6 g⋅L−1 in batch shake flask, corresponding to ethanol yields of 90.5%, 86.1%, and 80.8% at solids loadings of 20.0 wt %, 21.5 wt %, and 23.0 wt %, respectively. Ethanol yields remained at over 90% despite reducing enzyme loading to only 10 mg protein⋅g glucan−1 [∼6.5 filter paper units (FPU)], revealing that the enduring factors limiting further ethanol production were reduced cell viability and glucose uptake by D5A and not loss of enzyme activity or mixing issues, thereby demonstrating an SSF-based process that was limited by a strain’s metabolic capabilities and tolerance to ethanol.

Cellulose-hemicellulose interactions at elevated temperatures increase cellulose recalcitrance to biological conversion

It has been previously shown that cellulose-lignin droplets’ strong interactions, resulting from lignin coalescence and redisposition on cellulose surface during thermochemical pretreatments, increase cellulose recalcitrance to biological conversion, especially at commercially viable low enzyme loadings. However, information on the impact of cellulose–hemicellulose interactions on cellulose recalcitrance following relevant pretreatment conditions are scarce. Here, to investigate the effects of plausible hemicellulose precipitation and re-association with cellulose on cellulose conversion, different pretreatments were applied to pure Avicel® PH101 cellulose alone and Avicel mixed with model hemicellulose compounds followed by enzymatic hydrolysis of resulting solids at both low and high enzyme loadings. Solids produced by pretreatment of Avicel mixed with hemicelluloses (AMH) were found to contain about 2 to 14.6% of exogenous, precipitated hemicelluloses and showed a remarkably much lower digestibility (up to 60%) than their respective controls. However, the exogenous hemicellulosic residues that associated with Avicel following high temperature pretreatments resulted in greater losses in cellulose conversion than those formed at low temperatures, suggesting that temperature plays a strong role in the strength of cellulose–hemicellulose association. Molecular dynamics simulations of hemicellulosic xylan and cellulose were found to further support this temperature effect as the xylan–cellulose interactions were found to substantially increase at elevated temperatures. Furthermore, exogenous, precipitated hemicelluloses in pretreated AMH solids resulted in a larger drop in cellulose conversion than the delignified lignocellulosic biomass containing comparably much higher natural hemicellulose amounts. Increased cellulase loadings or supplementation of cellulase with xylanases enhanced cellulose conversion for most pretreated AMH solids; however, this approach was less effective for solids containing mannan polysaccharides, suggesting stronger association of cellulose with (hetero) mannans or lack of enzymes in the mixture required to hydrolyze such polysaccharides.

Temperature-dependent phase behaviour of tetrahydrofuran-water alters solubilization of xylan to improve co-production of furfurals from lignocellulosic biomass

Xylan is an important polysaccharide found in the hemicellulose fraction of lignocellulosic biomass that can be hydrolysed to xylose and further dehydrated to the furfural, an important renewable platform fuel precursor. Here, pairing molecular simulation and experimental evidence, we reveal how the unique temperature-dependent phase behaviour of water–tetrahydrofuran (THF) co-solvent can delay xylan solubilization to synergistically improve catalytic co-processing of biomass to furfural and 5-HMF. Our results indicate, based on polymer correlations between polymer conformational behaviour and solvent quality, that both co-solvent and aqueous environments serve as ‘good’ solvents for xylan. Interestingly, the simulations also revealed that unlike other cell-wall components (i.e., lignin and cellulose), the make-up of the solvation shell of xylan in THF–water is dependent on the temperature-phase behaviour. At temperatures between 333 K and 418 K, THF and water become immiscible, and THF is evacuated from the solvation shell of xylan, while above and below this temperature range, THF and water are both present in the polysaccharides solvation shell. This suggested that the solubilization of xylan in THF–water may be similar to aqueous-only solutions at temperatures between 333 K and 418 K and different outside this range. Experimental reactions on beachwood xylan corroborate this hypothesis by demonstrating 2-fold reduction of xylan solubilization in THF–water within a miscible temperature regime (445 K) and unchanged solubilization within an immiscible regime (400 K). Translating this phase-dependent behaviour to processing of maple wood chips, we demonstrate how the weaker xylan solvation in THF–water under miscible conditions can delay furfural production from xylan, allowing 5-HMF production from cellulose to “catch-up” such that their high yield production from biomass can be synergized in a single pot reaction.

Characterization of fractional cuts of co-solvent enhanced lignocellulosic fractionation lignin isolated by sequential precipitation

Lignin valorization is significantly hindered by the intrinsic heterogeneity of its complex structures and variability of biomass feedstocks. Fractionation of lignin can overcome these challenges by producing functionally distinct lignin cuts that can be further tailored to end products. Herein, lignin was extracted and depolymerized from poplar by the co-solvent enhanced lignocellulosic fractionation method with renewable THF to obtain CELF lignin. Several solvents were screened to separate soluble and insoluble fractions from the parent CELF lignin. The ethanol soluble portion was then fractionated into different molecular weight cuts via sequential precipitation of the lignin by reducing the concentration of THF. The physicochemical structures of different CELF lignin cuts were elucidated by GPC and NMR techniques. These results suggest that CELF lignin cuts with lower molecular weight contain progressively higher phenolic and carboxylic acid OH groups, which can be more suitable as green antioxidants than the parent lignin.