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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Development of energetic and enzymatic limitations on microbial carbon cycling in soils

Soil organic carbon (SOC) constitutes an important reservoir in the global carbon cycle that is vulnerable to transformation and loss from land use and climate change. Anoxic conditions protect SOC from microbial degradation through limiting the energetics of respiration and inhibiting extracellular oxidative enzymes. Given growing evidence of prevalent anaerobic microsites in upland soils, we designed an experiment testing the development of dissolved organic carbon (DOC) signatures of energetic and enzymatic limitations on microbial carbon utilization across simulated soil aggregates or peds. Reactors comprised a soil column “aggregate” underlying an advective “macropore” channel. Soils received downward diffusive inputs of aerated porewater media with added nitrate, sulfate, or no amendment—where native ferrihydrite served as dominant anaerobic terminal electron acceptor (TEA). After 40 days, added nitrate resulted in highest bulk respiration and DOC production while sulfate did not differ from the control. Nominal oxidation state of carbon (NOSC) was higher (more favorable) with added TEAs at soil surfaces and decreased with depth, while NOSC in the non-amended soil remained lower and constant with depth. DOC generally increased with depth, which along with decreasing NOSC values indicates joint electron-donor and acceptor control over respiration energetics. Of all organic compound classes, only the relative abundance of phenolics increased between 0 and 0.5 cm depth, which aligns with the oxic-anoxic transition and suggests oxidative enzyme inhibition. Furthermore, our results suggest that oxygen limitation within upland soil aggregates may preserve SOC via both energetic and enzymatic C protection mechanisms, which are vulnerable upon exposure to oxygen.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Stepwise allomorphic transformations by alkaline and ethylenediamine treatments on bamboo crystalline cellulose for enhanced enzymatic digestibility

Efficient modification and decrystallization of cellulose Iβ in lignocellulosic biomass are potential ways to overcome biomass recalcitrance and facilitates enzymatic degradation. Herein, we have treated moso bamboo and microcrystalline cellulose separately by either alkali or ethylenediamine (EDA) treatments, as well as using both in succession. The pretreatments successfully transformed the initial cellulose Iβ allomorph into cellulose II, III I , and cellulose III II . Crystal structures and crystallinities were determined by Rietveld X-ray diffraction and CP/MAS 13 C NMR. Comparatively, the samples that were sequentially treated by EDA and NaOH had low crystallinity (~50%), larger d-spacings of ~4.4 Å for the hydrophobic (110) planes, and less condensed microfibrillar aggregation. The factors simultaneously contributed to as high enzymatic hydrolysis yield as 89.5% and 91.7% respectively, followed by the yields of mercerized cellulose II and cellulose III II allomorph. Here, the stepwise allomorphic transformations may provide new strategies on improving efficiency of saccharification process and biomass utilization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Aqueous buffer solution-induced crystallization competes with enzymatic depolymerization of pre-treated post-consumer poly (ethylene terephthalate) waste

In this paper, the complex relationship between kinetics and substrate morphology during enzymatic depolymerization of melt processed poly(ethylene terephthalate) (PET) is explored and the effects of competing transformations are analyzed. Extruded PET substrates from post-consumer recycled PET (RPET) bottles flakes subjected to enzymatic depolymerization are examined to reveal increases in crystallinity from ~10% post-extrusion to >30% after 3 days of depolymerization as well as increases in glass transition temperature (T g ) from ~66°C to > 80°C. Further investigation into this behavior shows that post-extrusion RPET substrates do not exhibit changes in crystallinity when subjected to dry annealing at 65°C in an oven over 7 days, but they do experience annealing in depolymerization buffer solution with no enzymes at 65°C within 3 days. This difference may be attributed to plasticization of PET in the presence of water, also known as solvent induced crystallization. The impact of this plasticized annealing behavior is demonstrated by subjecting RPET substrates to increasing enzyme-to-substrate loads. As enzyme load increases, overall conversion of substrates increases despite crystallinity also increasing to similar levels regardless of the initial enzyme loading. The competition between depolymerization and crystallization suggests that the rate at which PET substrates are depolymerized at the operational temperature is integral to achieving high conversion to monomeric product. This, in turn, also suggests that savings from lowered enzyme loadings may be more detrimental than helpful in pursuing the most cost-effective recycling systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
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 OSTI2022

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on pre-oxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. Here, by offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Identifying transcription factors that reduce wood recalcitrance and improve enzymatic degradation of xylem cell wall in Populus

Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.

09 BIOMASS FUELS↗
DOE OSTI2025

Nitrous oxide production via enzymatic nitroxyl from the nitrifying archaeon Nitrosopumilus maritimus

Ammonia oxidizing archaea (AOA) are among the most abundant microorganisms on earth and are known to be a major source of nitrous oxide (N 2 O) emissions, although biochemical origins of this N 2 O remain unknown. Enzymological details of AOA nitrogen metabolism are broadly unavailable. We report the recombinant expression, purification, and characterization of a multicopper oxidase, Nmar_1354, from the AOA Nitrosopumilus maritimus . We show that Nmar_1354 selectively produces nitroxyl (HNO) by coupling the oxidation of the obligate nitrification intermediate hydroxylamine (NH 2 OH) to dioxygen (O 2 ) reduction. This HNO undergoes several downstream reactions, although the major fates are production of N 2 via reaction with NH 2 OH and dimerization with itself to yield N 2 O. These results afford one plausible enzymatic origin for N 2 O release by AOA. Moreover, these results reveal a physiologically relevant enzymatic reaction for producing HNO, an enigmatic nitrogen oxide speculated to be operative in cellular signaling and in energy transduction.

Voland, Robert W. (ORCID:0000000201061257)↗
DOE OSTI2024

Substitution Models of Protein Evolution with Selection on Enzymatic Activity

Abstract Substitution models of evolution are necessary for diverse evolutionary analyses including phylogenetic tree and ancestral sequence reconstructions. At the protein level, empirical substitution models are traditionally used due to their simplicity, but they ignore the variability of substitution patterns among protein sites. Next, in order to improve the realism of the modeling of protein evolution, a series of structurally constrained substitution models were presented, but still they usually ignore constraints on the protein activity. Here, we present a substitution model of protein evolution with selection on both protein structure and enzymatic activity, and that can be applied to phylogenetics. In particular, the model considers the binding affinity of the enzyme–substrate complex as well as structural constraints that include the flexibility of structural flaps, hydrogen bonds, amino acids backbone radius of gyration, and solvent-accessible surface area that are quantified through molecular dynamics simulations. We applied the model to the HIV-1 protease and evaluated it by phylogenetic likelihood in comparison with the best-fitting empirical substitution model and a structurally constrained substitution model that ignores the enzymatic activity. We found that accounting for selection on the protein activity improves the fitting of the modeled functional regions with the real observations, especially in data with high molecular identity, which recommends considering constraints on the protein activity in the development of substitution models of evolution.

Ferreiro, David↗
NASA NTRS1974

Enzymatic regeneration of adenosine triphosphate cofactor

Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

Marshall, D. L.↗
NASA NTRS1993

Evaluation of Enzymatic Hydrolysis of CELSS Wheat Residue Cellulose at a Scale Environment to NASA's KSC Breadboard Project

Biomass processing at the Kennedy Space Center CELSS breadboard project has focused on the evaluation of breadboard-scale enzymatic hydrolysis of wheat residue cellulose (25%, w/w). Five replicate runs of cellulase production by Trichoderma reesei (QM9414) and enzymatic hydrolysis of residue cellulose were completed. Enzymes were produced in 1 0 days (5 L, 25 g (dry weight) residue). Cellulose hydrolysis (12 L, 50 g (dry weight) residue) using these enzymes produced 5.5 to 6.0 g glucose liter(exp -1) in 7 days. Cellulose conversion efficiency was 29%. These processes are feasible technically on a breadboard scale, but would only increase the edible wheat yield 10%.

Strayer, Richard F.↗
DOE OSTI2022

The bamboo delignification saturation point in alkaline hydrogen peroxide pretreatment and its association with enzymatic hydrolysis

A delignification saturation point (DSP) was observed for bamboo alkaline hydrogen peroxide pretreatment (AHP). Lignin removal was increased from 52.23% to ~70% when increasing H 2 O 2 dosage from 0% to 2% at the optimum pH, but it cannot be further reinforced as increasing the H 2 O 2 . With partial lignin preserved, the glucan hydrolysis yield was found to have a ceiling of ~80%. This work indicated a strong association between enzymatic digestibility and lignin removal. Anatomical analysis by fluorescence microscope and confocal Raman microscope revealed that the undegradable lignin was mainly existing in the cell corner of sclerenchyma fibers, causing the DSP in the bamboo AHP. Finally, the residual lignin in pretreated bamboo was characterized with GPC, HSQC NMR, and 31 P NMR, which revealed the nature of DSP. This study could help to understand the lignin modification during the AHP and further contribute to the establishment of a chemical-saving biorefinery.

09 BIOMASS FUELS↗
DOE OSTI2023

Highly Stable Fe/Co-TPY-MIL-88(NH 2 ) Metal–Organic Framework (MOF) in Enzymatic Cascade Reactions for Chemiluminescence-Based Detection of Extracellular Vesicles

Metal-Organic Frameworks (MOFs) can deliver many advantages when acting as enzyme mimics to assist with signal amplification in molecular detection: they have abundant active catalytic sites per unit volume of the material; their structures and elemental compositions are highly tunable, and their high specific surface area and porous property can assist with target separation and enrichment. In the present work, we have demonstrated that, by adding the pore partition agent, 2,4,6-tris(4-pyridyl)pyridine (TPY) during synthesis of the bimetallic Fe/Co-MIL-88(NH 2 ) MOF to block the open metal sites, a highly porous MOF of Fe/Co-TPY-MIL-88(NH 2 ) can be produced. This material also exhibits high stability in basic solutions and biofluids and possesses high peroxidase-mimicking activity, which can be utilized to produce long-lasting chemiluminescence (CL) from luminol and H 2 O 2 . Moreover, acting as the peroxidase-mimic, the Fe/Co-TPY-MIL-88(NH 2 ) MOF can form the enzymatic cascade with glucose oxidase (GOx) for biomarker detection. Further, when applied to detect extracellular vesicles (EVs), the MOF material and GOx are brought to the proximity on the EVs through two surface proteins, which triggers the enzyme cascade to produce high CL from glucose and luminol. EVs within the concentration range of 5 × 10 5 - 4 ×10 7 particles/mL can be detected, and the method can be used to analyze EV contents in human serum without sample preparation and EV purification. Overall, our work demonstrates that the high versatility and tunability of the MOF structures could bring in significant benefits to biosensing and enable ultrasensitive detection of biomarkers with judicious material designs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

The effect of switchgrass plant cell wall properties on its deconstruction by thermochemical pretreatments coupled with fungal enzymatic hydrolysis or Clostridium thermocellum consolidated bioprocessing

A combination of thermochemical pretreatment and biological digestion technologies is usually required to overcome lignocellulosic recalcitrance and accomplish effective biomass deconstruction. In this study, we aimed to understand switchgrass breakdown by hydrothermal, dilute acid, dilute alkali, and co-solvent enhanced lignocellulosic fractionation (CELF) pretreatments followed by application of traditional fungal enzymatic hydrolysis (EH) and Clostridium thermocellum consolidated bioprocessing (CBP) to the resulting solids. Unpretreated and pretreated switchgrass and their EH and CBP residues were characterized by a suite of analytical techniques to understand structural changes that occurred during deconstruction. CELF pretreated solids showed the highest accessibility and digestibility by both EH and CBP followed by dilute alkali and then dilute acid/hydrothermal pretreated solids. Lignin removal from biomass had a more positive impact on substrate accessibility and digestibility than did xylan removal, while xyloglucan removal by pretreatment appeared essential for cellulose digestion by fungal enzymes. The extent of CBP digestion of cellulose and non-cellulosic glycans was larger than that by EH. Unlike dilute alkali pretreatment, cellulose crystallinity increased for acid-based pretreatments in the following order: hydrothermal, dilute acid, and CELF. Acid-based pretreatments also substantially reduced cellulose degree of polymerization. All thermochemical and biological digestion approaches increased syringyl to guaiacyl lignin (S/G) ratio and reduced β-O-4 lignin interunit linkage and hydroxycinnamates content from levels in unpretreated switchgrass. The substantial increase in S/G ratio after hydrothermal and dilute alkali preatreatments suggested that high temperatures or alkali removed a large portion of G lignin from switchgrass.

09 BIOMASS FUELS↗
DOE OSTI2022

Solvent‐free enzymatic esterification of free fatty acids with glycerol for biodiesel application: Optimized using the Taguchi experimental method

Abstract Presence of free fatty acids along with glycerides poses a technical difficulty for biodiesel production. This work used a Taguchi L9 design to optimize the solvent‐free enzymatic process to result in the esterification of oleic acid with glycerol. Under optimal conditions the esterification reaction temperature of 60°C, enzyme dose of 5 wt%, glycerol: oleic acid molar ratio of 5:1, and reaction time of 3 h, a 75.235 ± 2.19% conversion of oleic acid to esters was achieved. With the addition of molecular sieves, the conversion increased to 86.73% ± 1.09%. However, using the parameters predicted by Taguchi design (60°C, 5 wt%, 5:1, and 4.5 h), 88.5% ± 1.11% of oleic acid could be converted to esters derivative. Diglycerides were the major product, and the reaction equilibrium was attained after 4 h. The immobilized enzyme could be used up to seven times with only a 10% reduction in the conversion. Thus, the process can efficiently reduce the free fatty acid content of oil to make it suitable for biodiesel production.

09 BIOMASS FUELS↗
DOE OSTI2022

Enzymatic synthesis of kraft lignin-acrylate copolymers using an alkaline tolerant laccase

Abstract Softwood kraft lignin is a major bioresource relevant to the production of sustainable bio-based products. Continued challenges to lignin valorization, however, include poor solubility in organic solvents and in aqueous solutions at neutral pH. Herein, an alkaline tolerant laccase was used to graft acrylate functionalities onto softwood kraft lignin, which is expected to enhance the reactivity of lignin with isocyanate when producing bio-based polyurethanes. Proton nuclear magnetic resonance, Fourier-transform infrared spectroscopy, and high-performance liquid chromatography were used to confirm successful grafting of the acrylate monomer onto lignin and verify the importance of including tert-butyl hydroperoxide as an initiator in the grafting reaction. Laccase-mediated grafting of softwood kraft lignin under alkaline conditions produced lignin products with approximately 30% higher hydroxyl value and higher reactivity toward isocyanate. The reported enzymatic and aqueous process presents an opportunity for the sustainable valorization of softwood kraft lignin. Key points • Softwood kraft lignin displayed high phenolic hydroxyl content, polydispersity index and average molecular weight • Grafting hydroxyethyl acrylate (HEA) monomer onto kraft lignin by laccase was successful at 60 °C and alkaline conditions • Lignin-HEA grafted copolymer showed an increase in total OH value and an increase in average molecular weight

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A combination of deep eutectic solvent and ethanol pretreatment for synergistic delignification and enhanced enzymatic hydrolysis for biorefinary process

Here, a novel pretreatment system containing deep eutectic solvents and ethanol (DES-E) for synergistic carbohydrate conversion and delignification was reported in this study. The DES-E pretreatment resulted in an enhanced glucose yield compared to individual DES and ethanol pretreatment for the three tested biomass, including Broussonetia papyrifera, corn stover and pine. To further explore the delignification mechanism, the solubilized lignin and residual lignin from Broussonetia papyrifera was recovered and extracted, then thoroughly characterized. The highest total OH content was found in the DES-E solubilized lignin, which could be used as antioxidant. The presence of ethanol in pretreatment liquor could protect the β-O-4 substructure from breakage and reduce lignin condensation, which favors the subsequent enzymatic hydrolysis. Comparable glucose yield and delignification performance was achieved by recycled DES. DES-E pretreatment offers a promising method for lignin isolation and cellulose digestibility improvement simultaneously.

09 BIOMASS FUELS↗
DOE OSTI2023

A novel mineral-acid free biphasic deep eutectic solvent/γ-valerolactone system for furfural production and boosting the enzymatic hydrolysis of lignocellulosic biomass

The failure of hemicellulose valorization in a deep eutectic solvent (DES) pretreatment has become a bottleneck that challenges its further development. Here, to address this issue, this study developed a DES/GVL (γ-valerolactone) biphasic system for effective hemicellulose-furfural conversion, enhanced cellulose saccharification and lignin isolation. The results indicated that the biphasic system could significantly improve the lignin removal (as high as 89.1%), 86.0% higher than the monophasic DES, accompanied by ~100% hemicellulose degradation. Notably, the GVL in the biphasic solvent restricted the condensation of hemicellulose degradation products, which as a result generated large amount of furfural in the pretreatment liquid with a yield of 68.6%. With the removal of hemicellulose and lignin, cellulose enzymatic hydrolysis yield was boosted and reached near 100%. This study highlighted that the novel DES/GVL is capable of fractionating the biomass and benefiting their individual utilization, which could provide a new biorefinery configuration for a DES pretreatment.

09 BIOMASS FUELS↗