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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Saccharide analysis of onion outer epidermal walls

Abstract Background Epidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics. Results Epidermal walls were depolymerized by acidic methanolysis combined with 2M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per- O -trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by 13 C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes). Conclusions Although exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~ 50%), cellulose (~ 20%), galactan (~ 10%), xyloglucan (~ 10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.

09 BIOMASS FUELS↗
DOE OSTI2020

Deacetylation and Mechanical Refining (DMR) and Deacetylation and Dilute Acid (DDA) Pretreatment of Corn Stover, Switchgrass, and a 50/50 Corn Stover/Switchgrass Blend

The most economical feedstock available to future lignocellulosic biorefineries may be a feedstock blend. Corn stover, switchgrass, and a 50:50 blend of corn stover and switchgrass were subjected to three different dilute alkali deacetylation conditions in a 90 L reactor prior to either mechanical refining (DMR) in a disc refiner followed by a Szego mill or dilute acid pretreatment (DDA) at five different conditions in a 4 L batch steam explosion reactor. The forty-five DDA and nine DMR slurries were subjected to high solids enzymatic hydrolysis at an enzyme loading of 15 mg protein per gram cellulose using Novozymes Cellic CTec3 (DDA) or a 4:1 mix of Novozymes Cellic CTec3:HTec 3 (DMR). The highest glucose yield was achieved using DMR processed corn stover from the high severity deacetylation condition (92 °C and 100 kg NaOH/ODMT) at 95.9% ± 0.8%, while the greatest monomeric glucose yields for switchgrass were between 75% to 80%. The glucose yields from saccharification of the 50:50 corn stover/switchgrass blend was approximately the average of the two individual feedstocks for both DDA and DMR processes. Statistical analysis showed that for a given feedstock type dilute alkali deacetylation severity had a significant impact (p < 0.05) on enzymatic hydrolysis yields, and for a given deacetylation severity the feedstock type had a significant effect on glucose conversion with the addition of switchgrass resulting in lower glucose yields. A blending strategy to maximize enzymatic hydrolysis sugar yields while reducing carbohydrate solubilization during the alkaline treatment step was proposed that allows deacetylation of feedstocks with different recalcitrance at optimal conditions for that feedstock, then blending prior to enzymatic hydrolysis.

BIOMASS FUELS,INORGANIC, ORGANIC, PHYSICAL, AND AN↗
DOE OSTI2020

Sustainable Seaweed Biotechnology Solutions for Carbon Capture, Composition, and Deconstruction

Seaweeds or macroalgae are attractive candidates for carbon capture, while also supplying a sustainable photosynthetic bioenergy feedstock, thanks to their cultivation potential in offshore marine farms. Seaweed cultivation requires minimal external nutrient requirements and allows for year-round production of biomass. Despite this potential, there remain significant challenges associated with realizing large-scale, sustainable agronomics, as well as in the development of an efficient biomass deconstruction and conversion platform to fuels and products. Recent biotechnology progress in the identification of enzymatic deconstruction pathways, tailored to complex polymers in seaweeds, opens up opportunities for more complete utilization of seaweed biomass components. Effective, scalable, and economically viable conversion processes tailored to seaweed are discussed and gaps are identified for yield and efficiency improvements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Background: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Results: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H 2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. Conclusion: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O 2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

TEA Modeling to Quantify Economic Implications for Biorefinery Processing of Isolated Anatomical Fractions of Corn Stover

The Feedstock-Conversion Interface Consortium (FCIC; https://www.energy.gov/sites/prod/files/2020/01/f70/beto-fcic-overview-web.pdf), a collaboration of nine national laboratory partners, seeks to understand impacts of feedstock attributes on biorefinery performance. It is hypothesized that different individual anatomical fractions of corn stover vary in composition and recalcitrance, such that processing each fraction on its own through dedicated campaigns may enable better biorefinery economics overall relative to processing the whole stover material. This presentation focuses on techno-economic analysis (TEA) modeling to quantify the yield and cost ramifications for processing isolated anatomical fractions of corn stover through a low-temperature conversion biorefinery, reflecting a biochemical processing pathway consisting of biomass deconstruction through pretreatment and enzymatic hydrolysis, sugar fermentation and upgrading to hydrocarbon fuels, and lignin upgrading to value-added coproducts. Commercial-scale process simulation and economic evaluation leveraged experimental and analytical data from FCIC researchers for conversion of whole corn stover plus three individual anatomical fractions (cobs, husks, and stalks) across key steps of the conversion process. Our assessment found encouraging potential for biorefinery economic gains that may be achieved through this approach. TEA results indicated fuel yields varying from 29-44 gallons gasoline equivalent (GGE)/dry ton for the individual anatomical fractions compared to whole stover at 34 GGE/ton, equating to minimum fuel selling prices (MFSPs) between $6.37-$10.18/GGE for the fractions versus $8.76/GGE for whole stover (when lignin is burned), or $9.15-$15.19/GGE for the fractions versus $13.11/GGE for whole stover (when lignin is upgraded to coproducts, based on current experimental performance levels). Cobs and husks demonstrated the ability to achieve the highest fuel yields and lowest MFSPs, outperforming whole stover, while stalks led to the opposite result, as a composite reflection of compositional differences and process convertibility. Notably, even when taking the weighted average of the results reflecting each anatomical fraction weighted by its corresponding makeup of corn stover, this feasibility TEA screening supports feedstock cost allowances on the order of roughly $22-$29/ton as may reflect accommodating additional biomass fractionation equipment during feedstock pre-processing upstream of the conversion biorefinery gate to separate corn stover into such constituent fractions. Or viewed differently, the weighted average MFSP for the fractions was found to be $0.31-$0.32/GGE lower than the MFSP for the whole stover basis across either lignin scenario, when maintaining a fixed biomass feedstock cost. These findings highlight favorable implications for biorefinery economics as may be achieved by moving to a staged campaign approach for processing different corn stover fractions sequentially. Further opportunities exist for future work to fill in data gaps for remaining anatomical constituents (e.g. leaves) that were not included in the initial experimental studies, though are expected to maintain similar trends.

biochemical processing pathway↗
DOE OSTI2026

Engineering Enantiocomplementary Protoglobins for Stereoconvergent Construction of N -Alkylated α-Aminoketones

The synthesis of enantiopure compounds from a mixture of E/Z alkenes represents a notable challenge in synthetic chemistry. While enzymes excel in achieving unparalleled selectivity, their inherent specificity often confines activity to a single stereoisomeric substrate, consequently restricting the overall efficiency of such transformations. Here, we demonstrate that protoglobin-derived hemoproteins can catalyze stereoconvergent intermolecular amination using simple N-alkyl hydroxylamines as nitrene precursors, a transformation which remains elusive in synthetic chemistry. These engineered enzymes process E/Z mixtures of silyl enol ethers, enabling the precise incorporation of N-alkyl amino moieties (−NHAlkyl) into diverse molecular structures (up to 79% yield and 95% ee). Two complementary protoglobin variants were engineered using directed evolution to enable enantiodivergent synthesis of both enantiomers of α-aminoketones. This enzymatic platform achieves stereoconvergent and enantiodivergent transformations, facilitating the conversion of simple chemicals into an array of valuable pharmaceutical compounds featuring aminoketone functionalities.

Alcohols↗
DOE OSTI2023

Techno-Economic Case Study: Low-Temperature Conversion Performance Based on Isolated Anatomical Fractions of Corn Stover

This report summarizes analysis conducted to support a case study under the Feedstock-Conversion Interface Consortium (FCIC) focused on techno-economic analysis (TEA) modeling to quantify the yield and cost ramifications for processing isolated anatomical fractions of corn stover through a low-temperature conversion (biochemical pathway) biorefinery. It is hypothesized that different individual anatomical fractions of corn stover vary in composition and recalcitrance, such that processing each fraction on its own e.g. through dedicated campaigns may enable economic benefits relative to processing the whole stover material. To investigate this concept, we worked with FCIC researchers to obtain data on compositional analysis, as well as processing conditions and yields, for conversion of whole corn stover plus three individual anatomical fractions (cobs, husks, and stalks) across key steps of the biorefinery conversion process within FCIC research scope, namely pretreatment and enzymatic hydrolysis. After running the data through the TEA models, fractionated cobs and husks achieved higher fuel yields and lower minimum fuel selling prices than whole corn stover, but stalks fared worse than whole stover on both metrics. The most direct takeaway from this assessment is that a biorefinery would stand to benefit economically from maximizing the use of cob and husk fractions while avoiding or minimizing the use of stalks, though recognizing practical constraints with this approach in maintaining an equivalent processing capacity (fixed at 2,000 dry metric tonne per day in all cases).

09 BIOMASS FUELS↗
DOE OSTI2023

Techno-Economic Case Study: Low-Temperature Conversion Performance Based on Isolated Anatomical Fractions of Corn Stover

This report summarizes analysis conducted to support a case study under the Feedstock Conversion Interface Consortium (FCIC) focused on techno-economic analysis (TEA) modeling to quantify the process yield and resulting process cost impacts for processing isolated anatomical fractions of corn stover through a low-temperature conversion (biochemical) pathway. It is hypothesized that different individual anatomical fractions of corn stover vary in both composition and recalcitrance, giving biorefineries options in whether and how to deal with fractionated or whole biomass feedstock. By quantifying the techno-economic impacts of this variability, we provide actionable information for end users to understand tradeoffs in conversion system yields and economics in considering feedstock processing decisions at the biorefinery gate. For this study, we worked with FCIC researchers to obtain data on the compositional analysis and conversion performance of whole corn stover alongside three individual anatomical fractions (cobs, husks, and stalks) across key steps of the biorefinery conversion process within FCIC’s research scope—pretreatment and enzymatic hydrolysis. This TEA screening assessment highlighted biorefinery economic trade-offs observed through this approach. Namely, relative to processing whole stover biomass, two of the three anatomical fractions for which composition/conversion data were available (cobs and husks) demonstrated the ability to achieve higher fuel yields and lower minimum fuel selling prices (MFSPs), while the third fraction (stalks) led to the opposite result, as a composite reflection of compositional differences and process convertibility.

cost impacts↗
DOE OSTI2024

Enzymatic depolymerization of polyester: Foaming as a pretreatment to increase specific surface area

Abstract Poly(ethylene terephthalate) (PET) is widely used for its high strength‐to‐weight ratio, gas barrier properties, and chemical resistance. The growing PET use highlights the demand for a better recycling system. Enzymatic recycling, alongside mechanical and chemical methods, is eco‐friendly and yields properties similar to virgin PET. Substrate properties ( T g , crystallinity, and specific surface area [SSA]) and enzyme stability significantly impact conversion efficiency. Higher SSA and lower crystallinity tend to yield improved depolymerization when employing leaf compost‐cutinase (LCC‐ICCG) enzymes. This study explored melt extrusion and foaming as pretreatment techniques to modify PET structural properties, using a low‐cost chemical foaming agent (CFA). The monomer conversion rate and efficiency during depolymerization were measured and related to the processing, extrudate micro‐ and meso‐structure, and polyester type. Pretreated PET substrates showed reduced T g , crystallinity, density, and enhanced SSA, resulting in a 90% mass loss for foamed RPET and VPET substrates within 2 days. In contrast, PET with ~30% of cyclohexanedimethanol comonomer exhibited a nearly 50% lower depolymerization rate, with zero BHET production. It indicates that the combination of low crystallinity, low T g , and high SSA leads to improved monomer conversion. These findings emphasize the significance of amorphization and foaming in enhancing PET enzymatic depolymerization.

42 ENGINEERING↗
DOE OSTI2026

Urea-to-Ammonia Conversion at Proteus mirabilis Modified Pt–Ni/BDD Electrodes

Efficient wastewater recycling technologies are essential for long-duration space missions and sustainable water management on Earth. Here, a bioelectrochemical system integrating Proteus mirabilis with a platinum–nickel-modified boron-doped diamond electrode (Pt–Ni/BDDE) for urea-to-ammonia conversion in synthetic urine is presented. Immobilized P. mirabilis catalyzes enzymatic ureolysis, converting urea into ammonia, which is subsequently oxidized electrochemically, and no direct electrochemical urea oxidation is observed. Cyclic voltammetry (CV) of P. mirabilis on Pt–Ni/BDDE in 0.1 M urea and synthetic urine revealed a broad anodic oxidation peak at approximately 0.55–0.75 V vs Ag/AgCl (sat. KCl), corresponding to ammonia oxidation. Control experiments using bare BDDE and Pt–Ni/BDDE in synthetic urine showed no oxidation peak, establishing that the bioelectrocatalytic response originates exclusively from the bioelectrode interface. Chronoamperometry studies revealed that immobilization potential and time critically influenced bacterial adhesion and electrochemical response, with optimal conditions yielding a maximum current density of 0.0055 mA·cm –2 . These quantitative results establish that microbial ureolysis can be efficiently coupled with advanced electrode materials for urea-to-ammonia conversion, offering a promising self-sustaining strategy for urine processing and water recovery in closed-loop life support systems.

Ammonia↗
DOE OSTI2019

A two-phase substrate model for enzymatic hydrolysis of lignocellulose: application to batch and continuous reactors

Abstract Background Enzymatic hydrolysis continues to have a significant projected production cost for the biological conversion of biomass to fuels and chemicals, motivating research into improved enzyme and reactor technologies in order to reduce enzyme usage and equipment costs. However, technology development is stymied by a lack of accurate and computationally accessible enzymatic-hydrolysis reaction models. Enzymatic deconstruction of cellulosic materials is an exceedingly complex physico-chemical process. Models which elucidate specific mechanisms of deconstruction are often too computationally intensive to be accessible in process or multi-physics simulations, and empirical models are often too inflexible to be effectively applied outside of their batch contexts. In this paper, we employ a phenomenological modeling approach to represent rate slowdown due to substrate structure (implemented as two substrate phases) and feedback inhibition, and apply the model to a continuous reactor system. Results A phenomenological model was developed in order to predict glucose and solids concentrations in batch and continuous enzymatic-hydrolysis reactors from which liquor is continuously removed by ultrafiltration. A series of batch experiments were performed, varying initial conditions (solids, enzyme, and sugar concentrations), and best-fit model parameters were determined using constrained nonlinear least-squares methods. The model achieved a good fit for overall sugar yield and insoluble solids concentration, as well as for the reduced rate of sugar production over time. Additionally, without refitting model coefficients, good quantitative agreement was observed between results from continuous enzymatic-hydrolysis experiments and model predictions. Finally, the sensitivity of the model to its parameters is explored and discussed. Conclusions Although the phenomena represented by the model correspond to behaviors that emerge from clusters of mechanisms, and hence a set of model coefficients are unique to the substrate and the enzyme system, the model is efficient to solve and may be applied to novel reactor schema and implemented in computational fluid dynamics (CFD) simulations. Hence, this modeling approach finds the right balance between model complexity and computational efficiency. These capabilities have broad application to reactor design, scale-up, and process optimization.

09 BIOMASS FUELS↗
DOE OSTI2025

Insights from designing an artificial cascade catalysis system using principles from substrate channeling in enzymes

Generalizing the key requirements of highly-selective, multi-step chemical conversions involving spatially separated reaction centers remains one of the grand challenges of chemistry. Much work towards this effort has focused on decomposing multi-step conversions into their constituent reactions, whose intermediates are successively upgraded in a chemical cascade via diffusion from center to center. This approach for synthesizing more complex molecules takes its cues from biochemical networks, where near-unit conversion of even complex carbohydrates is achieved by upgrading chemical precursors via enzymatic cascades. In this computational study we examine a simple cascade involving coupled Ag and Cu catalysts that sequentially converts CO2 to CO and then CO2 and CO to reduced products, generically named CO2Product and COProduct. The system architecture is inspired by the phenomenon of biological substrate channeling, and components are examined to evaluate their effects on conversion efficiency in the cascade. Aside from a substrate channel linking two reaction centers, we find efficient cascades must also incorporate directional substrate diffusion, compartmentalization of the reaction centers, and proper timing of substrate arrival at the active center. We make explicit linkages between these requirements and chemical conversion in known biological systems, revealing additional control elements that could be incorporated.

CO 2 reduction↗
DOE OSTI2020

Effects of the advanced organosolv pretreatment strategies on structural properties of woody biomass

Plants have evolved their lignocellulosic cell walls through complex structural and chemical mechanisms to protect itself against microbial attacks, which makes native lignocellulosic biomass recalcitrant to enzymatic deconstruction. Pretreatment is a crucial step in the biological conversion of biomass as it can render structural changes in the plant cell wall to reduce the biomass recalcitrance, thus enhancing its sugar release performance. There have been many efforts to develop effective pretreatment technologies to overcome the biomass recalcitrance with a primary focus on the efficient conversion of biomass carbohydrates to liquid fuels, while lignin is significantly underutilized despite its bulk amount and high-value opportunities. In this study, the effects of two recent organosolv pretreatment strategies, co-solvent enhanced lignocellulosic fractionation (CELF) and γ-Valerolactone (GVL) pretreatments, on physicochemical properties of poplar were investigated and compared with the effects of conventional ethanol organosolv pretreatment. Diverse physicochemical properties of biomass including chemical compositions, molecular weights of cellulose and lignin, aromatics and inter-unit linkages of lignin, lignin hydroxyl group contents, cellulose crystallinity, and accessible surface area of cellulose were analyzed before and after pretreatments. The results revealed how each organic solvent pretreatment system affected biomass structural characteristics and recalcitrance.

Meng, Xianzhi↗
DOE OSTI2020

Physico-chemical properties of lignin fractions from acid pretreated corn stover and their effects on enzymatic hydrolysis of microcrystalline cellulose

Lignin has been shown to be a recalcitrance factor in many biomass conversion studies. To better understand the effects of lignin on cellulose conversion, different lignin fractions were extracted from the same dilute acid pretreated corn stover by three sequential isolation methods, namely ethanol extraction, dioxane extraction, and enzyme purification. The physicochemical properties of each lignin fraction including molecular weight distribution, surface area, surface charge, and other structural features varied, depending on the isolation methods. All three lignin fractions had negative surface charges, and ethanol-extracted lignin carried the highest surface charges, followed by dioxane-extracted lignin and cellulase-purified residual lignin. These physicochemical properties of lignin fractions also resulted in different extent of inhibitory effects on enzymatic hydrolysis of microcrystalline cellulose (MCC). Dioxane-extracted lignin exhibited the highest inhibitory effect on glucose release from MCC, followed by the cellulase-purified residual lignin fraction and ethanol-extracted lignin. Furthermore, lignin fractions with higher contents of syringyl (S) substructure and β-O-4 aryl ether interunit linkages showed a stronger negative effect on cellulase hydrolysis of MCC.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Cross-kingdom comparative genomics reveal the metabolic potential of fungi for lignin turnover in deadwood

Deadwood is a major carbon source in forests, and yet the fate of this carbon remains a gap in our understanding of global carbon cycling. Lignin, the most recalcitrant biopolymer in wood, is mainly decayed through extracellular enzymatic and chemical processes initiated by white-rot fungi. However, the intracellular conversion of lignin decay products has been overlooked in the fungal kingdom. Here we integrate comparative genomic and phylogenetic analyses to understand the distribution and evolution of enzymes responsible for modifying lignin-related aromatic compounds—such as decarboxylases, hydroxylases, dioxygenases and other downstream ring-cleavage enzymes—that funnel carbon to central metabolism across the bacterial and the fungal kingdoms. We demonstrate that specific fungal lineages conserve these enzyme families, and that the abilities to enzymatically depolymerize lignin and catabolize lignin-related aromatic compounds are not necessarily coupled. Our analyses also reveal an expanded substrate specificity of aromatic ring-cleavage enzymes during fungal evolution, as well as a clade of extracellular enzymes among them, broadening the spatial range of these biochemical capabilities. Together, our results highlight a large diversity of fungal enzymes and hosts that warrant further investigation for inclusion into carbon cycling models and biotechnological applications for the conversion of aromatic compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Data for Tolerance of Engineered Rhodosporidium toruloides to Sorghum Hydrolysates During Batch and Fed-Batch Lipid Production

Oleaginous yeasts are a promising candidate for the sustainable conversion of lignocellulosic feedstocks into fuels and chemicals, but their growth on these substrates can be inhibited as a result of upstream pretreatment and enzymatic hydrolysis conditions. Previous studies indicate a high citrate buffer concentration during hydrolysis inhibits downstream cell growth and ethanol fermentation in Saccharomyces cerevisiae . In this study, an engineered Rhodosporidium toruloides strain with enhanced lipid accumulation was grown on sorghum hydrolysate with high and low citrate buffer concentrations. Both hydrolysis conditions resulted in similar sugar recovery rates and concentrations. No significant differences in cell growth, sugar utilization rates, or lipid production rates were observed between the two citrate buffer conditions during batch fermentation of R. toruloides . Under fed-batch growth on low-citrate hydrolysate a lipid titer of 16.7 g/L was obtained. Citrate buffer was not found to inhibit growth or lipid production in this engineered R. toruloides strain, nor did reducing the citrate buffer concentration negatively affect sugar yields in the hydrolysate. As this process is scaled-up, $131 per ton of hydrothermally pretreated biomass can be saved by use of the lower citrate buffer concentration during enzymatic hydrolysis.

Conversion↗
DOE OSTI2025

Nanoscale Examination of Chemical and Enzymatic Degradation of Plant Cell Walls

Lignocellulosic materials present the largest source of biomass for biotechnology and green energy. This study aimed at better understanding the cell wall disintegration mechanisms relevant for the biochemical conversion of biomass to carbohydrates. Herein, we examined nanoscale changes in cell wall structure and composition upon industrially relevant chemical and enzymatic treatments to achieve the desired level of breakdown. One treatment involved hydrogen peroxide and acetic acid to remove lignin. Another modification used the cellulase enzyme for cell wall degradation. Band excitation contact resonance atomic force microscopy was used to visualize and mechanically characterize cell wall layers. After cellulase treatments, we detected microcracks across the cell wall. Wet-chemical, Fourier-transform infrared and Raman spectroscopic analyses confirmed the removal of lignin and extractives through acid bleaching, while the enzymatic treatment minimally affected the biopolymer composition. Delignification resulted in cell wall delamination and reduced stiffness. In conclusion, X-ray diffraction revealed changes in cellulose structure and crystallinity.

Soini, Steven A. [Florida Atlantic Univ., Boca Rat↗
DOE OSTI2023

Light‐Driven Metabolic Pathways in Non‐Photosynthetic Biohybrid Bacteria

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. Furthermore, this proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗