Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries.

The valorization of lignin, a currently underutilized component of lignocellulosic biomass, has attracted attention to promote a stable and circular bioeconomy. Successful approaches including thermochemical, biological, and catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino-refineries and lignocellulosic biorefineries. Although significant progress in lignin valorization has been made, this review describes unexplored opportunities in chemical and biological routes for lignin depolymerization and thereby contributes to economically and environmentally sustainable lignin-utilizing biorefineries. This review also highlights the integration of chemical and biological lignin depolymerization and identifies research gaps while also recommending future directions for scaling processes to establish a lignino-chemical industry.

An Engineered Laccase from Fomitiporia mediterranea Accelerates Lignocellulose Degradation.

Laccases from white-rot fungi catalyze lignin depolymerization, a critical first step to upgrading lignin to valuable biodiesel fuels and chemicals. In this study, a wildtype laccase from the basidiomycete Fomitiporia mediterranea (Fom_lac) and a variant engineered to have a carbohydrate-binding module (Fom_CBM) were studied for their ability to catalyze cleavage of ß-O-4' ether and C-C bonds in phenolic and non-phenolic lignin dimers using a nanostructure-initiator mass spectrometry-based assay. Fom_lac and Fom_CBM catalyze ß-O-4' ether and C-C bond breaking, with higher activity under acidic conditions (pH < 6). The potential of Fom_lac and Fom_CBM to enhance saccharification yields from untreated and ionic liquid pretreated pine was also investigated. Adding Fom_CBM to mixtures of cellulases and hemicellulases improved sugar yields by 140% on untreated pine and 32% on cholinium lysinate pretreated pine when compared to the inclusion of Fom_lac to the same mixtures. Adding either Fom_lac or Fom_CBM to mixtures of cellulases and hemicellulases effectively accelerates enzymatic hydrolysis, demonstrating its potential applications for lignocellulose valorization. We postulate that additional increases in sugar yields for the Fom_CBM enzyme mixtures were due to Fom_CBM being brought more proximal to lignin through binding to either cellulose or lignin itself.

Maximizing microbial bioproduction from sustainable carbon sources using iterative systems engineering.

Maximizing the production of heterologous biomolecules is a complex problem that can be addressed with a systems-level understanding of cellular metabolism and regulation. Specifically, growth-coupling approaches can increase product titers and yields and also enhance production rates. However, implementing these methods for non-canonical carbon streams is challenging due to gaps in metabolic models. Over four design-build-test-learn cycles, we rewire Pseudomonas putida KT2440 for growth-coupled production of indigoidine from para-coumarate. We explore 4,114 potential growth-coupling solutions and refine one design through laboratory evolution and ensemble data-driven methods. The final growth-coupled strain produces 7.3 g/L indigoidine at 77% maximum theoretical yield in para-coumarate minimal medium. The iterative use of growth-coupling designs and functional genomics with experimental validation was highly effective and agnostic to specific hosts, carbon streams, and final products and thus generalizable across many systems.

Lignin deconstruction by anaerobic fungi.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

Funneled Depolymerization of Ionic Liquid-Based Biorefinery "Heterogeneous" Lignin into Guaiacols over Reusable Palladium Catalyst.

The efficient utilization of lignin, the direct source of renewable aromatics, into value-added renewable chemicals is an important step towards sustainable biorefinery practices. Nevertheless, owing to the random heterogeneous structure and limited solubility, lignin utilization has been primarily limited to burning for energy. The catalytic depolymerization of lignin has been proposed and demonstrated as a viable route to sustainable biorefinery, however, low yields and poor selectivity of products, high char formation, and limited to no recycling of transition-metal-based catalyst involved in lignin depolymerization demands attention to enable practical-scale lignocellulosic biorefineries. In this study, we demonstrate the catalytic depolymerization of ionic liquid-based biorefinery poplar lignin into guaiacols over a reusable zirconium phosphate supported palladium catalyst. The essence of the study lies in the high conversion (>80 %), minimum char formation (7-16 %), high yields of guaiacols (up to 200 mg / g of lignin), and catalyst reusability. Both solid residue, liquid stream, and gaseous products were thoroughly characterized using ICP-OES, PXRD, CHN analysis, GC-MS, GPC, and 2D NMR to understand the hydrogenolysis pathway.

Advanced one-pot deconstruction and valorization of lignocellulosic biomass into triacetic acid lactone using Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides is capable of co-utilization of complex carbon sources and robust growth from lignocellulosic hydrolysates. This oleaginous yeast is therefore an attractive host for heterologous production of valuable bioproducts at high titers from low-cost, deconstructed biomass in an economically and environmentally sustainable manner. Here we demonstrate this by engineering R. toruloides to produce the polyketide triacetic acid lactone (TAL) directly from unfiltered hydrolysate deconstructed from biomass with minimal unit process operations. RESULTS: Introduction of the 2-pyrone synthase gene into R. toruloides enabled the organism to produce 2.4 g/L TAL from simple media or 2.0 g/L from hydrolysate produced from sorghum biomass. Both of these titers are on par with titers from other better-studied microbial hosts after they had been heavily engineered. We next demonstrate that filtered hydrolysates produced from ensiled sorghum are superior to those derived from dried sorghum for TAL production, likely due to the substantial organic acids produced during ensiling. We also demonstrate that the organic acids found in ensiled biomass can be used for direct synthesis of ionic liquids within the biomass pretreatment process, enabling consolidation of unit operations of in-situ ionic liquid synthesis, pretreatment, saccharification, and fermentation into a one-pot, separations-free process. Finally, we demonstrate this consolidation in a 2 L bioreactor using unfiltered hydrolysate, producing 3.9 g/L TAL. CONCLUSION: Many steps involved in deconstructing biomass into fermentable substrate can be combined into a distinct operation, and directly fed to cultures of engineered R. toruloides cultures for subsequent valorization into gram per liter titers of TAL in a cost-effective manner.

Renewable Schiff-Base Ionic Liquids for Lignocellulosic Biomass Pretreatment.

Growing interest in sustainable sources of chemicals and energy from renewable and reliable sources has stimulated the design and synthesis of renewable Schiff-base (iminium) ionic liquids (ILs) to replace fossil-derived ILs. In this study, we report on the synthesis of three unique iminium-acetate ILs from lignin-derived aldehyde for a sustainable "future" lignocellulosic biorefinery. The synthesized ILs contained only imines or imines along with amines in their structure; the ILs with only imines group exhibited better pretreatment efficacy, achieving >89% sugar release. Various analytical and computational tools were employed to understand the pretreatment efficacy of these ILs. This is the first study to demonstrate the ease of synthesis of these renewable ILs, and therefore, opens the door for a new class of "Schiff-base ILs" for further investigation that could also be designed to be task specific.

Solubility Studies of Cyclosporine Using Ionic Liquids.

Six ionic liquids (ILs) were selected based on their chemical and physical properties to study the solubility of cyclosporine A. Of these, cyclosporine exhibited higher room temperature solubility in 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) than in acetone, an effective molecular solvent used to solubilize and purify cyclosporine. The solubility of cyclosporine in the ILs dramatically increased at higher temperatures, a critical factor that cannot be varied in a wide range with low boiling molecular solvents. The differences in solubility were explored for cyclosporine purification. Cyclosporine was purified up to ∼93% with n-butylammonium acetate ([C4NH3][OAc]) and could be further purified to 95% using an IL/organic solvent biphasic system. After purification, cyclosporine was recovered as an amorphous solid using the ILs.

Azolate Anions in Ionic Liquids: Promising and Under-Utilized Components of the Ionic Liquid Toolbox.

Owing to their ease of synthesis, diffuse positive charge, and chemical stability, 1-alkyl-3-methylimidazolium cations (i.e., [Cn mim]+ ) are one of the most routinely utilized and historically important components in ionic liquid (IL) chemistry. However, while this is a routinely encountered member of the IL family as cations, relatively few workers have explored the versatile chemistry of azoles to allow their use as an anionic component in ILs, as azolates. Azolate anions possess many of the desired properties for IL formation, including a diffuse ionic charge, tailorable asymmetry, and synthetic flexibility, with the added advantages of not relying on halogen atoms for electron-withdrawing effects, as is commonly encountered with IL anions such as hexafluorophosphate. This review explores the 122 azolate-containing ILs known in the literature (prepared from only 39 disparate azolate anions), with a view to highlighting not only their demonstrated utility as an IL component, but the ways in which the larger scientific community may utilize their advantageous properties for new tailored materials.

Ionic liquids in cross-coupling reactions: "liquid" solutions to a "solid" precipitation problem.

To alleviate the problem of solid salt precipitation when using inorganic bases in cross-coupling reactions, basic anions were combined with the trihexyl(tetradecyl)phosphonium ([P66614]+) cation to ensure an ionic liquid byproduct. If the starting base is also an ionic liquid, as is the case for [P66614][OH]·4MeOH, it can be used as both base and solvent.

Selective Oxidation of 1,6-Hexanediol to 6-Hydroxycaproic Acid over Reusable Hydrotalcite-Supported Au-Pd Bimetallic Catalysts.

Selective oxidation of 1,6-hexanediol into 6-hydroxycaproic acid was achieved over hydrotalcite-supported Au-Pd bimetallic nanoparticles as heterogeneous catalyst using aqueous H2 O2 . N,N-dimethyldodecylamine N-oxide (DDAO) was used as an efficient capping agent. Spectroscopic analyses by UV/Vis, TEM, XPS, and X-ray absorption spectroscopy suggested that interactions between gold and palladium atoms are responsible for the high activity of the reusable Au40 Pd60 -DDAO/HT catalyst.

Direct synthesis of 1,6-hexanediol from HMF over a heterogeneous Pd/ZrP catalyst using formic acid as hydrogen source.

A new approach is developed for hydrogenolytic ring opening of biobased 5-hydroxymethylfurfural (HMF), dehydration product of hexoses, towards 1,6-hexanediol (HDO) under atmospheric pressure. The highest yield of HDO, 43%, is achieved over reusable Pd/zirconium phosphate (ZrP) catalyst at 413 K in the presence of formic acid as hydrogen source. In comparison with various Brønsted and/or Lewis acidic supports, the specific Brønsted acidity on ZrP support effectively accelerated the cleavage of C-O bond in a furan ring.