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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Unveiling Feedstock Variability: Insights into Corn Stover Conversion - Part I: Physicochemical Properties and Self-Degradation

Transforming agricultural waste into biofuels and bioproducts is crucial to advancing a low-carbon bioeconomy. However, the inherent variability in the composition and quality introduces uncertainties in the conversion efficiency and poses challenges in process development. Through integrating a high-throughput conversion system, material characterization techniques, and advanced data analysis tools, this study investigates the variability of corn stover and its subsequent impacts on carbohydrate conversion. The findings reveal that indoor storage substantially reduces the moisture and ash content and soil contamination, while other properties remain largely unchanged. Self-degradation due to microbial activity during storage decreases the carbohydrate content of corn stover but enhances glucose and xylose yields. A negative correlation is observed between sugar yields and lignin content across samples with varying ash and moisture content. The inhibitory effect of lignin diminishes in self-degraded samples likely due to the disrupted cell wall structure. Although self-degradation slightly increases cellulose crystallinity, no strong correlation was observed between the crystallinity and sugar yield. Hot water pretreatment under mild conditions effectively mitigates inherent variability, consistently improving the sugar yield from corn stover by up to 50%. By elucidating the feedstock variability and its impact on convertibility, these findings offer valuable insights into appropriate feedstock handling and management, highlighting potential strategies to address variability challenges.

09 BIOMASS FUELS↗
DOE OSTI2024

Expanded genome and proteome reallocation in a novel, robust Bacillus coagulans strain capable of utilizing pentose and hexose sugars

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans’ robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60%–65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and tricarboxylic acid (TCA) cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate.

carbohydrate metabolism↗
DOE OSTI2024

Insights into the action of phylogenetically diverse microbial expansins on the structure of cellulose microfibrils

Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp. Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2021

Determination of Cellulosic Glucan Content in Starch Containing Feedstocks (Laboratory Analytical Procedure)

Carbohydrates make up a major portion of biomass samples. These carbohydrates are polysaccharides constructed primarily of glucose, xylose, arabinose, galactose, and mannose monomeric subunits. One such polysaccharide is starch, which is present in certain types of biomass. Starch will contribute to the overall glucose measurement when it breaks into the glucose subunit. Starch is often a more readily accessible source of glucose than structurally bound glucose polymers. Due to the different chemical nature of the glucan species, it is often advantageous to determine the amount of cellulosic glucose separately from the starch glucan.

09 BIOMASS FUELS↗
DOE OSTI2026

Functional characterization of glycosyltransferases in duckweed to enable predictive biology

Glycosyltransferases (GTs) catalyze the formation of glycosidic linkages to produce almost all complex carbohydrates. This project used a multi-disciplinary, high-throughput (HTP) biochemical and computational biology approach focused on duckweed as a model energy crop, to study carbohydrate metabolic processes. To achieve this, developed and carried out out high-throughput (HTP) functional characterization of plant glycosyltransferases (GTs) role of enzymatic microenvironments be assessed through a combined proteomic and computational biology approach, and the combined data was used to populate deep-learning frameworks to predict plant GT function. Functional validation achieved through this research is being used to assign gene function and study plant processes at the systems level to efficiently link the genome sequence with gene function. Together, the combined approaches used within this study provide a foundation for how computational prediction, in combination with high-throughput functional validation, can be used to study plant processes at the systems level and translate knowledge gained to efficiently link genome sequence with gene function in a species agnostic manner.

09 BIOMASS FUELS↗
DOE OSTI2023

Cyanobacteria as cell factories for the photosynthetic production of sucrose

Biofuels and other biologically manufactured sustainable goods are growing in popularity and demand. Carbohydrate feedstocks required for industrial fermentation processes have traditionally been supplied by plant biomass, but the large quantities required to produce replacement commodity products may prevent the long-term feasibility of this approach without alternative strategies to produce sugar feedstocks. Cyanobacteria are under consideration as potential candidates for sustainable production of carbohydrate feedstocks, with potentially lower land and water requirements relative to plants. Several cyanobacterial strains have been genetically engineered to export significant quantities of sugars, especially sucrose. Sucrose is not only naturally synthesized and accumulated by cyanobacteria as a compatible solute to tolerate high salt environments, but also an easily fermentable disaccharide used by many heterotrophic bacteria as a carbon source. In this review, we provide a comprehensive summary of the current knowledge of the endogenous cyanobacterial sucrose synthesis and degradation pathways. We also summarize genetic modifications that have been found to increase sucrose production and secretion. Finally, we consider the current state of synthetic microbial consortia that rely on sugar-secreting cyanobacterial strains, which are co-cultivated alongside heterotrophic microbes able to directly convert the sugars into higher-value compounds (e.g., polyhydroxybutyrates, 3-hydroxypropionic acid, or dyes) in a single-pot reaction. We summarize recent advances reported in such cyanobacteria/heterotroph co-cultivation strategies and provide a perspective on future developments that are likely required to realize their bioindustrial potential.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Sn doping on partially dealuminated Beta zeolite by solid state ion exchange for 5‐hydroxymethylfurfural ( 5‐HMF ) production from glucose

Abstract BACKGROUND The conversion of glucose into 5‐hydroxymethylfurfural (5‐HMF) involves the isomerization of glucose to fructose catalyzed by Lewis acid and subsequent dehydration of fructose catalyzed by Brönsted acid. In this work, in order to obtain a high yield of 5‐HMF, a series of Sn‐Al‐Beta catalysts containing both Lewis and Brönsted acid sites were prepared by a convenient solid state ion exchange (SSIE) method using partially dealuminated Beta zeolite and tin(II) acetate. RESULTS The obtained bifunctional catalysts were investigated for one‐pot production of 5‐HMF from glucose in the ionic liquid (IL) [C 4 mim]Cl. Among various catalysts, Sn‐Al‐Beta‐ 4 ‐ 8 exhibited the best catalytic performance, giving a 5‐HMF yield of 54% under optimal reaction conditions (393 K, 2 h), along with a glucose conversion of 81%. The Sn‐Al‐Beta‐ 4‐8 catalyst and IL were regenerated by the extraction of 5‐HMF, and the reactivity was reserved after five runs of recycling. In addition to glucose, the Sn‐Al‐Beta‐ 4‐8 /[C 4 mim]Cl system was also proven to have the potential to effectively convert other carbohydrates into 5‐HMF. CONCLUSION The results suggested that the catalyst is convenient to prepare and has preferable efficiency and stability in the conversion of carbohydrates into 5‐HMF. © 2022 Society of Chemical Industry (SCI).

Zhang, Wei↗
DOE OSTI2020

High-throughput screening of environmental polysaccharide-degrading bacteria using biomass containment and complex insoluble substrates

Abstract Carbohydrate degradation by microbes plays an important role in global nutrient cycling, human nutrition, and biotechnological applications. Studies that focus on the degradation of complex recalcitrant polysaccharides are challenging because of the insolubility of these substrates as found in their natural contexts. Specifically, current methods to examine carbohydrate-based biomass degradation using bacterial strains or purified enzymes are not compatible with high-throughput screening using complex insoluble materials. In this report, we developed a small 3D printed filter device that fits inside a microplate well that allows for the free movement of bacterial cells, media, and enzymes while containing insoluble biomass. These devices do not interfere with standard microplate readers and can be used for both short- (24–48 h) and long-duration (> 100 h) experiments using complex insoluble substrates. These devices were used to quantitatively screen in a high-throughput manner environmental isolates for their ability to grow using lignocellulose or rice grains as a sole nutrient source. Additionally, we determined that the microplate-based containment devices are compatible with existing enzymatic assays to measure activity against insoluble biomass. Overall, these microplate containment devices provide a platform to study the degradation of complex insoluble materials in a high-throughput manner and have the potential to help uncover ecologically important aspects of bacterial metabolism as well as to accelerate biotechnological innovation.

3D printing↗
DOE OSTI2020

Evaluation of near-ambient algal biomass fractionation conditions for bioproduct development

This contribution describes an algal fractionation scheme based on cell lysing and carbohydrate hydrolysis under acidic conditions, coupled with solvent extraction, that produces algal lipids, carbohydrates, and proteinaceous solid from partially dewatered algal biomass. Here, a design of experiments analysis was employed to identify the effect of fractionation conditions on the yields of the three product streams. By selection of appropriate conditions, the process can be steered from simple lipid extraction to near complete fractionation of the biomass. Lipid purification and upgrading were respectively achieved with a low-cost adsorbent and an inexpensive Ni-based catalyst that deoxygenated the lipids via decarboxylation/decarbonylation, an approach offering several advantages over the hydrodeoxygenation-based processes typically employed to convert lipids to hydrocarbons. The proteinaceous solids obtained were found to have much lower ash content as well as higher protein content relative to the untreated algae, enhancing the suitability of this material as a feedstock for the production of bioplastics.

09 BIOMASS FUELS↗
DOE OSTI2022

DISCOVR strain pipeline screening – Part II: Winter and summer season areal productivities and biomass compositional shifts in climate-simulation photobioreactor cultures

Assessing the seasonal biomass productivity and compositional shift dynamics under simulated outdoor culture conditions of the top 22 algae strains selected during Tier I flask screening is an important step in the further prioritization of strains with regard to outdoor pond cultivation. These top 22 strains were subjected to Tier II testing in the PNNL Laboratory Environmental Algae Pond Simulator (LEAPS) photobioreactors, simulating light and temperature conditions of 20 cm deep outdoor ponds during the Arizona winter and summer season. All strains were grown in two consecutive nutrient-replete batch culture experiments at their particular optimal medium salinity to quantify their respective seasonal linear-phase areal biomass productivities. To determine biomass compositional shifts in response to nutrient-depletion, the LEAPS cultures were allowed to enter a 9-day nutrient depletion phase at the end of the second batch run. The following strains were evaluated in winter-season climate-simulated cultures and are listed in the order from highest (7.9 g m -2 day -1 ) to lowest (2.3 g m -2 day -1 ) areal N-replete biomass productivity: Monoraphidium minutum 26B-AM, Tetraselmis striata LANL1001, Chlorella vulgaris LRB AZ-1201, Micractinium reisseri NREL14-F2, Monoraphidium sp. MONOR1, Chlorella vulgaris NREL4-C12, Scenedesmus obliquus UTEX393, Scenedesmus acutus LRB-AP-0401, Nannochloropsis oceanica CCAP849/10, and Stichococcus minutus CCALA727. The following strains were evaluated in summer-season climate-simulated cultures and are listed in the order from highest (31.8 g m -2 day -1 ) to lowest (14.2 g m -2 day -1 ) areal N-replete biomass productivity: Picochlorum renovo NREL39-A8, Scenedesmus obliquus UTEX393, Porphyridium cruentum CCMP675, Picochlorum celeri TG2-WT-CSM/EMRE, Chlorella sorokiniana DOE1116, Stichococcus minor CCMP819, Picochlorum oklahomensis CCMP2329, Chlorella sorokiniana DOE1412 (UTEXB3016), Scenedesmus rubescens NREL46B-D3, Picochlorum soloecismus DOE101, Tetraselmis striata LANL1001, Scenedesmus obliquus DOE 0152.z, and Agmenellum quadruplicatum UTEX2268. All LEAPS cultures experienced a significant reduction in areal biomass productivity in response to nutrient-depletion, from 7-16% in the winter season simulation and up to 1-60% in the summer season simulation. Finally, for 10 of the strains tested, the carbohydrate content more than doubled upon nutrient depletion, and for 9 strains, the lipid content increased by over 50% of the initial content.

09 BIOMASS FUELS↗
DOE OSTI2022

Plant biomass fermentation by the extreme thermophile Caldicellulosiruptor bescii for co-production of green hydrogen and acetone: Technoeconomic analysis

A variety of chemical and biological processes have been proposed for conversion of sustainable low-cost feedstocks into industrial products. Here, a biorefinery concept is formulated, modeled, and analyzed in which a naturally (hemi)cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii, is metabolically engineered to convert the carbohydrate content of lignocellulosic biomasses (i.e., soybean hulls, transgenic poplar) into green hydrogen and acetone. Experimental validation of C. bescii fermentative performance demonstrated 82% carbohydrate solubilization of soybean hulls and 55% for transgenic poplar. A detailed technical design, including equipment specifications, provides the basis for an economic analysis that establishes metabolic engineering targets. This robust industrial process leveraging metabolically engineered C. bescii yields 206 kg acetone and 25 kg H 2 per metric ton of soybean hull, or 174 kg acetone and 21 kg H 2 per metric ton transgenic poplar. Beyond this specific case, the model demonstrates industrial feasibility and economic advantages of thermophilic fermentation.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2023

Applications of biomass-derived solvents in biomass pretreatment – Strategies, challenges, and prospects

We report biomass pretreatment is considered a key step in the 2nd generation biofuel production from lignocellulosic biomass. Research on conventional biomass pretreatment solvents has mainly been focused on carbohydrate conversion efficiency, while their hazardousness and/or carbon intensity were not comprehensively considered. Recent sustainability issues request further consideration for eco-friendly and sustainable alternatives like biomass-derived solvents. Carbohydrate and lignin-derived solvents have been proposed and investigated as green alternatives in many biomass processes. In this review, the applications of different types of biomass pretreatment solvents, including organic, ionic liquid, and deep eutectic solvents, are thoroughly discussed. The role of water as a co-solvent in these pretreatment processes is also reviewed. Finally, current research challenges and prospects of utilizing biomass-derived pretreatment solvents for pretreatment are discussed. Given bioethanol's market potential and increasing public awareness about environmental concerns, it will be a priority adopting sustainable and green biomass pretreatment solvents in biorefinery.

09 BIOMASS FUELS↗
DOE OSTI2024

Biocompatible organosolv fractionation via a novel alkaline lignin-first strategy towards lignocellulose valorization

Simultaneous valorization of both carbohydrate and lignin fractions in lignocellulose remains a great challenge. Herein, a novel lignin-first strategy using triethylene glycol (TEG) under alkaline conditions for effective biomass fractionation producing highly digestible carbohydrates and reactive lignin was developed. Delignification was over 80% and fermentable sugar yields were close to 90% after pretreatment at 90°C. The biocompatibility of TEG allowed direct enzymatic hydrolysis of the solid residue without washing, thus minimizing wastewater generation. Furthermore, the obtained lignin (TEGL) had an uncondensed structure with well-preserved β-O-4 linkage, leading to near-equal aromatic monomer yields compared to cellulolytic enzyme lignin after catalytic-free pyrolysis, demonstrating high valorization potential. Finally, the proposed TEG solvent system is promising for a green and sustainable biorefinery process to achieve the complete utilization of lignocellulose.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Reactive oxygen species alter chemical composition and adsorptive fractionation of soil organic matter

Reactive oxygen species (ROS), formed during redox fluctuations in iron-rich soils, have been known to stimulate lignin degradation, although not much is known about how they alter soil organic matter (SOM) composition and interaction with mineral surfaces. We conducted a laboratory experiment to see how ROS altered SOM composition and adsorptive fractionation onto Fe-mineral surfaces. We reacted water extracts of SOM with ·OH, produced by the Fenton reaction, and then conducted a sorption experiment of the extracts with goethite to analyze the amount and quality of SOM adsorbed. The Fenton reaction preferentially consumed low-O, mostly aromatic molecules, and new high-O molecules were detected post-Fenton, nearly half of which were carbohydrate-like. Although the amount of C adsorbed did not change after oxidation, the post-Fenton adsorbed molecules were more oxidized. Pre-Fenton adsorption was dominated by aromatic molecules (90%), but post-Fenton, the adsorbed molecules were 75% aromatic and 25% carbohydrate-like. We show that the ·OH radical oxidized SOM and shifted patterns of adsorption to a more oxidized pool. Because adsorption to minerals is thought to stabilize SOM, our results suggest that ROS may alter the availability and stabilization patterns of SOM.

Patel, Kaizad F.↗
DOE OSTI2024

Structural characterization of strawberry pomace

Strawberries are a nutrient dense food rich in vitamins, minerals, non-nutrient antioxidant phenolics, and fibers. Strawberry fiber bioactive structures are not well characterized and limited information is available about the interaction between strawberry fiber and phenolics. Therefore, we analyzed commercial strawberry pomace in order to provide a detailed carbohydrate structural characterization, and to associate structures with functions. The pomace fraction, which remained after strawberry commercial juice extraction, contained mostly insoluble (49.1 % vs. 5.6 % soluble dietary fiber) dietary fiber, with pectin, xyloglucan, xylan, β-glucan and glucomannan polysaccharides; glucose, fructose, xylose, arabinose, galactose, fucose and galacturonic acid free carbohydrates; protein (15.6 %), fat (8.34 %), and pelargonidin 3-glucoside (562 μg/g). Oligosaccharides from fucogalacto-xyloglucan, methyl-esterified rhamnogalacturonan I with branched arabinogalacto-side chains, rhamnogalacturonan II, homogalacturonan and β-glucan were detected by MALDI-TOF MS, NMR and glycosyl-linkage analysis. Previous reports suggest that these oligosaccharide and polysaccharide structures have prebiotic, bacterial pathogen anti-adhesion, and cholesterol-lowering activity, while anthocyanins are well-known antioxidants. A strawberry pomace microwave acid-extracted (10 min, 80 °C) fraction had high molar mass (2376 kDa) and viscosity (3.75 dL/g), with an extended rod shape. A random coil shape, that was reported previously to bind to phenolic compounds, was observed for other strawberry microwave-extracted fractions. These strawberry fiber structural details suggest that they can thicken foods, while the polysaccharide and polyphenol interaction indicates great potential as a multiple-function bioactive food ingredient important for gut and metabolic health.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Towards the sustainable conversion of corn stover into bioenergy and bioproducts through biochemical route: Technical, economic and strategic perspectives

Corn stover (CS) is one of the most abundant agricultural wastes and is ubiquitous around the world that is left over after grain harvest and accounts for 47–50% dry mass of the total grain yield. The global CS yield is 1661.25 million tons/year, which is 27.2% of the total agricultural waste. CS consists of about 34.5% stems, 32.3% leaves, 14.3% husks, 12.3% cobs and 6.6% flowers, with up to 45% cellulose, 30% hemicellulose and 20% lignin. Further, due to its favorable economic and environmental potential, CS is considered an ideal raw material for producing biofuels and biobased chemicals, which can largely be divided into carbohydrate and lignin platforms. However, the techno-economic perspectives of CS-based biorefineries have remained questionable due to the inadequate supply chain logistics, lack of cost-effective conversion technologies, limited scale-up of the product-specific technologies, and lower competitiveness in the market compared to their counterparts, such as sugar and starch-based biorefineries. While CS is converted by thermochemical and biochemical approaches, the latter is considered to be more sustainable for its selective conversion under mild conditions using microorganisms. This review aimed to critically discuss the latest research and developments on the biochemical conversion of CS into biofuels and chemicals. In particular, this paper covers the market potential of biofuels and chemicals to which CS can make a significant contribution, technological developments in the microbial conversion of CS, major biofuels and chemicals produced from CS-derived carbohydrates and lignin, and the technoeconomic perspectives of CS-based biorefinery.

09 BIOMASS FUELS↗
DOE OSTI2021

Lignin-enzyme interaction: A roadblock for efficient enzymatic hydrolysis of lignocellulosics

Efficiently producing second-generation biofuels from biomass is of strategic significance and meets sustainability targets, but it remains a long-term challenge due to the existence of biomass recalcitrance. Lignin contributes significantly to biomass recalcitrance by physically limiting the access of enzymes to carbohydrates, and this could be partially overcome by applying a pretreatment step to directly target lignin. However, lignin typically cannot be completely removed, and its structure is also significantly altered during the pretreatment. As a result, lignin residue in the pretreated materials still significantly hindered a complete conversion of carbohydrate to its monosugars by interacting with cellulase enzymes. The non-productive adsorption driven by hydrophobic, electrostatic, and/or hydrogen bonding interactions is widely considered as the major mechanism of action governing the unfavored lignin-enzyme interaction. One could argue this type of interaction between lignin residue and the activated enzymes is the major roadblock for efficient enzymatic hydrolysis of pretreated lignocellulosics. To alleviate the negative effects of lignin on enzyme performance, a deep understanding of lignin structural transformation upon different types of pretreatments as well as how and where does lignin bind to enzymes are prerequisites. In the last decade, the progress toward a fundamental understanding of lignin-enzyme interaction, structural characterization of lignin during pretreatment and/or conformation change of enzyme during hydrolysis is resulting in advances in the development of methodologies to mitigate the negative effect of lignin. Here in this review, the lignin structural transformation upon different types of pretreatments and the inhibition mechanism of lignin in the bioconversion of lignocellulose to bioethanol are summarized. Some technologies to minimize the adverse impact of lignin on the enzymatic hydrolysis, including chemical modification of lignin, adding blocking additives, and post-treatment to remove lignin were also introduced. The production of liquid biofuels from lignocellulosic biomass has shown great environmental benefits such as reducing greenhouse gas emissions and mitigate climate change. By addressing the root causes of lignin-enzyme interaction and how to retard this interaction, it is our hope that this comprehensive review will pave the way for significantly reducing the high cost associated with the enzymatic hydrolysis process, and ultimately achieving a cost-effective and sustainable biorefinery system.

09 BIOMASS FUELS↗