Production and characterization of stabilized lignin

Lignocellulosic biomass is a sustainable source of renewable carbon, and it is the most abundant form of terrestrial biomass. The three main constituents of lignocellulosic biomass are cellulose, hemicellulose, and lignin. The monomers from these biopolymers can be valuable feedstocks for a future sustainable chemical industry, and they include glucose from cellulose, predominantly xylose from hemicellulose, and aromatic molecules from lignin. Even though both glucose and xylose are already established feedstocks for further upgrading in biorefineries, the upgrading of lignin to higher value-added chemicals has not achieved the same success, despite the tremendous need for renewable aromatic molecules. Developing methods for the high-yield depolymerization of lignin can dramatically increase the efficiency and profitability of biorefineries. The main limiting factor in producing high-quality lignin is the inter-unit carbon-carbon linkages in the chemical structure of lignin, and also the formation of these bonds during the extraction process, known as repolymerization or condensation. In the first part of my thesis, I study the possibility of avoiding the condensation of lignin by preventing the formation of C-C linkages. This is achieved by adding formaldehyde, as a protection group, to the pretreatment of lignocellulosic biomass. The results show a near theoretical yield of monomers from the stabilized lignin after depolymerization by hydrogenolysis. This method was then further expanded by screening other protecting groups in order to develop a comprehensive picture of the chemistry involved in this process, and to optimize the process notably to be able to yield a narrower product distribution. Even though this method provides high-quality lignin, there is a need for developing a fractionation process for large-scale production of isolated lignin. This can facilitate the implementation of the process in biorefineries and also further studies on developing new upgrading pathways. The second part of this thesis presents a protocol that fractionates lignocellulosic biomass into pure parts of three main constituents in large laboratory-scale with high extraction yields. The results show extraction yields higher than 95% for each biopolymer. The highly digestible cellulose fraction and high-quality lignin can be depolymerized into their constituent monomers with conventional methods. However, the hemicellulose fraction is obtained as functionalized sugars due to its reaction with aldehyde during pretreatment. The third part of my work presents a non-destructive method for the prediction of the potential yield of monomers from the depolymerization of isolated lignin. A novel 2D heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) spectroscopy technique can be used for simultaneous identification and quantification of chemical groups in the structure of lignin. Standard 2D-HSQC NMR spectroscopy does not provide accurate quantification for polymers and oligomers such as lignin due to errors that are principally caused by differences in relaxation times for different parts of the oligomer chain. However, these errors can be avoided by extrapolation to time-zero 13C HSQC (HSQC0) for series of spectrums, acquired with different repetition times. The prediction results show agreement within a few percentage points with experimental results determined by gas chromatography (GC) after hydrogenolysis.

Developing structure-oriented models for enzymatic hydrolysis of lignocellulosic biomass

Jessica Charlène Rohrbach

With the unsustainable use of fossil fuels increasing strains on human institutions and ecosystems, the development of a renewable energy alternative is of paramount importance. Second generation biorefineries, based on the production of fuels and chemicals from lignocellulosic biomass, appear as attractive alternative to their non-renewable counterparts. Within the multiplicity of existing valorisation routes, enzymatic hydrolysis of lignocellulosic biomass into its constituent sugars has generated considerable interest, notably in the context of biofuels production. However, major hurdles stemming from the intricate structure of the lignocellulosic feedstocks still impede large-scale deployment of this process. The multiplicity of highly intertwined spatiotemporal factors impacting the enzymatic hydrolysis of lignocellulosic biomass represents a challenge to rationally design efficient hydrolysis process. Here, we propose a theory-based modelling framework relying on pore-diffusion and surface reaction to explore the effects of recognised bottlenecks on the enzymatic hydrolysis of lignocellulosic substrates. The model is based on a set of partial differential equations describing the evolution of the substrate morphology to investigate the interplay between experimental conditions and the physical characteristics of biomass particles as the reaction proceeds. The overall quantity of cellulase present in the hydrolysis mixture is carefully considered to investigate its interplay with the available accessible cellulose surface. Also, non-uniformity in terms of cellulose accessibility and cellulose digestibility are introduced in the model to weight their influence on observed hydrolysis rates. Finally, deactivation mechanisms are considered through unproductive adsorption of cellulases on both cellulose and lignin fractions, with the existence of such phenomena alleged to be critical in the efficiency of the hydrolysis process. Based on predictions of our model, we were able to confirm the critical role of cellulose accessibility, as defined by the combination of particle size, porosity and accessible cellulose surface, in dictating early reaction rates for a range of pretreated beech wood substrates. While high biomass loading should be favoured to improve enzyme penetration in the substrates, high enzyme loadings going beyond the initial number of cellulose adsorption appeared beneficial in notably two cases: (i) to promote internal diffusion in large particles and (ii) counteract undesired enzyme adsorption on lignin. For the latter, a relatively low increase in enzyme loading was sufficient to offset the resulting slowdown. We also showed that the existence of structural heterogeneities, and in particular non-uniform pore volume distribution within the lignocellulosic samples, contribute to the rate slowdown observed at later stage of the hydrolysis, while not explaining it in its entirety. Unproductive adsorption to cellulose, coupled to decrease in the cellulase efficiency at the cellulose surface, appeared as major contributor to the rate slowdown. Overall, we show how the use of a theory-based model can help decouple and evaluate the effects of key factors in the enzymatic hydrolysis of lignocellulosic biomass. As such, our model can help pave the way towards efficient integrated rational design strategies for enzyme process engineering for biomass conversion.

EPFL2021

Extraction and controlled functionalization of lignin for materials applications

Stefania Bertella

Lignin is a renewable aromatic polymer that due to its abundance and unique chemical structure is a promising candidate to replace aromatic materials that are currently sourced from fossil oil. The same structure of lignin poses however drawbacks for its valorization. The high temperature and strong pH that are generally used to isolate lignin, make this polymer prone to reactions of condensation during its extraction. This translates into isolated lignins where most of their native functional groups are eliminated in favor of the formation of interunit carbon-carbon linkages. The lack of control on lignin's chemical functionalities ultimately hinders the use of this polymer for the development of new materials. Many research groups have been trying to tackle these challenges by developing new processes that could allow for the efficient isolation of lignin without compromising on its functional groups. Amongst these, the Aldehyde-Assisted Fractionation (AAF), which relies on the formation of acetal groups between lignin's ß-O-4 units and an added aldehyde, was proven to be efficient in isolating high yields of lignin, that could be then quantitatively depolymerized into small aromatic monomers. Inspired by this research, the aim of my work is to take advantage of the AAF to quantitatively isolate lignin from lignocellulose, at the same time introducing on the biopolymer scaffold new functional groups that could allow for more effective development of lignin-based materials. My work is based on a Multifunctional-AAF, in which the multifunctional aldehydes can react with the lignin via the formation of acetals, at the same time leaving on the biopolymer additional functionalities. In the first part of my thesis, I show how the extraction and simultaneous functionalization of lignin can be achieved by using terephthalic aldehyde (TALD). In addition to successfully functionalizing the lignin, I demonstrate how the degree of functionalization can be precisely controlled and monitored via 1H NMR, yielding a material with high reactivity towards phenolation. In the second part, I show how the isolation and controlled functionalization of lignin can be translated to the use of other aldehydes, namely glyoxylic acid (GA), to functionalize lignin with carboxylates, quantified via 31P NMR. The isolated lignin offers in this case unique avenues for the development of more sustainable surfactants. In the last chapter of the thesis, I show that the Multifunctional-AAF can be expanded to isolate and functionalize lignin with multiple functionalities by using mixtures of aldehydes (TALD and GA), demonstrating again that each functional group can be easily quantified. These lignins, extracted with different ratios of the two functional groups, showed dissimilar properties in terms of solubility and reactivity towards gelatin. We used these lignins to develop lignin-gelatin based hydrogels, showing how the bi-functionalization was pivotal for the formation of hydrogels with improved rheological, mechanical, self-healing and adhesive properties. In summary, this thesis shows the development of a new approach for lignin extraction and simultaneous valorization. The versatility of this process enables more efficient functionalization, allowing the obtainment of lignins which are "tailor-made" for their end use. The control over lignin's chemical structure and functionalization will pave the way for the use of lignin in high-end application.

EPFL2022

Topology of Pretreated Wood Fibers Using Dynamic Nuclear Polarization

Michel Bardet, Pierrick Berruyer, David Lyndon Emsley, Wu Lan, Jeremy Luterbacher, Arthur César Pinon, Jasmine Viger-Gravel

In the continuously developing field of lignocellulosic biomass, high-yield lignin depolymerization processes are sought to optimize its productivity and profitability. Recently, formaldehyde stabilization during lignin extraction and biomass pretreatment has been found to drastically enhance subsequent lignin upgradeability but can affect cellulose digestibility. The exact role and/or form of formaldehyde on the residual biomass surface is still not fully understood. Here, we use magic angle spinning (MAS) dynamic nuclear polarization (DNP) methods to characterize the components that remain inside the residual cell wall after the lignin extraction process and reveal the topochemistry of the solid residue. The regioselectivity of relayed DNP allows the observation of hyperpolarization in a range of 40-200 nm from the surface of the cell wall for poplar wood materials. That regioselectivity allows us to distinguish between the external secondary cell wall and the inner middle lamellae. In that respect, for the untreated wood, we confirm that there is less lignin in the outer part of the cell wall than deeper inside. In treated wood, we determine that the role of dioxane during the process is to enable the extraction of the modified products from the cell wall. We show that the modified lignins which were not extracted in the absence of dioxane accumulate in a 40 nm region at the surface of the cell wall. Also, using carbon-13 enriched formaldehyde during the process, we show that 1% of the total amount of carbon in the material is assigned to self-polymerization and that no covalent bonds to cellulose are observed.