Integrated Conversion of Lignocellulosic Biomass to Bio-Based Amphiphiles using a Functionalization-Defunctionalization Approach.

Concerns over the sustainability and end-of-life properties of fossil-derived surfactants have driven interest in bio-based alternatives. Lignocellulosic biomass with its polar functional groups is an obvious feedstock for surfactant production but its use is limited by process complexity and low yield. Here, we present a simple two-step approach to prepare bio-based amphiphiles directly from hemicellulose and lignin at high yields (29 % w/w based on the total raw biomass and >80 % w/w of these two fractions). Acetal functionalization of xylan and lignin with fatty aldehydes during fractionation introduced hydrophobic segments and subsequent defunctionalization by hydrogenolysis of the xylose derivatives or acidic hydrolysis of the lignin derivatives produced amphiphiles. The resulting biodegradable xylose acetals and/or ethers, and lignin-based amphiphilic polymers both largely retained their original natural structures, but exhibited competitive or superior surface activity in water/oil systems compared to common bio-based surfactants.

Quantification of Native Lignin Structural Features with Gel-Phase 2D-HSQC0 Reveals Lignin Structural Changes During Extraction.

Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin's structural features. Here, we developed a rapid elevated-temperature 1H-13C Heteronuclear Single-Quantum Coherence Zero (HSQC0) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC0 measurements with yield measurements following depolymerisation, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde-assisted fractionation.

Aldehyde-Assisted Lignocellulose Fractionation Provides Unique Lignin Oligomers for the Design of Tunable Polyurethane Bioresins.

Thanks to chemical stabilization, aldehyde-assisted fractionation (AAF) of lignocellulosic biomass has recently emerged as a powerful tool for the production of largely uncondensed lignin. Depolymerization of AAF lignin via ether cleavage provides aromatic monomers at near theoretical yields based on ether cleavage and an oligomeric fraction that remains largely unexploited despite its unique material properties. Here, we present an in-depth analytical characterization of AAF oligomers derived from hardwood and softwood in order to elucidate their molecular structures. These bioaromatic oligomers surpass technical Kraft lignin in terms of purity, solubility, and functionality and thus cannot even be compared to this common feedstock directly for material production. Instead, we performed comparative experiments with Kraft oligomers of similar molecular weight (Mn ∼ 1000) obtained through solvent extraction. These oligomers were then formulated into polyurethane materials. Substantial differences in material properties were observed depending on the amount of lignin, the botanical origin, and the biorefining process (AAF vs Kraft), suggesting new design principles for lignin-derived biopolymers with tailored properties. These results highlight the surprising versatility of AAF oligomers towards the design of new biomaterials and further demonstrate that AAF can enable the conversion of all biomass fractions into value-added products.

Consolidated bioprocessing of lignocellulosic biomass to lactic acid by a synthetic fungal-bacterial consortium.

Consolidated bioprocessing (CBP) of lignocellulosic feedstocks to platform chemicals requires complex metabolic processes, which are commonly executed by single genetically engineered microorganisms. Alternatively, synthetic consortia can be employed to compartmentalize the required metabolic functions among different specialized microorganisms as demonstrated in this work for the direct production of lactic acid from lignocellulosic biomass. We composed an artificial cross-kingdom consortium and co-cultivated the aerobic fungus Trichoderma reesei for the secretion of cellulolytic enzymes with facultative anaerobic lactic acid bacteria. We engineered ecological niches to enable the formation of a spatially structured biofilm. Up to 34.7 gL-1 lactic acid could be produced from 5% (w/w) microcrystalline cellulose. Challenges in converting pretreated lignocellulosic biomass include the presence of inhibitors, the formation of acetic acid and carbon catabolite repression. In the CBP consortium hexoses and pentoses were simultaneously consumed and metabolic cross-feeding enabled the in situ degradation of acetic acid. As a result, superior product purities were achieved and 19.8 gL-1 (85.2% of the theoretical maximum) of lactic acid could be produced from non-detoxified steam-pretreated beech wood. These results demonstrate the potential of consortium-based CBP technologies for the production of high value chemicals from pretreated lignocellulosic biomass in a single step.

Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C). Enzyme bound predominantly to areas with low autofluorescence, where structure loss and lignin removal had occurred during pretreatment; this confirms the importance of these processes for successful hydrolysis. The overall shape of both untreated and pretreated hardwood and switchgrass particles showed little change during enzymatic hydrolysis beyond a drop in autofluorescence intensity. The permanence of shape along with a relatively constant bound enzyme signal throughout hydrolysis was similar to observations previously made for filter paper, and was consistent with a modeling geometry of a hollowing out cylinder with widening pores represented as infinite slits. Modeling estimates of available surface areas for pretreated biomass were consistent with previously reported experimental results.

Modeling enzymatic hydrolysis of lignocellulosic substrates using confocal fluorescence microscopy I: filter paper cellulose.

Enzymatic hydrolysis is one of the critical steps in depolymerizing lignocellulosic biomass into fermentable sugars for further upgrading into fuels and/or chemicals. However, many studies still rely on empirical trends to optimize enzymatic reactions. An improved understanding of enzymatic hydrolysis could allow research efforts to follow a rational design guided by an appropriate theoretical framework. In this study, we present a method to image cellulosic substrates with complex three-dimensional structure, such as filter paper, undergoing hydrolysis under conditions relevant to industrial saccharification processes (i.e., temperature of 50°C, using commercial cellulolytic cocktails). Fluorescence intensities resulting from confocal images were used to estimate parameters for a diffusion and reaction model. Furthermore, the observation of a relatively constant bound enzyme fluorescence signal throughout hydrolysis supported our modeling assumption regarding the structure of biomass during hydrolysis. The observed behavior suggests that pore evolution can be modeled as widening of infinitely long slits. The resulting model accurately predicts the concentrations of soluble carbohydrates obtained from independent saccharification experiments conducted in bulk, demonstrating its relevance to biomass conversion work.

A pore-hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass.

Until now, most efforts to improve monosaccharide production from biomass through pretreatment and enzymatic hydrolysis have used empirical optimization rather than employing a rational design process guided by a theory-based modeling framework. For such an approach to be successful a modeling framework that captures the key mechanisms governing the relationship between pretreatment and enzymatic hydrolysis must be developed. In this study, we propose a pore-hindered diffusion and kinetic model for enzymatic hydrolysis of biomass. When compared to data available in the literature, this model accurately predicts the well-known dependence of initial cellulose hydrolysis rates on surface area available to a cellulase-size molecule. Modeling results suggest that, for particles smaller than 5 × 10(-3) cm, a key rate-limiting step is the exposure of previously unexposed cellulose occurring after cellulose on the surface has hydrolyzed, rather than binding or diffusion. However, for larger particles, according to the model, diffusion plays a more significant role. Therefore, the proposed model can be used to design experiments that produce results that are either affected or unaffected by diffusion. Finally, by using pore size distribution data to predict the biomass fraction that is accessible to degradation, this model can be used to predict cellulose hydrolysis with time using only pore size distribution and initial composition data.

Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy.

Understanding the depolymerization mechanisms of cellulosic substrates by cellulase cocktails is a critical step towards optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 ß-glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), which was immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A, which served as a reporter to track cellulase binding onto the physical structure of the cellulosic substrate. Both micro-scale imaging and bulk experiments were conducted. All reported experiments were conducted at 50 °C, the optimal temperature for maximum hydrolytic activity of the enzyme cocktail. BMCC structure was observed throughout degradation by labeling it with a fluorescent dye. This method allowed us to measure the binding of cellulases in situ and follow the temporal morphological changes of cellulose during its depolymerization by a commercial cellulase mixture. Three kinetic models were developed and fitted to fluorescence intensity data obtained through confocal microscopy: irreversible and reversible binding models, and an instantaneous binding model. The models were successfully used to predict the soluble sugar concentrations that were liberated from BMCC in bulk experiments. Comparing binding and kinetic parameters from models with different assumptions to previously reported constants in the literature led us to conclude that exposing new binding sites is an important rate-limiting step in the hydrolysis of crystalline cellulose.

Selecting Suitable Near-Native Lignins for Research.

There are several methods to isolate near-native lignins, including milled-wood lignin, enzymatic lignin, cellulolytic enzyme lignin, and enzymatic mild-acidolysis lignin. Which one is the most representative of the native lignin? Herein, near-native lignins were isolated from different plant groups and structurally analyzed to determine how well these lignins represented their native lignin counterparts. Analytical methods were applied to understand the molecular weight, monomer composition, and distribution of interunit linkages in the structure of the lignins. The results indicated that either enzymatic lignin or cellulolytic enzyme lignin may be used to represent native lignin in softwoods and hardwoods. None of the lignins, however, appeared to represent native lignins in grasses (monocot plants) because of substantial syringyl/guaiacyl differences. Complicating the understanding of grass lignin structure, large amounts of hydroxycinnamates acylate their polysaccharides and, when released, are often conflated with actual lignin monomers.

Two-temperature stage biphasic CO2-H2O pretreatment of lignocellulosic biomass at high solid loadings.

Most biomass pretreatment processes for monosaccharide production are run at low-solid concentration (<10 wt%) and use significant amounts of chemical catalysts. Biphasic CO(2) -H(2) O mixtures could provide a more sustainable pretreatment medium while using high-solid contents. Using a stirred reactor for high solids (40 wt%, biomass water mixture) biphasic CO(2)-H(2) O pretreatment of lignocellulosic biomass allowed us to explore the effects of particle size and mixing on mixed hardwood and switchgrass pretreatment. Subsequently, a two-temperature stage pretreatment was introduced. After optimization, a short high-temperature stage at 210°C (16 min for hardwood and 1 min for switchgrass) was followed by a long low-temperature stage at 160°C for 60 min. Glucan to glucose conversion yields of 83% for hardwood and 80% for switchgrass were obtained. Total molar sugar yields of 65% and 55% were obtained for wood and switchgrass, respectively, which consisted of a 10% points improvement over those obtained during our previous study despite a 10-fold increase in particle size. These yields are similar to those obtained with other major pretreatment technologies for wood and within 10% of major technologies for switchgrass despite the absence of chemical catalysts, the use of large particles (0.95 cm) and high solid contents (40 wt%).

Extraction and Surfactant Properties of Glyoxylic Acid-Functionalized Lignin.

The amphiphilic chemical structure of native lignin, composed by a hydrophobic aromatic core and hydrophilic hydroxy groups, makes it a promising alternative for the development of bio-based surface-active compounds. However, the severe conditions traditionally needed during biomass fractionation make lignin prone to condensation and cause it to lose hydrophilic hydroxy groups in favour of the formation of C-C bonds, ultimately decreasing lignin's abilities to lower surface tension of water/oil mixtures. Therefore, it is often necessary to further functionalize lignin in additional synthetic steps in order to obtain a surfactant with suitable properties. In this work, multifunctional aldehyde-assisted fractionation with glyoxylic acid (GA) was used to prevent lignin condensation and simultaneously introduce a controlled amount of carboxylic acid on the lignin backbone for its further use as surfactant. After fully characterizing the extracted GA-lignin, its surface activity was measured in several water/oil systems at different pH values. Then, the stability of water/mineral oil emulsions was evaluated at different pH and over a course of 30 days by traditional photography and microscopy imaging. Further, the use of GA-lignin as a surfactant was investigated in the formulation of a cosmetic hand cream composed of industrially relevant ingredients. Contrary to industrial lignins such as Kraft lignin, GA-lignin did not alter the color or smell of the formulation. Finally, the surface activity of GA-lignin was compared with other lignin-based and fossil-based surfactants, showing that GA-lignin presented similar or better surface-active properties compared to some of the most commonly used surfactants. The overall results showed that GA-lignin, a biopolymer that can be made exclusively from renewable carbon, can successfully be extracted in one step from lignocellulosic biomass. This lignin can be used as an effective surfactant without further modification, and as such is a promising candidate for the development of new bio-based surface-active products.

Sustainable polyesters via direct functionalization of lignocellulosic sugars.

The development of sustainable plastics from abundant renewable feedstocks has been limited by the complexity and efficiency of their production, as well as their lack of competitive material properties. Here we demonstrate the direct transformation of the hemicellulosic fraction of non-edible biomass into a tricyclic diester plastic precursor at 83% yield (95% from commercial xylose) during integrated plant fractionation with glyoxylic acid. Melt polycondensation of the resulting diester with a range of aliphatic diols led to amorphous polyesters (Mn = 30-60 kDa) with high glass transition temperatures (72-100 °C), tough mechanical properties (ultimate tensile strengths of 63-77 MPa, tensile moduli of 2,000-2,500 MPa and elongations at break of 50-80%) and strong gas barriers (oxygen transmission rates (100 µm) of 11-24 cc m-2 day-1 bar-1 and water vapour transmission rates (100 µm) of 25-36 g m-2 day-1) that could be processed by injection moulding, thermoforming, twin-screw extrusion and three-dimensional printing. Although standardized biodegradation studies still need to be performed, the inherently degradable nature of these materials facilitated their chemical recycling via methanolysis at 64 °C, and eventual depolymerization in room-temperature water.

High-solids biphasic CO2-H2O pretreatment of lignocellulosic biomass.

A high pressure (200 bar) CO(2)-H(2)O process was developed for pretreating lignocellulosic biomass at high-solid contents, while minimizing chemical inputs. Hardwood was pretreated at 20 and 40 (wt.%) solids. Switchgrass, corn stover, big bluestem, and mixed perennial grasses (a co-culture of big bluestem and switchgrass) were pretreated at 40 (wt.%) solids. Operating temperatures ranged from 150 to 250 degrees C, and residence times from 20 s to 60 min. At these conditions a biphasic mixture of an H(2)O-rich liquid (hydrothermal) phase and a CO(2)-rich supercritical phase coexist. Following pretreatment, samples were then enzymatically hydrolyzed. Total yields, defined as the fraction of the theoretical maximum, were determined for glucose, hemicellulose sugars, and two degradation products: furfural and 5-hydroxymethylfurfural. Response surfaces of yield as a function of temperature and residence time were compared for different moisture contents and biomass species. Pretreatment at 170 degrees C for 60 min gave glucose yields of 77%, 73%, and 68% for 20 and 40 (wt.%) solids mixed hardwood and mixed perennial grasses, respectively. Pretreatment at 160 degrees C for 60 min gave glucan to glucose yields of 81% for switchgrass and 85% for corn stover.

A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose.

Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes. Clostridium tyrobutyricum, Veillonella criceti, or Megasphaera elsdenii were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals.

Fractionation of lignocellulosic biomass to produce uncondensed aldehyde-stabilized lignin.

Lignin is one of the most promising sources of renewable aromatic hydrocarbons. Current methods for its extraction from lignocellulosic biomass-which include the kraft, sulfite, and organosolv processes-result in the rapid formation of carbon-carbon bonds, leading to a condensed lignin that cannot be effectively depolymerized into its constituent monomers. Treatment of lignocellulosic biomass with aldehydes during lignin extraction generates an aldehyde-stabilized lignin that is uncondensed and can be converted into its monomers at near-theoretical yields. Here, we outline an efficient, reproducible, and scalable process for extracting and purifying this aldehyde-stabilized lignin as a solid, which can easily be re-dissolved in an organic solvent. Upon exposure to hydrogenolysis conditions, this material provides near-theoretical yields of aromatic monomers (~40-50% of the Klason lignin for a typical hardwood). Cellulose and hemicellulose are also efficiently fractionated. This protocol requires 6-7 h for the extraction of the stabilized lignin and a basic proficiency in synthetic chemistry.

Formaldehyde stabilization facilitates lignin monomer production during biomass depolymerization.

Practical, high-yield lignin depolymerization methods could greatly increase biorefinery productivity and profitability. However, development of these methods is limited by the presence of interunit carbon-carbon bonds within native lignin, and further by formation of such linkages during lignin extraction. We report that adding formaldehyde during biomass pretreatment produces a soluble lignin fraction that can be converted to guaiacyl and syringyl monomers at near theoretical yields during subsequent hydrogenolysis (47 mole % of Klason lignin for beech and 78 mole % for a high-syringyl transgenic poplar). These yields were three to seven times those obtained without formaldehyde, which prevented lignin condensation by forming 1,3-dioxane structures with lignin side-chain hydroxyl groups. By depolymerizing cellulose, hemicelluloses, and lignin separately, monomer yields were between 76 and 90 mole % for these three major biomass fractions.

A lignocellulosic ethanol strategy via nonenzymatic sugar production: process synthesis and analysis.

The work develops a strategy for the production of ethanol from lignocellulosic biomass. In this strategy, the cellulose and hemicellulose fractions are simultaneously converted to sugars using a γ-valerolactone (GVL) solvent containing a dilute acid catalyst. To effectively recover GVL for reuse as solvent and biomass-derived lignin for heat and power generation, separation subsystems, including a novel CO2-based extraction for the separation of sugars from GVL, lignin and humins have been designed. The sugars are co-fermented by yeast to produce ethanol. Furthermore, heat integration to reduce utility requirements is performed. It is shown that this strategy leads to high ethanol yields and the total energy requirements could be satisfied by burning the lignin. The integrated strategy using corn stover feedstock leads to a minimum selling price of $5 per gallon of gasoline equivalent, which suggests that it is a promising alternative to current biofuels production approaches.

Solvent-enabled nonenyzmatic sugar production from biomass for chemical and biological upgrading.

We recently reported a nonenzymatic biomass deconstruction process for producing carbohydrates using homogeneous mixtures of γ-valerolactone (GVL) and water as a solvent. A key step in this process is the separation of the GVL from the aqueous phase, enabling GVL recycling and the production of a concentrated aqueous carbohydrate solution. In this study, we demonstrate that phenolic solvents-sec-butylphenol, nonylphenol, and lignin-derived propyl guaiacol-are effective at separating GVL from the aqueous phase using only small amounts of solvent (0.5 g per g of the original water, GVL, and sugar hydrolysate). Furthermore, using nonylphenol, we produced a hydrolysate that supported robust growth and high yields of ethanol (0.49 g EtOH per g glucose) at an industrially relevant concentration (50.8 g L(-1) EtOH). These results suggest that using phenolic solvents could be an interesting solution for separating and/or detoxifying aqueous carbohydrate solutions produced using GVL-based biomass deconstruction processes.

Nonenzymatic sugar production from biomass using biomass-derived γ-valerolactone.

Widespread production of biomass-derived fuels and chemicals will require cost-effective processes for breaking down cellulose and hemicellulose into their constituent sugars. Here, we report laboratory-scale production of soluble carbohydrates from corn stover, hardwood, and softwood at high yields (70 to 90%) in a solvent mixture of biomass-derived γ-valerolactone (GVL), water, and dilute acid (0.05 weight percent H2SO4). GVL promotes thermocatalytic saccharification through complete solubilization of the biomass, including the lignin fraction. The carbohydrates can be recovered and concentrated (up to 127 grams per liter) by extraction from GVL into an aqueous phase by addition of NaCl or liquid CO2. This strategy is well suited for catalytic upgrading to furans or fermentative upgrading to ethanol at high titers and near theoretical yield. We estimate through preliminary techno-economic modeling that the overall process could be cost-competitive for ethanol production, with biomass pretreatment followed by enzymatic hydrolysis.